U.S. patent application number 15/437927 was filed with the patent office on 2017-09-14 for multilayer compositions, coated devices and use thereof.
The applicant listed for this patent is Massachusetts Institute of Technology. Invention is credited to Peter C. DeMuth, Paula T. Hammond, Darrell Irvine, Younjin Min.
Application Number | 20170258738 15/437927 |
Document ID | / |
Family ID | 50478072 |
Filed Date | 2017-09-14 |
United States Patent
Application |
20170258738 |
Kind Code |
A1 |
DeMuth; Peter C. ; et
al. |
September 14, 2017 |
Multilayer Compositions, Coated Devices And Use Thereof
Abstract
The present invention provides, among other things, multilayer
film coating compositions, coated substrates and methods thereof.
In some embodiments, a structure, comprising a substrate and a
multilayer film on the substrate, wherein the multilayer film
comprises a release layer and one or more layer-by-layer films. In
some embodiments, a structure comprising a microneedle substrate
and a multilayer film coated on at least portion of the microneedle
substrate, wherein the multilayer film comprises an agent for
delivery.
Inventors: |
DeMuth; Peter C.;
(Cambridge, MA) ; Min; Younjin; (Cambridge,
MA) ; Irvine; Darrell; (Arlington, MA) ;
Hammond; Paula T.; (Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology |
Cambridge |
MA |
US |
|
|
Family ID: |
50478072 |
Appl. No.: |
15/437927 |
Filed: |
February 21, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14435057 |
Apr 10, 2015 |
9610252 |
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PCT/US2013/064530 |
Oct 11, 2013 |
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15437927 |
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61713457 |
Oct 12, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/5084 20130101;
A61K 9/7007 20130101; A61K 2039/55511 20130101; A61K 39/21
20130101; A61K 9/0021 20130101; A61K 2039/53 20130101; A61K 47/32
20130101; C12N 15/89 20130101; A61K 47/34 20130101; C12N 7/00
20130101; B05D 1/36 20130101; C12N 2740/16234 20130101 |
International
Class: |
A61K 9/70 20060101
A61K009/70; A61K 9/00 20060101 A61K009/00 |
Claims
1. A structure, comprising: a substrate, comprising a microneedle
array; a release layer disposed on the substrate; and a controlled
delivery layer disposed on the release layer, wherein the
controlled delivery layer comprises one or more layer-by-layer
(LBL) films.
2. The structure of claim 1, wherein the release layer comprises a
polymer, the polymer being a photocleavable polymer, convertible
into a photocleaved polymer.
3. The structure of claim 1, wherein the controlled delivery layer
comprises one or more pharmaceutical agents.
4. The structure of claim 3, wherein the one or more pharmaceutical
agent is a protein, a polypeptide.
5. The structure of claim 3, wherein the one or more pharmaceutical
agent is a radionuclide, a fluorescent dye, a luminescent dye, a
magnetic imaging agent, or a nutraceutical agent.
6. The structure of claim 3, wherein the one or more pharmaceutical
agent is an anti-viral agent, an anesthetic agent, an anticoagulant
agent, an anti-cancer agent, a decongestant, an antihypertensive
agent, a sedative agent, a birth control agent, a progestational
agent, an anti-cholinergic agent, an anti-depressant agent, an
anti-psychotic agent, a .beta.-adrenergic blocking agent, a
diuretic agent, a cardiovascular active agent, or a vasoactive
agent.
7. The structure of claim 3, wherein the one or more pharmaceutical
agent is an antibiotic.
8. The structure of claim 7, wherein the antibiotic is ampicillin,
aziocillin, aztreonam, carbenicillin, cefoperazone, ceftriaxone,
cephaloridine, cephalothin, cloxacillin, moxalactam, penicillin G,
piperacillin, ticarcillin, a macrolide antibiotic, a monobactam
antibiotic, a rifamycin antibiotic, a tetracycline antibiotic, a
chloramphenicol antibiotic, a clindamycin antibiotic, a lincomycin
antibiotic, fusidic acid, a novobiocin antibiotic, a fosfomycin
antibiotic, a capreomycin antibiotic, a colistimethate antibiotic,
a gramicidin antibiotic, minocycline, doxycycline, bacitracin,
erythromycin, nalidixic acid, vancomycin, and trimethoprim.
9. The structure of claim 3, wherein the pharmaceutical agent is a
non-steroidal anti-inflammatory drugs (NSAIDs), a corticosteroid
agent, a cycloplegic agent, or an immune selective
anti-inflammatory derivative (ImSAIDs).
9. The structure of claim 2, wherein the photocleaved polymer is pH
sensitive, so that it is stable in a predetermined pH range but
unstable at or near physiological pH.
10. The structure of claim 2, wherein the photocleaved polymer is
substantially less soluble at pH of 6.5 or below than at a pH of
6.5 or greater.
11. The structure of claim 2, wherein the photocleavable polymer is
a terpolymer of a hydrophobic monomer, a hydrophilic monomer, and
an additional monomer having a side group represented by the
following structural formula: ##STR00005##
12. The structure of claim 5, wherein the hydrophobic monomer is
selected from methyl methacrylate, ethyl methacrylate, n-butyl
methacrylate, n-decyl methacrylate, 2-ethylhexyl methacrylate,
N-(n-octadecyl)acrylamide, n-tert-octylacrylamide, stearyl
acrylate, stearyl methacrylate, and vinyl stearate.
13. The structure of claim 5, wherein the hydrophilic monomer is
selected from hydroxyethylmethacrylate, hydroxyethyl acrylate,
4-hydroxybutyl methacrylate, N-(2-hydroxypropyl)methacrylamide,
n-methylmethacrylamide, acrylamide, poly(ethylene glycol)
monomethyl ether methacrylates, poly(ethylene glycol) methacrylate,
poly(ethylene glycol) methacrylates, and n-vinyl-2-pyrrolidone.
14. The structure of claim 5, wherein the additional monomer is
photocleavable to a carboxyl group.
15. The structure of claim 2, wherein the photocleavable polymer is
a terpolymer of methyl methacrylate, poly(ethylene glycol)
methacrylate, and o-nitrobenzyl methacrylate.
16. The structure of claim 1, wherein the LBL films comprises a
first plurality of a first unit.
17. The structure of claim 10, wherein the LBL films further
comprise a second plurality of a second unit.
18. The structure of claim 1, wherein at least a portion of the LBL
films comprises alternating polycationic and polyanionic layers,
and degradation of the LBL films is characterized by hydrolytic
degradation of at least a portion of one of the polycationic
layers, one of the polyanionic layers, or both.
19. The structure of claim 1, wherein at least a portion of the LBL
films comprises a degradable polyelectrolyte.
20. The structure of claim 13, wherein the degradable
polyelectrolyte comprises a polymer selected from polyester,
polyanhydride, polyorthoester, polyphosphazene, and
polyphosphoester, or any combination thereof.
21. The structure of claim 14, wherein the degradable
polyelectrolyte comprises the polyester selected from a group
consisting of poly(.beta.-amino ester)s,
poly(L-lactide-co-L-lysine), poly(serine ester),
poly(4-hydroxy-L-proline ester), and
poly[.alpha.-(4-aminobutyl)-L-glycolic acid], or any combination
thereof.
22. The structure of claim 15, wherein the polyester is the
poly(.beta.-amino ester) selected from the group consisting of
##STR00006## wherein: linker A and linker B are each independently
selected from the group consisting of carbon chains of 1 to 30
carbon atoms, heteroatom-containing carbon chains of 1 to 30 atoms,
and carbon chains and heteroatom-containing carbon chains with at
least one substituent selected from the group consisting of
branched and unbranched alkyl, branched and unbranched alkenyl,
branched and unbranched alkynyl, amino, alkylamino, dialkylamino,
trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic,
cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide,
carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl,
alkylthioether, and thiol groups; R1 and R2 are each independently
selected from the group consisting of hydrogen, branched and
unbranched alkyl, branched and unbranched alkenyl, branched and
unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl,
carboxyl ester, carbonyldioxyl, amide, thiohydroxyl,
alkylthioether, amino, alkylamino, dialkylamino, trialkylamino,
cyano, ureido, a substituted alkanoyl group, cyclic, cyclic
aromatic, heterocyclic, and aromatic heterocyclic groups, each of
which may be substituted with at least one substituent selected
from the group consisting of branched and unbranched alkyl,
branched and unbranched alkenyl, branched and unbranched alkynyl,
amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido,
heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic,
halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic
acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiol
groups; and n is an integer greater than or equal to 5.
23. The structure of claim 15, wherein the polyester is the
poly(.beta.-amino ester) selected from the group consisting of
##STR00007## wherein: linker B is independently selected from the
group consisting of carbon chains of 1 to 30 carbon atoms,
heteroatom-containing carbon chains of 1 to 30 atoms, and carbon
chains and heteroatom-containing carbon chains with at least one
substituent selected from the group consisting of branched and
unbranched alkyl, branched and unbranched alkenyl, branched and
unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino,
aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic
cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl,
carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether,
and thiol groups; R is selected from the group consisting of
hydrogen, branched and unbranched alkyl, branched and unbranched
alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl,
alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide,
thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino,
trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic,
cyclic aromatic, heterocyclic, and aromatic heterocyclic groups,
each of which may be substituted with at least one substituent
selected from the group consisting of branched and unbranched
alkyl, branched and unbranched alkenyl, branched and unbranched
alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl,
ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic
cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl,
carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether,
and thiol groups; and n is an integer greater than or equal to
5.
24. The structure of claim 15, wherein the poly(.beta.-amino ester)
is selected from the group consisting of ##STR00008##
25. The structure of claim 1, wherein the nucleic acid comprises a
therapeutic gene.
26. The structure of claim 1, wherein the nucleic acid comprises a
plasmid DNA.
27. The structure of claim 1, wherein the nucleic acid comprises a
plasmid DNA, and wherein the controlled delivery layer further
includes a polymeric transfection agent and an adjuvant.
28. A method of making a structure, comprising a substrate,
comprising a microneedle array; a release layer disposed on the
substrate; and a controlled delivery layer disposed on the release
layer, wherein the controlled delivery layer comprises one or more
layer-by-layer (LBL) films and at least one pharmaceutical agent,
the method comprising a steps of: coating the substrate with the
release layer; and coating the release layer with the controlled
delivery layer.
29. The method of claim 22, further comprising altering the
property of the release layer to allow the release of the
controlled delivery layer under certain conditions.
30. The method of claim 23, wherein the release layer comprises a
photocleavable polymer and the step of altering the property of the
release layer comprises exposing the release layer to UV to obtain
a photocleaved polymer.
31. The method of claim 24, wherein the photocleaved polymer is pH
sensitive, so that it is stable in a predetermined pH range but
unstable at or near physiological pH.
32. The method of claim 23, wherein the step of altering the
property of the release layer is performed before the step of
coating the release layer with the controlled delivery layer.
33. The method of claim 22, further comprising incorporating at
least one pharmaceutical agent into the controlled delivery
layer.
34. A method of delivering at least one pharmaceutical agent to a
subject in need thereof, the method comprising: providing a
structure, comprising: a substrate, comprising a microneedle array;
a release layer disposed on the substrate; and a controlled
delivery layer disposed on the release layer, wherein the
controlled delivery layer comprises one or more layer-by-layer
(LBL) films and at least one pharmaceutical agent; and contacting
an application site on the subject with the structure so that the
release layer releases the controlled delivery layer from the
substrate into the application site.
35. The method of claim 28, further including removing the
substrate from the application site.
Description
RELATED APPLICATION(S)
[0001] This application is a continuation of U.S. application Ser.
No. 14/435,057, filed Apr. 10, 2015, which is the U.S. National
Stage of International Application No. PCT/US2013/064530, filed on
Oct. 11, 2013, published in English, which claims the benefit of
U.S. Provisional Application No. 61/713,457, filed on Oct. 12,
2012. The entire teachings of the above applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Layer-by-Layer (LBL) assembly of multilayer film coatings is
driven by the alternating deposition of materials (e.g., polymers
with complementary electrostatic functionalities). The LBL assembly
process produces nanometer to micron scale thin film coatings. A
major benefit of LBL assembly is the potential to achieve
controlled and sequential delivery of agents by tuning the
deposition of these agents at specific layers within the film.
[0003] There is a particular interest in achieving delivery of
vaccines and/or therapeutic agents by taking advantage of LBL
films. Delivery through the skin (i.e., transcutaneous delivery) is
a focus of much research. Thus, there is a need in the art for
versatile platform for delivery, particularly transcutaneous
delivery of drugs and other agents that is effective, generally
applicable, safe, pain-free, and/or cost effective.
SUMMARY OF THE INVENTION
[0004] The present invention provides, among other things, a
release layer. Various structures comprising substrates coated with
such a release layer alone or in combination with other
films/layers (e.g., LBL films) are provided. In some embodiments, a
release layer is or comprises a polymer, which is stable during
deposition/assembly onto a substrate and can be converted and
become unstable when exposed in a liquid medium under certain
conditions for releasing.
[0005] In one aspect, the invention provides certain multilayer
films comprising LBL films and at least one release layer, for
example as a coating composition on a substrate. In some
embodiments, such multilayer films, for example, at least a portion
of LBL films, are associated with one or more agents for delivery.
Multilayer film compositions can be particularly useful for DNA
delivery in certain embodiments.
[0006] In one aspect, the invention provides a structure comprising
a substrate arranged and constructed for contact with a biological
tissue; such a substrate being coated with a multilayer film
coating composition. In some embodiments, such a substrate is or
comprises a microneedle or a microneedle array.
[0007] Among other things, the present invention demonstrates and
achieves various improvements in microneedle devices, and
particularly in delivery of nucleic acids together with other
agents such as transfection and/or immunological agents from the
devices.
[0008] It is recognized in the present invention that a underlying
release layer enables a rapid release of LBL films or other outer
layers/films from a coated substrate. For example, coated
microneedles can be used to rapidly implant drug delivery films by
brief application to a tissue (e.g., skin), which allows the
kinetics of the agent for delivery from the films in the tissue to
be tailored separately from the time required for microneedles to
be kept in contact with the tissue.
[0009] The present invention also encompasses the recognition that,
in many cases, combining the flexible and highly tunable nature of
provided multilayer films with microneedle devices provides a
versatile platform for delivery of a variety of agents.
[0010] The present invention also provides methods of making and
using provided structures and/or multilayer films.
[0011] Other features, objects, and advantages of the present
invention are apparent in the detailed description, drawings and
claims that follow. It should be understood, however, that the
detailed description, the drawings, and the claims, while
indicating embodiments of the present invention, are given by way
of illustration only, not limitation. Various changes and
modifications within the scope of the invention will become
apparent to those skilled in the art.
Definitions
[0012] In order for the present disclosure to be more readily
understood, certain terms are first defined below. Additional
definitions for the following terms and other terms are set forth
throughout the specification.
[0013] In this application, the use of "or" means "and/or" unless
stated otherwise. As used in this application, the term "comprise"
and variations of the term, such as "comprising" and "comprises,"
are not intended to exclude other additives, components, integers
or steps. As used in this application, the terms "about" and
"approximately" are used as equivalents. Any numerals used in this
application with or without about/approximately are meant to cover
any normal fluctuations appreciated by one of ordinary skill in the
relevant art. In certain embodiments, the term "approximately" or
"about" refers to a range of values that fall within 25%, 20%, 19%,
18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%,
4%, 3%, 2%, 1%, or less in either direction (greater than or less
than) of the stated reference value unless otherwise stated or
otherwise evident from the context (except where such number would
exceed 100% of a possible value).
[0014] "Associated": As used herein, the term "associated"
typically refers to two or more entities in physical proximity with
one another, either directly or indirectly (e.g., via one or more
additional entities that serve as a linking agent), to form a
structure that is sufficiently stable so that the entities remain
in physical proximity under relevant conditions, e.g.,
physiological conditions. In some embodiments, associated moieties
are covalently linked to one another. In some embodiments,
associated entities are non-covalently linked. In some embodiments,
associated entities are linked to one another by specific
non-covalent interactions (i.e., by interactions between
interacting ligands that discriminate between their interaction
partner and other entities present in the context of use, such as,
for example, streptavidin/avidin interactions, antibody/antigen
interactions, etc.). Alternatively or additionally, a sufficient
number of weaker non-covalent interactions can provide sufficient
stability for moieties to remain associated. Exemplary non-covalent
interactions include, but are not limited to, affinity
interactions, metal coordination, physical adsorption, host-guest
interactions, hydrophobic interactions, pi stacking interactions,
hydrogen bonding interactions, van der Waals interactions, magnetic
interactions, electrostatic interactions, dipole-dipole
interactions, etc.
[0015] "Biocompatible": The term "biocompatible", as used herein is
intended to describe materials that do not elicit a substantial
detrimental response in vivo. In certain embodiments, the materials
are "biocompatible" if they are not toxic to cells. In certain
embodiments, materials are "biocompatible" if their addition to
cells in vitro results in less than or equal to 20% cell death,
and/or their administration in vivo does not induce inflammation or
other such adverse effects. In certain embodiments, materials are
biodegradable.
[0016] "Biodegradable": As used herein, "biodegradable" materials
are those that, when introduced into cells, are broken down by
cellular machinery {e.g., enzymatic degradation) or by hydrolysis
into components that cells can either reuse or dispose of without
significant toxic effects on the cells. In certain embodiments,
components generated by breakdown of a biodegradable material do
not induce inflammation and/or other adverse effects in vivo. In
some embodiments, biodegradable materials are enzymatically broken
down. Alternatively or additionally, in some embodiments,
biodegradable materials are broken down by hydrolysis. In some
embodiments, biodegradable polymeric materials break down into
their component polymers. In some embodiments, breakdown of
biodegradable materials (including, for example, biodegradable
polymeric materials) includes hydrolysis of ester bonds. In some
embodiments, breakdown of materials (including, for example,
biodegradable polymeric materials) includes cleavage of urethane
linkages.
[0017] "Hydrolytically degradable": As used herein, "hydrolytically
degradable" materials are those that degrade by hydrolytic
cleavage. In some embodiments, hydrolytically degradable materials
degrade in water. In some embodiments, hydrolytically degradable
materials degrade in water in the absence of any other agents or
materials. In some embodiments, hydrolytically degradable materials
degrade completely by hydrolytic cleavage, e.g., in water. By
contrast, the term "non-hydrolytically degradable" typically refers
to materials that do not fully degrade by hydrolytic cleavage
and/or in the presence of water (e.g., in the sole presence of
water).
[0018] "Nucleic acid": The term "nucleic acid" as used herein,
refers to a polymer of nucleotides. In some embodiments, nucleic
acids are or contain deoxyribonucleic acids (DNA); in some
embodiments, nucleic acids are or contain ribonucleic acids (RNA).
In some embodiments, nucleic acids include naturally-occurring
nucleotides (e.g., adenosine, thymidine, guanosine, cytidine,
uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and
deoxycytidine). Alternatively or additionally, in some embodiments,
nucleic acids include non-naturally-occurring nucleotides
including, but not limited to, nucleoside analogs (e.g.,
2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,
3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine,
C5-bromouridine, C5-fluorouridine, C5-iodouridine,
C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine,
8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and
2-thiocytidine), chemically modified bases, biologically modified
bases (e.g., methylated bases), intercalated bases, modified sugars
(e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and
hexose), or modified phosphate groups. In some embodiments, nucleic
acids include phosphodiester backbone linkages; alternatively or
additionally, in some embodiments, nucleic acids include one or
more non-phosphodiester backbone linkages such as, for example,
phosphorothioates and 5'-N-phosphoramidite linkages. In some
embodiments, a nucleic acid is an oligonucleotide in that it is
relatively short (e.g., less that about 5000, 4000, 3000, 2000,
1000, 900, 800, 700, 600, 500, 450, 400, 350, 300, 250, 200, 150,
100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10 or fewer
nucleotides in length.
[0019] "Physiological conditions": The phrase "physiological
conditions", as used herein, relates to the range of chemical
(e.g., pH, ionic strength) and biochemical (e.g., enzyme
concentrations) conditions likely to be encountered in the
intracellular and extracellular fluids of tissues. For most
tissues, the physiological pH ranges from about 7.0 to 7.4.
[0020] Polyelectrolyte": The term "polyelectrolyte", as used
herein, refers to a polymer which under some set of conditions
(e.g., physiological conditions) has a net positive or negative
charge. Polyelectrolytes includes polycations and polyanions.
Polycations have a net positive charge and polyanions have a net
negative charge. The net charge of a given polyelectrolyte may
depend on the surrounding chemical conditions, e.g., on the pH.
[0021] "Polypeptide": The term "polypeptide" as used herein, refers
to a string of at least three amino acids linked together by
peptide bonds. Polypeptides such as proteins may contain only
natural amino acids, although non-natural amino acids (i.e.,
compounds that do not occur in nature but that can be incorporated
into a polypeptide chain; see, for example,
http://www.cco.caltech.edu/-dadgrp/Unnatstruct.gif, which displays
structures of non-natural amino acids that have been successfully
incorporated into functional ion channels) and/or amino acid
analogs as are known in the art may alternatively be employed.
Also, one or more of the amino acids in a protein may be modified,
for example, by the addition of a chemical entity such as a
carbohydrate group, a phosphate group, a farnesyl group, an
isofarnesyl group, a fatty acid group, a linker for conjugation,
functionalization, or other modification, etc.
[0022] "Polysaccharide": The term "polysaccharide" refers to a
polymer of sugars. Typically, a polysaccharide comprises at least
three sugars. In some embodiments, a polypeptide comprises natural
sugars (e.g., glucose, fructose, galactose, mannose, arabinose,
ribose, and xylose); alternatively or additionally, in some
embodiments, a polypeptide comprises one or more non-natural amino
acids (e.g, modified sugars such as 2'-fluororibose,
2'-deoxyribose, and hexose).
[0023] "Small molecule": As used herein, the term "small molecule"
is used to refer to molecules, whether naturally-occurring or
artificially created (e.g., via chemical synthesis), that have a
relatively low molecular weight. Typically, small molecules are
monomeric and have a molecular weight of less than about 1500
g/mol. Preferred small molecules are biologically active in that
they produce a local or systemic effect in animals, preferably
mammals, more preferably humans. In certain preferred embodiments,
the small molecule is a drug. Preferably, though not necessarily,
the drug is one that has already been deemed safe and effective for
use by the appropriate governmental agency or body. For example,
drugs for human use listed by the FDA under 21 C.F.R.
.sctn..sctn.330.5, 331 through 361, and 440 through 460; drugs for
veterinary use listed by the FDA under 21 C.F.R. .sctn..sctn.500
through 589, incorporated herein by reference, are all considered
acceptable for use in accordance with the present application.
[0024] "Substantially": As used herein, the term "substantially",
and grammatic equivalents, refer to the qualitative condition of
exhibiting total or near-total extent or degree of a characteristic
or property of interest. One of ordinary skill in the art will
understand that biological and chemical phenomena rarely, if ever,
go to completion and/or proceed to completeness or achieve or avoid
an absolute result.
[0025] "Treating": As used herein, the term refers to any method
used to partially or completely alleviate, ameliorate, relieve,
inhibit, prevent, delay onset of, reduce severity of and/or reduce
incidence of one or more symptoms or features of a particular
disease, disorder, and/or condition. Treatment may be administered
to a subject who does not exhibit signs of a disease and/or
exhibits only early signs of the disease for the purpose of
decreasing the risk of developing pathology associated with the
disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1A through FIG. 1C collectively illustrate
quick-release multi-layer film architecture and rapid delamination
behavior. FIG. 1A: (1) PLLA microneedles are coated with PNMP
through spraying in 1,4-dioxane. (2) UV-irradiation converts PNMP
from hydrophobic to hydrophilic with pH-sensitive dissolution
behavior, forming a uv-PNMP `release-layer`. (3) Overlying
multi-layer films are constructed using LbL at pH 5.0. (4)
Microneedle application to skin and exposure to pH 7.4 gives rapid
release-layer dissolution, mediating overlying film delamination
and implantation following array removal. (5) Implanted films
provide controlled/sustained release through hydrolytic PBAE
degradation forming polyplexes for transfection and
immune-modulation. FIG. 1B: Micrograph and surface profilometer
trace showing heterogeneous film architecture for silicon-based
(LPEI/PAA).sub.3-(BPEI/PAA).sub.30 films, with an underlying
uv-PNMP release layer (arrow, scale bar-100 .mu.m). FIG. 1C:
Time-lapse micrographs showing release-layer dissolution and
multi-layer delamination following exposure to PBS, pH 7.4. Shown
are micrographs for 1, 4, and 30 minutes incubation (scale bar
--500 .mu.m).
[0027] FIG. 2A through FIG. 2H collectively illustrate that
multi-layer film deposition is controllable and modular. FIG. 2A:
Film architecture for uv-PNMP-(PS/SPS).sub.20-(PBAE/pLUC).sub.n,
multi-layers. FIG. 2B: Film growth for (poly-1/pLUC)n and
(poly-2/pLUC).sub.n multi-layers assembled following uv-PNMP and
(PS/SPS).sub.20 deposition on silicon. Shown is the thickness of
the underlying uv-PNMP release layer, and the overlying (PS/SPS),
and (PBAE/pLUC).sub.n films. FIG. 2C: Representative confocal
images of a SAv488-bPNMP-(PS/SPS).sub.20-(poly-1/Cy5-pLUC).sub.35
coated PLLA microneedles (left-transverse sections, right-lateral
sections, 100 .mu.m interval, scale --200 .mu.m, blue
-Sav488-uv-bPNMP, yellow--Cy5-pLUC). FIG. 2D: Quantification of
encapsulated Cy5-pLUC and Sav488-bPNMP in PLLA microneedle-based
SAv488-bPNMP-(PS/SPS).sub.20-(poly-1/Cy5-pLUC).sub.n films through
confocal image analysis (left axis, n=15) and total film elution
(right axis, n=3). FIG. 2E: Film architecture for
uv-PNMP-(PS/SPS).sub.20-(Poly-1/pLUC).sub.n-(Poly-1/poly(I:C)).sub.n
multi-layers. FIG. 2F: Representative confocal images of
SAv488-uv-bPNMP-(PS/SPS).sub.20-(poly-1/TMR-poly(I:C)).sub.15-(poly-1/Cy5-
-pLUC).sub.15 coated PLLA microneedle (left--transverse sections,
right--lateral sections, 100 .mu.m interval, scale--200 .mu.m,
blue--Sav488-uv-bPNMP, yellow--Cy5-pLUC, red--TMR-poly(LC)). FIG.
2G: Quantification of encapsulated Cy5-pLUC, TMR-poly(LC), and
SAv488-bPNMP in PLLA microneedle-based
SAv488-bPNMP-(PS/SPS).sub.20-(poly-1/TMR-poly(I:C)).sub.n-(poly-1/Cy5-pLU-
C).sub.n films through confocal image analysis (n=15) and FIG. 2H:
total film elution (right axis, n=3).
[0028] FIG. 3A through FIG. 3G collectively demonstrate that
microneedle-based films are rapidly implanted at penetration sites
in vivo. FIG. 3A: Optical micrograph of ear skin showing
microneedle penetration pattern stained using trypan blue (scale
bar--500 .mu.m). FIG. 3B: Quantitation of confocal imaging (n=15)
showing UV-dependent loss of Sav488-bPNMP and Cy5-pLUC signal upon
application to skin. FIG. 3C: Representative confocal images of an
SAv488-bPNMP-(PS/SPS).sub.20-(poly-1/Cy5-pLUC).sub.35 coated PLLA
microneedle with UV treatment, before application (lateral
sections, 100 .mu.m interval, scale--200 .mu.m, left,
blue--Sav488-bPNMP, yellow--Cy5-pLUC), after 15 min application
(middle), and without UV treatment, after 15 min application
(right), FIG. 3D: Representative confocal image of treated murine
skin showing film implantation after 15 min (green--MHC II-GFP,
yellow--Cy5-pLUC, penetration site outlined, scale bar--100 .mu.m).
FIG. 3E: Facial and profile confocal images showing depth of
Cy5-pLUC film deposition after 15 minute microneedle application
(green--MHC II-GFP, yellow--Cy5-pLUC, penetration site outlined,
scale bar--200 .mu.m). FIG. 3F: Representative confocal image of
treated murine skin showing TMR-poly(LC) film implantation after 15
minute microneedle application (green--MHC II-GFP,
red--TMR-poly(LC), penetration site outlined, scale bar--100
.mu.m)). FIG. 3G: Colocalization and uptake of TMR-poly(LC) by MHC
II-GFP.sup.+ APCs at microneedle insertion site 24 hrs following
film implantation (green--MHC II-GFP, red--TMR-poly(LC),
yellow--overlay, scale bar--50 .mu.m)). ***, p<0.0001, analyzed
by unpaired t-test.
[0029] FIG. 4A through FIG. 4E collectively illustrate that
implanted films control and sustain release of pDNA and poly(LC) in
vivo, FIG. 4A: In vitro release of poly(LC) from
(PS/SPS).sub.20-(PBAE/poly(I:C)).sub.35 films on silicon, FIG. 4B:
Whole animal fluorescence images of TMR-poly(I:C) retention at
application site 1, 3, 10, and 14 days after 15 minute
uv-PNMP-(PS/SPS).sub.20-(PBAE/TMR-poly(I:C)).sub.35 coated PLLA
microneedle array application, for poly-1 and poly-2. FIG. 4C:
Quantification of fluorescence imaging of TMR-poly(LC) clearance
from application site, FIG. 4D: Whole animal bioluminescence images
of pLUC expression at application site 1 h, 3, 10, or 20 days after
15 minute uv-PNMP-(PS/SPS).sub.20-(PBAE/pLUC).sub.35 coated PLLA
microneedle array application, for poly-1 and poly-2. FIG. 4E:
Quantification of bioluminescence intensity at application
site.
[0030] FIG. 5A through FIG. 5F collectively demonstrate that
microneedle-film delivery gives potent cellular and humoral
immunity against HIV-Gag. FIG. 5A: C57B1/6 mice were immunized with
20 .mu.g pGag and 10 .mu.g poly(LC) on day 0 and 28 intramuscularly
(IM.+-.EP) in the quadriceps, intradermally (ID) in the dorsal ear
skin, or by 15 minute application of
uv-PNMP-(PS/SPS).sub.20-(poly-1/poly(I:C)).sub.35-(poly-1/pLUC).sub.35
coated microneedles (MN) at the dorsal ear skin, FIG. 5B:
Enzyme-linked-immunosorbent assay analysis of total Gag-specific
IgG in sera at d42. FIG. 5C: Frequency of Gag-specific T cells in
peripheral blood assessed by flow cytometry analysis of
tetramer.sup.+ CD8.sup.+ T cells. Shown are representative
cytometry plots from individual mice at d42 and FIG. 5D: mean
tetramer.sup.+ values from d21 and d42. FIG. 5E: Analysis of T-cell
effector/central memory phenotypes in peripheral blood by
CD44/CD62L staining on tetramer+cells from peripheral blood. Shown
are representative cytometry plots from individual mice at d49 and
FIG. 5F: mean percentages of tetramerCD44.sup.+ CD62L.sup.+ among
CD8.sup.+ T cells at d28 and d49. **, p<0.005, analyzed by
two-way analysis of variance.
[0031] FIG. 6A through FIG. 6F collectively illustrate exemplary
microneedle fabrication, FIG. 6A: PDMS is laser ablated to form
micron-scale cavities, FIG. 6B: PLLA is added to the surface of the
PDMS mold, FIG. 6C: PLLA is melted under vacuum and then cooled
before FIG. 6D: removal of PLLA microneedle arrays, FIG. 6E: SEM
and FIG. 6F: optical micrograph of PLLA microneedle arrays produced
through PDMS melt-casting (scale bar--500 .mu.m).
[0032] FIG. 7A through FIG. 7C collectively show chemical structure
of polymers, FIG. 7A: Structure of biotinylated-PNMP (bPNMP,
MW.about.17,000 Da) in which a pendant biotin is conjugated to the
free hydroxyl terminus of the PEG-methacrylate monomer unit, FIG.
7B: Chemical structure of poly-1 (MW.about.15,000 Da) and FIG. 7C:
poly-2 (MW.about.21,000 Da) used in this study.
[0033] FIG. 8A through FIG. 8D collectively illustrate that
multi-layer deposition is controllable and modular, FIG. 8A: Film
growth for (poly-1/poly(I:C)).sub.n and (poly-2/poly(I:C)).sub.n
multi-layers assembled following uv-PNMP and (PS/SPS).sub.20
deposition on silicon, FIG. 8B: Representative confocal images of
an SAv488-bPNMP-(PS/SPS).sub.20-(poly-1/TMR-poly(I:C)).sub.35
coated microneedle (left--transverse sections, right--lateral
sections, 100 .mu.m interval, scale--200 .mu.m, blue--Sav488-bPNMP,
red--TMR-poly(LC)). FIG. 8C: Quantification of encapsulated
TMR-poly(LC) dosage through confocal image analysis (left axis,
n=15) and film elution (right axis, n=3). FIG. 8D: Film growth for
(poly-1/pLUC)n and (poly-1/poly(I:C)).sub.n multi-layers assembled
following uv-PNMP and (PS/SPS).sub.20 deposition on silicon.
[0034] FIG. 9A through FIG. 9B collectively illustrate that
microneedle-based films rapidly delaminate in vitro FIG. 9A:
Representative confocal images of an
SAv488-bPNMP-(PS/SPS).sub.20-(poly-1/Cy5-pLUC).sub.35 coated PLLA
microneedle with UV treatment, before application (lateral
sections, 100 .mu.m interval, scale--200 .mu.m, left,
blue--Sav488-bPNMP, yellow--Cy5-pLUC), after 15 min application
(middle), and without UV treatment, after 15 min application
(right), FIG. 9B: Quantitation of confocal imaging (n=15) showing
UV-dependent loss of SAV488-bPNMP and Cy5-pLUC signal upon
application to skin.
[0035] FIG. 10 illustrates that microneedle-based films are rapidly
implanted at penetration sites in vivo. Representative facial and
profile confocal images showing depth of film deposition in treated
murine skin after 15 min (green--MHC II-GFP, red--TMR-poly(LC),
penetration site outlined, scale bar--200 .mu.m).
[0036] FIG. 11 illustrates that multi-layer films control release
of pLUC in vitro. In vitro release of pLUC from
(PS/SPS).sub.20-(PBAE/pLUC).sub.35 films on silicon.
[0037] FIG. 12A through FIG. 12B collectively illustrate that
implanted films control and sustain release of pDNA in vivo. Whole
animal bioluminescence images of pLUC expression at application
site 1, 3, 10, or 20 days following a 15 min application of
SAv488-bPNMP-(PS/SPS).sub.20-(poly-1/pLUC).sub.35 coated
microneedle array without UV pretreatment.
[0038] FIG. 13A through FIG. 13C collectively illustrate that in
vitro multi-layer delamination is rapid. Time-lapse optical
microscopic images of multi-layer delamination and release from
silicon. The delamination of an (LPEI/PAA).sub.3-(BPEI/PAA).sub.30
multi-layer film constructed on an underlying uv-PNMP release layer
is shown from t=0 (before immersion) to t=30 min (30 min following
immersion in PBS at pH 7.4). PBS was added at t=1 sec.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0039] In various embodiments, compositions and methods for
assembling at least one release layer and LBL films associated with
one or more agents for delivery are disclosed. Provided film
composition and methods can be used to coat a substrate for
controlled delivery of one or more agents.
LBL Films
[0040] LBL films may have various film architecture, film
thickness, film materials, surface chemistry, and/or incorporation
of agents according to the design and application of coated
devices.
[0041] In general, LBL films comprise multiple layers. In many
embodiments, LBL films are comprised of multilayer units; each unit
comprising individual layers. In accordance with the present
disclosure, individual layers in an LBL film interact with one
another. In particular, a layer in an LBL film comprises an
interacting moiety, which interacts with that from an adjacent
layer, so that a first layer associates with a second layer
adjacent to the first layer, each contains at least one interacting
moiety.
[0042] In some embodiments, adjacent layers are associated with one
another via non-covalent interactions. Exemplary non-covalent
interactions include, but are not limited to, hydrogen bonding,
affinity interactions, metal coordination, physical adsorption,
host-guest interactions, hydrophobic interactions, pi stacking
interactions, hydrogen bonding interactions, van der Waals
interactions, magnetic interactions, dipole-dipole interactions and
combinations thereof.
[0043] In some embodiments, an interacting moiety is a charge,
positive or negative. In some embodiments, an interacting moiety is
a hydrogen bond donor or acceptor. In some embodiments, an
interacting moiety is a complementary moiety for specific binding
such as avidin/biotin. In various embodiments, more than one
interactions can be involved in the association of two adjacent
layers. For example, an electrostatic interaction can be a primary
interaction; a hydrogen bonding interaction can be a secondary
interaction between the two layers.
[0044] LBL films may be comprised of multilayer units with
alternating layers of opposite charge, such as alternating anionic
and cationic layers.
[0045] In some embodiments, the present invention provides the
insight that at least some potential layer materials, including
potential agents for delivery that could otherwise be utilized as
layer materials do not and/or cannot carry sufficient charge to
mediate stable electrostatic interactions. In addition to
electrostatic interaction or alternatively, they can be associated
via non-electrostatic interaction in a coated device in accordance
with the present invention.
[0046] According to the present disclosure, LBL films may be
comprised of one or more multilayer units. In some embodiments, an
LBL film may include a plurality of a single unit (e.g., a bilayer
unit, a tetralayer unit, etc.). In some embodiments, an LBL film is
a composite that includes more than one unit. For example, more
than one unit possessing different film architecture (e.g.,
bilayers, tetralayer, etc.), film thickness, film materials (e.g.,
polymers), surface chemistry, and/or agents that are associated
with one of the units. In some embodiments, an LBL film is a
composite that includes more than one bilayer unit, more than one
tetralayer unit, or any combination thereof. In some embodiments,
an LBL film is a composite that includes a plurality of a single
bilayer unit and/or a plurality of a single tetralayer unit.
[0047] In some embodiments, the number of a multilayer unit is 3,
5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400 or
even 500.
[0048] LBL films may have various thickness depending on design,
methods of fabricating, and applications. In some embodiments, an
LBL film has an average thickness in a range of about 1 nm and
about 100 .mu.m. In some embodiments, an LBL film has an average
thickness in a range of about 1 .mu.m and about 50 .mu.m. In some
embodiments, an LBL film has an average thickness in a range of
about 2 .mu.m and about 5 .mu.m. In some embodiments, the average
thickness of an LBL film is or more than about 1 nm, about 100 nm,
about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600
nm, about 700 nm, about 800 nm, about 900 nm, about 1 .mu.m, about
1.5 .mu.m, about 2 .mu.m, about 3 .mu.m, about 4 .mu.m, about 5
.mu.m, about 10 .mu.m, about 20 .mu.m, about 50 .mu.m, about 100
.mu.m. In some embodiments, an LBL film has an average thickness in
a range of any two values above.
[0049] An individual layer of an LBL film can contain a polymeric
material. In some embodiments, a polymer is degradable or
non-degradable. In some embodiments, a polymer is natural or
synthetic.
[0050] In some embodiments, a polymer is a polyelectrolyte.
[0051] In some embodiment, a polymer is a polypeptide. In some
embodiments, a polymer has a relatively small molecule weight. In
some embodiments, a polymer is an agent for delivery. For example,
poly-1 and poly-2 were used as transfection agents in Example 1
below.
[0052] LBL films can be decomposable. In many embodiments, a
polymer of an individual layer includes a degradable
polyelectrolyte. In some embodiments, decomposition of LBL films is
characterized by substantially sequential degradation of at least a
portion of the polyelectrolyte layers that make up the LBL films.
Degradation may be at least partially hydrolytic, at least
partially enzymatic, at least partially thermal, and/or at least
partially photolytic. Degradable polyelectrolytes and their
degradation byproducts may be biocompatible so as to make LBL films
amenable to use in vivo.
[0053] Degradable polyelectrolytes can be used in an LBL film
disclosed herein, including, but not limited to, hydrolytically
degradable, biodegradable, thermally degradable, and photolytically
degradable polyelectrolytes. Hydrolytically degradable polymers
known in the art include for example, certain polyesters,
polyanhydrides, polyorthoesters, polyphosphazenes, and
polyphosphoesters. Biodegradable polymers known in the art,
include, for example, certain polyhydroxyacids,
polypropylfumerates, polycaprolactones, polyamides, poly(amino
acids), polyacetals, polyethers, biodegradable polycyanoacrylates,
biodegradable polyurethanes and polysaccharides. For example,
specific biodegradable polymers that may be used include but are
not limited to polylysine, poly(lactic acid) (PLA), poly(glycolic
acid) (PGA), poly(caprolactone) (PCL), poly(lactide-co-glycolide)
(PLG), poly(lactide-co-caprolactone) (PLC), and
poly(glycolide-co-caprolactone) (PGC). Those skilled in the art
will recognize that this is an exemplary, not comprehensive, list
of biodegradable polymers. Of course, co-polymers, mixtures, and
adducts of these polymers may also be employed.
[0054] Anionic polyelectrolytes may be degradable polymers with
anionic groups distributed along the polymer backbone. Anionic
groups, which may include carboxylate, sulfonate, sulphate,
phosphate, nitrate, or other negatively charged or ionizable
groupings, may be disposed upon groups pendant from the backbone or
may be incorporated in the backbone itself. Cationic
polyelectrolytes may be degradable polymers with cationic groups
distributed along the polymer backbone. Cationic groups, which may
include protonated amine, quaternary ammonium or
phosphonium-derived functions or other positively charged or
ionizable groups, may be disposed in side groups pendant from the
backbone, may be attached to the backbone directly, or can be
incorporated in the backbone itself
[0055] For example, a range of hydrolytically degradable amine
containing polyesters bearing cationic side chains have been
developed. Examples of these polyesters include
poly(L-lactide-co-L-lysine), poly(serine ester),
poly(4-hydroxy-L-proline ester), and
poly[.alpha.-(4-aminobutyl)-L-glycolic acid].
[0056] In addition, poly(.beta.-amino ester)s (PBAE), which can be
prepared from the conjugate addition of primary or secondary amines
to diacrylates, are suitable for use. Typically, poly(.beta.-amino
ester)s have one or more tertiary amines in the backbone of the
polymer, preferably having one or two per repeating backbone unit.
Alternatively, a co-polymer may be used in which one of the
components is a poly(.beta.-amino ester). Poly(.beta.-amino ester)s
are described in U.S. Pat. Nos. 6,998,115 and 7,427,394, entitled
"Biodegradable poly(.beta.-amino esters) and uses thereof and Lynn
et al., J. Am. Chem. Soc. 122: 10761-10768, 2000, the entire
contents of both of which are incorporated herein by reference.
[0057] In some embodiments, a poly(.beta.-amino ester) can have a
formula below:
##STR00001##
where A and B are linkers which may be any substituted or
unsubstituted, branched or unbranched chain of carbon atoms or
heteroatoms. The molecular weights of the polymers may range from
1000 g/mol to 20,000 g/mol, preferably from 5000 g/mol to 15,000
g/mol. In certain embodiments, B is an alkyl chain of one to twelve
carbons atoms. In other embodiments, B is a heteroaliphatic chain
containing a total of one to twelve carbon atoms and heteroatoms.
The groups R.sub.1 and R.sub.2 may be any of a wide variety of
substituents. In certain embodiments, R.sub.1 and R.sub.2 may
contain primary amines, secondary amines, tertiary amines, hydroxyl
groups, and alkoxy groups. In certain embodiments, the polymers are
amine-terminated; and in other embodiments, the polymers are
acrylated terminated. In some embodiments, the groups R.sub.1
and/or R.sub.2 form cyclic structures with the linker A.
[0058] Exemplary poly(.beta.-amino esters) include
##STR00002##
[0059] Exemplary R groups include hydrogen, branched and unbranched
alkyl, branched and unbranched alkenyl, branched and unbranched
alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl
ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino,
alkylamino, dialkylamino, trialkylamino, cyano, ureido, a
substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic,
and aromatic heterocyclic groups, each of which may be substituted
with at least one substituent selected from the group consisting of
branched and unbranched alkyl, branched and unbranched alkenyl,
branched and unbranched alkynyl, amino, alkylamino, dialkylamino,
trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic,
cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide,
carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl,
alkylthioether, and thiol groups.
[0060] Exemplary linker groups B includes carbon chains of 1 to 30
carbon atoms, heteroatom-containing carbon chains of 1 to 30 atoms,
and carbon chains and heteroatom-containing carbon chains with at
least one substituent selected from the group consisting of
branched and unbranched alkyl, branched and unbranched alkenyl,
branched and unbranched alkynyl, amino, alkylamino, dialkylamino,
trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic,
cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide,
carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl,
alkylthioether, and thiol groups. The polymer may include, for
example, between 5 and 10,000 repeat units.
[0061] In some embodiments, poly(.beta.-amino ester)s are selected
from the group consisting of
##STR00003##
derivatives thereof, and combinations thereof.
[0062] Alternatively or additionally, zwitterionic polyelectrolytes
may be used. Such polyelectrolytes may have both anionic and
cationic groups incorporated into the backbone or covalently
attached to the backbone as part of a pendant group. Such polymers
may be neutrally charged at one pH, positively charged at another
pH, and negatively charged at a third pH. For example, an LBL film
may be constructed by LBL deposition using dip coating in solutions
of a first pH at which one layer is anionic and a second layer is
cationic. If such an LBL film is put into a solution having a
second different pH, then the first layer may be rendered cationic
while the second layer is rendered anionic, thereby changing the
charges on those layers.
[0063] The composition of degradable polyeletrolyte layers can be
fine-tuned to adjust the degradation rate of each layer within the
film, which is believe to impact the release rate of drugs. For
example, the degradation rate of hydro lyrically degradable
polyelectrolyte layers can be decreased by associating hydrophobic
polymers such as hydrocarbons and lipids with one or more of the
layers. Alternatively, polyelectrolyte layers may be rendered more
hydrophilic to increase their hydrolytic degradation rate. In
certain embodiments, the degradation rate of a given layer can be
adjusted by including a mixture of polyelectrolytes that degrade at
different rates or under different conditions.
[0064] In other embodiments, polyanionic and/or polycationic layers
may include a mixture of degradable and non-degradable
polyelectrolytes. Any non-degradable polyelectrolyte can be used.
Exemplary non-degradable polyelectrolytes that could be used in
thin films include poly(styrene sulfonate) (SPS), poly(acrylic
acid) (PAA), linear poly(ethylene imine) (LPEI),
poly(diallyldimethyl ammonium chloride) (PDAC), and poly(allylamine
hydrochloride) (PAH).
[0065] Alternatively or additionally, the degradation rate may be
fine-tuned by associating or mixing non-biodegradable, yet
biocompatible polymers with one or more of the polyanionic and/or
polycationic layers. Suitable non-biodegradable, yet biocompatible
polymers are well known in the art and include polystyrenes,
certain polyesters, non-biodegradable polyurethanes, polyureas,
poly(ethylene vinyl acetate), polypropylene, polymethacrylate,
polyethylene, polycarbonates, and poly(ethylene oxide)s.
[0066] Polymers used herein in accordance with the present
disclosure generally can be biologically derived or natural.
Polymers that may be used include charged polysaccharides. In some
embodiments, polysaccharides include glycosaminoglycans such as
heparin, chondroitin, dermatan, hyaluronic acid, etc. (Some of
these terms for glycoasminoglycans are often used interchangeably
with the name of a sulfate form, e.g., heparan sulfate, chondroitin
sulfate, etc. It is intended that such sulfate forms are included
among a list of exemplary polymers used in accordance with the
present invention.).
[0067] Additionally or alternatively, polymers can be a natural
acid.
[0068] LBL films may be exposed to a liquid medium (e.g.,
intracellular fluid, interstitial fluid, blood, intravitreal fluid,
intraocular fluid, gastric fluids, etc.). In some embodiments, an
LBL film comprises at least one polycationic layer that degrades
and at least one polyanionic layer that delaminates sequentially.
Releasable agents are thus gradually and controllably released from
the LBL film. It will be appreciated that the roles of the layers
of an LBL film can be reversed. In some embodiments, an LBL film
comprises at least one polyanionic layer that degrades and at least
one polycationic layer that delaminates sequentially.
Alternatively, polycationic and polyanionic layers may both include
degradable polyelectrolytes.
Release Layer
[0069] In accordance with some embodiments of the present
disclosure, LBL films can be used with at least one release layer.
In many embodiments, at least one release layer and one or more LBL
films are assembled and/or deposited on a substrate. When a release
layer is removed from a substrate, for example, by dissolution in a
liquid medium, LBL films outside the release layer will be released
from the substrate. In addition, any other films or layers can be
used to coat a substrate. In certain embodiments, those
films/layers can be assembled and/or deposited in between a release
layer and a substrate, in between a release layer and a LBL film,
in between LBL films, or on the most outer film/layer.
[0070] In various embodiments, a release layer is or comprises a
polymer. Such a polymer, in some embodiments, is stable during
deposition/assembly and can be converted to become unstable when
exposed in a liquid medium for releasing. In some embodiments, a
polymer comprises hydrophobic moieties, which renders the polymer
stable and not soluble under certain conditions during its
deposition onto a substrate (e.g., a microneedle/microneedle
array).
[0071] In some embodiments, conversion is conducted by exposing a
polymer to UV to photocleave hydrophobic moieties. A polymer can be
a photocleavable polymer. A photocleaved polymer can be a pH
sensitive polymer, so that the photocleaved polymer is stable at a
predetermined pH or in a predetermined pH range, but unstable at or
near physiological pH.
[0072] In some embodiments, a photocleaved polymer is substantially
less soluble in liquid mediums having a predetermined pH and below
than in liquid mediums having a pH greater than the predetermined
pH. A predetermined pH can be about 5, about 6, about 6.5, about 7,
or about 7.4.
[0073] In certain embodiments, a terpolymer of a hydrophobic
monomer, a hydrophilic monomer, and a monomer having a sidegroup
that is photocleavable to produce a carboxyl side chain can be used
as a polymer of a release layer in accordance with the present
disclosure. To give an example, Poly(o-Nitro benzyl
methacrylate-co-Methyl methacrylate-co-Poly(ethylene glycol)
methacrylate) (PNMP) was used in Example 1 for the release layer.
More details of a terpolymer can be found in US Patent Application
No. 20060194145, the contents of which are incorporated herein by
reference.
[0074] In some embodiments, a terpolymer is a random co-polymer of
methyl methacrylate (MMA), poly(ethylene glycol) methacrylate
(PEGMA), and o-nitrobenzyl methacrylate (ONBMA). The ratios of the
three co-monomers may be adjusted to manipulate the solubility of
the terpolymer. In general, sufficient ONBMA may be present to
create sufficient carboxylic acid groups after UV exposure to
promote solubility. The amount of ONBMA may be at least 30%, at
least 35%, at least 40%, at least 45%, or at least 50%. The amount
of PEGMA may be adjusted to provide sufficient hydrophilicity that
the terpolymer does not dewet from the substrate while not
dissolving prematurely in aqueous buffers and to prevent excessive
hydrogen bonding between PEGMA and the photogenerated carboxylic
acid. The amount of PEGMA will partially depend on the amount of
ONBMA and may be 30% or less, 25% or less, 20% or less, 15% or
less, or 10% or less. The amount of MMA may be adjusted to provide
sufficient hydrophobicity to prevent premature dissolution while
not being so great that a terpolymer dewets from substrates. The
amount of MMA may be at least at least 30%, at least 35%, at least
40%, at least 45%, or at least 50%. In certain embodiments, PEGMA
units also serve as a barrier to nonspecific protein binding to
terpolymers.
[0075] One skilled in the art will recognize that the composition
of the co-monomers may also be varied. Monomers may be substituted
for any of MMA, PEGMA, or ONBMA. In general, monomers may be chosen
that do not significantly absorb at the wavelength used for
cleavage of the photoreactive group. For example, upon UV exposure,
the ONBMA is cleaved from a pH sensitive carboxylic acid. The
compositions of monomers may be varied using the same
considerations (e.g., balancing hydrophobicity and hydrophilicity,
minimizing hydrogen bonding, etc.) as described above for the
relative ratios of the co-monomers. Exemplary monomers that may be
substituted for MMA include ethyl methacrylate, n-butyl
methacrylate, n-decyl methacrylate, 2-ethylhexyl methacrylate,
N-(n-octadecyl)acrylamide, n-tert-octylacrylamide, stearyl
acrylate, stearyl methacrylate, and vinyl stearate. Exemplary
monomers that may be substituted for the PEGMA include
hydroxyethylmethacrylate, hydroxyethyl acrylate, 4-hydroxybutyl
methacrylate, N-(2-hydroxypropyl)methacrylamide,
n-methylmethacrylamide, acrylamide, poly(ethylene glycol)
monomethyl ether methacrylates, poly(ethylene glycol)
methacrylates, and n-vinyl-2-pyrrolidone. The PEG chain on PEGMA
may have 6 mers. More or fewer mers may be employed as well, for
example, 3-9 mers. A longer PEG chain will increase hydrogen
bonding and vice versa.
[0076] Alternative photocleavable groups of a general structure
##STR00004##
that leave behind a carboxyl group after photocleavage may also be
substituted for the o-nitrobenzyl group on the ONBMA. For example,
the position R.sub.2 may be substituted with benzoyl, hydrogen,
benzyl, alkyl, alkenyl, aryl, or cycloalkyl. Three groups may in
turn be substituted, for example, with benzoyl, benzyl, alkyl,
alkenyl, aryl, or cycloalkyl. Alternatively, or in addition,
R.sub.1 may be hydrogen or nitro. In one embodiment, benzoin
(R.sub.1=H, R.sub.2=benzoyl), which is photocleavable at 350 nm, is
employed. Alternatively or in addition, photocleavage may occur
after exposure to about 1350 mJ/cm.sup.2 to about 2025 mJ/cm.sup.2
or more of UV radiation. One skilled in the art will recognize that
the energy required for photocleavage may depend on a variety of
factors, including layer composition and thickness. The required
energy for a particular layer may be determined by "titrating" the
film with various amounts of energy.
[0077] The thickness of a release layer may vary depending on
methods of deposition and applications. In some embodiments, a
release layer has an average thickness in a range of about 1 nm and
about 10 .mu.m. In some embodiments, a release layer has an average
thickness in a range of about 10 nm and about 1 .mu.m. In some
embodiments, a release layer has an average thickness in a range of
about 100 nm and about 200 nm. In some embodiments, the average
thickness of an LBL film is or more than about 10 nm, about 50 nm,
about 80 nm, about 100 nm, about 150 nm, about 200 nm, about 300
nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about
800 nm, about 900 nm, about 1 .mu.m, about 5 .mu.m, or about 10
.mu.m. In some embodiments, a release layer has an average
thickness in a range of any two values above.
Agents for Delivery
[0078] Film compositions and/or film-coated substrates utilized in
accordance with the present invention can comprise one or more
agents for delivery. In some embodiments, one or more agents are
independently associated with a substrate, an LBL film coating the
substrate, or both in a coated substrate.
[0079] In some embodiments, an agent can be associated with
individual layers of an LBL film for incorporation, affording the
opportunity for exquisite control of loading and release from the
film. In certain embodiments, an agent is incorporated into an LBL
film by serving as a layer.
[0080] In some embodiments, an agent for delivery is released when
one or more layers of a LBL film are decomposed. Additionally or
alternatively, an agent is released by diffusion.
[0081] In theory, any agents may be associated with the LBL film
disclosed herein to be released, which includes, for example,
therapeutic agents (e.g. antibiotics, NSAIDs, glaucoma medications,
angiogenesis inhibitors, neuroprotective agents), cytotoxic agents,
diagnostic agents (e.g. contrast agents; radionuclides; and
fluorescent, luminescent, and magnetic moieties), prophylactic
agents (e.g. vaccines), transfection agents, immunological agents
(e.g., adjuvant), nutraceutical agents (e.g. vitamins, minerals,
etc.), and/or other substances that may be suitable for
introduction to biological tissues, including pharmaceutical
excipients and substances for tattooing, cosmetics, and the
like.
[0082] In some embodiments, an agent can be small molecules, large
(i.e., macro-) molecules, or a combination thereof. Exemplary
agents include, but are not limited to, small molecules (e.g.
cytotoxic agents), nucleic acids (e.g., siRNA, RNAi, and microRNA
agents), proteins (e.g. antibodies), peptides, lipids,
carbohydrates, hormones, metals, radioactive elements and
compounds, drugs, vaccines, transfection agents, immunological
agents, etc., and/or combinations thereof. In some embodiments, an
agent can be a drug formulation including various forms, such as
liquids, liquid solutions, gels, hydrogels, solid particles (e.g.,
microparticles, nanoparticles), or combinations thereof.
[0083] In some embodiments, compositions and methods in accordance
with the present disclosure are particularly useful for vaccination
and/or therapeutic by releasing of one or more nucleic acids. In
certain embodiments, a nucleic acid is a plasmid DNA.
[0084] In addition to nucleic acids or alternatively, various other
agents may be associated with compositions in accordance with the
present disclosure. In some embodiments, transfection agents and/or
immunological agents can be used in combination with nucleic acids.
In certain embodiments, a transfection agent is a cationic polymer.
For example, poly(-amino ester) (PBAE) as discussed herein can be
used as a transfection agent. In certain embodiments, an
immunological agent is an adjuvant molecule (e.g., poly(I:C) as
used in Example 1).
[0085] Compositions in many embodiments of the present disclosure
can comprise therapeutic agents for delivery. In some embodiments,
a therapeutic agent is a small molecule and/or organic compound
with pharmaceutical activity. In some embodiments, a therapeutic
agent is a clinically-used drug. In some embodiments, a therapeutic
agent to be delivered is an agent useful in combating inflammation
and/or infection. In some embodiments, a therapeutic agent is or
comprises an antibiotic, anti-viral agent, anesthetic,
anticoagulant, anti-cancer agent, inhibitor of an enzyme, steroidal
agent, anti-inflammatory agent, anti-neoplastic agent, antigen,
vaccine, antibody, decongestant, antihypertensive, sedative, birth
control agent, progestational agent, anti-cholinergic, analgesic,
anti-depressant, anti-psychotic, .beta.-adrenergic blocking agent,
diuretic, cardiovascular active agent, vasoactive agent,
anti-glaucoma agent, neuroprotectant, angiogenesis inhibitor,
etc.
[0086] In some embodiments, a therapeutic agent may be a mixture of
pharmaceutically active agents. For example, a local anesthetic may
be delivered in combination with an antiinflammatory agent such as
a steroid. Local anesthetics may also be administered with
vasoactive agents such as epinephrine. To give but another example,
an antibiotic may be combined with an inhibitor of the enzyme
commonly produced by bacteria to inactivate the antibiotic {e.g.,
penicillin and clavulanic acid).
[0087] In some embodiments, a therapeutic agent may be an
antibiotic. Exemplary antibiotics include, but are not limited to,
.beta.-lactam antibiotics, macrolides, monobactams, rifamycins,
tetracyclines, chloramphenicol, clindamycin, lincomycin, fusidic
acid, novobiocin, fosfomycin, fusidate sodium, capreomycin,
colistimethate, gramicidin, minocycline, doxycycline, bacitracin,
erythromycin, nalidixic acid, vancomycin, and trimethoprim. For
example, .beta.-lactam antibiotics can be ampicillin, aziocillin,
aztreonam, carbenicillin, cefoperazone, ceftriaxone, cephaloridine,
cephalothin, cloxacillin, moxalactam, penicillin G, piperacillin,
ticarcillin and any combination thereof.
[0088] In some embodiments, a therapeutic agent may be an
anti-inflammatory agent. Anti-inflammatory agents may include
corticosteroids (e.g., glucocorticoids), cycloplegics, nonsteroidal
anti-inflammatory drugs (NSAIDs), immune selective
anti-inflammatory derivatives (ImSAIDs), and any combination
thereof. Exemplary NSAIDs include, but not limited to, celecoxib
(Celebrex.RTM.); rofecoxib (Vioxx.RTM.), etoricoxib (Arcoxia.RTM.),
meloxicam (Mobic.RTM.), valdecoxib, diclofenac (Voltaren.RTM.,
Cataflam.RTM.), etodolac (Lodine.RTM.), sulindac (Clinori.RTM.),
aspirin, alclofenac, fenclofenac, diflunisal (Dolobid.RTM.),
benorylate, fosfosal, salicylic acid including acetylsalicylic
acid, sodium acetylsalicylic acid, calcium acetylsalicylic acid,
and sodium salicylate; ibuprofen (Motrin), ketoprofen, carprofen,
fenbufen, flurbiprofen, oxaprozin, suprofen, triaprofenic acid,
fenoprofen, indoprofen, piroprofen, flufenamic, mefenamic,
meclofenamic, niflumic, salsalate, rolmerin, fentiazac, tilomisole,
oxyphenbutazone, phenylbutazone, apazone, feprazone, sudoxicam,
isoxicam, tenoxicam, piroxicam (Feldene.RTM.), indomethacin
(Indocin.RTM.), nabumetone (Relafen.RTM.), naproxen
(Naprosyn.RTM.), tolmetin, lumiracoxib, parecoxib, licofelone
(ML3000), including pharmaceutically acceptable salts, isomers,
enantiomers, derivatives, prodrugs, crystal polymorphs, amorphous
modifications, co-crystals and combinations thereof.
[0089] Those skilled in the art will recognize that this is an
exemplary, not comprehensive, list of agents that can be released
using compositions and methods in accordance with the present
disclosure.
Substrate
[0090] A coated device in accordance with the present invention
comprises one or more multilayer films coated on at least one
surface of a substrate.
[0091] A variety of materials can be used as a substrate. In some
embodiments, a material of a substrate is metals (e.g., gold,
silver, platinum, and aluminum); metal-coated materials; metal
oxides; and combinations thereof. In some embodiments, a material
of a substrate is plastics, ceramics, silicon, glasses, mica,
graphite or combination thereof. In some embodiments, a material of
a substrate is a polymer. Exemplary polymers include, but are not
limited to, polyamides, polyphosphazenes, polypropylfumarates,
polyethers, polyacetals, polycyanoacrylates, polyurethanes,
polycarbonates, polyanhydrides, polyorthoesters, polyhydroxyacids,
polyacrylates, ethylene vinyl acetate polymers and other cellulose
acetates, polystyrenes, poly(vinyl chloride), poly(vinyl fluoride),
poly(vinyl imidazole), poly(vinyl alcohol), poly(ethylene
terephthalate), polyesters, polyureas, polypropylene,
polymethacrylate, polyethylene, poly(ethylene oxide)s and
chlorosulphonated polyolefms; and combinations thereof. In some
embodiments, a substrate may comprise more than one material to
form a composite.
[0092] A substrate can be a medical device. Some embodiments of the
present disclosure comprise various medical devices, such as
sutures, bandages, clamps, valves, intracorporeal or extracorporeal
devices (e.g., catheters), temporary or permanent implants, stents,
vascular grafts, anastomotic devices, aneurysm repair devices,
embolic devices, and implantable devices (e.g., orthopedic
implants) and the like.
Microneedle Substrates
[0093] Microneedle substrates, for example, can be used in
accordance with the present invention. Coated microneedle
substrates and methods for coating are described herein, enabling
various multilayer films containing agents to be controllably
coated onto microneedle substrates. Such coated microneedle
substrates can be contacted with biological tissues, particularly
for transdermal delivery of agents.
[0094] In some embodiments, a microneedle substrate is provided
which includes at least one microneedle having a base, a tip end,
and a shaft portion therebetween, and a multilayer film coating on
at least a portion of the surface of the microneedle. In some
embodiments, the multilayer film coating includes at least one
releasable agents. Such multilayer film coatings can be a
homogeneous or a heterogeneous composition.
[0095] A microneedle substrate can be formed/constructed of
different biocompatible materials, including metals, glasses,
semi-conductor materials, ceramics, or polymers. Examples of
suitable metals include pharmaceutical grade stainless steel, gold,
titanium, nickel, iron, tin, chromium, copper, and alloys thereof.
In some embodiments, stainless steel is an attractive material for
microneedle fabrication because it is FDA approved for medical
devices and is inexpensive.
[0096] In some embodiments, a microneedle substrate may include or
be formed of a polymer. A polymer can be biodegradable or
non-biodegradable. Examples of suitable biocompatible,
biodegradable polymers include polylactides, polyglycolides,
polylactide-co-glycolides (PLGA), polyanhydrides, polyorthoesters,
polyetheresters, polycaprolactones, polyesteramides, poly(butyric
acid), poly(valeric acid), polyurethanes and copolymers and blends
thereof. Representative non-biodegradable polymers include
polyacrylates, polymers of ethylene-vinyl acetates and other acyl
substituted cellulose acetates, non-degradable polyurethanes,
polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl
imidazole), chlorosulphonate polyolefms, polyethylene oxide, blends
and copolymers thereof. Biodegradable microneedles can provide an
increased level of safety compared to nonbiodegradable ones, such
that they are essentially harmless even if inadvertently broken off
into the biological tissue following contact.
[0097] In some embodiments, a microneedle substrate includes a
substantially planar foundation from which one or more microneedles
extend, typically in a direction normal (i.e., perpendicular or
`out-of-plane`) to the foundation. Additionally or alternatively,
microneedles may be fabricated on the edge of a substrate
`in-plane` with the substrate. In some embodiments, a single
microneedle can be fabricated on a substrate surface or edge. In
some embodiments, microneedles are fabricated on a flexible base
substrate. It would be advantageous in some circumstances to have a
base substrate that can bend to conform to the shape of the surface
of a biological tissue in which the substrate contacts. In some
embodiments, the microneedles are fabricated on a curved base
substrate. The curvature of the base substrate typically would be
designed to conform to the shape of the tissue surface.
[0098] Microneedles in theory can be of any shape or design. A
microneedle may be solid or hollow. A microneedle can be porous or
non-porous. A microneedles may be planar, cylindrical, or
conical.
[0099] In some embodiments, the dimensions of a microneedle, or
array thereof, are designed for the particular way in which it is
to be used. In various embodiments, the microneedle may have a
dimension in a range of between about 50 .mu.m and about 5000
.mu.m, about 100 .mu.m and about 1500 .mu.m, or between about 200
.mu.m and about 1000 .mu.m.
[0100] In some embodiments, a microneedle substrate includes a
single microneedle or an array of two or more microneedles. The
microneedles can be fabricated as, or combined to form microneedle
arrays. For example, a microneedle substrate may include an array
of between 2 and 1000 (e.g., between 2 and 100) microneedles. In
some embodiments, a microneedle substrate may include an array of
between 2 and 10 microneedles. An array of microneedles may include
a mixture of different microneedles. For instance, an array may
include microneedles having various lengths, base portion
diameters, tip portion shapes, spacings between microneedles, drug
coatings, etc.
Methods and Uses
[0101] There are several advantages to LBL assembly techniques used
to coat a substrate in accordance with the present disclosure,
including mild aqueous processing conditions (which may allow
preservation of biomolecule function); nanometer-scale conformal
coating of surfaces; and the flexibility to coat objects of any
size, shape or surface chemistry, leading to versatility in design
options. According to the present disclosure, one or more LBL films
in addition to at least one release layer can be assembled and/or
deposited on a substrate to provide a coated device.
[0102] In many embodiments, a coated device having one or more
agents for delivery associated within LBL films, such that
decomposition of layers of LBL films results in release of the
agents. LBL films can be different in film architecture (e.g.,
bilayers, tetralayer, etc.), film thickness, film materials (e.g.,
polymers), surface chemistries, and/or agent association depending
on methods and/or uses. In many embodiments, a coated device in
accordance with the present disclosure is for medical use.
[0103] It will be appreciated that an inherently charged surface of
a substrate can facilitate LBL assembly of an LBL film on the
substrate. In addition, a range of methods are known in the art
that can be used to charge the surface of a substrate, including
but not limited to plasma processing, corona processing, flame
processing, and chemical processing, e.g., etching, micro-contact
printing, and chemical modification.
[0104] In some embodiments, a substrate can be coated with a base
layer. Additionally or alternatively, substrates can be primed with
specific polyelectrolyte bilayers such as, but not limited to,
LPEI/SPS, PDAC/SPS, PAH/SPS, LPEI/PAA, PDAC/PAA, and PAH/PAA
bilayers, that form readily on weakly charged surfaces and
occasionally on neutral surfaces. Exemplary polymers can be used as
a primer layer include poly(styrene sulfonate) and poly(acrylic
acid) and a polymer selected from linear poly(ethylene imine),
poly(diallyl dimethyl ammonium chloride), and poly(allylamine
hydrochloride). It will be appreciated that primer layers provide a
uniform surface layer for further LBL assembly and are therefore
particularly well suited to applications that require the
deposition of a uniform thin film on a substrate that includes a
range of materials on its surface, e.g., an implant or a complex
tissue engineering construct.
[0105] The assembly/deposition of release layers and LBL films can
be performed separately. In some embodiments, assembly/deposition
of multilayer films may involve a series of dip coating steps in
which a substrate is dipped in alternating solutions. In some
embodiments, assembly/deposition of multilayer films may involve
mixing, washing or incubation steps to facilitate interactions of
layers, in particular, for non-electrostatic interactions.
Additionally or alternatively, it will be appreciated that
assembly/deposition of multilayer films may also be achieved by
spray coating, dip coating, brush coating, roll coating, spin
casting, or combinations of any of these techniques. In some
embodiments, spray coating is performed under vacuum. In some
embodiments, spray coating is performed under vacuum of about 10
psi, 20 psi, 50 psi, 100 psi, 200 psi or 500 psi. In some
embodiments, spray coating is performed under vacuum in a range of
any two values above.
[0106] In some embodiments, a release layer is deposited on a
substrate and then converted to alter its property to become
releasable under certain conditions. For example, conversion can be
conducted by exposing a release layer to UV. Such a step of
converting can be performed before or after the step of assembling
LBL films.
[0107] Certain characteristics of a coated device may be modulated
to achieve desired functionalities for different applications. Dose
(e.g., loading capacity) may be modulated, for example, by changing
the number of multilayer units that make up the film, the type of
degradable polymers used, the type of polyelectrolytes used, and/or
concentrations of solutions of agents used during construction of
LBL films. Similarly, release kinetics (both rate of release and
release timescale of an agent) may be modulated by changing any or
a combination of the aforementioned factors.
[0108] In some embodiments, the total amount of agent released per
square centimeter is about or greater than about 1 mg/cm2. In some
embodiments, the total amount of agent released per square
centimeter in an LBL film is about or more than about 100
.mu.g/cm2. In some embodiments, the total amount of agent released
per square centimeter in an LBL film is about or more than about 50
.mu.g/cm2. In some embodiments, the total amount of agent released
per square centimeter in an LBL film is about or more than about 10
mg/cm2, about 1 mg/cm2, 500 .mu.g/cm2, about 200 .mu.g/cm2, about
100 .mu.g/cm2, about 50 .mu.g/cm2, about 40 .mu.g/cm 2, about 30
.mu.g/cm2, about 20 .mu.g/cm2, about 10 .mu.g/cm2, about 5
.mu.g/cm2, or about 1 .mu.g/cm2. In some embodiments, the total
amount of agent released per square centimeter in an LBL film is in
a range of any two values above.
[0109] Release of a releasable agent may follow linear kinetics
over a period of time. Release of multiple drugs from a multilayer
film may be complicated by interactions between layers, and/or
drugs. Such a release profile may be desirable to effect a
particular dosing regimen. During all or a part of the time period
of release, release may follow approximately linear kinetics.
[0110] A release timescale (e.g., t.sub.50%, t.sub.85%, t.sub.99%)
of an agent for delivery can vary depending on applications. In
some embodiments, a release timescale of an agent for delivery is
less or more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours,
10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 40 hours, 50
hours, 75 hours, 100 hours, 150 hours, or 200 hours. In some
embodiments, a release timescale of an agent for delivery is less
or more than about 1 day, 2 days, about 5 days, about 10 days,
about 12 days, about 20 days, about 30 days, 50 or about 100 days.
In some embodiments, a release timescale of an agent for delivery
is in a range of any two values above.
EXAMPLES
Example 1
[0111] In this Example, rapid hypodermic-needle-free DNA delivery
was demonstrated, using microneedle patches that implant
multi-layer polymer films into the epidermis following a brief
application to skin. Biodegradable multi-layer films carrying DNA,
a polymeric transfection agent, and adjuvant molecules implanted in
murine skin both efficiently transfected cells in the local tissue
and elicited immune responses comparable to in vivo electroporation
of plasmid DNA (pDNA), one of the most promising current
technologies for DNA vaccine delivery.
[0112] To substantially enhance DNA vaccine delivery, an approach
was designed to simultaneously (i) improve targeting of DNA to
tissues rich in immune response-governing dendritic cells, (ii)
promote sustained transfection without toxicity, (iii) and provide
supporting inflammatory cues to enhance the induction of a potent
immune response. To this end, a strategy was developed using
microneedles to rapidly and painlessly implant biodegradable drug
delivery films into the skin, which continuously released DNA
polyplexes and adjuvant molecules in this immunologically-rich
tissue over a tunable and sustained period of time (FIG. 1a).
Relative to other potential materials that might achieve this goal,
PolyElectrolyte Multi-layers (PEMs) have a number of advantages
including their ability to be assembled by mild aqueous processing,
easily embed diverse cargos in their nanostructure, carry large
weight fractions of functional cargo (e.g., DNA, up to 40% of total
film mass), and exhibit controlled release characteristics
predetermined by film architecture/composition. In addition, PEMs
composed of nucleic acids and polymeric transfection agents have
been previously been demonstrated to transfect cells in vitro and
in vivo, through the continuous release of in szYw-formed
polyplexes.
[0113] To translate this concept in vivo for vaccination, an
approach for polymer multilayer "tattooing" was developed using
microneedle arrays coated with vaccine-loaded PEMs that are
released from the microneedle surface and remain implanted into the
skin as the patch is removed (FIG. 1a). It is contemplated here
that multi-layer release can be achieved via an underlying
polymeric `release layer` designed to instantly dissolve on
hydration by interstitial fluid in the skin (FIG. 1a). This design
allows the kinetics of DNA/adjuvant release in the tissue to be
separately tailored from the time required for a microneedle patch
to be kept on the skin. However, a water-soluble release layer
would by definition be unstable during aqueous Layer-by-Layer (LBL)
coating of the microneedle array with the vaccine multi-layer. To
solve this problem, we employed a photo-sensitive polymer,
Poly(o-Nitro benzyl methacrylate-co-Methyl
methacrylate-co-Poly(ethylene glycol) methacrylate) (PNMP) for the
release layer (FIG. 1a). PNMP is organic soluble, but on brief
exposure to UV, cleavage of the pendant o-nitrobenzyl groups
converts the polymer to a weak polyelectrolyte (uv-PNMP) that is
soluble in water above pH .about.6.5 but stable in aqueous
solutions below this pH. It is demonstrated here that the organic
solubility of PNMP permitted facile deposition on microneedles by
spray deposition from volatile organic solvents, while the
selective water solubility of uv-PNMP allowed both aqueous LBL
assembly of an overlying PEM from mildly acidic pH buffers and
rapid release of the PEM when dried films were exposed to
near-neutral pH solutions. Further, as shown below,
photo-switchable PNMP solubility provided the means to prove that
PEM film implantation depended on release layer dissolution.
[0114] To first demonstrate the use of PNMP copolymer coatings to
serve as a pH-responsive base for PEM assembly and subsequent
release from an underlying substrate, the PNMP thin films (130 nm
in thickness) were cast on silicon, UV-irradiated to induce the
organic-to-aqueous solubility transition in the polymer, and then
constructed overlying model PEM films composed of linear
poly(ethylineimine) (LPEI)/poly(acrylic acid) (PAA) and branched
PEI (BPEI)/PAA bilayers using LBL assembly from aqueous polymer
solutions at pH 5. After scratching .about.1500 nm thick dried
(PNMP)-(LPEI/PAA)3(BPEI/PAA)30 films with a razor blade, the
underlying intact PNMP release layer could be directly observed by
light microscopy, and surface profilometry at the edge of these
defects showed that the thickness of the underlying uv-PNMP layer
was unchanged (131.+-.3 nm) following
(PEI/PAA).sub.3-(BPEI/PAA).sub.30 film deposition (FIG. 1b). Next,
the film-coated substrates were immersed in pH 7.4
phosphate-buffered saline (PBS) and observed by time-lapse optical
microscopy. The uv-PNMP layer was observed to dissolve within 1 min
of exposure to PBS, followed by the formation and coalescence of
aqueous bubbles under the PEM film and macroscopic delamination of
intact films without any agitation after approximately 15 min (FIG.
1c and FIG. 13). Physical agitation greatly accelerated this
process, with delamination observed nearly instantaneously
following immersion of dry films and agitation by pipetting (not
shown). Multi-layer delamination was dependent upon UV exposure of
the PNMP prior to PEM assembly and the pH of the immersion buffer,
as no PNMP dissolution or film delamination was observed for
composite films that did not receive UV pre-treatment or that were
incubated in buffered solutions at pH <6.5 (data not shown).
[0115] Given these promising initial results, an analogous process
was developed for fabrication of microneedle arrays coated with
PNMP release layers underlying a DNA vaccine-carrying PEM film.
Skin patches were first fabricated by melt-molding poly(L-lactide)
(PLLA) on poly(dimethyl siloxane) (PDMS) masters to obtain arrays
of microneedles each 250 .mu.m in diameter at their base and 650
.mu.m in height (FIG. 6). Biotinylated PNMP (bPNMP), (FIG. 7) films
were coated on the microneedles by spray deposition from
1,4-dioxane solutions, followed by staining with fluorescent
streptavidin (SAv488) to permit visualization of the release layer
by microscopy. Next, LBL deposition was used to construct an
overlying PEM film composed of Cy5 -labeled pDNA encoding
luciferase (Cy5-pLUC) and the transfection agent poly-1 (FIG. 7), a
biodegradable poly(.beta.-amino ester) (PBAE). PEM films were
initiated by depositing 20 bilayers of protamine sulfate (PS) and
poly(4-styrene sulfonate) (PSS) to provide a uniform charge
density, followed by iterative adsorption of poly-1 and Cy5-pLUC
(FIG. 2a). Profilometry measurements performed on PEMs constructed
in parallel on Si substrates showed linear multi-layer growth with
increasing deposition cycles as previously reported for PBAE/pDNA
films and intact underlying uv-PNMP films (FIG. 2b).
Cross-sectional confocal imaging of microneedles coated with
composite uv-PNMP-(poly-1/Cy5-pLUC).sub.35 PEM films showed
conformal co-localized fluorescence from SAv-labeled uv-bPNMP and
Cy5-pLUC over the surface of each PLLA microneedle (FIG. 2c).
(Individual uv-bPNMP and PEM films were too thin to resolve as
distinct layers). When analyzed at each stage of PEM film
deposition, the mean total SAv488-bPNMP fluorescence signal from
single microneedles was stable but Cy5-pLUC fluorescence linearly
increased with each round of bilayer deposition, confirming linear
film growth on the microneedles as well (FIG. 2d). In line with
this result, the total dosage of DNA recovered from microneedle
coatings disrupted by treatment with sodium chloride showed a
linear increase in plasmid dosage with increasing bilayer number
(.about.4.2 .mu.g/bilayer per cm.sup.2 microneedle array area, FIG.
2d). Similar analyses of microneedles coated with PNMP and
multi-layers comprised of poly-1 or poly-2 and the nucleic acid
adjuvant poly(LC) also showed conformal film deposition and
approximately linear film growth with increasing number of
deposited bilayers (FIG. 8). Finally, sequential assembly of PEM
films comprising layers of (poly-1/poly(I:C)) followed by layers of
(poly-1/pLUC) generated microneedles coated with complete vaccine
films containing pDNA, a transfection agent, and a strong adjuvant
(FIG. 2e-h and FIG. 8).
[0116] To test PEM film release from microneedle arrays, dried
composite SAv-labeled
uv-bPNMP-(PS/SPS).sub.20(poly-1/Cy5-pLUC).sub.35 coatings (referred
to henceforth as PNMP/PEM films) were immersed in pH 7.4 PBS for
varying times in vitro and imaged by confocal microscopy to
quantitate uv-bPNMP and Cy5-pLUC fluorescence remaining on the
microneedle surfaces over time. Mirroring the results obtained with
model films on flat Si substrates, we observed a significant loss
of both SAv488-wv-bPNMP and Cy5-pLUC fluorescence from microneedle
arrays after only 15 min incubation in PBS without agitation (FIG.
9). Delamination plateaued after 30 min with approximately 60% loss
of both SAv488-uv-bPNMP and Cy5-pLUC signal. By contrast, no film
release was observed if PEMs were assembled onto PNMP coatings that
had not been irradiated to photo-switch the release layer's
solubility. However, in agreement with our previous report of
biodegradable (poly-1/pDNA) microneedle coatings, composite films
prepared without UV-treatment of the release layer showed complete
loss of Cy5-pLUC signal due to hydrolysis of poly-1 and degradation
of the PEM by 24 hr, while the SAv488-uv-bPNMP signal remained
unchanged (FIG. 9). Thus, DNA-carrying PEM films assembled onto a
pH-sensitive PNMP release layer are rapidly released from the
surfaces of microneedles when exposed to physiological saline
solution, coincident with rapid PNMP film dissolution.
[0117] To test whether microneedles coated with "quick release" PEM
films would permit rapid PEM film implantation in vivo, we applied
dry PNMP-PEM-coated PLLA microneedles to the dorsal ear skin of
C57B1/6-MHC II-GFP mice, transgenic animals expressing green
fluorescent protein (GFP) fused to class II major
histocompatibility complex (MHC) molecules. The MHC II-GFP fusion
protein provides an in situ marker for the viable epidermis, as
fluorescent epidermal MHC II+ Langerhans cells (LCs) residing in
the tissue are readily detected by confocal microscopy. As expected
from prior studies of microneedle arrays fabricated from PLLA,
trypan blue staining of skin following microneedle patch
application with gentle pressure showed consistent and uniform
microneedle penetration (FIG. 3a). Mirroring our in vitro
observations, confocal imaging of microneedles after application of
the patches to murine skin showed that both uv-bPNMP and Cy5-pLUC
fluorescence was rapidly lost from coated microneedles, but only if
PNMP films were irradiated before microneedle coating to prime for
rapid dissolution of the release layer (FIGS. 3b, c). To determine
whether PEM films released from microneedles were in fact deposited
in the skin, we performed confocal optical sectioning on skin
samples following application of PNMP-PEM-coated microneedle
arrays. Application of microneedles to skin for 15 min when the
release layer was not UV primed resulted in no detectable Cy5-pLUC
delivery into the skin (data not shown). By contrast, application
of UV-primed microneedles for the same time led to significant
transfer of Cy5-pLUC both in the upper epidermis co-localized with
GFP+ Langerhans cells (FIG. 3d) and up to 400 .mu.m deep into the
skin (FIG. 3e). Similarly, microneedles carrying poly(I:C)-loaded
PEM films deposited fluorescently-labeled poly(LC) into the skin,
colocalizing in the same z-plane with MHC II-expressing cell
populations (FIGS. 3f, g and FIG. 10). In both cases, confocal
z-stacks of treated skin indicated the consistent deposition of
pLUC and poly(LC) 300-400 .mu.m below the skin surface at sites of
microneedle penetration (FIG. 3e and FIG. 10). Thus, the uv-PNMP
release layer promotes rapid implantation of DNA- or RNA-loaded
films into the skin.
[0118] We also tested whether the in vivo kinetics of nucleic acid
release into the surrounding tissue could be controlled via the
composition of the multi-layers implanted in the skin. Past studies
have demonstrated the ability of PEM films to provide controlled
release of nucleic acids and promote transfection by released DNA
in vitro, and multi-layers composed of pDNA assembled with the
PBAEs poly-1 or poly-2 have previously been shown to mediate the
release of pDNA with varying kinetics, and to generate polymer-pDNA
polyplexes in situ. Consistent with these prior studies,
(PS/SPS).sub.20-(poly-1/poly(I:C)).sub.35 and
(PS/SPS).sub.20-(poly-1/pLUC).sub.35 multi-layers constructed on Si
substrates and incubated in PBS at 37.degree. C. in vitro exhibited
a substantial burst release of .about.80% pLUC or poly(I:C) within
24 hr, while analogous films constructed with poly-2 showed a lower
burst release of .about.35% followed by nearly zero-order release
kinetics for 10 days (FIG. 4a and FIG. 11). Dynamic light
scattering analysis of eroded films revealed large aggregates
(50-300 nm, data not shown) consistent with previous evidence of in
situ polyplex formation. To determine whether the composition of
PBAE films implanted via microneedle delivery could mediate similar
tunable release of nucleic acid therapeutics in vivo, we
constructed multi-layer films composed of Cy5-poly(I:C) assembled
with either poly-1 or poly-2 on PNMP-coated microneedles as before.
Following application of the coated microneedles to the skin of
C57B1/6 mice for 15 min, we monitored the fluorescence signal of
Cy5-poly(I:C) implanted in the skin over time using whole animal
fluorescence imaging (FIG. 4b). The results show that similar to
the in vitro trend, films encapsulating Cy5-poly(I:C) with poly-1
are cleared from the application site within 3 days. Conversely,
Cy5-poly(LC) signal was observed in mice treated with poly-2 film
variants for 10 days following application, with clearance
following kinetics similar to those seen in vitro (FIG. 4c). Thus,
the composition of the PEM films delivered by microneedle tattooing
can directly control the release and clearance of their nucleic
acid cargos in the skin.
[0119] Polymers, poly-1 and poly-2, as exemplary polycation
components of these PEM coatings were selected because of not only
their biodegradable nature and ability to regulate the rate of
release of nucleic acid cargos from films, but also their important
role in directly promoting transfection of released pDNA, via the
in situ formation of polyplexes during film degradation and plasmid
release. To test whether the composition of PBAE/pDNA multi-layers
could predetermine the kinetics of the DNA cargo bioactivity in
vivo (i.e. transfection), we used whole animal bioluminescence
imaging to longitudinally monitor expression of luciferase
following pLUC delivery by microneedles. Microneedles were prepared
with PNMP-(PBAE/pLUC) coatings, with or without UV priming of the
PNMP release layer. Application of control microneedles (where the
release layer was not UV primed) to the skin of mice for 15 minutes
led to no detectable expression of pLUC (FIG. 12), consistent with
the lack of detectable film transfer into skin under this
condition. By contrast, the skin of mice treated with microneedles
coated with uv-PNMP/(PBAE/pLUC) films showed significant levels of
bioluminescence one day after application, demonstrating
transfection of cells in situ (FIGS. 4d, e). Further, the kinetics
of pLUC expression varied greatly depending on the PBAE selected as
the complimentary polycation. In the case of skin tattooed with
(poly-1/pLUC) multi-layers, luciferase expression peaked sharply
after three days and declined to background levels 10 days after
treatment, while implantation of slower-degrading (poly-2/pLUC)
films showed a slower increase in bioluminescence, peaking and
remaining consistent from day 3-7, and then slowly decreasing to
background levels by day 22 (FIG. 4e). Together these results
demonstrate the potential to control the level and duration of
plasmid expression in vivo through selection of constituent
polymers with varying half-lives of degradation.
[0120] It is contemplated herein that polymer multi-layer tattooing
could be utilized for enhancing the efficacy of DNA vaccines, by
(1) targeting DNA to skin tissue rich in antigen-presenting cells,
(2) promoting transfection of antigen-encoding DNA via in situ
polyplex formation/release from multi-layer films, and (3)
co-incorporation of nucleic-acid based adjuvant molecules to
promote the immune response. To test this idea, microneedles were
coated with PEMs of poly-1 co-assembled with poly(LC) and pGag, a
plasmid encoding a model HIV gag antigen, for immunization of
animals transcutaneously via microneedle tattooing (FIG. 5a).
Multi-layer tattooing was compared to two control immunizations:
injection of "naked" pGag plasmid DNA solutions, the most common
experimental strategy for DNA immunization in mice and humans,
which relies on spontaneous uptake of DNA following injection in
tissues; and in vivo electroporation, where DNA is administered in
the presence of an electric field via two locally-applied needle
electrodes to promote DNA uptake. Naked DNA immunization elicits
immune responses in small animal models but has showed low potency
in non-human primates and humans; in vivo electroporation is
currently one of the most potent alternative strategies for
promoting DNA transfection and vaccine responses, though it lacks
practicality for mass vaccination. We immunized groups of animals
on days 0 and 28 with 20 .mu.g pGag with10 .mu.g poly(LC) via
multi-layer tattooing with microneedles (MN) applied to the dorsal
ear skin. Control mice received equivalent doses of soluble pGag
and poly(I:C) intradermally (ID) in the ear skin, or soluble DNA/R
A given intramuscularly with or without in vivo electroporation
(IM.+-.EP). Two weeks following the boost, we measured total
Gag-specific IgG titers in sera and observed a significant increase
in microneedle treated mice over those given IM.+-.EP and ID
immunizations (FIG. 5b). Similarly, we compared the frequency of
antigen-specific CD8+ T lymphocytes as well as long-lived CD44+
CD62L+ central memory T cells two weeks and three weeks following
prime and boost, respectively. As seen in FIGS. 5c-d, IM and ID
administration produced only weak antigen-specific CD8+ T cell
response, while the microneedle treated groups showed robust
expansion of Gag-reactive T cells exceeding 5% of the circulating
CD8+ population two weeks following boost. This was quantitatively
similar to frequencies observed for IM+EP immunized mice.
Additionally, microneedle administration effectively generated
greater frequencies of CD44+ CD62L+ central memory T cells, a
population shown to be important for recall immunity and long-term
protection, three weeks following prime and boost (FIGS. 5e-f).
This was true even relative to mice receiving IM+EP treatment.
Thus, DNA vaccination via polymer multi-layer tattooing shows the
potential to match the potency of in vivo electroporation, using a
skin patch that can be stored in a dry state, is painlessly applied
with no extraneous apparatus, and could be self-applied in
minutes.
[0121] In summary, we have demonstrated here a new approach to
therapeutic delivery in skin via polymer thin film "tattooing",
using microneedles to rapidly implant biodegradable drug-loaded
multi-layers in the skin enabling the kinetics of release and
functionality of diverse drug cargos to be manipulated in situ. DNA
vaccination was a test-bed application here, due to the relevance
of needle-free vaccines for global health and the need for enhanced
DNA vaccination strategies. As one with ordinary skill in the art
will appreciate that the adaptability of LBL films for
incorporation and controlled release of therapeutics ranging from
small-molecule drugs to large macromolecules suggests this approach
can be applicable to diverse drug delivery applications. Further,
the pH-sensitive release layer strategy employed here is a
generalizable approach to create selectively-released multi-layer
films. The data shown here demonstrated that multi-layer tattooing
is useful to enhance the efficacy of DNA vaccines, a platform
technology can be applied universally in vaccine development.
Materials
[0122] PNMP was synthesized and biotinylated (bPNMP) as previously
reported, and analyzed by NMR and GPC. Poly-1 and poly-2 were
synthesized, and analyzed by GPC. pLUC and pGag were a gift from
Dr. Daniel Barouch, Beth Israel Deaconess Medical Center.
Phycoerythrin-conjugated AL-1 1/H-2 Kb peptide-MHC II tetramers
were provided by the NIH tetramer core facility.
PLLA Microneedle Fabrication
[0123] PDMS molds (Sylgard 184, Dow Corning) were fabricated by
laser ablation using a Clark-MXR CPA-2010 micromachining system
(VaxDesign Inc.). PLLA pellets (IV 1.9 dL/g, Lakeshore
Biomaterials) were melted over the molds under vacuum (.about.25
in. Hg) at 200.degree. C. for 40 min, and then cooled to
-20.degree. C. before separating the cast microneedle arrays. The
resulting microneedle arrays were then treated at 140.degree. C.
for 4 hr. Microneedles were characterized using a
JEOL 6700F FEG-SEM.
[0124] PNMP Release Layer Deposition
[0125] For film deposition on atomically flat Si substrates, a 3 wt
% PNMP solution in 1,4-dioxane was deposited by spin coating using
a Specialty Coating Systems P6700, and then dried under vacuum for
12 hr. For deposition on PLLA microneedles, a 0.25 wt % bPNMP
solution was deposited using a modified air-brush as previously
described (0.2 mL/s, 15 cm range, 10 s), then dried under vacuum
for 12 hr. bPNMP release-layers were labeled using SAv488
(Sigma-Aldrich) at 10 .mu.g/mL in 1.times. PBS, pH 6.0. Film
deposition was characterized using a Veeco Dektak surface
profilometer and a JEOL 6700F FEG-SEM.
Polymer Multi-Layer Film Preparation
[0126] All LbL films were assembled using a Carl Ziess HMS DS50
slide stainer. Films were constructed on Si wafers and PLLA
microneedle arrays following deposition of bPNMP release layers.
Prior to multi-layer assembly, the solubility of the PNMP release
layer was photoswitched via UV irradiation of coated Si or
microneedle substrates (254 nm, 2.25 mW/cm2, UVP) for 15 min. To
build (PS/SPS) baselayers, substrates were alternatingly dipped
into PS (2 mg/mL, 1.times. PBS, Sigma-Aldrich) and SPS (5 mM,
1.times. PBS, Sigma-Aldrich) solutions for 10 min, separated by two
sequential 1 min rinses in PBS. (PBAE/pLUC) and (PBAE/poly(I:C))
multilayers were deposited similarly, alternating 5 min dips in
poly-1/2 (2 mg/mL, 1.times. PBS) and either pLUC, pGag (1 mg/mL,
1.times. PBS) or poly(LC) (1 mg/mL, 1.times. PBS, Invivogen)
solutions separated by two sequential 30 sec rinsing steps in lx
PBS. Fluorescent pLUC and poly(LC) were prepared using Cy5 and
tetramethyl-rhodamine (TMR) Label-IT reagent (Minis Bio
Corporation). All solutions were adjusted to pH 5.0 and filtered
(0.2 .mu.m, except pLUC, pGag and poly(LC)) prior to dipping. Films
were characterized using a Veeco Dektak surface profilometer and a
Zeiss LSM 510. Data analysis was performed using Image J. Film
loading was determined using a SpectraMax 250 spectrophotometer
following elution of films in 1.times. PBS, pH 7.4, 2 M NaCl for 24
hours.
In Vitro/In Vivo Delamination/Delivery
[0127] For in vitro delamination assays, coated Si wafers were
incubated in PBS, pH 7.4 and time-lapse microscopy was performed
using a Leica DMXR instrument. For in vitro release
(PS/SPS)2o-(PBAE/pLUC)35 or (PS/SPS)20-(PBAE/poly(I:C))35 films on
silicon were incubated in PBS at 37.degree. C. and aliquots were
assayed for released pLUC or poly(I:C) using picogreen or ribogreen
detection kits (Invitrogen) For in vitro delivery, coated
microneedle arrays were incubated in PBS, pH 7.4, dried, and imaged
by confocal microscopy. In vivo delivery experiments were performed
on anesthetized C57BL/6 mice (Jackson Laboratories) and MHC II-GFP
transgenic mice (a gift from Prof. Hidde Ploegh). Ears were rinsed
briefly with PBS on the dorsal side and dried before application of
microneedle arrays by gentle pressure. Following application,
microneedles were dried and imaged by confocal. Treated mice were
sacrificed and excised ears were stained with trypan blue before
imaging for needle penetration. Ears collected from mice treated
with Cy5-pLUC- or TMR-poly(I:C)-coated microneedles
(.+-.UV-treatment) were mounted on glass slides and imaged by
confocal. Clearance of fluorescent poly(LC) and transfection in
mice treated with pLUC-coated arrays (.+-.UV-treatment) was
measured using an IVIS Spectrum 200 (Caliper Lifesciences) to
detect fluorescence and bioluminescence respectively.
Bioluminescence was measured following IP injection of luciferin
and data analysis was performed using the Living Image Software
package.
Vaccinations
[0128] All animal studies were approved by the MIT IUCAC and
animals were cared for in the USDA-inspected MIT Animal Facility
under federal, state, local, and NIH guidelines for animal care.
Groups of 4 C57B1/6 mice were immunized with 20 .mu.g pGag and 10
.mu.g poly(LC) by intramuscular injection (15 .mu.l in the
quadriceps) with or without in vivo electroporation (performed
according to the manufacturer's instructions, Harvard Apparatus
ECM830, 2.times.60 ms pulses, 200 V/cm), intradermal injection (15
.mu.l in the dorsal caudal ear skin) or by microneedle array (15
min application of
(PS/SPS).sub.20-(poly-1/poly(I:C)).sub.35-(poly-1/pGag).sub.35 on
UV-treated PNMP coated PLLA arrays). Frequencies of Gag-specific
CD8+ T-cells and their phenotypes elicited by immunization were
determined by flow cytometry analysis of peripheral blood
mononuclear cells at selected time points following staining with
DAPI (to discriminate live/dead cells), anti-CD8.alpha., anti-CD44,
anti-CD62L, and phycoerythrin-conjugated AL-11/H-2K.sup.b
peptide-MHC tetramers. Anti-Gag IgG titers, defined as the dilution
of sera at which 450 nm OD reading was 0.25, were determined by
ELISA analysis of sera from immunized mice. Animals were cared for
following NIH, state, and local guidelines.
Statistical Analysis
[0129] Statistical analysis was carried out with Graphpad Prism (La
Jolla, CA). Data was analyzed using two-way analysis of variance or
t-test. / -values less than 0.05 were statistically significant.
All values are reported as mean .+-.s.e.m.
Other Embodiments and Equivalents
[0130] While the present disclosures have been described in
conjunction with various embodiments and examples, it is not
intended that they be limited to such embodiments or examples. On
the contrary, the disclosures encompass various alternatives,
modifications, and equivalents, as will be appreciated by those of
skill in the art. Accordingly, the descriptions, methods and
diagrams of should not be read as limited to the described order of
elements unless stated to that effect.
[0131] Although this disclosure has described and illustrated
certain embodiments, it is to be understood that the disclosure is
not restricted to those particular embodiments. Rather, the
disclosure includes all embodiments that are functional and/or
equivalents of the specific embodiments and features that have been
described and illustrated.
* * * * *
References