U.S. patent application number 13/520775 was filed with the patent office on 2013-06-06 for multifunctional degradable nanoparticles with control over size and functionalities.
This patent application is currently assigned to Vanderbilt University. The applicant listed for this patent is David J. Calkins, Eva M. Harth, Alice E. Van Der Ende. Invention is credited to David J. Calkins, Eva M. Harth, Alice E. Van Der Ende.
Application Number | 20130142733 13/520775 |
Document ID | / |
Family ID | 56682866 |
Filed Date | 2013-06-06 |
United States Patent
Application |
20130142733 |
Kind Code |
A1 |
Harth; Eva M. ; et
al. |
June 6, 2013 |
MULTIFUNCTIONAL DEGRADABLE NANOPARTICLES WITH CONTROL OVER SIZE AND
FUNCTIONALITIES
Abstract
In one aspect, the invention relates to polymers, crosslinked
polymers, functionalized polymers, nanoparticles, and
functionalized nanoparticles and methods of making and using same.
In one aspect, the invention relates to degradable polymers and
degradable nanoparticles. In one aspect, the invention relates to
methods of preparing degradable nanoparticles and, more
specifically, methods of controlling particle size during the
preparation of degradable nanoparticles. In one aspect, the
degradable nanoparticles are useful for complexing, delivering, and
releasing payloads, including pharmaceutically active payloads.
This abstract is intended as a scanning tool for purposes of
searching in the particular art and is not intended to be limiting
of the present invention.
Inventors: |
Harth; Eva M.; (Nashville,
TN) ; Calkins; David J.; (Nashville, TN) ; Van
Der Ende; Alice E.; (Dickson, TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Harth; Eva M.
Calkins; David J.
Van Der Ende; Alice E. |
Nashville
Nashville
Dickson |
TN
TN
TN |
US
US
US |
|
|
Assignee: |
Vanderbilt University
Nashville
TN
|
Family ID: |
56682866 |
Appl. No.: |
13/520775 |
Filed: |
January 4, 2011 |
PCT Filed: |
January 4, 2011 |
PCT NO: |
PCT/US11/20148 |
371 Date: |
January 28, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12651710 |
Jan 4, 2010 |
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13520775 |
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PCT/US2008/082529 |
Jan 5, 2008 |
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12651710 |
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60985608 |
Nov 5, 2007 |
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Current U.S.
Class: |
424/9.6 ;
424/489; 424/78.3 |
Current CPC
Class: |
A61K 9/5153 20130101;
A61K 47/58 20170801; A61K 49/0093 20130101; C08G 63/912 20130101;
A61K 9/0048 20130101; A61K 47/593 20170801; A61K 47/59 20170801;
A61K 49/0041 20130101; A61K 47/595 20170801; C08G 63/08 20130101;
A61K 49/0056 20130101; C08G 63/78 20130101 |
Class at
Publication: |
424/9.6 ;
424/78.3; 424/489 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 9/51 20060101 A61K009/51; A61K 49/00 20060101
A61K049/00 |
Goverment Interests
ACKNOWLEDGMENT
[0002] This invention was made with government support under a
CAREER Award CHE-0645737 awarded by the National Science
Foundation. The United States government has certain rights in the
invention.
Claims
1. A method of treating a ophthalmic disorder comprising
administering to a subject an effective amount of a degradable
polyester nanoparticle pharmaceutical or biologically active agent
complex (nanoparticle complex).
2. The method of claim 1, wherein treating comprises inhibiting
VEGF activity.
3. The method of claim 1, wherein treating comprises inhibiting
carboninc anhydrase activity.
4. The method of claim 1, wherein the nanoparticle complex targets
a cell in an ocular tissue.
5. The method of claim 4, wherein the tissue is selected from
neuronal, fibrous, blood, gangloid, dermal, muscular, amacrine,
bipolar, horizontal, connective, epithelial, and vitreal fluid.
6. The method of claim 5, wherein the tissue is from a region of
the eye selected from sclera, cornea, retina, vitrius fluid, rods,
cones, iris, zonular fibers, aqueous humour, choroid, ciliary
muscle, optic disc, dura mater, optic nerve, fovea, and macula.
7. The method of claim 4, wherein the cell is a neuronal,
epidermal, muscular, or gangloid cell.
8. The method of claim 1, wherein the ophthalmic disorder is
selected from glaucoma, macular degeneration, keratoconus,
Stargardt's disease, scleritis, keratitis, corneal ulcer, and
Thygeson's superficial punctate keratopathy.
9. The method of claim 1, wherein the nanoparticle complex
comprises one or more pharmaceutical or biologically active agents
encapsulated by a degradable polyester nanoparticle.
10. The method of claim 9, wherein the pharmaceutical agent is a
alpha agonist, beta blocker, prostaglandin analog, carbonic
anhydrase inhibitor, or cholinergic.
11. The method of claim 10, wherein the pharmaceutical agent is
selected from triamcinolone, ranibizumab, bevacizumab, pegaptanib
(MACUGEN.RTM.), travoprost, bimatoprost, methazolamide,
brinzolamide, dorzolamide HCl, scetazolamide, memantine, timolol
maleate, betaxolol HCl, levobunolol HCl, metipranolol, timolol
hemihydrate, pilocarpine HCl, carbachol, brimonidine tartrate,
apraclonidine HCl, and latanoprost (XALATAN.RTM.).
12. The method of claim 9, wherein the nanoparticle complex
comprises two or more pharmaceutical agents.
13. The method of claim 12, wherein the two pharmaceutical agents
are dorzolomide HCl and timolol maleate or brimonidine tartrate and
timolol maleate.
14. The method of claim 1, wherein the nanoparticle complex is
administered to the subject via a route selected from ophthalmic,
irrigation, topical, drops, epicutaneous, intravitreal,
intraocular, conjunctival, subconjuctival, intracorneal,
retrobulbar, intravenous, and intramuscular.
15. A composition comprising a degradable polyester nanoparticle
and, encapsulated therein, a biologically active agent, a
pharmaceutically active agent, or an imaging agent.
16. The composition of claim 15, wherein the biologically active
agent is encapsulated within the nanoparticle.
17. The composition of claim 15, wherein the pharmaceutically
active agent is encapsulated within the nanoparticle.
18. The composition of claim 15, wherein the imaging agent is
encapsulated within the nanoparticle.
19. The composition of claim 15, wherein the degradable polyester
nanoparticle comprises a crosslinked degradable nanoparticle having
a polyester backbone and one or more crosslinks having a structure
selected from: ##STR00176## wherein Y is O, S, or N--R, wherein R
is C1-C4 alkyl; ##STR00177## and ##STR00178## wherein L is a
divalent alkyl chain or alkyloxyalkyl chain.
20. The composition of claim 15, wherein the nanoparticle is
produced by crosslinking a polymer comprising: (a) at least one
monomer residue having an optionally substituted structure
represented by a formula: ##STR00179## wherein m is an integer from
0 to 6, and wherein n is an integer from 0 to 2; or (b) at least
one propargyl-functionalized monomer residue having an optionally
substituted structure represented by a formula: ##STR00180##
wherein m.sup.1 is an integer from 0 to 6, and wherein n.sup.1 is
an integer from 0 to 2; or (c) at least one monomer residue having
an optionally substituted structure represented by a formula:
##STR00181## wherein n.sup.2 is an integer from 0 to 2; or (d) at
least one keto-functionalized monomer residue having an optionally
substituted structure represented by a formula: ##STR00182##
wherein n.sup.3 is an integer from 0 to 2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. application Ser.
No. 12/651,710, filed Jan. 4, 2010, which is hereby incorporated
herein by reference in its entirety.
BACKGROUND
[0003] Biodegradable nanoparticles have received increasing
attention as versatile drug delivery scaffolds to enhance the
efficacy of therapeutics. Effectiveness of delivery, however, can
be influenced by the particle size and morphology, as these
parameters can greatly affect the biological function and fate of
the material. [Zweers, M. L. T.; Grijpma, D. W.; Engbers, G. H. M.;
Feijen, J., J. Controlled Release 2003, 87, 252-254.] Narrowly
dispersed particles are highly preferred for use in delivery or
sensing applications with respect to monitoring and predicting
their behavior as their exhibit a more constant response to
external stimuli. [Lubetkin, S.; Mulqueen, P.; Paterson, E. Pesti.
Sci. 1999, 55, 1123-1125.]
[0004] One disadvantage of conventional methods is the
irreproducibility in the size and shape of the particles, since
these can be profoundly influenced by the stabilizer and the
solvent used. [Kumar, M. N. V. R.; Bakowsky, U.; Lehr, C. M.,
Biomaterials 2004, 25, 1771-1777.] Another major drawback of
conventional biodegradable nanoparticles, based on
poly(.epsilon.-caprolactone) and other aliphatic polyesters, is the
lack of pendant functional groups, which can make physiochemical,
mechanical, and biological properties difficult to modify. [(a)
Riva, R.; Lenoir, S.; Jerome, R.; Lecomte, P. Polymer 2005, 46,
8511-8518. (b) Sasatsu, M.; Onishi, H.; Machida, Y. Inter. J.
Pharm. 2006, 317, 167-174.] The availability of functional groups
is a desirable means of tailoring the properties of a particle,
including hydrophilicity, biodegradation rate, and bioadhesion.
[0005] Therefore, there remains a need for methods and compositions
that overcome these deficiencies and that effectively provide
functionalized, degradable nanoparticles with reproducibility in
particle size and shape.
SUMMARY
[0006] In accordance with the purpose(s) of the invention, as
embodied and broadly described herein, the invention, in one
aspect, relates to polymers, crosslinked polymers, functionalized
polymers, nanoparticles, and functionalized nanoparticles and
methods of making and using same.
[0007] Disclosed are methods of administering a pharmaceutical or
biologically active agent to a cell comprising contacting the cell
with a degradable polyester nanoparticle-agent complex
(nanoparticle complex) thereby administering the pharmaceutical or
biologically active agent to the cell.
[0008] Also disclosed are methods of modulating a receptor on a
cell comprising contacting the receptor with a degradable polyester
nanoparticle pharmaceutical or biologically active agent complex,
wherein one or more pharmaceutical agents is encapsulated by a
degradable polyester nanoparticle.
[0009] Also disclosed are methods of inhibiting VEGF activity in an
eye in a subject comprising administering to the subject a
degradable polyester nanoparticle pharmaceutical or biologically
active agent complex (nanoparticle complex).
[0010] Also disclosed are methods of inhibiting carboninc anhydrase
activity in an eye in a subject comprising administering to the
subject an effective amount of a degradable polyester nanoparticle
pharmaceutical or biologically active agent complex (nanoparticle
complex).
[0011] Also disclosed are methods of treating a ophthalmic disorder
comprising administering to a subject an effective amount of a
degradable polyester nanoparticle pharmaceutical or biologically
active agent complex (nanoparticle complex).
[0012] Also disclosed are crosslinked degradable nanoparticle a
having a polyester backbone and one or more crosslinks having a
structure selected from:
##STR00001##
wherein Y is O, S, or N--R, wherein R is C1-C4 alkyl;
##STR00002##
wherein L is a divalent alkyl chain or alkyloxyalkyl chain.
[0013] Also disclosed are compositions comprising a degradable
polyester nanoparticle and, encapsulated therein, a biologically
active agent, a pharmaceutically active agent, or an imaging
agent.
[0014] Also disclosed are kits comprising a first degradable
polyester nanoparticle and a first biologically active agent, first
pharmaceutically active agent, or first imaging agent encapsulated
within the first nanoparticle, and one or more of: a second
biologically active agent, second pharmaceutically active agent, or
second imaging agent encapsulated within the first nanoparticle,
wherein the first biologically active agent, first pharmaceutically
active agent, or first imaging agent is different from the second
biologically active agent, second pharmaceutically active agent, or
second imaging agent; or a second degradable polyester nanoparticle
and a second biologically active agent, second pharmaceutically
active agent, or second imaging agent encapsulated within the
second nanoparticle, wherein the first biologically active agent,
first pharmaceutically active agent, or first imaging agent is
different from the second biologically active agent, second
pharmaceutically active agent, or second imaging agent; a
pharmaceutically acceptable carrier; or instructions for treating a
disorder known to be treatable by the first biologically active
agent or first pharmaceutically active agent.
[0015] Also disclosed are the products of the disclosed
methods.
[0016] Also disclosed are methods of intracellular delivery
comprising administering an effective amount of a disclosed
nanoparticle to a subject.
[0017] Also disclosed are methods for the manufacture of a
medicament for delivery of a biologically active agent, a
pharmaceutically active agent, and/or an imaging moiety comprising
combining at least one disclosed polymer or at least one disclosed
nanoparticle with a pharmaceutically acceptable carrier.
[0018] Also disclosed are uses of a disclosed polymer or a
disclosed nanoparticle to deliver a biologically active agent, a
pharmaceutically active agent, and/or an imaging moiety.
[0019] Also disclosed are pharmaceutical compositions comprising a
therapeutically effective amount of one or more disclosed polymer
and/or one or more disclosed nanoparticle and a pharmaceutically
acceptable carrier for administration in a subject, for example, a
mammal.
[0020] Also disclosed are pharmaceutical compositions for
diagnosing, treating, and/or preventing ophthalmic disorders, the
compositions comprising a therapeutically effective amount of one
or more disclosed polymer and/or one or more disclosed nanoparticle
and a pharmaceutically acceptable carrier for administration in a
subject, for example, a mammal. In one aspect, the compositions can
be administered transcorneally.
[0021] Also disclosed are microparticles, and/or larger networks,
for use as materials for tissue engineering and biogels in
biomedical devices.
[0022] While aspects of the present invention can be described and
claimed in a particular statutory class, such as the system
statutory class, this is for convenience only and one of skill in
the art will understand that each aspect of the present invention
can be described and claimed in any statutory class. Unless
otherwise expressly stated, it is in no way intended that any
method or aspect set forth herein be construed as requiring that
its steps be performed in a specific order. Accordingly, where a
method claim does not specifically state in the claims or
descriptions that the steps are to be limited to a specific order,
it is no way intended that an order be inferred, in any respect.
This holds for any possible non-express basis for interpretation,
including matters of logic with respect to arrangement of steps or
operational flow, plain meaning derived from grammatical
organization or punctuation, or the number or type of aspects
described in the specification.
BRIEF DESCRIPTION OF THE FIGURES
[0023] The accompanying figures, which are incorporated in and
constitute a part of this specification, illustrate several aspects
and together with the description serve to explain the principles
of the invention.
[0024] FIG. 1 shows hydrolytic biodegradation studies of
(.tangle-solidup.) 725.1.+-.94.3 nm poly(vl-evl) nanoparticles to
(.diamond-solid.) 30.71.+-.2.21 nm AB nanoparticles. All particles
are non-emulsified.
[0025] FIG. 2 shows cytotoxicity of vitamin E TPGS formulated
nanoparticles on HeLa cells after 24 h incubation using the MTT
assay. Fitted curve shows cell viability of the HeLa cell line.
[0026] FIG. 3 shows in vitro degradation profile of vitamin E TPGS
formulated poly(vl-evl-avl-opd) nanoparticles of 53 nm in DPBS at
pH 7.4 and 37.degree. C. over a period of 384 h (16 days).
[0027] FIG. 4 shows in vitro release profile of paclitaxel from
particles loaded with 11.3% paclitaxel prepared with the
emulsification process. The drug release was performed in DPBS at
pH 7.4 and 37.degree. C. for 60 days. The cumulative release
profile shows a desirable controlled and sustained release of
paclitaxel from the nanoparticles.
[0028] FIG. 5 shows transmission electron microscopy (TEM) images
of (A) nanoparticles without taxol with a size of 53 nm and (B)
nanoparticles encapsulated with 11.3% taxol with a size dimension
of 57 nm.
[0029] FIG. 6 shows synthesis of a targeted, water-soluble
nanoparticle drug delivery system involving thiol-ene "click"
chemistries and drug loading via developed emulsification process
after post-modification.
[0030] FIG. 7 shows encapsulation of brimonidine in
nanoparticles.
[0031] FIG. 8 shows drug release of disclosed nanoparticles in
comparison to other polyester based nanoparticle systems.
[0032] FIG. 9 shows synthesis and validation of optimized
nanoparticles differentiated by size, release kinetics,
incorporated drug, targeting parameter, and/or imaging
modality.
[0033] FIG. 10 shows multifunctional linear polyester precursors
with epoxide cross-linking entity.
[0034] FIG. 11 shows TEM images of AB nanoparticles; (1) 2
equivalents of amine; (2) 5 equivalents of amine; (3) 8 equivalents
of amine.
[0035] FIG. 12 shows polynomial increase of nanoparticle diameter
(nm) with increase of diamine cross-linker; (.box-solid.) ABD
nanoparticles; (.diamond-solid.) AB nanoparticles; ( ) ABC
nanoparticles.
[0036] FIG. 13 shows .sup.1H NMR overlay for poly(vl-evl)
nanoparticles with increasing cross-linking.
[0037] FIG. 14 shows polynomial increase of nanoparticle diameter
(nm) with increase of diamine cross-linker for (.diamond-solid.) AB
nanoparticles from FIG. 12.
[0038] FIG. 15 shows polynomial increase of nanoparticle diameter
(nm) with increase of diamine cross-linker for (.box-solid.) ABD
nanoparticles from FIG. 12.
[0039] FIG. 16 shows polynomial increase of nanoparticle diameter
(nm) with increase of diamine cross-linker for ( ) ABC
nanoparticles from FIG. 12.
[0040] FIG. 17 shows polynomial increase of nanoparticle diameter
(nm) with increase of diamine cross-linker for AB nanoparticles
from poly(vl-evl) (2% evl) (.box-solid.).
[0041] FIG. 18 shows polynomial increase of nanoparticle diameter
(nm) with increase of diamine cross-linker for AB nanoparticles
from poly(vl-evl) (7% evl) (.diamond-solid.).
[0042] FIG. 19 shows polynomial increase of nanoparticle diameter
(nm) with increase of diamine cross-linker for AB nanoparticles
from poly(vl-evl) (19% evl) (.diamond-solid.).
[0043] FIG. 20 shows a schematic representation of the structures
for FD-1 and FD-2.
[0044] FIG. 21 shows time course of internalization of (a) FD-1 and
(b) FD-2 into NIH-3T3 Fibroblasts at 37.degree. C. The conjugate
concentration was 10 .mu.M.
[0045] FIG. 22 shows the effect of temperature on (a) FD-1 and (b)
FD-2 internalization. The human microvascular endothelial cells
(HMEC) cells were incubated with conjugates (10 uM) for 2.5 min at
4.degree. C. or at 37.degree. C.
[0046] FIG. 23 shows the effect of temperature on (a) FD-1 and (b)
FD-2 internalization. The HMEC cells were incubated with conjugates
(1 uM) for 30 min at 4.degree. C. or at 37.degree. C.
[0047] FIG. 24 shows control experiments: (a) The HMEC cells were
incubated with free FITC conjugates (10 uM) for 60 min at
37.degree. C. (b) The HMEC cells were incubated with Boc-protected
guanidinylated FD-2 (10 uM) for 60 min at 37.degree. C.
[0048] FIG. 25A and FIG. 25B show an exemplary synthetic scheme for
the preparation of FD-1, FD-2, and intermediates thereof.
[0049] FIG. 26 shows a schematic of exemplary multimodal
nanoparticles.
[0050] FIG. 27 shows an exemplary conjugation of a disclosed
dendrimeric material with a disclosed cross-linked organic
nanoparticle.
[0051] FIG. 28 shows a schematic illustrating a disclosed delivery
system (e.g., gene delivery).
[0052] FIG. 29 illustrates preparation of a disclosed delivery
system (e.g., gene delivery).
[0053] FIG. 30 shows micrographs demonstrating mitrochondrial
localization of the disclosed delivery systems (e.g., gene
delivery).
[0054] FIG. 31 shows micrographs demonstrating uptake of a
disclosed delivery system (e.g., gene delivery) in ciEndothelial
cells.
[0055] FIG. 32 demonstrates the flexibility of assembly of the
disclosed delivery systems.
[0056] FIG. 33 shows micrographs of HeLa cells exposed 10 .mu.M
FD-1 for 1 h, fixed with 3.3% paraformaldehyde, stained with 100 nM
Mitotracker.RTM. Red 580 FM. The illuminated regions show cell
penetration (left), mitochondria location (center), and overlap
(right).
[0057] FIG. 34 shows micrographs of HeLa cells exposed 20 .mu.M
FD-2 for 1 h, fixed with 3.3% paraformaldehyde, stained with 100 nM
Mitotracker.RTM. Red 580 FM. The illuminated regions show cell
penetration (left), mitochondria location (center), and overlap
(right).
[0058] FIG. 35 shows micrographs demonstrating intercellular
transport of an aprotinin-fluorophore-transporter conjugate (FD-1,
illustrated) into HAEC cells.
[0059] FIG. 36 shows micrographs demonstrating intercellular
transport of an aprotinin-fluorophore-transporter conjugate (FD-2)
into HAEC cells.
[0060] FIG. 37 shows micrographs demonstrating no uptake (i.e., no
intercellular transport into HAEC cells) of a control
aprotinin-fluorophore conjugate (illustrated).
[0061] FIG. 38 illustrates several chemical strategies for binding
transporter moities to various protein functional groups (e.g.,
amine, thiol, carbonyl).
[0062] FIG. 39 presents strategies for vaccine development by
incorporation of aprotinin through conjugation to
carbonyl-functionalized proteins (e.g., tyrosine residues) by
Mannich reaction.
[0063] FIG. 40 illustrates incorporation of fluorophores through
conjugation to carbonyl-functionalized proteins (e.g., tyrosine
residues) by Mannich reaction.
[0064] FIG. 41 illustrates incorporation of transporter moieties
through conjugation to carbonyl-functionalized proteins.
[0065] FIG. 41 shows TEM analysis of the nanoparticles (225.6 nm)
produced from crosslinking of poly(vl-evl-avl-opd) (ABbD).
[0066] FIG. 43 shows the particle size distribution measured by
dynamic light scatter analysis of "one-pot" nanoparticles
(272.3.+-.23.3 nm) produced from crosslinking of
poly(vl-evl-avl-opd) (ABbD).
[0067] FIG. 44 shows a scheme for a thiol exchange reaction with an
IgG antibody to form an IgGMT bioconjugate.
[0068] FIG. 45 shows microscopy images of uptake of IgGMT into
HEp-2 cells for 10 min, 30 min, 1 h, 2 h, 6 h and negative control
experiment (NC) with Alexa Fluor.RTM. 568 labeled IgG.
[0069] FIG. 46 shows microscopy images of HEp-2 cells infected with
RSV for 24 h, washed and imaged 48 h after infection for the
fluorescence of GFP (c). HEp-2 cells infected with RSV for 24 h,
incubated for 30 min with IgGMT and imaged after 48 h for the
fluorescence of GFP (a) and Alexa Fluor.RTM. 568 of the IgGMT (b),
merged images (a) and (b) (merged a+b).
[0070] FIG. 47 shows microscopy images of HEp-2 cells infected with
RSV for 24 h, incubated for 30 min with IgGMT and imaged
immediately for the green fluorescence of the GFP (a) and the red
fluorescence of the IgGMT conjugate (b), merged images of (a) and
(b) (a+b merged). HEp-2 cells infected with RSV for 24 h, incubated
for 30 min with IgGMT and imaged after 48 h for the fluorescence of
GFP (a) and Alexa Fluor.RTM. 568 of the IgGMT conjugate (b), merged
images (a) and (b) (merged a+b).
[0071] FIG. 48 shows results for a radiation guided
Nanoparticle-peptide targeting in a Lewis-Lung Carcinoma Tumor
Model.
[0072] FIG. 49 shows a scheme for delivery of a biological active
substance.
[0073] FIG. 50 shows fluorescence microscopy images of portions of
the eye of a rat after administration of a nanoparticle
bioconjugate comprising an imaging agent.
[0074] FIG. 51 shows a schematic of nanoparticle formation from
poly(vl-opd) via reductive amination.
[0075] FIG. 52 shows a transmission electron microscopy (TEM) image
of nanoparticles formed from poly(vl-opd) via reductive
amination.
[0076] FIG. 53 shows deposition of DiO dye on the retinal surface
over time after a single injection of DiO nanoparticle complex.
[0077] FIG. 54 shows deposition of DiO dye in ganglion cells over
time after a single injection of DiO nanoparticle complex.
[0078] FIG. 55 shows "nanosponges," which are three-dimensional
nano-networks formed from degradable materials, in particular,
formed by crosslinking degradable linear polyesters. FIG. 55A is a
schematic representation of a 50 nm degradable nanoparticle
(nanosponge), 7% cross-linking density, loaded with 1.3% travatan,
0.38 mg/mL. FIG. 55B is a schematic representation of a 400 nm
degradable nanoparticle (nanosponge), 14% cross-linking density,
loaded with 22.4% bimatoprost, 3.58 mg/mL. FIG. 55C is a schematic
representation of a 700 nm degradable nanoparticle (nanosponge),
14% cross-linking density, loaded with 29.35% bimatoprost, 4.7
mg/mL. In a separate example, a more crystalline 700 nm degradable
nanoparticle (nanosponge), 14% cross-linking density, was loaded
with 25.41% bimatoprost, 4.07 mg/mL.
[0079] FIG. 56 summarizes hypotensive drug trials with a 50 nm
"nanosponge" (7% cross-linking density, 1.3% travatan, 0.38 mg/mL).
The upper panel is a graph of intraocular pressure as a function of
time after intravitreal administration of the nanosponge
(intravitreal travatan nanoparticles) (- -) versus time after
intravitreal administration of PBS (-.gradient.-). The lower panel
is a graph of intraocular pressure as a function of time after
intravitreal administration of topical travatan (- -) versus time
after intravitreal administration of the nanosponge (intravitreal
travatan nanoparticles) (-.gradient.-).
[0080] FIG. 57 summarizes hypotensive drug trials with a Lumigan
(Bimatoprost Ophthalmic)-loaded 400 nm "nanosponge" (14%
cross-linking density, 22.4% bimatoprost, 3.58 mg/mL), with a 700
nm "nanosponge" (14% cross-linking density, 29.35% bimatoprost, 4.7
mg/mL), and with a 700 nm "nanosponge" (14% cross-linking density,
25.41% bimatoprost, 4.07 mg/mL). The upper panel is a graph of
intraocular pressure as a function of time after intravitreal
administration of control (PBS) (- -) versus time after
intravitreal administration of the 400 nm nanosponge (intravitreal
bimatoprost nanoparticles) (-.gradient.-). The lower panel is a
graph of intraocular pressure as a function of time after
intravitreal administration of control (PBS) (- -) versus time
after intravitreal administration of the 700 nm nanosponge
(intravitreal bimatoprost nanoparticles) (-.gradient.-).
[0081] Additional advantages of the invention will be set forth in
part in the description which follows, and in part will be obvious
from the description, or can be learned by practice of the
invention. The advantages of the invention will be realized and
attained by means of the elements and combinations particularly
pointed out in the appended claims. It is to be understood that
both the foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive of the invention, as claimed.
DESCRIPTION
[0082] The present invention can be understood more readily by
reference to the following detailed description of the invention
and the Examples included therein.
[0083] Before the present compounds, compositions, articles,
systems, devices, and/or methods are disclosed and described, it is
to be understood that they are not limited to specific synthetic
methods unless otherwise specified, or to particular reagents
unless otherwise specified, as such may, of course, vary. It is
also to be understood that the terminology used herein is for the
purpose of describing particular aspects only and is not intended
to be limiting. Although any methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present invention, example methods and materials are
now described.
[0084] All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited. The publications
discussed herein are provided solely for their disclosure prior to
the filing date of the present application. Nothing herein is to be
construed as an admission that the present invention is not
entitled to antedate such publication by virtue of prior invention.
Further, the dates of publication provided herein can be different
from the actual publication dates, which can require independent
confirmation.
A. Definitions
[0085] As used herein, nomenclature for compounds, including
organic compounds, can be given using common names, IUPAC, IUBMB,
or CAS recommendations for nomenclature. When one or more
stereochemical features are present, Cahn-Ingold-Prelog rules for
stereochemistry can be employed to designate stereochemical
priority, E/Z specification, and the like. One of skill in the art
can readily ascertain the structure of a compound if given a name,
either by systemic reduction of the compound structure using naming
conventions, or by commercially available software, such as
CHEMDRAW.TM. (Cambridgesoft Corporation, U.S.A.).
[0086] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a functional group," "an alkyl," or "a residue"
includes mixtures of two or more such functional groups, alkyls, or
residues, and the like.
[0087] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another aspect includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another aspect. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that each unit between two particular units are
also disclosed. For example, if 10 and 15 are disclosed, then 11,
12, 13, and 14 are also disclosed.
[0088] References in the specification and concluding claims to
parts by weight of a particular element or component in a
composition denotes the weight relationship between the element or
component and any other elements or components in the composition
or article for which a part by weight is expressed. Thus, in a
compound containing 2 parts by weight of component X and 5 parts by
weight component Y, X and Y are present at a weight ratio of 2:5,
and are present in such ratio regardless of whether additional
components are contained in the compound.
[0089] A weight percent (wt. %) of a component, unless specifically
stated to the contrary, is based on the total weight of the
formulation or composition in which the component is included.
[0090] As used herein, the terms "optional" or "optionally" means
that the subsequently described event or circumstance can or can
not occur, and that the description includes instances where said
event or circumstance occurs and instances where it does not.
[0091] As used herein, the term "treatment" refers to the medical
management of a patient with the intent to cure, ameliorate,
stabilize, or prevent a disease, pathological condition, or
disorder. This term includes active treatment, that is, treatment
directed specifically toward the improvement of a disease,
pathological condition, or disorder, and also includes causal
treatment, that is, treatment directed toward removal of the cause
of the associated disease, pathological condition, or disorder. In
addition, this term includes palliative treatment, that is,
treatment designed for the relief of symptoms rather than the
curing of the disease, pathological condition, or disorder;
preventative treatment, that is, treatment directed to minimizing
or partially or completely inhibiting the development of the
associated disease, pathological condition, or disorder; and
supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the
associated disease, pathological condition, or disorder.
[0092] As used herein, the term "diagnosed" means having been
subjected to a physical examination by a person of skill, for
example, a physician, and found to have a condition that can be
diagnosed or treated by the compounds, compositions, or methods
disclosed herein. For example, "diagnosed with an occular disorder"
means having been subjected to a physical examination by a person
of skill, for example, a physician, and found to have a disorder of
the eye or eyes prior to treatment. As a further example,
"diagnosed with glaucoma" means having been subjected to a physical
examination by a person of skill, for example, a physician, and
found to have glaucoma (e.g., "open angle" or "closed angle") prior
to treatment.
[0093] As used herein, the phrase "identified to be in need of
treatment for a disorder," or the like, refers to selection of a
subject based upon need for treatment of the disorder. For example,
a subject can be identified as having a need for treatment of a
disorder (e.g., an occular disorder, glaucoma, "open angle"
glaucoma, or "closed angle" glaucoma) based upon an earlier
diagnosis by a person of skill and thereafter subjected to
treatment for the disorder. As a further example, a subject can be
identified as having a need for treatment of a disorder after
administration by recognition of the subject's response to the
treatment (i.e., alleviation of symptoms or prevention of
disorder). It is contemplated that the identification can, in one
aspect, be performed by a person different from the person making
the diagnosis. It is also contemplated, in a further aspect, that
the administration can be performed by one who subsequently
performed the administration.
[0094] As used herein, the term "prevent" or "preventing" refers to
precluding, averting, obviating, forestalling, stopping, or
hindering something from happening, especially by advance action.
It is understood that where reduce, inhibit or prevent are used
herein, unless specifically indicated otherwise, the use of the
other two words is also expressly disclosed.
[0095] As used herein, the terms "administering" and
"administration" refer to any method of providing a pharmaceutical
preparation to a subject. Such methods are well known to those
skilled in the art and include, but are not limited to, oral
administration, transdermal administration, administration by
inhalation, nasal administration, topical administration (such as,
for example, eye drops, creams, salves, and irrigation),
intravaginal administration, ophthalmic administration, intraaural
administration, intracerebral administration, rectal
administration, and parenteral administration, including injectable
such as intravenous administration, intra-arterial administration,
intramuscular administration, and subcutaneous administration.
Administration can be continuous or intermittent. In various
aspects, a preparation can be administered therapeutically; that
is, administered to treat an existing disease or condition. In
further various aspects, a preparation can be administered
prophylactically; that is, administered for prevention of a disease
or condition. It is further contemplated that administration
methods include parenteral methods such as intravitreal,
subcutaneous, intradermal, intravenous, epicutaneous, intraocular,
conjunctival, subconjuctival, intracorneal, retrobulbar, and
intramuscular injections.
[0096] As used herein, the term "subject" refers to a target of
administration. The subject of the herein disclosed methods can be
a vertebrate, such as a mammal, a fish, a bird, a reptile, or an
amphibian. Thus, the subject of the herein disclosed methods can be
a human, non-human primate, horse, pig, rabbit, dog, sheep, goat,
cow, cat, guinea pig or rodent. The term does not denote a
particular age or sex. Thus, adult and newborn subjects, as well as
fetuses, whether male or female, are intended to be covered. A
patient refers to a subject afflicted with a disease or disorder.
The term "patient" includes human and veterinary subjects.
[0097] As used herein, the terms "effective amount" and "amount
effective" refer to an amount that is sufficient to achieve a
desired result or to have an effect on undesired symptoms, but is
generally insufficient to cause adverse side affects. The specific
effective dose level for any particular patient will depend upon a
variety of factors including the disorder being treated and the
severity of the disorder; the specific composition employed; the
age, body weight, general health, sex and diet of the patient; the
time of administration; the route of administration; the rate of
excretion of the specific compound employed; the duration of the
treatment; drugs used in combination or coincidental with the
specific compound employed and like factors well known in the
medical arts. For example, it is well within the skill of the art
to start doses of a compound at levels lower than those required to
achieve the desired effect and to gradually increase the dosage
until the desired effect is achieved. If desired, the effective
daily dose can be divided into multiple doses for purposes of
administration. Consequently, single dose compositions can contain
such amounts or submultiples thereof to make up the daily dose. The
dosage can be adjusted by the individual physician in the event of
any contraindications. Dosage can vary, and can be administered in
one or more dose administrations daily, for one or several days.
Guidance can be found in the literature for appropriate dosages for
given classes of pharmaceutical products. In a further aspect, a
preparation can be administered in a "diagnostically effective
amount"; that is, an amount effective for diagnosis of a disease or
condition. In a further aspect, a preparation can be administered
in a "therapeutically effective amount"; that is, an amount
effective for treatment of a disease or condition. In a further
aspect, a preparation can be administered in a "prophylactically
effective amount"; that is, an amount effective for prevention of a
disease or condition.
[0098] As used herein, the term "pharmaceutically acceptable
carrier" refers to sterile aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, as well as sterile powders
for reconstitution into sterile injectable solutions or dispersions
just prior to use. Examples of suitable aqueous and nonaqueous
carriers, diluents, solvents or vehicles include water, ethanol,
polyols (such as glycerol, propylene glycol, polyethylene glycol
and the like), carboxymethylcellulose and suitable mixtures
thereof, vegetable oils (such as olive oil) and injectable organic
esters such as ethyl oleate. Proper fluidity can be maintained, for
example, by the use of coating materials such as lecithin, by the
maintenance of the required particle size in the case of
dispersions and by the use of surfactants. These compositions can
also contain adjuvants such as preservatives, wetting agents,
emulsifying agents and dispersing agents. Prevention of the action
of microorganisms can be ensured by the inclusion of various
antibacterial and antifungal agents such as paraben, chlorobutanol,
phenol, sorbic acid and the like. It can also be desirable to
include isotonic agents such as sugars, sodium chloride and the
like. Prolonged absorption of the injectable pharmaceutical form
can be brought about by the inclusion of agents, such as aluminum
monostearate and gelatin, which delay absorption. Injectable depot
forms are made by forming microencapsule matrices of the drug in
biodegradable polymers such as polylactide-polyglycolide,
poly(orthoesters) and poly(anhydrides). Depending upon the ratio of
drug to polymer and the nature of the particular polymer employed,
the rate of drug release can be controlled. Depot injectable
formulations are also prepared by entrapping the drug in liposomes
or microemulsions which are compatible with body tissues. The
injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter or by incorporating
sterilizing agents in the form of sterile solid compositions which
can be dissolved or dispersed in sterile water or other sterile
injectable media just prior to use. Suitable inert carriers can
include sugars such as lactose. Desirably, at least 95% by weight
of the particles of the active ingredient have an effective
particle size in the range of 0.01 to 10 micrometers.
[0099] As used herein, the term "biologically active agent" or
"bioactive agent" means an agent that is capable of providing a
local or systemic biological, physiological, or therapeutic effect
in the biological system to which it is applied. For example, the
bioactive agent can act to control infection or inflammation,
enhance cell growth and tissue regeneration, control tumor growth,
act as an analgesic, promote anti-cell attachment, and enhance bone
growth, among other functions. Other suitable bioactive agents can
include anti-viral agents, vaccines, hormones, antibodies
(including active antibody fragments sFv, Fv, and Fab fragments),
aptamers, peptide mimetics, functional nucleic acids, therapeutic
proteins, peptides, or nucleic acids. Other bioactive agents
include prodrugs, which are agents that are not biologically active
when administered but, upon administration to a subject are
converted to bioactive agents through metabolism or some other
mechanism. Additionally, any of the compositions of the invention
can contain combinations of two or more bioactive agents. It is
understood that a biologically active agent can be used in
connection with administration to various subjects, for example, to
humans (i.e., medical administration) or to animals (i.e.,
veterinary administration).
[0100] As used herein, the term "pharmaceutically active agent"
includes a "drug" or a "vaccine" and means a molecule, group of
molecules, complex or substance administered to an organism for
diagnostic, therapeutic, preventative medical, or veterinary
purposes. This term include externally and internally administered
topical, localized and systemic human and animal pharmaceuticals,
treatments, remedies, nutraceuticals, cosmeceuticals, biologicals,
devices, diagnostics and contraceptives, including preparations
useful in clinical and veterinary screening, prevention,
prophylaxis, healing, wellness, detection, imaging, diagnosis,
therapy, surgery, monitoring, cosmetics, prosthetics, forensics and
the like. This term may also be used in reference to agriceutical,
workplace, military, industrial and environmental therapeutics or
remedies comprising selected molecules or selected nucleic acid
sequences capable of recognizing cellular receptors, membrane
receptors, hormone receptors, therapeutic receptors, microbes,
viruses or selected targets comprising or capable of contacting
plants, animals and/or humans. This term can also specifically
include nucleic acids and compounds comprising nucleic acids that
produce a bioactive effect, for example deoxyribonucleic acid (DNA)
or ribonucleic acid (RNA). Pharmaceutically active agents include
the herein disclosed categories and specific examples. It is not
intended that the category be limited by the specific examples.
Those of ordinary skill in the art will recognize also numerous
other compounds that fall within the categories and that are useful
according to the invention. Examples include a radiosensitizer, the
combination of a radiosensitizer and a chemotherapeutic, a steroid,
a xanthine, a beta-2-agonist bronchodilator, an anti-inflammatory
agent, an analgesic agent, a calcium antagonist, an
angiotensin-converting enzyme inhibitors, a beta-blocker, a
centrally active alpha-agonist, an alpha-1-antagonist, carbonic
anhydrase inhibitors, prostaglandin analogs, a combination of an
alpha agonist and a beta blocker, a combination of a carbonic
anhydrase inhibitor and a beta blocker, an
anticholinergic/antispasmodic agent, a vasopressin analogue, an
antiarrhythmic agent, an antiparkinsonian agent, an
antiangina/antihypertensive agent, an anticoagulant agent, an
antiplatelet agent, a sedative, an ansiolytic agent, a peptidic
agent, a biopolymeric agent, an antineoplastic agent, a laxative,
an antidiarrheal agent, an antimicrobial agent, an antifungal
agent, or a vaccine. In a further aspect, the pharmaceutically
active agent can be coumarin, albumin, bromolidine, steroids such
as betamethasone, dexamethasone, methylprednisolone, prednisolone,
prednisone, triamcinolone, budesonide, hydrocortisone, and
pharmaceutically acceptable hydrocortisone derivatives; xanthines
such as theophylline and doxophylline; beta-2-agonist
bronchodilators such as salbutamol, fenterol, clenbuterol,
bambuterol, salmeterol, fenoterol; antiinflammatory agents,
including antiasthmatic anti-inflammatory agents, antiarthritis
antiinflammatory agents, and non-steroidal antiinflammatory agents,
examples of which include but are not limited to sulfides,
mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically
acceptable diclofenac salts, nimesulide, naproxene, acetominophen,
ibuprofen, ketoprofen and piroxicam; analgesic agents such as
salicylates; calcium channel blockers such as nifedipine,
amlodipine, and nicardipine; angiotensin-converting enzyme
inhibitors such as captopril, benazepril hydrochloride, fosinopril
sodium, trandolapril, ramipril, lisinopril, enalapril, quinapril
hydrochloride, and moexipril hydrochloride; beta-blockers (i.e.,
beta adrenergic blocking agents) such as sotalol hydrochloride,
timolol maleate, timol hemihydrate, levobunolol hydrochloride,
esmolol hydrochloride, carteolol, propanolol hydrochloride,
betaxolol hydrochloride, penbutolol sulfate, metoprolol tartrate,
metoprolol succinate, acebutolol hydrochloride, atenolol, pindolol,
and bisoprolol fumarate; centrally active alpha-2-agonists (i.e.,
alpha adrenergic receptor agonist) such as clonidine, brimonidine
tartrate, and apraclonidine hyrochloride; alpha-1-antagonists such
as doxazosin and prazosin; anticholinergic/antispasmodic agents
such as dicyclomine hydrochloride, scopolamine hydrobromide,
glycopyrrolate, clidinium bromide, flavoxate, and oxybutynin;
vasopressin analogues such as vasopressin and desmopressin;
prostaglandin analogs such as latanoprost, travoprost, and
bimatoprost; cholinergics (i.e., acetylcholine receptor agonists)
such as pilocarpine hydrochloride and carbachol; glutamate receptor
agonists such as the N-methyl D-aspartate receptor agonist
memantine; anti-Vascular endothelial growth factor (VEGF) aptamers
such as pegaptanib; anti-VEGF antibodies (including but not limited
to anti-VEGF-A antibodies) such as ranibizumab and becacizumab;
carbonic anhydrase inhibitors such as methazolamide, brinzolamide,
dorzolamide hydrochloride, and acetazolamide; antiarrhythmic agents
such as quinidine, lidocaine, tocamide hydrochloride, mexiletine
hydrochloride, digoxin, verapamil hydrochloride, propafenone
hydrochloride, flecamide acetate, procainamide hydrochloride,
moricizine hydrochloride, and disopyramide phosphate;
antiparkinsonian agents, such as dopamine, L-Dopa/Carbidopa,
selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine,
and bromocryptine; antiangina agents and antihypertensive agents
such as isosorbide mononitrate, isosorbide dinitrate, propranolol,
atenolol and verapamil; anticoagulant and antiplatelet agents such
as coumadin, warfarin, acetylsalicylic acid, and ticlopidine;
sedatives such as benzodiazapines and barbiturates; ansiolytic
agents such as lorazepam, bromazepam, and diazepam; peptidic and
biopolymeric agents such as calcitonin, leuprolide and other LHRH
agonists, hirudin, cyclosporin, insulin, somatostatin, protirelin,
interferon, desmopres sin, somatotropin, thymopentin, pidotimod,
erythropoietin, interleukins, melatonin,
granulocyte/macrophage-CSF, and heparin; antineoplastic agents such
as etoposide, etoposide phosphate, cyclophosphamide, methotrexate,
5-fluorouracil, vincristine, doxorubicin, cisplatin, hydroxyurea,
leucovorin calcium, tamoxifen, flutamide, asparaginase,
altretamine, mitotane, and procarbazine hydrochloride; laxatives
such as senna concentrate, casanthranol, bisacodyl, and sodium
picosulphate; antidiarrheal agents such as difenoxine
hydrochloride, loperamide hydrochloride, furazolidone,
diphenoxylate hdyrochloride, and microorganisms; vaccines such as
bacterial and viral vaccines; antimicrobial agents such as
penicillins, cephalosporins, and macrolides, antifungal agents such
as imidazolic and triazolic derivatives; and nucleic acids such as
DNA sequences encoding for biological proteins, and antisense
oligonucleotides. It is understood that a pharmaceutically active
agent can be used in connection with administration to various
subjects, for example, to humans (i.e., medical administration) or
to animals (i.e., veterinary administration).
[0101] As used herein, the term "ophthalmic disorders" and/or
"ophthalmic conditions" refers to ophthalmic diseases, conditions,
and/or disorders including, without limitation, those associated
with the anterior chamber of the eye (i.e., hyphema, synechia); the
choroid (i.e., choroidal detachment, choroidal melanoma, multifocal
choroidopathy syndromes); the conjunctiva (i.e., conjunctivitis,
cicatricial pemphigoid, filtering Bleb complications, conjunctival
melanoma, Pharyngoconjunctival Fever, pterygium, conjunctival
squamous cell carcinoma); connective tissue disorders (i.e.,
ankylosing spondylitis, pseudoxanthoma elasticum, corneal abrasion
or edema, limbal dermoid, crystalline dystrophy keratits,
keratoconjunctivitis, keratoconus, keratopathy (including but not
limited to Thygeson's superficial punctuate keratopathy),
megalocornea, corneal ulcer); dermatologic disorders (i.e.,
ecrodermatitis enteropathica, atopic dermatitis, ocular rosacea,
psoriasis, Stevens-Johnson syndrome); endocrine disorders (i.e.,
pituitary apoplexy); extraocular disorders (i.e., Abducens Nerve
Palsy, Brown syndrome, Duane syndrome, esotropia, exotropia,
oculomotor nerve palsy); genetic disorders (i.e., albinism, Down
syndrome, Peters Anomaly); the globe (i.e., anophthalmos,
endophthalmitis); hematologic and cardiovascular disorders (i.e.,
Giant Cell Arteritis, hypertension, leukemias, Ocular Ischemic
syndrome, sickle cell disease); infectious diseases (i.e.,
actinomycosis, botulism, HIV, diphtheria, Escherichia coli,
Tuberculosis, ocular manifestations of syphilis); intraocular
pressure (i.e., glaucoma, ocular hypotony, Posner-Schlossman
syndrome), the iris and ciliary body (i.e., aniridia, iris prolaps,
juvenile xanthogranuloma, ciliary body melanoma, iris melanoma,
uveitis); the lacrimal system (i.e., alacrima, Dry Eye syndrome,
lacrimal gland tumors); the lens (i.e., cataract, ectopia lentis,
intraocular lens decentration or dislocation); the lid (i.e.,
blepharitis, dermatochalasis, distichiasis, ectropion, eyelid
coloboma, Floppy Eye syndrome, trichiasis, xanthelasma); metabolic
disorders (i.e., gout, hyperlipoproteinemia, Oculocerebrorenal
syndrome); neurologic disorders (i.e., Bell Palsy, diplopia,
multiple sclerosis); general ophthalmologic (i.e., red eye,
cataracts, macular degeneration, red eye, macular degeneration);
the optic nerve (i.e., miningioma, optic neuritis, optic
neuropathy, papilledema); the orbit (i.e., orbital cellulits,
orbital dermoid, orbital tumors); phakomatoses (i.e.,
ataxia-telangiectasia, neurofibromatosis-1); presbyopia; the pupil
(i.e., anisocoria, Horner syndrome); refractive disorders (i.e.,
astigmatism, hyperopia, myopia); the retina (i.e., Coats disease,
Eales disease, macular edema, retinitis, retinopathy); and the
sclera (i.e., episcleritis, scleritis).
[0102] As used herein, the terms "imaging moiety" and "imaging
agent" refer to any chemical groups or substance useful for imaging
applications, as known to those of skill in the art. Examples of
imaging agents include radioconjugate, cytotoxin, cytokine,
Gadolinium-DTPA or a quantum dot, iron oxide, manganese oxide, and
fluorescent agents such as Alexa Fluor dyes and Neuro DiO. In one
aspect, an imaging agent can be provided in nanoparticular form or
in microparticular form. In a further aspect, an imaging agent
comprises Gadolinium-DTPA and iron oxide nanoparticles (magnetite),
as specific MRI contrast agents. In a yet further aspect, an
imaging agent comprises at least one near infrared dye, for example
near infrared dyes based on a porphyrin and/or a phthalocyanine.
See Ghoroghchian et al., Near-infrared-emissive polymersomes:
Self-assembled soft matter for in vivo optical imaging, PNAS, 2005,
vol. 102, no. 8, 2922-2927.
[0103] As used herein, the term "polymer" refers to a relatively
high molecular weight organic compound, natural or synthetic, whose
structure can be represented by a repeated small unit, the monomer
(e.g., polyethylene, rubber, cellulose). Synthetic polymers are
typically formed by addition or condensation polymerization of
monomers.
[0104] As used herein, the term "copolymer" refers to a polymer
formed from two or more different repeating units (monomer
residues). By way of example and without limitation, a copolymer
can be an alternating copolymer, a random copolymer, a block
copolymer, or a graft copolymer. It is also contemplated that, in
certain aspects, various block segments of a block copolymer can
themselves comprise copolymers.
[0105] As used herein, the term "oligomer" refers to a relatively
low molecular weight polymer in which the number of repeating units
is between two and ten, for example, from two to eight, from two to
six, or form two to four. In one aspect, a collection of oligomers
can have an average number of repeating units of from about two to
about ten, for example, from about two to about eight, from about
two to about six, or form about two to about four.
[0106] As used herein, the term "reactive residue" refers to a
moiety (e.g., a monomer residue) capable of undergoing chemical
reaction at a reaction temperature and/or in response to a stimulus
to form a reactive intermediate. In one aspect, a reactive residue
is a moiety capable undergoing an intramolecular cross-linking
reaction to provide intramolecular chain collapse.
[0107] As used herein, the term "polymerizable group" refers to a
group (i.e., a chemical functionality) capable of undergoing a
polymerization reaction at a polymerization temperature and/or in
response to a polymerization initiator to form a polymer or an
oligomer. In one aspect, the polymerization reaction is a radical
polymerization (e.g., a vinyl polymerization). It is understood
that catalysts can be employed in connection with the
polymerization reaction. It is contemplated that, in various
aspects, polymerizable groups can be used in step-growth or chain
growth reactions. Exemplary polymerizable groups include residues
of vinyl, styryl, acryloyl, methacryloyl, aryl, and heteroaryl
compounds.
[0108] A residue of a chemical species, as used in the
specification and concluding claims, refers to the moiety that is
the resulting product of the chemical species in a particular
reaction scheme or subsequent formulation or chemical product,
regardless of whether the moiety is actually obtained from the
chemical species. Thus, an ethylene glycol residue in a polyester
refers to one or more --OCH.sub.2CH.sub.2O-- units in the
polyester, regardless of whether ethylene glycol was used to
prepare the polyester. Similarly, a sebacic acid residue in a
polyester refers to one or more --CO(CH.sub.2).sub.8CO-- moieties
in the polyester, regardless of whether the residue is obtained by
reacting sebacic acid or an ester thereof to obtain the
polyester.
[0109] As used herein, the term "substituted" is contemplated to
include all permissible substituents of organic compounds. In a
broad aspect, the permissible substituents include acyclic and
cyclic, branched and unbranched, carbocyclic and heterocyclic, and
aromatic and nonaromatic substituents of organic compounds.
Illustrative substituents include, for example, those described
below. The permissible substituents can be one or more and the same
or different for appropriate organic compounds. For purposes of
this disclosure, the heteroatoms, such as nitrogen, can have
hydrogen substituents and/or any permissible substituents of
organic compounds described herein which satisfy the valences of
the heteroatoms. This disclosure is not intended to be limited in
any manner by the permissible substituents of organic compounds.
Also, the terms "substitution" or "substituted with" include the
implicit proviso that such substitution is in accordance with
permitted valence of the substituted atom and the substituent, and
that the substitution results in a stable compound, e.g., a
compound that does not spontaneously undergo transformation such as
by rearrangement, cyclization, elimination, etc.
[0110] In defining various terms, "A.sup.1," "A.sup.2," "A.sup.3,"
and "A.sup.4" are used herein as generic symbols to represent
various specific substituents. These symbols can be any
substituent, not limited to those disclosed herein, and when they
are defined to be certain substituents in one instance, they can,
in another instance, be defined as some other substituents.
[0111] The term "alkyl" as used herein is a branched or unbranched
saturated hydrocarbon group of 1 to 24 carbon atoms, such as
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl,
t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl,
octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl,
tetracosyl, and the like. The alkyl group can also be substituted
or unsubstituted. The alkyl group can be substituted with one or
more groups including, but not limited to, optionally substituted
alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro,
silyl, sulfo-oxo, or thiol, as described herein. A "lower alkyl"
group is an alkyl group containing from one to six carbon
atoms.
[0112] Throughout the specification "alkyl" is generally used to
refer to both unsubstituted alkyl groups and substituted alkyl
groups; however, substituted alkyl groups are also specifically
referred to herein by identifying the specific substituent(s) on
the alkyl group. For example, the term "halogenated alkyl"
specifically refers to an alkyl group that is substituted with one
or more halide, e.g., fluorine, chlorine, bromine, or iodine. The
term "alkoxyalkyl" specifically refers to an alkyl group that is
substituted with one or more alkoxy groups, as described below. The
term "alkylamino" specifically refers to an alkyl group that is
substituted with one or more amino groups, as described below, and
the like. When "alkyl" is used in one instance and a specific term
such as "alkylalcohol" is used in another, it is not meant to imply
that the term "alkyl" does not also refer to specific terms such as
"alkylalcohol" and the like.
[0113] This practice is also used for other groups described
herein. That is, while a term such as "cycloalkyl" refers to both
unsubstituted and substituted cycloalkyl moieties, the substituted
moieties can, in addition, be specifically identified herein; for
example, a particular substituted cycloalkyl can be referred to as,
e.g., an "alkylcycloalkyl." Similarly, a substituted alkoxy can be
specifically referred to as, e.g., a "halogenated alkoxy," a
particular substituted alkenyl can be, e.g., an "alkenylalcohol,"
and the like. Again, the practice of using a general term, such as
"cycloalkyl," and a specific term, such as "alkylcycloalkyl," is
not meant to imply that the general term does not also include the
specific term.
[0114] The term "cycloalkyl" as used herein is a non-aromatic
carbon-based ring composed of at least three carbon atoms. Examples
of cycloalkyl groups include, but are not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. The
term "heterocycloalkyl" is a type of cycloalkyl group as defined
above, and is included within the meaning of the term "cycloalkyl,"
where at least one of the carbon atoms of the ring is replaced with
a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur,
or phosphorus. The cycloalkyl group and heterocycloalkyl group can
be substituted or unsubstituted. The cycloalkyl group and
heterocycloalkyl group can be substituted with one or more groups
including, but not limited to, optionally substituted alkyl,
cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl,
sulfo-oxo, or thiol as described herein.
[0115] The term "polyalkylene group" as used herein is a group
having two or more CH.sub.2 groups linked to one another. The
polyalkylene group can be represented by the formula
--(CH.sub.2).sub.a--, where "a" is an integer of from 2 to 500.
[0116] The terms "alkoxy" and "alkoxyl" as used herein to refer to
an alkyl or cycloalkyl group bonded through an ether linkage; that
is, an "alkoxy" group can be defined as --OA.sup.1 where A.sup.1 is
alkyl or cycloalkyl as defined above. "Alkoxy" also includes
polymers of alkoxy groups as just described; that is, an alkoxy can
be a polyether such as --OA.sup.1-OA.sup.2 or
--OA.sup.1-(OA.sup.2).sub.a-OA.sup.3, where "a" is an integer of
from 1 to 200 and A.sup.1, A.sup.2, and A.sup.3 are alkyl and/or
cycloalkyl groups.
[0117] The term "alkenyl" as used herein is a hydrocarbon group of
from 2 to 24 carbon atoms with a structural formula containing at
least one carbon-carbon double bond. Asymmetric structures such as
(A.sup.1A.sup.2)C.dbd.C(A.sup.3A.sup.4) are intended to include
both the E and Z isomers. This can be presumed in structural
formulae herein wherein an asymmetric alkene is present, or it can
be explicitly indicated by the bond symbol C.dbd.C. The alkenyl
group can be substituted with one or more groups including, but not
limited to, optionally substituted alkyl, cycloalkyl, alkoxy,
alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,
aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy,
ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described
herein.
[0118] The term "cycloalkenyl" as used herein is a non-aromatic
carbon-based ring composed of at least three carbon atoms and
containing at least one carbon-carbon double bound, i.e., C.dbd.C.
Examples of cycloalkenyl groups include, but are not limited to,
cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl,
cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term
"heterocycloalkenyl" is a type of cycloalkenyl group as defined
above, and is included within the meaning of the term
"cycloalkenyl," where at least one of the carbon atoms of the ring
is replaced with a heteroatom such as, but not limited to,
nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and
heterocycloalkenyl group can be substituted or unsubstituted. The
cycloalkenyl group and heterocycloalkenyl group can be substituted
with one or more groups including, but not limited to, optionally
substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl,
alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino,
carboxylic acid, ester, ether, halide, hydroxy, ketone, azide,
nitro, silyl, sulfo-oxo, or thiol as described herein.
[0119] The term "alkynyl" as used herein is a hydrocarbon group of
2 to 24 carbon atoms with a structural formula containing at least
one carbon-carbon triple bond. The alkynyl group can be
unsubstituted or substituted with one or more groups including, but
not limited to, optionally substituted alkyl, cycloalkyl, alkoxy,
alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,
aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy,
ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described
herein.
[0120] The term "cycloalkynyl" as used herein is a non-aromatic
carbon-based ring composed of at least seven carbon atoms and
containing at least one carbon-carbon triple bound. Examples of
cycloalkynyl groups include, but are not limited to, cycloheptynyl,
cyclooctynyl, cyclononynyl, and the like. The term
"heterocycloalkynyl" is a type of cycloalkenyl group as defined
above, and is included within the meaning of the term
"cycloalkynyl," where at least one of the carbon atoms of the ring
is replaced with a heteroatom such as, but not limited to,
nitrogen, oxygen, sulfur, or phosphorus. The cycloalkynyl group and
heterocycloalkynyl group can be substituted or unsubstituted. The
cycloalkynyl group and heterocycloalkynyl group can be substituted
with one or more groups including, but not limited to, optionally
substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl,
alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino,
carboxylic acid, ester, ether, halide, hydroxy, ketone, azide,
nitro, silyl, sulfo-oxo, or thiol as described herein.
[0121] The term "aryl" as used herein is a group that contains any
carbon-based aromatic group including, but not limited to, benzene,
naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The
term "aryl" also includes "heteroaryl," which is defined as a group
that contains an aromatic group that has at least one heteroatom
incorporated within the ring of the aromatic group. Examples of
heteroatoms include, but are not limited to, nitrogen, oxygen,
sulfur, and phosphorus. Likewise, the term "non-heteroaryl," which
is also included in the term "aryl," defines a group that contains
an aromatic group that does not contain a heteroatom. The aryl
group can be substituted or unsubstituted. The aryl group can be
substituted with one or more groups including, but not limited to,
optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl,
cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde,
amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,
azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The
term "biaryl" is a specific type of aryl group and is included in
the definition of "aryl." Biaryl refers to two aryl groups that are
bound together via a fused ring structure, as in naphthalene, or
are attached via one or more carbon-carbon bonds, as in
biphenyl.
[0122] The term "aldehyde" as used herein is represented by the
formula --C(O)H. Throughout this specification "C(O)" is a short
hand notation for a carbonyl group, i.e., C.dbd.O.
[0123] The terms "amine" or "amino" as used herein are represented
by the formula NA.sup.1A.sup.2A.sup.3, where A.sup.1, A.sup.2, and
A.sup.3 can be, independently, hydrogen or optionally substituted
alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl,
aryl, or heteroaryl group as described herein.
[0124] The term "carboxylic acid" as used herein is represented by
the formula --C(O)OH.
[0125] The term "ester" as used herein is represented by the
formula --OC(O)A.sup.1 or --C(O)OA.sup.1, where A.sup.1 can be an
optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,
alkynyl, cycloalkynyl, aryl, or heteroaryl group as described
herein. The term "polyester" as used herein is represented by the
formula -(A.sup.1O(O)C-A.sup.2-C(O)O).sub.a-- or
-(A.sup.1O(O)C-A.sup.2-OC(O)).sub.a--, where A.sup.1 and A.sup.2
can be, independently, an optionally substituted alkyl, cycloalkyl,
alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl
group described herein and "a" is an integer from 1 to 500.
"Polyester" is as the term used to describe a group that is
produced by the reaction between a compound having at least two
carboxylic acid groups with a compound having at least two hydroxyl
groups.
[0126] The term "ether" as used herein is represented by the
formula A.sup.1OA.sup.2, where A.sup.1 and A.sup.2 can be,
independently, an optionally substituted alkyl, cycloalkyl,
alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl
group described herein. The term "polyether" as used herein is
represented by the formula -(A.sup.1O-A.sup.2O).sub.a--, where
A.sup.1 and A.sup.2 can be, independently, an optionally
substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl,
cycloalkynyl, aryl, or heteroaryl group described herein and "a" is
an integer of from 1 to 500. Examples of polyether groups include
polyethylene oxide, polypropylene oxide, and polybutylene
oxide.
[0127] The term "halide" as used herein refers to the halogens
fluorine, chlorine, bromine, and iodine.
[0128] The term "heterocycle," as used herein refers to single and
multi-cyclic aromatic or non-aromatic ring systems in which at
least one of the ring members is other than carbon. Heterocycle
includes pyridinde, pyrimidine, furan, thiophene, pyrrole,
isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole,
oxazole, including, 1,2,3-oxadiazole, 1,2,5-oxadiazole and
1,3,4-oxadiazole, thiadiazole, including, 1,2,3-thiadiazole,
1,2,5-thiadiazole, and 1,3,4-thiadiazole, triazole, including,
1,2,3-triazole, 1,3,4-triazole, tetrazole, including
1,2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridine, pyridazine,
pyrimidine, pyrazine, triazine, including 1,2,4-triazine and
1,3,5-triazine, tetrazine, including 1,2,4,5-tetrazine,
pyrrolidine, piperidine, piperazine, morpholine, azetidine,
tetrahydropyran, tetrahydrofuran, dioxane, and the like.
[0129] The term "hydroxyl" as used herein is represented by the
formula --OH.
[0130] The term "ketone" as used herein is represented by the
formula A.sup.1C(O)A.sup.2, where A.sup.1 and A.sup.2 can be,
independently, an optionally substituted alkyl, cycloalkyl,
alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl
group as described herein.
[0131] The term "azide" as used herein is represented by the
formula --N.sub.3.
[0132] The term "nitro" as used herein is represented by the
formula --NO.sub.2.
[0133] The term "nitrile" as used herein is represented by the
formula --CN.
[0134] The term "silyl" as used herein is represented by the
formula --SiA.sup.1A.sup.2A.sup.3, where A.sup.1, A.sup.2, and
A.sup.3 can be, independently, hydrogen or an optionally
substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl,
alkynyl, cycloalkynyl, aryl, or heteroaryl group as described
herein.
[0135] The term "sulfo-oxo" as used herein is represented by the
formulas --S(O)A.sup.1, --S(O).sub.2A.sup.1, --OS(O).sub.2A.sup.1,
or --OS(O).sub.2OA.sup.1, where A.sup.1 can be hydrogen or an
optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,
alkynyl, cycloalkynyl, aryl, or heteroaryl group as described
herein. Throughout this specification "S(O)" is a short hand
notation for S.dbd.O. The term "sulfonyl" is used herein to refer
to the sulfo-oxo group represented by the formula
--S(O).sub.2A.sup.1, where A.sup.1 can be hydrogen or an optionally
substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl,
cycloalkynyl, aryl, or heteroaryl group as described herein. The
term "sulfone" as used herein is represented by the formula
A.sup.1S(O).sub.2A.sup.2, where A.sup.1 and A.sup.2 can be,
independently, an optionally substituted alkyl, cycloalkyl,
alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl
group as described herein. The term "sulfoxide" as used herein is
represented by the formula A.sup.1S(O)A.sup.2, where A.sup.1 and
A.sup.2 can be, independently, an optionally substituted alkyl,
cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or
heteroaryl group as described herein.
[0136] The term "thiol" as used herein is represented by the
formula --SH.
[0137] The term "organic residue" defines a carbon containing
residue, i.e., a residue comprising at least one carbon atom, and
includes but is not limited to the carbon-containing groups,
residues, or radicals defined hereinabove. Organic residues can
contain various heteroatoms, or be bonded to another molecule
through a heteroatom, including oxygen, nitrogen, sulfur,
phosphorus, or the like. Examples of organic residues include but
are not limited alkyl or substituted alkyls, alkoxy or substituted
alkoxy, mono or di-substituted amino, amide groups, etc. Organic
residues can preferably comprise 1 to 18 carbon atoms, 1 to 15,
carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6
carbon atoms, or 1 to 4 carbon atoms. In a further aspect, an
organic residue can comprise 2 to 18 carbon atoms, 2 to 15, carbon
atoms, 2 to 12 carbon atoms, 2 to 8 carbon atoms, 2 to 4 carbon
atoms, or 2 to 4 carbon atoms
[0138] A very close synonym of the term "residue" is the term
"radical," which as used in the specification and concluding
claims, refers to a fragment, group, or substructure of a molecule
described herein, regardless of how the molecule is prepared. For
example, a 2,4-thiazolidinedione radical in a particular compound
has the structure
##STR00003##
regardless of whether thiazolidinedione is used to prepare the
compound. In some embodiments the radical (for example an alkyl)
can be further modified (i.e., substituted alkyl) by having bonded
thereto one or more "substituent radicals." The number of atoms in
a given radical is not critical to the present invention unless it
is indicated to the contrary elsewhere herein.
[0139] "Organic radicals," as the term is defined and used herein,
contain one or more carbon atoms. An organic radical can have, for
example, 1-26 carbon atoms, 1-18 carbon atoms, 1-12 carbon atoms,
1-8 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms. In a
further aspect, an organic radical can have 2-26 carbon atoms, 2-18
carbon atoms, 2-12 carbon atoms, 2-8 carbon atoms, 2-6 carbon
atoms, or 2-4 carbon atoms. Organic radicals often have hydrogen
bound to at least some of the carbon atoms of the organic radical.
One example, of an organic radical that comprises no inorganic
atoms is a 5,6,7,8-tetrahydro-2-naphthyl radical. In some
embodiments, an organic radical can contain 1-10 inorganic
heteroatoms bound thereto or therein, including halogens, oxygen,
sulfur, nitrogen, phosphorus, and the like. Examples of organic
radicals include but are not limited to an alkyl, substituted
alkyl, cycloalkyl, substituted cycloalkyl, mono-substituted amino,
di-substituted amino, acyloxy, cyano, carboxy, carboalkoxy,
alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide,
substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl,
thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl,
haloalkoxy, aryl, substituted aryl, heteroaryl, heterocyclic, or
substituted heterocyclic radicals, wherein the terms are defined
elsewhere herein. A few non-limiting examples of organic radicals
that include heteroatoms include alkoxy radicals, trifluoromethoxy
radicals, acetoxy radicals, dimethylamino radicals and the
like.
[0140] "Inorganic radicals," as the term is defined and used
herein, contain no carbon atoms and therefore comprise only atoms
other than carbon. Inorganic radicals comprise bonded combinations
of atoms selected from hydrogen, nitrogen, oxygen, silicon,
phosphorus, sulfur, selenium, and halogens such as fluorine,
chlorine, bromine, and iodine, which can be present individually or
bonded together in their chemically stable combinations. Inorganic
radicals have 10 or fewer, or preferably one to six or one to four
inorganic atoms as listed above bonded together. Examples of
inorganic radicals include, but not limited to, amino, hydroxy,
halogens, nitro, thiol, sulfate, phosphate, and like commonly known
inorganic radicals. The inorganic radicals do not have bonded
therein the metallic elements of the periodic table (such as the
alkali metals, alkaline earth metals, transition metals, lanthanide
metals, or actinide metals), although such metal ions can sometimes
serve as a pharmaceutically acceptable cation for anionic inorganic
radicals such as a sulfate, phosphate, or like anionic inorganic
radical. Inorganic radicals do not comprise metalloids elements
such as boron, aluminum, gallium, germanium, arsenic, tin, lead, or
tellurium, or the noble gas elements, unless otherwise specifically
indicated elsewhere herein.
[0141] Compounds described herein can contain one or more double
bonds and, thus, potentially give rise to cis/trans (E/Z) isomers,
as well as other conformational isomers. Unless stated to the
contrary, the invention includes all such possible isomers, as well
as mixtures of such isomers.
[0142] Unless stated to the contrary, a formula with chemical bonds
shown only as solid lines and not as wedges or dashed lines
contemplates each possible isomer, e.g., each enantiomer and
diastereomer, and a mixture of isomers, such as a racemic or
scalemic mixture. Compounds described herein can contain one or
more asymmetric centers and, thus, potentially give rise to
diastereomers and optical isomers. Unless stated to the contrary,
the present invention includes all such possible diastereomers as
well as their racemic mixtures, their substantially pure resolved
enantiomers, all possible geometric isomers, and pharmaceutically
acceptable salts thereof. Mixtures of stereoisomers, as well as
isolated specific stereoisomers, are also included. During the
course of the synthetic procedures used to prepare such compounds,
or in using racemization or epimerization procedures known to those
skilled in the art, the products of such procedures can be a
mixture of stereoisomers.
[0143] Disclosed are the components to be used to prepare the
compositions of the invention as well as the compositions
themselves to be used within the methods disclosed herein. These
and other materials are disclosed herein, and it is understood that
when combinations, subsets, interactions, groups, etc. of these
materials are disclosed that while specific reference of each
various individual and collective combinations and permutation of
these compounds can not be explicitly disclosed, each is
specifically contemplated and described herein. For example, if a
particular compound is disclosed and discussed and a number of
modifications that can be made to a number of molecules including
the compounds are discussed, specifically contemplated is each and
every combination and permutation of the compound and the
modifications that are possible unless specifically indicated to
the contrary. Thus, if a class of molecules A, B, and C are
disclosed as well as a class of molecules D, E, and F and an
example of a combination molecule, A-D is disclosed, then even if
each is not individually recited each is individually and
collectively contemplated meaning combinations, A-E, A-F, B-D, B-E,
B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any
subset or combination of these is also disclosed. Thus, for
example, the sub-group of A-E, B-F, and C-E would be considered
disclosed. This concept applies to all aspects of this application
including, but not limited to, steps in methods of making and using
the compositions of the invention. Thus, if there are a variety of
additional steps that can be performed it is understood that each
of these additional steps can be performed with any specific
embodiment or combination of embodiments of the methods of the
invention.
[0144] It is understood that the compositions disclosed herein have
certain functions. Disclosed herein are certain structural
requirements for performing the disclosed functions, and it is
understood that there are a variety of structures that can perform
the same function that are related to the disclosed structures, and
that these structures will typically achieve the same result.
B. Dendrimeric Compounds
[0145] Dendrimers can be ideal building blocks for biomedical
applications, because of their precise architecture, high loading
capacity, tunable solubility, immunogenicity, and bioconjugation
capability. [Gillies, E. R.; Frechet, J. M. J. Drug Discov. Today
2005, 10, 35.; Lee, C. C.; MacKay, J. A.; Frechet, J. M. J.; Szoka,
F. C. Nat. Biotechnol. 2005, 23, 1517.]The combination of the
unique properties of dendrimers with membrane-permeable guanidino
groups can lead to a more efficient-synthesis of membrane-permeable
carrier molecules possessing high efficiency, for example, for bulk
production.
[0146] The compounds of the invention are desirably based upon a
compact, high branching multiplicity dendrimer, for example, the
classic Newkome-type dendrimer. [Newkome, G. R.; Behera, R. K.;
Moorefield, C. N.; Baker, G. R. J. Org. Chem. 1991, 56, 7162.]
Newkome type dendrimers are typically 1.fwdarw.3 C-branched
polyamide macromolecules, built from "Behera's Amine" monomer or
its derivatives, and can be attached to a variety of starting
cores, surfaces, and polymers.
[0147] It is also understood that the compounds of the invention
can be tailored to enhance accumulation in specific sublocations of
cells, such as the nucleus, the cytosol, or the mitochondria.
Tailoring can be the selection of chemical moieties or groups
having an affinity for a targeted subcellular region of a cell, for
example an organelle, and the functionalization of the compounds
with the selected chemical moieties or groups. Such tailoring of
the compound structure can be accomplished using organic synthetic
methodology know to those of skill in the art.
[0148] In one aspect, the invention relates to compounds comprising
the structure:
##STR00004##
and at least one guanidinium residue, wherein m is zero or a
positive integer. In certain aspects, m can be 0, 1, 2, 3, 4, 5, or
6 and each residue can be substituted or unsubstituted. In a
further aspect, m is 1.
[0149] In one aspect, the invention relates to compounds comprising
the structure:
##STR00005##
wherein n and o are, independently, zero or a positive integer;
wherein R.sup.1 and R.sup.2 are, independently, hydrogen, oxygen,
alkyl, acyl, thioacyl, or carbonyl; wherein R.sup.3 is hydrogen,
alkyloxycarbonyl, or alkyl; R.sup.4 is hydrogen, or
alkyloxycarbonyl; wherein R.sup.5 and R.sup.6 are, independently,
hydrogen, or alkyl; and wherein R.sup.7 is hydrogen or
alkyloxycarbonyl.
[0150] In a further aspect, the compounds can comprise the
structure:
##STR00006##
wherein n is an integer from 1 to 9; wherein R.sup.1 and R.sup.2
are, independently, hydrogen, oxygen, nitrogen, alkyl, acyl,
thioacyl, carbonyl, or amine; wherein R.sup.3 is hydrogen or alkyl;
and wherein R.sup.4 is hydrogen, or alkyloxycarbonyl, alkyl, or
acyl. In certain aspects, n can be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9.
In a further aspect, n is 1 or 5. In a further aspect, R.sup.4 can
be hydrogen or alkyloxycarbonyl. In a further aspect, R.sup.2 is
Boc, for example, t-Boc.
[0151] In one aspect, the compound comprises the structure:
##STR00007##
wherein n is an integer from 1 to 9; wherein R.sup.1 and R.sup.2
are, independently, hydrogen, amino, hydroxyl, alkyl, alkoxyl,
acyl, carbonyl, or thioacyl; wherein R.sup.3 is hydrogen or alkyl;
and wherein R.sup.4 is hydrogen, or alkyloxycarbonyl.
C. Methods of Making Dendrimeric Compounds
[0152] The disclosed methods typically employ a divergent method to
prepare a G-1 dendrimer scaffold with nine end functionalities.
Although the Newkome type dendrimer is well known, one of the
drawbacks for a broader application of conventional methods is the
elaborate synthesis of the monomer. In contrast, the "Behera's
amine" gives the most compact, low molecular weight polyamide
dendrimer possible; achieving the necessary nine end
functionalities in just one generation of dendritic growth. As set
forth below and in the Experimental section, following synthesis of
the monomer through improved hydrogenation and work-up procedures,
the G-1 dendritic nona-acid scaffold can be prepared in high yields
(see FIGS. 6A and 6B).
[0153] In order to introduce the guanidinium groups to the
dendrimer exterior as shown in FIG. 20, the nine carboxylic acid
groups were first converted into nine protected amine groups, by
reaction with, for example, N-Boc ethylendiamine and
N-Boc-1,6-diaminehexane through amide coupling reactions. After
removal of the protecting groups, the nine free amines can be
reacted with a guandinylating reagent [Feichtinger, K.; Sings, H.
L.; Baker, T. J.; Matthews, K.; Goodman, M. J. Org. Chem. 1998, 63,
8432.] to give a guanidinylated dendritic scaffold in high
yield.
[0154] For uptake evaluation and imaging function, a fluorophore
can be conjugated to the focal point of the molecular transporter.
The attachment of a fluorescein isothiocyanate (FITC) moiety to the
guanidinylated scaffold can be achieved with a reduction of the
nitro group at the focal point to an amino group via hydrogenation
at room temperature in quantitative yields, followed by direct
reaction with FITC to form the Boc-protected FITC-labeled
guanidino-dendrimer. After deprotection of the Boc-protected
guanidine groups, FITC-labeled dendritic molecules can be obtained
and further purified by dialysis or HPLC.
[0155] In one aspect, the invention relates to methods of preparing
compounds having the structure:
##STR00008##
wherein n is an integer from 1 to 9, wherein R.sup.3 is hydrogen or
alkyl, wherein R.sup.4 and R.sup.7 are, independently, hydrogen,
alkyloxycarbonyl, alkyl, or acyl; wherein R.sup.7 is hydrogen,
alkyl, or acyl; wherein Y comprises a nitro group, an amine group,
an amide group, azide group, or an alkyloxycarbonyl protected amine
group or a derivative thereof, the method comprising the steps of
providing a first compound comprising the structure:
##STR00009##
wherein X comprises OH, halogen, or OC(O)-alkyl; coupling the first
compound with at least about three molar equivalents of a second
compound comprising the structure:
##STR00010##
wherein G.sup.1 is an ester-protecting group; removing the
ester-protecting group; reacting the product of step (c) with at
least about three molar equivalents of a third compound comprising
the structure:
##STR00011##
wherein G.sup.2 is an amine-protecting group; removing the
amine-protecting group; and functionalizing the product of step (e)
with at least three molar equivalents of a guanidine-providing
agent.
[0156] In a further aspect, the guanidine-providing agent comprises
at least one of N,N'-diBoc-N''-triflylguanidine,
N,N'-diCbz-N''-triflylguanidine, N,N'-dialloc-N''-triflylguanidine,
N,N'-ditroc-N''-triflylguanidine,
1,3-diboc-2-(2-hydroxyethyl)guanidine,
N,N'-diBoc-1H-pyrazole-1-carboxamidine,
N,N'-diCbz-1H-pyrazole-1-carboxamidine, 1H-pyrazole-1-carboxamidine
hydrochloride, 1,3-diboc-2-(2-hydroxyethyl)guanidine,
2-(2-aminoethyl)-1,3-diboc-guandine, or
1,3-diboc-2-(carboxymethyl)guanidine
[0157] In a further aspect, the method further comprises the step
of transforming Y into an amine to provide a compound comprising
the structure:
##STR00012##
[0158] In a further aspect, the method further comprises the step
of removing R.sup.7. The removing step can be, for example,
treatment with one or more reagents known to those of skill in the
art for removing protecting groups.
[0159] In one aspect, the providing step comprises synthesis of the
starting materials.
[0160] Each starting material can be obtained commercially and/or
prepared by those of skill in the art from commercially available
compounds. For example, the nitroester shown below can be prepared
using methodology from Newkone, G. R.; Behera, R. K.; Moorefield,
C. N.; Baker, G. R.; J. Org. Chem. 1991, 56, 7162:
##STR00013##
[0161] In a further aspect, the ester-protecting group comprises
methyl, ethyl, or t-butyl.
[0162] In a further aspect, the amine-protecting group comprises a
butyloxycarbonyl group, a trifluoroacyl group, a
9-fluorenylmethyloxycarbonyl group, an alloc group, or a
carbobenzyloxy group.
[0163] In a further aspect, the method further comprises the step
of acylating the amine with a compound comprising the
structure:
##STR00014##
wherein o and p are, independently, zero or a positive integer. In
a yet further aspect, the method further comprises the step of
reacting the product of the acylating step with a payload compound
comprising at least one amine group and at least one of a
luminescent group, a biologically active group, or a
pharmaceutically active group.
[0164] In a further aspect, the method further comprises the step
of acylating the amine with a fourth compound comprising the
structure:
##STR00015##
wherein o and p are, independently, zero or a positive integer, and
wherein G.sup.3 is an thiol-protecting group.
[0165] In a further aspect, the thiol protecting group comprises
the structure:
##STR00016##
and wherein the fourth compound comprises the structure:
##STR00017##
[0166] In a further aspect, the thiol-protecting group comprises
the structure:
##STR00018##
[0167] In a further aspect, the method further comprises the step
of removing the thiol-protecting group, thereby providing a
deprotected thiol. In a yet further aspect, the method further
comprises the step of attaching the deprotected thiol to a
thiol-functionalized payload. In a still further aspect, the
thiol-functionalized payload comprises at least one of a
luminescent group, a biologically-active group, or a
pharmaceutically-active group.
D. Compositions
[0168] In one aspect, the invention relates to compositions
comprising one or more compounds of the invention or one or more
products of the methods of the invention.
[0169] 1. Intracellular Delivery Compositions
[0170] In one aspect, the invention relates to intracellular
delivery compositions comprising the general structure P-L-B-F,
wherein P is payload moiety; wherein L is a linking moiety
comprising the structure:
##STR00019##
wherein o and p are, independently, zero or a positive integer;
wherein B is a branching moiety comprising the structure:
##STR00020##
and wherein F is a functional moiety comprising at least one
guanidinium residue. In a further aspect, p is an integer from 0 to
6, for example, 0, 1, 2, 3, 4, 5, or 6. In a further aspect, the
composition comprises at least six guanidinium residues, at least
seven guanidinium residues, at least eight guanidinium residues, or
at least nine guanidinium residues.
[0171] In one aspect, L-B-F comprises the structure:
##STR00021##
wherein n is an integer from 1 to 9; wherein R.sup.3 is hydrogen or
alkyl; wherein R.sup.4 is hydrogen, alkyl or acyl; and wherein
R.sup.7 is hydrogen, alkyl or acyl.
[0172] In a further aspect, P-L-B-F comprises the structure:
##STR00022##
wherein n is an integer from 1 to 9; wherein R.sup.3 is hydrogen or
alkyl; wherein R.sup.4 is hydrogen, alkyl or acyl; wherein R.sup.7
is hydrogen, alkyl or acyl; and wherein R.sup.8 comprises the
structure:
##STR00023##
[0173] a. Payloads
[0174] Typically, the compounds of the invention can be
functionalized to carry a payload. In various aspects, a payload
compound can be attached or associated with a compound of the
invention by covalent bonding, by ionic bonding, by coordination
bonding, or by hydrogen bonding. In further aspects, a payload
compound can be associated with a compound of the invention by
hydrophilic interactions or hydrophobic interactions. In certain
aspects, a payload compound is part of a compound of the invention,
while in certain further aspects, payload compound is a separate
compound from of a compound of the invention.
[0175] In one aspect, the payload moiety bears a thiol moiety. In a
further aspect, the payload moiety is a luminescent group. For
example, the luminescent group can comprise the structure:
##STR00024##
[0176] In certain aspects, the luminescent group is selected from a
dansyl group, a coumarin group, an FITC group, a DOTA group, a
catechol group, or a DPTA group. DOTA, catechol, and/or DPTA groups
can be used for complexing, for example, lanthanides. Catechol can
be used for complexing, for example, quantum dots, lanthanides,
metals (such as iron or copper (e.g., radioactive Cu)), ironoxides,
metal oxides, and/or platinum (e.g., cis-platinum).
[0177] In a further aspect, the payload moiety is a
biologically-active group. For example, the biologically-active
group can be selected from one or more of an oligonucleotide, a
plasmid DNA, a protein, an immunoglobulin, an antisense oligoDNA, a
peptide nucleic acid (PNA), or a peptide. For example, in various
aspects, the biologically-active group can comprise one or more of
.beta.-galactosidase, horseradish peroxidase, RNase, anti-apoptotic
proteins Bcl-X(L)/PEA-15, catalase, green fluorescence protein,
heat shock protein 70, human glutamate dehydrogenase, ovalbumin,
neuroptotectant Bcl-xL, E2 protein, phosphorothioate antisense
oligonucleotides, anti-tetanus F(ab').sub.2, G protein,
p16.sup.INK4a, caspase-3, p14.sup.INK4a, p27.sup.kipl, Bak BH3
domain peptide, cGPK-I.alpha. inhibitory peptide, IKK.beta.
C-terminal peptide, PKA inhibitory peptide, MEK 1 N-terminal
peptide, luciferin, RhoA, APO-BEC-1, Cre recombinase, H-Ras,
Filmin-1, p16, HPC-1/syntaxin, Cdk2, E2f-1/p73/p53, influenza
virus, antibodies, single chain antibodies, si-RNA, RNA
derivatives, peptide 46, peptide 15, peptides that influence the
imunresponse, mitochondrial DNA, bacteria, birdflu virus, and/or
bacteria.
[0178] In a further aspect, the payload moiety is a
pharmaceutically-active group. For example, the
pharmaceutically-active group is selected from a small molecular
weight drug, a silica nanoparticle, a metal nanoparticle, a
protein, a peptide, a linear polymer backbone, a hydrogel, a
collapsed nanoparticle, a dendrimers, or a hyperbranched polymeric
structure. For example, in various aspects, the
pharmaceutically-active group can comprise one or more of
superparamagnetic iron oxide particles, doxorubicin, methotrexate,
liposome, multiple sclerosis agents, cis-platinum, paclitaxel,
hormones, antioxidants, antimicrobials, antibacterial agents,
antidepressants, sedatives, antihypertensive drugs, antibodies, a
carbohydrate-based drug, cardioprotective EPKC agonist peptide, Fab
fragments of the anti-melanoma antibody NRML-05, pan-carcinoma
antibody NRLU-10, anti-CEA immunotoxin, liposome drugs,
bromonidine, fusogenic, dendritic cell vaccines, VHL tumor
suppressor peptide, HER-2, Pro-apotoxic Smac peptide, viralcapsids,
and/or bacteria.
[0179] A doxorubicin biocongujate, for example, can be synthesized
by the scheme shown below:
##STR00025##
[0180] In a still further aspect, the payload is an antibody, an
intrabody, DNA, RNA, siRNA, among other biologically significant
conjugates. For example, an antibody can be attached to the core of
a disclosed dendrimer, through disclosed methods. Such compositions
can be used to treat viral related disorders, such as, for example,
HIV or influenza, among others. A specific example of an antibody
suitable for use with the disclosed dendimers is an IgG
antibody.
[0181] A disclosed dendrimer can also be attached to a protein is
associated with a number of disorders, including cancer. For
example, a disclosed protein-dendrimer can conjugate can be used to
treat a cancer. An example is a p53 (tumor suppressor protein)
dendrimer conjugate which can be capable of restoration of a mutant
p53 transcriptional activity, to trigger apoptosis and stop tumor
progression through the cytoplasm. A further example is a
dendrimer-Huntingtin (protein responsible of Huntington's disease)
conjugate which can aid in the inhibition of aberrant protein
aggregation in a cellular model of Huntington's disease, by
targeting huntingtin to the nucleus, through the action of the
dendritic molecular transporter.
[0182] Further examples of conjugates that can be used in
combination with the disclosed dendritic transporters include M and
N intrabodies for RSV, RV6-26 Fab Rotavirus, Tat
(HIV-1-transcription activator) for the inhibition of viral
replication by sequestering Tat in the cytoplasm.
[0183] b. Intracellular Delivery
[0184] In one aspect, the invention relates to methods of
intracellular delivery comprising administering an effective amount
of one or more compounds of the invention or one or more
compositions of the invention to a subject. In one aspect, The
subject is a mammal, for example, a human. In a further aspect, the
subject is a cell. The delivery can be, for example, oral,
transmucosal, rectal, or subcutaneous administration or, for
example, intravenous, intrathecal, intramuscular, intranasal,
intraperitonel, or intraocular injection.
[0185] 2. Pharmaceutical Compositions
[0186] A pharmaceutical composition comprising a therapeutically
effective amount of one or more compounds of the invention or one
or more compositions of the invention and a pharmaceutically
acceptable carrier for administration in a mammal, for example, a
human. The compositions can be, for example, granules, powders,
tablets, or capsules.
[0187] a. Dosage
[0188] The specific therapeutically effective dose level for any
particular patient will depend upon a variety of factors including
the compound or composition being administered; the disorder being
treated and the severity of the disorder; the specific composition
employed; the age, body weight, general health, sex and diet of the
patient; the time of administration; the route of administration;
the rate of excretion of the specific compound employed; the
duration of the treatment; drugs used in combination or
coincidental with the specific compound employed and like factors
well known in the medical arts. If desired, the effective daily
dose can be divided into multiple doses for purposes of
administration. Consequently, single dose compositions can contain
such amounts or submultiples thereof to make up the daily dose. The
dosage can be adjusted by the individual physician in the event of
any contraindications. Dosage can vary, and can be administered in
one or more dose administrations daily, for one or several
days.
[0189] b. Carriers
[0190] A "pharmaceutically acceptable carrier" refers to sterile
aqueous or nonaqueous solutions, dispersions, suspensions or
emulsions, as well as sterile powders for reconstitution into
sterile injectable solutions or dispersions just prior to use.
Examples of suitable aqueous and nonaqueous carriers, diluents,
solvents or vehicles include water, ethanol, polyols (such as
glycerol, propylene glycol, polyethylene glycol and the like),
carboxymethylcellulose and suitable mixtures thereof, vegetable
oils (such as olive oil) and injectable organic esters such as
ethyl oleate. Proper fluidity may be maintained, for example, by
the use of coating materials such as lecithin, by the maintenance
of the required particle size in the case of dispersions and by the
use of surfactants. These compositions may also contain adjuvants
such as preservatives, wetting agents, emulsifying agents, and
dispersing agents. Prevention of the action of microorganisms may
be ensured by the inclusion of various antibacterial and antifungal
agents such as paraben, chlorobutanol, phenol, sorbic acid, and the
like. It can also be desirable to include isotonic agents such as
sugars, sodium chloride, and the like. Prolonged absorption of the
injectable pharmaceutical form may be brought about by the
inclusion of agents, such as aluminum monostearate and gelatin,
which delay absorption. Injectable depot forms are made by forming
microencapsule matrices of the drug in biodegradable polymers such
as polylactide-polyglycolide, poly(orthoesters) and
poly(anhydrides). Depending upon the ratio of drug to polymer and
the nature of the particular polymer employed, the rate of drug
release can be controlled. Depot injectable formulations are also
prepared by entrapping the drug in liposomes or microemulsions
which are compatible with body tissues. The injectable formulations
may be sterilized, for example, by filtration through a
bacterial-retaining filter or by incorporating sterilizing agents
in the form of sterile solid compositions which can be dissolved or
dispersed in sterile water or other sterile injectable media just
prior to use. Suitable inert carriers can include sugars such as
lactose. Desirably, at least 95% by weight of the particles of the
active ingredient have an effective particle size in the range of
0.01 to 10 micrometers.
E. Synthesis and Characterization of "Bow-Tie" Dendritic Molecular
Transporters by Orthogonal and Click Approach
[0191] Disclosed is the synthesis and characterization of "Bow-Tie"
dendritic architectures with orthogonally reactive groups, defined
composition and functionality, which can be used as multi-drug
carries for specific intracellular delivery. Huisgen cycloadditions
or so called "click" reactions have been shown to be extremely
versatile tools for advanced macromolecular design. However, little
attempt has been made to utilize this approach to prepare
multifunctional dendritic structures. In the disclosed approach,
two orthogonal protected dendritic structures are combined by
utilizing the "click" reaction. This strategy allows the controlled
deprotection of the trifluoro protecting group to selectively
attach the dithiopyridylpropionic acid the periphery of the
macromolecule. In a further step, the BOC groups of the second
dendritic scaffolds are deprotected to be guanydilated to the
ethyl- or hexyl linker of the system. The bow-tie structure is the
first of its kind that consists of a molecular transporter part and
drug delivery entity on the other. The chemistry applied for the
construction is high-yielding and, thus, gives the bow-tie delivery
structure in the most straightforward approach. In this fashion,
nine drug molecules, for example peptides, genes and
oligonucleotides can be transported across cellular membranes.
[0192] Synthetic Pathway of Acid-Labile Azide-Linker-Dendron:
##STR00026##
[0193] Synthetic Pathway for Base-Labile Alkyne-Linker-Dendron:
##STR00027## ##STR00028##
[0194] Bifunctional Bow-Tie Synthesis by Click Reaction:
##STR00029## ##STR00030##
[0195] Further Functionalization for Synthesis of Cell-Permeable
Multi-Drug Carrier Conjugates:
##STR00031## ##STR00032## ##STR00033## ##STR00034##
[0196] In one aspect, the invention relates to compounds comprising
the structure:
##STR00035##
wherein each m is independently zero or a positive integer, and
wherein L is a linking moiety comprising optionally substituted
alkyl, optionally substituted alkoxyl, optionally substituted
heteroalkyl, or optionally substituted heteroaryl.
[0197] In a further aspect, L comprises a structure:
##STR00036##
wherein each n is independently selected from 0-8. That is, L can
comprise the reaction product of a "click" reaction.
[0198] In a further aspect, the compound can comprise a
structure
##STR00037##
wherein n is an integer from 1 to 9; wherein R.sup.3 is hydrogen or
alkyl; wherein R.sup.4 is hydrogen, or alkyloxycarbonyl, alkyl, or
acyl; and wherein R.sup.7 is hydrogen or alkyloxycarbonyl.
[0199] In a yet further aspect, the compound can comprise the
structure:
##STR00038##
wherein n is an integer from 1 to 9; wherein R.sup.3 is hydrogen or
alkyl; wherein R.sup.4 is hydrogen, or alkyloxycarbonyl, alkyl, or
acyl; and wherein R.sup.7 is hydrogen or alkyloxycarbonyl.
[0200] In a still further aspect, the compound can comprise the
structure:
##STR00039##
wherein n is an integer from 1 to 9; wherein R.sup.3 is hydrogen or
alkyl; wherein R.sup.4 is hydrogen, or alkyloxycarbonyl, alkyl, or
acyl; and wherein R.sup.7 is hydrogen or alkyloxycarbonyl.
[0201] In an even further aspect, the compound can comprise the
structure:
##STR00040##
wherein each n is independently an integer from 0 to 9; wherein
R.sup.3 is hydrogen or alkyl; wherein R.sup.4 is hydrogen, or
alkyloxycarbonyl, alkyl, or acyl; and wherein R.sup.7 is hydrogen
or alkyloxycarbonyl.
[0202] It is demonstrated that the disclosed transporter (e.g.,
FD-2, hexyl linker) shows selectivity towards the mitochondria of a
cell. (see FIG. 33) The PD-1 shows selectivity towards the cell
nucleus (see FIG. 32). A common obstacle in macromolecular drug
delivery is the cellular uptake into cell compartments that do not
release the drug delivery vector into the cytosol or mitochondria
in which the drug becomes effective. Most other delivery pathways
into the cell end up in the lysosome and do not get released
(endocytosis). The therapeutic efficacy of drug molecules typically
depends on its ability to reach desired target tissues, cells and
intracellular organelles.
[0203] The mitochondria play a key role in apoptosis (cancer
therapy), familial amyotrophic lateral sclerosis (ALS, Lou Gehrig's
disease), Leber hereditary optic neuropathy (LHON), lactic
acidosis, strokelike syndrome (MELAS) Huntington's disease, and
Alzheimer's disease, Kearns-Sayre Syndrome (KSS), myoclonic
epilepsy, ragged-red fibers (MERRF), cluster of metabolic diseases
(SyndromeX), progressive external ophthalmophlegia (PEO) and
antioxidants. By targeting the mitochondria, the disclosed
compounds, compositions, and methods can play a role in therapy or
prevention of disease processes relating to mitochondria
function.
[0204] When the disclosed transporter is attached to the disclosed
nanoparticle, it can enter the cell and also can achieve
localization in the entire cell, including the mitochondria. The
nanoparticle allows delivering a high drug load and, thus, can
facilitate delivery of small and other molecules, such as peptides,
nucleotides and such. The structures can be further modified with
amines to allow complexation with plasmic DNA and covalent
attachment s though covalent approaches. (See FIGS. 34-39).
[0205] A nanoparticle with a number of molecular transporter
moieties conjugated to the periphery crosses the plasmic membrane
and localizes in the cytosol and, particularly, in the mitochondria
of the cells. Techniques are disclosed that allow the attachment of
the molecular transporter the scaffolds that increase the drug load
significantly. Attachment to the "bow-tie" structure and/or the
attachment to nanoparticles from intramolecular chain collapse
techniques also increase the drug load significantly.
[0206] The dendritic transporter allows the conjugation of nine
bioactive conjugates and the drug load is increased nine fold by
attaching a dendric molecule to the focal point of the dendritic
molecular transporter (bow-tie). A well-defined macromolecule is
designed, that is "clicked" together in a Huisgen type reaction.
The deprotection of the basic/acidic protecting groups allows the
modification to a delivery system with a short ethyl linker or
hexyl linker before guanidylation to maintain uptake into specific
subcellular locations. The disulfide linker is only one of the
examples of a linker chemistry attached to the drug part of the
bow-tie structure. All other linkers presented can be applied as
well.
[0207] The drug load can be increased nine-fold by attaching a
dendric molecule to the focal point of the dendritic molecular
transporter (bow-tie). A well-defined macromolecule is designed,
that is "clicked" together in a Huisgen type reaction. The
deprotection of the basic/acidic protecting groups allows the
modification to a delivery system with a short ethyl linker or
hexyl linker before guanidylation to maintain uptake into specific
subcellular locations.
[0208] Here, the drug load can be increased to a theoretical amount
of 100-300 positions to conjugate small molecule drugs, peptides,
oligonucleotides and more. The functionalization of the particle
with a varied amount of amines allows together with the attachment
of transporter allows the development of a gene delivery system. A
"drug" can also be conjugated though a disulfide bond in a covalent
conjugation approach. For example, proteins can be delivered. (See
FIGS. 47, 48, 50, and 51).
F. Crosslinked Degradable Polymeric Nanoparticles
[0209] Traditional polyester nanoparticle delivery systems are
typically self-assembled from linear polyesters chains driven by
the polarity of the solvent, emulsion composition and addition
techniques. These procedures predetermine the drug loading during
nanoparticle formation and limit post-modification chemistries in
organic and aqueous solutions. Furthermore, the result of this
self-assembly process is mirrored in the morphology and degradation
properties of the release systems. It has been recognized that the
degradation behavior of the nanoparticles and release profile of
the entrapped drug molecules are factors to establish predictable
pharmacokinetic profiles in effective multidrug cancer therapies.
So far, release kinetics are challenged by a rapid release of the
drug molecules in the first 24-48 h followed by a slower release,
referred to as a "burst-effect." These release profiles typically
prevent the establishment of reliable dosages and contribute to
developing multidrug resistance, often times the result of
non-optimized drug concentrations at tumor sites.
[0210] In contrast, actively targeted drug delivery carriers can
entrap high concentrations of hydrophobic therapeutics and maintain
a linear release profile, which can be tuned to the demands of the
tumor type as a result of the adjustable supramolecular
architecture accomplished through an intermolecular cross-linking
technique. The disclosed methods of preparing polyester particles
utilize a controlled cross-linking mechanism of linear polyester
precursors that contain pendant functional groups as one of the
cross-linking units with a difunctionalized linker that acts as the
second cross-linking partner. To achieve control over a series of
different nanoparticle size dimensions, the amount of the
difunctionalized linker is added in a series of varying
equivalencies to the pendant functionalities of the linear
polyester precursor. Nanoparticles can be produced, depending on
the linker amount present in the reaction, with unique sizes and
standard deviations of only 10%. These "nano-networks," depending
on their nanoparticle size and cross-linking density, influence
their crystallinity, but the particles are amorphous at the
temperature of use (37.degree. C.). To determine if the amorphous
properties of poly(valerolactoneepoxyvalerolactone), poly(vl-evl)
particles have a positive effect on the degradation behavior, a
series of degradation studies in buffer at pH 7.4 at 37.degree. C.
were performed, investigating particles from a completed series of
linear precursors and increasing amounts of difunctionalized
cross-linkers with controlled nanoscopic dimensions (FIG. 1).
[0211] Degradation of the particles was monitored by the change of
the absolute molecular weight, as determined through static light
scattering (SLS). Linear degradation profiles were observed for all
particles investigated, with the highest loss of molecular weight
for the 725 nm nanoparticle with 17.5% of its total molecular mass
remaining after 10 days. Smaller particles with a slightly higher
degree of crystallinity of 20.6% were degraded to 26% of the
original molecular weight. The observed linear degradation kinetics
are a parameter that determines the quality of the developed
particles towards applications as controlled release systems.
[0212] The capacity to encapsulate small molecule drugs, such as
paclitaxel (taxol), can also be evaluated. Traditional polyester
particles, produced with salting-out or nanoprecipitation methods,
typically do not exceed a drug loading over 5% that is facilitated
during nanoparticle formation. However, the disclosed nanoparticles
consist of crosslinked supramolecular structures that are readily
soluble in organic solvents without affecting the 3-D architecture.
This property provides the opportunity to load the particles after
formation by dissolving the particles in dimethyl sulfoxide (DMSO)
together with cancer therapeutics, such as paclitaxel (taxol), and
precipitating into water.
[0213] Determination of drug loading capacity was performed with
particles of 53 nm in diameter from linear precursors,
poly(-valerolactone-epoxyvalerolactone-allylvalerolactone-oxepanedione),
poly(vl-evl-avl-opd), containing 11% epoxide and crosslinked with 2
equivalents of diamines per epoxide (FIG. 3). In preparation for in
vivo experiments, the encapsulation method was designed to also
increase the homogenity of the particle dispersion in water for a
practical administration of the drug loaded particles by injection.
An emulsification process with vitamin E TPGS (D-a-tocopherol
polyethylene glycol 1000 succinate) was used, which achieves a
homogenous dispersion of the loaded or un-loaded particles in water
or buffer. The resulting particles are analyzed by UV-Vis with a
NanoDrop Spectrophotometer at 254 nm, and along with a calibration
curve, the drug loading with paclitaxel was found to be 15.7% for
an aimed 20% drug load and 11.3% for a 15% drug load, respectively.
With this process, it is not only possible to load therapeutic drug
molecules to a higher degree into prepared nanoparticles, but it is
also possible to solubilize hydrophobic cancer therapeutics in
aqueous solutions.
[0214] Side effects known to be caused by adjuvant agents, such as
Cremophor EL (50:50 ethanol-polyoxyethylated castor oil) to
solubilize hydrophobic drug molecules for intravenous injections,
can be avoided. To ensure that no cellular toxicity is caused by
the vitamin E TPGS formulated particles prior to drug loading, the
cell viability was assessed by utilizing a MTT assay (FIG. 2). The
cellular toxicity was determined by incubating HeLa cells with
varying concentrations of particles in triplicate ranging from 5
mg/ml to 0.001 mg/ml. Following 24 h of incubation with particles,
cell viability was assessed. As seen in FIG. 2, the nanoparticles
did not cause significant cytotoxicity against the HeLa cell line.
The experimental TC50 value for the formulated particles was found
to be 1.0 mg/ml. Moreover, emulsification had an effect on the
degradation profile and was found to correlate with the in vitro
release studies. Over the period of 16 days, the particles
experienced a low controlled degradation, as seen by the linear
degradation profile, finishing with 70% of its original molecular
weight remaining (FIG. 3). Without wishing to be bound by theory,
it is believed that the slower degradation rate can be attributed
to the well-defined structure of the nanoparticle and the vitamin E
TPGS that remains at the surface to stabilize the particles.
Consequently, this gradual constant degradation profile of the
particles is a desirable feature, as it translates into the
controlled and sustained release of therapeutics.
[0215] The paclitaxel release kinetics from vitamin E TPGS
formulated nanoparticles were assessed by monitoring the cumulative
release of taxol at 37.degree. C. in DPBS at pH 7.4. At particular
time intervals, the samples were centrifuged, and the supernatant
was taken for analysis of paclitaxel concentration by NanoDrop
spectrophotometry (254 nm). FIG. 4 depicts the cumulative release
of paclitaxel from the particles. The profile shows a collective
release of 4.4% and 7.4% taxol in the first 2 and 6 h respectively,
followed by a slow and sustained release over 60 days, which again
confirmed the efficient encapsulation of paclitaxel within the
cross-linked nanoparticles. Without wishing to be bound by theory,
it is believed that the initial instant release of paclitaxel in
the first several hours is due to the dissolution or diffusion of
the drug that was absorbed onto the nanoparticle surface, while the
linear slow continuous release is attributed to the diffusion of
the drug encapsulated in the nanoparticle during degradation. In
contrast, traditional poly(lactic-co-glycolic acid) (PLGA)
nanoparticles experience an erratic nonlinear drug release, that
includes a "burst-effect" in which about 40% of taxol is released
in the first day, followed by a fast release of about 10-30% in the
next 2-5 days and then finally a slow release till no paxlitaxel
(taxol) remains. In is noteworthy that the release kinetics can be
adjusted to faster or slower release, governed by the density of
cross-linking and the particle size.
[0216] In preparation for in vivo studies, in which the particle
sizes can play an role in the interaction with the tumor
vasculature, the influence of formulation and encapsulation of
small molecule drugs to the diameter of the nanoparticles was
evaluated. It was found that based on the 3-D crosslinked network
structure, the size dimension slightly changes from 53 nm to 57 nm
and indicates the conformity of the 3-D network structure upon
encapsulation, as seen by transmission electron microscopy (TEM),
(FIG. 5), with 2-8 times more drug incorporated compared to
traditional polyester nanoparticle systems. Drug release profiles
for conventional polyester based nanoparticle systems, compared to
the disclosed nanoparticle systems, are shown in FIG. 8.
[0217] Thus, the disclosed nanoparticle synthesis pathways allow
for the introduction of functional groups, such as alkyne, allyl or
keto functionalities, that are not affected by the cross-linking
reactions and nanoparticle formation. In particular, thiol-ene
"click" reactions allow for the conjugation of peptides with
integrated cysteines added to the sequence near the N-terminus.
Such mild reaction conditions do not require the addition of
radical starters and use slightly elevated temperatures of
37.degree. C. To synthesize the drug delivery systems, the linear
poly(vl-evl-avl-opd) precursor was prepared, which was cross-linked
with 2 equivalents of diamines per epoxide to form a nanoparticle
of 53 nm in size. The remaining allyl groups were then
functionalized with peptides to target radiated and nonradiated
tumor vasculature, such as the reported peptides with recognition
units HVGGSSV and cRGD, respectively (FIG. 6). The bioconjugates
were analyzed via NMR, DLS and SLS and were then loaded with
paclitaxel and formulated with vitamin E. Using UV-Vis, the loading
capacity was found to be 11%, aiming for a 15% drug load.
[0218] It can also be demonstrated that the linear release kinetics
are in fact adjustable. With decreasing cross-linking density, the
drug molecule is released at a higher rate. By decreasing the
cross-linking density (.about.50%), from 7% to 2%, the release rate
increased by around 50%. These data point indicate that a 15%
cross-linking can decrease the release by another 50% with 140 days
for 100% release and would afford a very slow release rate. To
afford a faster release of the drug molecule than the 2%
cross-liking, a longer cross-linker (MW 2003) can be used to
prepare a particle with a wider network architecture for an even
faster release profile. With a 7% cross-linking density, 40% of the
drug is released in 6 days. It is understood that with the decrease
of the cross-linking density to 2%, the release can be increased to
3 days (40%). This is represented schematically in FIG. 9.
[0219] 1. Polymers
[0220] It is understood that the disclosed polymers can be used in
connection with the disclosed nanoparticles and disclosed methods.
Unless stated to the contrary, the disclosed structures can be used
in connection with the disclosed methods, the disclosed polymers,
and the disclosed nanoparticles.
[0221] a. Epdxide-Functionalized Polymers
[0222] In one aspect, the invention relates to a polymer comprising
at least one monomer residue having an optionally substituted
structure represented by a formula:
##STR00041##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m
is an integer from 0 to 6; wherein n is an integer from 0 to 2; and
wherein the monomer residue comprises less than about 10% by weight
of the monomer residue of halogen selected from chlorine, bromine,
and iodine. In further aspects, the monomer residue can comprise
less than about 8%, less than about 5%, less than about 4%, less
than about 3%, less than about 2%, or less than about 1% of halogen
selected from chlorine, bromine, and iodine, by weight of the
monomer residue.
[0223] In a further aspect, an epoxide-functionalized polymer can
further comprise at least one monomer residue selected from a
propargyl-functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00042##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein
m.sup.1 is an integer from 0 to 6, and wherein n.sup.1 is an
integer from 0 to 2; a monomer residue having an optionally
substituted structure represented by a formula:
##STR00043##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.2 is an integer from 0 to 2; and a keto-functionalized
monomer residue having an optionally substituted structure
represented by a formula:
##STR00044##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.3 is an integer from 0 to 2.
[0224] In one aspect, Z is O. That is, the polymer residue can be a
polyester residue. In a further aspect, the polymer is a polyester.
In a further aspect, the polymer is a co-polyester.
[0225] In a further aspect, the Z is NR, wherein R is H or C1 to C6
alkyl. In one aspect, the polymer residue can be a polyamide
residue. In a further aspect, the polymer is a polyamide. In a
further aspect, the polymer is a co-polyamide. The alkyl can be
optionally further substituted. R can be C1 to C6, C2 to C6, C1 to
C5, C2 to C5, C1 to C4, C2 to C4, C1, C2, C3, C4, C5, or C6
alkyl.
[0226] In one aspect, the polymer comprises at least one monomer
residue having an optionally substituted structure represented by a
formula:
##STR00045##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein Y
is O, S, or NR, wherein R is H or C1 to C6 alkyl; wherein R.sup.L
is selected from optionally substituted alkyl and optionally
substituted alkoxylene; wherein m is an integer from 0 to 6; and
wherein n is an integer from 0 to 2.
[0227] In a further aspect, the polymer further comprises at least
one monomer residue selected from a propargyl-functionalized
monomer residue having an optionally substituted structure
represented by a formula:
##STR00046##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein
m.sup.1 is an integer from 0 to 6, and wherein n.sup.1 is an
integer from 0 to 2; a monomer residue having an optionally
substituted structure represented by a formula:
##STR00047##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.2 is an integer from 0 to 2; and a keto-functionalized
monomer residue having an optionally substituted structure
represented by a formula:
##STR00048##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.3 is an integer from 0 to 2.
[0228] In a further aspect, the polymer comprises an optionally
substituted structure represented by a formula:
##STR00049##
wherein m and m' are independently integers from 0 to 6; wherein n
and n' are independently integers from 0 to 2; and wherein Z and Z'
are independently O or NR, wherein R is H or C1 to C6 alkyl;
wherein Y and Y' are independently O, S, or NR, wherein R is H or
C1 to C6 alkyl; and wherein R.sup.L is selected from optionally
substituted alkyl and optionally substituted alkoxylene.
[0229] In various aspects, m can be an integer from 0 to 6, from 1
to 6, from 0 to 5, from 1 to 5, from 0 to 4, from 1 to 4, from 0 to
3, from 1 to 3, from 0 to 2, from 1 to 2, 0, 1, 2, 3, 4, 5, or 6.
In various aspects, m' can be an integer from 0 to 6, from 1 to 6,
from 0 to 5, from 1 to 5, from 0 to 4, from 1 to 4, from 0 to 3,
from 1 to 3, from 0 to 2, from 1 to 2, 0, 1, 2, 3, 4, 5, or 6. In
various aspects, m.sup.1 can be an integer from 0 to 6, from 1 to
6, from 0 to 5, from 1 to 5, from 0 to 4, from 1 to 4, from 0 to 3,
from 1 to 3, from 0 to 2, from 1 to 2, 0, 1, 2, 3, 4, 5, or 6.
[0230] In various aspects, n can be an integer from 0 to 2, from 1
to 2, from 0 to 1, 0, 1, or 2. In various aspects, n' can be an
integer from 0 to 2, from 1 to 2, from 0 to 1, 0, 1, or 2. In
various aspects, n.sup.1 can be an integer from 0 to 2, from 1 to
2, from 0 to 1, 0, 1, or 2. In various aspects, n.sup.2 can be an
integer from 0 to 2, from 1 to 2, from 0 to 1, 0, 1, or 2. In
various aspects, n.sup.3 can be an integer from 0 to 2, from 1 to
2, from 0 to 1, 0, 1, or 2.
[0231] R.sup.L can be selected from optionally substituted alkyl
and optionally substituted alkoxylene. Suitable alkyls include
divalent organic radicals selected from ethyl, propyl, butyl,
pentyl, hexyl, heptyl, octyl, decyl, dodecyl, hexadecyl, and higher
alkyl. Suitable alkoxylene include divalent organic radicals
selected from groups having a structure represented by a
formula:
##STR00050##
Further suitable alkoxylene include divalent organic radicals
selected from groups having a structure represented by a
formula:
##STR00051##
Further suitable alkoxylene include a divalent organic radical
having a structure represented by a formula:
##STR00052##
which can be derived from 2,2-(ethylenedioxy)bis(ethylamine).
[0232] The polymers and copolymers typically have a number average
molecular weight (Mn) of from about 3500-4800 Daltons with a narrow
polydispersity of from about 1.17 to about 1.27. It is understood
that the molecular weight can be higher or lower and that one of
skill in the art can readily manipulate reaction conditions to
achieve a different desired molecular weight.
[0233] b. Multifunctional Polymers
[0234] In one aspect, a polymer can be a multifunctional polymer.
That is, the polymer comprises monomer residues selected from two
or more of an epoxide-functionalized monomer residue having an
optionally substituted structure represented by a formula:
##STR00053##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m
is an integer from 0 to 6; wherein n is an integer from 0 to 2; and
a propargyl-functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00054##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein
m.sup.1 is an integer from 0 to 6, and wherein n.sup.1 is an
integer from 0 to 2; and a keto-functionalized monomer residue
having an optionally substituted structure represented by a
formula:
##STR00055##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.3 is an integer from 0 to 2.
[0235] In one aspect, the epoxide-functionalized monomer residue is
present and comprises less than about 10% by weight of the monomer
residue of halogen selected from chlorine, bromine, and iodine. In
further aspects, the monomer residue can comprise less than about
8%, less than about 5%, less than about 4%, less than about 3%,
less than about 2%, or less than about 1% of halogen selected from
chlorine, bromine, and iodine, by weight of the monomer
residue.
[0236] In a further aspect, the polymer further comprises at least
one monomer residue having an optionally substituted structure
represented by a formula:
##STR00056##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.2 is an integer from 0 to 2.
[0237] In one aspect, a polymer comprises at least one monomer
residue having an optionally substituted structure represented by a
formula:
##STR00057##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein Y
is O, S, or NR, wherein R is H or C1 to C6 alkyl, wherein R.sup.L
is selected from optionally substituted alkyl and optionally
substituted alkoxylene, wherein m is an integer from 0 to 6, and
wherein n is an integer from 0 to 2; and one or more of: a
propargyl-functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00058##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein
m.sup.1 is an integer from 0 to 6, and wherein n.sup.1 is an
integer from 0 to 2; and a keto-functionalized monomer residue
having an optionally substituted structure represented by a
formula:
##STR00059##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.3 is an integer from 0 to 2. In a further aspect, the polymer
further comprises at least one monomer residue having an optionally
substituted structure represented by a formula:
##STR00060##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.2 is an integer from 0 to 2. In one aspect, the at least one
monomer residue has an optionally substituted structure represented
by a formula:
##STR00061##
wherein m and m' are independently integers from 0 to 6; wherein n
and n' are independently integers from 0 to 2; and wherein Z and Z'
are independently O or NR, wherein R is H or C1 to C6 alkyl;
wherein Y and Y' are independently O, S, or NR, wherein R is H or
C1 to C6 alkyl; and wherein R.sup.L is selected from optionally
substituted alkyl and optionally substituted alkoxylene.
[0238] In one aspect, at least one monomer residue has an
optionally substituted structure represented by a formula:
##STR00062##
wherein m and m.sup.1' are independently integers from 0 to 6;
wherein n and n.sup.1' are independently integers from 0 to 2; and
wherein Z and Z' are independently O or NR, wherein R is H or C1 to
C6 alkyl; wherein Y is O, S, or NR, wherein R is H or C1 to C6
alkyl; and wherein R.sup.L is selected from optionally substituted
alkyl and optionally substituted alkoxylene.
[0239] In various aspects, m can be an integer from 0 to 6, from 1
to 6, from 0 to 5, from 1 to 5, from 0 to 4, from 1 to 4, from 0 to
3, from 1 to 3, from 0 to 2, from 1 to 2, 0, 1, 2, 3, 4, 5, or 6.
In various aspects, m' can be an integer from 0 to 6, from 1 to 6,
from 0 to 5, from 1 to 5, from 0 to 4, from 1 to 4, from 0 to 3,
from 1 to 3, from 0 to 2, from 1 to 2, 0, 1, 2, 3, 4, 5, or 6. In
various aspects, m.sup.1 can be an integer from 0 to 6, from 1 to
6, from 0 to 5, from 1 to 5, from 0 to 4, from 1 to 4, from 0 to 3,
from 1 to 3, from 0 to 2, from 1 to 2, 0, 1, 2, 3, 4, 5, or 6. In
various aspects, m.sup.1' can be an integer from 0 to 6, from 1 to
6, from 0 to 5, from 1 to 5, from 0 to 4, from 1 to 4, from 0 to 3,
from 1 to 3, from 0 to 2, from 1 to 2, 0, 1, 2, 3, 4, 5, or 6.
[0240] In various aspects, n can be an integer from 0 to 2, from 1
to 2, from 0 to 1, 0, 1, or 2. In various aspects, n' can be an
integer from 0 to 2, from 1 to 2, from 0 to 1, 0, 1, or 2. In
various aspects, n.sup.1 can be an integer from 0 to 2, from 1 to
2, from 0 to 1, 0, 1, or 2. In various aspects, n.sup.1 can be an
integer from 0 to 2, from 1 to 2, from 0 to 1, 0, 1, or 2. In
various aspects, n.sup.2 can be an integer from 0 to 2, from 1 to
2, from 0 to 1, 0, 1, or 2. In various aspects, n.sup.3 can be an
integer from 0 to 2, from 1 to 2, from 0 to 1, 0, 1, or 2.
[0241] R.sup.L can be selected from optionally substituted alkyl
and optionally substituted alkoxylene. Suitable alkyls include
divalent organic radicals selected from ethyl, propyl, butyl,
pentyl, hexyl, heptyl, octyl, decyl, dodecyl, hexadecyl, and higher
alkyl. Suitable alkoxylene include divalent organic radicals
selected from groups having a structure represented by a
formula:
##STR00063##
Further suitable alkoxylene include divalent organic radicals
selected from groups having a structure represented by a
formula:
##STR00064##
Further suitable alkoxylene include a divalent organic radical
having a structure represented by a formula:
##STR00065##
which can be derived from 2,2-(ethylenedioxy)bis(ethylamine) or
2,2-(ethylenedioxy)bis(ethylazide).
[0242] In one aspect, a polymer can comprise at least one monomer
residue having an optionally substituted structure represented by a
formula:
##STR00066##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein
R.sup.L is selected from optionally substituted alkyl and
optionally substituted alkoxylene, wherein m.sup.1 is an integer
from 0 to 6, and wherein n.sup.1 is an integer from 0 to 2; and one
or more of: an epoxide-functionalized monomer residue having an
optionally substituted structure represented by a formula:
##STR00067##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m
is an integer from 0 to 6; wherein n is an integer from 0 to 2; and
a keto-functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00068##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.3 is an integer from 0 to 2. In a further aspect, the
epoxide-functionalized monomer residue is present and comprises
less than about 10% by weight of the monomer residue of halogen
selected from chlorine, bromine, and iodine.
[0243] In one aspect, the polymer further comprises at least one
monomer residue having an optionally substituted structure
represented by a formula:
##STR00069##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.2 is an integer from 0 to 2.
[0244] In a further aspect, at least one monomer residue has an
optionally substituted structure represented by a formula:
##STR00070##
wherein m.sup.1 and m.sup.1' are independently integers from 0 to
6; wherein n.sup.1 and n.sup.1' are independently integers from 0
to 2; and wherein Z and Z' are independently O or NR, wherein R is
H or C1 to C6 alkyl; and wherein R.sup.L is selected from
optionally substituted alkyl and optionally substituted
alkoxylene.
[0245] In one aspect, at least one monomer residue has an
optionally substituted structure represented by a formula:
##STR00071##
wherein m.sup.1 and m' are independently integers from 0 to 6;
wherein n.sup.1 and n' are independently integers from 0 to 2; and
wherein Z and Z' are independently O or NR, wherein R is H or C1 to
C6 alkyl; and wherein Y' is O, S, or NR, wherein R is H or C1 to C6
alkyl; wherein R.sup.L is selected from optionally substituted
alkyl and optionally substituted alkoxylene.
[0246] 2. Nanoparticles
[0247] It is understood that the disclosed nanoparticles can be
used in connection with the disclosed polymers and disclosed
methods. Unless stated to the contrary, the disclosed structures
can be used in connection with the disclosed methods, the disclosed
polymers, and the disclosed nanoparticles.
[0248] In one aspect, the invention relates to a degradable
polymeric nanoparticle comprising at least one monomer residue
having an optionally substituted structure represented by a
formula:
##STR00072##
wherein m and m' are independently integers from 0 to 6; wherein n
and n' are independently integers from 0 to 2; and wherein Z and Z'
are independently O or NR, wherein R is H or C1 to C6 alkyl;
wherein Y and Y' are independently O, S, or NR, wherein R is H or
C1 to C6 alkyl; and wherein R.sup.L is selected from optionally
substituted alkyl and optionally substituted alkoxylene, wherein
the nanoparticle has a particle size of from about 5 nm to about
850 nm.
[0249] In one aspect, the nanoparticle further comprises at least
one monomer residue selected from a propargyl-functionalized
monomer residue having an optionally substituted structure
represented by a formula:
##STR00073##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein
m.sup.1 is an integer from 0 to 6, and wherein n.sup.1 is an
integer from 0 to 2; a keto-functionalized monomer residue having
an optionally substituted structure represented by a formula:
##STR00074##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.3 is an integer from 0 to 2; and a monomer residue having an
optionally substituted structure represented by a formula:
##STR00075##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.2 is an integer from 0 to 2. In a further aspect, Z and Z'
are O.
[0250] In one aspect, the nanoparticle further comprises at least
one epoxide-functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00076##
[0251] In a further aspect, the nanoparticle further comprises at
least one functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00077##
wherein X is OH, SH, NH.sub.2, or NHR, wherein R is H or C1 to C6
alkyl; and wherein R.sup.1 is an optionally substituted organic
radical comprising 1 to 24 carbon atoms.
[0252] In a further aspect, R.sup.1 is further substituted with at
least one biologically active agent, at least one pharmaceutically
active agent, and/or at least one imaging moiety.
[0253] In one aspect, the nanoparticle further comprises at least
one nucleophile-functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00078##
[0254] In a further aspect, the nanoparticle further comprises at
least one functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00079##
wherein R.sup.1 is an optionally substituted organic radical
comprising 1 to 24 carbon atoms.
[0255] In a further aspect, R.sup.1 is further substituted with at
least one biologically active agent, at least one pharmaceutically
active agent, and/or at least one imaging moiety.
[0256] In one aspect, the invention relates to a degradable
polymeric nanoparticle comprising at least one monomer residue
having an optionally substituted structure represented by a
formula:
##STR00080##
wherein m.sup.1 and m.sup.1' are independently integers from 0 to
6; wherein n.sup.1 and n.sup.1' are independently integers from 0
to 2; and wherein Z and Z' are independently O or NR, wherein R is
H or C1 to C6 alkyl; and wherein R.sup.L is selected from
optionally substituted alkyl and optionally substituted alkoxylene,
wherein the nanoparticle has a particle size of from about 5 nm to
about 850 nm.
[0257] In a further aspect, the nanoparticle further comprises at
least one monomer residue selected from: an epoxide-functionalized
monomer residue having an optionally substituted structure
represented by a formula:
##STR00081##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m
is an integer from 0 to 6; wherein n is an integer from 0 to 2; a
keto-functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00082##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.3 is an integer from 0 to 2; and a monomer residue having an
optionally substituted structure represented by a formula:
##STR00083##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.2 is an integer from 0 to 2. In a further aspect, Z and Z'
are O.
[0258] In a further aspect, the nanoparticle further comprises at
least one propargyl-functionalized monomer residue having an
optionally substituted structure represented by a formula:
##STR00084##
[0259] In a further aspect, the nanoparticle further comprises at
least one functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00085##
wherein R.sup.1 is an optionally substituted organic radical
comprising 1 to 24 carbon atoms. In a further aspect, R.sup.1 is
further substituted with at least one biologically active agent, at
least one pharmaceutically active agent, and/or at least one
imaging moiety.
[0260] In a further aspect, the nanoparticle further comprises at
least one azide-functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00086##
[0261] In a further aspect, the nanoparticle further comprises at
least one functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00087##
wherein R.sup.1 is an optionally substituted organic radical
comprising 1 to 24 carbon atoms. In a further aspect, R.sup.1 is
further substituted with at least one biologically active agent, at
least one pharmaceutically active agent, and/or at least one
imaging moiety.
[0262] In one aspect, the invention relates to crosslinked
degradable nanoparticles having a polyester backbone and one or
more crosslinks having a structure selected from:
##STR00088##
wherein Y is O, S, or N--R, wherein R is C1-C4 alkyl;
##STR00089##
wherein L is a divalent alkyl chain or alkyloxyalkyl chain.
[0263] In a further aspect, the one or more crosslinks are produced
by a nucleophilic epoxide ring opening reaction. In a further
aspect, the one or more crosslinks are produced by a reductive
amination reaction. In a further aspect, the one or more crosslinks
are produced by an azide alkyne cycloaddition.
[0264] In a further aspect, the nanoparticle further comprises one
or more biologically active agents or pharmaceutically active
agents.
[0265] In a further aspect, the nanoparticle is produced by
crosslinking a polymer comprising at least one monomer residue
having an optionally substituted structure represented by a
formula:
##STR00090##
wherein m is an integer from 0 to 6, and wherein n is an integer
from 0 to 2; or at least one propargyl-functionalized monomer
residue having an optionally substituted structure represented by a
formula:
##STR00091##
wherein m.sup.1 is an integer from 0 to 6, and wherein n.sup.1 is
an integer from 0 to 2; or at least one monomer residue having an
optionally substituted structure represented by a formula:
##STR00092##
wherein n.sup.2 is an integer from 0 to 2; or at least one
keto-functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00093##
wherein n.sup.3 is an integer from 0 to 2.
[0266] In one aspect, the invention relates to compositions
comprising a degradable polyester nanoparticle and, encapsulated
therein, a biologically active agent, a pharmaceutically active
agent, or an imaging agent. In a further aspect, the biologically
active agent is encapsulated within the nanoparticle. In a further
aspect, the pharmaceutically active agent is encapsulated within
the nanoparticle. In a further aspect, the imaging agent is
encapsulated within the nanoparticle.
[0267] In a further aspect, the degradable polyester nanoparticle
comprises a crosslinked degradable nanoparticle having a polyester
backbone and one or more crosslinks having a structure selected
from:
##STR00094##
wherein Y is O, S, or N--R, wherein R is C1-C4 alkyl;
##STR00095##
##STR00096##
wherein L is a divalent alkyl chain or alkyloxyalkyl chain.
G. Preparation Methods
[0268] It is understood that the disclosed methods can be used in
connection with the disclosed polymers and disclosed nanoparticles.
Unless stated to the contrary, the disclosed structures can be used
in connection with the disclosed methods, the disclosed polymers,
and the disclosed nanoparticles.
[0269] 1. Methods of Making Polymer
[0270] To address the deficiencies of conventional nanoparticle
compositions and methods, the availability of novel functional
polyesters that allow orthogonal modification approaches was
addressed. Additionally, controlled chain cross-linking strategies
for obtaining distinct nanoparticles in a variety of nanoscopic
dimensions are disclosed. In contrast to investigating
emulsification-solvent techniques [Hans, M. L.; Lowman, A. M. Curr.
Opin. Solid State Mater. Sci. 2002, 6, 319-327.] or emulsion
diffusion methods [Kallinteri, P.; Higgins, S.; Hutcheon, G. A.;
St. Pourcain, C. B.; Garnett, M. C. Biomacromolecules 2005, 6,
1885-1894.] that need surfactants or salts, the disclosed methods
and compositions involve controlled cross-linking techniques.
[0271] A clean and non-toxic cross-linking entity can be provided
from epoxide groups that react with dinucleophiles (e.g., diamines)
to form alkane --OFT groups. While this crosslinking unit has been
employed to form acrylate based microparticles [Burke, S. K.;
Slatopolsky, E. A.; Goldberg, D. I., Nephrol. Dial. Transplant.
1997, 12, (8), 1640-1644.], it has been never investigated in the
formation of degradable nanoparticles due to the lack of suitable
linear precursors.
[0272] The epoxide entity for the formation of discrete
cross-linked nanoparticles can be integrated by polymerization of a
low molecular weight linear copolymer, Ab, with pendant allyl
groups. See FIG. 10. Pendant allyl groups represent valuable
intermediates to many functional groups and can be incorporated
into the polymer backbone by copolymerizing
.alpha.-allyl-.delta.-valerolactone, (b), and commercially
available .delta.-valerolactone, (A), via ring-opening
polymerization (ROP). [Parrish, B.; Quansah, J. K.; Emrick, T. J.
Polym. Sci. Part A: Polym. Chem. 2002, 40, 1983-1990.] Upon
copolymerization, the pendant allyl groups can be oxidized by a
Baeyer-Villiger oxidation with meta-chloroperbenzoic acid (m-CPBA)
to convert the double bonds to epoxide rings, which then became a
coupling group in the preparation of the nanoparticles. [(a)
Mecerreyes, D.; Miller, R. D.; Hedrick, J. L.; Detrembleur, C.;
Jerome, R. J. Polym. Sci. Part A: Polym. Chem. 2000, 38, 870-875.
(b) Latere, J. P.; Lecomte, P.; Dubois, P.; Jerome, R.
Macromolecules 2002, 35, 7857-78591 To introduce additional
functional groups into the nanoparticle, additional monomers can be
synthesized, for example .alpha.-propargyl-.delta.-valerolactone,
(C), and 2-oxepane-1,5-dione, (D). These monomers can then be
individually copolymerized with (B) and .delta.-valerolactone, (A),
in a similar manner as Ab, to give rise to linear polyesters with
additional propargyl or keto functionalities respectively. To
increase the number of functionalities that allow orthogonal
modification approaches, (C) and (D) were copolymerized together
with (b) and .delta.-valerolactone (A), as summarized in FIG. 10.
The copolymers were typically obtained in molecular weight ranges
of 3500-4800 Da with narrow polydispersities of 1.17-1.27.
[0273] In one aspect, the invention relates to a method of
preparing a polymer comprising the step of copolymerizing a mixture
of two or more of an alkene-functionalized monomer providing a
residue having an optionally substituted structure represented by a
formula:
##STR00097##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m
is an integer from 0 to 6; wherein n is an integer from 0 to 2; a
propargyl-functionalized monomer providing a residue having an
optionally substituted structure represented by a formula:
##STR00098##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein
m.sup.1 is an integer from 0 to 6, and wherein n.sup.1 is an
integer from 0 to 2; and a keto-functionalized monomer providing a
residue having an optionally substituted structure represented by a
formula:
##STR00099##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.3 is an integer from 0 to 2.
[0274] In a further aspect, the mixture further comprises at least
one monomer providing a residue having an optionally substituted
structure represented by a formula:
##STR00100##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.2 is an integer from 0 to 2.
[0275] In one aspect, the alkene-functionalized monomer is present
and the method further comprises the step of oxidizing the
resultant polymer to provide an epoxide-functionalized monomer
residue having an optionally substituted structure represented by a
formula:
##STR00101##
[0276] In a further aspect, the alkene-functionalized monomer is
present and has an optionally substituted structure represented by
a formula:
##STR00102##
[0277] In a further aspect, the propargyl-functionalized monomer is
present and has an optionally substituted structure represented by
a formula:
##STR00103##
[0278] In a further aspect, the keto-functionalized monomer is
present and has an optionally substituted structure represented by
a formula:
##STR00104##
[0279] In a further aspect, the monomer providing a residue having
an optionally substituted structure represented by a formula:
##STR00105##
has an optionally substituted structure represented by a
formula:
##STR00106##
[0280] In one aspect, the invention relates to a method of
preparing an epoxide-functionalized polymer comprising the step of
oxidizing a polymer having at least one monomer residue having an
optionally substituted structure represented by a formula:
##STR00107##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m
is an integer from 0 to 6, and wherein n is an integer from 0 to
2.
[0281] In a further aspect, the polymer further comprises at least
one monomer residue selected from:
##STR00108##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein
m.sup.1 is an integer from 0 to 6, and wherein n.sup.1 is an
integer from 0 to 2;
##STR00109##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein
n.sup.2 is an integer from 0 to 2; and
##STR00110##
wherein n.sup.3 is an integer from 0 to 2.
[0282] In a further aspect, at least one monomer residue has an
optionally substituted structure represented by a formula:
##STR00111##
wherein m is an integer from 0 to 6, and wherein n is an integer
from 0 to 2. For example, in one aspect, m is 1, and n is 0,
providing an optionally substituted structure represented by a
formula:
##STR00112##
[0283] In a further aspect, the epoxide-functionalized polymer has
an optionally substituted structure represented by a formula:
##STR00113##
[0284] 2. Methods of Crosslinking
[0285] In one aspect, the invention relates to a method of
crosslinking a polymer comprising the step of reacting a polymer
comprising at least one monomer residue selected from an
epoxide-functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00114##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m
is an integer from 0 to 6; wherein n is an integer from 0 to 2; and
a propargyl-functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00115##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein
m.sup.1 is an integer from 0 to 6, and wherein n.sup.1 is an
integer from 0 to 2; with a cross-linker having a structure
represented by a formula X--R.sup.L--X', wherein X and X' are
independently N.sub.3, OH, SH, NH.sub.2, or NHR, wherein R is H or
C1 to C6 alkyl, and wherein R.sup.L is selected from optionally
substituted alkyl and optionally substituted alkoxylene.
[0286] In one aspect, the linker groups can be bis-nucleophilic
(e.g., diamine) compounds derived from alkylene oxides (e.g.,
diamino poly(ethylene oxides)) and/or alkyls (e.g.,
1,8-diaminooctane; Jeffamines) and their derivatives.
[0287] In a further aspect, the linker groups can be thiols. For
example, the dinucleophile can have a structure X--R.sup.L--X',
wherein X and X' are each SH, wherein R is H or C1 to C6 alkyl, and
wherein R.sup.L is selected from optionally substituted alkyl,
optionally substituted alkoxylene, and optionally substituted
esters.
[0288] Thiols suitable for crosslinking include mono- and di-thiol
analogues of compounds derived from alkylene oxides (e.g., diamino
poly(ethylene oxides)) and/or alkyls (e.g., 1,8-diaminooctane;
Jeffamines) and their derivatives. Other suitable dithiols for
cross-linking include:
##STR00116##
[0289] An example crosslinking reaction, and example product
thereof, is shown below:
##STR00117##
[0290] In one aspect, the cross-linker reacts with two polymer
strands. In a further aspect, X--R.sup.L--X' reacts with two
epoxide-functionalized monomer residues. In a further aspect,
X--R.sup.L--X' reacts with two propargyl-functionalized monomer
residues. In a further aspect, X--R.sup.L--X' reacts with one
epoxide-functionalized monomer residue and one
propargyl-functionalized monomer residue. In a further aspect,
X.dbd.X'. In a further aspect, X.dbd.X'.dbd.NH.sub.2. In a further
aspect, R.sup.L comprises two or more residues of ethylene oxide or
trimethylene oxide. In a further aspect, X--R.sup.L--X' is
2,2-(ethylenedioxy)bis(ethylamine). In a further aspect,
X.dbd.X'.dbd.N.sub.3.
[0291] In one aspect, the polymer comprises at least one monomer
residue having an optionally substituted structure represented by a
formula:
##STR00118##
and wherein X.dbd.X'.dbd.NH.sub.2. In one aspect, the polymer and
the crosslinker are reacted in a ratio of about 1:1
(polymer:cross-linker). In a further aspect, the polymer and the
crosslinker are reacted in a ratio of about >1:1
(polymer:cross-linker) to provide a polymer with excess
epoxide-functionalization. In a further aspect, the polymer and the
crosslinker are reacted in a ratio of about <1:1
(polymer:cross-linker) to provide a polymer with excess
amino-functionalization.
[0292] In one aspect, the polymer comprises at least one monomer
residue having an optionally substituted structure represented by a
formula:
##STR00119##
and wherein X.dbd.X'.dbd.N.sub.3. In a further aspect, the polymer
and the crosslinker are reacted in a ratio of about 1:1
(polymer:cross-linker). In a further aspect, the polymer and the
crosslinker are reacted in a ratio of about >1:1
(polymer:cross-linker) to provide a polymer with excess
alkyne-functionalization. In a further aspect, the polymer and the
crosslinker are reacted in a ratio of about <1:1
(polymer:cross-linker) to provide a polymer with excess
azide-functionalization.
[0293] In a further aspect, the polymer further comprises a
keto-functionalized monomer providing a residue having an
optionally substituted structure represented by a formula:
##STR00120##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.3 is an integer from 0 to 2.
[0294] In a further aspect, the polymer further comprises at least
one monomer providing a residue having an optionally substituted
structure represented by a formula:
##STR00121##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.2 is an integer from 0 to 2.
[0295] 3. Methods of Functionalizing Polymers
[0296] In one aspect, the invention relates to a method of
functionalizing a polymer comprising the step of reacting an
epoxide-functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00122##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m
is an integer from 0 to 6; wherein n is an integer from 0 to 2;
with a nucleophile having a structure represented by a formula
X--R.sup.1, wherein X is OH, SH, NH.sub.2, or NHR, wherein R is H
or C1 to C6 alkyl; and wherein R.sup.1 is an optionally substituted
organic radical comprising 1 to 24 carbon atoms.
[0297] Organic radicals suitable for use as R.sup.1 include
substituted or unsubstituted monovalent organic radicals selected
from ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, decyl,
dodecyl, hexadecyl, and higher alkyl. The alkyl can be linear or
branched and can be cyclic or acyclic. In a further aspect, R.sup.1
can comprise an optionally substituted alkoxylene. Suitable
alkoxylene include substituted or unsubstituted monovalent organic
radicals selected from groups having a structure represented by a
formula:
##STR00123##
wherein R.sup.3 comprises C1 to C6 alkyl.
[0298] In a further aspect, R.sup.1 is further substituted with at
least one biologically active agent, at least one pharmaceutically
active agent, and/or at least one imaging moiety, thus providing a
convenient method for functionalizing the polymer with one or more
biologically active agents, pharmaceutically active agents, and/or
imaging moieties via a nucleophilic substitution reaction. That is,
R.sup.1 can comprise at least one biologically active agent, at
least one pharmaceutically active agent, and/or at least one
imaging moiety. In a further aspect, R.sup.1 can comprise a portion
of the at least one biologically active agent, at least one
pharmaceutically active agent, and/or at least one imaging moiety.
In a further aspect, R.sup.1 can be covalently bonded to at least
one biologically active agent, at least one pharmaceutically active
agent, and/or at least one imaging moiety.
[0299] In one aspect, the invention relates to a method of
functionalizing a polymer comprising the step of reacting a
propargyl-functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00124##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein
m.sup.1 is an integer from 0 to 6, and wherein n.sup.1 is an
integer from 0 to 2; with an azide having a structure represented
by a formula N.sub.3--R.sup.1, wherein R.sup.1 is an optionally
substituted organic radical comprising 1 to 24 carbon atoms. In a
further aspect, R.sup.1 is further substituted with at least one
biologically active agent, at least one pharmaceutically active
agent, and/or at least one imaging moiety.
[0300] In one aspect, the invention relates to a method of
functionalizing a polymer comprising the steps of reacting a
keto-functionalized monomer providing a residue having an
optionally substituted structure represented by a formula:
##STR00125##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.3 is an integer from 0 to 2; with an amine having a structure
represented by a formula H.sub.2N--R.sup.1, wherein R.sup.1 is an
optionally substituted organic radical comprising 1 to 24 carbon
atoms; and reducing the resulting imine. In a further aspect, the
reacting step and the reducing step are performed simultaneously.
In a further aspect, R.sup.1 is further substituted with at least
one biologically active agent, at least one pharmaceutically active
agent, and/or at least one imaging moiety.
[0301] In one aspect, the invention relates to a method of
functionalizing a polymer comprising the step of reacting a
nucleophile-functionalized monomer residue having an optionally
substituted structure represented by a formula:
##STR00126##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m
is an integer from 0 to 6;
[0302] wherein n is an integer from 0 to 2; wherein Y and Y' are
independently O, S, or NR, wherein R is H or C1 to C6 alkyl; and
wherein R.sup.L is selected from optionally substituted alkyl and
optionally substituted alkoxylene; with an electrophile having a
structure represented by a formula E-R.sup.1, wherein E is an
electrophilic moiety; and wherein R.sup.1 is an optionally
substituted organic radical comprising 1 to 24 carbon atoms.
[0303] In a further aspect, Y' is NH.sub.2 or NHR. In a further
aspect, wherein Y.dbd.Y'. In a further aspect, the electrophilic
moiety is selected from alkyl halide, alkyl pseudohalide, and
carboxyl derivative. In a further aspect, R.sup.1 is further
substituted with at least one biologically active agent, at least
one pharmaceutically active agent, and/or at least one imaging
moiety.
[0304] 4. Methods of Making Nanoparticles
[0305] The formation of nanoparticles in controlled size dimensions
can proceed from linear polymers containing pendant epoxide groups
which crosslink with 2,2'-(ethylenedioxy)bis(ethylamine). To
evaluate the particle formation under controlled conditions,
reactions in which the equivalents of diamine cross-linker were
linearly increased with respect to the reactive epoxide groups of
the polymers were studied.
[0306] To achieve a high degree of cross-linking between the
individual polyester chains, the polymer solution with the pendant
expoxide entities can be added in a dropwise fashion to a refluxing
solution of different equivalents of dinucleophile (e.g., diamine)
in dichloromethane. In this strategy, the difunctional amine is in
high excess during the addition (13 mL/min) of the linear polymer
solution (0.5 M) and thus provides optimum cross-linking reactions
(Table 1; particle size reported in nm diameter by dynamic light
scattering (DLS) in relation to varying amine ratios).
TABLE-US-00001 TABLE 1 Nanoparticle Size Dimensions (nm) Diameter
(nm) Diameter (nm) Diameter (nm) Amine/ Poly(vl-evl)
Poly(v1-evl-opd) Poly(vl-evl-pvl) 1 Epoxide AB ABD ABC 1 30.71 .+-.
2.21 34.29 .+-. 3.22 21.40 .+-. 2.90 2 58.06 .+-. 6.20 63.46 .+-.
7.68 41.70 .+-. 5.36 3 82.1 .+-. 5.73 118.3 .+-. 13.6 114.9 .+-.
8.9 4 115.6 .+-. 25.4 164.9 .+-. 65.7 148.3 .+-. 25.2 5 255.7 .+-.
60.3 292.7.+-. 80.3 186.1 .+-. 37.5 6 342.2 .+-. 52.2 341.0 .+-.
86.6 253.9 .+-. 41.4 8 425.1 .+-. 100 525.0 .+-. 100 472.1 .+-.
103.1 10 725.1 .+-. 94.3 800.0.+-. 135 675.0 .+-. 126.1 Amine/
Diameter (nm) Mw,RI M.sub.w 1 Epoxide AB.sub.1 nanoparticles.sup.a
(g/mol) .sup.b PDI.sup.r (kg/mol).sup.d 1 30.71 .+-. 2.21 3403 1.16
60.5 .+-. 3.5 2 58.06 .+-. 6.20 3445 1.16 81.5.+-. 4.6 3 82.61 .+-.
5.73 3544 1.17 96.1 .+-. 4.9 4 115.6 .+-. 12.5 3860 1.18 112 .+-. 6
5 255.7 .+-. 26.9 4005 1.18 187 .+-. 8 6 342.2 .+-. 42.2 4267 1.21
222 .+-. 11 8 425.1 .+-. 44.6 4470 1.21 328 .+-. 15 10 725.1 .+-.
94.3 4887 1.22 525 .+-. 28
[0307] The first trial was employed with polymer (AB) and
implemented 1 to 10 equivalents of amine functionalities to the
pendant epoxide cross-linking entity. The resulting particles were
characterized by transmission electron microscopy (TEM) that
provides the actual size, and by dynamic light scattering (DLS), to
obtain the hydrodynamic diameter as a representative measure of the
particle under physiological conditions. Micrographs of
representative nanoparticles are shown in FIG. 11. It is also
contemplated that reaction stoichiometry can be selected to utilize
in excess of ten (10) equivalents, thereby providing
microparticles, materials for us in tissue engineering and biogels
in biomedical applications and devices.
[0308] As illustrated in FIG. 12, the particle size increase with a
polynominal trend as the equivalents of amine rises. For example,
two equivalents of amine yielded 58 nm particles, and five
equivalents produced particles with 255 nm dimensions (Table 1).
Synthesized linear polymers containing additional functionalities
(ABC and ABD) were found to respond in the same way to the
controlled intermolecular chain crosslinking conditions, as with
polymer (AB) from the original trial, and well-defined
nanoparticles were obtained (Table 1). As shown in FIG. 13,
characterization of the particles with .sup.1H NMR confirmed the
nanoparticle formation for each trial with an increase of signals
at 3.5 and 2.89 ppm corresponding to protons neighboring the
secondary amine of the polyethylene glycol (PEG)-linker after
successful crosslinking event. In particular, a shift in resonance
from 2.86 to 2.89 ppm was observed due to the change of the primary
amine to the secondary amine after cross-linking. As a consequence,
the continuous increase in amine cross-linker equivalents not only
extends the particle size, but it also introduces additional amine
functionalities connected to short PEG linker that are available
for further modification strategies.
[0309] In one aspect, the invention relates to a method of
preparing a degradable nanoparticle comprising the step of adding a
polymer comprising at least one monomer residue having a structure
represented by a formula:
##STR00127##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m
is an integer from 0 to 6, and wherein n is an integer from 0 to 2;
to a solution of from about 1 to about 10 molar equivalents of a
dinucleophile (nucleophilic moiety:epoxide functionality) having a
structure X--R.sup.L--X', wherein X and X' are independently OH,
SH, NH.sub.2, or NHR, wherein R is H or C1 to C6 alkyl, and wherein
R.sup.L is selected from optionally substituted alkyl and
optionally substituted alkoxylene. In a further aspect, the monomer
residue comprises less than about 10% by weight of the monomer
residue of halogen selected from chlorine, bromine, and iodine. In
a further aspect, Z is O.
[0310] In one aspect, the solution comprises from about 1 molar
equivalent of a dinucleophile (nucleophilic moiety:epoxide
functionality) and the resultant nanoparticle has a particle size
of from about 5 nm to about 55 nm. In a further aspect, the
solution comprises from about 1 molar equivalent of a dinucleophile
(nucleophilic moiety:epoxide functionality) and the resultant
nanoparticle has a particle size of from about 5 nm to about 55 nm.
In a further aspect, the solution comprises from about 2 molar
equivalents of a dinucleophile (nucleophilic moiety:epoxide
functionality) and the resultant nanoparticle has a particle size
of from about 30 nm to about 80 nm. In a further aspect, the
solution comprises from about 3 molar equivalents of a
dinucleophile (nucleophilic moiety:epoxide functionality) and the
resultant nanoparticle has a particle size of from about 70 nm to
about 120 nm. In a further aspect, the solution comprises from
about 4 molar equivalents of a dinucleophile (nucleophilic
moiety:epoxide functionality) and the resultant nanoparticle has a
particle size of from about 110 nm to about 170 nm. In a further
aspect, the solution comprises from about 5 molar equivalents of a
dinucleophile (nucleophilic moiety:epoxide functionality) and the
resultant nanoparticle has a particle size of from about 175 nm to
about 300 nm. In a further aspect, the solution comprises from
about 6 molar equivalents of a dinucleophile (nucleophilic
moiety:epoxide functionality) and the resultant nanoparticle has a
particle size of from about 250 nm to about 350 nm. In a further
aspect, the solution comprises from about 8 molar equivalents of a
dinucleophile (nucleophilic moiety:epoxide functionality) and the
resultant nanoparticle has a particle size of from about 400 nm to
about 550 nm. In a further aspect, the solution comprises from
about 10 molar equivalents of a dinucleophile (nucleophilic
moiety:epoxide functionality) and the resultant nanoparticle has a
particle size of from about 650 nm to about 850 nm. It is also
contemplated that reaction stoichiometry can be selected to utilize
in excess of ten (10) molar equivalents, thereby providing higher
particle sizes.
[0311] In one aspect, the invention relates to a method of
preparing a degradable nanoparticle comprising the step of adding a
polymer comprising at least one monomer residue having a structure
represented by a formula:
##STR00128##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein
m.sup.1 is an integer from 0 to 6, and wherein n.sup.1 is an
integer from 0 to 2; to a solution of from about 1 to about 10
molar equivalents of a bis-azide (azide moiety:alkyne
functionality) having a structure N.sub.3--R.sup.L--N.sub.3,
wherein R.sup.L is selected from optionally substituted alkyl and
optionally substituted alkoxylene. In a further aspect, the monomer
residue comprises less than about 10% by weight of the monomer
residue of halogen selected from chlorine, bromine, and iodine. In
a further aspect, Z is O.
[0312] In one aspect, the invention relates to a method of
controlling particle size during the preparation of a degradable
nanoparticle comprising the step of adding an
epoxide-functionalized polymer to a solution of a dinucleophilic
cross-linker, wherein the stoichiometry of the cross-linker (ratio
of nucleophilic moiety:epoxide functionality) is selected to
provide a desired particle size according to one or more of the
graphs shown in FIG. 14-FIG. 19.
[0313] 5. Methods of Functionalizing Nanoparticles
[0314] In one aspect, the invention relates to a method of
functionalizing a nanoparticle comprising the step of reacting a
nanoparticle comprising at least one epoxide-functionalized monomer
residue having an optionally substituted structure represented by a
formula:
##STR00129##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m
is an integer from 0 to 6; wherein n is an integer from 0 to 2;
with a nucleophile having a structure represented by a formula
X--R.sup.1, wherein X is OH, SH, NH.sub.2, or NHR, wherein R is H
or C1 to C6 alkyl; and wherein R.sup.1 is an optionally substituted
organic radical comprising 1 to 24 carbon atoms. In a further
aspect, R.sup.1 is further substituted with at least one
biologically active agent, at least one pharmaceutically active
agent, and/or at least one imaging moiety.
[0315] In one aspect, the invention relates to a method of
functionalizing a nanoparticle comprising the step of reacting a
nanoparticle comprising at least one propargyl-functionalized
monomer residue having an optionally substituted structure
represented by a formula:
##STR00130##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein
m.sup.1 is an integer from 0 to 6, and wherein n.sup.1 is an
integer from 0 to 2; with an azide having a structure represented
by a formula N.sub.3--R.sup.1, wherein R.sup.1 is an optionally
substituted organic radical comprising 1 to 24 carbon atoms. In a
further aspect, R.sup.1 is further substituted with at least one
biologically active agent, at least one pharmaceutically active
agent, and/or at least one imaging moiety.
[0316] In one aspect, the invention relates to a method of
functionalizing a nanoparticle comprising the steps of reacting a
nanoparticle comprising at least one keto-functionalized monomer
providing a residue having an optionally substituted structure
represented by a formula:
##STR00131##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein
n.sup.3 is an integer from 0 to 2; with an amine having a structure
represented by a formula H.sub.2N--R.sup.1, wherein R.sup.1 is an
optionally substituted organic radical comprising 1 to 24 carbon
atoms; and reducing the resulting imine. In a further aspect, the
reacting step and the reducing step are performed simultaneously.
In a further aspect, R.sup.1 is further substituted with at least
one biologically active agent, at least one pharmaceutically active
agent, and/or at least one imaging moiety.
[0317] In one aspect, the invention relates to a method of
functionalizing a nanoparticle comprising the step of reacting a
nanoparticle comprising at least one nucleophile-functionalized
monomer residue having an optionally substituted structure
represented by a formula:
##STR00132##
wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m
is an integer from 0 to 6; wherein n is an integer from 0 to 2;
wherein Y and Y' are independently O, S, or NR, wherein R is H or
C1 to C6 alkyl; and wherein R.sup.L is selected from optionally
substituted alkyl and optionally substituted alkoxylene; with an
electrophile having a structure represented by a formula E-R.sup.1,
wherein E is an electrophilic moiety; and wherein R.sup.1 is an
optionally substituted organic radical comprising 1 to 24 carbon
atoms. In a further aspect, Y' is NH.sub.2 or NHR. In a further
aspect, Y.dbd.Y'. In a further aspect, the electrophilic moiety is
selected from alkyl halide, alkyl pseudohalide, and carboxyl
derivative. In a further aspect, R.sup.1 is further substituted
with at least one biologically active agent, at least one
pharmaceutically active agent, and/or at least one imaging
moiety.
[0318] 6. Methods of Degrading Nanoparticles
[0319] In one aspect, the invention relates to a method of
degrading a degradable nanoparticle comprising subjecting the
nanoparticle to reaction conditions sufficient to hydrolyze an
ester. In a further aspect, the conditions are biological
conditions. In a further aspect, the conditions involve exposure to
an esterase. In a further aspect, the conditions exist within an
organism.
[0320] In one aspect, the invention relates to a method of
degrading a degradable polymer comprising subjecting the polymer to
reaction conditions sufficient to hydrolyze an ester. In certain
aspects, the degradable polymer is a disclosed polymer or a product
of a disclosed method.
H. Functionalized Polymers and Nanoparticles
[0321] In one aspect, the disclosed nanoparticles can be
functionalized with, for example, the disclosed dendrimeric
compounds. That is, in one aspect, the invention relates to a
nanoparticle-dendrimer conjugate. In a further aspect, the
nanoparticle can be a disclosed organic quantum dots via
intramolecular chain collapse. In a further aspect, the
nanoparticle can be a disclosed degradable nanoparticle. In a
further aspect, the dendrimer can be a disclosed intracellular
delivery composition.
[0322] As disclosed herein, certain nanoparticles can bear
electrophilic (e.g., ketone) functionalities. Vinylsulfonyl
functionality can be introduced to the disclosed nanoparticles.
Thus, a vinylsulfonyl linker moiety was prepared that can be
attached in a reductive amination procedure to a keto groups of the
particle. The synthesis of such a linker appears in Scheme 1. It is
understood that the alkyl chain can be homologated by selection of
appropriate reagents.
##STR00133##
[0323] The vinylsulfonyl moiety readily reacts with a nucleophile
(e.g., a thiol) to form a covalent bond, thereby further
functionalizing a nanoparticle. These linkers can be used to attach
peptides that are labeled with dye molecules at the focal point of
the peptide or other amines groups of the peptide. The thiol groups
of cysteines can be used to attach to the vinyl function of the
vinyl sulfonyl linker. Also, the thiol group in the focal point of
the disclosed dendritic molecular transporters can be attached to
the vinyl sulfonyl (or allyl) group, thus allowing a transporter to
be attached to any post-modified nanoparticle.
[0324] The same reaction can be used to attach peptides that are
not labeled with dye. In such cases, the particle can be labeled
with dye or not labeled.
[0325] Peptides (or other amines) can also be attached directly
through the amine terminus of the peptide to the keto group through
reductive amination. See Scheme 2. Here, it is preferred that the
peptide contains only one amine group. Before the reductive
amination is performed, the particle can be labeled with a dye that
adds to the amine functionality of the particle. After the
reaction, residual dyes can be quenched so as to not interfere with
the following reductive amination.
##STR00134##
##STR00135##
[0326] Similar systems can be constructed with particles from
intramolecular cross-linking reactions. Replacing N-BED with an
ethylenoxide equivalent enhances the solubility of the system.
[0327] Another approach that can enable formation of
nanoparticle-dendrimer conjugate involves direct attachment of
nucleophile-functionalized moieties (e.g., peptides or disclosed
intracellular delivery compositions) to an allylic function on
disclosed degradable nanoparticles. As shown in Scheme 4, direct
attachment of a thiol with an allyl functionalized polymer or
nanoparticle can bypass use of the disclosed SVEC linker.
##STR00136##
[0328] In one aspect, an allylic function on disclosed degradable
nanoparticles can be provided via incomplete oxidation of epoxide
functionalities, as shown in Scheme 5a.
##STR00137## ##STR00138##
[0329] Still other examples of linear precursors can be prepared
according to Scheme 5b.
##STR00139## ##STR00140##
[0330] An allyl functionality is thus available for
functionalization and allows very mild conditions for the
attachment of peptides and other moieties that contain nucleophilic
(e.g., thiol) groups. The allyl groups from Ab linear precursors
can be partially preserved by partial oxidation to the epoxide that
is needed for cross-linking to the nanoparticle to from AbB linear
polymer. This chemistry is also compatible with the
keto-group-containing ABD linear precursor to from AbBD.
[0331] The nanoparticle formation does not take part in the
cross-linking reaction and is therefore available for further
modification. The allyl group is inert under the conditions used
during the cross-linking process. The crosslinking reaction is
illustrated in Scheme 6.
##STR00141##
[0332] Again, the thiol group of the focal point of the dendritic
molecular transporter can be attached to the ally group. One
advantage of such attachment is that it requires no other reagent.
This can permit the transporter to be attached to any already
post-modified nanoparticle because of the mild reaction
conditions.
[0333] In order to track the drug delivery system and study the
uptake into tissues, an imaging moiety (e.g., a dye molecule such
as rodamine or other dye) that has functionality to react with
amines such as NHS-ester or isothiocyanates can be attached to the
free amine groups that result from the cross-linking reaction, as
shown in Scheme 7. The allyl groups or all other groups introduced
are not affected.
##STR00142##
[0334] The allyl groups can then be reacted with thiol groups of
the focal point of the dendritic transporter, as illustrated in
Scheme 8, thereby providing multiply functionalized degradable
nanoparticles.
##STR00143## ##STR00144##
[0335] The number of molecular transporter(s) bonded to the
nanoparticle can be selected by varying the stoichiometry of the
reagents added to the allyl groups. The same reaction can be
performed with thiol groups attached to peptides. It was found that
elevated temperatures such as 37.degree. C. speed up the reaction
but do not destroy the peptide.
[0336] In a further aspect, a nanoparticle can be attached to a
disclosed dendritic molecular transporter through an exemplary
strategy shown in Scheme 8b.
##STR00145## ##STR00146## ##STR00147##
[0337] The dendritic transporter shown in Scheme 8 can be further
functionalized according to Scheme 8c.
##STR00148## ##STR00149## ##STR00150##
[0338] The nanoparticle of the intramolecular chain collapse
reaction can be reacted with the commercially available N-Boc
ethylenoxide amine. The amine can be deprotected via acid cleavage
with HCl or formic acid. Some of the free amines can be labeled
with dye via NHS-ester reaction or thioisocyanide reaction. An SVEC
moiety can then be connected trough an NHS ester reaction. After
the reaction the residual amine groups are being quenched. The
thiol groups are attached to the vinylsulfone groups of the SVEC.
The thiol groups of the molecular transporter can also be attached
in the same fashion as the peptides, as shown in Scheme 9a.
##STR00151## ##STR00152##
[0339] Another example of attaching a peptide to a nanoparticle
core is shown in Scheme 9b.
##STR00153##
[0340] Imaging moieties (e.g., dyes or DOTA moieties) that can
function as therapeutic and tracking units can also be attached via
a nucleophilic functionality, as shown below.
##STR00154## ##STR00155##
##STR00156## ##STR00157##
##STR00158## ##STR00159##
[0341] In a further aspect, a disclosed nanoparticle can be
functionalized with a dye for imaging the eye in a subject. For
example, such a method can be accomplished conveniently by Scheme
12b.
##STR00160##
[0342] In a further aspect, analogous chemistry can be used to
prepare a drug delivery system comprising a drug molecule that is
attached to a pH sensitive linker and includes a hydrazide linker
and doxorubicin. The synthesis is illustrated in Schemes 13-15.
##STR00161##
##STR00162##
##STR00163##
[0343] A novel c-RGD has been prepared and can be attached to the
nanoparticles and used for targeting of the disclosed delivery
systems (See Scheme 16).
##STR00164##
[0344] The synthesis of the c-RGD that contains free amine and
thiol unit for attachment to SVEC of the particle from the
intra-molecular chain collapse and the SVEC or the allyl group of
the polyester particles is detailed in Scheme 17.
##STR00165##
[0345] The attachment of the molecular transporter to the
maleiminde of the intra-molecular chain collapse particle has also
been investigated to create a system that transports peptides to
intracellular location and across biological barriers. See Scheme
18.
##STR00166##
[0346] Further modifications of the nanoparticle-dendrimer
conjugate systems have also been investigated. See Schemes 19-20.
The disclosed modifications, as well as analogous transformations,
results in a collection of compounds available for use in
intracellular transport.
##STR00167##
##STR00168##
I. Nanosponges
[0347] In one aspect, the invention relates to intravitreal
drug-delivery nanoparticles ("nanosponges"), which are
three-dimensional nano-networks formed from degradable materials,
in particular, formed by crosslinking degradable linear polyesters.
In various aspects, nanosponges can refer to compositions
comprising one or more disclosed compounds of the invention or one
or more products of the disclosed methods. In particular,
nanosponges can refer to disclosed compounds or products
encapsulating one or more pharmaceutically active agent or
biologically active agent, for example, agents disclosed
herein.
[0348] In a further aspect, a nanosponge is an ocular delivery
platform (degradable polyester nanoparticle pharmaceutical or
biologically active agent complex, which can be also referred to as
a nanoparticle complex, and can comprise one or more degradable
crosslinked polyester nanoparticles and one or more biologically
active agents, one or more pharmaceutically active agents, and/or
one or more imaging agents, as disclosed herein. In a particular
aspect, a nanosponge is an ocular delivery platform for treatment
and/or prevention of eye diseases (e.g., glaucoma) and cancer
(e.g., intraocular melanoma).
[0349] Nanosponges can offer significant advantages over
conventional drug delivery systems. For example, nanosponges can be
prepared using practical synthetic methods in suitable nanoscopic
dimensions. In one aspect, nanosponges can be prepared for
treatment of eye disease (e.g., 400 nm and 700 nm) or for treatment
of cancer (e.g., 50 nm and, optionally, modified with targeting
unit that only targets cancer site).
[0350] In one aspect, nanosponges can encapsulate hydrophobic,
potent drugs as well as solubilize them in high concentrations.
This leads to a larger pool of drugs available for drug discovery
efforts. It is observed that there is no accumulation of
nanosponges in other organs. As disclosed herein, nanosponges can
be tailored to facilitate treatment of cancer type and disease
stage. For example, drug release can be tailored (e.g., fast,
medium, slow), which can be important for fast and slow growing
cancer types (e.g., beast, prostate, lung, and brain). Nanosponges
can be prepared for release of the encapsulated drug at a constant
rate, which can be important for the development of clinical
protocols.
[0351] In one aspect, the disclosed nanosponges can be used in
connection with treatment of eye diseases such as glaucoma (4th
major cause of blindness): Inter Ocular Pressure (IOP) can be
controlled over a period of two months with ONE treatment, so far
limited or no treatment possible.
J. Methods of Administration
[0352] The disclosed compositions are useful for the deposition of
pharmaceutical agents encapsulated within the degradable polyester
nanoparticle. Thus, disclosed herein are methods of administering a
pharmaceutical or biologically active agent to a cell comprising
contacting the cell with a degradable polyester
nanoparticle-pharmaceutical or biologically active agent complex
(nanoparticle complex) thereby administering the pharmaceutical
biologically active agent to the cell. It is contemplated herein
that the nanoparticles can release the pharmaceutical agents over
time as the particle degrades resulting in the time release of the
agent.
[0353] It is understood the nanoparticle-pharmaceutical agent
complex can be administered to any cell type desired. For example,
the cell can be a neuron (e.g., a photoreceptor neuron), ganglion
cell, cone cell, rod cell, epithelial cell, muscle cell, adipose
cell, hepatic cell, erythrocyte, leukocyte, mast cell, fibroblast
(e.g., a corneal fibroblast). Such cells can be part of a larger
tissue such as neuronal, fibrous, blood, gangloid, dermal,
muscular, amacrine, bipolar, horizontal, connective, epithelial,
and vitreal fluid. It is further understood that the cells to which
the nanoparticle-pharmaceutical agent complex is applied can be
located in a region of an organ such as the eye. Examples of such
regions include by are not limited to a region of the eye selected
from the group consisting of sclera, cornea, retina, vitrius fluid,
rods, cones, iris, zonular fibers, aqueous humour, choroid, ciliary
muscle, optic disc, dura mater, optic nerve, fovea, and macula.
Because the nanoparticle-pharmaceutical agent complex can be
delivered to living tissue, organs, or cells, it is further
contemplated herein that said complexes have particular uses for
administration of a pharmaceutical agent to a subject.
[0354] Due to the size of the nanoparticle complexes disclosed
herein, it is understood that the degradable polyester
nanoparticles can be used to deliver a pharmaceutical agent
directly to the interior of a cell. Thus contemplated herein are
methods of administration, wherein the nanoparticle compex is
administered to anorganelle of a cell such as for example
mitochondria, the nucleus, the golgi apparatus, endoplasmic
reticulum, ribosomes, lysosomes, or centrioles. Thus, for example,
disclosed herein are methods of administering a pharmaceutical or
biologically active agent to the nucleus comprising contacting a
cell with a degradable polyester nanoparticle-pharmaceutical or
biologically active agent complex. It is understood that the
complex can be taken up by the cell or can pass through a molecular
channel such that the pharmaceutical or biologically active agent
is internalized into the cell. It is further understood that the
nanpaticle complex can further pass through organelle membranes to
enter mitochondria or the nucleus of the cell.
[0355] It is understood that there are circumstances where one of
skill in the art would want to monitor the deposition of the
pharmaceutical or biologically active agent following the
administration of the degradable polyester nanoparticle
pharmaceutical or biologically active complexes. Therefore, it is
contemplated herein that the nanoparticle complexes can further
comprise a mechanism for detection. Detection can occur the use of
imaging agents such as labels and dyes, but can also occur through
the measure of physical characteristics such as measuring
interocular pressure (TOP) or visualization such as electron
microscopy. Where a dye or label is used, the means of detection
can employ any method known in the art including but not limited to
microscopy such as immunofluorescence, radioimmunoassay, ELISAs,
ELISpot, and flow cytometry. As used herein, a label can include
radiolabels, pigment dyes, a fluorescent dye, a member of a binding
pair, such as biotin/streptavidin, a metal (e.g., gold), or an
epitope tag that can specifically interact with a molecule that can
be detected, such as by producing a colored substrate or
fluorescence. Substances suitable for detectably labeling proteins
include fluorescent dyes (also known herein as fluorochromes and
fluorophores) and enzymes that react with colorometric substrates
(e.g., horseradish peroxidase). The use of fluorescent dyes is
generally preferred in the practice of the invention as they can be
detected at very low amounts. Furthermore, in the case where
multiple antigens are reacted with a single array, each antigen can
be labeled with a distinct fluorescent compound for simultaneous
detection. Labeled spots on the array are detected using a
fluorimeter, the presence of a signal indicating an antigen bound
to a specific antibody.
[0356] Fluorophores are compounds or molecules that luminesce.
Typically fluorophores absorb electromagnetic energy at one
wavelength and emit electromagnetic energy at a second wavelength.
Representative fluorophores include, but are not limited to, 1,5
IAEDANS; 1,8-ANS; 4-Methylumbelliferone;
5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);
5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine
(5-TAMRA); 5-Hydroxy Tryptamine (5-HAT); 5-ROX
(carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;
7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);
7-Hydroxy-4-I methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine
(ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine
Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin
(Photoprotein); AFPs--AutoFluorescent Protein--(Quantum
Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350.TM.; Alexa Fluor
430.TM.; Alexa Fluor 488.TM.; Alexa Fluor 532.TM.; Alexa Fluor
546.TM.; Alexa Fluor 568.TM.; Alexa Fluor 594.TM.; Alexa Fluor
633.TM.; Alexa Fluor 647.TM.; Alexa Fluor 660.TM.; Alexa Fluor
680.TM.; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC);
AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin
D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7;
APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R;
Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG.TM.
CBQCA; ATTO-TAG.TM. FQ; Auramine; Aurophosphine G; Aurophosphine;
BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH);
Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP(Y66H);
Blue Fluorescent Protein; BFP/GFP FRET; Bimane; Bisbenzemide;
Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; Blancophor SV;
BOBO.TM.-1; BOBO.TM.-3; Bodipy492/515; Bodipy493/503;
Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy 542/563;
Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591;
Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl;
Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR;
Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP;
Bodipy TR-X SE; BO-PRO.TM.-1; BO-PRO.TM.-3; Brilliant Sulphoflavin
FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson-; Calcium
Green; Calcium Green-1 Ca.sup.2+ Dye; Calcium Green-2 Ca.sup.2+;
Calcium Green-5N Ca.sup.2+; Calcium Green-C18 Ca.sup.2+; Calcium
Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade
Blue.TM.; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA;
CFP (Cyan Fluorescent Protein); CFP/YFP FRET; Chlorophyll;
Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine;
Coelenterazine cp; Coelenterazine f; Coelenterazine fcp;
Coelenterazine h; Coelenterazine hcp; Coelenterazine ip;
Coelenterazine n; Coelenterazine O; Coumarin Phalloidin;
C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan; Cy2.TM.;
Cy3.1 8; Cy3.5.TM.; Cy3.TM.; Cy5.1 8; Cy5.5.TM.; Cy5.TM.; Cy7.TM.;
Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl
Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl
fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3'DCFDA; DCFH
(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydrorhodamine
123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP);
Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer;
DiD (DilC18(5)); DIDS; Dihydrorhodamine 123 (DHR); Dil (DilC18(3));
I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF
(high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS;
EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC;
Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin;
EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen
(Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC;
Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein
Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine);
Fluor-Ruby; Fluor X; FM 1-43.TM.; FM 4-46; Fura Red.TM. (high pH);
Fura Red.TM./Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red
B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl
Yellow 5GF; GeneBlazer; (CCF2); GFP(S65T); GFP red shifted (rsGFP);
GFP wild type' non-UV excitation (wtGFP); GFP wild type, UV
excitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue;
Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580;
HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold);
Hydroxytryptamine; Indo-1, high calcium; Indo-1 low calcium;
Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf;
JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751
(RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine
Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer;
LOLO-1; LO-PRO-1;; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker
Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker
Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue;
Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2;
Mag-Fura-5; Mag-1ndo-1; Magnesium Green; Magnesium Orange;
Malachite Green; Marina Blue; I Maxilon Brilliant Flavin 10 GFF;
Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin;
Mitotracker Green FM; Mitotracker Orange; Mitotracker Red;
Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH);
Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD
Amine; Neuro DiO; Nile Red; Nitrobenzoxedidole; Noradrenaline;
Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliant lavin E8G;
Oregon Green.TM.; Oregon Green.TM. 488; Oregon Green.TM. 500;
Oregon Green.TM. 514; Pacific Blue; Pararosaniline (Feulgen); PBFI;
PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin
B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite
RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin
R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1;
POPO-3; PO-PRO-1; PO-I PRO-3; Primuline; Procion Yellow; Propidium
lodid (P1); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant
Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2;
Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine
6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB;
Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine:
Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal;
R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L;
S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B;
Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron Orange;
Sevron Yellow L; sgBFP.TM. (super glow BFP); sgGFP.TM. (super glow
GFP); SITS (Primuline; Stilbene Isothiosulphonic Acid); SNAFL
calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green;
SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ
(6-methoxy-N-(3 sulfopropyl)quinolinium); Stilbene; Sulphorhodamine
B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14;
SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO
23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44;
SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO
80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX
Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC);
Texas Red.TM.; Texas Red-X.TM. conjugate; Thiadicarbocyanine
(DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin
S; Thioflavin TON; Thiolyte; Thiozole Orange; Tinopol CBS
(Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1;
TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate;
True Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW
781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow
GFP; YFP; YO-PRO-1; YO-PRO3; YOYO-1; YOYO-3; Sybr Green; Thiazole
orange (interchelating dyes); semiconductor nanoparticles such as
quantum dots; or caged fluorophore (which can be activated with
light or other electromagnetic energy source), or a combination
thereof.
[0357] A modifier unit such as a radionuclide can be incorporated
into or attached directly to any of the compounds described herein
by halogenation. Examples of radionuclides useful in this
embodiment include, but are not limited to, tritium, iodine-125,
iodine-131, iodine-123, iodine-124, astatine-210, carbon-11,
carbon-14, nitrogen-13, fluorine-18. In another aspect, the
radionuclide can be attached to a linking group or bound by a
chelating group, which is then attached to the compound directly or
by means of a linker. Examples of radionuclides useful in the apset
include, but are not limited to, Tc-99m, Re-186, Ga-68, Re-188,
Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62. Radiolabeling
techniques such as these are routinely used in the
radiopharmaceutical industry.
[0358] The radiolabeled compounds are useful as imaging agents to
diagnose neurological disease (e.g., a neurodegenerative disease)
or a mental condition or to follow the progression or treatment of
such a disease or condition in a mammal (e.g., a human). The
radiolabeled compounds described herein can be conveniently used in
conjunction with imaging techniques such as positron emission
tomography (PET) or single photon emission computerized tomography
(SPECT).
[0359] Labeling can be either direct or indirect. In direct
labeling, the detecting antibody (the antibody for the molecule of
interest) or detecting molecule (the molecule that can be bound by
an antibody to the molecule of interest) include a label. Detection
of the label indicates the presence of the detecting antibody or
detecting molecule, which in turn indicates the presence of the
molecule of interest or of an antibody to the molecule of interest,
respectively. In indirect labeling, an additional molecule or
moiety is brought into contact with, or generated at the site of,
the immunocomplex. For example, a signal-generating molecule or
moiety such as an enzyme can be attached to or associated with the
detecting antibody or detecting molecule. The signal-generating
molecule can then generate a detectable signal at the site of the
immunocomplex. For example, an enzyme, when supplied with suitable
substrate, can produce a visible or detectable product at the site
of the immunocomplex. ELISAs use this type of indirect
labeling.
[0360] As another example of indirect labeling, an additional
molecule (which can be referred to as a binding agent) that can
bind to either the molecule of interest or to the antibody (primary
antibody) to the molecule of interest, such as a second antibody to
the primary antibody, can be contacted with the immunocomplex. The
additional molecule can have a label or signal-generating molecule
or moiety. The additional molecule can be an antibody, which can
thus be termed a secondary antibody. Binding of a secondary
antibody to the primary antibody can form a so-called sandwich with
the first (or primary) antibody and the molecule of interest. The
immune complexes can be contacted with the labeled, secondary
antibody under conditions effective and for a period of time
sufficient to allow the formation of secondary immune complexes.
The secondary immune complexes can then be generally washed to
remove any non-specifically bound labeled secondary antibodies, and
the remaining label in the secondary immune complexes can then be
detected. The additional molecule can also be or include one of a
pair of molecules or moieties that can bind to each other, such as
the biotin/avadin pair. In this mode, the detecting antibody or
detecting molecule should include the other member of the pair.
[0361] Other modes of indirect labeling include the detection of
primary immune complexes by a two step approach. For example, a
molecule (which can be referred to as a first binding agent), such
as an antibody, that has binding affinity for the molecule of
interest or corresponding antibody can be used to form secondary
immune complexes, as described above. After washing, the secondary
immune complexes can be contacted with another molecule (which can
be referred to as a second binding agent) that has binding affinity
for the first binding agent, again under conditions effective and
for a period of time sufficient to allow the formation of immune
complexes (thus forming tertiary immune complexes). The second
binding agent can be linked to a detectable label or
signal-generating molecule or moiety, allowing detection of the
tertiary immune complexes thus formed. This system can provide for
signal amplification.
[0362] Immunoassays that involve the detection of as substance,
such as a protein or an antibody to a specific protein, include
label-free assays, protein separation methods (i.e.,
electrophoresis), solid support capture assays, or in vivo
detection. Label-free assays are generally diagnostic means of
determining the presence or absence of a specific protein, or an
antibody to a specific protein, in a sample. Protein separation
methods are additionally useful for evaluating physical properties
of the protein, such as size or net charge. Capture assays are
generally more useful for quantitatively evaluating the
concentration of a specific protein, or antibody to a specific
protein, in a sample. Finally, in vivo detection is useful for
evaluating the spatial expression patterns of the substance, i.e.,
where the substance can be found in a subject, tissue or cell.
[0363] Provided that the concentrations are sufficient, the
molecular complexes ([Ab-Ag] n) generated by antibody-antigen
interaction are visible to the naked eye, but smaller amounts may
also be detected and measured due to their ability to scatter a
beam of light. The formation of complexes indicates that both
reactants are present, and in immunoprecipitation assays a constant
concentration of a reagent antibody is used to measure specific
antigen ([Ab-Ag]n), and reagent antigens are used to detect
specific antibody ([Ab-Ag]n). If the reagent species is previously
coated onto cells (as in hemagglutination assay) or very small
particles (as in latex agglutination assay), "clumping" of the
coated particles is visible at much lower concentrations. A variety
of assays based on these elementary principles are in common use,
including Ouchterlony immunodiffusion assay, rocket
immunoelectrophoresis, and immunoturbidometric and nephelometric
assays. The main limitations of such assays are restricted
sensitivity (lower detection limits) in comparison to assays
employing labels and, in some cases, the fact that very high
concentrations of analyte can actually inhibit complex formation,
necessitating safeguards that make the procedures more complex.
Some of these Group 1 assays date right back to the discovery of
antibodies and none of them have an actual "label" (e.g. Ag-enz).
Other kinds of immunoassays that are label free depend on
immunosensors, and a variety of instruments that can directly
detect antibody-antigen interactions are now commercially
available. Most depend on generating an evanescent wave on a sensor
surface with immobilized ligand, which allows continuous monitoring
of binding to the ligand. Immunosensors allow the easy
investigation of kinetic interactions and, with the advent of
lower-cost specialized instruments, may in the future find wide
application in immunoanalysis.
[0364] The deposition of the nanoparticle-pharmaceutical agent
complex can be direct or indirect depending on the needs of the
particular situation. For example, the nanoparticle-pharmaceutical
agent complexes disclosed herein can be applied directly to the
sclera of an eye. Also, by way of example, the
nanoparticle-pharmaceutical agent complexes can be injected into
the vitreal fluid whereby the complexes can then come into contact
with cells on the retina. One of skill in the art understands that
the particle method of applying the nanoparticle-pharmaceutical
agent complex depends
[0365] In one aspect, the invention relates to a method of
intracellular delivery comprising administering an effective amount
of a disclosed nanoparticle to a subject. In a further aspect, the
nanoparticle is further substituted with at least one biologically
active agent, at least one pharmaceutically active agent, and/or at
least one imaging moiety. In a further aspect, the method further
comprises the step of degrading the nanoparticle.
[0366] In a further aspect, the invention relates to a method of
intracellular delivery comprising administering an effective amount
of a disclosed polymer or product of a disclosed method to a
subject. In a further aspect, the polymer or product of a disclosed
method is further substituted with at least one biologically active
agent, at least one pharmaceutically active agent, and/or at least
one imaging moiety. In a further aspect, the method further
comprises the step of degrading the polymer or product of a
disclosed method.
K. Methods of Treatment
[0367] It is contemplated herein that degradable polyester
nanoparticles disclosed herein will slowly release any agent
encapsulated within the nanoparticle at a rate equivalent to the
degradation of the particle. Such release over time is particularly
useful for the time release of pharmaceutical or biologically
active agents that can be used to treat various diseases or
conditions. Such diseases and conditions can include but are not
limited to ophthalmic disorders. Thus, disclosed herein are method
of treating any of the ophthalmic disorder disclosed herein (e.g.,
glaucoma) comprising administering to a subject a degradable
polyester nanoparticle pharmaceutical or biologically active agent
complex (nanoparticle complex).
[0368] The disclosed treatment methods may be used with any
pharmaceutical or biologically active agent known for use as a
treatment for the given ophthalmic disorder to be treated. Thus,
for example, the pharmaceutical or biologically active agent can be
an aptamer, an antibody, an alpha agonist, beta blocker,
prostaglandin analog, carbonic anhydrase inhibitor, cholinergic, or
any other agent disclosed herein.
[0369] Such agents are well known to those of skill in the art, but
can include for example, triamcinolone, ranibizumab, bevacizumab,
pegaptanib (MACUGEN.RTM.), travoprost, bimatoprost, methazolamide,
brinzolamide, Dorzolamide HCl, Acetazolamide, Timolol Maleate,
Betaxolol HCl, Levobunolol HCl, Metipranolol, Timolol hemihydrate,
Pilocarpine HCl, Carbachol, brimonidine tartrate, memantine,
Apraclonidine HCl, or latanoprost (XALATAN.RTM.). It is understood
that the particular agent used will be suited to the medicinal
purpose of the skilled artisan. For example, for treatment of
macular degeneration, one or more pharmaceutical or biologically
active agent such as ranibizumab or bevacizumab can be used. For
the treatment of diabetic related disorders, triamcinolone can be
used. For the treatment of glaucoma, one or more agents such as
pegaptanib (MACUGEN.RTM.), travoprost, bimatoprost, methazolamide,
brinzolamide, Dorzolamide HCl, Acetazolamide, Timolol Maleate,
Betaxolol HCl, Levobunolol HCl, Metipranolol, Timolol hemihydrate,
Pilocarpine HCl, Carbachol, brimonidine tartrate, Apraclonidine
HCl, memantine, or latanoprost (XALATAN.RTM.) can be used in the
disclosed methods. Thus, disclosed herein are methods of treating
an ophthalmic disorder comprising administering to a subject one or
more of the pharmaceutical or biologically active agents selected
from the group consisting of triamcinolone, ranibizumab,
bevacizumab, pegaptanib (MACUGEN.RTM.), travoprost, bimatoprost,
methazolamide, brinzolamide, Dorzolamide HCl, Acetazolamide,
Timolol Maleate, Betaxolol HCl, Levobunolol HCl, Metipranolol,
Timolol hemihydrate, Pilocarpine HCl, memantine, Carbachol,
brimonidine tartrate, Apraclonidine HCl, and latanoprost
(XALATAN.RTM.).
[0370] It is disclosed herein that the treatment of any of the
ophthalmic disorders disclosed herein may be treated by the use of
more than one pharmaceutical or biologically active agent used in
combination in the nanoparticle complexes. For example, disclosed
herein are methods of treating glaucoma comprising administering to
a subject a degradable polyester nanoparticle pharmaceutical agent
complex wherein the complex comprises at least two pharmaceutical
agents. It is understood that the disclosed methods of treatment or
modulating receptor or enzymatic treatment can utilize within the
nanoparticle complex any comprising a combination of at least two
or more pharmaceutical or biologically active agents disclosed
herein. For example, a combination of pharmaceutical agents may
comprise an alpha agonist and a beta blocker such as Brimonidine
Tartrate and Timolol Maleate or a beta blocker and a carbonic
anhydrase inhibitor such as Dorzolomide HCl and Timolol Maleate.
Other combinations contemplated herein include two or more alpha
agonists, two or more beta blockers, two or more cholinergics, two
or more carbonic anhydrases, two or more prostaglandin analogs, two
or more antibodies, an alpha agonist and a beta blocker, an alpha
agonist and a carbonic anhydrase inhibitor, an alpha agonist and a
cholinergic, an alpha agonist and a carbonic anhydrase inhibitor,
an alpha agonist and a prostaglandin analog, an alpha agonist an
antibody, a beta blocker and a carbonic anhydrase inhibitor, a beta
blocker and a prostaglandin analog, a beta blocker and an antibody,
a beta blocker an a cholinergic, a carbonic anhydrase inhibitor and
a prostaglandin analog, a carbonic anhydrase inhibitor and a
cholinergic, a carbonic anhydrase inhibitor and an antibody, a
cholinergic and a prostaglandin analog, a cholinergic and an
antibody, and a prostaglandin analog and an antibody.
[0371] The mechanism by which the agents for use in the disclosed
nanoparticles have their effect are known to those of skill in the
art. For example, those of skill in the art know that the alpha
agonist disclosed herein such as brimonidine tartrate and
Apraclonidine HCl, function by interacting with a G coupled protein
receptor known as the alpha adrenergic receptor. Similarly, the
mechanism by which beta blockers function is two inhibit the
functioning of a G coupled protein receptor referred to as the beta
adrenergic receptor. Inhibitors of the beta adrenergic receptor
include but are not limited to Timolol Maleate, Betaxolol HCl,
Levobunolol HCl, Metipranolol, and Timolol hemihydrate. It is
further understood that some agents which act as modulators of G
coupled protein receptors are analogs to the natural ligand for the
receptor. For example, latanoprost, travoprost, and bimatoprost are
prostaglandin receptor analogs which modulate the activity of the
prostaglandin F2 (FP) receptor. The activity of other receptors
such as the acetylcholine receptor can also be modulated by the
activity of the agents disclosed herein. For example, Pilocarpine
HCl or Carbachol modulate acetylcholine receptor activity. Thus,
disclosed herein are methods of modulating a receptor on a cell
comprising contacting the receptor with a degradable polyester
nanoparticle pharmaceutical biologically active agent complex
(nanoparticle complex), wherein one or more pharmaceutical or
biologically active agents is encapsulated by a degradable
polyester nanoparticle. Also disclosed are methods of modulating a
receptor wherein the receptor is a G coupled protein receptor such
as the alpha adrenergic receptor, the beta adrenergic receptor, or
prostaglandin F2 (FP) receptor comprising administering to a
subject the nanoparticle complexes disclosed herein. Additionally,
disclosed are methods of modulating a receptor wherein the receptor
is the acetylcholine receptor comprising administering to a subject
the nanoparticle complexes disclosed herein.
[0372] It is further contemplated herein that not all of the agents
disclosed herein are known to those of skill in the art for use in
the methods of treatment function by modulating the activity of a
receptor. Some of the agents described herein have their medicinal
effect through the ability to change the effect of an enzyme. For
example, VEGF and in particular VEGF-A effects the outflow of
vitreal fluid. Agents such as triamcinolone (a steroid) or
pegaptanib (an aptamer) bind and inhibit VEGF whereas ranibizumab
or bevacizumab are antibodies with a more specific action of
inhibiting VEGF-A. Other agents such as methazolamide,
brinzolamide, Dorzolamide HCl, and Acetazolamide inhibit carbonic
anhydrase. Therefore disclosed herein are methods of modulating the
activity of an enzyme such as VEGF, VEGF-A, or carbonic anhydrase
comprising administering to a subject comprising administering to a
subject the nanoparticle complexes disclosed herein. Due to the
effect of enzyme activity on viteous outflow or other biological
activity associated with ophthalmic disorders, disclosed herein are
methods of treating an ophthalmic disorder (e.g., glaucoma, macular
degeneration or diabetic odema) comprising administering to a
subject a nanoparticle compex, wherein one or more pharmaceutical
or biologically active agent encapsulated by the nanoparticle
modulates that activity of VEGF, VEGF-A, or carbonic anhydrase.
L. Uses
[0373] Also provided are uses of the disclosed polymers,
nanoparticles, and products. In one aspect, the invention relates
to a use of a disclosed polymer or a disclosed nanoparticle to
deliver a biologically active agent, a pharmaceutically active
agent, and/or an imaging moiety. The disclosed compounds,
compositions, and conjugates and practical synthesis of same
provide approaches for applications in cancer treatment and drug
delivery across biological barriers such as the cornea, tissues,
skin, and the blood brain barrier.
[0374] These degradable polymers find application in controlled
release technologies that have to penetrate tissues and cellular
membranes. Thus, the nanoparticle-dendrimer conjugates comprising a
disclosed degradable nanoparticle and a disclosed intracellular
delivery composition can hold and deliver therapeutics ranging from
small molecules to larger peptides, proteins, and antibodies.
[0375] In a further aspect, the invention relates to a use of a
disclosed polymer or a disclosed nanoparticle for trancorneal
delivery of a biologically active agent, a pharmaceutically active
agent, and/or an imaging moiety.
[0376] Many regions of the eye are relatively inaccessible to
systemically administered agents. For example, orally administered
agents pass through the liver before reaching estrogen sensitive
tissues. Because the liver contains enzymes that can inactivate the
agent, the agent that eventually reaches tissue targeted for
treatment can be virtually ineffective. Moreover, systemic
administration risks production of undesirable side effects. It can
also be problematic to deliver a biologically active agent, a
pharmaceutically active agent, and/or an imaging moiety into the
eye via invasive procedures such as injection. Further still,
patient compliance can be low in cases of invasive
administration.
[0377] As a result, topical drug delivery remains the preferred
route of administration to the eye. There are a variety of factors
that affect the absorption of drugs into the eye. These factors
include: the instillation volume of the drug, the frequency of
instilled drug administration, the structure and integrity of the
cornea, the protein level in tears, the level of enzymes in tears,
lacrimal drainage and tear turnover rate, as well the rate of
adsorption and absorption of a drug by the conjunctiva, sclera, and
eyelids. A potential way of reducing or even eliminating systemic
side effects is to improve ocular targeting that would allow for
the use of reduced doses of the biologically active agent in the
ophthalmic drug formation.
[0378] A major barrier to ocular drug penetration is the cornea.
The cornea is composed of three layers: a lipid-rich epithelium, a
lipid-poor soma, and a lipid-rich endothelium. Therefore, an agent
must possess both lipophilic-hydrophilic balance for adequate
transcorneal penetration and, thus, ocular bioavailability (Akers,
H. J., "Ocular bioavailability of topically applied ophthalmic
drugs," Am Pharm, NS23:33-36 (1983)).
[0379] Thus, in one aspect, the disclosed compounds provide
improved physicochemical properties including, but not limited to,
favorable ocular bioavailability and facile transcorneal
penetration.
[0380] In another aspect, the disclosed compounds treat and/or
protect against various ocular diseases. That is, the disclosed
compounds can be used to diagnose, prevent, and/or treat ophthalmic
disorders. Preferred disclosed compounds can be effective in
treating and/or preventing maladies associated with
vision-threatening intraocular damage due to pathophysiological
predispositions. Preferred disclosed compounds include those which
treat retinal infection, glaucoma, and/or macular degeneration.
M. Manufacture of a Medicament
[0381] Also provided is a method for the manufacture of a
medicament. In one aspect, the invention relates to a method for
the manufacture of a medicament for delivery of a biologically
active agent, a pharmaceutically active agent, and/or an imaging
moiety comprising combining at least one disclosed polymer or at
least one disclosed nanoparticle with a pharmaceutically acceptable
carrier.
[0382] In a further aspect, the pharmaceutical composition relates
to a composition for preventing and/or treating ophthalmic
disorders.
N. Pharmaceutical Compositions
[0383] In one aspect, the invention relates to pharmaceutical
compositions comprising the disclosed compositions. That is, a
pharmaceutical composition can be provided comprising a
therapeutically effective amount of one or more disclosed polymer
and/or one or more products of a disclosed method and/or one or
more disclosed nanoparticle and a pharmaceutically acceptable
carrier for administration in a mammal. In a further aspect, the
one or more disclosed polymer and/or one or more products of a
disclosed method and/or the one or more disclosed nanoparticle is
further substituted with at least one biologically active agent, at
least one pharmaceutically active agent, and/or at least one
imaging moiety.
[0384] The disclosed pharmaceutical compositions can further
comprise other therapeutically active compounds, which are usually
applied in the treatment of the above mentioned pathological
conditions. It is understood that the disclosed compositions can be
employed in the disclosed methods of using.
O. Kits
[0385] Also provided are kits related to the disclosed
compositions. In one aspect, the invention relates to a kit
comprising at least one disclosed polymer, at least one disclosed
nanoparticle or at least one product of a disclosed method. It is
understood that the disclosed kits can be used in connection with
the disclosed methods of using.
[0386] Thus, in one aspect, the invention related to kits
comprising a first degradable polyester nanoparticle and a first
biologically active agent, first pharmaceutically active agent, or
first imaging agent encapsulated within the first nanoparticle, and
one or more of: a second biologically active agent, second
pharmaceutically active agent, or second imaging agent encapsulated
within the first nanoparticle, wherein the first biologically
active agent, first pharmaceutically active agent, or first imaging
agent is different from the second biologically active agent,
second pharmaceutically active agent, or second imaging agent; or a
second degradable polyester nanoparticle and a second biologically
active agent, second pharmaceutically active agent, or second
imaging agent encapsulated within the second nanoparticle, wherein
the first biologically active agent, first pharmaceutically active
agent, or first imaging agent is different from the second
biologically active agent, second pharmaceutically active agent, or
second imaging agent; a pharmaceutically acceptable carrier; or
instructions for treating a disorder known to be treatable by the
first biologically active agent or first pharmaceutically active
agent.
[0387] In a further aspect, at least one agent is brominidine
tartrate. In a further aspect, at least one agent is an inhibitor
of VEGF. In a further aspect, at least one agent is an inhibitor of
VEGF-A. In a further aspect, at least one agent is a alpha agonist,
beta blocker, prostaglandin analog, carbonic anhydrase inhibitor,
antibody, aptamer, or cholinergic. In a further aspect, at least
one agent is selected from triamcinolone, ranibizumab, bevacizumab,
pegaptanib (MACUGEN.RTM.), travoprost, bimatoprost, methazolamide,
brinzolamide, dorzolamide HCl, acetazolamide, memantine, timolol
maleate, betaxolol HCl, levobunolol HCl, metipranolol, timolol
hemihydrate, pilocarpine HCl, carbachol, brimonidine tartrate,
apraclonidine HCl, and latanoprost (XALATAN.RTM.).
P. Experimental
[0388] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary of the invention and are not
intended to limit the scope of what the inventors regard as their
invention. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
[0389] 1. Characterization Methods
[0390] .sup.1H NMR spectra were obtained from a Bruker AC300
Fourier Transform Spectrometer, with CDCl.sub.3 in TMS as the
solvent. .sup.13C NMR spectra were obtained from a Bruker AC400
Fourier Transform Spectrometer with CDCl.sub.3 as the solvent.
[0391] Gel-permeation chromatography (GPC) was performed on a
Waters chromatograph equipped with a Waters 2414 refractive index
detector, a Waters 2481 dual X absorbance detector, a Waters 1525
binary HPLC pump, and four 5 mm Waters columns (300 mm.times.7.7
mm), connected in series with increasing pore size (100, 1000,
100,000 and 1,000,000 .ANG. respectively). All runs were performed
with tetrahydrofuran (THF) as the eluent at a flow rate of 1
mL/min.
[0392] For dynamic light scattering (DLS) a Zetasizer Nano Series
instrument with a CGS-3 compact goniometer system by Malvern
Instruments (Malvern Zetasizer Nanoseries, Malvern, UK) was
employed at a fixed angle of 90.degree. at 25.degree. C., taking
the average of three measurements. The particles were diluted with
toluene to a concentration of 5-6 mg/mL, which gave the desired
number of counts in order to obtain a good signal-to-noise
ratio.
[0393] Samples for transmission electron microscopy (TEM) imaging
were prepared by dissolving 0.5 mg nanoparticles in 1 mL
isopropanol and 0.3 mL acetonitrile. The samples were sonicated for
5 min and were stained with 2 drops of 3% phosphotungstic acid. The
carbon grids were prepared by placing a drop of dispersed particles
onto an Ultrathin Carbon Type-A 400 Mesh Copper Grid (Ted Pella,
Inc., Redding, Calif.) and drying at ambient temperature. A Philips
CM20T transmission electron microscope operating at 200 kV in
bright-field mode was used to obtain TEM micrographs of the
polymeric nanoparticles.
[0394] Samples were centrifuged at 600 rpm on a Model CS
International Centrifuge from International Equipment Company
(Boston, Mass.).
[0395] 2. Materials
[0396] Reagent chemicals were purchased from Aldrich (Milwaukee,
Wis.), EMD, Alfa-Aesar, Fisher Scientific, and Acros and used as
received, unless otherwise stated. Spectra/Por.RTM. Dialysis
membrane and SnakeSkin.RTM. Pleated Dialysis Tubing, regenerated
cellulose, were purchased from Spectrum Laboratories Inc. and
Pierce Biotechnology, respectively. Analytical TLC was performed on
commercial Merck plates coated with silica gel GF254 (0.24 mm
thick). Silica gel for flash chromatography was Merck Kieselgel 60
(230-400 mesh, ASTM) or Sorbent Technologies 60 .ANG. (40-63 .mu.m,
technical grade). MAL-dPeg.TM..sub.4-t-boc-hydrazide was obtained
from Quanta Biodesign, Ltd. (Powell, Ohio) and used as received.
Cy3 NHS dye and PD-10 Desalting columns were received from GE
Healthcare (Piscataway, N.J.). Spectra/Por.RTM. Biotech Cellulose
Ester (CE) Dialysis Membranes (1,000 MWCO) obtained from Spectrum
Laboratories, Inc. (Rancho Dominguez, Calif.). SnakeSkin.RTM.
Pleated Dialysis Tubing (10,000 MWCO) was obtained from Pierce
Biotechnology, Inc. (Rockford, Ill.). Absolute molecular weight was
determined with static light scattering.
[0397] 3. Synthesis of .alpha.-allyl-.DELTA.-valerolactone (avl)
(B)
[0398] A 500 mL round bottom flask, equipped with stir bar, was
sealed with a septum, purged with nitrogen for 30 min and cooled in
a dry ice/acetone bath. A solution of lithium diisopropylamine (2.0
M in THF/heptane/ethyl benzene, 33 mL, 66 mmol) was added to the
round bottom flask. A nitrogen purged solution of
.delta.-valerolactone (5.43 mL, 60 mmol) in THF (60 mL) was added
dropwise via syringe over 1.5 h. After an additional 30 min of
stirring, a solution of allyl bromide (6.21 mL, 72 mmol) in
hexamethylphosphoramide (12.51 mL, 72 mmol) was added dropwise via
syringe over 30 min. The reaction mixture was warmed up to
-40.degree. C. using a dry ice/acetone bath and stirred for 3 h.
The reaction was quenched with excess NH.sub.4Cl solution and
warmed to room temperature. The crude product was washed twice with
brine, dried with anhydrous magnesium sulfate and concentrated via
rotary evaporator. Column chromatography using CH.sub.2Cl.sub.2
gave a viscous yellow product. Yield: 3.4262 g (41%). .sup.1H NMR
(300 MHz, CDCl.sub.3/TMS, ppm) .delta.: 5.7 (m, 1H,
H.sub.2C.dbd.CH--), 5.08 (m, 2H, H.sub.2C.dbd.CH--), 4.28 (m, 2H,
--C(O)OCH.sub.2--), 2.53-2.58 (m, 2H, H.sub.2C.dbd.CHCH.sub.2--),
2.27 (m, 1H, H.sub.2C.dbd.CHCH.sub.2CH--), 2.06 (m, 1H,
H.sub.2C.dbd.CHCH.sub.2CHCH.sub.2--), 1.89 (m, 2H,
C(O)OCH.sub.2CH.sub.2--), 1.55 (m, 1H,
H.sub.2C.dbd.CHCH.sub.2CHCH.sub.2--); .sup.13C NMR (400 MHz,
CDCl.sub.3, ppm) .delta.: 173.8 (--C(O)O--), 135.0
(H.sub.2C.dbd.CH--), 117.4 (H.sub.2C.dbd.CH--), 68.4
(--C(O)OCH.sub.2--), 39.2 (H.sub.2C.dbd.CHCH.sub.2CH--), 35.4
(H.sub.2C.dbd.CHCH.sub.2--), 24.0 (--CH.sub.2CH.sub.2CH.sub.2--),
21.9 (--CH.sub.2CH.sub.2CH.sub.2--).
[0399] 4. Synthesis of Copolymer poly(vl-avl) (Ab)
[0400] A 50 mL 3-necked round bottom flask, equipped with stir bar,
was sealed with two septa and a gas inlet. The flask was evacuated
and refilled with nitrogen three times. Stock solutions of 1.7 M
ethanol (EtOH) in THF and 3.7.times.10.sup.-2 M tin(II)
2-ethylhexanoate (Sn(Oct).sub.2) in THF were made in sealed N.sub.2
purged flasks. Solutions of EtOH (0.32 mL, 5.410.times.10.sup.-1
mmol) and Sn(Oct).sub.2 (0.30 mL, 1.12.times.10.sup.-2 mmol) were
combined in the nitrogen purged 50 mL flask. After stirring the
mixture for 30 min, .alpha.-allyl-.delta.-valerolactone (1.16 g,
8.32 mmol) and .delta.-valerolactone (vl, 2.5 g, 24.97 mmol) were
added. The reaction vessel stirred in a 105.degree. C. oil bath for
48 h. Residual monomer and catalyst were removed by dialyzing with
Spectra/Por.RTM. dialysis membrane (MWCO=1000) against
CH.sub.2Cl.sub.2 to give a golden brown polymer. Yield: 3.2398 g
(88%). M.sub.w=4834 Da, PDI=1.17; .sup.1H NMR (300 MHz,
CDCl.sub.3/TMS, ppm) .delta.: 5.7 (m, H.sub.2C.dbd.CH--), 5.09 (m,
H.sub.2C.dbd.CH--), 4.09 (m, --CH.sub.2--O--), 3.65 (m,
CH.sub.3CH.sub.2O--), 2.35 (m, vl, --CH.sub.2CH.sub.2C(O)O--, avl,
H.sub.2C.dbd.CHCH.sub.2CH--, H.sub.2C.dbd.CHCH.sub.2CH--), 1.68 (m,
avl & vl, --CHCH.sub.2CH.sub.2--), 1.25 (t,
CH.sub.3CH.sub.2O--); .sup.13C NMR (400 MHz, CDCl.sub.3, ppm)
.delta.: 174.6 (avl, --C(O)--), 172.7 (vl, --C(O)--), 134.6
(H.sub.2C.dbd.CH--), 116.4 (H.sub.2C.dbd.CH--), 63.3, 44.3, 35.9,
33.1, 27.5, 25.9, 23.6, 20.9.
[0401] 5. Synthesis of .alpha.-propargyl-.DELTA.-valerolactone
(pvl) (C)
[0402] A 250 mL round bottom flask, equipped with stir bar, was
sealed with a septum, purged with nitrogen for 30 min and cooled in
a dry ice/acetone bath. A solution of lithium diisopropylamine (2.0
M in THF/heptane/ethyl benzene, 22 mL, 44 mmol) was added to the
flask. A nitrogen purged solution of .delta.-valerolactone (3.62
mL, 40 mmol) in THF (40 mL) was added dropwise via syringe over 1.5
h. After an additional 30 min of stirring, a solution of propargyl
bromide (4.34 mL, 48 mmol) in hexamethylphosphoramide (8.4 mL, 48
mmol) was added dropwise via syringe over 20 min. The reaction
mixture was warmed up to -30.degree. C. using a dry ice/acetone
bath and stirred for 3 h. The reaction was quenched with excess
NH.sub.4Cl solution and warmed to room temperature. The crude
product was washed twice with brine, dried with anhydrous magnesium
sulfate and concentrated via rotary evaporator. Column
chromatography with CH.sub.2Cl.sub.2 gave a viscous yellow product.
Yield: 2.8194 g (50.6%). .sup.1H NMR (300 MHz, CDCl.sub.3/TMS, ppm)
.delta.: 4.35 (m, 2H, --C(O)OCH.sub.2--), 2.69 (m, 2H,
--C(O)CHCH.sub.2CCH), 2.53 (m, 1H--C(O)CHCH.sub.2CCH), 2.29 (m, 1H,
--CHCH.sub.2CH.sub.2--), 2.05 (s, 1H, HC.ident.CCH.sub.2--), 1.96
(m, 2H, --CHCH.sub.2CH.sub.2--), 1.74 (m, 1H,
--CHCH.sub.2CH.sub.2--); .sup.13C NMR (400 MHz, CDCl.sub.3, ppm)
.delta.: 172.8, 80.8, 70.1, 68.5, 38.5, 23.8, 21.7, 20.4.
[0403] 6. Synthesis of Copolymer poly (vl-avl-pvl) (AbC)
[0404] A 50 mL 3-necked round-bottom flask, equipped with stir bar,
was sealed with two septa and a gas inlet. The flask was evacuated
and refilled with nitrogen three times. Stock solutions of 1.7 M
ethanol in THF and 3.7.times.10.sup.-2M Sn(Oct).sub.2 in THF were
made in sealed N.sub.2 purged flasks. Solutions of ethanol (0.21
mL, 3.69.times.10.sup.-1 mmol) and Sn(Oct).sub.2 (0.20 mL,
5.41.times.10.sup.-3 mmol) were combined in the nitrogen purged 50
mL flask. After stirring the mixture for 30 min,
.alpha.-allyl-.delta.-valerolactone (0.8 g, 5.7 mmol),
.delta.-valerolactone (1.26 g, 12.6 mmol) and
.alpha.-propargyl-.delta.-valerolactone (0.63 g, 4.6 mmol) were
added. The reaction vessel stirred in a 105.degree. C. oil bath for
48 h. Residual monomer and catalyst were removed by dialyzing with
Spectra/Por.RTM. dialysis membrane (MWCO=1000) against
CH.sub.2Cl.sub.2 to give a golden brown polymer. Yield: 2.25 g
(84%). M.sub.w=3500 Da, PDI=1.26; .sup.1H NMR (300 MHz,
CDCl.sub.3/TMS, ppm) .delta.: 5.71 (m, H.sub.2C.dbd.CH--), 5.03 (m,
H.sub.2C.dbd.CH--), 4.08 (m, --CH.sub.2O--), 3.65 (m,
CH.sub.3CH.sub.2O--), 2.55 (m, pvl, --C(O)CH--, --CHCH.sub.2CCH),
2.45 (m, --CH.sub.2CCH), 2.34 (m, vl, --CH.sub.2CH.sub.2C(O)O--,
avl, H.sub.2C.dbd.CHCH.sub.2CH--, H.sub.2C.dbd.CHCH.sub.2CH--),
2.02 (m, pvl, --C.ident.CH), 1.68 (m, pvl, avl & vl,
--CHCH.sub.2CH.sub.2--), 1.259 (t, CH.sub.3CH.sub.2O--); .sup.13C
NMR (400 MHz, CDCl.sub.3, ppm) .delta.: 174.6, 172.7, 133.6, 117.2,
80.7, 69.9, 63.3, 44.3, 35.9, 33.1, 27.5, 25.9, 23.6, 20.9.
[0405] 7. Synthesis of 2-oxepane-1,5-dione (opd) (D)
[0406] A 100 mL round bottom flask, equipped with stir bar, was
charged with 1,4-cyclohexanedione (2.0 g, 17.84 mmol) and
3-chloroperoxybenzoic acid (4.5 g, 26.08 mmol). Dichloromethane (22
mL) was added and the reaction mixture stirred and refluxed for 3 h
at 40.degree. C. The reaction mixture was cooled to room
temperature and dried with anhydrous MgSO.sub.4. Solvent was
removed via rotary evaporation. The crude product was washed three
times with cold diethyl ether (100 mL for each wash) and dried in
vacuo at room temperature. Yield: 1.4814 g (64.7%). .sup.1H NMR
(300 MHz, CDCl.sub.3/TMS, ppm) .delta.: 4.4 (t, 2H,
--C(O)OCH.sub.2CH.sub.2C(O)--), 2.84 (dd, 2H, --CH.sub.2C(O)O--),
2.72 (m, 4H, --CH.sub.2C(O)CH.sub.2--); .sup.13C NMR (400 MHz,
CDCl.sub.3, ppm) .delta.: 204.9 (--C(O)--), 173.3 (--C(O)O--), 63.3
(--CH.sub.2O--), 44.7 (--OCH.sub.2CH.sub.2C(O)--), 38.6
(--C(O)CH.sub.2CH.sub.2C(O)--), 27.9 (--CH.sub.2C(O)O--).
[0407] 8. Synthesis of Copolymer poly(vl-avl-opd) (AbD)
[0408] To a 50 mL 3-necked round bottom flask, equipped with stir
bar, condenser, nitrogen purge and septa, 2-oxepane-1,5-dione
(0.6987 g, 5.45 mmol) and dry toluene (4 mL) was added. The mixture
stirred in an oil bath at 70.degree. C. to dissolve the monomer.
Upon dissolving, .delta.-valerolactone (1.5 g, 14.98 mmol),
.alpha.-allyl-.delta.-valerolactone (0.9546 g, 6.81 mmol), absolute
ethanol (0.0205 g, 4.4.times.10.sup.-1 mmol) and Sn(Oct).sub.2
(0.0119 g, 2.73.times.10.sup.-2 mmol) were then added to the
reactor and the mixture was heated for 48 h at 110.degree. C.
Residual monomer and catalyst were removed by dialyzing with
Spectra/Por.RTM. dialysis membrane (MWCO=1000) against
CH.sub.2Cl.sub.2 to give a golden brown polymer. Yield: 2.6894 g
(85%). M.sub.w=4858 Da, PDI=1.27; .sup.1H NMR (300 MHz,
CDCl.sub.3/TMS, ppm) .delta.: 5.72 (m, H.sub.2C.dbd.CH--), 5.06 (m,
H.sub.2C.dbd.CH--), 4.34 (m,
--CH.sub.2CH.sub.2C(O)CH.sub.2CH.sub.2O--), 4.08 (m,
--CH.sub.2O--), 3.67 (m, --OCH.sub.2CH.sub.3), 2.78 (m, opd,
--OC(O)CH.sub.2CH.sub.2C(O)CH.sub.2--), 2.58 (m, opd,
--OC(O)CH.sub.2CH.sub.2C(O)CH.sub.2--), 2.34 (m, vl,
--CH.sub.2CH.sub.2C(O)O--, avl, H.sub.2C.dbd.CHCH.sub.2CH--,
H.sub.2C.dbd.CHCH.sub.2CH--), 1.66 (m, avl & vl,
--CHCH.sub.2CH.sub.2--), 1.25 (t, --CH.sub.2CH.sub.3); .sup.13C NMR
(400 MHz, CDCl.sub.3, ppm) .delta.: 204.9, 175.2, 173.7, 173.2,
135.0, 117.0, 63.9, 44.8, 36.4, 33.6, 28.0, 26.3, 21.3.
[0409] 9. Synthesis of Copolymer poly(vl-avl-pvl-opd) (AbCD)
[0410] To a 25 mL 3-necked round bottom flask, equipped with stir
bar, 2-oxepane-1,5-dione (0.2626 g, 2.05 mmol) was added and the
flask was sealed with two septa and a gas inlet. The flask was
evacuated and refilled with argon three times. Dry toluene (1.25
mL) was added and the mixture stirred in an oil bath at 70.degree.
C. to dissolve the monomer. Upon dissolving, Sn(Oct).sub.2 (0.0018
g, 4.41.times.10.sup.-3 mmol in 0.15 mL dry toluene), absolute
ethanol (12.8 .mu.L, 2.22.times.10.sup.-1 mmol),
.delta.-valerolactone (0.62 g, 6.2 mmol),
.alpha.-allyl-.delta.-valerolactone (0.38 g, 2.69 mmol), and
.alpha.-propargyl-.delta.-valerolactone (0.38 g, 2.73 mmol) were
added. The temperature of the oil bath was increased to 105.degree.
C. and the mixture stirred for 50 h. Residual monomer and catalyst
were removed by dialyzing with Spectra/Por.RTM. dialysis membrane
(MWCO=1000) against CH.sub.2Cl.sub.2 to give a golden brown
polymer. Yield: 1.31 g (80%). M.sub.w=3525 Da, PDI=1.27; .sup.1H
NMR (300 MHz, CDCl.sub.3/TMS, ppm) .delta.: 5.86 (m,
H.sub.2C.dbd.CH--), 5.09 (m, H.sub.2C.dbd.CH--), 4.34 (m, opd,
--CH.sub.2CH.sub.2C(O)CH.sub.2CH.sub.2O--), 4.08 (m, avl, pvl &
vl, --CH.sub.2O--), 3.65 (m, --OCH.sub.2CH.sub.3), 2.74 (m, opd,
--OC(O)CH.sub.2CH.sub.2C(O)--), 2.60 (m, opd,
--CH.sub.2CH.sub.2C(O)CH.sub.2CH.sub.2--, pvl, --OC(O)CH--,
--CHCH.sub.2CCH), 2.50 (m, CHCH.sub.2CCH), 2.34 (m, vl,
--CH.sub.2CH.sub.2C(O)O--, avl, H.sub.2C.dbd.CHCH.sub.2CH--,
H.sub.2C.dbd.CHCH.sub.2CH--), 2.02 (m, HC.ident.C--), 1.68 (m, pvl,
avl & vl, --CHCH.sub.2CH.sub.2--), 1.25 (m,
--CH.sub.2CH.sub.3).
[0411] 10. General Procedure for Oxidation of Copolymers
[0412] In a 200 mL round bottom flask, equipped with stir bar,
poly(vl-avl) (2.7389 g, 6.12 mmol) was dissolved in 37 mL of
CH.sub.2Cl.sub.2. To this solution, 3-chloroperoxybenzoic acid
(2.0903 g, 12.11 mmol) was added slowly. The mixture was stirred
for 72 h at room temperature and then concentrated via rotary
evaporator. The crude product was dissolved in a minimal amount of
THF (5 mL) and poured into a round-bottomed flask containing 1 L
diethyl ether. The solution was kept overnight at 0.degree. C. and
a white solid was obtained. The solution was decanted off and the
solid was dried in vacuo to obtain poly(vl-evl). Yield: 1.9467 g
(71%). .sup.1H NMR (300 MHz, CDCl.sub.3/TMS, ppm) .delta.: The
significant change is the disappearance of the allylic protons at
5.7 and 5.09 ppm and the appearance of small broad resonance peaks
at 2.96, 2.75 and 2.47 ppm due to the formation of the epoxide
ring. All other aspects of the spectrum are similar.
[0413] 11. General Procedure for Nanoparticle Formation
[0414] In a 100 mL three-necked round bottom flask equipped with
stir bar, condenser and septa, a solution of
2,2'-(ethylenedioxy)diethylamine (39.3 .mu.L, 2.68.times.10.sup.4
mol) in 27.6 mL CH.sub.2Cl.sub.2. A solution of poly(vl-evl)
(0.1330 g, M.sub.w=4834 Da, PDI=1.17) dissolved in CH.sub.2Cl.sub.2
(0.18 mL) was added dropwise via a peristaltic pump at 13 mL/min
with vigorous stirring. The mixture was heated at reflux for a
total of 12 h. Residual diamine was removed by dialyzing with
SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000) against
dichloromethane. .sup.1H NMR (300 MHz, CDCl.sub.3/TMS, ppm)
.delta.: The significant change is the disappearance of the epoxide
protons at 2.96, 2.75 and 2.47 ppm and the appearance of signals at
3.5 and 2.89 ppm corresponding to the protons neighboring the
secondary amine of the PEG linker after cross-linking. All other
aspects of the spectrum are similar.
[0415] 12. Determination of Amine Content
[0416] Nanoparticles can be titrated with a strong acid to
determine amine content. As shown in Table 2, several poly(vl-evl)
(AB) nanoparticle samples were titrated with perchloric acid to
determine the weight percentages (wt %) of primary amine and
secondary amine in the three samples that we analyzed with
transmission electron microscopy. The three samples (shown in Table
2) titrated have the following size dimensions by DLS: 58.06, 255.7
and 425.1 nm.
TABLE-US-00002 TABLE 2 Correlation of particle size and amine
content AB Nanoparticle Primary Secondary size (nm) amine wt %
amine wt % 58.06 0.008% 0.031% 255.7 0.025% 0.098% 425.1 0.055%
0.20%
[0417] 13. Nanoparticles Formed by Co-Polymerization
[0418] While nanoparticles are typically prepared with a single
type of polymer or copolymer, nanoparticles have also been
successfully produced from a mixture of poly(vl-evl-pvl) and
poly(vl-evl-opd). Such nanoparticles are tabulated in Table 3.
TABLE-US-00003 TABLE 3 Nanoparticles formed from two polymers
Diameter (nm) Amine/1 Poly(vl-evl-pvl) with Epoxide
poly(vl-evl-opd) 4 43.7 .+-. 4.50 8 94.15 .+-. 6.85
[0419] 14. Varying Comonomer Content
[0420] The properties of nanoparticles can be further tailored by
incorporating different percentages of epoxy-.delta.-valerolactone
(evl) into the polymer backbone. The data summarized in Table 4,
below, shows the nanoparticles made from the linear poly(vl-evl)
with 2% evl, 7% evl, and 19% evl. These data show that, as the %
evl is decreased to 2% in the linear polymer, smaller nanoparticles
can be obtained. As the % evl is increase to 19%, the resulting
nanoparticles are larger but have a small deviation in comparison
to the larger nanoparticles made from poly(vl-evl) with 7% evl.
TABLE-US-00004 TABLE 4 Effect of varying comonomer content Diameter
(nm) Diameter (nm) Poly(vl-evl) Diameter (nm) Amine/ Poly(vl-evl)
7% evl Poly(vl-evl) 1 Epoxide 2% evl AB 19% evl 3 7.02 .+-. 1.05
82.1 .+-. 5.73 179.9 .+-. 18.0 4 19.04 .+-. 1.32 115.6 .+-. 25.4
225.6 .+-. 22.5 5 33.55 .+-. 1.93 255.7 .+-. 60.3 299.0 .+-. 31.2 6
48.66 .+-. 3.18 342.2 .+-. 52.2 409.1 .+-. 42.7 8 84.89 .+-. 10.47
425.1 .+-. 100 843.3 .+-. 88.0
[0421] The relationship between reaction stoichiometry and particle
size for varying comonomer content is further illustrated in FIG.
17-FIG. 19.
[0422] 15. Addition of ethylenediamine 2-vinylsulfonyl-ethyl
carbonate to ABD (poly(vl-evl-opd)) Nanoparticles
[0423] In a 100 mL round bottom flask, equipped with stir bar, ABD
nanoparticles (0.0846 g, 2.45.times.10.sup.-4 mmol) were dissolved
in 12.5 mL of CH.sub.2Cl.sub.2. To this solution, ethylenediamine
2-(vinylsulfonyl)-ethyl carbonate in methanol (0.0152 g in 69 .mu.L
methanol, 5.89.times.10.sup.-2 mmol) was added. Sodium
cyanoborohydride (0.0111 g, 1.76.times.10.sup.-1 mmol) was
dissolved in 12.5 mL methanol and added to the round bottom flask.
The pH of the reaction mixture was adjusted to a pH of 6.5 with
aqueous 1 M NaOH and 1 M HCl. The mixture was stirred for 25 h at
room temperature and then dialyzed with SnakeSkin.RTM. Pleated
Dialysis Tubing (MWCO=10,000) against 1:1 dichloromethane/methanol.
Successful attachment of the linker was observed by the appearance
of signals 6.7 ppm and 6.9 ppm (.sup.1H NMR, 300 MHz,
CDCl.sub.3/TMS) due to the vinyl protons of the linker.
[0424] 16. Attachment of GV-13-Alexafluor750 to ABD
Nanoparticles
[0425] In a small vial, equipped with a stir bar, linker modified
nanoparticles (L-ABD) (29.9 mg) were dissolved in 800 .mu.l, PBS
buffer (pH 7.2) and 700 .mu.L dimethylformamide. To this solution,
251 .mu.L GV-13-Alexafluor (0.44 mg in 150 .mu.L PBS buffer and
26.5 .mu.L DMF) was added to the vial via micropipette. After 45
min of stirring at room temperature, GV-13 (2.08 mg,
1.9.times.10.sup.-3 mmol) dissolved in 200 .mu.L PBS buffer was
added. The reaction mixture stirred for 24 h in aluminum covered
beaker. The resulting mixture was purified with concentrating tubes
(MWCO=10,000) to remove excess GV-13 and GV-13-Alexafluor. The
purified product was concentrated via rotary evaporator. Successful
attachment of peptide and dye was observed by the presence of a
bright blue color due to the dye. .sup.1H NMR also shows the
presence of the peptide.
[0426] General.
[0427] Commercial reagents were obtained from commercial sources
(Aldrich, EMD, Alfa-Aesar, Fisher Scientific, and Acros) and used
without further purification. Analytical TLC was performed on
commercial Merck plates coated with silica gel GF254 (0.24 mm
thick) and spots located by UV light (254 and 366 nm). Silica gel
for flash chromatography was Merck Kieselgel 60 (230-400 mesh,
ASTM) or Sorbent Technologies 60 .ANG. (40-63 .mu.m, technical
grade). MAL-dPeg.TM..sub.4-t-boc-hydrazide was obtained from Quanta
Biodesign, Ltd. (Powell, Ohio) and used as received. Cy3 NHS dye
and PD-10 Desalting columns were received from GE Healthcare
(Piscataway, N.J.). Spectra/Por.RTM. Biotech Cellulose Ester (CE)
Dialysis Membranes (1,000 MWCO) obtained from Spectrum
Laboratories, Inc. (Rancho Dominguez, Calif.). SnakeSkin.RTM.
Pleated Dialysis Tubing (10,000 MWCO) was obtained from Pierce
Biotechnology, Inc. (Rockford, Ill.).
[0428] Instrumentation:
[0429] Samples were centrifuged at 600 rpm on a Model CS
International Centrifuge from International Equipment Company
(Boston, Mass.). Reverse-phase high performance liquid
chromatography (RP-HPLC) was carried out with a Varian Prostar
HPLC. The products were eluted using a solvent gradient (solvent
A=0.05% TFA/H.sub.2O; solvent B=0.05% TFA/CH.sub.3CN). Nuclear
magnetic resonance was performed on Bruker AC300 and AC400 Fourier
Transform Spectrometers using deuterated solvents and the solvent
peak as a reference. Gel permeation chromatography was performed in
tetrahydrofuran (THF) with the eluent at a flow rate of 1 mL/min on
a Waters chromatograph equipped with four 5 mm Waters columns (300
mm.times.7.7 mm) connected in series with increasing pore size
(100, 1000, 100,000 and 1,000,000 .ANG. respectively). A Waters
2487 Dual X Absorbance Detector and a 2414 Refractive Index
Detector were employed. Dynamic light scattering was performed on a
Malvern Zetasizer Nanoseries instrument with a CGS-3 compact
goniometer system.
[0430] 17. Synthesis of Compound 1
[0431] To a solution of dimethoxyethane (40 mL) was added
MeNO.sub.2 (11.37 mL, 200 mmol) followed by Triton B (2 mL). The
mixture was heated to 67.degree. C. and then tert-butyl acrylate
(91.83 mL, 620 mmol) was added to maintain the temperature at
75.degree. C. When the temperature started to decrease, additional
Triton B (1 mL) was added. After the addition was completed, the
solution was heated to maintain at 75.degree. C. for 2 hours. The
solvent was removed in vacuo and the residue was dissolved in
CHCl.sub.3 and the resulting organic solution was washed with 10%
HCl, brine, and dried over anhydrous Na.sub.2SO.sub.4. Removal of
the solvent in vacuo gave a crude solid that was further purified
by recrystallization from EtOH to obtain a colorless crystal (95%
yield). .sup.1H NMR (CDCl.sub.3): .delta.1.44 (s, CH.sub.3, 27H),
2.21 (m, CH.sub.2, 12H). .sup.13C NMR (CDCl.sub.3): 27.93
(CH.sub.3), 29.68 (CH.sub.2CO), 30.22 (CCH.sub.2), 81.02
(CCH.sub.3), 92.09 (CNH.sub.2), 170.97 (CO.sub.2).
[0432] 18. Synthesis of Compound 2
[0433] A solution of compound I (6.0 g, 0.0135 mol) in a mixture of
ethanol (140 mL) and dichloromethane (20 mL) was added to a Parr
hydrogenation bottle. Then, 4 grams of Raney-nickel was added. The
mixture was hydrogenated at 50 psi and room temperature. The
reaction was monitored by thin-layer chromatography (TLC) until the
starting material disappeared. The catalyst was carefully filtered
through Celite, and the solvent was removed in vacuo yielding a
crude solid. The residue was dissolved in dichloromethane and
washed with saturated NaHCO.sub.3 and water, and then dried over
anhydrous Na.sub.2SO.sub.4. Removal of dichloromethane gave a white
solid (93%). .sup.1H NMR (CDCl.sub.3): .delta.1.44 (s, CH.sub.3,
27H), 1.95 (t, CH.sub.2, 6H), 2.43 (t, CH.sub.2, 6H); .sup.13C NMR
(CDCl.sub.3): 27.98 (CH.sub.3), 29.46 (CH.sub.2CO), 31.47
(CCH.sub.2), 56.99 (CNH.sub.2), 80.96 (CCH.sub.3), 172.30
(CO.sub.2).
[0434] 19. Synthesis of Compound 4
[0435] To a solution of compound 3 (0.65 g, 2.35 mmol) in 50 mL dry
THF the following reagents were added 1-hydrobenzotriazole (HOBt)
(0.96 g, 7.10 mmol), DCC (1.46 g, 7.10 mmol) and then 2 (3.54 g,
8.5 mmol). The solution was stirred at room temperature and the
reaction was monitored by TLC. After 40 hrs, the white precipitate
was filtered and the solution was concentrated to yield a crude
residue. The product was purified by column chromatography (silica
gel, hexane:ethyl acetate=3:2) yielding a white solid (85%).
.sup.1H NMR (CDCl.sub.3): .delta.1.44 (m, CH.sub.3, 81H), 1.95 (m,
CH.sub.2, 18 H), 2.21 (m, CH.sub.2, 30H), 6.20 (s, NH, 3H);
.sup.13C NMR (CDCl.sub.3): 28.04, 29.74, 29.85, 31.28, 57.56,
80.69, 92.47, 170.46, 172.76.
[0436] 20. Synthesis of Compound 5
[0437] A solution of compound 4 (1.47 g, 1 mmol) in 15 mL of formic
acid was stirred at room temperature overnight. After the solution
was concentrated, toluene was added and the solution was evaporated
to remove any residue of formic acid to give a white solid (100%).
.sup.1H NMR (DMSO): .delta. 1.81 (m, CH.sub.2, 18 H), 2.11 (m,
CH.sub.2, 30H), 7.29 (s, NH, 3H), 12.10 (br, COOH); .sup.13C NMR
(DMSO): 28.03, 29.03, 30.08, 56.41, 93.31, 170.43, 174.42.
[0438] 21. Synthesis of Compound 6
[0439] To a solution of compound 5 (2.12 g, 0.0022 mol) in DMF (30
mL), HOBt (2.68 g, 0.0198 mol) and DCC (4.09 g, 0.0198 mol) were
added. The mixture was chilled to 0.degree. C. with ice-water bath.
Then, a solution of N-Boc-ethylenediamine (3.49 g, 0.0218 mol) in
DMF (5 mL) was added dropwise at 0.degree. C. The reaction mixture
was stirred at room temperature for 48 hrs. The solution was then
filtered and 200 mL of dichloromethane was added, and washed with
1N HCl, saturated NaHCO.sub.3, and water. The organic phase was
dried over anhydrous Na.sub.2SO4 and evaporated to yield a crude
residue. The product was purified by column chromatography (eluted
first with 2% methanol in dichloromethane, then with 6% methanol in
dichloromethane, followed by 10% methanol in dichloromethane) to
obtain a white solid (51%). .sup.1H NMR (CD.sub.3OD): .delta. 1.44
(m, CH.sub.3, 81H), 1.80-2.10 (m, CH.sub.2, 48 H), 3.0-3.2 (m,
CH.sub.2, 36H), 6.20 (m, NH, 3H), 6.46 (m, NH, 8H), 7.71 (m, NH,
8H); .sup.13C NMR (CD.sub.3OD): 28.40, 31.24, 31.44, 31.80, 32.09,
40.66, 40.97, 59.14, 80.13, 94.42, 158.48, 173.48, 175.91. This
white solid was then dissolved in 40 mL of 1,4-dioxane. At
0.degree. C., 40 mL of 4 M HCl in dioxane was added to the solution
under Ar atmosphere and stirred at room temperature for 1 hr.
Removal of the solvent gave a white solid as the deprotected HCl
salt (100%). .sup.1H NMR (D.sub.2O): .delta. 1.70-2.15 (m,
CH.sub.2, 48 H), 3.30 (m, CH.sub.2, 18H), 3.36 (m, CH.sub.2, 18H);
.sup.13C NMR (D.sub.2O): 27.61, 27.98, 28.86, 35.11, 37.41, 56.29,
92.01, 171.84, 174.98. 1.53 g (0.92 mmol) of the resulting HCl salt
was dissolved in 80 mL of methanol. At 0.degree. C., 3.5 mL of
Et.sub.3N was added to the solution, followed by the addition of
N,N'-diBoc-N''-triflylguanidine (4.2 g, 10.73 mmol). The solution
was stirred at room temperature for 24 hr. After removal of the
solvent, the residue was dissolved in dichloromethane and washed
with water, 1N HCl, saturated NaHCO.sub.3, and water. The organic
layer was dried over anhydrous Na.sub.2SO.sub.4 and removed in
vacuo. The residue product was purified by column chromatography
(eluted with 2% methanol in dichloromethane, then 10% methanol in
dichloromethane) to give a white solid (90%) as compound 6. .sup.1H
NMR (CD.sub.3OD): .delta. 1.45 (m, CH.sub.3, 81H), 1.51 (m,
CH.sub.3, 81H), 1.90-2.25 (m, CH.sub.2, 48 H), 3.30-3.52 (m,
CH.sub.2, 36H); .sup.13C NMR (CD.sub.3OD): 28.37, 28.67, 31.32,
31.67, 32.06, 39.74, 41.24, 59.02, 80.23, 84.35, 94.31, 153.91,
157.737, 164.38, 173.33, 175.87.
[0440] 22. Synthesis of Compound 7
[0441] To a solution of compound 5 (1.2, 0.001245 mol), HOBt (1.514
g, 0.0112 mol) and DCC (2.311 g, 0.0112 mol) were added in 20 mL of
DMF. Then, N-Boc-1,6-diaminohexane (2.66 g, 0.0123 mol) was
dissolved in 5 mL of DMF dropwise at 0.degree. C. The solution was
then stirred at room temperature for 48 hrs. The solution was then
filtered and 200 mL of dichloromethane was added, and washed with
1N HCl, saturated NaHCO.sub.3, and water. The organic phase was
dried over anhydrous Na.sub.2SO.sub.4 and evaporated to yield a
crude residue. The product was purified by column chromatography
(eluted first with 2% methanol in dichloromethane, then with 5%
methanol in dichloromethane, followed by 10% methanol in
dichloromethane) to obtain a white solid (45%). .sup.1H NMR
(CD.sub.3OD): .delta. 1.2-1.6 (m, CH.sub.3, CH.sub.2, 153H),
1.80-2.10 (m, CH.sub.2, 48H), 3.0-3.2 (m, CH.sub.2, 36H); .sup.13C
NMR (CD.sub.3OD): 27.54, 28.85, 30.37, 30.90, 31.28, 31.60, 32.14,
40.58, 41.24, 59.13, 79.30, 94.30, 158.49, 173.50, 175.56. This
white solid was then dissolved in 40 mL of 1,4-dioxane. At
0.degree. C., 40 mL of 4 M HCl in dioxane was added to the solution
under Ar atmosphere and stirred at room temperature for 1 hr.
Removal of the solvent gave a white solid as the deprotected HCl
salt (100%). .sup.1H NMR (D.sub.2O): .delta. 1.10-1.60 (m,
CH.sub.2, 72 H), 1.7-2.2 (m, CH.sub.2, 48H), 3.30 (m, CH.sub.2,
18H), 3.36 (m, CH.sub.2, 18H). 0.838 g (0.385 mmol) of the
resulting HCl salt was dissolved in 80 mL of methanol. At 0.degree.
C., 1.45 mL of Et.sub.3N was added to the solution, followed by the
addition of N,N'-diBoc-N''-triflylguanidine (1.765 g, 4.51 mmol).
The solution was stirred at room temperature for 24 hr. After
removal of the solvent, the residue was dissolved in
dichloromethane and washed with water, 1N HCl, and water. The
organic layer was dried over anhydrous Na.sub.2SO.sub.4 and removed
in vacuo. The residue product was purified by column chromatography
(eluted with 2% methanol in dichloromethane, then 10% methanol in
dichloromethane) to give a white solid (90%) as compound 7. .sup.1H
NMR (CD.sub.3OD): .sup.1H NMR of 9 (CD.sub.3OD): .delta. 1.15-1.55
(m, 234H), 1.70-2.15 (m, CH.sub.2, 48H), 3.29-3.30 (m, CH.sub.2,
36H); .sup.13C NMR (CD.sub.3OD): 27.70, 27.62, 28.33, 28.67, 30.08,
30.33, 31.30, 31.60, 40.48, 40.62, 41.27, 54.5, 59.14, 80.25,
84.40, 154.22, 157.49, 164.53, 173.50, 175.53.
[0442] 23. Synthesis of Compound 8 and 9
[0443] Compound 6 (or 7, 0.10 mmol) was dissolved in 40 mL of
ethanol and transferred into a hydrogenation bottle containing 5 g
of Raney-Nickel catalyst. The solution was hydrogenated at room
temperature at 65 psi and monitored by TLC. The catalyst was
filtered through Celite. The solvent was removed in vacuo to give a
white solid 8 or 9 (80%). .sup.1H NMR of 8 (CD.sub.3OD): .delta.
1.46 (m, CH.sub.3, 81H), 1.51 (m, CH.sub.3, 81H), 1.90-2.25 (m,
CH.sub.2, 48H), 3.30-3.55 (m, CH.sub.2, 36H); .sup.13C NMR
(CD.sub.3OD): 28.37, 28.67, 31.40, 31.76, 39.76, 41.27, 54.0,
58.86, 80.32, 84.37, 153.97, 157.81, 164.4, 175.61, 176.02. .sup.1H
NMR of 9 (CD.sub.3OD): .delta. 1.20-1.70 (m, 234H), 1.85-2.40 (m,
CH.sub.2, 48 H), 3.10-3.50 (m, CH.sub.2, 36H); .sup.13C NMR
(CD.sub.3OD): 27.01, 27.18, 28.27, 28.53, 29.42, 29.71, 30.15,
30.88, 31.19, 40.03, 41.23, 54.3, 58.21, 79.93, 83.84, 153.62,
156.65, 163.83, 175.77.
[0444] 24. Synthesis of Compound FD-1
[0445] FITC (0.14 g, 0.36 mmol), dissolved in 1 mL of DMF, was
added to a solution of compound 8 (0.23 g, 0.066 mmol) in a mixture
of DMF and dichloromethane. The solution was chilled to 0.degree.
C., to which Et.sub.3N (0.092 mL, 0.66 mmol) was added. The mixture
was stirred overnight at room temperature. After removal of DMF in
vacuo, the residue was dissolved in dichloromethane and washed with
1N HCl and water. The dichloromethane layer was dried over
anhydrous Na.sub.2SO.sub.4 and concentrated to obtain a yellow
solid. .sup.1H NMR (CD.sub.3OD): .delta. 1.46 (m, CH.sub.3, 81H),
1.51 (m, CH.sub.3, 81H), 1.90-2.25 (m, CH.sub.2, 48 H), 3.30-3.55
(m, CH.sub.2, 36H), 6.52-6.72 (br, 4H), 7.15 (br, 1H), 7.5 (br,
2H), 7.72 (br, 1H), 8.4 (br, 1H). The resulting yellow solid (200
mg, 0.052 mmol) was dissolved in 10 mL of 1,4-dioxane. At 0.degree.
C., 10 mL of 4 M HCl in dioxane was added to the solution under Ar
protection and stirred at room temperature overnight. After
evaporation of the solvent in vacuo, the product was dissolved in
water and the insoluble precipitate was filtered. Removal of water
yielded a crude yellow solid, which was further purified by RP-HPLC
using a solvent gradient (solvent A=0.05% TFA/H.sub.2O; solvent
B=0.05% TFA/CH.sub.3CN) to obtain compound 10. .sup.1H NMR
(D.sub.2O): .delta. 1.85-2.30 (m, CH.sub.2, 48 H), 3.10-3.30 (m,
CH.sub.2, 36H), 6.9 (br, 2H), 7.10-7.2 (m, 3H), 7.4 (s, 2H), 7.5
(br, 1H), 8.1 (s, 1H).
[0446] 25. Synthesis of Compound FD-2
[0447] FITC (0.016 g, 0.0376 mmol), dissolved in 1 mL of DMF, was
added to a solution of compound 9 (0.050 g, 0.0125 mmol) in a
mixture of DMF and dichloromethane (1:1). The solution was chilled
to 0.degree. C., to which Et.sub.3N (12 .mu.L) was added. The
mixture was stirred overnight at room temperature. After removal of
DMF in vacuo, the residue was dissolved in dichloromethane and
washed with 1N HCl and water. The dichloromethane layer was dried
over anhydrous Na.sub.2SO.sub.4 and concentrated to obtain a solid.
The product was dissolved in methanol and purified by dialysis with
Spectro.RTM.Por Biotech RC membranes (MWCO 3500). After removal of
the methanol, a yellow solid was obtained. .sup.1H NMR
(CD.sub.3OD): .delta. 1.20-1.7 (m, CH.sub.3, CH.sub.2, 234H),
1.89-2.30 (m, CH.sub.2, 48 H), 3.10-3.40 (m, CH.sub.2, 36H),
6.52-6.72 (br, 4H), 7.15 (br, 1H), 7.5-7.72 (br, 3H), 8.1 (br, 1H).
The resulting yellow solid (200 mg, 0.052 mmol) was dissolved in 10
mL of 1,4-dioxane. At 0.degree. C., 10 mL of 4 M HCl in dioxane was
added to the solution under Ar protection and stirred at room
temperature overnight. The precipitate was filtered out and dried
in vacuo. The obtained yellow solid was dissolved in water and
lyophilized to yield compound 11. .sup.1H NMR (D.sub.2O): .delta.
1.1-1.50, (m, CH.sub.2, 72H), 1.50-2.20 (m, CH.sub.2, 48 H),
3.10-3.30 (m, CH.sub.2, 36H), 6.5-6.7 (br, 6H), 7.10 (m, 1H), 7.5
(br, 3H).
[0448] 26. Examples FD-1 and FD-2
[0449] As examples of the compounds of the invention, two
non-peptidic fluorescently labeled Newkome-type dendrimers,
differentiated over a varied alkylspacer with guanidine end
moieties, were designed and synthesized. The assessment of
internalization into mammalian cells using NIH-3T3 fibroblasts and
human microvascular endothelial cells (HMEC) showed that the spacer
length at the terminal generation of the dendrimers can affect
direction of cargo molecules precisely into specific subcellular
compartments (e.g., nucleus or cytosol). Such direction can be
particularly advantageous for the controlled intracellular delivery
of bioactive cargo molecules into targeted locations.
[0450] The two exemplary FITC-dendrimer conjugates were found to be
highly water soluble and were further investigated for their
capability to translocate through the cell membrane.
Internalization of FD-1 and FD-2 in mammalian cells was assessed
using two different cell lines and a previously described method
[Futaki, S.; Nakase, I.; Suzuki, T.; Youjun, Y.; Sugiura, Y.
Biochemistry 2002, 41, 79251 with NIH-3T3 fibroblasts and HMEC
(human microvascular endothelial cells) and a Zeiss LSM 510
confocal microscope. FIG. 21 shows the time course of uptake of
FD-1 and FD-2 into NIH-3T3 Fibroblasts at 37.degree. C. The
fluorescence was clearly observed within the cells 2.5 min after
the addition of conjugates to the medium, which is comparable to
the uptake rate of Tat-peptide. [Futaki, S.; Nakase, I.; Suzuki,
T.; Youjun, Y.; Sugiura, Y. Biochemistry 2002, 41, 7925.; Vives,
E.; Brodin, P.; Lebleu, B. J. Biol. Chem. 1997, 272, 160101
Furthermore, the extent of internalization increased in an
incubation time-dependent manner, and it was observed that after
just 10 min, the fluorescence intensity of cells treated with FD-2
was near saturation. However, the fluorescence intensity of cells
treated with FD-1 did not approach saturation until the longer time
points (45 min .about.2 hr). Additionally, FD-1 and FD-2 exhibited
differential patterns of subcellular localization, as FD-1 appeared
to concentrate in the nucleus while FD-2 appeared to concentrate in
the cytosol. Without wishing to be bound by theory, it is believed
that the length of the spacer at the terminal generation of the
dendrimer can not only control the uptake rate, [Wender, P. A.;
Kreider, E.; Pelkey, E. T.; Steinman, L.; Rothbard, J. B.;
VanDeusen, C. L. Org. Lett. 2005, 7, 48151 but also regulate the
subcellular localization of the molecule and its putative cargo.
For instance, the uptake levels of FD-2 appeared to be generally
stronger than those of FD-1 after the same incubation time at the
same concentration. Therefore, the dendrimer with a hexyl spacer
crosses the cell membrane faster than the molecule with an ethyl
chain. On the other hand, the localization patterns can also be
controlled by the length of the spacer. FD-1 with the short spacer
appeared to be localized everywhere in the cell, but highly
concentrated in the nucleus. However, FD-2, with its longer spacer,
was observed to reside mainly in the cytosol. These translocation
features of guanidinlyated dendritic scaffolds as carriers can be
important for intracellular delivery of cargo molecules to specific
subcellular compartments (e.g., cytosol or nucleus). For example, a
translocation approach that does not saturate the nucleus can be
highly attractive as it can be both less cytotoxic and could afford
cytosolic-targeted cargos with greater accuracy in delivery, and
therefore higher efficacy. Without wishing to be bound by theory,
it is believed that the differential uptake patterns by ED-1 and
FD-2 are due to the presence of a hexyl spacing chain in FD-2,
resulting in a greater hydrophobicity of the entire conjugate as
compared with FD-1. Additionally, the uptake of FD-1 and FD-2
conjugates by HMEC was also conducted. Entry of the two conjugates
into HMEC shows a similar internalization pattern to that seen in
fibroblasts.
[0451] In control experiments, cells treated with free FITC and
Boc-protected guanidinylated FITC-dendrimer showed no or extremely
weak fluorescence, respectively. Therefore, the guanidino groups
play an important role in the cell permeability of these molecules,
while the length of the spacing chain determines both the
differential rate of uptake and subcellular localization patterns.
Although the mechanism of Tat translocation remains to be
understood, it has been demonstrated that the rate of uptake is not
temperature dependent. [Futaki, S.; Nakase, I.; Suzuki, T.; Youjun,
Y.; Sugiura, Y. Biochemistry 2002, 41, 7925.; Vives, E.; Brodin,
P.; Lebleu, B. J. Biol. Chem. 1997, 272, 16010.] This indicates
that endocytosis does not play a crucial role in the translocation
process. Evaluation of the effect of temperature on the
internalization of FD-1 and FD-2 indicated that the two conjugates
are able to get into cells not only at 37.degree. C., but also at
4.degree. C., even at a lower dendrimer concentration (1 .mu.M)
(see FIGS. 3 and 4 in contrast to control experiments, as shown in
FIG. 24). No significant decrease in fluorescence intensity of
cells treated with FD-1 or FD-2 was observed, indicating that the
uptake process does not occur via endocytosis.
[0452] 27. Synthesis of dendrimer B11.
[0453] A three-neck round bottom flask was flame-dried under argon,
to which nitrotriacid B3 (3.192 g, 0.0115 mmol),
1-hydrobenzotriazole (HOBt) (5.609 g, 0.0415 mol), DCC (8.560 g,
0.0415 mol) and 100 mL THF were added sequentially. After 2 hours
activation, aminotriester B2 (17.216 g, 0.0415 mol) was added. The
solution was stirred at room temperature for 40 h, and the crude
product was purified by flash column chromatography, eluting first
with hexane/ethyl acetate (10:1) and then hexane/ethyl acetate
(3:2) to yield dendrimer B11 (15.91 g, 94.1%). .sup.1H NMR (400
MHz, CDCl.sub.3): .delta.=1.44 (m, CH.sub.3, 81H), 1.95 (m,
CH.sub.2, 18 H), 2.21 (m, CH.sub.2, 30H), 6.20 (s, NH, 3H);
.sup.13C NMR (400 MHz, CDCl.sub.3): .delta.=28.04, 29.74, 29.85,
31.28, 57.56, 80.69, 92.47, 170.46, 172.76.
[0454] 28. Synthesis of Dendrimer B12.
[0455] A solution of B11 (10.0 g, 0.0 mol) in 150 mL of absolute
ethanol in the presence of 8 grams of Raney-Nickel was hydrogenated
at 60 psi of hydrogen at room temperature for 24 h. The suspension
was carefully filtered through Celite and removal of the solvent
under reduced pressure yielded B12 (9.86 g, 98.5%). .sup.1H NMR
(400 MHz, CD.sub.3OD): .delta.=1.44 (m, CH.sub.3, 81H), 1.61 (m,
CH.sub.2, 6 H), 1.95 (m, CH.sub.2, 12 H), 2.21 (m, CH.sub.2, 30 H);
.sup.13C NMR (400 MHz, CD.sub.3OD): .delta.=28.42, 30.24, 30.47,
32.02, 36.24, 53.53, 58.37, 81.18, 173.96, 175.39.
[0456] 29. Synthesis of B25.
[0457] To a room temperature stirred solution of 6-bromohexanoic
acid (2.0 g, 0.0102 mol) in 7 mL of DMF was added NaN.sub.3 (1.30
g, 0.020 mol). The reaction mixture was heated and stirred at
85.degree. C. for 5 h. After DMF was removed, DCM was added to
dissolve the residue. The mixture was washed with 0.1 N HCl and
dried over anhydrous NaSO.sub.4. Removal of the solvent gave a
crude oil that was purified by flash column chromatography, eluting
first with DCM and then ethyl acetate/DCM (3:7) to yield B25 (1.67
g, 69.07%). .sup.1H NMR (400 MHz, MeOD): .delta.=1.38-1.49 (m,
CH.sub.2, 2H), 1.54-1.70 (m, CH.sub.2, 4 H), 2.32 (t, CH.sub.2, 2
H), 3.30 (t, CH.sub.2, 2 H); .sup.13C NMR (400 MHz, MeOD):
.delta.=25.57, 27.32, 29.62, 34.72, 52.27, 177.38.
[0458] 30. Synthesis of Dendrimer B13.
[0459] To a stirred solution of B25 (1.29 g, 8.22 mmol) in
anhydrous THF (50 mL) were added DCC (1.70 g, 8.22 mmol) and HOBt
(1.112 g, 8.22 mmol) at room temperature. The mixture was stirred
for 2 h, then dendrimer B12 (9.86 g, 6.85 mmol) was added and the
resulting solution was stirred for 40 h. After filtration and
removal of THF, the product was purified by flash column
chromatography, eluting with hexane/ethyl acetate (1:1) to yield
B13 (8.50 g, 78.53%). .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta.=1.44 (m, CH.sub.3, CH.sub.2, 83 H), 1.95 (m, CH.sub.2, 18
H), 2.21 (m, CH.sub.2, 32 H), 3.30 (m, CH.sub.2, 2 H); .sup.13C NMR
(400 MHz, CD.sub.3OD): .delta.=26.47, 27.47, 28.43, 29.62, 30.35,
30.61, 32.07, 32.23, 37.56, 52.28, 58.63, 58.77, 81.54, 174.21,
175.35, 175.66.
[0460] 31. Synthesis of Dendrimer B14.
[0461] To a 0.degree. C. stirred solution of nona-amine B5 (4.06 g,
2.43 mmol) in a methanol/acetonitrile (25 mL/15 mL) were added
triethylamine (6.87 g, 68.0 mmol) and ethyl trifluoroacetate (9.32
g, 65.6 mmol) and the reaction mixture was stirred at 0.degree. C.
for 1 h and then at room temperature overnight. The solvent was
removed in vacuo and the residue was taken up in ethyl acetate, and
the resulting organic solution was washed with 1N HCl and brine and
dried over anhydrous NaSO.sub.4. Removal of the solvent in vacuo
gave a crude solid that was purified by flash chromatography
(EtOAc/Methanol gradient) to yield a solid (3.02 g, 56.3%). .sup.1H
NMR (400 MHz, CD.sub.3OD): .delta.=1.85-2.10 (m, CH.sub.2, 18 H),
2.11-2.35 (m, CH.sub.2, 30H), 3.24-3.48 (m, CH.sub.2, 36 H);
.sup.13C NMR (400 MHz, CD.sub.3OD): .delta.=31.08, 31.26, 31.75,
32.01, 39.42, 40.42, 58.93, 94.33, 111.74, 115.54, 119.33, 123.13,
158.57, 159.06, 159.55, 160.04, 173.57, 176.14. The resulting white
solid (1.0 g, 0.453 mmol) was dissolved in ethanol (45 mL) and
transferred into a hydrogenation vessel containing Raney-Nickel
catalyst (5 g) and the suspension was stirred at 80 psi of hydrogen
at 50.degree. C. for 48 h. After filtration through Celite, the
solvent was removed under reduced pressure to give a B14 as a white
solid (0.964 g, 97.7%). .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta.=1.67 (m, CH.sub.2, 6 H), {tilde over (1)}986 (m, CH.sub.2,
12H), 2.188 (m, CH.sub.2, 30 H), 3.30-3.55 (m, CH.sub.2, 36 H);
.sup.13C NMR (400 MHz, CD.sub.3OD) .delta.=31.20, 32.11, 36.17,
39.38, 40.52, 54.06, 58.80, 111.79, 115.53, 119.36, 123.10, 158.58,
159.04, 159.50, 160.11, 175.55, 176.24.
[0462] 32. Synthesis of Dendrimer B15.
[0463] To a stirred solution of 6-heptynoic acid (0.3022 g, 2.40
mmol) in anhydrous THF (50 mL) were added DCC (0.4952 g, 2.40 mmol)
and HOBt (0.3245 g, 2.40 mmol) at room temperature. The mixture was
stirred for 2 h, then dendrimer B14 (1.0432 g, 0.48 mmol) was added
and the resulting solution was stirred for 40 h. After filtration
and removal of THF, the product was purified by flash column
chromatography, eluting with ethyl acetate/methanol gradient to
yield B15 (0.620 g, 56.57%). .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta.=1.53 (m, CH.sub.2, 2 H), 1.71 (m, CH.sub.2, 3 H), 1.890-2.5
(m, CH.sub.2, 50 H), 3.30 (m, CH.sub.2, 36 H); .sup.13C NMR (400
MHz, CD.sub.3OD): .delta.=18.81, 26.14, 29.43, 31.27, 31.80, 37.37,
39.35, 40.43, 58.83, 59.05, 69.95, 83.4, 111.74, 115.57, 119.37,
123.13, 158.55, 159.07, 159.53, 159.99, 175.60, 176.25.
[0464] 33. Synthesis of Dendrimer B16.
[0465] Azide dendron B13 (100 mg, 0.044 mmol) and alkyne dendron
B15 (70 mg, 0.044 mmol) were dissolved in THF/H.sub.2O (4:1) and
DIPEA (0.017 g, 0.132 mmol, 3 equiv) followed by
Cu(PPh.sub.3).sub.3Br (0.0042 g, 0.0044 mmol) were added. The
reaction mixture was placed in the microwave reactor (Biotage) and
irradiated at 120.degree. C. for 20 min. After completion of the
reaction, THF was removed and the residue was taken up in DCM. The
organic layer was washed with water once and dried over anhydrous
Na.sub.2SO.sub.4. .sup.1H NMR of B16 (400 MHz, CD.sub.3OD):
.delta.=1.43 (m, CH.sub.3, 81H), 1.71 (m, CH.sub.2, 8 H), 1.890-2.5
(m, CH.sub.2, 96 H), 2.71 (m, CH.sub.2, 2 H), 3.30 (m, CH.sub.2, 36
H), 4.38 (m, CH.sub.2, 2 H), 7.75 (s, 1H).
[0466] 34. Synthesis of B17 and B18.
[0467] The "Bow-Tie" B16 was stirred in formic acid overnight at
room temperature. After the solvent was evaporated under reduced
pressure, toluene was added and concentrated in vacuo to remove any
residue of formic acid to give a white nonacid (100%). To a
solution of the above resulting solid in DMF, HOBt and DCC were
added and the solution was cooled to 0.degree. C.
N-Boc-ethylenediamine or N-Boc-hexyldiamine was added dropwise and
the mixture was stirred for 48 h at room temperature, filtered and
concentrated under reduced pressure. The residue was dissolved in
dichloromethane and the resulting organic solution was washed
sequentially with 1N HCl, water and dried over anhydrous
Na.sub.2SO.sub.4. The solvent was evaporated under reduced pressure
and the crude residue was purified by flash column chromatography
to yield B17 or B18.
[0468] 35. Synthesis of B19 and B20.
[0469] Potassium carbonate was added to B17 or B18 in
methanol/water, the mixture was stirred at room temperature for 6
h. The crude product was purified by dialysis against methanol with
Spectra.RTM.Por Biotech regenerated cellulose membranes (MWCO=3500)
for 24 h to give B19 or B20.
[0470] 36. Synthesis of B21 and B22.
[0471] The above B19 or B20 was then dissolved in 1,4-dioxane and
the solution cooled 0.degree. C., 4 M HCl in dioxane was added and
stirred for 1 hr at room temperature. Removal of the solvent under
reduced pressure gave a white solid. The resulting HCl salt was
dissolved in methanol and the solution was cooled to 0.degree. C.
Et.sub.3N was added, followed by N,N'-diBoc-N''-triflylguanidine
and the mixture was stirred for 24 h at room temperature. After the
solvent was evaporated under reduced pressure, the residue was
dissolved in dichloromethane and the solution was washed with 1N
HCl water, and dried over anhydrous Na.sub.2SO.sub.4. After removal
of the solvent under reduced pressure, the crude product was
purified by dialysis against methanol with Spectra.RTM.Por Biotech
regenerated cellulose membranes (MWCO=3500) for 24 h to give B21 or
B22.
[0472] 37. Synthesis of B23 and B24.
[0473] The resulting solid B21 or B22 was dissolved in 1,4-dioxane
and the solution cooled to 0.degree. C., 4 M HCl in dioxane was
added and the solution stirred overnight at room temperature. The
precipitate was filtered off and dried to give a crude product. The
solid was re-dissolved in water and insoluble precipitate was
filtered off and the filtrate was dialyzed against water with
Spectra.RTM.Por Biotech cellulose ester membranes (MWCO=1000) for
48 hrs and lyophilized to yield a water-soluble B23 or B24.
[0474] Synthesis of Copolymer poly(vl-avl-opd) (AbD).
[0475] To a 25 mL 3-necked round bottom flask, equipped with stir
bar, gas inlet and 2 rubber septa, 2-oxepane-1,5-dione (0.7000 g,
5.46 mmol) was added. The round bottom flask was purged with argon.
After purging for 30 min, dry toluene (4 mL) was added. The mixture
stirred in an oil bath at 80.degree. C. to dissolve the monomer.
Upon dissolving, Sn(Oct).sub.2 (0.011 g, 2.73.times.10.2 mmol) in
0.5 mL dry toluene, absolute ethanol (0.020 g, 4.4.times.10.1
mmol), .alpha.-allyl-.delta.-valerolactone (1.15 g, 8.2 mmol) and
.delta.-valerolactone (1.37 g, 13.7 mmol) were then added to the
reactor and the mixture was heated for 48 h at 105.degree. C.
Residual monomer and catalyst were removed by dialyzing with
Spectra/Por.RTM. dialysis membrane (MWCO=1000) against
CH.sub.2Cl.sub.2 to give a golden brown polymer. Yield: 1.7 g.
M.sub.w=3287 Da, PDI=1.17; .sup.1H NMR (300 MHz, CDCl.sub.3/TMS,
ppm) .delta.: 5.72 (m, H.sub.2C.dbd.CH--), 5.06 (m,
H.sub.2C.dbd.CH--), 4.34 (m,
--CH.sub.2CH.sub.2C(O)CH.sub.2CH.sub.2O--), 4.08 (m,
--CH.sub.2O--), 3.67 (m, --OCH.sub.2CH.sub.3), 2.78 (m, opd,
--OC(O)CH.sub.2CH.sub.2C(O)CH.sub.2--), 2.58 (m, opd,
--OC(O)CH.sub.2CH.sub.2C(O)CH.sub.2--), 2.34 (m, vl,
--CH.sub.2CH.sub.2C(O)O--, avl, H.sub.2C.dbd.CHCH.sub.2CH--,
H.sub.2C.dbd.CHCH.sub.2CH--), 1.66 (m, avl & vl,
--CHCH.sub.2CH.sub.2--), 1.25 (t, --CH.sub.2CH.sub.3); .sup.13C NMR
(400 MHz, CDCl.sub.3, ppm) .delta.: 204.9, 175.2, 173.7, 173.2,
135.0, 117.0, 63.9, 44.8, 36.4, 33.6, 28.0, 26.3, 21.3. (10.39%
avl, 7.97% evl, 6.42% opd and 75.21% vl).
[0476] 38. Synthesis of poly(vl-evl-avl-opd) (ABbD)).
[0477] In a 200 mL round bottom flask, equipped with stir bar,
poly(vl-avl-opd) (1.7 g, 1.56 mmol) was dissolved in 30 mL
CH.sub.2Cl.sub.2. To this solution, 3-chloroperoxybenzoic acid
(0.2210 g, 1.28 mmol) was added slowly. The mixture was stirred for
72 h at room temperature and then concentrated via rotary
evaporator. The crude product was dissolved in a minimal amount of
THF (5 mL) and poured into a round-bottomed flask containing 1 L
diethyl ether. The solution was kept overnight at 0.degree. C. and
a white solid was obtained. The solution was decanted off and the
solid was dried in vacuo to obtain poly(avl-evl-vl-opd). Yield: 1.2
g (71%). .sup.1H NMR (300 MHz, CDCl.sub.3/TMS, ppm) .delta.: 5.72
(m, H.sub.2C.dbd.CH--), 5.06 (m, H.sub.2C.dbd.CH--), 4.34 (m,
--CH.sub.2CH.sub.2C(O)CH.sub.2CH.sub.2O--), 4.08 (m,
--CH.sub.2O--), 3.67 (m, --OCH.sub.2CH.sub.3), 2.96 (m, epoxide
proton), 2.78 (m, evl epoxide proton, opd,
--OC(O)CH.sub.2CH.sub.2C(O)CH.sub.2--), 2.58 (m, opd,
--OC(O)CH.sub.2CH.sub.2C(O)CH.sub.2--), 2.47 (epoxide proton), 2.34
(m, vl, --CH.sub.2CH.sub.2C(O)O--, avl,
H.sub.2C.dbd.CHCH.sub.2CH--, H.sub.2C.dbd.CHCH.sub.2CH--), 1.66 (m,
avl & vl, --CHCH.sub.2CH.sub.2--), 1.25 (t,
--CH.sub.2CH.sub.3).
[0478] 39. Nanoparticle Formation from poly(vl-evl-avl-opd).
[0479] In a 250 mL three-necked round bottom flask equipped with
stir bar, condenser and septa, a solution of
2,2'-(ethylenedioxy)diethylamine (26.4 .mu.L, 0.18 mmol) in 55.6 mL
CH.sub.2Cl.sub.2 was heated at 44.degree. C. A solution of
poly(avl-evl-vl-opd) (0.2500 g, M.sub.w=3287 Da, PDI=1.17)
dissolved in CH.sub.2Cl.sub.2 (0.36 mL) was added dropwise via a
peristaltic pump at 13 mL/min with vigorous stirring. The reaction
mixture was heated for 12 h. Residual diamine was removed by
dialyzing with SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000)
against dichloromethane. .sup.1H NMR (300 MHz, CDCl.sub.3/TMS, ppm)
.delta.: The significant change is the disappearance of the epoxide
protons at 2.96, 2.75 and 2.47 ppm and the appearance of signals at
3.5 and 2.89 ppm corresponding to the protons neighboring the
secondary amine of the PEG linker after cross-linking. All other
aspects of the spectrum are similar. To demonstrate the reactivity
of the ally groups to thiols, in a model reaction we added benzyl
mercaptan to the allyl groups. We found a high reactivity using no
other reactant. We also added the molecular transporter in the same
fashion.
[0480] 40. Attachment of Benzyl Mercaptan to poly(vl-evl-avl-opd)
Nanoparticles (General Procedure to Attach Thiol Functionalized
Compounds Including "Molecular Transporter" and Peptides)
[0481] In a vial equipped with a stir bar, poly(avl-evl-vl-opd)
nanoparticles (0.030 g, 0.0268 mmol) and benzyl mercaptan (9.48 mg,
0.0764 mmol) were dissolved in 0.6 mL toluene. The reaction mixture
was heated for 72 h at 30.degree. C. The remaining toluene was
removed in vacuo and residual benzyl mercaptan was removed by
dialyzing with SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000)
against dichloromethane. .sup.1H NMR (300 MHz, CDCl.sub.3/TMS, ppm)
.delta.: The significant change is the disappearance of the allyl
protons at 5.72 and 5.06 ppm and the appearance of signals at 3.52
ppm and 7.30 ppm corresponding to the methylene and benzene protons
respectively of the attached benzyl mercaptan. All other aspects of
the spectrum are similar.
[0482] 41. Attachment of N-Boc-ethylenediamine to succinimidyl
2-vinylsulfonylmethyl carbonate (SVEC).
[0483] To a solution of SVEC (1.03 g, 3.72 mmol) in acetonitrile
(50 mL), N-boc-ethylenediamine (0.77 mL, 4.86 mmol) and water (50
mL) were added. Sodium bicarbonate (0.4066 g, 4.84 mmol) was added
and the reaction stirred for 4 h at room temperature. The
acetonitrile was removed in vacuo and the remaining aqueous phase
was diluted with brine (45 mL). The aqueous phase was extracted
three times with dichloromethane (90 mL). The organic phases were
combined, washed with brine, dried with MgSO.sub.4, and
concentrated in vacuo. The crude product was purified by flash
chromatography (eluent: ethyl acetate) to give a white solid in 90%
yield. .sup.1H NMR (300 MHz, CDCl.sub.3/TMS, ppm) .delta.: 6.6 (m,
H.sub.2C.dbd.CH--), 6.4 & 6.17 (m, H.sub.2C.dbd.CH--), 4.43 (t,
--CH.sub.2CH.sub.2OC(O)--), 3.3 (t, --CH.sub.2CH.sub.2OC(O)--),
3.24 (m, --NHCH.sub.2CH.sub.2NHC(O)--), 1.41 (s,
--NHC(O)OC(CH.sub.3).sub.3).
[0484] 42. Attachment of Sulfonyl Linker to Nanoparticles from
poly(vl-evl-opd).
[0485] In a 100 mL round bottom flask, equipped with stir bar,
poly(vl-evl-opd) (ABD) nanoparticles (84.6 mg, 2.45.times.10.sup.-7
mol) were dissolved in 12.5 mL CH.sub.2Cl.sub.2. To this solution,
sulfonyl linker (69 .mu.L of 0.85 M linker in methanol,
5.89.times.10.sup.-5 mol), NaCNBH.sub.3 (0.0111 g in 0.1 mL
methanol, 1.77.times.10.sup.-4 mol) and methanol (12.4 mL) were
added. The pH was adjusted to 6.5 using 0.1 M hydrochloric acid
aqueous solution and 0.1 M sodium hydroxide aqueous solution. The
reaction mixture stirred for 25 h at room temperature and was
purified by dialyzing with SnakeSkin.RTM. Pleated Dialysis Tubing
(MWCO=10,000) against 1:1 dichloromethane/methanol. .sup.1H NMR
(300 MHz, CDCl.sub.3/TMS, ppm) .delta.: The significant change is
the appearance of the following peaks: 6.8 (m, CH.sub.2.dbd.CH--),
6.5 & 6.3 (m, CH.sub.2.dbd.CH--), 4.5 (m,
CH.sub.2.dbd.CHSO.sub.2CH.sub.2CH.sub.2--), 3.3 (m,
--NHCH.sub.2CH.sub.2NH--), 3.1 (m,
CH.sub.2.dbd.CHSO.sub.2CH.sub.2CH.sub.2--). All other aspects of
the spectrum are similar.
[0486] 43. General Procedure for Attachment of Peptide-Alexa
Fluor.RTM. 750 to Linker Conjugated Nanoparticles.
[0487] In a small vial, equipped with stir bar, peptide (33 .mu.L
of 0.013 mg/mL peptide in phosphate buffer-pH 7.2) and Alexa
Fluor.RTM. 750 (26.5 .mu.L of 20 mg/mL Alexa Fluor.RTM. in
dimethylformamide were added. The reaction stirred for 24 h in an
aluminum foiled. In a small vial, poly(vl-evl-opd) (ABD)
nanoparticles (29.9 mg) were dissolved in 800 .mu.L phosphate
buffer (pH=7.2) and 700 .mu.L dimethylformamide. To the
peptide-Alexa Fluor.RTM. solution, 251 .mu.L of dissolved
nanoparticles was added. After stifling for 45 min at room
temperature, additional peptide (2 mg, 1.84.times.10.sup.-6 mol)
was added. The reaction mixture was purified using concentrator
tubes with a molecular weight cut-off of 10,000 Da.
[0488] 44. Attachment of Alexa Fluor.RTM. 750 to poly(vl-evl-opd)
Nanoparticles.
[0489] In a 25 mL round bottom flask, poly(vl-evl-opd)
nanoparticles (63.55 mg, 1.92.times.10.sup.-7 mol) was dissolved in
6.4 mL tetrahydrofuran. The round bottom flask was sealed with a
rubber septum and purged with argon. To the purged solution, Alexa
Fluor.RTM. 750 (5 mg in 0.5 mL anhydrous dimethylformamide was
added. The reaction mixture stirred for 24 h at room temperature.
After 24 h, N-acetoxy succinimide (50 mg, 0.3 mmol) was added to
quench the remaining unreacted amines. .sup.1H NMR (300 MHz,
CDCl.sub.3/TMS, ppm) .delta.: The significant change is the
appearance of the following peaks: 7.12, 5.6, 5.5, 5.1, 3.81, 1.90
ppm. The structure of Alexa Fluor.RTM. 750 is not publicly known.
All other aspects of the spectrum are similar.
[0490] 45. General Reductive Amination for the Attachment of
Peptides to Alexa Fluor.RTM. Conjugated Nanoparticles.
[0491] In a small vial, equipped with stir bar, peptide (2.6 mg,
2.4.times.10.sup.-6 mol) was dissolved in 2 mL tetrahydrofuran. To
this solution, dye conjugated nanoparticles (0.0923 g,
2.8.times.10.sup.-8 mol, in 0.5 mL tetrahydrofuran) and
NaCNBH.sub.3 (2.23 .mu.L of 1.0 M NaCNBH.sub.3 in tetrahydrofuran)
were added. The reaction mixture stirred for 12 h at room
temperature. The reaction mixture was purified by dialyzing with
SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000) against
tetrahydrofuran. .sup.1H NMR (300 MHz, CDCl.sub.3/TMS, ppm)
.delta.: The significant change is the appearance of the following
peaks: 5.2, 5, 4.8, 2.6, 2.45, 2.0, 1.22 and 0.89 ppm.
[0492] 46. Attachment of
N-(Boc)-2,2(ethylenedioxy)diethylamine.
[0493] A 50 mL 3-neck round bottom flask was flame-dried under
argon. The deprotected nanoparticles (27.6 mg, 0.79 mmol) were
dissolved in DriSolv DMF and transferred to the sealed flask, which
was then cooled to 0.degree. C. via an ice bath. N-methylmorpholine
(6.37 mg, 0.063 mmol) followed by isobutyl chloroformate (9.46 mg,
0.0693 mmol) was added to the cooled solution and allowed to
activate for 1.5 h. Next, N-(boc)-2,2(ethylenedioxy)diethylamine
(15.6 mg, 0.063 mmol) was added, the ice bath was removed and the
reaction was allowed to stir overnight. The reaction was
concentrated in vacuo, the residue was dissolved in MeOH,
transferred to SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10
000), and was dialysed against MeOH.
[0494] 47. Deprotection of Nanoparticles Containing
N-(Boc)-2,2(ethlenedioxy)diethylamine.
[0495] The nanoparticles were dissolved in 2 M HCl/Dioxane (15 mL).
The reaction was allowed to stir overnight. The reaction was
dissolved in MeOH/H.sub.2O and transferred to SnakeSkin.RTM.
Pleated Dialysis Tubing (MWCO=10 000), and was dialysed against
MeOH/H.sub.2O.
[0496] 48. Attachment of Alexa Fluor 750.RTM..
[0497] PBS Buffer (pH 7.3) was purged with argon for 1 h. The Alexa
Fluor.RTM. 750 (3 mg, 2.3 .mu.mol) in 0.3 mL DMF was added to a
solution of deprotected nanoparticles (15.8 mg) in PBS Buffer (1.2
mL) and was allowed to stir for 24 h. The reaction was diluted with
H.sub.2O, was transferred to SnakeSkin.RTM. Pleated Dialysis Tubing
(MWCO=10 000), and was dialyzed against H.sub.2O.
[0498] 49. Attachment of SVEC.
[0499] The nanoparticles were dissolved in 4 mL of H.sub.2O
followed by the addition of sodium bicarbonate (2.7 mg, 0.0318
mmol). Next, the SVEC was added in 1 mL of ACN followed by an
additional 3 mL of ACN. The reaction was allowed to proceed for 2 h
at which time acetoxysuccinimide (127 mg, 0.79 mmol) was added in
order to quench any remaining amines. This reaction was allowed to
proceed for 2 h. The reaction was diluted with H.sub.2O and was
transferred to SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10
000), and was dialyzed against H.sub.2O (pH 4.5).
[0500] 50. Targeting Peptide Attachment.
[0501] The modified nanoparticles (2 mg) were dissolved in 0.2 mL
of PBS Buffer (pH 7.3) and to that a solution of GCGGGNHVGGSSV
(11.4 mg, 0.0105 mmol) in 0.4 mL of PBS Buffer (pH 7.3) was added.
This reaction was allowed to proceed for 24 h. The reaction was
diluted with H.sub.2O and was transferred to SnakeSkin.RTM. Pleated
Dialysis Tubing (MWCO=10 000), and was dialyzed against
H.sub.2O.
[0502] 51. Control Peptide Attachment.
[0503] The modified nanoparticles (2 mg) were dissolved in 0.2 mL
of PBS Buffer (pH 7.3) and to that a solution of GCGGGSGVSGHNG
(11.0 mg, 0.0105 mmol) in 0.4 mL of PBS Buffer (pH 7.3) was added.
This reaction was allowed to proceed for 24 h. The reaction was
diluted with H.sub.2O and was transferred to SnakeSkin.RTM. Pleated
Dialysis Tubing (MWCO=10 000), and was dialyzed against
H.sub.2O.
[0504] 52. Attachment of
N-(Boc)-2,2(ethylenedioxy)diethylamine.
[0505] A 50 mL 3-neck round bottom flask was flame-dried under
argon. The deprotected nanoparticles (27.6 mg, 0.79 .mu.mol) were
dissolved in DriSolv DMF and transferred to the sealed flask, which
was then cooled to 0.degree. C. via an ice bath. N-methylmorpholine
(6.37 mg, 0.063 mmol) followed by isobutyl chloroformate (9.46 mg,
0.0693 mmol) was added to the cooled solution and allowed to
activate for 1.5 h. Next, N-(boc)-2,2(ethylenedioxy)diethylamine
(15.6 mg, 0.063 mmol) was added, the ice bath was removed and the
reaction was allowed to stir overnight. The reaction was
concentrated in vacuo, the residue was dissolved in MeOH,
transferred to SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10
000), and was dialysed against MeOH.
[0506] 53. Deprotection of Nanoparticles Containing
N-(Boc)-2,2(ethlenedioxy)diethylamine.
[0507] The nanoparticles were dissolved in 2 M HCl/Dioxane (15 mL).
The reaction was allowed to stir overnight. The reaction was
dissolved in MeOH/H.sub.2O, was transferred to SnakeSkin.RTM.
Pleated Dialysis Tubing (MWCO=10 000), and was dialysed against
MeOH/H2O.
[0508] 54. Attachment of
1,4,7,10-tetraazacyclododecane-1,4,7-tris(T-butyl
acetate)-10-succinimidyl acetate (DOTA).
[0509] The nanoparticles were dissolved in DMF followed by the
addition of triethylamine (TEA). To this solution, DOTA was added
and the reaction was allowed to stir overnight. The reaction was
concentrated in vacuo, the residue was dissolved in MeOH/H.sub.2O,
transferred to SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10
000), and dialyzed against MeOH/H.sub.2O.
[0510] 55. Deprotection of Nanoparticles Containing T-Butyl
Protected DOTA.
[0511] The nanoparticles were dissolved in 2 M HCl/Dioxane (15 mL).
The reaction was allowed to stir overnight. The reaction was
dissolved in MeOH/H.sub.2O, transferred to SnakeSkin.RTM. Pleated
Dialysis Tubing (MWCO=10 000), and dialyzed against
MeOH/H.sub.2O.
[0512] 56. Attachment of SVEC.
[0513] The nanoparticles were dissolved in H.sub.2O followed by the
addition of sodium bicarbonate. Next, the SVEC was added in ACN
followed by an additional ACN. The reaction was allowed to proceed
for 2 h at which time acetoxysuccinimide was added in order to
quench any remaining amines. This reaction was allowed to proceed
for 2 h. The reaction was diluted with H.sub.2O, was transferred to
SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10 000), and was
dialyzed against H.sub.2O (pH 4.5).
[0514] 57. Modification of Alexa Fluor 750.RTM..
[0515] Alexa Fluor 750.RTM. (1.43 mg, 1.1 .mu.mol) was dissolved in
143 .mu.L DMSO and added to cysteamine (0.077 mg, 1.0 .mu.mol) in
30.9 .mu.L of PBS Buffer (pH 7.5). The reaction was allowed to
proceed overnight.
[0516] 58. Simultaneous Attachment of Modified Alexa Fluor 750.RTM.
and Peptide.
[0517] The modified nanoparticles were dissolved in PBS Buffer (pH
7.3), which had been purged with argon for 20 min. Next, the
modified Alexa Fluor 750.RTM. and one equivalent of GCGGGNHVGGSSV
was added and allowed to react for 2 h. An additional 4 equivalents
of peptide was then added and the reaction stirred overnight. The
solution was diluted in H.sub.2O, transferred to SnakeSkin.RTM.
Pleated Dialysis Tubing (MWCO=10 000), and dialyzed against
H.sub.2O.
[0518] 59. Synthesis of Linear RGD.
[0519] A typical Fmoc solid phase peptide synthesis was performed
to synthesize the linear peptide. A cysteine preloaded
2-chlorotrityl resin was employed. HOBt:HBTU:DIPEA (1:1:2) in DMF
was used as the coupling reagent and amino acids were double
coupled. A 20% piperidine (v/v) in DMF employed to deprotect the
Fmoc. An amino-hexyl spacer was coupled to the cystine on the
resin, followed by glutamic acid, aspartic acid, glycine, arginine,
phenylalanine, and finally lysine.
[0520] 60. Cyclization of RGD.
[0521] The peptide was cyclized by utilizing an ODmab group, which
allows for the selective deprotection carboxylic acid side chain of
the glutamic acid, which can then be coupled to the N-terminus. The
ODmab was deprotected using 2% v/v hydrazine-H.sub.2O/DMF added to
the resin and allowed to react for 7 min. Next it was washed with
20 mL of DMF followed by 10 mL of a 5% v/v DIPEA/DMF solution which
was allowed to shake for 10 min. Carboxy activation was achieved
through the use of DCC (44.6 mg, mmol) and HOBt (29.2 mg, mmol) was
added to 10 mL of DMF and then added to the resin and allowed to
shake for 18 h.
[0522] Reagent R was used to deprotect all side groups and cleave
the cyclic peptide from the resin. Reagent R was prepared by
combining 5.4 mL TFA, 0.3 mL thioanisole, 0.18 mL anisole, and 0.12
mL ethanedithiol. This was allowed to react for 3 hours at which
time the resin was filtered off. The supernatant was cooled to
0.degree. C. and the peptide was precipitated using cold diethyl
ether. It was collected through centrifugation and then washed
three times using diethyl ether. The pellet was dissolved in 0.6 mL
H.sub.2O and 0.4 mL ACN with 0.3% TFA and purified using HPLC.
[0523] 61. Synthesis of N-Boc-N-Tfa-ethylenediamine.
[0524] To a solution of N-boc-ethylenediamine (5.0 g, 31.2 mmol) in
20 mL THF, ethyl trifluoroacetate (3.72 mL, 31.2 mmol) was added
dropwise and the reaction stirred overnight. The reaction solution
was concentrated to yield a white crystalline product (8.0 g,
100%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.44 (s, 9H,
CH.sub.3), 3.37 (dd, 2H, J=5.4 Hz, J=10.2 Hz, CH.sub.2), 3.46 (dd,
2H, J=5.1 Hz, J=10.4 Hz, CH.sub.2), 5.01 (s, 1H, NH), 7.85 (s, 1H,
NH); .sup.13C NMR (400 MHz, CDCl.sub.3) .delta. 28.2, 39.1, 42.2,
80.6, 140.6, 151.2, 157.7.
[0525] 62. Boc Deprotection of N-Boc-N-Tfa-ethylenediamine.
[0526] N-Boc-N-Tfa-ethylenediamine (8.0 g, 31.5 mmol) was dissolved
in 50 mL formic acid and stirred for 14 h at room temperature.
After the solvent was evaporated under reduced pressure, toluene
was added and concentrated to remove any residual formic acid,
yielding an orange oil (4.90 g, 99.7%). .sup.1H NMR (400 MHz, MeOD)
.delta. 2.31 (s, 2H, NH.sub.2), 3.15 (t, 2H, J=6.1 Hz, CH.sub.2),
3.61 (t, 2H, J=6.1 Hz, CH.sub.2), 8.35 (s, 1H, NH); .sup.13C NMR
(400 MHz, MeOD) .delta. 38.5, 39.7, 113.1, 115.9, 118.8, 121.6,
159.7, 160.1.
[0527] 63. Attachment of N-Tfa-ethylenediamine.
[0528] The deprotected nanoparticles (162.3 mg, 4.58 .mu.mol) in
DriSolv DMF (10.0 mL) were stirred under argon at 0.degree. C. with
N-methylmorpholine (47.8 mg, 472.6 .mu.mol) followed by dropwise
addition of isobutyl chloroformate (71.0 mg, 519.8 .mu.mol) in
DriSolv DMF (0.75 mL). After 1.5 h, a solution of
N-Tfa-ethylenediamine (73.8 mg, 472.6 .mu.mol) in DriSolv DMF (2.5
mL) was added dropwise. The reaction was allowed to warm to room
temperature and stirred overnight. After removal of DMF in vacuo,
the product was dissolved in methanol and dialyzed against methanol
with SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000). .sup.1H
NMR (400 MHz, MeOD) .delta. 7.61-6.45 (br m, aromatic from
crosslinker), 3.15-3.00 (br m, N-Tfa-ethylenediamine), 3.00-2.69
(br m, backbone and N-Tfa-ethylenediamine), 2.69-1.34 (br m,
backbone).
[0529] 64. Deprotection of MAL-dPeg.TM..sub.4-t-boc-hydrazide.
[0530] In a 100 mL round bottomed flask,
MAL-dPeg.TM..sub.4-t-boc-hydrazide (127.1 mg, 239.5 .mu.mol) was
dissolved in 80.0 mL of formic acid and stirred over night at room
temperature. After the solvent was evaporated under reduced
pressure, toluene was added and concentrated to remove any residual
formic acid to give MAL-dPeg.TM..sub.4-hydrazide (103.1 mg,
100%).
[0531] 65. Attachment of MAL-dPeg.TM..sub.4-hydrazide.
[0532] The deprotected nanoparticles (141.1 mg, 3.13 .mu.mol) in
DriSolv DMF (10.0 mL) were stirred under argon at 0.degree. C. with
N-methylmorpholine (17.1 mg, 169.1 .mu.mol) followed by dropwise
addition of isobutyl chloroformate (25.4 mg, 86.0 .mu.mol) in
DriSolv DMF (0.7 mL). After 1.5 h, a solution of
MAL-dPeg.TM..sub.4-hydrazide (103.1 mg, 239.5 .mu.mol) was added
dropwise. The reaction was allowed to warm to room temperature and
stirred overnight. After removal of DMF in vacuo, the product was
dissolved in methanol and dialyzed against methanol with
SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000). .sup.1H NMR
(400 MHz, MeOD) .delta. 7.67-6.47 (br m, aromatic from crosslinker
and maleimide linker), 3.89-3.48 (br t, maleimide linker),
3.21-3.02 (br m, N-Tfa-ethylenediamine), 3.02-2.69 (br m, backbone
and N-Tfa-ethylenediamine), 2.69-1.01 (br m, backbone).
[0533] 66. Hydrogenation of G1.
[0534] A solution of G1 (8.36 g, 5.69 mmol) in ethanol (214 mL) in
a Parr hydrogenation bottle with Raney-Nickel (3.49 g) was shaken
at 65 psi for 3 days at room temperature. Another 1 g of
Raney-Nickel was added to the reaction and it was again shaken at
65 psi for 3 days at room temperature. The reaction was filtered
through Celite, and the removal of the solvent under reduced
pressure gave the crude product. The residue was dissolved in ethyl
acetate and subsequently washed with saturated sodium bicarbonate
solution (2.times., 100 mL) and brine (2.times., 100 mL) then the
organic layer was dried over anhydrous sodium sulfate. The solution
was filtered and concentrated under reduced pressure to yield amine
G1 (8.19 g, 93.7%).
[0535] 67. PDPOH Attachment to G1.
[0536] PDPOH (91.46 mg, 4.25 mmol) in dry THF (100 mL) was stirred
under argon at room temperature with 1-hydrobenzotriazole (HOBt)
(68.90 mg, 5.10 mmol) and DCC (1.05 g, 5.10 mmol). After 1 h, amine
G1 (7.34 g, 5.10 mmol) was added to the solution and the reaction
proceeded for 48 h, after which, it was filtered and concentrated
under reduced pressure. The crude material was purified via flash
column chromatography eluting with 10:1 hexanes:ethyl acetate
increasing to 100% ethyl acetate to give white SS-G1 (4.67 g,
67.1%).
[0537] 68. SSG1 Deprotection Via Formic Acid.
[0538] SSG1 (4.67 g, 2.85 mmol) was dissolved with stirring in
formic acid (100 mL) and the reaction proceeded at room temperature
overnight. Upon completion, the formic acid was removed
azeotropically with toluene under reduced pressure to yield the
product (3.29 g, 100.0%).
[0539] 69. N-Boc-1,6-diaminohexane Attachment to SSG1.
[0540] SSG1OH (3.29 g, 2.91 mmol) in anhydrous THF (100 mL) was
stirred under argon at room temperature with HOBt (4.23 g, 31.25
mmol) and DCC (6.45 g, 31.25 mmol). After one hour,
N-boc-1,6-diaminohexane (6.76 g, 31.25 mmol) was added to the
solution and the reaction proceeded for 48 h at room temperature.
Upon completion, the reaction solution was filtered to remove the
DCC salt and the filtrate concentrated and purified via flash
column chromatography eluting with 1% methanol in dichloromethane
and gradually increasing to 10% methanol in dichloromethane to
yield a white solid (4.42 g, 52.0%).
[0541] The resulting solid was dissolved in 1,4-dioxane (20 mL),
the solution was cooled to 0.degree. C., and 4 M HCl in 1,4-dioxane
(20 mL) was added and the reaction stirred for 24 h at room
temperature. Removal of the solvent under pressure gave a white
solid (3.55 g, 100.0%).
[0542] 70. Attachment of Goodman's Reagent to SSG1LL.
[0543] The resulting SSG1LL HCl salt (3.55 g, 1.51 mol) was
dissolved in methanol (50 mL), and the solution was cooled to
0.degree. C. Triethylamine (TEA) (3.41 mL, 24.56 mmol) was added
followed by N,N'-diboc-N''-triflylguanidine (6.94 g, 17.74 mmol)
and the reaction was stirred 24 h at room temperature. After
removal of the solvent under reduced pressure, the crude product
was purified via flash column chromatography eluting with 1%
methanol in dichloromethane and gradually increasing to 10%
methanol in dichloromethane to yield a white solid (838.2 mg,
13.13%). .sup.1H NMR (300 MHz, MeOD) .delta. 1.33-1.47 (m, 246H,
CH.sub.2, CH.sub.3), 2.03 (d, 48H, J=65.3 Hz, CH.sub.2), 3.15 (td,
30H, J=6.3 Hz, J=12.7 Hz, CH.sub.2), 3.29 (m, 45H, CH.sub.2), 7.42
(m, 1H, ArH), 7.67 (d, 1H, J=8.2 Hz, ArH), 7.79 (d, 1H, J=8.1 Hz,
ArH), 8.02 (s, 1H, ArH).
[0544] 71. Cleavage of Disulfide Bridge on Molecular
Transporter.
[0545] The disulfide linker hexyl molecular transporter (257.8 mg,
61.41 .mu.mol) in DriSolv DMF (5 mL) was stirred under argon and a
solution of DL-dithiothreitol (740.0 mg, 4.80 mmol) in DMF (5 mL)
was added dropwise and the reaction proceeded for 2 h at room
temperature. After removal of DMF in vacuo, the reaction was
purified using a Sephadex LH-20 column, eluting with DMF and
concentrating the fractions in vacuo again yielding the product
(251.0 mg, 100%).
[0546] 72. Attachment of Molecular Transporter to
Nanoparticles.
[0547] The nanoparticles (147.4 mg, 3.07 .mu.mol) in DriSolv DMF
(10.0 mL) were stirred under argon and the free thiol hexyl
molecular transporter (251.0 mg, 61.4 .mu.mol) in DriSolv DMF (10.0
mL) was added dropwise followed by the addition of a catalytic
amount of N-methylmorpholine. After removal of DMF in vacuo, the
product was dissolved in methanol and dialyzed against a 1:1
methanol:water solution, eventually dialyzing against pure methanol
with SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000). .sup.1H
NMR (400 MHz, MeOD) .delta. 7.55-6.21 (br m, aromatic from
crosslinker), 3.85-3.49 (br t, maleimide linker), 3.22-3.00 (br m,
N-Tfa-ethylenediamine and molecular transporter), 3.00-2.70 (br m,
backbone, N-Tfa-ethylenediamine, and molecular transporter),
2.70-1.00 (br m, backbone and molecular transporter).
[0548] 73. Deprotection of Trifluoroacetyl Protected Amines on
Modified Particles.
[0549] The nanoparticles (142.0 mg, 1.54 .mu.mol) were dissolved in
methanol (5.0 mL) and a 10% K2CO3 solution of 5:3 methanol:water
(13.0 mL) was added to the solution and the reaction proceeded
overnight at room temperature. The reaction was purified by
dialysis with SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10 000)
against a 5:3 methanol:water solution, eventually dialyzing against
pure methanol then dialyzing against a 1:1 methanol THF solution,
eventually dialyzing against pure THF. .sup.1H NMR (400 MHz, THF
d8) .delta. 8.26-6.53 (br m, aromatic from crosslinker), 3.94-3.52
(br m, maleimide linker), 3.28-3.12 (br t, ethylenediamine and
molecular transporter), 3.12-2.68 (br m, backbone, ethylenediamine,
and molecular transporter), 2.68-1.05 (br m, backbone and molecular
transporter).
[0550] 74. Attachment of 3-(pyridine-2-yl disulfanyl)propanoic acid
Nanoparticles.
[0551] A solution of 3-(pyridine-2-yl disulfanyl)propanoic acid
(16.8 mg, 77.9 .mu.mol) in anhydrous THF (2.5 mL) was stirred under
argon at 0.degree. C. with N-methylmorpholine (7.88 mg, 77.9
.mu.mol) followed by dropwise addition of isobutyl chloroformate
(11.7 mg, 85.7 .mu.mol). After 1.5 h, a solution of the deprotected
nanoparticles (111.0 mg, 1.30 .mu.mol) in anhydrous THF (35.0 mL)
was added dropwise. The reaction was allowed to warm to room
temperature and stirred for 24 h. The reaction was diluted and
purified by dialysis with SnakeSkin.RTM. Pleated Dialysis Tubing
(MWCO=10,000) against methanol, eventually dialyzing against a 3:1
THF:MeOH solution. .sup.1H NMR (400 MHz, THF d.sub.8) .delta.
7.58-6.22 (br m, aromatic from crosslinking and disulfide linker),
3.87-3.67 (br m, maleimide linker), 3.24-3.16 (br m, disulfide
linker), 3.15-3.04 (br m, diamine and molecular transporter),
2.93-2.83 (br m, diamine and molecular transporter), 2.78-2.62 (br
m, disulfide linker), 2.62-1.06 (br m, backbone).
[0552] 75. Attachment of Alexa Fluor.RTM. 568.
[0553] To a solution of multifunctional nanoparticles (10.0 mg,
106.0 nmol) in DriSolv DMF (3.0 mL), a solution of Alexa Fluor.RTM.
568 (3.78 mg, 4.77 .mu.mol) in anhydrous DMSO (377.7 .mu.L) and
triethylamine (50.0 .mu.L, 358.7 .mu.mol) was added to the solution
and the reaction proceeded in the dark for 24 h at room
temperature. The reaction was diluted with THF and purified by
dialysis with SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000)
against 1% H.sub.2O in THF eventually dialyzing against pure
THF.
[0554] 76. Capping of the Remaining Amines.
[0555] Upon completion of the Alexa Fluor 568 addition to the
nanoparticles, a solution of N-acetoxysuccinimide (47.1 mg, 299.5
.mu.mol) in DriSolv DMF (1.0 mL) was added to the reaction
solution. The reaction was allowed to proceed for 3 h at RT. After
removal of DMF in vacuo, the product was dissolved in methanol and
dialyzed against methanol with SnakeSkin.RTM. Pleated Dialysis
Tubing (MWCO=10,000). .sup.1H NMR (400 MHz, MeOD) .delta. 1.29-2.43
(br m, backbone and molecular transporter), 2.59-2.83 (br m,
disulfide linker), 2.84-2.95 (br m, disulfide linker), 2.98-3.02
(br m, disulfide linker), 3.04-3.09 (br m, disulfide linker), 3.16
(br t, diamine and molecular transporter), 3.67 (br t, maleimide
linker), 6.53-7.98 (br m, aromatic from crosslinking, disulfide
linker, and FITC).
[0556] 77. Boc Deprotection of Modified Nanoparticles.
[0557] Modified nanoparticles (30.0 mg, 434.0 nmol) were dissolved
in anhydrous 1,4-dioxane (10 mL) and chilled to 0.degree. C. A
solution of 4 M HCl in 1,4-dioxane (10 mL) was added dropwise to
the stirring nanoparticles and the reaction was allowed to proceed
overnight at room temperature. The nanoparticle solution was
diluted to three times the original volume with water and dialyzed
against water with SnakeSkin.RTM. Pleated Dialysis Tubing
(MWCO=10,000). Upon completion of dialysis, the aqueous solution
was lyophilized to yield a yellow solid. .sup.1H NMR (400 MHz,
D.sub.2O) .delta. 1.18-2.37 (br m, backbone and molecular
transporter), 2.71-2.79 (br m, disulfide linker), 2.81-2.86 (br m,
disulfide linker), 2.89-2.93 (br m, disulfide linker), 2.94-2.99
(br m, disulfide linker), 3.12 (br t, diamine and molecular
transporter), 3.69 (br t, maleimide linker), 6.53-8.41 (br m,
aromatic from crosslinking, disulfide linker, and FITC).
[0558] 78. Synthesis of Copolymer poly(vl-avl) (Ab).
[0559] A 50 mL 3-necked round bottom flask, equipped with stir bar,
was sealed with two septa and a gas inlet. The flask was evacuated
and refilled with nitrogen three times. Stock solutions of 1.7 M
ethanol (EtOH) in THF and 3.7.times.10.sup.-2M tin(II)
2-ethylhexanoate (Sn(Oct).sub.2) in THF were made in sealed N.sub.2
purged flasks. Solutions of EtOH (0.32 mL, 5.41.times.10.sup.-1
mmol) and Sn(Oct).sub.2 (0.30 mL, 1.12.times.10.sup.-2 mmol) were
combined in the nitrogen purged 50 mL flask. After stirring the
mixture for 30 min, .alpha.-allyl-.delta.-valerolactone (1.16 g,
8.32 mmol) and .delta.-valerolactone (vl, 2.50 g, 24.97 mmol) were
added. The reaction vessel stirred at 105.degree. C. for 48 h.
Residual monomer and catalyst were removed by dialyzing with
Spectra/Por.RTM. dialysis membrane (MWCO=1000) against
CH.sub.2Cl.sub.2 to give a golden brown polymer. Yield: 3.24 g
(88%). M.sub.w=3042 Da, PDI=1.18; .sup.1H NMR (300 MHz,
CDCl.sub.3/TMS, ppm) .delta.: 5.7 (m, H.sub.2C.dbd.CH--), 5.09 (m,
H.sub.2C.dbd.CH--), 4.09 (m, --CH.sub.2--O--), 3.65 (m,
CH.sub.3CH.sub.2O--), 2.35 (m, vl, --CH.sub.2CH.sub.2C(O)O--, avl,
H.sub.2C.dbd.CHCH.sub.2CH--, H.sub.2C.dbd.CHCH.sub.2CH--), 1.68 (m,
avl & vl, --CHCH.sub.2CH.sub.2--), 1.25 (t,
CH.sub.3CH.sub.2O--); .sup.13C NMR (400 MHz, CDCl.sub.3, ppm)
.delta.: 174.6 (avl, --C(O)--), 172.7 (vl, --C(O)--), 134.6
(H.sub.2C.dbd.CH--), 116.4 (H.sub.2C.dbd.CH--), 63.3, 44.3, 35.9,
33.1, 27.5, 25.9, 23.6, 20.9.
[0560] 79. Nanoparticle Formation from Ab.
[0561] A solution of Ab (0.0804 g, M.sub.w=3042 Da, PDI=1.18)
dissolved in CH.sub.2Cl.sub.2 (0.18 mL) was added to a solution of
3,6-dioxa-1,8-octanedithiol (30.0 .mu.L, 0.18 mmol) in
CH.sub.2Cl.sub.2 (28.4 mL) at 44.degree. C. The reaction mixture
was heated for 12 h. Residual dithiol was removed by dialyzing with
SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000) against
dichloromethane. Yield: 0.078 g. .sup.1H NMR (300 MHz,
CDCl.sub.3/TMS) .delta.: The significant change is the reduction of
the allyl protons at 5.06 and 5.77 ppm and the appearance of
signals at 3.65 and 2.71 ppm corresponding to the protons
neighboring the thiols of the PEG linker after cross-linking. All
other aspects of the spectrum are similar to that of Ab. The
reaction can also be conducted with photoinitiators at RT in
organic solvents. The particle sizes of the resulting particles
correspond to those produced in analogous epoxide/amine
procedures.
[0562] When reaction times are increased to from about 24 h to
about 48 h, the particle sizes increase due to the total
consumption of allyl moieties. Reaction at room temperature was
found to be sufficient. Addition of radical starters or other
photoinitiators does not significantly increase the quality of the
particles.
[0563] 80. One Pot Synthesis of Nanoparticles from
poly(vl-evl-avl-opd) (ABbD).
[0564] In a 25 mL three-necked round bottom flask equipped with
stir bar, condenser and septa, 2,2'-(ethylenedioxy)diethylamine
(18.3 .mu.L, 1.25.times.10.sup.-4 mol), 17.1 mL CH.sub.2Cl.sub.2
and a solution of poly(vl-evl-were added. A solution of
poly(vl-evl-avl-opd), ABbD, (0.0781 g, M.sub.w=3500 Da, PDI=1.29).
The mixture was heated at 44.degree. C. for 12 h. Residual diamine
was removed by dialyzing with SnakeSkin.RTM. Pleated Dialysis
Tubing (MWCO=10,000) against dichloromethane. .sup.1H NMR (300 MHz,
CDCl.sub.3/TMS, ppm) .delta.: The significant change is the
disappearance of the epoxide protons at 2.94, 2.75 and 2.47 ppm and
the appearance of signals at 3.64 and 2.97 ppm corresponding to the
protons neighboring the secondary amine of the PEG linker after
cross-linking. All other aspects of the spectrum are similar. TEM
analysis of the resulting nanoparticles is shown in FIG. 41. The
particle size distribution, with unusually narrow polydispersity,
of the resulting nanoparticles is shown in FIG. 43. To increase
particle sizes, reaction times can be increased to from about 24 h
to about 48 h.
[0565] 81. Uptake Experiment Protocol.
[0566] Fluorescent multifunctional nanoparticle, negative control
particle, FD-1, and FD-2 uptake by mammalian cells was assessed
using HeLa cells, cancer cells, grown in uncoated, 14 mm diameter
Microwell, No. 1.5 MatTek Dishes and a Zeiss LSM 510 META confocal
microscope. HeLa cells were grown in Dulbecco's Modified Eagle's
Medium-Low Glucose (DMEM) (Sigma Aldrich) supplemented with 10%
(v/v) fetal bovine serum (Gibco) and 1% (v/v)
antibiotic-antimycotic (Gibco). The cells were treated with the
multifunctional nanoparticles, negative control particles, FD-1, or
FD-2 for one hour, washed three times with Ca.sup.2+/Mg.sup.2+ free
Phosphate Buffered Saline with EDTA (PBS), fixed with 3.3%
paraformaldehyde at room temperature for 10 minutes, and analyzed
using confocal microscopy.
[0567] 82. Bioconjugate Molecular Transporter.
[0568] To prepare an exemplary antibody conjugated molecular
transporter, a G1-Newkome dendrimer that contains nine t-butyl end
functionalities and a primary amine group at the focal point was
prepared using disclosed methods (FIG. 44). The amine functionality
was reacted with 3-(2-pyridinyldithio)propanoic acid via amide
coupling reactions with DCC/HOBt to form a protected dendimer with
a reactive core. The t-butyl ester groups on the periphery of the
dendritic scaffold were deprotected with formic acid to give free
carboxylic acid groups that were coupled with
N-Boc-1,6-diaminohexane. After deprotection of the Boc protecting
groups with 2M HCl in dioxane the free amines were transformed into
guanidine groups with N,N-diBoc-N-triflylguanidine and the
subsequent deprotection of the Boc groups using 2 M HCl in dioxane
gave the desired compound (Scheme 21).
##STR00169## ##STR00170##
[0569] The final compound is designed to localize in the cytoplasm
of the cells, as an integrated a hexyl alkyl spacer is present at
the periphery of the dendrimer scaffold that was previously found
to be a feature for the specificity of its subcellular location.
Furthermore, the pyidinyldithio linker at the focal point allows
for the exchange with sulfhydryl groups to form bioconjugates that
are connected over a disulfide bond to afford a reductive cleavable
linker that can maintain activity of the biomolecule in cells. The
IgG molecular transporter conjugate (IgGMT) was formed by the mild
reaction of Alexa Fluor.RTM. 568 labeled IgG antibody in PBS buffer
at RT with the dendrimer (FIG. 44). Five transporter dendrimers
were attached to the IgG structure which has a molecular weight of
148 kDa. The conjugate was dialyzed against PBS buffer to remove
any unreacted dendrons and the concentration in the dialysis tubing
was chosen to be 1 mg ml.sup.-1 IgGMT, that allowed for the use of
the solution directly for the uptake and neutralization
experiments.
[0570] First, the uptake efficiency of the IgGMT conjugate into
human epithelial cells (HEp-2) was tested. The 60% confluently
grown cells were incubated with IgGMT initially for 10 min then for
30 min, 1, 2 and 6 h. The uptake efficiency was studied with
confocal microscopy and the uptake of the bioconjugate could be
observed as early as 10 min. Later time points showed an increase
of red fluorescence of the IgGMT conjugate, progressing from the
cell surface membrane to localize intracellularly in the
perinuclear area at time points of 2 and 6 h. Contrary to the
affinity and uptake of the IgGMT bioconjugate, the Alexa Fluor.RTM.
568 labeled, unmodified IgG did not enter the cell at all times
points investigated (FIG. 45). After the uptake into HEp-2 cells
was confirmed with no evidence of cellular damage, the activity of
the conjugate in RSV infected cells that expressed green
fluorescent protein GFP as a result of RSV infection was examined.
First, HEp-2 cells were infected for 24 h with recombinant RSV-GFP,
washed and allowed to incubate for an additional 48 h. The cells
were then imaged with confocal microscopy at a total of 72 h after
initial infection (FIG. 46). The typical syncytia formation was
observed, a combination and fusion of the infected cells, together
with the expression of the green fluorescent protein (GFP). To
study the neutralization effect of the IgGMT, HEp-2 cells infected
for 24 h with RSV-GFP were incubated for 30 min with a solution of
IgGMT in PBS buffer, washed and imaged 48 h later (FIGS. 46a and
b). By confocal microscopy it was observed that a significant
reduction of the green fluorescence of GFP (a) and a strong red
fluorescence of the Alexa Fluor.RTM. 568 labeled IgGMT conjugate
(b). The merged images of (a) and (b) combined with differential
interference contrast (DIC) also gave evidence of healthier cells
with significantly less syncytia formation than the untreated
infected cells at the same time period (FIG. 46c). This result
illustrated the significant reduction of GFP in treated cells in
contrast to the untreated cells observed at a total incubation time
of 72 h after infection with RSV for 24 h. Besides the presence of
neutralized cells that showed only the red fluorescence of the
conjugate, cells that showed the coexistence of RSV-GFP and the red
fluorescence of the IgGMT, appearing in the center of the cells
(FIG. 46 and FIG. 47, a+b merged) were also observed. Imaging
directly after the 30 min incubation of the RSV infected cells with
the IgGMT, showed the green fluorescence localized intracellularly,
whereas the red fluorescence was observed on the cell-surface
membranes (FIG. 47, a+b merged). These images documented the high
affinity of the conjugate with the cells surface directly after the
incubation period followed by the uptake into the Hep-2 cells after
an additional incubation time of 48 h. Parallel investigations of
the RSV titres of the supernatant showed a significant reduction by
80-90% in viral replication when compared to cells not exposed to
the IgGMT conjugate. Without wishing to be bound by theory, the
intracellular delivery of IgG antibody directed to the surface
protein inhibits the syncytial formation mediated by the F protein
and has an effect on total virus production when added 24 h after
the initiation of RSV infection.
[0571] 83. Tailored Polyester Nanoparticles.
[0572] In this example, polyester nanoparticles in controlled
nanoscopic dimensions have been prepared through a one-pot
procedure that contains amine, keto, and allyl groups and is
tailored towards the conjugation of bioactive building blocks, such
as a dendritic molecular transporter to facilitate cellular uptake,
or peptides and dyes to accomplish targeting and imaging. In
several examples of bioconjugate synthesis, demonstrated is the
versatility and the orthogonal attachment strategies involving high
yielding thiol-ene reactions under mild conditions and reductive
amination reactions, circumventing the integration of linker and
multi-step post-modification pathways. Several linear nanoparticle
precursors were prepared according to Scheme 22.
##STR00171##
[0573] After the collapse of the above linear precursors using
disclosed methods, the nanoparticle can be functionalized with a
desired moiety. In contrast to reported strategies that form amide
bonds with polyester scaffolds using EDC activation that are
typically not very high yielding and require a high excess of
expensive peptides, in this example the N-terminus of the targeting
unit (e.g., a peptide) was reacted with the keto group, integrated
in the polymer backbone of the developed polyester particle. In a
model reaction, the successful reductive amination of
N-Boc-ethylenediamine with keto groups of the particle has been
shown and applied these reaction conditions to test the addition of
peptidic units. Here, the targeting peptide sequence GCGGGNHVGGSSV
was tested and chosen for the reaction with the ABD nanoparticle,
with the nanoscopic dimension of 118 nm prepared from the ABD
linear precursor polymer with 1.5 equivalents of
2,2'-(ethylenedioxy)bis(ethylamine) cross-linking units with the
conditions as described above (Scheme 22). The amine groups of the
nanoparticle were first capped with N-acetoxysuccinimide and the
modified nanoparticle and the peptide were solubilized in
tetrahydrofuran with NaCNBH3 as the reducing reagent.
[0574] After purification through dialysis the modified particles,
3, were characterized with .sup.1H NMR and DLS. The increase in
hydrodynamic diameter from 118.+-.10 nm to 120.+-.10 nm indicated
the addition of peptides to the polyester backbone and further
investigations with .sup.1H NMR showed the conjugation of peptides
with the characteristic resonance peaks at 4.39 and 7.42 ppm. With
additional analysis through static light scattering (SLS) we could
determine the amount of peptide attached to the nanoparticle that
was estimated to be between 36 of the intended attachment of 40
peptides per particle.
[0575] This result confirmed the efficiency of the reductive
amination reactions with the N-terminus of the selected peptidic
units. Targeting units, however, that contain more than one amine
group give mixed conjugation products and an alternative strategy
has to be developed. For this reason, we wanted to pursue thiol-ene
type reactions that will be performed between cysteine units,
integrated into the sequence close to the N-terminus, and double
bonds that we find in maleiimides, vinylsulfones or allyl groups.
To integrate the reaction partner for the thiol/cysteine containing
units, such as peptides, or oligonucleotides, into the
nanoparticles, either a suitable linker that would be attached to
the prepared nanoparticle was synthesized or a method that would
circumvent the conjugation of a linker molecule to facilitate the
attachment of thiol containing entities was found. Therefore, the
integration of allyl groups in the polyester backbone as pendant
functional units that would be already present in the linear
polyester precursor before nanoparticle formation was studied. The
available allyl groups that stem from the
.alpha.-allyl-.delta.-valerolactone of the linear polyester
precursors were oxidized and converted entirely into epoxide groups
to provide units that would cross-link with the diamine. However,
with partial oxidation of the allyl group, linear polyester
precursors containing epoxide units and remaining allyl groups,
could be accomplished. In the next step, a linear polyester AbD
that was partially oxidized to comprise 16% of allyl units and 11%
of epoxide units was cross-linked with 1.5 equivalents of diamine,
using the novel one-pot reaction procedure to examine the
compatibility of the allyl groups to the conditions of nanoparticle
formation. The investigation of the resulting particles with DLS
showed that hydrodynamic diameters corresponded to the size and
solubility of the particles that did not contain any allyl groups.
The allyl resonance peaks were still present in the .sup.1H NMR
spectra of the particles and were found to be analogous to the
resonances of the allyl functionalities in the linear
precursor.
[0576] After attaching a fluorescent probe, a disclosed cyclic
peptide was attached, as shown in Scheme 23.
##STR00172##
[0577] In the next step, a combined dendritic, peptidic,
nanoparticle scaffold was synthesized according to Scheme 24.
##STR00173##
[0578] For the first approach, linear peptides GCGGGNHVGGSSV with
the recognition unit HVGGSSV with protected amines after capping
with N-acetoxysuccinimide, were conjugated to the allyl
functionality of a ABbD nanoparticle of 126.6 nm through the thiol
of the cysteine unit as discussed above. In a following reaction,
the imaging reagent Alexa Fluor.RTM.594 was introduced to label
around 20 of the incorporated amine units of the nanoparticle. In a
sequential thiolene reaction, the conjugation of 30 dendritic
transporter molecules was achieved (Scheme 7), as was confirmed via
.sup.1H NMR spectroscopy. The sequential conjugation of the
bioactive compounds can be followed with an overlay of the .sup.1H
NMR spectra that show the addition of first the peptide and the
remaining allyl groups of the nanoparticle and the characteristic
peaks of the molecular transporter molecule at 2.0 and 3.2 ppm.
[0579] The reaction sequence was changed to obtain a similar
bioconjugate product that was only differentiated by the peptidic
targeting unit. The amine groups of the c-RGD unit were not capped
to avoid inactivation of the Arginine.RTM. recognition unit.
Therefore the conjugation strategy included that the amine groups
of the nanoparticle were first labeled with the NHS Alex Fluor dye
followed by the thiol-ene reaction with the targeting unit as shown
in Scheme 6. In the last step, same as in the previous reaction,
the dendritic transporter unit was added in a sequential thiol-ene
reaction (Scheme 25).
##STR00174##
[0580] In a third and last reaction sequence, we could demonstrate
the versatility of the provided functional units of the
nanoparticle and proceeded with an orthogonal conjugation approach.
The free amine groups of the nanoparticle are capped with
N-acetoxysuccinimide to not interfere with the following reductive
amination reaction between the keto group of the polyester backbone
and the N-terminus of the unmodified targeting peptide HVGGSSV.
After the reductive amination reaction was completed in the same
fashion as described for compound 3, a thiolene reaction between
the allyl groups of the nanoparticle and the thiol group of the
molecular transporter could achieve the attachment of 30 units
according to 111 NMR spectroscopy analysis. The additional final
characterization of the modified particles with static light
scattering (SLS) the number of conjugated peptides peptides could
determine the addition of 36 peptides to the particle. In a last
step, the NHS ester Alexa Fluor dye was modified with thiol
ethylamine (Scheme 26) to label exclusively the particle through a
thiol-ene reaction to image the system in vitro. The Alexa Fluor
594 dye proved to be stable under the conditions and another
example of the chemical versatility of the system was given.
##STR00175##
TABLE-US-00005 TABLE 6 Summary of nanoparticle conjugates with
definition of particle type depending on linear polymer precursora
and connected targeting peptideb: `c` for capped N-terminus of
peptide with HVGGSSV recognition unit via N-acetoxysuccinimide and
`c` for cyclic RGD. c Dendritic molecular transporter is
abbreviated as MT, and the compound name is given in the order of
the attachment d. Alexa Dendritic Particle Targeting Fluor .RTM.
Molecular Compound Type.sup.a Peptides.sup.b Dye Transporter.sup.c
Compound Name.sup.d Class ABD HVGGSSV -- -- ABD-NP-HVGGSSV (3) NP-P
ABbD HVGGSSV -- -- ABbD-NP-HVGGSSV (14) NP-P ABbD cHVGGSSV 594 --
ABbD-NP-cHVGGSSV-594 (8) NP-P-dye ABbD cRGD 594 -- ABbD-NP-594-cRGD
(10) NP-P-dye ABbD -- 594 MT ABbD-NP-594-MT (6) NP-MT-dye ABbD
cHVGGSSV 594 MT ABbD-NP-cHVGGSSV-594-MT (11) NP-P-MT-dye ABbD cRGD
594 MT ABbD-NP-594-cRGD-MT (12) NP-P-MT-dye ABbD HVGGSSV 594 MT
ABbD-NP-594-MT (16) NP-P-MT-dye
[0581] Below are the experimental procedures relevant to Example
123.
[0582] Synthesis of Copolymer poly(vl-avl-opd) (AbD).
[0583] To a 25 mL 3-necked round bottom flask, equipped with stir
bar, gas inlet and 2 rubber septa, 2-oxepane-1,5-dione (0.70 g,
5.46 mmol) was added. The round bottom flask was purged with argon.
After purging for 30 min, dry toluene (4 mL) was added. The mixture
stirred in an oil bath at 80.degree. C. to dissolve the monomer.
Upon dissolving, Sn(Oct).sub.2 (11.1 mg, 27.3 .mu.mol) in 0.5 mL
dry toluene, absolute ethanol (20.5 mg, 440 .mu.mol),
.alpha.-allyl-.delta.-valerolactone (1.15 g, 8.19 mmol) and
.delta.-valerolactone (1.37 g, 13.7 mmol) were then added to the
reactor and the mixture was heated for 48 h at 105.degree. C.
Residual monomer and catalyst were removed by dialyzing with
Spectra/Por.RTM. dialysis membrane (MWCO=1000) against CH2Cl2 to
give a golden brown polymer. Yield: 2.70 g (85%). Mw=3287 Da,
PDI=1.17; .sup.1H NMR (300 MHz, CDCl.sub.3/TMS, ppm) .delta.: 5.72
(m, H2C.dbd.CH--), 5.06 (m, H2C.dbd.CH--), 4.34 (m,
--CH2CH2C(O)CH2CH2O--), 4.08 (m, --CH2O--), 3.67 (m, --OCH2CH3),
2.78 (m, opd, --OC(O)CH2CH2C(O)CH2-), 2.58 (m, opd,
--OC(O)CH2CH2C(O)CH2-), 2.34 (m, vl, --CH2CH2C(O)O--, avl,
H2C.dbd.CHCH2CH--, H2C.dbd.CHCH2CH--), 1.66 (m, avl & vl,
--CHCH2CH2-), 1.25 (t, --CH2CH3); .sup.13C NMR (400 MHz,
CDCl.sub.3, ppm) .delta.: 204.9, 175.2, 173.7, 173.2, 135.0, 117.0,
63.9, 44.8, 36.4, 33.6, 28.0, 26.3, 21.3.
[0584] Synthesis of poly(vl-evl-opd) (ABD).
[0585] To a solution of AbD (2.70 g, 4.67 mmol) in CH.sub.2Cl.sub.2
(37 mL), 3-chloroperoxybenzoic acid (1.46 g, 8.48 mmol) was added.
The mixture stirred for 72 h at room temperature and then
concentrated via rotary evaporator. The crude product was dissolved
in a minimal amount of tetrahydrofuran (THF) (5 mL) and dropped
into a round bottom flask containing 1 L diethyl ether. The
solution was kept overnight at 0.degree. C. and a white solid was
obtained. The solution was decanted off and the solid was dried in
vacuo to obtain ABD. Yield: 1.95 g (72%). Mw=3392 Da, PDI=1.19.
.sup.1H NMR (300 MHz, CDCl.sub.3/TMS) .delta.: The significant
change is the disappearance of the allylic protons at 5.74 and 5.09
ppm and the appearance of small broad resonance peaks at 2.94, 2.75
and 2.47 ppm due to the formation of the epoxide ring. All other
aspects of the spectrum are similar.
[0586] Nanoparticle Formation from Poly(vl-evl-opd) (ABD).
[0587] A solution of ABD (0.11 g, Mw=3392 Da, PDI=1.19) dissolved
in CH.sub.2Cl.sub.2 (0.26 mL) was added dropwise via a peristaltic
pump at 13 mL/min with vigorous stirring to a solution of
2,2'-(ethylenedioxy)diethylamine (76.4 .mu.L, 0.52 .mu.mol) in
CH.sub.2Cl.sub.2 (40.3 mL) at 44.degree. C. The mixture was heated
for 12 h. Residual diamine was removed by dialyzing with
SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000) against
dichloromethane. Yield: 0.17 g (91%). DLS: DH=118.3.+-.9.6 nm. SLS:
Mw=323,000. .sup.1H NMR (300 MHz, CDCl.sub.3/TMS) .delta.: The
significant change is the disappearance of the epoxide protons at
2.94, 2.75 and 2.47 ppm and the appearance of signals at 3.54 and
2.97 ppm corresponding to the protons neighboring the secondary
amine of the PEG linker after cross-linking. All other aspects of
the spectrum are similar.
[0588] N-Boc-Ethylenediamine (NBED) Conjugated ABD
Nanoparticles.
[0589] To a solution of ABD nanoparticles (20 mg, 0.06 .mu.mol) in
THF (2 mL), N-acetoxysuccinimide (0.02 g, 0.13 mmol) was added. The
reaction mixture stirred for 3 h. Residual N-acetoxysuccinimide was
removed by dialyzing with SnakeSkin.RTM. Pleated Dialysis Tubing
(MWCO=10,000) against THF. Once the product was concentrated and
dried, the nanoparticles (18 mg, 0.05 .mu.mol) were dissolved in a
mixture of CH.sub.2Cl.sub.2 and CH3OH (1:1, v/v, 2 mL). To this
solution, N-Boc-ethylenediamine (4.6 .mu.L of 1.59 M NBED in
CH.sub.3OH) and NaCNBH3 (21.8 .mu.L of 1.0 M NaCNBH3 in THF) were
added. The reaction mixture stirred for 12 h at room temperature
and then was purified by dialyzing with SnakeSkin.RTM. Pleated
Dialysis Tubing (MWCO=10,000) against 1:1
CH.sub.2Cl.sub.2/CH.sub.3OH. Yield: 18 mg (88%). DLS:
DH=119.5.+-.10.3 nm; original particle DH=118.3.+-.9.6 nm. .sup.1H
NMR (300 MHz, CDCl.sub.3/TMS): The significant change is the
appearance of the peak at 1.43 ppm due to the Boc protecting group.
All other aspects of the spectrum are similar to that of the ABD
nanoparticles.
[0590] General Procedures for the Synthesis of HVGGSSV Peptide
(1).
[0591] The HVGGSSV peptide was synthesized by solid-phase peptide
synthesis using standard Fmoc chemistry on a Model 90 Peptide
Synthesizer (Advanced ChemTech). General procedure: Attachment of
N-Fmoc amino acids to resin. After swelling with dichloromethane
(20 mL) for 20 min, H-val-2-Cl-Trt resin (0.20 g, 1.03 mmol/g, 0.21
mmol surface amino acids) was treated with a solution of
Fmoc-protected amino acids (4.4 equiv, 0.9 mmol) in
dimethylformamide (DMF) (9 mL). The amino acids were attached to
the resin using double coupling with a solution (9 mL) consisting
of N-hydroxybenzotriazole monohydrate (HOBt) (0.9 mmol, 0.14 g)
o-(benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
(0.9 mmol, 0.34 g), N,N'-diisopropylethylamine (DIPEA) (1.8 mmol,
0.31 mL) in 9 mL DMF. The reaction mixture was shaken for 60 min
and washed with DMF (4.times.10 mL), methanol (4.times.10 mL) and
DMF (4.times.10 mL). The end of the coupling was controlled by the
Ninhydrin test. A 20% (v/v) piperidine in DMF solution was used to
deprotect the Fmoc groups. The amino acids were attached to the
resin in the following sequence: Ser, Ser, Gly, Gly, Val, His, Asn,
Gly, Gly, Gly, Cys, and Gly.
[0592] General Procedure: Cleavage from Resin.
[0593] The resin was treated with Reagent R, a solution of TFA,
thioanisole, anisole, and ethanedithiol (90:5:3:2, 6 mL), for 4 h.
After removal of the resin by filtration, the filtrate was
concentrated to precipitate the peptide with cold diethyl ether.
Crude peptides were purified by RP-HPLC and lyophilized. Peptide
identity was confirmed by MALDI-MS (m/z: 1087.1).
[0594] HVGGSSV Conjugated ABD Nanoparticles (3).
[0595] To a solution of ABD nanoparticles (20.0 mg, 0.06 .mu.mol)
in THF (2 mL), N-acetoxysuccinimide (3 mg, 18.1 .mu.mol) was added.
The reaction mixture stirred for 3 h. Residual N-acetoxysuccinimide
was removed by dialyzing with SnakeSkin.RTM. Pleated Dialysis
Tubing (MWCO=10,000) against 1:1 THF/CH.sub.3OH to give amine
capped ABD nanoparticles, 2. To a solution of 2 (0.0174 g, 0.05
.mu.mol, in 3 mL THF), 1 (3.5 mg, 3.18 .mu.mol) dissolved in DMSO
(2 mL) and NaCNBH3 (6.36 .mu.L 1.0 M NaCNBH3 in THF) were added.
The reaction mixture stirred for 12 h at room temperature. The
reaction mixture was purified by dialyzing with SnakeSkin.RTM.
Pleated Dialysis Tubing (MWCO=10,000) against 1:1 THF/CH3CN. Yield:
19 mg (88%) DLS: DH=120.5.+-.10.2 nm; original particle
DH=118.3.+-.9.6 nm. SLS: Mw=362,000; original particle Mw=323,000.
.sup.1H NMR (600 MHz, (CD3)2SO) .delta.: The significant change is
the appearance of the following peaks: 8.26-7.87, 7.42, 6.90, 4.39,
and 4.25 ppm due to the attachment of the peptide. All other
aspects of the spectrum are similar to that of the ABD
nanoparticles.
[0596] Synthesis of poly(vl-evl-avl-opd) (ABbD).
[0597] To a solution of AbD (1.70 g, 1.56 mmol) in CH.sub.2Cl.sub.2
(30 mL), 3-chloroperoxybenzoic acid (0.22 g, 1.28 mmol) was added.
The mixture stirred for 72 h at room temperature and then was
concentrated via rotary evaporator. The crude product was dissolved
in a minimal amount of THF (5 mL) and poured into a round bottom
flask containing 1 L diethyl ether. The solution was kept overnight
at 0 C and a white solid was obtained. The solution was decanted
off and the solid was dried in vacuo to obtain ABbD. Yield: 1.2 g
(71%). Mw=3356 Da, PDI=1.18. 1H NMR (300 MHz, CDCl.sub.3/TMS, ppm)
.delta.: 5.72 (m, H2C.dbd.CH--), 5.06 (m, H2C.dbd.CH--), 4.34 (m,
--CH2CH2C(O)CH2CH2O--), 4.08 (m, --CH2O--), 3.67 (m, --OCH2CH3),
2.96 (m, epoxide proton), 2.78 (m, evl epoxide proton, opd,
--OC(O)CH2CH2C(O)CH2-), 2.58 (m, opd, --OC(O)CH2CH2C(O)CH2-), 2.47
(epoxide proton), 2.34 (m, vl, --CH2CH2C(O)O--, avl,
H2C.dbd.CHCH2CH--, H2C.dbd.CHCH2CH--), 1.66 (m, avl & vl,
--CHCH2CH2-), 1.25 (t, --CH2CH3).
[0598] Nanoparticle Formation from ABbD.
[0599] A solution of ABbD (0.21 g, Mw=3356 Da, PDI=1.18) dissolved
in CH.sub.2Cl.sub.2 (0.39 mL) was added dropwise via a peristaltic
pump at 13 mL/min with vigorous stirring to a solution of
2,2'-(ethylenedioxy)diethylamine (42.6 .mu.L, 0.29 mmol) in CH2Cl2
(60 mL) at 44.degree. C. The reaction mixture was heated for 12 h.
Residual diamine was removed by dialyzing with SnakeSkin.RTM.
Pleated Dialysis Tubing (MWCO=10,000) against dichloromethane.
Yield: 0.24 g (96%). DLS: DH=123.4.+-.9.22 nm. SLS: Mw=345,000.
.sup.1H NMR (300 MHz, CDCl.sub.3/TMS) .delta.: The significant
change is the disappearance of the epoxide protons at 2.96, 2.75
and 2.47 ppm and the appearance of signals at 3.56 and 2.98 ppm
corresponding to the protons neighboring the secondary amine of the
PEG linker after crosslinking. All other aspects of the spectrum
are similar to that of ABbD.
[0600] One Pot Synthesis of Nanoparticles from ABbD.
[0601] To a solution of 2,2'(ethylenedioxy)diethylamine (26.2
.mu.L, 0.18 mmol) in CH.sub.2Cl.sub.2 (34.6 mL), a solution of ABbD
(0.13 g, Mw=3356 Da, PDI=1.18) in CH.sub.2Cl.sub.2 (0.24 mL) was
added. The mixture was heated at 44.degree. C. for 12 h. Residual
diamine was removed by dialyzing with SnakeSkin.RTM.Pleated
Dialysis Tubing (MWCO=10,000) against CH2Cl2. Yield: 0.15 g (94%).
DLS: DH=126.6.+-.9.3 nm. SLS: Mw=350,000. 1H NMR (300 MHz,
CDCl3/TMS) .delta.: The significant change is the disappearance of
the epoxide protons at 2.94, 2.75 and 2.47 ppm and the appearance
of signals at 3.54 and 2.97 ppm corresponding to the protons
neighboring the secondary amine of the PEG linker after
cross-linking. All other aspects of the spectrum are similar to
that of ABbD.
[0602] General Procedure for the Attachment of Benzyl Mercaptan to
ABbD Nanoparticles.
[0603] To a solution of ABbD nanoparticles (15 mg, 0.04 .mu.mol) in
toluene (0.5 mL), benzyl mercaptan (3.5 .mu.L, 29 .mu.mol) was
added. The reaction mixture was heated for 72 h at 35.degree. C.
The remaining toluene was removed in vacuo and residual benzyl
mercaptan was removed by dialyzing with SnakeSkin.RTM. Pleated
Dialysis Tubing (MWCO=10,000) against CH2Cl2. 1H NMR (300 MHz,
CDCl.sub.3/TMS) .delta.: The significant change is the reduction of
the allyl protons at 5.72 and 5.06 ppm and the appearance of
signals at 3.73 and 7.30 ppm corresponding to the methylene and
benzene protons respectively of the attached benzyl mercaptan. All
other aspects of the spectrum are similar to that of ABbD
nanoparticles.
[0604] Deprotection of molecular transporter (MT) (5) (contribution
of Sharon Hamilton). To a solution of LL-MT (15 mg, 4.56 .mu.mol)
in CH.sub.3OH (0.4 mL), a solution of D,L-dithiothreitol in
CH.sub.3OH (0.2 mL) was added. The reaction mixture stirred for 3 h
at room temperature. Residual dithiothreitol was removed by
purification with Sephadex LH-20. The product was immediately
attached to ABbD nanoparticles.
[0605] Model reaction of attachment of MT to ABbD nanoparticles. To
a solution of ABbD nanoparticles (15 mg, 0.04 .mu.mol) in
CH.sub.3OH (0.2 mL), 5 (11 mg, 3.35 .mu.mol) in CH.sub.3OH (0.4 mL)
was added. The reaction mixture was heated for 72 h at 37.degree.
C. Residual 5 was removed by dialyzing with SnakeSkin.RTM. Pleated
Dialysis Tubing (MWCO=10,000) against methanol. Yield: 31.3 mg
(89%). DLS: DH=128.9.+-.10.2 nm; original particle DH=126.6.+-.9.3
nm. .sup.1H NMR (300 MHz, CD3OD) .delta.: The significant change is
the reduction of the allyl protons at 5.72 and 5.06 ppm and the
appearance of signals at 2.20-1.98 (CH2), 1.57 (CH2) and 1.39 (CH2)
ppm due to the dendritic backbone of the MT. All other aspects of
the spectrum are similar to that of ABbD nanoparticles.
[0606] Alexa Fluor.RTM. 594 conjugated ABbD nanoparticles (4). To a
solution of ABbD nanoparticles (0.021 g, 0.06 mmol) in dry THF (1.5
mL), Alexa Fluor.RTM. 594 (0.14 mL of 10 mg/mL Alexa Fluor.RTM. 594
in DMF, 1.7 mop was added. The reaction mixture stirred for 24 h at
room temperature. Residual Alexa Fluor.RTM. 594 was removed by
dialyzing with SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000)
against CH.sub.3OH. Yield: 15.2 mg (88%). 1H NMR (300 MHz, CD3OD)
.delta.: The significant change is the appearance of the following
peaks due to Alexa Fluor.RTM. 594: 7.14-7.20, 6.78, 5.48, 4.48,
3.62, 3.43, and 1.24 ppm. 1H NMR (600 MHz, (CD3)2SO) .delta.: The
significant change is the appearance of the following peaks due to
Alexa Fluor.RTM. 594: 7.52, 7.47, 7.08, 5.32, 4.44, 4.35, 3.58,
3.16, 2.03, and 1.25 ppm. All other aspects of the spectrum are
similar to that of ABbD nanoparticles.
[0607] Attachment of MT to Alexa Fluor.RTM. 594 Conjugated ABbD
Nanoparticles, NP-594-MT (6).
[0608] To a solution of 4 (8 mg, 0.89 .mu.mol) in CH.sub.3OH (0.2
mL), 5 (7.5 mg, 2.27 .mu.mol) in CH.sub.3OH (0.4 mL) was added. The
reaction mixture was heated for 72 h at 37.degree. C. Residual 5
was removed by dialyzing with SnakeSkin.RTM. Pleated Dialysis
Tubing (MWCO=10,000) against CH3OH. Yield: 10.0 mg (91%). DLS:
DH=129.4.+-.9.8 nm; original particle DH=126.6.+-.9.3 nm. 1H NMR
(300 MHz, CD3OD) .delta.: The significant change is the reduction
of the allyl protons at 5.72 and 5.06 ppm and the appearance of
signals at 2.20-1.98 (CH2), 1.57 (CH2) and 1.39 (CH2) ppm due to
the dendritic backbone of the MT. All other aspects of the spectrum
are similar to that of 4.
[0609] N-Acetoxysuccinimide Conjugated HVGGSSV Peptide, cHVGGSSV
(7).
[0610] To a solution of 1 (29.4 mg, 2.7.times.10.sup.-5 mol)
dissolved in CH3CN (3 mL), N-acetoxysuccinimide (0.42 g,
2.7.times.10.sup.-3 mol) was added. The reaction mixture stirred
for 3 h at room temperature. After removal of the solvent under
reduced pressure, the crude product was purified by RP-HPLC.
MALDI-MS: m/z=(M+H+) 1174.2.
[0611] Capped HVGGSSV Conjugated Alexa Fluor.RTM. 594-ABbD
Nanoparticles, NP-cHVGGSSV-594.
[0612] To a solution of ABbD nanoparticles (0.021 g, 0.06 mmol) in
dimethylsulfoxide (0.7 mL), 7 (6.4 mg, 5.46 mmol) was added. The
reaction mixture was heated for 72 h at 33.degree. C. To this
solution, Alexa Fluor.RTM. 594 (0.14 mL of 10 mg/mL Alexa
Fluor.RTM. 594 in DMF, 1.7 .mu.mol) was added. Residual Alexa
Fluor.RTM. 594 and peptide were removed by dialyzing with
SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000) against 1:1
CH.sub.3OH/CH3CN. Yield: 20.1 mg (80%). DLS: DH=128.9.+-.10.9 nm;
original particle DH=126.6.+-.9.3 nm. 1H NMR (600 MHz, (CD3)2SO)
.delta.: The significant change is the reduction of the allyl
protons at 5.72 and 4.97 ppm and the appearance of the following
sets of significant signals: 8.21, 7.83, 4.55, 3.73 and 0.80 ppm
due to the peptide, and 7.25, 7.16, 6.53, 5.32, 4.44, 4.37, and
1.25 ppm due to the Alexa Fluor.RTM. 594. All other aspects of the
spectrum are similar to that of ABbD nanoparticles.
[0613] Attachment of MT to cHVGGSSV Conjugated Alexa Fluor.RTM.
594-ABbD Nanoparticles, NP-cHVGGSSV-594-MT.
[0614] To a solution of 8 (6 mg, 0.02 .mu.mol) in DMSO (0.1 mL), 5
(2 mg, 0.88 .mu.mol) in CH.sub.3OH (0.3 mL) was added. The reaction
mixture was heated for 48 h at 33.degree. C. Residual 5 was removed
by dialyzing with SnakeSkin.RTM. Pleated Dialysis Tubing
(MWCO=10,000) against 1:1 CH3OH/CH3CN. Yield: 7.4 mg (93%). DLS:
DH=130.7.+-.9.4 nm; original particle DH=126.6.+-.9.3 nm. 1H NMR
(600 MHz, (CD3)2SO) .delta.: The significant change is the
reduction of the allyl protons at 5.72 and 4.97 ppm and the
appearance of signals at 3.06 (CH2), 2.96 (CH2), 1.97 (CH2), 1.77
(CH2), 1.41 (CH2) and 1.35 (CH2) ppm due to the dendritic backbone
of the MT. All other aspects of the spectrum are similar to that of
8.
[0615] Synthesis of Cyclic RGD, cRGD (9)
[0616] The RGD peptide was synthesized by solid-phase peptide
synthesis using standard Fmoc chemistry on a Model 90 Peptide
Synthesizer (Advanced ChemTech).
[0617] Synthesis of Linear RGD.
[0618] After swelling with dichloromethane (20 mL),
Fmoc-Cys-2-Cl-Trt resin (0.20 g, 0.9 mmol/g, 0.18 mmol surface
amino acids) was deprotected with a 20% (v/v) piperidine in DMF
solution and treated with a solution of Fmoc-protected amino acid
(4.4 equiv, 0.9 mmol) in dimethylformamide (DMF) (9 mL). The amino
acids were attached to the resin using double coupling with a
solution (9 mL) consisting of N-hydroxybenzotriazole monohydrate
(0.9 mmol, 0.14 g) o-(benzotriazole-N,N,N',N'-tetramethyluronium
hexafluorophosphate (0.9 mmol, 0.34 g), N,N'-diisopropylethylamine
(1.8 mmol, 0.31 mL) in 9 mL DMF. The reaction mixture was shaken
for 60 min and washed with DMF (4.times.10 mL), methanol
(4.times.10 mL) and DMF (4.times.10 mL). A 20% (v/v) piperidine in
DMF solution was used to deprotect the Fmoc groups. An amino-ahexyl
spacer was coupled to the cystine on the resin, followed by
glutamic acid, aspartic acid, glycine, arginine, phenylalanine, and
finally lysine.
[0619] Cyclization of RGD.
[0620] The peptide was cyclized by utilizing an ODmab group, which
allows for the selective deprotection carboxylic acid side chain of
the glutamic acid, which can then be coupled to the N-terminus. The
ODmab was deprotected using 2% v/v hydrazine monohydrate/DMF added
to the resin and shaken for 7 min. Next it was washed with 20 mL of
DMF followed by 10 mL of a 5% v/v DIPEA/DMF solution which was
allowed to shake for 10 min. Carboxy activation was achieved
through the use of N,N'-dicyclohexylcarboimide (DCC) (44.6 mg, 0.22
mmol) and hydroxybenzotriazole (HOBt) (29.2 mg, 0.22 mmol) which
was added to 10 mL of DMF and then added to the resin and allowed
to shake for 18 h.
[0621] General Procedure: Cleavage from Resin.
[0622] The resin was treated with Reagent R, a solution of TFA,
thioanisole, anisole, and ethanedithiol (90:5:3:2, 6 mL), for 3 h.
After removal of the resin by filtration, the filtrate was
concentrated to precipitate the peptide with cold diethyl ether.
The crude peptide was collected by centrifugation, purified by
RP-HPLC and lyophilized. Peptide identity was confirmed by MALDI-MS
(m/z: 945).
[0623] Attachment of cRGD to Alexa Fluor.RTM. 594 Conjugated ABbD
Nanoparticles, NP-594-cRGD (10).
[0624] To a solution of ABbD nanoparticles (23.0 mg, 0.07 .mu.mol)
in THF (2.3 mL), Alexa Fluor.RTM. 594 (0.15 mL of 10 mg/mL Alexa
Fluor.RTM. 594 in DMF, 1.83 .mu.mol) was added. After stirring the
reaction mixture for 24 h at room temperature, the solvent was
removed via rotary evaporator. To the Alexa Fluor.RTM. 594
conjugated nanoparticles, methanol (0.35 mL) and 9 (5.7 mg, 6.0
.mu.mol), dissolved in DMSO (0.35 mL), were added. The reaction
mixture was heated for 72 h at 33.degree. C. Residual Alexa
Fluor.RTM. 594 and peptide were removed by dialyzing with
SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000) against 1:1
CH3OH/CH3CN. Yield: 22.0 mg (81%). DLS: DH=129.8.+-.9.6 nm;
original particle DH=126.6.+-.9.3 nm. 1H NMR (600 MHz, (CD3)2SO)
.delta.: The significant change is the reduction of the allyl
protons at 5.72 and 4.97 ppm and the appearance of the following
sets of significant signals: 7.37, 4.79, 2.23 and 1.66 ppm due to
cRGD, and 7.25, 6.55, 5.31, 4.44, and 1.23 ppm due to the Alexa
Fluor.RTM. 594. All other aspects of the spectrum are similar to
that of ABbD nanoparticles.
[0625] Attachment of MT to cRGD Conjugated Alexa Fluor.RTM.
594-ABbD Nanoparticles, NP-594-cRGD-MT (12).
[0626] To a solution of 10 (7.8 mg, 0.02 .mu.mol) in DMSO (0.1 mL),
5 (1.4 mg, 0.67 mop in CH3OH (0.3 mL) was added. The reaction
mixture was heated for 48 h at 33.degree. C. Residual 5 was removed
by dialyzing with SnakeSkin.RTM. Pleated Dialysis Tubing
(MWCO=10,000) against 1:1 CH3OH/CH3CN. Yield: 7.6 mg (83%). DLS:
DH=131.9.+-.10.6 nm; original particle DH=126.6.+-.9.3 nm. .sup.1H
NMR (600 MHz, (CD3)2SO) .delta.: The significant change is the
reduction of the allyl protons at 5.72 and 4.97 ppm and the
appearance of signals at 3.04 (CH2), 2.98 (CH2), 1.98 (CH2), 1.75
(CH2), 1.41 (CH2), and 1.35 (CH2) ppm due to the dendritic backbone
of the MT. All other aspects of the spectrum are similar to that of
11.
[0627] HVGGSSV Conjugated ABbD Nanoparticles, NP-HVGGSSV (14).
[0628] To a solution of ABbD nanoparticles (50.0 mg, 0.14 mol) in
THF (2 mL), N-acetoxysuccinimide (7 mg, 44.5 .mu.mol) was added.
The reaction mixture stirred for 3 h. Residual N-acetoxysuccinimide
was removed by dialyzing with SnakeSkin.RTM. Pleated Dialysis
Tubing (MWCO=10,000) against 1:1 THF/CH.sub.3OH to give amine
capped ABbD nanoparticles, 13. To a solution of 13 (50.0 mg, 0.14
.mu.mol, in 3 mL THF), 1 (9.3 mg, 8.57 .mu.mol) dissolved in DMSO
(2 mL) and NaCNBH3 (17.1 .mu.L 1.0 M NaCNBH3 in THF) were added.
The reaction mixture stirred for 12 h at room temperature. The
reaction mixture was purified by dialyzing with SnakeSkin.RTM.
Pleated Dialysis Tubing (MWCO=10,000) against 1:1 THF/CH3CN. Yield:
43.2 mg (83%). DLS: DH=129.7.+-.9.5 nm; original particle
DH=126.6.+-.9.3 nm. SLS: Mw=391,000; original particle Mw=350,000.
.sup.1H NMR (600 MHz, (CD3)2SO, ppm) .delta.: The significant
change is the appearance of the following peaks: 8.21, 7.85, 4.55,
3.73 and 0.80 ppm due to the peptide. All other aspects of the
spectrum are similar to that of ABbD nanoparticles.
[0629] Thiolated Alexa Fluor.RTM. 594 (15).
[0630] To a solution of Alexa Fluor.RTM. 594 (0.2 mL of 10 mg/mL
Alexa Fluor.RTM. 594 in DMF, 2.4 .mu.mol), cystemaine (68.4 .mu.L
of 2.5 mg/mL cysteamine in DMSO, 2.2 .mu.mol) was added. The
reaction mixture stirred for 3 h at room temperature. The product
was immediately attached to 14.
[0631] Attachment of MT to HVGGSSV conjugated Alexa Fluor.RTM.
594-ABbD nanoparticles, NPHVGGSSV-594-MT (16). To a solution of 14
(16 mg, 0.04 .mu.mol) in DMSO (0.2 mL), 15 (2 mg, 1.95 .mu.mol) in
DMSO (0.2 mL) and 5 (2.7 mg, 1.2 .mu.mol) in CH3OH (0.4 mL) were
added. The reaction mixture was heated for 48 h at 33.degree. C.
Residual 5 and 15 were removed by dialyzing with SnakeSkin.RTM.
Pleated Dialysis Tubing (MWCO=10,000) against CH.sub.3OH. Yield:
18.5 mg (86%). DLS: DH=132.1.+-.9.3 nm; original particle
DH=126.6.+-.9.3 nm. .sup.1H NMR (600 MHz, (CD3)2SO) .delta.: The
significant change is the reduction of the allyl protons at 5.72
and 4.97 ppm and the appearance of the following sets of
significant signals: 3.08, 2.99, 1.97, 1.79, 1.43 and 1.34 ppm due
to the dendritic backbone of the MT, and 7.27, 7.07, 6.53, 5.32,
4.46, 4.37, and 1.24 ppm due to the Alexa Fluor.RTM. 594. All other
aspects of the spectrum are similar to that of 14.
[0632] 84. Paclitaxel Encapsulation in poly(vl-evl-avl-opd) (ABbD)
Nanoparticles.
[0633] To a 150 mL beaker containing D-.alpha.-tocopherol
polyethylene glycol 1000 succinate (0.39 g) dissolved in Lonza cell
culture water (78 mL), poly(vl-evl-avl-opd), ABbD, nanoparticles
(0.17 g) and paclitaxel (34.0 mg) dissolved in dimethyl sulfoxide
(0.75 mL) was added slowly with vigorous stirring. The solution was
split into two 50 mL centrifuge tubes. The paclitaxel loaded
nanoparticles were purified by applying two cycles of
centrifugation (8000 rpm for 1 h) and reconstitution with cell
culture water. The nanoparticle suspension was then lyophilized.
The loading ratio of paclitaxel for the encapsulation was
determined by NanoDrop UV/Vis and was found to be 11.34%.
[0634] 85. In Vivo Administration of Nanoparticle-Bioconjugate.
[0635] Five adult Sprague-Dawley rats were sacrificed by lethal
inhalation of CO.sub.2. At the moment of euthanasia, eight eyes of
four rats were treated with a solution of 2.times.10.sup.-2 M
nanoparticle conjugate in a molar ratio of 5:1 (dye:transporter) up
to 15 minutes, one rat served as the no treatment control. The
solution was dropped with a micropipette on to the cornea and
multiple drops were instilled in series to maintain a tear meniscus
over the cornea. The rats were kept in the dark in a cold room for
two hours after the treatment and underwent encleation of the globe
with optic nerve stump attached. The eye globes with attached optic
nerves were placed in 4% paraformaldehyde until paraffin embedding.
The paraffin blocks were cut into 4-.mu.m sections and were stained
with traditional DAPI dye. Slides were viewed at 40.times.'s
magnification using a digital fluorescent microscope Olympus Provis
AX70 digitally interfaced with a semi-cooled CCD camera to
visualize Alexa Fluor 594-labeled transporter. Background
autofluorescence was subtracted and the settings were held constant
for both the control and the treatment eyes. To proof and image the
intended eye region, images of the same location were measured
under the DAPI and Alexafluor wavelength with the
microscope-mounted camera (see FIG. 50, A-D).
[0636] 86. Synthesis of Copolymer poly(vl-opd).
[0637] To a 25 mL 3-necked round bottom flask, equipped with stir
bar, 2-oxepane-1,5-dione (0.7 g, 5.46 mmol) was added and the flask
was sealed with two septa and a gas inlet. The flask was evacuated
and refilled with argon three times. Dry toluene (4 mL) was added
and the mixture stirred in an oil bath at 70.degree. C. to dissolve
the monomer. Upon dissolving, Sn(Oct).sub.2 (20 mg,
5.48.times.10.sup.-2 mmol in 0.5 mL dry toluene), absolute ethanol
(51.1 .mu.L, 8.86.times.10.sup.-1 mmol), and .delta.-valerolactone
(2.87 mL, 30.7 mmol) were added. The temperature of the oil bath
was increased to 105.degree. C. and the mixture stirred for 48 h.
The crude product was dissolved in a minimal amount of THF (5 mL)
and poured into a round bottom flask containing 1 L diethyl ether.
The solution was kept overnight at 0.degree. C. and a white solid
was obtained. The solution was decanted off and the solid was dried
in vacuo to obtain poly(vl-opd). Yield: 2.31 g. M.sub.w=3525 Da,
PDI=1.27; .sup.1H NMR (300 MHz, CDCl.sub.3/TMS, ppm) .delta.: 4.34
(m, opd, --CH2CH2C(O)CH2CH2O--), 4.08 (m, vl, --CH2O--), 3.65 (m,
--OCH2CH3), 2.74 (m, opd, --OC(O)CH2CH2C(O)--), 2.60 (m, opd,
--CH2CH2C(O)CH2CH2-), 2.34 (m, vl, --CH2CH2C(O)O--), 1.68 (m, vl,
--CHCH2CH2-), 1.25 (m, --CH2CH3).
[0638] 87. Nanoparticle Formation from poly(vl-opd) Via Reductive
Amination.
[0639] In a 100 mL round bottom flask equipped with stir bar,
poly(vl-opd) (0.16 g) was dissolved in CH.sub.2Cl.sub.2 (11.5 mL).
After dissolving the polymer, tetrahydrofuran (11.5 mL),
2,2'-(ethylenedioxy)bisethylamine (16.9 .mu.L, 0.12 mmol), and
NaBH.sub.3CN (1.2 mL, 1.2 mmol) were added. The pH was adjusted to
6-7 using 1M NaOH and the reaction stirred for 12 h at room
temperature. Residual polymer, diamine and NaBH.sub.3CN were
removed by dialyzing with SnakeSkin.RTM. Pleated Dialysis Tubing
(MWCO=10,000) against 50/50 CH.sub.2Cl.sub.2/CH.sub.3OH. .sup.1H
NMR (300 MHz, CDCl.sub.3/TMS, ppm) .delta.: The significant change
is the appearance of signals at 3.66 ppm corresponding to the
protons neighboring the secondary amine of the PEG linker after
cross-linking. All other aspects of the spectrum are similar. The
particle size of the nanoparticles formed for various
stoichiometries was investigated by dynamic light scattering, as
tablated in Table 5, below.
TABLE-US-00006 TABLE 5 Size Analysis from Dynamic Light Scattering
Diameter (nm) Diameter (nm) Amine/ Ab.sub.1 nanoparticles Ab.sub.2
nanoparticles 1 Keto 7% keto 12% keto 2 11.3 .+-. 1.2 18.5 .+-. 1.9
3 20.7 .+-. 1.8 26.4 .+-. 2.4 4 38.1 .+-. 4.0 47.1 .+-. 4.9 6 77.4
.+-. 6.7 107.6 .+-. 8.9
[0640] A example preparation of degradable polyester nanoparticle
from copolymer poly(vl-opd) is illustrated in FIG. 51. A
transmission electron microscopy (TEM) image of particles formed is
provided in FIG. 52.
[0641] 88. General Nanoparticle Formation Utilizing
3,6-dioxa-1,8-octanedithiol.
[0642] A solution of poly(avl-vl) (0.14 g, M.sub.w=3042 Da,
PDI=1.18) dissolved in CH.sub.2Cl.sub.2 (0.16 mL) was added to a
solution of 3,6-dioxa-1,8-octanedithiol (19.6 .mu.L, 0.12 mmol) in
CH.sub.2Cl.sub.2 (24.6 mL). The reaction mixture was heated for 12
h at 45.degree. C. Residual dithiol was removed by dialyzing with
SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000) against
CH.sub.2Cl.sub.2. Yield: 0.13 g. DLS: D.sub.H=72.6.+-.2.8 nm.
.sup.1H NMR (300 MHz, CDCl.sub.3/TMS) .delta.: The significant
change is the reduction of the allyl protons at 5.06 and 5.77 ppm
and the appearance of signals at 3.65 and 2.71 ppm corresponding to
the protons neighboring the thiols of the PEG linker after
cross-linking. All other aspects of the spectrum are similar to
that of poly(avl-vl).
[0643] 89. General Nanoparticle Formation Utilizing Peg
Dithiol.
[0644] A solution of poly(avl-vl) (0.13 g, M.sub.w=3042 Da,
PDI=1.18) dissolved in CH.sub.2Cl.sub.2 (0.16 mL) was added to a
solution of PEG dithiol (0.13 g, 38.1 .mu.mol) in CH.sub.2Cl.sub.2
(23.5 mL). The reaction mixture was heated for 12 h at 45.degree.
C. Residual dithiol was removed by dialyzing with SnakeSkin.RTM.
Pleated Dialysis Tubing (MWCO=25,000) against CH.sub.2Cl.sub.2.
Yield: 0.11 g. DLS: D.sub.H=33.7.+-.3.8 nm. .sup.1H NMR (300 MHz,
CDCl.sub.3/TMS) .delta.: The significant change is the reduction of
the allyl protons at 5.06 and 5.77 ppm and the appearance of
signals at 3.65 and 2.71 ppm corresponding to the protons
neighboring the thiols of the PEG linker after cross-linking. All
other aspects of the spectrum are similar to that of
poly(avl-vl).
[0645] 90. Synthesis of Copolymer
poly(propargylvalerolactone-valerolactone) (poly(pvl-vl)).
[0646] A 25 mL 3-necked round bottom flask, equipped with stir bar,
was sealed with two septa and a gas inlet. The flask was evacuated
and refilled with argon three times. Stock solutions of 1.7 M
ethanol (EtOH) in THF and 3.7.times.10.sup.-2M tin(II)
2-ethylhexanoate (Sn(Oct).sub.2) in THF were made in sealed
Ar.sub.(g) purged flasks. Solutions of EtOH (0.13 mL, 0.22 mmol)
and Sn(Oct).sub.2 (0.12 mL, 4.3.times.10.sup.-3 mmol) were combined
in the Ar.sub.(g) purged 3-necked round bottom flask. After
stirring the mixture for 20 min,
.alpha.-propargyl-.delta.-valerolactone (pvl, 0.35 g, 2.5 mmol) and
.delta.-valerolactone (vl, 1.1 g, 10.0 mmol) were added. The
reaction vessel stirred at 105.degree. C. for 48 h. Residual
monomer and catalyst were removed by precipitating the polymer into
cold diethyl ether to give a golden brown polymer. Yield: 1.18 g
(81.4%). M.sub.w=3000 Da, PDI=1.18. .sup.1H NMR (300 MHz,
CDCl.sub.3/TMS): .delta. 4.10 (m, --CH.sub.2--O--), 3.64 (m,
CH.sub.3CH.sub.2O--), 2.59 (m, pvl, HC.ident.CCH.sub.2CH--), 2.35
(m, vl, --CH.sub.2CH.sub.2C(O)O--, pvl, HC.ident.CCH.sub.2CH--,
HC.ident.CCH.sub.2CH--), 2.03 (m, HC.ident.C--), 1.68 (m, pvl &
vl, --CHCH.sub.2CH.sub.2--), 1.25 ppm (t, CH.sub.3CH.sub.2O--).
.sup.13C NMR (400 MHz, CDCl.sub.3): .delta. 173.6 (pvl, --C(O)--),
172.5 (vl, --C(O)--), 78.2 (HC.ident.C--), 71.3, 68.4, 63.8, 36.7,
33.6, 29.7, 28.3, 24.6, 21.5, 19.0, 16.7 ppm.
[0647] 91. Click Reaction Conditions for Nanoparticle Formation
Utilizing polyoxyethylene bis(azide).
[0648] Poly(pvl-vl) (10 mg, M.sub.w=3000 Da, PDI=1.18) was added to
a vial, which was then sealed and purged with argon.
Polyoxyethylene bis(azide) (58.7 mg, 1.2.times.10.sup.-2 mmol)
dissolved in anhydrous dimethylformamide (0.5 mL) and copper (I)
bromide (23.4 .mu.L, 3.5.times.10.sup.-2 M solution in DMF) were
added. The reaction mixture stirred for 24 h at room temperature.
Residual azide and copper bromide were removed by dialyzing with
SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=25,000) against 50/50
dichloromethane/methanol. Yield: 43.4 mg. DLS: D.sub.H=21.9.+-.1.9
nm. .sup.1H NMR (300 MHz, CDCl.sub.3/TMS): .delta. The significant
change is the reduction of the alkyne proton at 2.03 ppm and the
appearance of signals at 3.65 and 3.40 ppm corresponding to the
protons of the PEG linker and the signal at 7.49 ppm due to the
protons from triazole formation as a result of cross-linking. All
other aspects of the spectrum are similar to that of
poly(vl-pvl).
[0649] 92. Click Reaction Conditions for Nanoparticle Formation
Utilizing 1,8-diazide-3,5-dioxaoctane.
[0650] Poly(pvl-vl) (40.8 mg, M.sub.w=3000 Da, PDI=1.18) was added
to a vial, which was then sealed and purged with argon. To the
vial, 1,8-diazide-3,5-dioxaoctane (19.5 mg, 9.7.times.10.sup.-2
mmol) dissolved in anhydrous dimethylformamide (0.8 mL) and copper
(I) bromide (115.3 .mu.L, 5.9.times.10.sup.-2 mM solution in DMF)
were added. The reaction mixture stirred for 24 h at 40.degree. C.
Residual azide and copper bromide were removed by dialyzing with
SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000) against 50/50
CH.sub.2Cl.sub.2/CH.sub.3OH. Yield: 37.8 mg. DLS:
D.sub.H=21.9.+-.1.9 nm .sup.1H NMR (300 MHz, CDCl.sub.3/TMS):
.delta. The significant change is the reduction of the alkyne
proton at 2.03 ppm and the appearance of signals at 3.65 and 3.40
ppm corresponding to the protons of the PEG linker and the signal
at 7.49 ppm due to the protons from triazole formation as a result
of cross-linking. All other aspects of the spectrum are similar to
that of poly(pvl-vl).
[0651] 93. General Procedure for Formulating Nanoparticles with
TPGS-Vitamin E.
[0652] To a 150 mL beaker containing D-.alpha.-tocopheryl
polyethylene glycol 1000 succinate (vitamin E TPGS) (0.28 g)
dissolved in Lonza cell culture water (55 mL), nanoparticles
(0.0977 g) dissolved in dimethyl sulfoxide (DMSO) (0.50 mL) were
added slowly with vigorous stirring. The solution was split equally
into two 50 mL centrifuge tubes. The nanoparticles were rinsed by
applying three cycles of centrifugation (8000 rpm for 30 min) and
reconstituted with cell culture water. The nanoparticle suspension
was then lyophilized.
[0653] 94. General Procedure for In Vitro Cytotoxicity of
Formulated Nanoparticles (MTT Assay).
[0654] The cytotoxicity of the formulated nanoparticles was
evaluated using an MTT assay. HeLa cells were cultured in Eagle's
Minimum Essential Medium supplemented with 10% heat inactivated
fetal bovine serum, L-glutamine, penicillin streptomycin sulfate
antibiotic-antimycotic mixture and gentamicin. Cells were
maintained at 37.degree. C. with 5% CO.sub.2 in a 95% humidity
incubator. The cells were seeded in a 96-well plate in 100 .mu.L,
media per well at a density of 10,000 cells/well and incubated for
24 h. The media was then replaced with 100 .mu.L of phenol red free
medium-containing nanoparticles at different concentrations in
triplicate and incubated for 24 h. After incubation, the
nanoparticle containing media was removed, the cells were rinsed
three times with DPBS, to avoid interference in the assays, and 100
.mu.L of fresh phenol red free media was added, followed by 10
.mu.L MTT solution (5 mg/mL). The cells were incubated for 4 h,
after which time the medium was carefully removed. To the resulting
purple crystals, 100 .mu.L DMSO was added to lyse the cells and was
incubated for 10 min at 37.degree. C. The MTT absorbance was
measured at 540 nm using a Synergy HT Multi-mode microplate reader
(B10 Tek Instruments, Winooski, Vt.). Optical densities measured
for wells containing cells that received no nanoparticle were
considered to represent 100% viability. Results are expressed as
the mean.+-.S.D. of viable cells.
[0655] 95. Encapsulation of Brimonidine in Nanoparticles.
[0656] To a 150 mL beaker containing D-.alpha.-tocopherol
polyethylene glycol 1000 succinate (0.15 g) dissolved in Lonza cell
culture water (30 mL), nanoparticles (60.5 mg) and brimonidine (6.1
mg) dissolved in dimethyl sulfoxide (0.50 mL) were added slowly
with vigorous stirring. The solution was split equally into two 50
mL centrifuge tubes. The brimonidine loaded nanoparticles were
purified by applying three cycles of centrifugation (8000 rpm for
30 min) and reconstituted with cell culture water. The nanoparticle
suspension was then lyophilized. The concentration of encapsulated
brimonidine was determined by NanoDrop.TM. UV-Vis at a wavelength
of 389 nm. Brimonidine standards (0.32-1.92 mg/mL) were measured by
UV-Vis and a calibration curve was rendered. With the calibration
curve, the concentration of encapsulated brimonidine was determined
by the absorbency of the brimonidine in the nanoparticle at 389 nm
and the loading ratio was found to be 6.5%.
[0657] 96. General Procedure for the Formation of Nanoparticles
from poly(vl-evl).
[0658] In a 100 mL three-necked round bottom flask equipped with
stir bar, condenser and septa, 2,2'-(ethylenedioxy)bisethylamine
(34.1 .mu.L, 2.32.times.10.sup.-4 mol), 28.7 mL CH.sub.2Cl.sub.2
and a solution of poly(vl-evl) (0.14 g, M.sub.w=3400 Da, PDI=1.16)
in 0.19 mL CH.sub.2Cl.sub.2 were added. The mixture was heated at
44.degree. C. for 12 h. Residual diamine was removed by dialyzing
with Snakeskin.RTM. Pleated Dialysis Tubing (MWCO=10,000) against
dichloromethane. DLS: D.sub.H=272.3.+-.23.3 nm. .sup.1H NMR (300
MHz, CDCl.sub.3/TMS) .delta.: The significant change is the
disappearance of the epoxide protons at 2.94, 2.75 and 2.47 ppm and
the appearance of signals at 3.64 and 2.97 ppm corresponding to the
protons neighboring the secondary amine of the PEG linker after
cross-linking. All other aspects of the spectrum are similar to
that of poly(vl-evl), as referenced in the literature..sup.8
[0659] 97. General Procedure for In Vitro Nanoparticle Degradation
Studies.
[0660] poly(vl-evl) nanoparticles (10 mg) were suspended in 2 mL of
Dulbecco's Phosphate Buffered Saline (pH 7.2) in 2 dram vials
equipped with stir bars. The vials were sealed to avoid evaporation
and the samples were maintained at 37.degree. C. under continuous
stirring. At 48 h intervals, samples were removed and
dichloromethane was added (3.times.4 mL) to extract remaining
nanoparticles and degradation products. The extraction solutions
were concentrated via rotary evaporator and dried in vacuo. The
degradation of the nanoparticles was monitored by the change in
molecular weight, as determined by static light scattering, with
incubation time.
[0661] 98. General Procedure for Nanoparticle Formation from
poly(vl-evl-avl-opd).
[0662] To a solution of 2,2'-(ethylenedioxy)diethylamine (23.4
.mu.L, 0.16 mmol) in CH.sub.2Cl.sub.2 (98.7 mL), a solution of
poly(vl-evl-avl-opd) (0.1840 g, M.sub.w=3440 Da) in
CH.sub.2Cl.sub.2 (0.64 mL) was added. The mixture was heated at
44.degree. C. for 12 h. Residual diamine was removed by dialyzing
with SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000) against
CH.sub.2Cl.sub.2. Yield: 0.15 g (94%). DLS: D.sub.H=52.9.+-.3.3 nm
SLS: M.sub.w=147,000 Da. .sup.1H NMR (300 MHz, CDCl.sub.3/TMS)
.delta.: The significant change is the disappearance of the epoxide
protons at 2.94, 2.75 and 2.47 ppm and the appearance of signals at
3.54 and 2.97 ppm corresponding to the protons neighboring the
secondary amine of the PEG linker after cross-linking. All other
aspects of the spectrum are similar to that of
poly(vl-evl-avl-opd), as referenced in the literature..sup.9
[0663] 99. Formulation of poly(vl-evl-avl-opd) Nanoparticles with
TPGS-Vitamin E.
[0664] To a 150 mL beaker containing D-.alpha.-tocopheryl
polyethylene glycol 1000 succinate (vitamin E TPGS) (0.28 g)
dissolved in Lonza cell culture water (55 mL), nanoparticles
(0.0977 g) dissolved in dimethyl sulfoxide (DMSO) (0.50 mL) were
added slowly with vigorous stirring. The solution was split equally
into two 50 mL centrifuge tubes. The nanoparticles were rinsed by
applying three cycles of centrifugation (8000 rpm for 30 min) and
reconstituted with cell culture water. The nanoparticle suspension
was then lyophilized.
[0665] 100. In Vitro poly(vl-evl-avl-opd) Nanoparticle Degradation
Studies.
[0666] TPGS formulated poly(vl-evl-avl-vl) nanoparticles (10 mg)
were suspended in 2 mL of Dulbecco's Phosphate Buffered Saline (pH
7.4) in 2 dram vials equipped with stir bars. The vials were sealed
to avoid evaporation and the samples were maintained at 37.degree.
C. under continuous stirring. At 48 h intervals, samples were
removed and dichloromethane was added (3.times.4 mL) to extract
remaining nanoparticles and degradation products. The extraction
solutions were concentrated via rotary evaporator and dried in
vacuo. The degradation of the nanoparticles was monitored by the
change in molecular weight, as determined by static light
scattering, with incubation time.
[0667] 101. In Vitro Cytotoxicity of Formulated
poly(vl-evl-avl-opd) Nanoparticles (MTT Assay).
[0668] The cytotoxicity of TPGS formulated nanoparticles was
evaluated using an MTT assay. HeLa cells were cultured in Eagle's
Minimum Essential Medium supplemented with 10% heat inactivated
fetal bovine serum, L-glutamine, penicillin streptomycin sulfate
antibiotic-antimycotic mixture and gentamicin. Cells were
maintained at 37.degree. C. with 5% CO.sub.2 in a 95% humidity
incubator. The cells were seeded in a 96-well plate in 100 .mu.L,
media per well at a density of 10,000 cells/well and incubated for
24 h. The media was then replaced with 100 .mu.L of phenol red free
medium-containing nanoparticles at different concentrations in
triplicate and incubated for 24 h. After incubation, the
nanoparticle containing media were removed, the cells were rinsed
three times with DPBS, to avoid interference in the assays, and 100
.mu.L of fresh phenol red free media was added, followed by 10
.mu.L MTT solution (5 mg/mL). The cells were incubated for 4 h,
after which time the medium was carefully removed. To the resulting
purple crystals, 100 .mu.L DMSO was added to lyse the cells and was
incubated for 10 min at 37.degree. C. The MTT absorbance was
measured at 540 nm using a Synergy HT Multi-mode microplate reader
(B10 Tek Instruments, Winooski, Vt.). Optical densities measured
for wells containing cells that received no nanoparticle were
considered to represent 100% viability. Results are expressed as
the mean.+-.S.D. of viable cells.
[0669] 102. In Vitro Release of Paclitaxel from
poly(vl-evl-avl-opd) Nanoparticles.
[0670] To a 150 mL beaker containing D-.alpha.-tocopherol
polyethylene glycol 1000 succinate (0.34 g) dissolved in Lonza cell
culture water (68 mL), poly(vl-evl-avl-opd) nanoparticles (56.5 mg)
and paclitaxel (8.5 mg) dissolved in dimethyl sulfoxide (0.50 mL)
were added slowly with vigorous stirring. The solution was split
equally into two 50 mL centrifuge tubes. The paclitaxel loaded
nanoparticles were purified by applying three cycles of
centrifugation (8000 rpm for 30 min) and reconstituted with cell
culture water. The nanoparticle suspension was then lyophilized.
The concentration of encapsulated paclitaxel was determined by
NanoDrop.TM. UV-Vis at a wavelength of 254 nm. Paclitaxel standards
(0.398-2.39 mg/mL) were measured by UV-Vis and a calibration curve
was rendered. With the calibration curve, the concentration of
encapsulated paclitaxel was determined by the absorbency of the
paclitaxel in the nanoparticle at 254 nm and the loading ratio was
found to be 11.3%. The release of paclitaxel from the nanoparticles
was measured in PBS (pH 7.4) at 37.degree. C. The paclitaxel-loaded
nanoparticles (20 mg) were suspended in PBS (20 mL). At particular
time intervals, the nanoparticle dispersion was centrifuged, the
supernatant was removed and the released paclitaxel was extracted
from the supernatant with CH.sub.2Cl.sub.2. The concentration of
released paclitaxel was determined by NanoDrop.TM. UV-Vis at a
wavelength of 254 nm as mentioned above.
[0671] 103. General Procedures for the Synthesis of HVGGSSV
Peptide.
[0672] The peptide was synthesized by solid-phase peptide synthesis
using standard Fmoc chemistry on a Model 90 Peptide Synthesizer
(Advanced ChemTech).
[0673] General Procedure: Attachment of N-Fmoc Amino Acids to
Resin.
[0674] After swelling with dichloromethane (20 mL) for 20 min,
H-Val-2-Cl-Trt resin (0.20 g, 1.03 mmol/g, 0.21 mmol surface amino
acids) was treated with a solution of Fmoc-protected amino acids (4
equiv, 0.9 mmol) in dimethylformamide (DMF) (6 mL). The amino acids
were attached to the resin using double coupling with a solution (9
mL) consisting of N-hydroxybenzotriazole monohydrate (HOBt) (0.9
mmol, 137.8 mg) o-(benzotriazole-N,N,N',N'-tetramethyluronium
hexafluorophosphate (0.9 mmol, 0.34 g), N,N'-diisopropylethylamine
(DIPEA) (1.8 mmol, 0.31 mL) in 9 mL DMF. The reaction mixture was
shaken for 60 min and washed with DMF (4.times.10 mL), methanol
(4.times.10 mL) and DMF (4.times.10 mL). A 20% (v/v) piperidine in
DMF solution was used to deprotect the Fmoc groups. The amino acids
were attached to the resin in the following sequence: Ser, Ser,
Gly, Gly, Val, His, Asn, Gly, Gly, Gly, Cys, and Gly.
[0675] General Procedure: Cleavage from Resin.
[0676] The resin was treated with Reagent R, a solution of TFA,
thioanisole, anisole, and ethanedithiol (90:5:3:2, 6 mL), for 4 h.
After removal of the resin by filtration, the filtrate was
concentrated to precipitate the peptide with cold diethyl ether.
Crude peptides were purified by RP-HPLC and lyophilized. Peptide
identity was confirmed by MALDI-MS (m/z: 1086.45).
[0677] 104. Attachment of HVGGSSV Peptide to Nanoparticles.
[0678] To a solution of nanoparticles (105.6 mg, 0.78 .mu.mol) in
DMSO (1 mL), HVGGSSV peptide (56 mg, 53.6 .mu.mol) in DMSO (2 mL)
was added. The reaction mixture was heated for 72 h at 34.degree.
C. Residual peptide was removed by dialyzing with SnakeSkin.RTM.
Pleated Dialysis Tubing (MWCO=10,000) against 50/50 THF/CH.sub.3CN.
Yield: 77 mg. DLS: D.sub.H=55.3.+-.3.6 nm; original particle
D.sub.H=52.9.+-.3.3 nm. SLS: M.sub.w=185,000 Da; original particle
M.sub.w=147,000 Da. .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta.:
The significant change is the reduction of the allyl protons at
5.69 and 5.00 ppm and the appearance of signals at 0.80, 1.39,
1.65, 2.74, 3.07, 3.75, 4.40 and 7.11-8.32 ppm due to the peptide.
All other aspects of the spectrum are similar to that of the
poly(vl-evl-avl-opd) nanoparticles.
[0679] 105. Encapsulation of Paclitaxel in HVGGSSV Conjugated
poly(vl-evl-avl-opd) Nanoparticles.
[0680] To a 150 mL beaker containing D-.alpha.-tocopherol
polyethylene glycol 1000 succinate (0.30 g) dissolved in Lonza cell
culture water (60 mL), HVGGSSV-nanoparticles (0.0681 g) and
paclitaxel (10.2 mg) dissolved in dimethyl sulfoxide (0.50 mL) were
added slowly with vigorous stirring. The solution was split into
two 50 mL centrifuge tubes. The paclitaxel loaded nanoparticles
were purified by applying two cycles of centrifugation (8000 rpm
for 30 min) and reconstituted with cell culture water. The
nanoparticle suspension was then lyophilized. The loading ratio of
paclitaxel for the encapsulation was determined by NanoDrop.TM.
UV-Vis at 254 nm as mentioned above and was found to be 11%.
[0681] 106. Lowering Intraocular Pressure (IOP) with Brimonidine
Nanoparticle Injection
[0682] Two groups of mice (N=3 each) were compared to determine the
effect of a single intravitreal injection of brimonidine-laced
nanoparticle relative to a single topical drop (eyedrop) of
clinical-grade brimonidine. For each mouse, intraocular pressure
(TOP) was acutely elevated by an injection (1 .mu.l) of polystyrene
microbeads into the anterior chamber of the eye. This induces a
35-40% elevation in IOP that persists for 3-4 weeks. One group had
a single topical application (1 .mu.l) of brimonidine; the other a
single intravitreal injection (1 .mu.l) of the
nanoparticle-brimonidine complex. IOP was tracked using TonoPen XL
measurements until any lowering effect was dissipated.
[0683] For the topical application group, microbead injection
induced a 35% elevation in IOP from a normal reading of 14 mmHG to
19-20 mmHG, one day after injection (FIG. 1). This elevation
persisted until day 4, when topical brimonidine was applied; the
application lowered IOP to normal levels one day later. Six days
following topical administration of brimonidine, IOP returned to
elevated levels.
[0684] For the nanoparticle group, microbead injection again
induced a 40% elevation in TOP one day after injection, from a
normal baseline of 15 mmHG to 21 mmHG (FIG. 2).
[0685] This elevation persisted until day 4, when a single
intravitreal injection (1 .mu.l) of the nanoparticle-brimonidine
complex was applied. The nanoparticle complex actually lowered IOP
below normal levels to 11.5 mmHG one day later; this depression was
significant (p<0.01). IOP remained below or at baseline for 6
days. For this period, IOP was indistinguishable from
pre-microbead/baseline levels (p=0.43). This is dramatically
different than topical application, which returned to elevated IOP
during the same period. For the nanoparticle group, IOP returned to
elevated levels by day 18. Control groups for both experiments
demonstrated continuously elevated IOP due to microbead injection
for the duration.
[0686] 107. Measuring Retinal Diffusion after Nanoparticle
Injection
[0687] For glaucoma, there is no FDA approved neuroprotective
therapy for preventing or treating retinal and optic nerve
degeneration. All available drugs have as their action IOP
lowering. Thus, a secondary use of the nanoparticle delivery system
would be to expose the retina and optic nerve to a slow-release of
directly neuroprotective compounds, such as memantine or
brimonidine, which is known to have secondary neuronal actions
independent of TOP lowering. For macular degeneration, the best
available practice is a monthly or biweekly intravitreal injection
of antiangiogenic compounds, such as LUCENTIS.RTM. (ramibizumab) or
AVASTIN.RTM. (bevaizumab). Again the nanoparticle delivery system
could ameliorate the need for such frequent injections.
[0688] To determine how much of the retina could be stained over
time with a common neuronal dye (DiO) interlaced into the
nanoparticle after a single intravitreal injection, the area of the
retina covered by DiO released from the nanoparticle complex was
measured as a function of time after the injection (N=2-3 mice for
each time). Deposition of DiO was defined very conservatively, as
that portion of the retina contained DiO signal intensity of 100%
contrast compared to background. The area of the retina represented
by DiO label was compared to the total surface area of the retina.
Retinas were examined at 3 days, 1 week, 2 weeks, and 4 weeks
post-nanoparticle injection.
[0689] Over a 4 week period, DiO deposition appeared to increase on
the retinal surface by about 15% compared to the initial
measurement at 3 days; this was not significant (p=0.50) (FIG. 53).
In between, deposition was statistically constant compared to the
initial measurement as well. This indicates that retinal uptake of
the DiO is fairly consistent and matched to its slow release from
the nanoparticle complex. Thus, retinal exposure to a released drug
would be constant in between nanoparticle injections. Additionally,
the ability of nanoparticles to pas through the inner limiting
membrane and deliver DiO to ganglion cells was measured at 3 days,
1 week, and 2 weeks following injection. Micrographs show that
deposition was observed in ganglion cells and maintained over the
observatory period (FIG. 54).
[0690] 108. Preparation of Nanoparticles from Linear Polymer
Precursor
[0691] a. Formation of 50 nm Nanoparticles
[0692] To a 100-mL round bottom flask equipped with a stir bar,
poly(vl-evl) (0.1001 g, M.sub.w=2350 Da, 7% cross-linking) and 20.2
mL CH.sub.2Cl.sub.2 were added, followed by
2,2'-(ethylenedioxy)diethylamine (9.6 .mu.L, 6.55.times.10.sup.-5
mol). The mixture was heated at reflux at 44.degree. C. for 12 h
and promptly transferred to SnakeSkin.RTM. Pleated Dialysis Tubing
(MWCO=10,000) and dialyzed against dichloromethane to remove
residual diamine. .sup.1H NMR (400 MHz), CDCl.sub.3/TMS, ppm)
.delta.: The significant change, proving conversion from the linear
polymer to the nanoparticle, is the disappearance of epoxide
protons at 2.96, 2.75, and 2.47 ppm and the appearance of signals
at 3.5 ppm and 2.9 ppm due to the protons near the secondary amine
of the PEG linker.
[0693] b. Formation of 400 nm Nanoparticles
[0694] To a 200-mL round bottom flask equipped with a stir bar,
poly(vl-evl) (0.1210 g, M.sub.w=2325 Da, 13% cross-linking) and
45.1 mL CH.sub.2Cl.sub.2 were added, followed by
2,2'-(ethylenedioxy)diethylamine (75.1 .mu.L, 5.13.times.10.sup.-4
mol). The mixture was heated at reflux at 44.degree. C. for 12 h
and promptly transferred to SnakeSkin.RTM. Pleated Dialysis Tubing
(MWCO=10,000) and dialyzed against dichloromethane to remove
residual diamine. .sup.1H NMR (400 MHz), CDCl.sub.3/TMS, ppm)
.delta.: The significant change is the disappearance of epoxide
protons at 2.93, 2.76, and 2.47 ppm and the appearance of signals
at 3.5 and 2.9 ppm, correlating to the protons of the PEG
linker.
[0695] c. Formation of 700 nm Nanoparticles
[0696] To a 200-mL round bottom flask equipped with a stir bar,
poly(vl-evl-avl) (0.1057 g, M.sub.w=7200 Da, 15% cross-linking) and
46.4 mL CH.sub.2Cl.sub.2 were added, followed by
2,2'-(ethylenedioxy)diethylamine (82.5 .mu.L, 5.64.times.10.sup.4
mol). The mixture was heated at reflux at 44.degree. C. for 12 h
and promptly transferred to SnakeSkin.RTM. Pleated Dialysis Tubing
(MWCO=10,000) and dialyzed against dichloromethane to remove
residual diamine. .sup.1H NMR (400 MHz), CDCl.sub.3/TMS, ppm)
.delta.: The significant change is the disappearance of epoxide
protons at 2.94, 2.75, and 2.48 ppm and the appearance of signals
at 3.5 and 2.9 ppm, correlating to the protons of the PEG
linker.
[0697] d. Formation of 700 nm Nanoparticles
[0698] To a 200-mL round bottom flask equipped with a stir bar,
poly(vl-evl-avl) (0.1001 g, M.sub.w=7200 Da, 15% cross-linking) and
43.9 mL CH.sub.2Cl.sub.2 were added, followed by
2,2'-(ethylenedioxy)diethylamine (39.1 .mu.L, 2.67.times.10.sup.4
mol) and 1,8-diaminooctane (38.5 mg, 2.67.times.10.sup.4 mol). The
mixture was heated at reflux at 44.degree. C. for 12 h and
transferred to SnakeSkin.RTM. Pleated Dialysis Tubing (MWCO=10,000)
and dialyzed against dichloromethane to remove residual diamines.
.sup.1H NMR (400 MHz), CDCl.sub.3/TMS, ppm) 6: The significant
change, confirming incorporation of 1,8-diaminooctane, is the
appearance of a signal at 1.32 ppm corresponding to the protons
between the secondary amines of the cross-linker The spectrum shows
otherwise similar shifts as the particles of 50 and 400 nm.
[0699] 109. General Procedure for Encapsulation of NP
[0700] The 700 nm nanoparticle with 15% cross-linking (100% PEG
linker) (16.7 mg) and bimatoprost
(7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-
-hept-5-enamide; a prostaglandin analog/prodrug used topically to
control the progression of glaucoma and in the management of ocular
hypertension; 5.0 mg) were accurately weighed together into a vial.
The two solids were dissolved in a minimal amount of DMSO (150
.mu.L) and added dropwise to a vigorously stirring solution of
water (8.3 mL) and vitamin E (0.125 g). The solution turned cloudy
and was immediately centrifuged at 8500 rpm for 20 min. The
supernatant was carefully removed, fresh water was added and the
pellet disturbed to ensure thorough washing of the drug-loaded
particles. The centrifugation wash was repeated for a total of
three washes. Finally, the particles were frozen and lyophilized to
yield the drug-loaded particles as a light and fluffy white solid
with 29.4% bimatoprost encapsulated.
[0701] The 700 nm nanoparticle with the 50:50 mixture of amorphous
and crystalline cross-linkers encapsulated 25.4% bimatoprost. The
400 nm nanoparticle encapsulated 22.4% bimatoprost, and the 50 nm
nanoparticle encapsulated 1.3% travatan (i.e., Travoprost,
propan-2-yl
7-[3,5-dihydroxy-2-[3-hydroxy-4-[3-(trifluoromethyl)phenoxy]-but-1-enyl]--
cyclopentyl]hept-5-enoate; topical medication used for controlling
the progression of glaucoma or ocular hypertension, by reducing
intraocular pressure), and the other 50 nm nanoparticle
encapsulated 3.3% brimonidine
(5-Bromo-N-(4,5-dihydro-1H-imidazol-2-yl)quinoxalin-6-amine; used
to treat open-angle glaucoma or ocular hypertension).
[0702] 110. Nanodrop to Determine % Drug Loading
[0703] About 0.4 mg of drug-loaded nanoparticles were weighed and
dissolved in 50 uL DMSO. 2 uL of sample solution was pipetted onto
the pedestal of a UV-VIS spectrometer (NanoDrop) and the absorbance
measured at 262 nm. A calibration curve between concentration of
drug and absorbance was made using a spread of samples with known
concentrations of drug. Using the calibration curve, the amount of
drug within the nanoparticle could be quantified and reported as a
weight percent.
[0704] 111. General Procedure for Mouse Study Preparation
[0705] The nanoparticle (1.20 mg, 700 nm amorphous) was accurately
weighed into an eppendorff tube and dispersed in PBS (75 .mu.L) for
an over-all concentration of 16 mg/mL, or 3.6 mg/mL
bimatoprost.
[0706] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
* * * * *