U.S. patent application number 14/700018 was filed with the patent office on 2016-01-14 for lipid-peptide-polymer conjugates and nanoparticles thereof.
The applicant listed for this patent is Lawrence Berkeley National Laboratory. Invention is credited to He Dong, Jessica Shu, Ting Xu.
Application Number | 20160009770 14/700018 |
Document ID | / |
Family ID | 44564161 |
Filed Date | 2016-01-14 |
United States Patent
Application |
20160009770 |
Kind Code |
A1 |
Xu; Ting ; et al. |
January 14, 2016 |
LIPID-PEPTIDE-POLYMER CONJUGATES AND NANOPARTICLES THEREOF
Abstract
The present invention provides a conjugate having a peptide with
from about 10 to about 100 amino acids, wherein the peptide adopts
a helical structure. The conjugate also includes a first polymer
covalently linked to the peptide, and a hydrophobic moiety
covalently linked to the N-terminus of the peptide, wherein the
hydrophobic moiety comprises a second polymer or a lipid moiety.
The present invention also provides helix bundles form by
self-assembling the conjugates, and particles formed by
self-assembling the helix bundles. Methods of preparing the helix
bundles and particles are also provided.
Inventors: |
Xu; Ting; (Berkeley, CA)
; Dong; He; (Potsdam, NY) ; Shu; Jessica;
(Apple Valley, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lawrence Berkeley National Laboratory |
Berkeley |
CA |
US |
|
|
Family ID: |
44564161 |
Appl. No.: |
14/700018 |
Filed: |
April 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13610637 |
Sep 11, 2012 |
9044514 |
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14700018 |
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PCT/US11/28198 |
Mar 11, 2011 |
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13610637 |
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61313522 |
Mar 12, 2010 |
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Current U.S.
Class: |
424/1.69 ;
514/44R; 514/773; 530/300; 530/324 |
Current CPC
Class: |
A61K 47/42 20130101;
A61K 47/542 20170801; B82Y 5/00 20130101; A61K 47/60 20170801; C07K
14/435 20130101; Y10S 977/773 20130101; A61K 47/6907 20170801; Y10S
977/906 20130101 |
International
Class: |
C07K 14/435 20060101
C07K014/435; A61K 47/42 20060101 A61K047/42 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] This invention was made with Government support under Grant
No. KC0202010, awarded by the Department of Energy-BES. The
Government has certain rights in this invention.
Claims
1.-17. (canceled)
18. A conjugate comprising: a three-helix bundle-forming peptide
having from about 10 to about 100 amino acids; a first polymer
covalently linked to the three-helix bundle-forming peptide,
wherein the first polymer is linked to the three-helix
bundle-forming peptide at an amino acid other than the N- or
C-terminus; and a hydrophobic moiety covalently linked to the
N-terminus of the peptide, wherein the hydrophobic moiety comprises
a second polymer or a lipid moiety comprising from 1 to 6
C.sub.10-20 alkyl groups.
19. The conjugate of claim 18, wherein the amino acids in the
three-helix bundle-forming peptide are characterized by a heptad
periodicity, -abcdefg-, wherein the amino acids at positions a and
d are hydrophobic amino acids, and wherein the amino acids at
positions e and g form salt bridges between adjacent helices.
20. The conjugate of claim 19, wherein each amino acid at position
a and position d is independently selected from the group
consisting of valine, leucine, isoleucine, methionine,
phenylalanine, and tryptophan.
21. The conjugate of claim 19, wherein each amino acid at position
e and position g is independently selected from the group
consisting of lysine, arginine, histidine, aspartate, and
glutamate.
22. The conjugate of claim 18, wherein the first polymer is a
hydrophilic polymer.
23. The conjugate of claim 18, wherein the first polymer is
polyethyleneglycol.
24. The conjugate of claim 18, wherein the second polymer comprises
polybutadiene.
25. The conjugate of claim 18, wherein the lipid moiety comprises
from 1 to 6 C.sub.10-20 alkyl groups.
26. The conjugate of claim 18, wherein the lipid moiety comprises
1, 2, or 4 C.sub.10-20 alkyl groups.
27. The conjugate of claim 18, further comprising an amino acid
residue covalently linked to the C-terminus of the three-helix
bundle-forming peptide.
28. The conjugate of claim 27, wherein the amino acid residue
comprises a member selected from the group consisting of GGG, HHH,
KK, EE, RGD and AYSSGAPPMPPF.
29. The conjugate of claim 18, wherein the amino acids in the
three-helix bundle-forming peptide are characterized by a heptad
periodicity, -abcdefg-, wherein each amino acid at position a and
position d is independently selected from the group consisting of
valine, leucine, isoleucine, methionine, phenylalanine, and
tryptophan, and each amino acid at position e and position g is
independently selected from the group consisting of lysine,
arginine, histidine, aspartate, and glutamate; the first polymer
comprises polyethylene glycol; the hydrophobic moiety comprises the
lipid moiety which comprises lysine and two C.sub.16 alkyl chains;
and an amino acid residue of from 2 to about 20 amino acids,
covalently linked to the C-terminus of the three-helix
bundle-forming peptide.
30. A helix bundle comprising 3 conjugates of claim 18.
31. A particle comprising from about 20 to about 200 conjugates of
claim 18.
32. The particle of claim 31, further comprising at least one
member selected from the group consisting of a therapeutic agent, a
diagnostic agent, DNA, and an oligonucleotide.
33. A method of forming a particle of claim 31, the method
comprising: contacting a plurality of conjugates of claim 18 such
that the conjugates self-assemble to form the particles of claim
31.
34. The method of claim 33, wherein the conjugates are at a
concentration of from about 1 nM to about 1 M.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/610,637, filed Sep. 11, 2012, now U.S. Pat.
No. 9,044,514, issued Jun. 2, 2015, which is a continuation-in-part
of PCT Application No. PCT/US11/28198, filed Mar. 11, 2011, which
claims priority to U.S. Provisional Application No. 61/313,522,
filed Mar. 12, 2010, which is incorporated in its entirety herein
for all purposes.
REFERENCE TO A SEQUENCE LISTING
[0003] This application includes a Sequence Listing as a text file
named "SEQTXT.sub.--77429.sub.--850121.sub.--007711US.txt" created
Sep. 10, 2012 and containing 2,207 bytes. The material contained in
this text file is incorporated by reference.
BACKGROUND OF THE INVENTION
[0004] Synthetic nanoparticles based on peptides/proteins, lipids
and polymers offer great promise for biomedical and pharmaceutical
applications, such as drug delivery, new vaccine formulations,
tissue engineering and protein therapeutics. They are also highly
desirable for the food and cosmetic industries. Various approaches
have been developed to prepare nanoparticles with different levels
of success such as liposomes, dendrimers, crosslinked polymeric
nanoparticles, polymersomes and synthetic virus-like nanoparticles
using recombinant proteins. The polymeric approach tends to give
large particles and there are limited reports to produce particles
with sizes around 10-20 nm. As the major carrier for many
therapeutic systems, liposomes can form nanoparticles with a wide
range of sizes down to 20-30 nm and are commonly used for drug and
gene delivery as well as skin cosmetics care and food industry. The
liposome formation process is not instantaneous and typically
involves multi-step procedures such as sonication, extrusion etc.
Yet, the liposomes tend to form large aggregates and require
optimization in particle size, polydispersity and shelf-life time.
Synthetic virus-like nanoparticles, such as Inflexal V.RTM., can be
made using recombinant proteins that self-assemble into 20-100 nm
diameter nanoparticles capable of displaying multiple antigenic
peptides on their surface. However, extensive purification is
required to remove residual compounds to avoid immune responses. In
addition, they require refrigeration to prevent protein
denaturation. Both limitations result in high cost and prevent
their extensive utilization. Thus, it still remains a significant
challenge to prepare monodisperse nanoparticles with diameters in
the range of tens of nanometers that are stable at room temperature
at low cost. Surprisingly, the present invention meets this and
other needs.
BRIEF SUMMARY OF THE INVENTION
[0005] In some embodiments, the present invention provides a
conjugate having a peptide with from about 10 to about 100 amino
acids, wherein the peptide adopts a helical structure. The
conjugate also includes a first polymer covalently linked to the
peptide, and a hydrophobic moiety covalently linked to the
N-terminus of the peptide, wherein the hydrophobic moiety comprises
a second polymer or a lipid moiety.
[0006] In some embodiments, the present invention provides a helix
bundle having from 2 to 6 conjugates of the present invention.
[0007] In some embodiments, the present invention provides a
particle having from about 20 to about 200 conjugates of the
present invention.
[0008] In some embodiments, the present invention provides a method
of forming particles of the present invention by contacting a
plurality of conjugates of the present invention such that the
conjugates self-assemble to form the particles of the present
invention.
[0009] In some embodiments, the present invention further provides
a method for delivering a diagnostic or therapeutic agent to a
subject comprising administering a particle to the subject. Thus,
the particle includes from about 20 to about 200 conjugates of the
present invention and the therapeutic agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1A shows a schematic of a lipid-peptide-polymer
conjugate of the present invention using polyethyleneglycol (PEG)
as the polymer.
[0011] FIG. 1B shows a schematic drawing of the lipidated 3-helix
bundle-forming peptide-polymer conjugate and its assembly into
higher order nanostructures.
[0012] FIG. 2A shows a Transmission Electron Micrograph (TEM) of
the particles prepared from the dC16-1coi-W-HHH conjugate 2.
[0013] FIG. 2B shows the MALDI-TOF of the dC16-1coi-W-HHH conjugate
2.
[0014] FIG. 3A shows a TEM image of the particles prepared from the
dC16-1CW-P2K-EE conjugate 10.
[0015] FIG. 3B shows the MALDI-TOF of the dC16-1CW-P2K-EE conjugate
10.
[0016] FIG. 4A shows a TEM image of the particles prepared from the
dC16-1CW-P2K-GB conjugate 12 with gold particles and
NaBH.sub.4.
[0017] FIG. 4B shows the MALDI-TOF of the dC16-1CW-P2K-GB conjugate
12.
[0018] FIG. 4C shows a TEM image of the particles prepared from the
dC16-1CW-P2K-GB conjugate 12 with gold particles only.
[0019] FIG. 5A shows a TEM image of the particles prepared from the
dC16-1CW-P2K-HHH conjugate 1.
[0020] FIG. 5B shows the small-angle X-ray scattering (SAXS) of the
particles prepared from the dC16-1CW-P2K-HHH conjugate 1 at both 4
wt. % (lower line) and 16 wt. % (upper line).
[0021] FIG. 5C shows the MALDI-TOF of the dC16-1CW-P2K-HHH
conjugate 1.
[0022] FIG. 6A shows a TEM image of the particles prepared from the
dC16-1CW-P2K-KK conjugate 5.
[0023] FIG. 6B shows the small-angle X-ray scattering (SAXS) of the
particles prepared from the dC16-1CW-P2K-KK conjugate 5 at both 4
wt. % (lower on the right) and 16 wt. % (upper on the right).
[0024] FIG. 6C shows the MALDI-TOF of the dC16-1CW-P2K-KK conjugate
5.
[0025] FIG. 7A shows a TEM image of the particles prepared from the
dC16-1CW-P2K-RGD conjugate 11.
[0026] FIG. 7B shows the small-angle X-ray scattering (SAXS) of the
particles prepared from the dC16-1CW-P2K-RGD conjugate 11 at 4 wt.
%.
[0027] FIG. 7C shows the MALDI-TOF of the dC16-1CW-P2K-RGD
conjugate 11.
[0028] FIG. 8A shows a TEM image of the particles prepared from the
dC16-1CW-P5K-HHH conjugate 3.
[0029] FIG. 8B shows the small-angle X-ray scattering (SAXS) of the
particles prepared from the dC16-1CW-P5K-HHH conjugate 3 at both 4
wt. % (upper on the right) and 8 wt. % (lower on the right).
[0030] FIG. 8C shows the MALDI-TOF of the dC16-1CW-P5K-HHH
conjugate 3.
[0031] FIG. 9A shows a TEM image of the particles prepared from the
dC16-BB-P2K conjugate 8.
[0032] FIG. 9B shows the MALDI-TOF of the dC16-BB-P2K conjugate
8.
[0033] FIG. 10A shows a TEM image of the particles prepared from
the PBD-1CW (end functionalization) conjugate 7.
[0034] FIG. 10B shows the MALDI-TOF of the PBD-1CW conjugate 7.
[0035] FIG. 11A shows a TEM image of the particles prepared from
the PS-BB (side functionalization) conjugate 9.
[0036] FIG. 1 lB shows the MALDI-TOF of the PS-BB conjugate 9.
[0037] FIG. 12A shows a TEM image of the particles prepared from
the sC14-1CW-P2K-DGR conjugate 13.
[0038] FIG. 12B shows the small-angle X-ray scattering (SAXS) of
the particles prepared from the sC14-1CW-P2K-DGR conjugate 13 at 16
wt. %.
[0039] FIG. 12C shows the MALDI-TOF of the sC14-1CW-P2K-DGR
conjugate 13.
[0040] FIG. 13A shows a TEM image of the particles prepared from
the sC16-1CW-P2K-KK conjugate 4.
[0041] FIG. 13B shows the small-angle X-ray scattering (SAXS) of
the particles prepared from the sC16-1CW-P2K-KK conjugate 4 at both
4 wt. % (lower on the right) and 16 wt. % (upper on the right).
[0042] FIG. 13C shows the MALDI-TOF of the sC16-1CW-P2K-KK
conjugate 4.
[0043] FIG. 14A shows a TEM image of the particles prepared from
the tC16-1CW-P2K-KK conjugate 6.
[0044] FIG. 14B shows the MALDI-TOF of the tC16-1CW-P2K-KK
conjugate 6.
[0045] FIG. 15 shows the MALDI for the dC16-SR-PEG2k conjugate
14.
[0046] FIG. 16 shows the MALDI for the dC18-1CW-PEG2k conjugate
15.
[0047] FIG. 17 shows the MALDI for the dC16-1CW-P2K-KKK.sub.F1
conjugate 17.
[0048] FIG. 18A shows the TEM of spherical nanoparticles prepared
from the dC18-1CW-P2K conjugate 15 with similar size to those
prepared from the dC16-1CW-P2K conjugate 16.
[0049] FIG. 18B shows DSC data indicating a much higher transition
temperature for the longer lipid chain with 35.degree. C., compared
with 17.degree. C. for particles prepared from the dC16-1CW-P2K
conjugate 16.
[0050] FIG. 19A shows the solution small angle x-ray scattering
analysis of the peptide amphiphile based--micelles. The small angle
x-ray scattering of particles prepared from the dC16-1CW-P2K-HHH
conjugate 1 at 5 mg/ml in 25 mM phosphate buffer. Fitting of the
data (solid line) to a core-shell spherical form factor yields a
core diameter of .about.3.8 nm, a shell thickness of .about.5.7 nm,
and polydispersity of .about.7%.
[0051] FIG. 19B is a vitreous ice cryogenic TEM of particles
prepared from the dC16-1CW-P2K-HHH conjugate 1 at 1 mg/ml in 25 mM
phosphate buffer at pH 7.5.
[0052] FIG. 19C shows a negatively stained TEM of particles
prepared from the dC16-1CW-P2K-HHH conjugate 1 at 1 mg/ml in 25 mM
phosphate buffer at pH 7.5.
[0053] FIG. 19D shows the sedimentation equilibrium analysis of
particles prepared from the dC16-1CW-P2K conjugate 16 at 100 .mu.M
in 25 mM phosphate buffer. Fitting of the data (solid line) into a
single-species model yields MW of 512 kDa corresponding to 26
trimolecular subunits.
[0054] FIG. 20A shows the thermal stability of the micelles. The
concentration dependent SAXS of samples prepared from the
dC16-1CW-P2K conjugate 16 in 25 mM phosphate buffer (16 wt %, first
from top; 8 wt %, second; 4 wt %, third; 0.5 wt %, bottom).
[0055] FIG. 20B shows the temperature dependent SAXS of samples
prepared from the dC16-1CW-P2K conjugate 16 in 25 mM phosphate
buffer upon heating from 25.degree. C. to 85.degree. C., at a ramp
rate of 1.degree. C./min and an equilibration time of 1 min prior
to measurement.
[0056] FIG. 20C shows the FRET spectra of a mixture of micelles
prepared from the dC16-1CW-P2K conjugate 16 encapsulating DIL and
DIO FRET pair dyes. The results demonstrate that minimal
fluorescence due to energy transfer after 44 hours, indicating the
absence of cargo leakage.
[0057] FIG. 21A shows the differential scanning calorimetry (DSC)
curves for the peptide amphiphiles based on peptide-polymer
conjugates. Distinct melting temperature of lipids in micelles
composed of subunits with different headgroups were observed. From
top to bottom: SR-dC16-PEG2K conjugate 14, 1coi-W-KK-dC16 conjugate
19, 1CW-dC16-PEG2K conjugate 16 and 1CW-dC16-PEG5K conjugate
20.
[0058] FIG. 21B shows DSC thermograms of 1CW-dC16-PEG2K with
different treatments. From top to bottom: freshly made, 16 hr
incubation at 20.degree. C., 1 week incubation at 20.degree. C.,
and annealed at 70.degree. C. and slowly cooled to 20.degree. C.
The schematic drawings on the right display the evolution process
of the headgroup arrangements.
[0059] FIG. 22 shows the time dependence of the fluorescence
recovery of fluorescein labeled nanoparticles upon the addition of
non-labeled nanoparticles. SR-dC 16-PEG2K refers to conjugate 14 in
Table 1, and 1CW-dC16-PEG2K refers to conjugate 16 in Table 1.
[0060] FIG. 23 shows a representative synthetic scheme for the
preparation of the amphiphilic peptide polymer conjugates.
[0061] FIG. 24 shows the critical micellar concentration
measurement by the pyrene encapsulation method.
[0062] FIG. 25 shows the size exclusion chromatography of samples
prepared from the dC16-1CW-P2K conjugate 16 at 1 mg/ml in 25 mM
phosphate buffer (pH=7.4).
[0063] FIG. 26 shows the small angle neutron scattering of samples
prepared from the dC16-1CW-P2K-HHH conjugate 1 with deuterated
alkyl tails, at 5 mg/ml in 25 mM pH 7.4 D.sub.2O phosphate buffer.
Based on best fit analysis of the data, the specific volume of the
micelle was estimated to be 0.877 ml/g.
[0064] FIG. 27 displays temperature dependent circular dichroism
analysis of samples prepared from the dC16-1CW-P2K conjugate 16.
The data shows that the peptides maintain high helicity in the
temperature range of 25.degree. C. to 85.degree. C. from
74-55%.
[0065] FIG. 28 shows SAXS profiles of samples prepared from the
dC16-1CW-P2K conjugate 16 that were freshly made (top line) and
incubated at 20.degree. C. for 16 hrs (middle line) and 2 months
(bottom line).
[0066] FIG. 29 shows a negatively stained TEM of the
dC16-1CW-P2K-HHH conjugate 1 in phosphate buffer (pH=7.4) stored at
room temperature for 9 months, showing a major fraction of
spherical micelles with .about.15 nm in diameter.
[0067] FIG. 30A shows the UV-vis spectra of heme titrations into a
.about.4.3 .mu.M solution of BB-dC16-P2K conjugate 8, a coiled-coil
4-helix bundle with four heme binding sites in the interior of the
bundle.
[0068] FIG. 30B shows the absorbance at 412 nm vs. the
[heme]/[4-helix bundle] ratio for BB-dC16-P2K conjugate 8.
[0069] Taken together, FIG. 30A and FIG. 30B show that the tertiary
structure of the peptide is retained upon self-assembly of the
amphiphiles to form micelles.
[0070] FIG. 31 shows the circular dichroism spectra of
SR-dC16-PEG2K conjugate 14 (top on right) and 1CW-dC16-PEGK
conjugate 16 (bottom on right). SR-dC16-PEG2K forms predominantly a
random coil, with a helical content of less than 5%, as compared to
1CW-dC6-PEG2K, which has a helical content of 74% under the same
conditions.
[0071] FIG. 32 shows the helix wheel of a typical de novo designed
three-helix bundle-forming peptide.
DETAILED DESCRIPTION OF THE INVENTION
I. GENERAL
[0072] The present invention provides conjugates of a peptide,
polymer and lipid moiety, where the conjugates self-assemble to
form trimers or tetramers, helix bundles, that then self-assemble
to form nanoparticles. The nanoparticles can be loaded with a
therapeutic or diagnostic agent for detection and/or treatment of a
disease or condition. The conjugates can also be modified with
another amino acid residue for binding to other biological moieties
or other particles.
[0073] A schematic drawing of the conjugate and its formation into
a higher order structure is depicted in FIG. 1. The conjugate is
composed of coiled-coil 3-helix bundle-forming peptides, with
hydrophobic di-alkyl tails conjugated to the N-terminus, and
hydrophilic PEG coupled to the side of the peptide, forming an
amphiphilic molecular building block with a cone-shaped geometry.
Upon dissolution of the conjugates in aqueous buffer, phase
separation occurs, leading to the formation of monodisperse
nanoparticle with diameters in the range of 10-20 nm. The
coiled-coils provide the chemical specificity and functionality
unavailable with liposomes and polymersomes, and have the potential
to order chemical cues laterally on the surface of the particle for
site-specific targeting. The peptide helix acts as a rigid rod and
determines the radial position of the polymer chains in a micelle.
When micelles form, the polymer chains are confined and forced into
close proximity and act like springs, affording a negative lateral
pressure that imparts enhanced stability to the discrete micelles,
much as repulsions can stabilize bulk assemblies of colloidal
particles. Chemical specificity, size, and shape can also be
tailored based on demand. Hydrophobic drugs can be encapsulated
into the lipid core of the nanoparticle, or drugs can be linked to
the peptide itself for high payloads. In a similar manner, imaging
agents and genetic material can be incorporated. Specific immune
responses can be elicited by presenting specific chemical cues on
the surface at a high areal density.
II. DEFINITIONS
[0074] "Conjugate" refers to a compound having a polymer, peptide
and lipid moiety all linked together. The conjugates are capable of
self-assembling to form helix bundles. The helix bundles are
prepared from 2 to 6 conjugates, typically 3 or 4.
[0075] "Polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. All three terms apply to amino acid polymers in which one
or more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymers. As used herein, the terms encompass amino acid
chains of any length, including full-length proteins, wherein the
amino acid residues are linked by covalent peptide bonds. The
peptides of the present invention are helical in structure and form
coiled-coil tertiary protein structure. The formation of
coiled-coil tertiary structure provides a structural scaffold to
position conjugated polymers and restrict the shape of individual
sub-units for the nanoparticle. The helices also enhances the
rigidity of the sub-unit and enable the geometric packing in a
manner similar to that of virus particles.
[0076] "Polymer" refers to a macromolecule having repeating units
connected by covalent bonds. Polymers can be hydrophilic,
hydrophobic or amphiphilic. Hydrophilic polymers are substantially
miscible with water and include, but are not limited to,
polyethyleneglycol. Hydrophobic polymers are substantially
immiscible with water and include, but are not limited to,
polybutadiene and polystyrene. Amphiphilic polymers have both
hydrophilic and hydrophobic properties and are typically block
copolymers of a hydrophilic and a hydrophobic polymer. Polymers
include homopolymers, random copolymers, block copolymers, and
others. Specific polymers useful in the present invention include
polyethyleneglycol, N-isopropylacrylamide (NIPAM), polybutadiene
and polystyrene, among others.
[0077] "Hydrophobic moiety" refers to polymers or small molecules
that are hydrophobic. Examples of hydrophobic moieties include, but
are not limited to, hydrophobic polymers such as polybutadiene and
polystyrene, as well as the lipid moieties of the present
invention.
[0078] "Lipid moiety" refers to a moiety having at least one lipid.
Lipids are small molecules having hydrophobic or amphiphilic
properties and are useful for preparation of vesicles, micelles and
liposomes. Lipids include, but are not limited to, fats, waxes,
fatty acids, cholesterol, phospholipids, monoglycerides,
diglycerides and triglycerides. The fatty acids can be saturated,
mono-unsaturated or poly-unsaturated. Examples of fatty acids
include, but are not limited to, butyric acid (C4), caproic acid
(C6), caprylic acid (C8), capric acid (C10), lauric acid (C12),
myristic acid (C14), palmitic acid (C16), palmitoleic acid (C16),
stearic acid (C18), isostearic acid (C18), oleic acid (C18),
vaccenic acid (C18), linoleic acid (C 18), alpha-linoleic acid (C
18), gamma-linolenic acid (C 18), arachidic acid (C20), gadoleic
acid (C20), arachidonic acid (C20), eicosapentaenoic acid (C20),
behenic acid (C22), erucic acid (C22), docosahexaenoic acid (C22),
lignoceric acid (C24) and hexacosanoic acid (C26). The lipid moiety
can include several fatty acid groups using branching groups such
as lysine and other branched amines.
[0079] "Anthracycline" refers to natural products of Streptomyces
peucetius and related derivatives. Anthracyclines are glycosides
containing an amino sugar and a fused, tetracyclic aglycone. Many
anthracyclines demonstrate antibiotic and antineoplastic activity.
Examples of anthracyclines include, but are not limited to,
daunorubicin, doxorubicin, epirubicin, and idarubicin.
[0080] "Macrolide" refers to compounds characterized by a large
(typically 14-to-16-membered) lactone ring substituted with pendant
deoxy sugars. Many macrolides demonstrate antibiotic and
immunomodulatory activity. Examples of macrolides include, but are
not limited to, rapamycin, clarithromycin, and erythromycin.
[0081] "Therapeutic agent" refers to an agent capable of treating
and/or ameliorating a condition or disease. Therapeutic agents
include, but are not limited to, compounds, drugs, peptides,
oligonucleotides, DNA, antibodies, and others.
[0082] "Diagnostic agent" refers to an agent capable of diagnosing
a condition or disease. Diagnostic agents include, but are not
limited to, dyes and radiolabels.
[0083] "Nucleic acid," "oligonucleotide," and "polynucleotide"
refer to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA)
and polymers thereof in either single- or double-stranded form.
Unless specifically limited, the term encompasses nucleic acids
containing known analogues of natural nucleotides that have similar
binding properties as the reference nucleic acid and are
metabolized in a manner similar to naturally occurring nucleotides.
The term nucleic acid is used interchangeably with gene, cDNA, and
mRNA encoded by a gene.
[0084] "Contacting" refers to the process of bringing into contact
at least two distinct species such that they can react. It should
be appreciated, however, the resulting reaction product can be
produced directly from a reaction between the added reagents or
from an intermediate from one or more of the added reagents which
can be produced in the reaction mixture.
[0085] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxygluatmate, and
O-phosphoserine.
[0086] "Amino acid analogs" refers to compounds that have the same
basic chemical structure as a naturally occurring amino acid, i.e.,
an .alpha. carbon that is bound to a hydrogen, a carboxyl group, an
amino group, and an R group, e.g., homoserine, norleucine,
methionine sulfoxide, methionine methyl sulfonium. Such analogs
have modified R groups (e.g., norleucine) or modified peptide
backbones, but retain the same basic chemical structure as a
naturally occurring amino acid.
[0087] "Unnatural amino acids" are not encoded by the genetic code
and can, but do not necessarily have the same basic structure as a
naturally occurring amino acid. Unnatural amino acids include, but
are not limited to azetidinecarboxylic acid, 2-aminoadipic acid,
3-aminoadipic acid, beta-alanine, aminopropionic acid,
2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid,
2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric
acid, 2-aminopimelic acid, tertiary-butylglycine,
2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid,
2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine,
homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline,
4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine,
N-methylglycine, N-methylisoleucine, N-methylpentylglycine,
N-methylvaline, naphthalanine, norvaline, ornithine, pentylglycine,
pipecolic acid and thioproline.
[0088] "Amino acid mimetics" refers to chemical compounds that have
a structure that is different from the general chemical structure
of an amino acid, but that functions in a manner similar to a
naturally occurring amino acid.
[0089] Amino acids may be referred to herein by either the commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, may be referred to by their commonly accepted
single-letter codes.
[0090] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, "conservatively modified variants" refers to those
nucleic acids that encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein that encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid that encodes a polypeptide is implicit in each described
sequence.
[0091] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid (i.e., hydrophobic, hydrophilic,
positively charged, neutral, negatively charged). Exemplified
hydrophobic amino acids include valine, leucine, isoleucine,
methionine, phenylalanine, and tryptophan. Exemplified aromatic
amino acids include phenylalanine, tyrosine and tryptophan.
Exemplified aliphatic amino acids include serine and threonine.
Exemplified basic amino acids include lysine, arginine and
histidine. Exemplified amino acids with carboxylate side-chains
include aspartate and glutamate. Exemplified amino acids with
carboxamide side chains include asparagines and glutamine.
Conservative substitution tables providing functionally similar
amino acids are well known in the art. Such conservatively modified
variants are in addition to and do not exclude polymorphic
variants, interspecies homologs, and alleles of the invention.
[0092] The following eight groups each contain amino acids that are
conservative substitutions for one another: [0093] 1) Alanine (A),
Glycine (G); [0094] 2) Aspartic acid (D), Glutamic acid (E); [0095]
3) Asparagine (N), Glutamine (Q); [0096] 4) Arginine (R), Lysine
(K); [0097] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine
(V); [0098] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
[0099] 7) Serine (S), Threonine (T); and [0100] 8) Cysteine (C),
Methionine (M) [0101] (see, e.g., Creighton, Proteins (1984)).
[0102] "Helix bundle" refers to a structure formed by the
self-assembly of a plurality of conjugates of the present
invention, where the hydrophobic moieties and peptides of each
conjugate are aligned with each other.
III. CONJUGATES, HELIX BUNDLES AND PARTICLES
[0103] The present invention provides conjugates of a peptide,
polymer and lipid moiety, where the conjugates self-assemble to
form trimers or tetramers of helix bundles, that then self-assemble
to form nanoparticles.
[0104] In some embodiments, the present invention provides a
conjugate having a peptide with from about 10 to about 100 amino
acids, wherein the peptide adopts a helical structure. The
conjugate also includes a first polymer covalently linked to the
peptide, and a hydrophobic moiety covalently linked to the
N-terminus of the peptide, wherein the hydrophobic moiety comprises
a second polymer or a lipid moiety.
[0105] Peptides useful in the conjugates of the present invention
are those that adopt a helical conformation. The peptides can be of
any suitable length, such as from about 10 to about 1000 amino
acids, or from about 10 to about 500 amino acids, or from about 10
to about 100 amino acids. In some embodiments, the peptide can be
SEQ ID NO: 1 (1CW), SEQ ID NO: 2 (BB) , SEQ ID NO: 4 (SR), and SEQ
ID NO: 5 (1coi-W).
[0106] FIG. 32 shows the helix wheel of a typical de novo designed
three-helix bundle. The peptide primary structure is characterized
by a heptad periodicity, -abcdefg-. Helix bundle formation is
driven by the hydrophobic interactions between amino acids at
positions a and d of each helix, forming a hydrophobic core. The
bundle is further stabilized by the salt bridges between amino
acids at positions e and g of adjacent helices.
[0107] In a preferred embodiment, the present invention comprises
peptide sequences that self-associate. In other embodiment, the
peptide sequence can be a de novo designed 3-helix bundle peptide,
such as, but not limited to SEQ ID NO: 1 (1CW). In particular
aspects, additional 1-50 amino acids can be appended to the
C-terminus of the peptide without interfering with micelle
formation. In some embodiments, the peptide includes an additional
1-25 amino acids at the C-terminus, preferably 1-10, more
preferably 1-5. In another embodiment, the peptide sequence can be
a control peptide sequence that form random coil, such as, but not
limited to SEQ ID NO: 4 (SR). The peptide can be designed based on
SEQ ID NO:5 (1coi-W), and have similar characteristics including PI
and hydrophobicity. In yet another embodiment, the peptide sequence
can be a heme-binding peptide that is able to form 4-helix bundles
such as SEQ ID NO: 2 (BB).
[0108] The conjugates of the present invention also include a first
polymer. The first polymer can be any suitable polymer. Exemplary
first polymers include hydrophilic, hydrophobic and amphiphilic
polymers. Some polymers useful as the first polymer of the present
invention include, but are not limited to, polyethyleneglycol (PEG
or P), poly(N-isopropylacrylamide) (NIPAM), polybutadiene (PBD) and
polystyrene (PS). In some embodiments, the first polymer is a
hydrophilic polymer. Hydrophilic polymers are miscible with water,
and include, but are not limited to, polyethyleneglycol, NIPAM, and
cellulose. In some other embodiments, the first polymer is
polyethyleneglycol.
[0109] The first polymer can be linked to any point of the peptide,
such as the N-terminus, the C-terminus and at any amino acid along
the peptide chain. The first polymer can be linked to the peptide
via covalent, ionic and other attachment means. In some
embodiments, the first polymer is linked to the peptide via
covalent bonds. In other embodiments, the first polymer is linked
to the peptide at an amino acid other than the N- or C-terminus.
Any suitable covalent linkage is useful for attaching the first
polymer to the peptide. For example, the covalent linkage can be
via an ester, amide, ether, thioether or carbon linkage. In some
embodiments, the first polymer can be modified with a maleimide
that reacts with a sulfhydryl group of the peptide, such as on a
cysteine. In other embodiments, the first polymer is linked to the
peptide via click chemistry, by reaction of an azide and an alkyne
to form a triazole ring.
[0110] In some embodiments, the hydrophobic moiety can be a second
polymer. Polymers useful as the hydrophobic moiety include
hydrophobic polymers which include, but are not limited to,
polybutadiene, polystyrene, polyacrylates, polymethacrylates,
polydiacetylene and others. In some other embodiments, the
hydrophobic moiety can be polybutadiene.
[0111] In other embodiments, the hydrophobic moiety can be a lipid
moiety. Lipid moieties useful in the present invention include from
1 to 20 long alkyl chains, from 1 to 10 alkyl chains, or from 1 to
6 alkyl chains, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alkyl chains.
The lipid moieties can be prepared from fatty acids, which include,
but are not limited to, capric acid (C10), lauric acid (C12),
myristic acid (C14), palmitic acid (C16), palmitoleic acid (C16),
stearic acid (C18), isostearic acid (C18), oleic acid (C18),
vaccenic acid (C18), linoleic acid (C 18), alpha-linoleic acid (C
18), gamma-linolenic acid (C 18), arachidic acid (C20), gadoleic
acid (C20), arachidonic acid (C20), eicosapentaenoic acid (C20),
behenic acid (C22), erucic acid (C22), docosahexaenoic acid (C22),
lignoceric acid (C24) and hexacosanoic acid (C26).
[0112] Exemplary alkyl groups in the lipid moieties include
C.sub.10-20 alkyl chains, such as C.sub.10, C.sub.12, C.sub.14,
C.sub.16, C.sub.18, or C.sub.20 alkyl groups. The alkyl groups can
be saturated or partially unsaturated. In some embodiments, the
lipid moieties have at least one C.sub.14 alkyl group, or at least
one C.sub.16 alkyl group. When the lipid moieties include more than
one alkyl group, the lipid moiety also includes a branched linker
providing for attachment of multiple alkyl groups. The branched
linkers useful in the present invention include, but are not
limited to, lysine, glutamic acid and other branched amines and
carboxylic acids. In some embodiments, the lipid moiety includes
from 1 to 6 C.sub.10-20 alkyl groups. The lipid moiety can include
1, 2, 3, 4, 5 or 6 C.sub.10-20 alkyl groups. In other embodiments,
the lipid moiety includes 1, 2, or 4 C.sub.10-20 alkyl groups. In
still other embodiments, the lipid moiety includes 1 C.sub.10-20
alkyl group. In yet other embodiments, the lipid moiety includes 2
C.sub.10-20 alkyl groups.
[0113] The hydrophobic moiety can be attached to the peptide at any
useful point on the peptide, such as at the N-terminus, C-terminus,
or anywhere along the length of the peptide. In some embodiments,
the hydrophobic moiety is linked to the peptide at the
N-terminus.
[0114] When the first polymer is linked to the peptide at a point
other than the N- or C-terminus, and the hydrophobic moiety is
linked to the N-terminus, the conjugates of the present invention
can also include a component linked to the C-terminus. The
component at the C-terminus can be any useful binding or labeling
moiety which can include, but is not limited to, an amino acid
residue, a oligonucleotide, a polypeptide, an antibody, a
diagnostic agent, a therapeutic agent, a polymer, and others. In
some embodiments, the conjugate includes an amino acid residue
linked to the C-terminus of the peptide. The amino acid residue can
have any suitable number of amino acids, such as from 2 to about
100, or from 2 to about 50, or from 2 to about 20 amino acids. In
other embodiments, the amino acid residue can be GGG, HHH, KK, EE,
RGD and AYSSGAPPMPPF, and combinations thereof. Other amino acid
residues are useful in the conjugates of the present invention.
[0115] In another embodiment, the conjugate includes the peptide of
SEQ ID NO: 1 (1CW), polyethyleneglycol as the first polymer, the
hydrophobic moiety having lysine and two C.sub.16 alkyl chains, and
an amino acid residue of from 2 to about 20 amino acids covalently
linked to the C-terminus of the peptide. In some other embodiments,
the conjugate includes the peptide of SEQ ID NO: 1 (1CW),
polyethyleneglycol as the first polymer, the hydrophobic moiety
having lysine and two C.sub.18 alkyl chains, and an amino acid
residue of from 2 to about 20 amino acids covalently linked to the
C-terminus of the peptide.
[0116] In another embodiment, the amphiphile is constructed by
covalently linking polyethylene glycol (PEG) of 2000 Da (PEG2k or
P2K) to the Cys14 of a 3-helix bundle-forming peptide of SEQ ID
NO:1 (1CW). Two C16 alkyl chains are attached to the peptide
N-terminus with a (6)-amino-hexanoic acid linker inserted between
the peptide and the double C16 tail. In another embodiment the
peptide amphiphile of SEQ ID NO: 5 (1coi-W) can be unconjugated to
any polymer.
[0117] The present invention also provides helix bundles, formed
from the self-assembly of a plurality of conjugates. The helix
bundles can be formed from 2, 3, 4, 5, 6, 7, 8, 9 or 10 conjugates.
In some embodiments, the present invention provides a helix bundle
having from 2 to 6 conjugates of the present invention. In other
embodiments, the helix bundles includes 3 conjugates. In some other
embodiments, the helix bundle includes 4 conjugates.
[0118] The present invention also provides particles formed from
the self-assembly of the helix bundles, such that the hydrophobic
moiety forms a micellar structure having a hydrophobic pocket, and
the peptide and first polymer are on the exterior of the micelle
formed by the hydrophobic moiety. The particles include any
suitable number of conjugates. In some embodiments, the present
invention provides a particle having from about 20 to about 200
conjugates of the present invention. The particles can be of any
suitable size. For example, the particles can be from about 5 nm to
about 500 nm in diameter, or from about 5 to about 100 nm in
diameter, or from about 5 nm to about 50 nm in diameter, or from
about 5 nm to about 25 nm in diameter.
[0119] The particles of the present invention can include cargo in
the hydrophobic interior of the particle. Cargo useful in the
particles of the present invention include, but are not limited to,
a therapeutic agent, a diagnostic agent, DNA and an
oligonucleotide. Examples of therapeutic agents include, but are
not limited to, anthracyclines (such as doxorubicin, daunorubicin,
epirubicin, and the like), macrolides (such as rapamycin,
fujimycin, pimecrolimus, and the like), alkylating agents (such as
temozolomide, procarbazine, altretamine, and the like), taxanes,
and vinca alkaloids. Examples of diagnostic agents include, but are
not limited to, chromophores, fluorophores, and radionuclides. The
conjugates, helix bundles and particles of the present invention
can be linked to other particles, such as gold nanoparticles and
magnetic nanoparticles that are typically a few nanometers in
diameter for imaging and manipulation purposes. In some
embodiments, the invention provides particles as described above,
wherein each additional agent is independently selected from a
fluorophore, a radionuclide, an anthracycline, and a macrolide. In
some embodiments, each additional agent is independently selected
from doxorubicin and rapamycin. Alternatively, the additional
agents be covalently or noncovalently bound to one of, a
combination of, or all of the peptide component, the first
polymeric component, and the second polymeric component of the
amphiphilic conjugates.
[0120] The conjugates, helix bundles and particles of the present
invention can be linked to other particles, such as gold
nanoparticles and magnetic nanoparticles that are typically a few
nanometers in diameter for imaging and manipulation purposes.
TABLE-US-00001 TABLE 1 Conjugates First C-terminus Amino Particle
Size Conjugate N-terminus.sup.1 Peptide.sup.2 Polymer.sup.3 Acid
Residue (nm) 1 dC16 1CW PEG2K HHH 16 nm 2 dC16 1coi-W -- HHH 8-10
nm 3 dC16 1CW PEG5K HHH 20-50 nm 4 sC16 1CW PEG2K KK 10-13 nm 5
dC16 1CW PEG2K KK 14-17 nm 6 tC16 1CW PEG2K KK nanorods 10 nm in
diameter 7 PBD 1CW -- -- 10-13 nm 8 dC16 BB PEG2K -- 10-15 nm 9
MeC(O) BB PS -- 10-12 nm 10 dC16 1CW PEG2K EE 14-17 nm 11 dC16 1CW
PEG2K RGD 14-17 nm 12 dC16 1CW PEG2K GB.sup.4 14-17 nm 13 sC14 1CW
PEG2K DGR 10-13 nm 14 dC16 SR PEG2K -- 10-15 nm 15 dC18 1CW PEG2K
-- 12-15 nm 16 dC16 1CW PEG2K -- 10-15 nm 17 dC16 1CW PEG2K
KK(K.sub.Fl).sup.5 -- 18 dC16 SR PEG2K KK(K.sub.Fl).sup.5 -- 19
dC16 1coi-W PEG2K KK -- 20 dC16 1CW PEG5K -- -- .sup.1The "s", "d"
and "t" refers to the number of fatty acid chains in the lipid
moiety: 1, 2, and 4, respectively. The "C16" refers to the number
of carbons in the fatty acid chain, such as myristic acid (C14)
palmitic acid (C16) and stearic acid (C18). PBD is polybutadiene.
.sup.21CW is SEQ ID NO: 1; BB is SEQ ID NO: 2; SR is SEQ ID NO: 4;
1coi-W is SEQ ID NO: 5. Conjugates 1-6, 10-12, and 17-18 contain a
GGG linker sequence between the peptide and the C-terminus amino
acid residue. .sup.3PEG is polyethyleneglycol; PS is polystyrene.
Polymer attached at Cys-14. .sup.4GB is AYSSGAPPMPPF, SEQ ID NO: 3.
.sup.5K.sub.Fl is lysine with carboxyfluorescein conjugated to the
side chain .epsilon.-amino group. As used throughout the instant
application, the conjugates of the present invention can be written
in shorthand as dC16-1CW-PEG2K or 1CW-dC16-PEG2K or any other
combination of the three components.
IV. METHODS OF PREPARING NANOPARTICLES
[0121] The nanoparticles of the present invention can be prepared
by any suitable method known to one of skill in the art. For
example, the nanoparticles can be prepared by first dissolving the
conjugates in a suitable solvent at any concentration from about 1
nM to about 1M, or from about 1 .mu.M to about 100 mM, or from
about 1 mM to about 100 mM. Alternatively, the conjugates can be
dissolved to form from about 0.1 to about 50 wt. % of the solution,
or from about 1 to about 50 wt. %, or from about 1 to about 25 wt.
%. The conjugates self-assemble to form the helix bundles of the
present invention. The helix bundles then self-assemble to form the
particles. In some embodiments, the present invention provides a
method of forming particles of the present invention by contacting
a plurality of conjugates of the present invention such that the
conjugates self-assemble to form the particles of the present
invention.
[0122] In an aqueous solvent, the conjugates of the present
invention can self-assemble such that the hydrophilic portion is
oriented towards the exterior of the nanocarrier and the
hydrophobic portion is oriented towards the interior, thus forming
a micelle. When a non-polar solvent is used, an inverse micelle can
be formed where the hydrophilic portion is oriented towards the
interior of the nanocarrier and the hydrophobic portion is oriented
towards the exterior of the nanocarrier.
[0123] The present invention also provides for particles prepared
by dissolving the conjugates of the present invention at a
concentration as described above, such that the conjugates
self-assemble to form helix bundles, and then allowing the helix
bundles to self-assemble to form the particles of the present
invention.
V. METHODS FOR DRUGS DELIVERY
[0124] In some embodiments, the present invention provides a method
for delivering a diagnostic or therapeutic agent to a subject
comprising administering a particle to the subject. In some
embodiments, the particle encapsulates the diagnostic or
therapeutic agent. In other embodiments, the diagnostic or
therapeutic agent is conjugated or coupled to the particle of the
present invention. Thus, the particle includes from about 20 to
about 200 conjugates of the present invention and the diagnostic or
therapeutic agent to be delivered. In some embodiments, the
therapeutic agent is selected from the group consisting of
doxorubicin, temzolomide, and rapamycin.
[0125] Delivery of the therapeutic agent can be conducted such that
drug-loaded micelles selectively accumulate at a desired site in a
subject, such as a specific organ or a tumor. In some cases,
micelle accumulation at a target site may be due to the enhanced
permeability and retention characteristics of certain tissues such
as cancer tissues. Accumulation in such a manner can arise, in
part, from the micelle size and may not require special targeting
functionality. In other cases, the micelles of the present
invention can also include ligands for active targeting as
described above. Target delivery can also be accomplished by
administering drug-loaded micelles directed to a desired site. In
some embodiments, delivery of a therapeutic agent can include
administering a particle of the present invention via intra-tumoral
infusion.
[0126] The nanoparticles of the present invention can be used to
deliver any suitable cargo in a targeted or untargeted fashion.
Suitable cargo includes, but is not limited to, vaccines, nucleic
acids such as DNA or RNA, peptides, proteins, imaging agents, and
drugs. The nanoparticles of the present invention are also useful
for gene therapy, the administration of an expressed or expressible
nucleic acid to a subject.
[0127] The nanocarrier cargo can be encapsulated within the
nanocarrier
Targeting Agents
[0128] Generally, the targeting agents of the present invention can
associate with any target of interest, such as a target associated
with an organ, tissues, cell, extracellular matrix, or
intracellular region. In certain embodiments, a target can be
associated with a particular disease state, such as a cancerous
condition. In some embodiments, the targeting component can be
specific to only one target, such as a receptor. Suitable targets
can include but are not limited to a nucleic acid, such as a DNA,
RNA, or modified derivatives thereof. Suitable targets can also
include but are not limited to a protein, such as an extracellular
protein, a receptor, a cell surface receptor, a tumor-marker, a
transmembrane protein, an enzyme, or an antibody. Suitable targets
can include a carbohydrate, such as a monosaccharide, disaccharide,
or polysaccharide that can be, for example, present on the surface
of a cell.
[0129] In certain embodiments, a targeting agent can include a
target ligand, a small molecule mimic of a target ligand, or an
antibody or antibody fragment specific for a particular target. In
some embodiments, a targeting agent can further include folic acid
derivatives, B-12 derivatives, integrin RGD peptides, NGR
derivatives, somatostatin derivatives or peptides that bind to the
somatostatin receptor, e.g., octreotide and octreotate, and the
like. The targeting agents of the present invention can also
include an aptamer. Aptamers can be designed to associate with or
bind to a target of interest. Aptamers can be comprised of, for
example, DNA, RNA, and/or peptides, and certain aspects of aptamers
are well known in the art. (See. e.g., Klussman, S., Ed., The
Aptamer Handbook, Wiley-VCH (2006); Nissenbaum, E. T., Trends in
Biotech. 26(8): 442-449 (2008)).
Therapeutic Agents
[0130] The therapeutic agent or agents used in the present
invention can include any agent directed to treat a condition in a
subject. In general, any therapeutic agent known in the art can be
used, including without limitation agents listed in the United
States Pharmacopeia (U.S.P.), Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 10.sup.th Ed., McGraw Hill,
2001; Katzung, Ed., Basic and Clinical Pharmacology,
McGraw-Hill/Appleton & Lange, 8.sup.th ed., Sep. 21, 2000;
Physician's Desk Reference (Thomson Publishing; and/or The Merck
Manual of Diagnosis and Therapy, 18.sup.th ed., 2006, Beers and
Berkow, Eds., Merck Publishing Group; or, in the case of animals,
The Merck Veterinary Manual, 9.sup.th ed., Kahn Ed., Merck
Publishing Group, 2005; all of which are incorporated herein by
reference.
[0131] Therapeutic agents can be selected depending on the type of
disease desired to be treated. For example, certain types of
cancers or tumors, such as carcinoma, sarcoma, leukemia, lymphoma,
myeloma, and central nervous system cancers as well as solid tumors
and mixed tumors, can involve administration of the same or
possibly different therapeutic agents. In certain embodiments, a
therapeutic agent can be delivered to treat or affect a cancerous
condition in a subject and can include chemotherapeutic agents,
such as alkylating agents, antimetabolites, anthracyclines,
alkaloids, topoisomerase inhibitors, and other anticancer agents.
In some embodiments, the agents can include antisense agents,
microRNA, siRNA and/or shRNA agents.
[0132] Therapeutic agents can include an anticancer agent or
cytotoxic agent including but not limited to avastin, doxorubicin,
temzolomide, rapamycin, platins such as cisplatin, oxaliplatin and
carboplatin, cytidines, azacytidines, 5-fluorouracil (5-FU),
gemcitabine, capecitabine, camptothecin, bleomycin, daunorubicin,
vincristine, topotecane or taxanes, such as paclitaxel and
docetaxel.
[0133] Therapeutic agents of the present invention can also include
radionuclides for use in therapeutic applications. For example,
emitters of Auger electrons, such as .sup.111In, can be combined
with a chelate, such as diethylenetriaminepentaacetic acid (DTPA)
or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),
and included in a nanoparticle to be used for treatment. Other
suitable radionuclide and/or radionuclide-chelate combinations can
include but are not limited to beta radionuclides (.sup.177Lu,
.sup.153Sm, .sup.88/90Y ) with DOTA, .sup.64Cu-TETA,
.sup.188/186Re(CO).sub.3-IDA; .sup.188/186Re(CO)triamines (cyclic
or linear), .sup.188/186Re(CO).sub.3-Enpy2, and
.sup.188/186Re(CO).sub.3-DTPA.
Diagnostic Agents
[0134] A diagnostic agent used in the present invention can include
any diagnostic agent known in the art, as provided, for example, in
the following references: Armstrong et al., Diagnostic Imaging,
5.sup.th Ed., Blackwell Publishing (2004); Torchilin, V. P., Ed.,
Targeted Delivery of Imaging Agents, CRC Press (1995);
Vallabhajosula, S., Molecular Imaging: Radiopharmaceuticals for PET
and SPECT, Springer (2009). A diagnostic agent can be detected by a
variety of ways, including as an agent providing and/or enhancing a
detectable signal that includes, but is not limited to,
gamma-emitting, radioactive, echogenic, optical, fluorescent,
absorptive, magnetic or tomography signals. Techniques for imaging
the diagnostic agent can include, but are not limited to, single
photon emission computed tomography (SPECT), magnetic resonance
imaging (MRI), optical imaging, positron emission tomography (PET),
computed tomography (CT), x-ray imaging, gamma ray imaging, and the
like.
[0135] In some embodiments, a diagnostic agent can include
chelators that bind to metal ions to be used for a variety of
diagnostic imaging techniques. Exemplary chelators include but are
not limited to ethylenediaminetetraacetic acid (EDTA), [4-(1,4,8,
11-tetraazacyclotetradec-1-yl) methyl]benzoic acid (CPTA),
cyclohexanediaminetetraacetic acid (CDTA),
ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA),
diethylenetriaminepentaacetic acid (DTPA), citric acid,
hydroxyethyl ethylenediamine triacetic acid (HEDTA), iminodiacetic
acid (IDA), triethylene tetraamine hexaacetic acid (TTHA), 1,4,7,
10-tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonic acid)
(DOTP), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid
(TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
(DOTA), and derivatives thereof.
[0136] A radioisotope can be incorporated into some of the
diagnostic agents described herein and can include radionuclides
that emit gamma rays, positrons, beta and alpha particles, and
X-rays. Suitable radionuclides include but are not limited to
.sup.225Ac, .sup.72As, .sup.211At, .sup.11B, .sup.128Ba,
.sup.212Br, .sup.75Br, .sup.77Br, .sup.14C, .sup.109Cd, .sup.62Cu,
.sup.64Cu, .sup.67Cu, .sup.18F, .sup.67Ga, .sup.68Ga, .sup.3H,
.sup.123I, .sup.125I, .sup.130I, .sup.131I, .sup.111In, .sup.177Lu,
.sup.13N, .sup.15O, .sup.32P, .sup.33P, .sup.212Pb, .sup.103Pd,
.sup.186Re, .sup.188Re, .sup.47Sc, .sup.153Sm, .sup.89Sr,
.sup.99mTc, .sup.88Y and .sup.90Y. In certain embodiments,
radioactive agents can include .sup.111In-DTPA,
.sup.99mTc(CO).sub.3-DTPA, .sup.99mTc(CO).sub.3-ENPy2,
.sup.62/64/67Cu-TETA, .sup.99mTc(CO).sub.3-IDA, and
.sup.99mTc(CO).sub.3triamines (cyclic or linear). In other
embodiments, the agents can include DOTA and its various analogs
with .sup.111In, .sup.177Lu, .sup.153Sm, .sup.88/90Y,
.sup.62/64/67Cu, or .sup.67/68Ga. In some embodiments, the micelles
can be radiolabeled, for example, by incorporation of chelating
groups, such as DTPA-lipid, as provided in the following
references: Phillips et al., Wiley Interdisciplinary Reviews:
Nanomedicine and Nanobiotechnology, 1(1): 69-83 (2008); Torchilin,
V. P. & Weissig, V., Eds. Liposomes 2nd Ed.: Oxford Univ. Press
(2003); Elbayoumi, T. A. & Torchilin, V. P., Eur. J. Nucl. Med.
Mol. Imaging 33:1196-1205 (2006); Mougin-Degraef, M. et al., Int'l
J. Pharmaceutics 344:110-117 (2007).
[0137] In other embodiments, the diagnostic agents can include
optical agents such as fluorescent agents, phosphorescent agents,
chemiluminescent agents, and the like. Numerous agents (e.g., dyes,
probes, labels, or indicators) are known in the art and can be used
in the present invention. (See, e.g., Invitrogen, The Handbook--A
Guide to Fluorescent Probes and Labeling Technologies, Tenth
Edition (2005)). Fluorescent agents can include a variety of
organic and/or inorganic small molecules or a variety of
fluorescent proteins and derivatives thereof. For example,
fluorescent agents can include but are not limited to cyanines,
phthalocyanines, porphyrins, indocyanines, rhodamines,
phenoxazines, phenylxanthenes, phenothiazines, phenoselenazines,
fluoresceins, benzoporphyrins, squaraines, dipyrrolo pyrimidones,
tetracenes, quinolines, pyrazines, corrins, croconiums, acridones,
phenanthridines, rhodamines, acridines, anthraquinones,
chalcogenopyrylium analogues, chlorins, naphthalocyanines, methine
dyes, indolenium dyes, azo compounds, azulenes, azaazulenes,
triphenyl methane dyes, indoles, benzoindoles, indocarbocyanines,
benzoindocarbocyanines, and BODIPY198 derivatives having the
general structure of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene,
and/or conjugates and/or derivatives of any of these. Other agents
that can be used include, but are not limited to, for example,
fluorescein, fluorescein-polyaspartic acid conjugates,
fluorescein-polyglutamic acid conjugates, fluorescein-polyarginine
conjugates, indocyanine green, indocyanine-dodecaaspartic acid
conjugates, indocyanine-polyaspartic acid conjugates, isosulfan
blue, indole disulfonates, benzoindole disulfonate,
bis(ethylcarboxymethyl)indocyanine,
bis(pentylcarboxymethyl)indocyanine, polyhydroxyindole sulfonates,
polyhydroxybenzoindole sulfonate, rigid heteroatomic indole
sulfonate, indocyaninebispropanoic acid, indocyaninebishexanoic
acid,
3,6-dicyano-2,5-[(N,N,N',N'-tetrakis(carboxymethy)amino]pyrazine,
3,6-[(N,N,N',N'-tetrakis(2-hydroxyethyl)amino]pyrazine-2,5-dicarboxylic
acid, 3,6-bis(N-azatedino)pyrazine-2,5-dicarboxylic acid,
3,6-bis(N-morpholino)pyrazine-2,5-dicarboxylic acid,
3,6-bis(N-piperazino)pyrazine-2,5-dicarboxylic acid,
3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid,
3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid S-oxide,
2,5-dicyano-3,6-bis(N-thiomorpholino)pyrazine S,S-dioxide,
indocarbocyaninetetrasulfonate, chloroindocarbocyanine, and
3,6-diaminopyrazine-2,5-dicarboxylic acid.
[0138] One of ordinary skill in the art will appreciate that
particular optical agents used can depend on the wavelength used
for excitation, depth underneath skin tissue, and other factors
generally well known in the art. For example, optimal absorption or
excitation maxima for the optical agents can vary depending on the
agent employed, but in general, the optical agents of the present
invention will absorb or be excited by light in the ultraviolet
(UV), visible, or infrared (IR) range of the electromagnetic
spectrum. For imaging, dyes that absorb and emit in the near-IR
(.about.700-900 nm, e.g., indocyanines) are preferred. For topical
visualization using an endoscopic method, any dyes absorbing in the
visible range are suitable.
[0139] In yet other embodiments, the diagnostic agents can include
but are not limited to magnetic resonance (MR) and x-ray contrast
agents that are generally well known in the art, including, for
example, iodine-based x-ray contrast agents, superparamagnetic iron
oxide (SPIO), complexes of gadolinium or manganese, and the like.
(See, e.g., Armstrong et al., Diagnostic Imaging, 5.sup.th Ed.,
Blackwell Publishing (2004)). In some embodiments, a diagnostic
agent can include a magnetic resonance (MR) imaging agent.
Exemplary magnetic resonance agents include but are not limited to
paramagnetic agents, superparamagnetic agents, and the like.
Exemplary paramagnetic agents can include but are not limited to
gadopentetic acid, gadoteric acid, gadodiamide, gadolinium,
gadoteridol , mangafodipir, gadoversetamide, ferric ammonium
citrate, gadobenic acid, gadobutrol, or gadoxetic acid.
Superparamagnetic agents can include but are not limited to
superparamagnetic iron oxide and ferristene. In certain
embodiments, the diagnostic agents can include x-ray contrast
agents as provided, for example, in the following references: H. S
Thomsen, R. N. Muller and R. F. Mattrey, Eds., Trends in Contrast
Media, (Berlin: Springer-Verlag, 1999); P. Dawson, D. Cosgrove and
R. Grainger, Eds., Textbook of Contrast Media (ISIS Medical Media
1999); Torchilin, V. P., Curr. Pharm. Biotech. 1:183-215 (2000);
Bogdanov, A. A. et al., Adv. Drug Del. Rev. 37:279-293 (1999);
Sachse, A. et al., Investigative Radiology 32(1):44-50 (1997).
Examples of x-ray contrast agents include, without limitation,
iopamidol, iomeprol, iohexol, iopentol, iopromide, iosimide,
ioversol, iotrolan, iotasul, iodixanol, iodecimol, ioglucamide,
ioglunide, iogulamide, iosarcol, ioxilan, iopamiron, metrizamide,
iobitridol and iosimenol. In certain embodiments, the x-ray
contrast agents can include iopamidol, iomeprol, iopromide,
iohexol, iopentol, ioversol, iobitridol, iodixanol, iotrolan and
iosimenol.
Gene Therapy
[0140] The nanoparticles of the present invention can also be used
to deliver any expressed or expressible nucleic acid sequence to a
cell for gene therapy or nucleic acid vaccination. The cells can be
in vivo or in vitro during delivery. The nucleic acids can be any
suitable nucleic acid, such as deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA). Moreover, any suitable cell can be used for
delivery of the nucleic acids.
[0141] Gene therapy can be used to treat a variety of diseases,
such as those caused by a single-gene defect or multiple-gene
defects, by supplementing or altering genes within the host cell,
thus treating the disease. Typically, gene therapy involves
replacing a mutated gene, but can also include correcting a gene
mutation or providing DNA encoding for a therapeutic protein. Gene
therapy also includes delivery of a nucleic acid that binds to a
particular messenger RNA (mRNA) produced by the mutant gene,
effectively inactivating the mutant gene, also known as antisense
therapy. Representative diseases that can be treated via gene and
antisense therapy include, but are not limited to, cystic fibrosis,
hemophilia, muscular dystrophy, sickle cell anemia, cancer,
diabetes, amyotrophic lateral sclerosis (ALS), inflammatory
diseases such as asthma and arthritis, and color blindness.
[0142] For general reviews of the methods of gene therapy, see
Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu,
1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and
Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May,
1993, TIBTECH 11(5): 155-215. Methods commonly known in the art of
recombinant DNA technology which can be used in the present
invention are described in Ausubel et al. (eds.), 1993, Current
Protocols in Molecular Biology, John Wiley & Sons, NY; and
Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,
Stockton Press, NY.
Formulation and Administration
[0143] When the nanocarriers are administered to deliver the cargo
as described above, the nanocarriers can be in any suitable
composition with any suitable carrier, i.e., a physiologically
acceptable carrier. As used herein, the term "carrier" refers to a
typically inert substance used as a diluent or vehicle for a drug
such as a therapeutic agent. The term also encompasses a typically
inert substance that imparts cohesive qualities to the composition.
Typically, the physiologically acceptable carriers are present in
liquid form. Examples of liquid carriers include physiological
saline, phosphate buffer, normal buffered saline, water, buffered
water, saline, glycine, glycoproteins to provide enhanced stability
(e.g., albumin, lipoprotein, globulin, etc.), and the like. Since
physiologically acceptable carriers are determined in part by the
particular composition being administered as well as by the
particular method used to administer the composition, there are a
wide variety of suitable formulations of pharmaceutical
compositions of the present invention (See, e.g., Remington's
Pharmaceutical Sciences, 17.sup.th ed., 1989).
[0144] Prior to administration, the nanocarrier compositions can be
sterilized by conventional, well-known sterilization techniques or
may be produced under sterile conditions. Aqueous solutions can be
packaged for use or filtered under aseptic conditions and
lyophilized, the lyophilized preparation being combined with a
sterile aqueous solution prior to administration. The compositions
can contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions, such as pH
adjusting and buffering agents, tonicity adjusting agents, wetting
agents, and the like, e.g., sodium acetate, sodium lactate, sodium
chloride, potassium chloride, calcium chloride, sorbitan
monolaurate, and triethanolamine oleate. Sugars can also be
included for stabilizing the compositions, such as a stabilizer for
lyophilized compositions.
[0145] The nanocarrier compositions can be made into aerosol
formulations (i.e., they can be "nebulized") to be administered via
inhalation. Aerosol formulations can be placed into pressurized
acceptable propellants, such as dichlorodifluoromethane, propane,
nitrogen, and the like.
[0146] Suitable formulations for rectal administration include, for
example, suppositories, which includes an effective amount of a
packaged composition with a suppository base. Suitable suppository
bases include natural or synthetic triglycerides or paraffin
hydrocarbons. In addition, it is also possible to use gelatin
rectal capsules which contain a combination of the composition of
choice with a base, including, for example, liquid triglycerides,
polyethylene glycols, and paraffin hydrocarbons.
[0147] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intratumoral, intradermal, intraperitoneal, and
subcutaneous routes, include aqueous and non-aqueous, isotonic
sterile injection solutions, which can contain antioxidants,
buffers, bacteriostats, and solutes that render the formulation
isotonic with the blood of the intended recipient, and aqueous and
non-aqueous sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives.
Injection solutions and suspensions can also be prepared from
sterile powders, granules, and tablets. In the practice of the
present invention, compositions can be administered, for example,
by intravenous infusion, topically, intraperitoneally,
intravesically, or intrathecally. Parenteral administration and
intravenous administration are the preferred methods of
administration. The formulations of nanocarrier compositions can be
presented in unit-dose or multi-dose sealed containers, such as
ampoules and vials.
[0148] The pharmaceutical preparation is preferably in unit dosage
form. In such form the preparation is subdivided into unit doses
containing appropriate quantities of the active component, e.g., a
nanocarrier composition. The unit dosage form can be a packaged
preparation, the package containing discrete quantities of
preparation. The composition can, if desired, also contain other
compatible therapeutic agents.
[0149] In therapeutic use, the nanocarrier compositions including a
therapeutic and/or diagnostic agent, as described above, can be
administered at the initial dosage of about 0.001 mg/kg to about
1000 mg/kg daily. A daily dose range of about 0.01 mg/kg to about
500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, or about 1 mg/kg
to about 100 mg/kg, or about 10 mg/kg to about 50 mg/kg, can be
used. The dosages, however, can be varied depending upon the
requirements of the patient, the severity of the condition being
treated, and the nanocarrier composition being employed. For
example, dosages can be empirically determined considering the type
and stage of cancer diagnosed in a particular patient. The dose
administered to a patient, in the context of the present invention,
should be sufficient to affect a beneficial therapeutic response in
the patient over time. The size of the dose will also be determined
by the existence, nature, and extent of any adverse side-effects
that accompany the administration of a particular nanocarrier
composition in a particular patient. Determination of the proper
dosage for a particular situation is within the skill of the
practitioner. Generally, treatment is initiated with smaller
dosages which are less than the optimum dose of the nanocarrier
composition. Thereafter, the dosage is increased by small
increments until the optimum effect under circumstances is reached.
For convenience, the total daily dosage can be divided and
administered in portions during the day, if desired.
Loading of Nanocarriers
[0150] Loading of the diagnostic and therapeutic agents can be
carried out through a variety of ways known in the art, as
disclosed for example in the following references: de Villiers, M.
M. et al., Eds., Nanotechnology in Drug Delivery, Springer (2009);
Gregoriadis, G., Ed., Liposome Technology: Entrapment of drugs and
other materials into liposomes, CRC Press (2006). In some
embodiments, one or more therapeutic agents can be loaded into the
nanocarriers. Loading of nanocarriers can be carried out, for
example, in an active or passive manner. For example, a therapeutic
agent can be included during the self-assembly process of the
nanocarriers in a solution, such that the therapeutic agent is
encapsulated within the nanocarrier. In certain embodiments, the
therapeutic agent may also be embedded in the lamellar layer. In
alternative embodiments, the therapeutic agent can be actively
loaded into the nanocarriers. For example, the nanocarriers can be
exposed to conditions, such as electroporation, in which the
lamellar membrane is made permeable to a solution containing
therapeutic agent thereby allowing for the therapeutic agent to
enter into the internal volume of the liposomes.
[0151] The diagnostic and therapeutic agents can also be covalently
or ionically linked to the surface of the nanocarrier, in the
interior of the micelle, or within the lamellar layer of the
micelle.
VI. METHODS FOR DISEASE TREATMENT
[0152] In some embodiments, the present invention provides a method
for treating a subject with a disease. The method includes
administering a therapeutically effective amount of a particle to
the subject. The particle includes from about 20 to about 200
conjugates of the present invention and a therapeutic agent. Thus,
the disease is treated.
[0153] Any suitable disease can be treated using the conjugates and
particles of the present invention. Representative diseases include
cancer and Parkinson's disease, among others. Cancers contemplated
for treatment using the methods of the present invention include
leukemia, lymphoma, skin cancers (including melanomas, basal cell
carcinomas, and squamous cell carcinomas), epithelial carcinomas of
the head and neck, lung cancers (including squamous or epidermoid
carcinoma, small cell carcinoma, adenocarcinoma, and large cell
carcinoma), breast cancer, gastrointestinal tract cancers,
malignant tumors of the thyroid, sarcomas of the bone and soft
tissue, ovarian cancer, carcinoma of the fallopian tube, uterine
cancer, cervical cancer, prostatic carcinoma, testicular cancer,
bladder cancer, renal cell carcinoma, pancreatic cancer, and
hepatocellular cancer. In some embodiments, the present invention
provides a method for treating a subject with a cancer
characterized by solid tumors. In some embodiments, the disease is
selected from the group consisting of a cancer and Parkinsons's
disease. In some embodiments, the cancer is Glioblastoma
multiforme.
[0154] In some embodiments, the present invention provides a method
for treating a subject with brain cancer. Brain cancers include
gliomas, meningiomas, pituitary adenomas, and nerve sheath tumors.
In some embodiments, the brain cancer is Gliobastoma multiforme.
Gliobastoma multiforme presents variants including giant cell
glioblastoma and gliosarcoma.
[0155] Any suitable therapeutic agent is useful in combination with
the conjugates and particles of the present invention. In some
embodiments, the therapeutic agent is selected from the group
consisting of doxorubicin, temzolomide, and rapamycin. In other
embodiments, the therapeutic agent is doxorubicin.
VII. EXAMPLES
[0156] Materials. Fmoc-protected amino acids,
2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU),
2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HCTU) were purchased from EMD biosciences and
used without further purification. The side chain protecting groups
of the Fmoc-protected amino acids were as follows: Lys(Boc),
Glu(OtBu), Asp(OtBu), Cys(Trt), Arg(Pbf), His(Trt), Trp(Boc),
Gln(Trt). In addition, Lys(Fmoc) was used for the conjugation of
two palmitic acids to each peptide, and a linker, Fmoc-6-Ahx-OH
(Sigma Aldrich) was appended between the peptide and the alkyl
tails. Peptide synthesis grade diisopropylethylpropylamine (DIPEA),
trifluoroacetic acid (TFA), triisopropylsilane, diethyl ether,
HPLC-grade dimethylformamide (DMF), dichloromethane (DCM) and
acetonitrile were purchased from Fisher and used without further
purification. Piperidine and palmitic acid were purchased from
Sigma Aldrich. Negative stain reagent phosphotungstic acid was
purchased from Ted Pella and prepared as the 2 wt % stock solution
in DI water.
Example 1
Preparation of Conjugates
[0157] The peptides, referred to hereafter as 1CW
(EVEALEKKVAALECKVQALEKKVEALEHGW), BB
(GGGEIWKLHEEFLKKFEELLKLHEERLKKM), SR
(EGKAGEKAGAALKCGVQELEKGAEAGEGGW), and 1coi-W
(EVEALEKKVAALESKVQALEKKVEALEHGW), were previously described in
detail. The C-terminus of peptides can be prepared with GGG, HHH,
KK, EE, RGD and AYSSGAPPMPPF, and combinations thereof, and other
peptide sequences through Fmoc-solid phase synthesis. The scheme in
FIG. 23 is typical of the procedures used for conjugate
synthesis.
[0158] The peptides were synthesized on a Protein Technologies
Prelude solid phase synthesizer using standard 9-fluorenylmethyl
carbamate (Fmoc) protection chemistry on Wang resin (Nova Biochem),
typically at 0.05 mmol scale. The side chain protecting groups were
as follows: Lys(Boc), Glu(OtBu), Asp(OtBu), Cys(Trt), Arg(Pbf),
His(Trt), Trp-(Boc), Ser(tBu), Gln(Trt). Peptides synthesized with
additional residues appended to the C-terminus optionally include
an interstitial GGG linking sequence. For the synthesis of 1CW-PEG
conjugates, the serine at position 14 was mutated to cysteine to
facilitate conjugation of maleimide end-functionalized PEG.
Similarly for BB, the lysine at position 15 was mutated to
cysteine. For the synthesis of dC16-1CW-P2K, Fmoc-hexanoic acid was
attached between the peptide and fatty acid as a linker.
Fmoc-Lys(Fmoc)-OH was then appended to the N-terminus to allow
simultaneous coupling of palmitic acid to the amine of the lysine
side chain and the N-terminus of the peptide, thus yielding a
double alkane tail, and the cysteine at position 15 was used for
coupling of maleimide-functionalized PEG of molecular weight 2000
g/mol and 5000 g/mol (Rapp Polymere) to the middle of the peptide
sequence. tC16-1CW-P2K was synthesized with two consecutive rounds
of coupling with Fmoc-Lys(Fmoc)-OH at the N-terminus generating
four branching points to attach palmitic acids. For peptide 9,
prior to peptide cleavage from the resin, the N-terminus was
acetylated using a 1:1 (v/v) acetic anhydride: pyridine solution
for 30 min. The peptides were cleaved from the resin and
simultaneously deprotected using 90:8:2 trifluoroacetic acid
(TFA)/ethanedithiol/water for 3.5 h. Crude peptides were
precipitated in cold ether and subsequently dissolved in water and
lyophilized. Maleimide end-functionalized PEG, purchased from Rapp
Polymere (Germany), was then coupled to the cysteine residues of
the peptides, which were in white powder form, in 25 mM potassium
phosphate buffer at pH 8 for 1 h.3 PEGs of three varying molecular
weights were utilized: 750, 2000, and 5000 Da. These are referred
henceforth as PEG750, PEG2K, and PEG5K, respectively.
[0159] For preparation of BB-PEG conjugates, the lysine at position
15 was mutated to cysteine to facilitate coupling of
maleimide-functionalized PEG of molecular weight 2000 g/mol and
5000 g/mol (Rapp Polymere) to the middle of the peptide sequence.
Peptides were synthesized on a Protein Technologies Prelude solid
phase synthesizer using standard 9-fluorenylmethyl carbamate (Fmoc)
protection chemistry on PEG-PAL resin (Applied Biosystems),
typically at 0.05 mmol scale. Peptides were dissolved in phosphate
buffer (25 mM, pH=8) at a concentration of 10 mg/ml. The solution
was purged with nitrogen for 5 mins before the addition of
maleimide functionalized polyethylene glycol. PEG-maleimide was
added in 7-8 equivalents of the peptides. The mixture was stirred
at room temperature for at least overnight before HPLC
purification.
[0160] The di-lipid conjugate dC16-1CW-P2K was prepared by reacting
Fmoc-hexanoic acid was attached between the peptide and fatty acid
as a linker. Fmoc-Lys(Fmoc)-OH was then appended to the N-terminus
to allow simultaneous coupling of palmitic acid to the amine of the
lysine side chain and the N-terminus of the peptide, thus yielding
a double alkane tail. Crude conjugates were precipitated in cold
ether and subsequently dissolved in solution and lyophilized,
resulting in a white powder.
[0161] The tetra-lipid conjugate tC16-1CW-P2K was synthesized with
two consecutive rounds of coupling with Fmoc-Lys(Fmoc)-OH at the
N-terminus generating four branching points to attach palmitic
acids. Peptides were cleaved from the resin and simultaneously
deprotected using 90:8:2 trifluoroacetic acid
(TFA)/ethanediol/water for 3.5 hr.
[0162] Peptides were also synthesized on a Protein Technologies
Prelude solid phase synthesizer using standard 9-fluorenylmethyl
carbamate (Fmoc) protection chemistry on PEG-PAL resin (Applied
Biosystems), typically at 0.05 mmol scale. Fmoc-Lys(Fmoc)-OH (EMD
Bioscience) was appended to the N-terminus to allow coupling of two
palmitic acid molecules to the N-terminus of the peptide.
Palmitoylated peptides were cleaved from the resin and
simultaneously deprotected using 90:8:2 trifluoroacetic acid
(TFA)/ethanedithiol/water for 3.5 hr. Crude peptides were
precipitated in cold ether and subsequently dissolved in solution
and lyophilized, resulting in a white powder. Cysteine at position
14 facilitates the site-specific coupling of
maleimide-functionalized PEG of molecular weight 2000 g/mol or 5000
g/mol (Rapp Polymere) to the middle of the peptide sequence.
[0163] Conjugates 1, 3, 5-6, 8 10-12, and 14-16 were prepared by
the method above. No additional amino acids were used for
conjugates 14-16. Conjugate 2 was prepared as described above,
without the step of attaching the polymer to the peptide. Conjugate
4 was prepared by coupling palmitic acid to the N-terminus of the
peptide without the interstitial Fmoc-Lys(Fmoc)-OH residue.
Conjugate 13 was by coupling palmitic acid to the N-terminus of the
peptide without interstitial aminohexanoic acid and
Fmoc-Lys(Fmoc)-OH residues.
[0164] Conjugate 7 was prepared by the following method: carboxyl
terminated polybutadiene (PBD, Polymer source) was dissolved in
mixture of dichloromethane and dimethyl formamide. In the presence
of HCTU and DIPEA, PBD was reacted with the amino group at the
N-terminus of the peptide (1-CW) through solid phase synthesis.
Upon completion of coupling reaction between PBD and peptide, the
conjugate was cleaved with 95% TFA, 2.5% TIS and 2.5% H2O for 1
hour. The crude PBD-1CW was purified by HPLC and characterized by
MALDI.
[0165] Conjugate 9 was prepared using BB with polystyrene on its
side by first preparing peptide such that it could be selectively
deprotected to result in free amino groups which could be utilized
for coupling carboxy terminated polymers to the peptide chain. More
specifically, Allyloxycarbonyl (Alloc) protected lysine,
Lys(Alloc), was used as the amino acid at 15.sup.th position of the
peptide chain. The removal of Alloc group was accomplished by
utilizing palladium catalyst. Resin bound peptide with the
N-terminus acylated as above was treated with tetrakis
(triphenylphosphine) palladium(0) Pd(PPh.sub.3).sub.4 catalyst and
radical trapping agent PhSiH.sub.3 in DCM. The reaction was
repeated two more times. In the next step, the resulting free amino
groups of lysine were utilized for conjugating carboxy terminated
polymer using HCTU/DIPEA chemistry. The reaction was performed at
room temperature for 48 hours. Polymer reacted resin was cleaved
and deprotected and the cleaved mixture was precipitated in cold
diethyl ether and purified by RP-HPLC. MALDI of the BB-PS conjugate
is shown in FIG. 11B. The molecular weight of the conjugate was
found to be 4720 Da.
[0166] Conjugates 17 and 18 were prepared by incorporating
allyloxycarbonyl (Alloc) protected lysine, Lys(Alloc), as the first
amino acid at the C-terminus of the peptide chain. Following
acylation of the N-terminus with linking groups and palmitic acid
as described above, the removal of the Alloc group was accomplished
by utilizing palladium catalyst. Resin bound peptide was treated
with tetrakis (triphenylphosphine) palladium(0) Pd(PPh.sub.3).sub.4
catalyst and radical trapping agent PhSiH.sub.3 in DCM. The
reaction was repeated two more times. In the next step, the
resulting free amino groups of lysine were utilized for conjugating
carboxy terminated fluorescein using HCTU/DIPEA chemistry. The
reaction was performed at room temperature for 12 hours and
repeated twice. The resin was cleaved and the cleaved mixture was
precipitated in cold diethyl ether. The crude fluorescein labeled
peptide was reacted with maleimide end functionalized PEG2000 in
phosphate buffer (pH=7.4) for overnight. The mixture was purified
by HPLC.
Example 2
Self-Assembly of Conjugates to Form Helix Bundles
[0167] Prepared as previously described in J. Y. Shu, C. Tan, W. F.
DeGrado, T. Xu, Biomacromolecules, 2008, 9(8), 2111-2117. The
peptide sequences are selected such that they form helix bundles
instantaneously upon dissolving them in aqueous buffered
solution.
Example 3
Self-Assembly of Helix Bundles to Form Nanoparticles
[0168] Lyophilized peptide-polymer conjugates were dissolved in
phosphate buffer (pH=8) at a concentration of 10 mg/ml (.about.1.5
mM). Upon sonication for 30 s, solutions were diluted to 1 mg/ml
(0.15 mM) or 0.1 mg/ml in phosphate buffer (pH=8). 5 .mu.l of the
diluted peptide solution were applied to a discharged holey
carbon-coated copper grid (Ted Pella Cu 400 mesh 01824) for 4
minutes before absorbing off excess solution using filter paper
(Whatman filter paper 1). The sample on the grid was negatively
stained with a 5 .mu.l 2% (w/v) phosphotungstic acid (adjusted to
pH=3.3 with 1 M NaOH) for 2 minutes. Excess stain solution was
wicked off. After complete drying, grids were examined by TEM at
120 kV (Philips/FEI Tecnai 12).
Example 4
Preparation of dC18-1CW-P2K (15)
[0169] To construct nanoparticles with more compact lipid packing
within hydrophobic core above room temperature, a new
peptide-polymer conjugate was prepared with two stearic acid chains
attached at the N-terminus of 1CW (referred as dC18-1CW-P2K,
Conjugate 15). TEM (FIG. 18A) showed the formation of spherical
nanoparticles with similar size to dC16-1CW-P2K. However, DSC study
(FIG. 18B) showed a much higher transition temperature for the
longer lipid chain with 35.degree. C., compared with 17.degree. C.
for dC16-1CW-P2K. No additional amino acids were attached at the
C-terminus. TEM shows particles size about .about.15 nm in
diameter.
Example 5
Characterization of Nanoparticles
Methods
[0170] Reversed-Phase High-Pressure Liquid Chromatography
(RP-HPLC). The amphiphilic conjugates were purified using RP-HPLC
(Beckman Coulter) on a C4 column (Vydac). The flow rate was 10
ml/min for semi-preparative runs and conjugates were injected at a
concentration of 10 mg/ml. Elution was monitored with a diode array
detector at wavelengths of 220 nm and 280 nm. Conjugates were
eluted with a linear AB gradient, where solvent A consisted of
water plus 0.1% (v/v) TFA and solvent B consisted of acetonitrile
plus 0.1% (v/v) TFA. A linear gradient of 30 to 100% B over 30 min
was used, with typical elution .about.85% B. Purification yield is
.about.40%.
[0171] MALDI-TOF Spectrometry. The identity and purity of the
peptides were verified by MALDI-TOF mass spectrometry using
.alpha.-cyano-4-hydroxycinnamic acid matrix. Mass spectra were
recorded on an Applied BioSystems Voyager-DE Pro.
[0172] Critical Micelle Concentration (CMC). The pyrene solubility
method was used to determine the critical micelle concentration. A
saturated solution of pyrene in PBS (.about.6.times.10.sup.-7
.mu.M) was prepared and used to dissolve the samples. Fluorescence
spectra were collected using a Jasco FP-6500 spectrofluorometer
with a bandwidth of 0.5 mm for both excitation and emission. For
fluorescence excitation spectra, .lamda..sub.em was 390 nm. When
solubilized in aqueous media at low peptide-polymer conjugate
concentrations, pyrene exhibits an excitation peak .about.333 nm.
As the concentration of the amphiphilic conjugate increases such
that micelles form, the peak at .about.333 nm shifts to .about.338
nm, which corresponds to the excitation of pyrene that has been
incorporated into the hydrophobic core of the micelles. The ratio
of the peaks at 338 and 333 nm was plotted to determine the cmc,
which corresponds to the intersection of the linear extrapolations
of the first two slopes in the data set.
[0173] Cryo Transmission Electron Microscopy. Cryo sample
preparation was done on a Vitrobot (FP5350/60). 5 .mu.l of peptide
solution were pipetted on a holey carbon grid and blotted for 2 s
to remove excess solution. The sample was quickly plunged into
liquid ethane and transferred to a cryo holder containing liquid
nitrogen. Samples were imaged on a JEOL 4000 microscope at
-177.degree. C. using low dose conditions.
[0174] Negatively Stained Transmission Electron Microscopy.
Lyophilized peptide powder was dissolved at 1 mg/ml in 25 mM
phosphate buffer at pH 7.4. 5 .mu.l of peptide solution was dropped
on a discharged holey carbon coated grid (Ted Pella 01824). After
removing excess peptide solution, 5 .mu.l of phosphotungstic acid
(2 wt %, pH=3.3) solution was then applied for 2 minutes. Samples
were dried in air and examined by a FEI Tecnai 12 transmission
electron microscope at 120 kV.
[0175] Small Angle X-ray Scattering (SAXS). SAXS was carried out at
beamline 7.3.3 at the Advanced Light Source, Lawrence Berkeley
National Laboratory. Samples were dissolved in 25 mM
KH.sub.2PO.sub.4, pH 7.4 buffer at a range of concentrations, from
0.5 wt % to 16 wt %. Samples of the lowest concentration were
measured in a homemade circulating flow cell with 0.025 mm thick
muscovite mica widows and counted for 5 s 50 times to garner the
form factor (Lipfert et al., Rev. Sci. Instrum. 77, (2006)).
Samples of higher concentration were measured in 2 mm boron-rich
thin-walled capillary tubes to investigate both the form and
structure factors. In-situ temperature studies were performed using
a capillary holder connected to a peltier device. Samples were
heated from 25.degree. C. to 85.degree. C. at a ramp rate of
1.degree. C./min and held for 1 min to ensure equilibrium before
acquisition of 10 images of 5 s exposures. The sample to detector
distance was .about.1.7 m, providing a q range of 0.01 to 0.3
.ANG..sup.-1, where q=4.pi. sin(.theta./2)/.lamda.,
.theta.=scattering angle, and .lamda.=1.24 .ANG.. The x-ray energy
was 10 keV. Scattering was collected with a PILATUS detector. 2D
diffraction patterns were radially integrated to garner a 1D
profile of the scattering intensity. Form factors were fit using
the core-shell sphere model included in the SANS software analysis
package provided by National Center for Neutron Research at
National Institute of Standards and Technology (NCNR-NIST). In more
detail, form factors were fit to a core-shell sphere model that
includes a Gaussian distribution in overall size of the micelle.
Data were fit with a limited q range between 0.04 and 0.15
.ANG..sup.-1 in order to garner the best fit in the q range of
interest. There appears to be a slight decrease in micellar size
over the first 16 hrs, as seen in the shift in the scattering
minimum at q.about.0.06 .ANG..sup.-1, possibly due to aging of the
micelles towards better subunit packing SAXS results confirmed
exceptional long-term stability of micelles, as there is no
aggregation or increases in micelle sizes over the course of 2
months.
[0176] Small Angle Neutron Scattering (SANS). SANS of micelles was
carried out at beamline CG-3 at High Flux Isotope Reactor, Oak
Ridge National Laboratory. Samples were dissolved in 25 mM
KH.sub.2PO.sub.4, pH 7.4 buffer at 5 mg/ml and measured in 1 mm
pathlength cylindrical cuvettes holding a sample volume of
.about.300 .mu.l. The sample to detector distance was .about.1.1 m,
providing a q range of 0.01 to 0.3 .ANG..sup.-1, where q=4.pi.
sin(.theta./2)/.lamda., .theta.=scattering angle, and .lamda.=6
.ANG.. Scattering was collected for 60 min on a 2D .sup.3He
detector and diffraction patterns were radially integrated to
garner a 1D profile of the scattering intensity. Form factors were
fit using a core-shell model with interfacial widths (Berndt et
al., Angew. Chem. Int. Ed., 45:1737-1741 (2006)). Fitting of the
data to a core-shell spherical form factor with interfacial widths
allowed estimation of the specific volume of the micelle to be
0.877 ml/g.
[0177] Analytical Ultracentrifugation. Sedimentation equilibrium
experiments were performed on a Beckman Optima XL-A at 25.degree.
C. with samples solubilized in 25 mM phosphate at pH 7.4. The path
length of the cells was 1.2 cm and the An-60Ti rotor was used.
Measurements at 5000, 7000, and 10000 rpm were taken after 10 h of
spinning at each speed to ensure equilibrium, which was verified by
matching the early and late data sets. The radial distribution of
absorbance was monitored at 280 nm. Sample concentrations were 100
.mu.M, and sample volumes were 120 .mu.l. The specific volume of
1CW-dC16-P2K was estimated to be 0.877 ml/g using the software
Sednterp (http://www.jphilo.mailway.com) and relying on the fit of
the SANS profile of 1CW-dC16-P2K to a core-shell model with
interfacial widths (FIG. S4) to estimate the number of water
molecules that penetrate the shell of the micelle. The density of
the buffer was 1.004 g/ml. Nonlinear global fits were made using
the UltraScan software program
(http://www.ultrascan.uthscsa.edu/).
[0178] Dynamic Light Scattering (DLS). DLS size measurements were
taken on a Malvern Zetasizer Nano-ZS with a 633 nm laser and a
scattering angle of 17.degree. to determine the hydrodynamic radius
of samples in solution. Samples were passed through 0.22 .mu.m
filters prior to the measurements.
[0179] Size Exclusion Chromatography (SEC). SEC was carried out on
a BioSep-SEC-S 3000 column (Phenomenex). The flow rate was 1 ml/min
with 25 mM phosphate buffer (pH=7.4) as the elution solvent. The
elution profile was monitored with a UV-vis detector at wavelengths
of 220 nm and 280 nm. The elution volume of the self-assembled
nanoparticles composed of 1CW-dC16-P2K is .about.6.5 ml,
corresponding to that of protein standards with a MW of 670
kDa.
[0180] Circular Dichroism (CD). Temperature dependent CD
measurements were made on a Jasco J810 spectropolarimeter. CD
spectra were collected from 260 to 190 nm at 0.2 nm intervals, a
rate of a 100 nm/min, a response time of 4 s, and a bandwidth of 1
nm. Temperature melt curves were measured using .about.200 .mu.M
solutions. The ellipticity was monitored at 222 nm as the
temperature increased from 5.degree. to 95.degree. C. in 5.degree.
C. increments at a rate of 1.degree. C./min, with a 1 min
equilibration time at each temperature before the measurement was
taken. One hundred percent helicity was estimated using the formula
[.theta.].sub.222=-40,000-[1-(2.5/n)].
[0181] Temperature dependent circular dichroism shows that peptides
maintain high helicity in the temperature range of 25.degree. C. to
85.degree. C. from 74-55%. For comparison, peptides without lipid
chain attached unfold significantly showing only .about.20%
helicity upon heating to 85.degree. C. (see, e.g., Forood et al.,
Proc. Natl. Acad. Sci. U.S.A., 90:838-842 (1993); Chen et al.,
Biochemistry, 13: 3350-3359 (1974)). Circular dichroism spectra of
SR-dC16-PEG2K and 1CW-dC16-PEG2K. SR-dC16-PEG2K forms predominantly
a random coil, with a helical content of less than 5%, as compared
to 1CW-dC16-PEG2K, which has a helical content of 74% under the
same conditions. This confirms that the scrambled peptide sequence
adopts a mostly random coil conformation, as designed.
[0182] Differential Scanning calorimetry (DSC). DSC was performed
on a VP-MicroCal MicroCal calorimeter (GE). .about.600 .mu.l of
sample and buffer were loaded into two parallel stainless steel
cells that were sealed tightly under the pressure of .about.27 psi
to prevent water evaporation during the heating cycle. The
temperature was increased from 5.degree. to 85.degree. C. at a rate
of 1.degree. C./min, with a 15 min equilibration time at 5.degree.
C. DSC thermograms were obtained after concentration normalization
and baseline correction using the Origin software provided by the
MicroCal.
[0183] Dynamics of Subunit Exchange Using Self-Quenching
Dye-labeled Micelles. Fluorescein-labeled nanoparticles (donor)
were prepared at a concentration of 16 .mu.M in 25 mM phosphate
buffer at pH 7.4. Non-labeled nanoparticles (acceptor) were
prepared at a concentration of 3.6 mM using the same buffer. The
two solutions were mixed in a 5:1 volume ratio, giving a
donor:acceptor molar ratio of 1:40. Time dependent fluorescence
intensity was recorded every 30 seconds upon mixing, with the
excitation wavelength at 488 nm and emission at 527 nm.
[0184] Forster Resonant Energy Transfer (FRET). A lipophilic FRET
pair, 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO, donor) and
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DiI, acceptor) were used to measure the energy transfer upon
mixing. DiO and DiI were dissolved in acetone to a concentration at
0.1 mg/ml, respectively. 50 .mu.l DiO and 50 .mu.l DiI were
independently added to 0.5 ml of peptide aqueous solution (1 mg/ml,
pH=7.4). After 24 hours stirring at room temperature, acetone was
evaporated with vials left open for 24 hours. The solutions were
then subject to centrifugation and spin dialysis to remove any
insoluble aggregates and soluble dyes in the supernatant. The
resulting dye-encapsulated nanoparticles were characterized by size
exclusion chromatography. Encapsulation of dye molecules within
nanoparticles were confirmed by the overlap of elution profiles
monitored at 220 nm and 490 nm, respectively for DiO, at 220 nm and
560 nm, respectively for DiI. Time dependent fluorescence intensity
was recorded for 44 hours upon mixing the nanoparticle solutions
with excitation wavelength at 488 nm.
[0185] Elastic Energy Estimation. In order to estimate the elastic
energy stored in the PEG chains when compressed in the confined
geometry of the micelle shell, we modeled the polymer chains to be
elastic springs described by an elastic spring constant,
.kappa.=3k.sub.BT/(Nb.sup.2), where k.sub.B is the Boltzmann
constant, N is the number of Kuhn monomers, and b is the Kuhn
length. The elastic energy is taken to be U=1/2.kappa.x.sup.2,
where x is the difference in radius between a PEG chain compressed
in the shell and that of an unperturbed PEG chain free in solution.
The radius of gyration of PEG of molecular weight of 2000 Da in
aqueous solution is tabulated to be .about.1.4 nm. The radius of
gyration of PEG in the micelle was estimated to be .about.0.5 nm by
comparing the conical volume available to each PEGylated 3-helix
bundle subunit and the volume occupied by the coiled-coil alone.
Both were measured using small angle x-ray scattering and small
angle neutron scattering. This yielded a stored elastic energy of
.about.10 kcal/mol of particle.
Results
[0186] Lyophilized peptide-polymer conjugates were dissolved in
phosphate buffer (pH=8) at a concentration of 10 mg/ml (.about.1.5
mM). Upon sonication for 30 s, solutions were diluted to 1 mg/ml
(0.15 mM) or 0.1 mg/ml in phosphate buffer (pH=8). 5 .mu.l of the
diluted peptide solution were applied to a discharged holey
carbon-coated copper grid (Ted Pella Cu 400 mesh 01824) for 4
minutes before absorbing off excess solution using filter paper
(Whatman filter paper 1).
[0187] Using the pyrene solubility method (Astafieva et al.,
Macromolecules, 26:7339-7352 (1993)), the critical micelle
concentration (CMC) of 1CW-dC16-PEG2K was found to be 4 .mu.M,
comparable to other peptide amphiphile systems (Mackay et al., Nat.
Mater., 8: 993-999 (2009). Above the CMC, dC16-1CW-P2K-HHH forms
uniform micelles. Solution small angle x-ray scattering (SAXS)
experiments (FIG. 19A) indicate the formation of core-shell
spherical micelles, .about.15 nm in diameter. The C16 alkyl tails
form the hydrophobic core, .about.3.8 nm in diameter, and the
1CW-PEG2K conjugates form a .about.5.7 nm thick hydrophilic shell.
FIG. 19B and C show the cryo-TEM image and the TEM image of
negatively stained dried nanoparticles, where micellar
nanoparticles can be clearly seen. The aggregation number is 78
which corresponds to 26 trimolecular subunit per micelle, as
determined by the analytical ultracentrifugation (AUC) results in
FIG. 19D and was further confirmed with size exclusion
chromatography (SEC) (see FIG. 25) for similar conjugates
dC16-1CW-PEG2K.
[0188] 1CW-dC16-PEG2K forms micelles spontaneously over a wide
range of amphiphile concentrations by simply dissolving the
lyophilized amphiphile in aqueous media. FIG. 20A shows a series of
SAXS profiles of 1CW-dC16-PEG2K solutions with concentrations
ranging from 0.5-16 wt %. Scattering profiles at q>0.08
.ANG..sup.-1 can be fit to a spherical core-shell model, similar to
that shown in FIG. 19A, confirming the integrity of individual
micelle and absence of random aggregates. As the volume fraction of
micelle increases to 34 vol % at 16 wt % of dC16-1CW-PEG2K, the
micelles start to co-assemble into structures with liquid-like
ordering reflected by the broad diffraction peak at q.about.0.035
.ANG..sup.-1 that corresponds to an inter-particle distance of
.about.18 nm.
[0189] The micelles exhibited excellent thermal stability. In-situ
SAXS profiles of a 16 wt % 1CW-dC16-PEG2K solution heated from
25.degree. C. to 85.degree. C. are shown in FIG. 20B. The peptide
helicity reduces from 74%-55%, but the headgroup remains mainly
helical (FIG. 27). The inter-particle distance decreases during
heating, due, more than likely, to an increase in micelle
concentration arising from the water condensation on the capillary
wall during the heating process. The scattering profiles for
q>0.08 .ANG..sup.-1 confirmed the formation of well-defined
micelles even at elevated temperatures. The micelles also exhibited
exceptional long-term stability with no storage requirements. The
SAXS profile of micelle solution remained the same after storing
for 2 months at room temperature (FIG. 28). In the case of
dC16-1CW-P2K-HHH micelles can still be clearly seen after 9 months
(FIG. 29).
[0190] The stability of micelles was further studied using Forster
resonant energy transfer (FRET) to quantify cargo leakage. A
lipophilic FRET pair, 3,3'-dioctadecyloxacarbocyanine perchlorate
(DiO, donor) and
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DiI, acceptor), were independently sequestered in 1CW-dC16-PEG2K
micelles. Minimal fluorescence due to energy transfer was seen and
essentially no cargo leakage was observed after over 44 hrs of
mixing at room temperature, consistent with the extremely slow
kinetics of subunit exchange (FIG. 20C).
[0191] The alkyl packing of the lipid moiety in the hydrophobic
core of the nanoparticles reflects the ordered organization of
helical peptides of the headgroups. FIG. 21A shows the differential
scanning calorimetry (DSC) curves for SR-dC16-PEG2K, 1CW-dC16,
1CW-dC16-P2K and 1CW-dC16-P5K upon heating from 5.degree. C. to
85.degree. C. All solutions were incubated at 20.degree. C. for 16
hrs before the DSC measurements. A sharp endothermic peak with a
melting temperature of 42.degree. C. can be seen for SR-dC16-PEG2K
and conjugating PEG to the side chain of a random coil does not
compromise the ordering of the alkyl chain. However, this is not
the case for designed amphiphiles based on helical peptide-polymer
conjugates. Two broad peaks centered at 17 and 32.degree. C. were
observed for 1CW-dC16-P2K and mainly one peak centered at
17.degree. C. was seen for 1CW-dC16-P5K when the molecular weight
of conjugated PEG increases to 5000 Da. Correlation of DSC results
with fluorescence self-quenching results indicate that the
exception stability of nanoparticles is achieved mainly through the
headgroup packing leading to significant repulsion energy stored
within nanoparticles, although this is realized with the sacrifice
of lipid chain packing.
[0192] dC16-1CW-PEG2K is based on a peptide that self-associates to
form 3-helix bundle. Upon dissolving, the peptide folds into a
helix instantaneously and a fraction of peptides form helix bundles
during micelle formation. As shown in FIG. 30, this was confirmed
by the heme-titration results of H10H24-dC16-PEG2K that forms
micelles, .about.12 nm in diameter. Over time, the peptides form
helix bundles via a lateral diffusion. FIG. 21 shows the DSC scans
of solutions of 1cw-dC16-PEG2K that were freshly made, incubated at
20.degree. C. for 16 hrs and 1 week, and heated to 70.degree. C.
and slowly cooled down, respectively. The endothermal peak centered
at 17.degree. C. intensifies with longer incubation and corresponds
to 1CW-dC16-PEG2K forming 3-helix bundle. This self-association
process of the headgroup is slow for 1CW-dC16-PEG2K due to the
crystallization of alkyl chains in the core and can be accelerated
by heating the solution to 70.degree. C., after which only one peak
at 17.degree. C. is seen as shown in FIG. 20. For 1CW-dC16-PEG5K,
longer PEG chain leads to a higher lateral pressure that splays the
alkyl chains and the headgroups arrange locally to form 3-helix
bundles during the incubation at 20.degree. C.
[0193] Without being bound to any particular theory, the stability
of the micelles is believed to arise from conjugation of PEG chains
to the exterior of the helix bundle. With PEG conjugation, all
micelle solutions appear to be stable for days, in contrast to
1CW-dC16 that forms large precipitates within a few hours. To
delineate the effects of protein structure, the kinetics of subunit
exchange was studied by monitoring the fluorescence recovery of a
self-quenching fluorophore, fluorescein, which was attached to the
peptide C-terminus. Fluorescein-labeled nanoparticles (donor) were
prepared at a concentration of 16 .mu.M in 25 mM phosphate buffer
at pH 7.4. Non-labeled nanoparticles (acceptor) were prepared at a
concentration of 3.6 mM using the same buffer. The two solutions
were mixed in a 11:1 volume ratio, giving a donor:acceptor molar
ratio of 1:40. Time dependent fluorescence intensity was recorded
every 30 seconds upon mixing, with the excitation wavelength at 488
nm and emission at 515 nm. Mixing preformed labeled nanoparticles
with non-labeled nanoparticles will result in fluorescence recovery
to varying degree depending on the kinetics of subunit desorption
from pre-existing nanoparticles. It was determined that even though
the hydrophobic core is mainly disordered, micelles of
1CW-dC16-PEG2K exhibit much slower subunit exchange kinetics than
that of SR-dC16-PEG2K (FIG. 22). This is strong evidence that it is
the PEG springs that stabilize micelles. The present studies show
that upon micelle formation, helical peptides determine the
position of PEG chains both radially and laterally in a micelle and
confine the PEG chains to enhance entropic repulsion.
[0194] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, one of skill in the art will appreciate that
certain changes and modifications may be practiced within the scope
of the appended claims. In addition, each reference provided herein
is incorporated by reference in its entirety to the same extent as
if each reference was individually incorporated by reference. Where
a conflict exists between the instant application and a reference
provided herein, the instant application shall dominate.
Sequences
TABLE-US-00002 [0195] SEQ ID NO: 1 EVEALEKKVAALECKVQALEKKVEALEHGW
SEQ ID NO: 2 GGGEIWKLHEEFLCKFEELLKLHEERLKKM SEQ ID NO: 3
AYSSGAPPMPPF SEQ ID NO: 4 EGKAGEKAGAALKCGVQELEKGAEAGEGGW SEQ ID NO:
5 EVEALEKKVAALESKVQALEKKVEALEHGW
Sequence CWU 1
1
5130PRTArtificial Sequencesynthetic helical coiled-coil peptide 1CW
1Glu Val Glu Ala Leu Glu Lys Lys Val Ala Ala Leu Glu Cys Lys Val1 5
10 15 Gln Ala Leu Glu Lys Lys Val Glu Ala Leu Glu His Gly Trp 20 25
30 230PRTArtificial Sequencesynthetic helical coiled-coil 4-helix
bundle heme-binding peptide BB 2Gly Gly Gly Glu Ile Trp Lys Leu His
Glu Glu Phe Leu Cys Lys Phe1 5 10 15 Glu Glu Leu Leu Lys Leu His
Glu Glu Arg Leu Lys Lys Met 20 25 30 312PRTArtificial
Sequencesynthetic C-terminal peptide GB 3Ala Tyr Ser Ser Gly Ala
Pro Pro Met Pro Pro Phe1 5 10 430PRTArtificial Sequencesynthetic
random coil peptide SR 4Glu Gly Lys Ala Gly Glu Lys Ala Gly Ala Ala
Leu Lys Cys Gly Val1 5 10 15 Gln Glu Leu Glu Lys Gly Ala Glu Ala
Gly Glu Gly Gly Trp 20 25 30 530PRTArtificial Sequencesynthetic
helical coiled-coil peptide 1coi-W 5Glu Val Glu Ala Leu Glu Lys Lys
Val Ala Ala Leu Glu Ser Lys Val1 5 10 15 Gln Ala Leu Glu Lys Lys
Val Glu Ala Leu Glu His Gly Trp 20 25 30
* * * * *
References