U.S. patent application number 12/248839 was filed with the patent office on 2009-04-23 for ultrathin multilayered films for controlled release of anionic reagents.
Invention is credited to Xianghui Liu, David M. Lynn, Jingtao Zhang.
Application Number | 20090105375 12/248839 |
Document ID | / |
Family ID | 40549840 |
Filed Date | 2009-04-23 |
United States Patent
Application |
20090105375 |
Kind Code |
A1 |
Lynn; David M. ; et
al. |
April 23, 2009 |
Ultrathin Multilayered Films for Controlled Release of Anionic
Reagents
Abstract
Multilayered films, particularly ultrathin multilayered films
comprising cationic polymers which are useful for controlled
release of anionic species, particularly for controlled release of
nucleic acids. The multilayer films herein are useful for temporal
controlled released of anionic species, particularly one or more
anionic peptides, proteins, nucleic acids or other anionic
biological agents. In one aspect, the invention relates to
multilayer films which release anionic species (anions) with
separate and/or distinct release profiles, particularly wherein the
anions are one or more anionic peptides, proteins or nucleic acids
or other anionic biological agents
Inventors: |
Lynn; David M.; (Middleton,
WI) ; Zhang; Jingtao; (Lansdale, PA) ; Liu;
Xianghui; (Waukegan, IL) |
Correspondence
Address: |
GREENLEE WINNER AND SULLIVAN P C
4875 PEARL EAST CIRCLE, SUITE 200
BOULDER
CO
80301
US
|
Family ID: |
40549840 |
Appl. No.: |
12/248839 |
Filed: |
October 9, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60978633 |
Oct 9, 2007 |
|
|
|
Current U.S.
Class: |
524/27 ;
525/55 |
Current CPC
Class: |
C08L 33/24 20130101;
A61K 9/7007 20130101; C08L 2666/02 20130101; C08L 33/24 20130101;
C08L 39/02 20130101 |
Class at
Publication: |
524/27 ;
525/55 |
International
Class: |
C08L 33/24 20060101
C08L033/24 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with United States government
support under Grants EB002746 and EB006820 awarded by the National
Institutes of Health. The U.S. government has certain rights in the
invention
Claims
1. A polyelectrolyte assembly comprising at least one
anion/cationic polymer bilayer where the cationic polymer is a
cationic polymer selected from: (a) a polymer of formula I:
##STR00016## where: n, m, l, k and i are zero or integers where
n+m+l+k+i=N, the total number of repeat units in the polymer;
A.sub.1-3, B.sub.1-3, C.sub.1-3, D.sub.1-3 and E.sub.1-3 are
linkers which may be the same or each may be different;
Z.sub.1-Z.sub.5 are most generally covalent bonds which may or may
not be degradable bonds; R.sub.1-4 and R.sub.11-19, independently,
can be hydrogen, or alkyl, alkenyl, alkynyl, cycloalkyl,
heterocyclic, aryl, or heteroaryl groups, all of which are
optionally substituted, with the exception that R.sub.1-4 are not
esters; and R.sub.5-R.sub.10 are linkers or covalent bonds which
may be the same or each may be different; or (b) a polymer of
formula II: ##STR00017## where n+m=N is the number of repeating
units in the polymer; each y, independently, is 1, 2 or 3; each x,
independently, is an integer ranging from 1-10; each R.sup.1, each
R.sup.2, each R.sup.3 and each R.sup.4, independently, is selected
from the group consisting of hydrogen, alkyl groups, alkenyl
groups, alkynyl groups, carbocyclic groups, heterocyclic groups,
aryl groups, heteroaryl groups, ether groups, all of which may be
substituted or unsubstituted.
2. The polyelectrolyte assembly of claim 1 wherein the polymer of
formula I has the formula: ##STR00018## where each r is an integer
ranging from 1-10.
3. The polyelectrolyte assembly of claim 2 comprising one or more
different anion/cationic polymer bilayers where at least a first
bilayer is formed from a cationic polymer of formula I and at least
a second bilayer is formed from a different cationic polymer of
formula I.
4. The polyelectrolyte assembly of claim 3 wherein the cationic
polymers of formula I differ from each other in the value of
(m+)/N.
5. The polyelectrolyte assembly of claim 4 wherein one of the
cationic polymers of formula I has a value of (m+)/N of 0.25 or
less and the other cationic polymer of formula I has a value of
(m+)/N of 0.5 or more.
7. The polyelectrolyte assembly of claim 4 wherein one of the
cationic polymers of formula I has a value of (m+)/N of 0.25 or
less and the other cationic polymer of formula I has a value of
(m+)/N of 0.75 or more.
8. The polyelectrolyte assembly of claim 4 wherein one of the
cationic polymers of formula I has a value of (m+)/N of 0.25 or
less and the other cationic polymer of formula I has a value of
(m+)/N of 1.0.
9. The polyelectrolyte assembly of any of claim 2, wherein in the
cationic polymers of formula I r is an integer from 1 to 3,
inclusive, and each R.sub.3 is a hydrogen or a C1-C3 alkyl group,
and each R.sub.11, R.sub.12 and R.sub.13 is a (C1-C6) alkyl
group.
10. The polyelectrolyte assembly of claim 1, further comprising one
or more additional anion/cationic polymer bilayers wherein the one
or more additional bilayers are formed by one or more cationic
polymers of formula I wherein each cationic polymer of formula I in
the assembly differs from each other cationic polymer of formula I
in the assembly in the value of (m+l)/N.
11. The polyelectrolyte assembly of claim 1 which comprises at
least one cationic polymer of formula I wherein the value of (m+)/N
is 0.25 or more.
12. The polyelectrolyte assembly of claim 1 which comprises at
least one cationic polymer of formula I wherein the value of (m+)/N
is 0.25 or less.
13. The polyelectrolyte assembly of claim 1 which comprises at
least one cationic polymer of formula I wherein the value of (m+)/N
is 0.25 or less and at least one cationic polymer of formula I
wherein the value of (m+)/N is 0.50 or more.
14. The polyelectrolyte assembly of claim 1, which comprises only
cationic polymer of formula I.
15. The polyelectrolyte assembly of claim 1 which comprises only
cationic polymers of formula II.
16. The polyelectrolyte assembly of claim 15 comprising two or more
different anion/cationic polymer bilayers where at least a first
bilayer is formed from a cationic polymer of formula II and at
least a second bilayer is formed from a different cationic polymer
of formula II.
17. The polyelectrolyte assembly of claim 16 wherein the cationic
polymers of formula II differ from each other in the value of
(n)/(n+m).
18. The polyelectrolyte assembly of claim 17 wherein at least one
of the cationic polymers of formula II has a value of (n)/(n+m) of
0.5 or more.
19. The polyelectrolyte assembly of claim 18 wherein at least one
of the cationic polymers of formula II has a value of (n)/(n+m) of
0.5 or less.
20. The polyelectrolyte assembly of claim 15, wherein in the
cationic polymers of formula II, all R.sup.2 and R.sup.4 are
hydrogens, all R.sup.1 are C1-C3 alkyl groups and each R.sup.3 is
independently hydrogens or C1-C3 alkyl groups.
21. The polyelectrolyte assembly of claim 1 comprising two or more
different anion/cationic polymer bilayers where at least a first
bilayer is formed from a cationic polymer of formula I and at least
a second bilayer is formed from a different cationic polymer of
formula II.
22. The polyelectrolyte assembly of claim 21 wherein the at least
one cationic polymer of formula I has a value of (m+)/N is 0.50 or
more.
23. The polyelectrolyte assembly of claim 21 wherein the at least
one cationic polymer of formula II has a value of n/n+m of 0.5 or
more.
24. The polyelectrolyte assembly of claim 23 wherein the at least
one cationic polymer of formula I has a value of (m+)/N is 0.50 or
more.
25. The polyelectrolyte assembly of claim 21, wherein in the
cationic polymers of formula I, r is an integer from 1 to 3,
inclusive, and each R.sub.3 is a hydrogen or a C1-C3 alkyl group,
and each R.sub.11, R.sub.12 and R.sub.13 is a (C1-C6) alkyl
group.
26. The polyelectrolyte assembly of claim 21, wherein in the
cationic polymers of formula II, all R.sup.2 and R.sup.4 are
hydrogens, all R.sup.1 are C1-C3 alkyl groups and each R.sup.3 is
independently hydrogens or C1-C3 alkyl groups.
27. The polyelectrolyte assembly of claim 1, wherein the anions of
the bilayers are nucleic acids.
28. The polyelectrolyte assembly of claim 1, wherein the anions of
the bilayers are nucleic acids which encode polypeptides.
29. The polyelectrolyte assembly of claim 1, wherein the
polyelectrolyte assembly is formed on a substrate.
30. The polyelectrolyte assembly of claim 1, wherein the
polyelectrolyte assembly is formed on one or mroe surfaces of an
implantable medical device.
31. A method for controlling the release of two or more anions from
a film which comprises the step of forming a polyelectrolyte
assembly of claim 1, wherein the polyelectrolyte assembly comprises
anion/cationic polymer bilayers having two or more different anions
and subjecting the polyelectrolyte assembly formed to conditions in
which ester functions in the polymer of formula I and in the
polymer of formula II are hydrolyzed to release the two or more
anions.
32. The method of claim 31 wherein the two or more anions are
released from the polyelectrolyte assembly with separate, distinct
or separate and distinct release profiles.
33. The method of claim 31 wherein the two or more anions are
released from the polyelectrolyte assembly such that at least one
anion is released short-term and at least one anion is released
long-term.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.
119(e) to U.S. Provisional Patent Application No. 60/978,633 filed
on Oct. 9, 2007, which is hereby incorporated by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0003] Materials that provide control over the release of multiple
chemical or biological agents are of interest in a broad range of
biomedical and biotechnological applications. [J. T. Santini, M. J.
Cima, R. Langer, Nature 1999, 397, 335; L. D. Shea, E. Smiley, J.
Bonadio, D. J. Mooney, Nat Biotechnol 1999, 17, 551; T. P.
Richardson, M. C. Peters, A. B. Ennett, D. J. Mooney, Nat
Biotechnol 2001, 19, 1029; W. M. Saltzman, W. L. Olbricht, Nat Rev
Drug Discov 2002, 1, 177; A. C. R. Grayson, I. S. Choi, B. M.
Tyler, P. P. Wang, H. Brem, M. J. Cima, R. Langer, Nat Mater 2003,
2, 767; J. M. Saul, M. P. Linnes, B. D. Ratner, C. M. Giachelli, S.
H. Pun, Biomaterials 2007, 28, 4705.] Temporal control over the
release of multiple biological cues, for example, will likely prove
critical in applications such as tissue engineering, for which
precise control over the administration of multiple different
growth factors and other signals is thought to be required to
promote the development of functional tissues. [Shea et al. 1999;
Richardson et al. 2001; Saltzman et al. 2002; Saul et al. 2007.]
Such sophisticated levels of control can also contribute to the
development of new tools for basic biomedical research and more
effective gene- and protein-based therapies. There is significant
interest in the controlled release of anionic species, particularly
anionic polypeptides and nucleic acids, including various forms of
RNA and DNA.
[0004] Several recent reports have demonstrated approaches to the
encapsulation of proteins or DNA in bulk matrices of degradable
polymers or the fabrication of devices that provide control over
the release of multiple agents [L. D. Shea, E. Smiley, J. Bonadio,
D. J. Mooney, Nat Biotechnol 1999, 17, 551; T. P. Richardson, M. C.
Peters, A. B. Ennett, D. J. Mooney, Nat Biotechnol 2001, 19, 1029;
J. M. Saul, M. P. Linnes, B. D. Ratner, C. M. Giachelli, S. H. Pun,
Biomaterials 2007, 28, 4705; Santini et al. 1999; Satlzman et al.
2002; Grayson et al. 2003.] Despite these advances, however, it has
proven difficult to design thin films and coatings that provide
control over the release of multiple proteins or DNA constructs
with separate and distinct release profiles (e.g., rapid release of
a first DNA construct, followed by the slower, sustained release of
a second DNA construct). This invention relates generally to
approaches to the fabrication of ultrathin polymer-based coatings
that can be exploited to provide temporal control of release of
anionic species. At least in part, the invention, relates to
approaches to the controlled release of two or more anionic species
with separate, distinct or both separate and distinct release
profiles.
[0005] The present work relates to the use of methods developed for
the layer-by-layer assembly of multilayered polyelectrolyte films
(or `polyelectrolyte multilayers`). These methods are entirely
aqueous and permit nanometer-scale control over the structures of
thin films fabricated from a wide variety of synthetic or natural
polyelectrolytes, including DNA. [G. Decher, Science 1997, 277,
1232; P. Bertrand, A. Jonas, A. Laschewsky, R. Legras, Macromol
Rapid Comm 2000, 21, 319; P. T. Hammond, Adv Mater 2004, 16, 1271;
Z. Y. Tang, Y. Wang, P. Podsiadlo, N. A. Kotov, Adv Mater 2006, 18,
3203; Y. Lvov, G. Decher, G. Sukhorukov, Macromolecules 1993, 26,
5396.]
[0006] Multilayers have been designed that release DNA and promote
surface-mediated cell transfection by fabricating films using DNA
and cationic polymers that are hydrolytically, enzymatically, or
reductively degradable. Approaches to the fabrication,
characterization, and application of DNA-containing multilayers
have been reviewed recently. [D. M. Lynn, Soft Matter 2006, 2, 269;
D. M. Lynn, Adv Mater 2007, 19, 4118.]
[0007] It has been reported that DNA can be incorporated into
polyelectrolyte multilayers using layer-by-layer methods of
assembly [Lvov, et al., 1993] and that it is possible to fabricate
films that erode and release DNA in aqueous environments if the
polycationic components of these assemblies are designed
appropriately. [J. Zhang, L. S. Chua, D. M. Lynn, Langmuir 2004,
20, 8015; C. M. Jewell, J. Zhang, N. J. Fredin, D. M. Lynn, J.
Control. Release. 2005, 106, 214; K. F. Ren, J. Ji, J. C. Shen,
Biomaterials 2006, 27, 1152; K. F. Ren, J. Ji, J. C. Shen,
Bioconjugate Chem. 2006, 17, 77; C. M. Jewell, J. Zhang, N. J.
Fredin, M. R. Wolff, T. A. Hacker, D. M. Lynn, Biomacromolecules
2006, 7, 2483; Blacklock, H. Handa, D. Soundara Manickam, G. Mao,
A. Mukhopadhyay, D. Oupicky, Biomaterials 2007, 28, 117; J. Chen,
S. Huang, W. Lin, R. Zhuo, Small 2007, 3, 636.] For example, it was
recently reported that polyelectrolyte multilayers fabricated from
plasmid DNA and hydrolytically degradable poly(beta-amino ester)s
erode when incubated in physiological media [Zhang, et al. 2004
supra; Jewell, et al. 2005, supra; Jewell, et al. 2006, supra and
D. M. Lynn, et al. 2006] and that objects coated with these
assemblies promote surface-mediated transfection when placed in
contact with mammalian cells. [Jewell et al. 2005, supra; Jewell et
al. 2006, supra.]
[0008] It has also been reported that enzymatically or reductively
degradable cationic polymers can be used to fabricate assemblies
that release DNA in the presence of enzymes, reducing agents, or
cells. [J. Zhang, et al. 2004; Jewell, et al. 2005; Zhang, et al.
2007; Ren, et al. Biomaterials 2006; K. F. Ren, J. Ji, J. C. Shen,
Bioconjugate Chem. 2006, 17, 77; Blacklock, et al. 2007; Chen et
al. 2007; N. Jessel, M. Oulad-Abdelghani, F. Meyer, P. Lavalle, Y.
Haikel, P. Schaaf, J. C. Voegel, Proc Natl Acad Sci USA 2006, 103,
8618.]. These studies report approaches to promoting film erosion
that involve the backbone degradation of cationic polymers and, in
general, lead to films that release DNA relatively rapidly (e.g.,
over several hours to several days).
[0009] The present invention, in part, relates to an alternative
approach to the disruption of ionic interactions in these
assemblies that provides a means to extend the release of DNA or
other anions over much longer periods (e.g., several months)
particularly in ways that are useful in applications that require
long-term exposure of cells or tissues to DNA. Additionally, the
invention relates to approaches for the controlled release of two
or more anions, particularly from a single multilayer film which
exhibit rapid short-term release of one anion combined with
long-term release of another anion.
[0010] U.S. Pat. No. 7,112,361 relates to decomposable films
comprising a plurality of polyelectrolyte bilayers. Related
published U.S. application 2007/0020469 reports decomposable films
comprising a plurality of polyelectrolyte layers wherein a portion
of the bilayers comprise a second entity selected from a
biomolecule, a small molecule, a bioactive agent, and any
combination thereof.
[0011] U.S. published application 20050027064 relates to
charge-dynamic polymers useful for the delivery of anionic
compounds including nucleic acids. The dynamic charge state
cationic polymers are designed to have cationic charge densities
that decrease by removal of removable functional groups from the
polymers. The application also relates to complexes containing the
polymers complexed to a polyanion and methods for using the
interpolyelectrolyte complexes to deliver anionic compounds. The
application describes compositions comprising a dynamic charge
state cationic polymer, having a polymeric backbone formed from
monomeric units, and having one or more removable functional groups
attached to the polymeric backbone. The cationic charge of the
dynamic charge state cationic polymer decreases when one or more of
the removable functional groups is removed from the polymer.
Specific dynamic charge state cationic polymers include those in
which the polymer backbone comprises a polyamine, acrylate or
methacrylate polymer, including polyethyleneimine, poly(propylene
imine), poly(allyl amine), poly(vinyl amine), poly(amidoamine), or
a dendrimer that is functionalized with terminal amine groups. The
application also describes a method for delivering an anionic
compound to a target cell by contacting a composition comprising a
interpolyelectrolyte complex comprising a dynamic charge state
cationic polymer complexed to one or more anions with the target
cell thereby allowing the target cell to uptake the composition.
After entry of the interpolyelectrolyte complex into the target
cell, one or more of the removable functional groups is removed
from the dynamic charge state cationic polymer decreasing the
cationic charge of the dynamic charge state cationic polymer and
promoting dissociation of the interpolyelectrolyte complex to
release one or more anions.
[0012] U.S. published application 20060251701 relates to delivery
of nucleic acids by polyelectrolyte assemblies formed by
layer-by-layer deposition of nucleic acid and polycation and
particularly to implantable medical devices coated with
polyelectrolyte assemblies. Such devices facilitate the local
delivery of a nucleic acid contained in the polyelectrolyte
assembly into a cell or tissue at an implantation site.
[0013] The following references relate to formation of
polyelectrolyte multilayers, dynamic charge state (charge shifting)
polymers, release of anionic polyelectrolytes and/or drug release
from thin films:
X. Liu, J. Zhang, and D. M. Lynn, "Polyelectrolyte Multilayers
Fabricated from `Charge-Shifting` Anionic Polymers: A New Approach
to Controlled Film Disruption and the Release of Cationic Agents
from Surfaces." Soft Matter 2008, 4, 1688-1695; C. M. Jewell and D.
M. Lynn, "Multilayered Polyelectrolyte Assemblies as Platforms for
the Delivery of DNA and Other Nucleic Acid-Based Therapeutics."
Advanced Drug Delivery Reviews 2008, 60, 979-999; N. J. Fredin, J.
Zhang, and D. M. Lynn, "Nanometer-Scale Decomposition of Ultrathin
Multilayered Polyelectrolyte Films." Langmuir 2007, 23, 2273-2276;
J. Zhang, S. I. Montanez, C. M. Jewell, and D. M. Lynn,
"Multilayered Films Fabricated from Plasmid DNA and a Side-Chain
Functionalized Poly(beta-amino ester): Surface-Type Erosion and
Sequential Release of Multiple Plasmid Constructs from Surfaces."
Langmuir 2007, 23, 11139-11146; J. Zhang and D. M. Lynn, "Ultrathin
Multilayered Films Assembled from `Charge-Shifting` Cationic
Polymers: Extended, Long-Term Release of Plasmid DNA from
Surfaces." Advanced Materials 2007, 19, 4218-4223; D. M. Lynn,
"Peeling Back the Layers: Controlled Erosion and Triggered
Disassembly of Multilayered Polyelectrolyte Thin Films." Advanced
Materials 2007, 19, 4118-4130; J. Zhang, N. J. Fredin, J. F. Janz,
B. Sun, and D. M. Lynn, "Structure/Property Relationships in
Erodible Multilayered Films: Influence of Polycation Structure on
Erosion Profiles and the Release of Anionic Polyelectrolytes."
Langmuir 2006, 22, 239-245; D. M. Lynn, "Layers of Opportunity:
Nanostructured Polymer Assemblies for the Delivery of
Macromolecular Therapeutics." Soft Matter 2006, 2, 269-273; K. C.
Wood, H. F. Chuang, R. D. Batten, D. M. Lynn, and P. T. Hammond,
"Controlling Interlayer Diffusion to Achieve Sustained, Multi-Agent
Delivery from Layer-by-Layer Films." Proceedings of the National
Academy of Sciences, USA 2006, 103, 10207-10212; J. Zhang, N. J.
Fredin, and D. M. Lynn, "Erosion of Multilayered Assemblies
Fabricated from Degradable Polyamines: Characterization and
Evidence in Support of a Mechanism that Involves Polymer
Hydrorysis." Journal of Polymer Science--Part A: Polymer Chemistry
2006, 44, 5161-5173; C. M. Jewell, J. Zhang, N. J. Fredin, M. R.
Wolff, T. A. Hacker, and D. M. Lynn, "Release of Plasmid DNA from
Intravascular Stents Coated with Ultrathin Multilayered
Polyelectrolyte Films." Biomacromolecules 2006, 7, 2483-2491; J.
Zhang and D. M. Lynn, "Multilayered Films Fabricated from
Combinations of Degradable Polyamines Tunable Erosion and Release
of Anionic Polyelectrolytes." Macromolecules 2006, 39, 8928-8935;
C. M. Jewell, J. Zhang, N. J. Fredin, and D. M. Lynn, "Multilayered
Polyelectrolyte Films Promote the Direct and Localized Delivery of
DNA to Cells." Journal of Controlled Release 2005, 106, 214-223; K.
Wood, J. Q. Boedicker, D. M. Lynn, and P. T. Hammond, "Tunable Drug
Release from Hydrolytically Degradable Layer-by-Layer Thin Films."
Langmuir 2005, 21, 1603-1609; N. J. Fredin, J. Zhang, and D. M.
Lynn, "Surface Analysis of Erodible Multilayered Polyelectrolyte
Films: Nanometer-Scale Structure and Erosion Profiles." Langmuir
2005, 21, 5803-5811; and X. Liu, J. W. Yang, A. D. Miller, E. A.
Nack, and D. M. Lynn, "Charge-Shifting Cationic Polymers that
Promote Self-Assembly and Self-Disassembly with DNA."
Macromolecules 2005, 38, 7907-7914.
[0014] There is a need in the art for materials and methods that
provide control over the release of multiple chemical or biological
agents, particularly for controlled release of nucleic acids.
SUMMARY OF THE INVENTION
[0015] The invention relates to multilayered films, particularly
ultrathin multilayered films comprising cationic polymers which are
useful for controlled release of anionic species, particularly for
controlled release of nucleic acids. The multilayer films herein
are useful for temporal controlled released of anionic species,
particularly one or more anionic peptides, proteins, nucleic acids
or other anionic biological agents
[0016] In one aspect, the invention relates to multilayer films
which release anionic species (anions) with separate and/or
distinct release profiles, particularly wherein the anions are one
or more anionic peptides, proteins or nucleic acids or other
anionic biological agents
[0017] In another aspect, the invention relates to multilayer films
which are useful for extended, long-term release of anions,
particularly one or more nucleic acids. In another aspect, the
invention relates to multilayer films which are useful for
achieving a combination of short-term and long-term controlled
release of anions, particularly one or more anionic peptides,
proteins or nucleic acids or other anionic biological agents. In a
specific embodiment, the short-term and long-term release of anions
occurs from a single multiple layer films.
[0018] Multilayer films of the invention comprise anion/cationic
polymer bilayers. The films can be formed using methods that are
entirely aqueous. Multilayer films can degrade in aqueous solution,
at least in part by degradation of ester linkages of the cationic
polymers of one or more bilayers. Degradation of films and release
of anions does not require the presence of enzymes or reducing
agents.
[0019] In one aspect, the invention relates to multilayer
polyelectrolyte films, also called polyelectrolyte assemblies,
which are formed from at least two different cationic charge
dynamic polymers, also called charge shifting polymers, selected
from polymers having Formula I:
##STR00001##
where: n, m, l, k and i are zero or integers where n+m+l+k+i=N, the
total number of repeat units in the polymer; A.sub.1-3, B.sub.1-3,
C.sub.1-3, D.sub.1-3 and E.sub.1-3 are linkers which may be the
same or each may be different; Z.sub.1-Z.sub.5 are most generally
covalent bonds which may or may not be degradable bonds; R.sub.1-4
and R.sub.11-19, independently, can be hydrogen, or alkyl, alkenyl,
alkynyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups, all
of which are optionally substituted, with the exception that
R.sub.1-4 are not esters; and R.sub.5-R.sub.10 are linkers or
covalent bonds which may be the same or each may be different.
[0020] In the polyelectrolyte films formed from cationic polymers
of formula I at least one of the polymers contains one or more
hydrolysable ester groups. In specific embodiments, at least one of
the polymers employed in the film is biodegradable and
biocompatible. In specific embodiments, all of the polymers
employed in the film are biodegradable and biocompatible.
[0021] A.sub.1-2, B.sub.1-2, C.sub.1-2, D.sub.1-2 and E.sub.1-2 are
linkers which can be the same or different and can be any
substituted or unsubstituted, branched or unbranched chain of
carbon atoms and heteroatoms with the exception that none of these
linkers is substituted with ester groups except as specifically
shown in the formula above. Linkers include those that are 1 to 30
atoms long, more preferably 1 to 15 atoms long and yet more
preferably 1 to 6 atoms long. The linkers may be substituted with
various substituents including, but not limited to, alkyl, alkenyl,
alkynyl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl,
alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic, cyano,
amide, carbamoyl, thioether, alkylthioether, thiol, and ureido
groups. As would be appreciated by one of skill in this art, each
of these groups may in turn be substituted. A.sub.1-2, B.sub.1-2,
C.sub.1-2, D.sub.1-2 and E.sub.1-2 can, for example, be selected
from --(CH.sub.2).sub.r--, --(CH.sub.2).sub.r--NR--,
--NR--(CH.sub.2).sub.r--, --(CH.sub.2).sub.r--O--,
--O--(CH.sub.2).sub.r--, --(CH.sub.2).sub.r--O--(CH.sub.2).sub.s--,
--(CH.sub.2).sub.r--S--, --S--(CH.sub.2).sub.r--,
--(CH.sub.2).sub.r--S--(CH.sub.2).sub.s, where r and s are integers
ranging from 1 to 30, and where r+s ranges from 2 to 30. More
preferably r and s range from 1-6 and r+s ranges from 2 to 12.
[0022] In specific embodiments of formula I, k and i are zero. In
specific embodiments n+m++k+i=N=5 to 100,000. More specifically, N
can range from 20 to 100,000, from 100 to 100,000, from 1,000 to
100,000 or from 10,000 to 100,000. In a specific embodiment, all of
n, m, k and i are zero. In a specific embodiment, m, n and are all
zero. In specific embodiments, (k+i)/N is 0.01 to 0.1, 0.05 to 0.2,
0.1 to 0.25, 0.25 to 0.50, 0.25 to 0.75, or 0.75 to 1. In specific
embodiments, (n+m+)/N is 0.01 to 0.1, 0.05 to 0.2, 0.1 to 0.25,
0.25 to 0.50, 0.25 to 0.75, or 0.75 to 1. In specific embodiments,
(m+)/N is 0.01 to 0.1, 0.05 to 0.2, 0.1 to 0.25, 0.25 to 0.50, 0.25
to 0.75, or 0.75 to 1.
[0023] In specific embodiments of formula I, 10% or more of the
groups bonded to the amine of the amine side chains of the polymer
are ester groups. In specific embodiments, 25% or more of the
groups bonded to the amine of the amine side chains of the polymer
are ester groups. In specific embodiments, 50% or more of the
groups bonded to the amine of the amine side chains of the polymer
are ester groups. In specific embodiments, 75% or more of the
groups bonded to the amine of the amine side chains of the polymer
are ester groups. In specific embodiments, 90% or more of the
groups bonded to the amine of the amine side chains of the polymer
are ester groups.
[0024] In other specific embodiments of formula I, 10%-25% of the
groups bonded to the amine of the amine side chains of the polymer
are ester groups. In specific embodiments, 25%-50% of the groups
bonded to the amine of the amine side chains of the polymer are
ester groups. In specific embodiments, 50%-75% of the groups bonded
to the amine of the amine side chains of the polymer are ester
groups. In specific embodiments, 75%-90% of the groups bonded to
the amine of the amine side chains of the polymer are ester groups.
In specific embodiments, 90%-100% of the groups bonded to the amine
of the amine side chains of the polymer are ester groups.
[0025] In specific embodiments of formula I, R.sub.1-4 are all
hydrogens. In other specific embodiments, R.sub.1-4 are
independently hydrogens or alkyl groups having 1-10 carbon atoms.
In other specific embodiments, R.sub.1-4 are independently
hydrogens or alkyl groups having 1-6 carbon atoms. In other
specific embodiments, R.sub.1-4 are independently hydrogens or
alkyl groups having 1-3 carbon atoms. In other specific
embodiments, R.sub.11-13 are independently alkyl groups having 1-10
carbon atoms. In other specific embodiments, R.sub.11-13 are
independently alkyl groups having 1-6 carbon atoms. In other
specific embodiments, R.sub.11-13 are independently alkyl groups
having 1-3 carbon atoms. In other specific embodiments, R.sub.11-13
are independently methyl or ethyl groups.
[0026] In specific embodiments of formula I, R.sub.14-19 are
independently alkyl groups having 1-10 carbon atoms. In specific
embodiments, R.sub.14-19 are independently alkyl groups having 1-6
carbon atoms. In specific embodiments, R.sub.14-19 are
independently alkyl groups having 1-3 carbon atoms.
[0027] In specific embodiments of formula I, R.sub.5-10 are
independently a covalent bond or alkylene chains (CH.sub.2).sub.p,
where each p is an integer ranging from 1-10. In specific
embodiments, R.sub.5-10 are independently a covalent bond or
alkylene chains (CH.sub.2).sub.p, where each p is an integer
ranging from 1-6. In specific embodiments, R.sub.5-10 are
independently a covalent bond or alkylene chains (CH.sub.2).sub.p,
where each p is an integer ranging from 1-3.
[0028] In specific embodiments of formula I, linker groups A.sub.3,
B.sub.3, C.sub.3, D.sub.3 and E.sub.3 are independently alkylene
chains (CH.sub.2).sub.q where q is an integer ranging from 1-10. In
specific embodiments, linker groups A.sub.3, B.sub.3, C.sub.3,
D.sub.3 and E.sub.3 are independently alkylene chains
(CH.sub.2).sub.q where q is an integer ranging from 1-6. In
specific embodiments, linker groups A.sub.3, B.sub.3, C.sub.3,
D.sub.3 and E.sub.3 are independently alkylene chains
(CH.sub.2).sub.q where p is an integer ranging from 1-3. In
specific embodiments, linker groups A.sub.3, B.sub.3, C.sub.3,
D.sub.3 and E.sub.3 are all --CH.sub.2-- groups.
[0029] In specific embodiments of formula I, Z.sub.1-Z.sub.5 are
all covalent bonds and the backbone of the polymer is not
hydrolytically degradable. In specific embodiments, the backbone of
the polymer is not enzymatically or hydrolytically degradable.
[0030] In another aspect, the invention relates to multilayer
polyelectrolyte films which are formed from cationic charge dynamic
polymers selected from polymers having formula II:
##STR00002##
where n+m=N is the number of repeating units in the polymer; each
y, independently, is 1, 2 or 3; each x, independently, is an
integer ranging from 1-10; each R.sup.1, each R.sup.2, each R.sup.3
and each R.sup.4, independently, is selected from the group
consisting of hydrogen, alkyl groups, alkenyl groups, alkynyl
groups, carbocyclic groups, heterocyclic groups, aryl groups,
heteroaryl groups, ether groups, all of which may be substituted or
unsubstituted. In specific embodiments, each y is the same and each
x is the same. In specific embodiments, x of the ester groups is
different from x on the amide groups. In specific embodiments, each
R.sup.1, each R.sup.2, each R.sup.3 and each R.sup.4,
independently, is selected from the group consisting of hydrogen,
C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, phenyl, and benzyl, each
of which is optionally substituted.
[0031] In specific embodiments, y is 1, x is 1-6, each R.sup.1 and
each R.sup.4, independently, are hydrogen or C1-C3 alkyl, R.sup.2
are all hydrogen, one R.sup.3 is hydrogen and two R.sup.3 are C1-C3
alkyl. In other specific embodiments, each R.sup.1 is a C1-C3
alkyl. In additional specific embodiments, each R.sup.4 is a
hydrogen. In yet other specific embodiments, each R.sup.1 is a
C1-C3 alkyl and each R.sup.4 is a hydrogen.
[0032] In specific embodiments of formula II, m is zero. In other
embodiments, n is zero. In specific embodiments N=5 to 100,000.
More specifically, N can range from 20 to 100,000, from 100 to
100,000, from 1,000 to 100,000 or from 10,000 to 100,000. In
specific embodiments, (n)/(m+n) ranges from 0.1 to 1, including
0.05 to 0.25, 0.25 to 0.50, 0.25 to 0.75, 0.75 to 1, 0.85 to 1,
0.90 to 1 or 0.95 to 1. In specific embodiments, (n)/(n+m) is 0.50
to 1. In other specific embodiments, (n)/(n+m) is 0.01 to 0.5.
[0033] In specific embodiments of formula II, 10% or more of the
groups bonded as side groups to the polymer are ester groups. In
specific embodiments, 25% or more of the groups bonded as side
groups to the polymer are ester groups. In specific embodiments,
50% or more of the groups bonded as side groups to the polymer are
ester groups. In specific embodiments, 75% or more of the groups
bonded as side groups to the polymer are ester groups. In specific
embodiments, 90% or more of the groups bonded as side groups to the
polymer are ester groups.
[0034] In other specific embodiments of formula II, 10%-25% of the
groups bonded as side groups to the polymer are ester groups. In
specific embodiments, 25%-50% of the groups bonded as side groups
to the polymer are ester groups. In specific embodiments, 50%-75%
of the groups bonded as side groups to the polymer are ester
groups. In specific embodiments, 75%-90% of the groups bonded as
side groups to the polymer are ester groups. In specific
embodiments, 90%-100% of the groups bonded as side groups to the
polymer are ester groups.
[0035] The polyelectrolyte films of this invention can provide for
release of anions with separate and/or distinct release profiles.
The polyelectrolyte films can release the same anion with separate
and distinct release profiles or preferably two or more different
anions with separate and distinct release profiles. More
specifically, the polyelectrolyte films can release two or more
different anions each exhibiting separate and distinct release
profiles. In specific embodiments, the anions are nucleic acids,
and in particular are nucleic acids which encode one or more
polypeptides. In specific embodiments, the nucleic acids are in a
form that is capable of expressing one or more polypeptides. In
specific embodiments, the nucleic acids are comprised in one or
more expression cassettes or expression vectors.
[0036] In a specific embodiment, the polyelectrolyte film includes
at least one anion/cationic polymer bilayer formed with a first
cationic polymer of formula I and at least one such bilayer formed
with a second cationic polymer of formula I.
[0037] In another specific embodiment, the polyelectrolyte film
includes at least one anion/cationic polymer bilayer formed with a
cationic polymer of formula II. In this embodiment, the film can
optionally include a combination of bilayers formed from two or
more different cationic polymers of formula II. In this embodiment,
the film can optionally include a combination of bilayers formed
from two or more different anions with the same or different
cationic polymers of formula II.
[0038] In another specific embodiment, the polyelectrolyte film
includes at least one anion/cationic polymer bilayer formed with a
cationic polymer of formula I and at least one such bilayer formed
with a cationic polymer of formula II. In this embodiment, the
bilayers formed from the cationic polymer of formula I and formula
II can contain the same or different anions.
[0039] In a specific embodiment, the polyelectrolyte film includes
at least one anion/cationic polymer bilayer formed with a first
cationic polymer of formula I and at least one such bilayer formed
with a second cationic polymer of formula I and at least one such
bilayer formed with a cationic polymer of formula II. In this
embodiment, the bilayers formed from the two or more different
cationic polymers of formula I and the cationic polymer of formula
II can each contain the same or different anions.
[0040] The polyelectrolyte film is preferably generated by
layer-by-layer deposition of the anion(s) and selected cationic
polymer(s). The polyelectrolyte film comprises a plurality of
anion/cationic polymer bilayers. In specific embodiments, the film
comprises a plurality of nucleic acid/cationic polymer
bilayers.
[0041] The first and second cationic polymers of formula I are
structurally distinct. In specific embodiments, the polyelectrolyte
film includes at least one anion/cationic polymer bilayer formed
with a first cationic polymer of formula I and a first anion and at
least one such bilayer formed with a second cationic polymer of
formula I and a second anion.
[0042] The first and second anions may be the same or different
anions. In specific embodiments, the anions are nucleic acids. In
specific embodiments, the first and second anions are nucleic acids
having different nucleic acid sequences. In specific embodiments,
the nucleic acids each encode one or more polypeptides. In specific
embodiments, the first and second nucleic acids encode first and
second polypeptides. In specific embodiments, the first and second
nucleic acids are comprised in first and second expression
cassettes or expression vectors.
[0043] In additional embodiments, the polyelectrolyte film includes
two or more anion/cationic polymer bilayers each of which is formed
from a different cationic polymer of formula I. In additional
embodiments, the polyelectrolyte film includes two or more
anion/cationic polymer bilayers each of which is formed from a
different cationic polymer of formula I and each of which is formed
with a different anion. In additional embodiments, the
polyelectrolyte film includes two, three or more nucleic
acid/cationic polymer bilayers each of which is formed from a
different cationic polymer of formula I and each of which is formed
with a different nucleic acid.
[0044] In additional embodiments, the polyelectrolyte film includes
two or more anion/cationic polymer bilayers one of which is formed
from a cationic polymer of formula I and the other of which is
formed from a cationic polymer of formula II. In additional
embodiments, the polyelectrolyte film includes two or more
anion/cationic polymer bilayers one of which is formed from a first
anion and a cationic polymer of formula I and one of which is
formed with a second anion and a cationic polymer of formula II. In
specific embodiments, the first and second anions are first and
second nucleic acids. In specific embodiments, the first and second
nucleic acids are comprises in a first and second expression
cassette or vector. In additional embodiments, the polyelectrolyte
film can include two, three or more anion/cationic polymer bilayers
(including nucleic acid/cationic polymer bilayers) each of which is
formed from one, two or more cationic polymers of formula II in
combinations with one, two or more different cationic polymers of
formula I wherein each of the bilayers is also formed with a
different anion (including different nucleic acids). In typical
embodiments, however a polyelectrolyte film of the invention will
be employed to carry and release one anion, two different anions or
three different anions, including one nucleic acid, two different
nucleic acids or three different nucleic acids.
[0045] In specific embodiments, the polyelectrolyte film is formed
on a substrate. The substrate can be the surface of an implantable
medical device from which in certain embodiments, one or two or
more different anion(s) can be delivered to tissues or cells with
separate and distinct release profiles. In more specific
embodiments, the anion(s) in the polyelectrolyte film on the
substrate, including the implantable medical device are nucleic
acids and the nucleic acids can be delivered to a tissue or cell.
In specific embodiments, delivery of nucleic acid to tissue or cell
results in expression of nucleic acid in the tissue or cell.
[0046] Polyelectrolyte assemblies or films of this invention
comprise a plurality of polyelectrolyte bilayers wherein at least
one bilayer comprises a cationic polymer of formula I or at least
one bilayer comprises a cationic polymer of formula II. In certain
embodiments, the polyelectrolyte assembly of the invention includes
multiple anion/polycation bilayers, preferably more than two
bilayers. In embodiments containing multiple bilayers, these
bilayers may alternatively differ from each other in their specific
anion composition and/or cationic polymer. Accordingly, respective
bilayers may incorporate one or more anions of different structure.
Bilayers may differ from each other in their specific polycation
makeup as in certain embodiments where differing polycations,
including polycations of formula I, formula II or both and
optionally polycations other than those of formula I or II, which
may be degradable and/or non-degradable, may be combined within a
single bilayer, or, alternatively, contained within distinct
bilayers.
[0047] In specific embodiments, polyelectrolyte films of this
invention comprise at least two different bilayers which are formed
with at least two different cationic polymers of formula I. In
specific embodiments, polyelectrolyte films of this invention
comprise a plurality of anion/cationic polymer bilayers and in at
least one and preferably in at least two such bilayers, the
cationic polymer is a polymer of formula I. In specific
embodiments, polyelectrolyte films of this invention comprise a
plurality of anion/cationic polymer bilayers wherein in all such
bilayers the cationic polymer is a polymer of formula I. In
specific embodiments, the polyelectrolyte films comprise a
plurality of first bilayers formed from an anion and a first
cationic polymer of formula I and a plurality of second bilayers
formed from an anion and a second cationic polymer of formula I.
The first and second pluralities of bilayers may be formed into a
layer configuration in which the first plurality of bilayers are
grouped together and the second plurality of bilayers are grouped
together and the first and second pluralities of bilayers are
optionally separated by one or more intermediate bilayers.
[0048] In other specific embodiments, polyelectrolyte films of this
invention comprise at least two different bilayers which are formed
with at a cationic polymer of formula I and a cationic polymer of
formula II. In specific embodiments, polyelectrolyte films of this
invention comprise a plurality of anion/cationic polymer bilayers
and in at least one and preferably in at least two such bilayers,
the cationic polymer is a cationic polymer of formula I or a
cationic polymer of formula II. In specific embodiments,
polyelectrolyte films of this invention comprise a plurality of
anion/cationic polymer bilayers wherein in all such bilayers the
cationic polymer is a polymer of formula I or a cationic polymer of
formula II. In specific embodiments, the polyelectrolyte films
comprise a plurality of first bilayers formed from an anion and a
cationic polymer of formula I and a plurality of second bilayers
formed from an anion and a cationic polymer of formula II. The
first and second pluralities of bilayers may be formed into a layer
configuration in which the first plurality of bilayers are grouped
together and the second plurality of bilayers are grouped together
and the first and second pluralities of bilayers are optionally
separated by one or more intermediate bilayers.
[0049] Each polyelectrolyte assembly of the invention can
optionally comprise one or more top protective bilayers and/or one
or more base bilayers. One or more base bilayers can be formed
between a substrate surface and an anion/polymer cation bilayer
where the anion is intended for controlled release. A plurality of
such base layers may intervene between the substrate surface and
any anion/cationic polymer bilayers. Base layers, if present, are
the bottom most layers in a polyelectrolyte assembly. An
intermediate bilayer or a plurality of intermediate layers may
intervene between bilayers or pluralities of bilayers of
anion/cationic polymers where the anion is intended for controlled
release. One or more top protective bilayers can be positioned as
the top most bilayers in a polyelectrolyte assembly. Intermediate,
top protective and base bilayers can comprise a cationic polymer of
formula I or formula II or both and an anion other than an anion
the release of which is intended to be temporally controlled.
Intermediate, top protective and base bilayers can comprise a
cationic polymer other than one of formula I or II, but which is
degradable. For example, the cationic polymer of the top, base or
intermediate layer may be a cationic polymer in which the polymer
backbone can degrade, such as a poly(beta-amino0 ester. When the
anions to be released from the films are one or more nucleic acids,
the anion of the intermediate, top protective and base layers are
anions other than nucleic acids which may be polymeric anions. In
specific embodiments, the anions of the intermediate, top
protective or base bilayers of the polyelectrolyte assembly are
poly(styrene sulfonate).
[0050] Generally, a polyelectrolyte film of this invention
comprises one bilayer or more than one sequential bilayers for each
different anion that is to be released with a separate and distinct
release profile. Each such different one or more sequential
bilayers is optionally separated from each other different one or
more sequential bilayers by one or more intermediate bilayers. In
specific embodiments, the polyelectrolyte film comprises one or
more sequential first bilayers and one or more sequential second
bilayers and optionally comprises one or more sequential third
bilayers, one or more sequential fourth layers, and one or more
sequential fifth bilayers. Each of the different one or more
sequential bilayers is optionally separated from each other
different one or more sequential bilayers by one or more
intermediate layer as noted above.
[0051] In specific embodiments, the polyelectrolyte film comprises
two, three, four or more different bilayers wherein the different
bilayers comprise at least two different cationic polymers of
formula I. In other specific embodiments, the polyelectrolyte film
comprises two, three, four or more different bilayers wherein the
different bilayers comprise at least one cationic polymers of
formula I and one cationic polymer of formula II. In general, a
bilayer can differ in cationic polymer(s) or in anion(s) present in
the bilayer. A given bilayer can comprise two or more different
cationic polymers (which may or may not be cationic polymers of
formula I or II) including at least one cationic polymer of formula
I or one cationic polymer of formula II. A given bilayer can
comprise two or more different anions which may be different
nucleic acids. In a preferred embodiment, a given bilayer contains
one cationic polymer of formula I and one anion or a given bilayer
contains one cationic polymer of formula II and one anion.
[0052] In specific embodiments, the polyelectrolyte assembly can
comprise a single anion a first portion of which is to be released
with separate or distinct release profile compared to a second
portion thereof. The polyelectrolyte film can comprises two or more
anions which are to be released with separate, distinct or both
separate and distinct release profiles. In specific embodiments, at
least one of such anions is a nucleic acid. In other embodiments,
at least two of such anions are different nucleic acids
[0053] In a specific embodiment, the invention provides a
polyelectrolyte assembly formed on a substrate. In specific
embodiments, the substrate is an implantable medical device. In
specific embodiments, wherein the polyelectrolyte assembly
comprises an anion that is a nucleic acid, the implantable medical
device is capable of localized delivery of nucleic acid to a cell.
In such an implantable medical device a polyelectrolyte assembly of
this invention coats at least a portion of a surface of the device.
This polyelectrolyte assembly includes at least one nucleic
acid/polycation bilayer fabricated by layer-by-layer deposition of
nucleic acid and a polycation of formula I, a polycation of formula
II or both.
[0054] A wide range of implantable devices are adaptable for use in
the present invention including, but not limited to, a stent, a
pacemaker, a defibrillator, an artificial joint, a prosthesis, a
neurostimulator, a ventricular assist device, congestive heart
failure device, an indwelling catheter, an insulin pump, an
incontinence device, a cochlear device, or an embolic filter.
[0055] Polyelectrolyte assemblies optionally comprise polycations
other than those of formula I or formula II which are
hydrolytically or enzymatically degradable polycations including,
but not limited to, poly(beta-amino ester)s,
poly(4-hydroxy-L-proline ester),
poly[alpha-(4-aminobutyl)-L-glycolic acid], and combinations
thereof.
[0056] Polyelectrolyte assemblies of this invention optionally
comprise polycations other than those of formula I or formula II
which are non-degradable polycations.
[0057] In certain embodiments, the polyelectrolyte assembly of the
invention includes multiple nucleic acid/polycation bilayers,
preferably more than two bilayers. In embodiments containing
multiple bilayers, these bilayers may alternatively differ from
each other in their specific composition of nucleic acid and/or
polycation. Accordingly, respective bilayers may incorporate varied
nucleic acids that differ by nucleic acid sequence and those
nucleic acids may, in alternative embodiments, be incorporated into
one or more expression vectors. Different nucleic acids may encode
the same polypeptide, but be under the control of different
regulatory sequences, which affect the level, location or timing of
expression of the polypeptide coding sequences. Different nucleic
acids may encode different polypeptides, but be under the control
of the same or similar regulatory sequences such that the level,
location or timing of expression of the polypeptide coding
sequences is the same or similar. Similarly, bilayers may differ
from each other in their specific polycation makeup as in certain
embodiments where differing cationic polymers, including cationic
polymers of formula I or formula II and optionally cationic
polymers other than those of formula I or formula II which may be
combined within a single bilayer, or, alternatively, contained
within distinct bilayers, either with or without the presence of
non-degradable cationic polymers
[0058] In some embodiments, the nucleic acid present in a bilayer
encodes a polypeptide such as, for example, endostatin,
angiostatin, an inhibitor of vasoactive endothelial growth factor
(VEGF), an inhibitor of a signal protein in a signaling cascade of
vascular endothelial growth factor, and inhibitor of basic
fibroblast growth factor (bFGF), an inhibitor of a signal protein
in a signaling cascade of bFGF, or combinations thereof.
[0059] In certain embodiments, the polyelectrolyte assembly
includes at least two nucleic acids that differ from each other in
nucleotide sequence. These respective nucleic acids typically
reside in different bilayers and, in carrying out the method, are
released from the polyelectrolyte assembly with separate, distinct
or both separate and distinct release profiles.
[0060] In specific embodiments, in which a polyelectrolyte assembly
(a multilayer film) comprises a plurality of first bilayers formed
with a cationic polymer of formula I and a plurality of second
bilayers formed with a cationic polymer of formula II, the same
anions or two or more different anions can be released such that a
first selected anion or mixture of anions is released relatively
rapidly over a period ranging from hours up to about 10 days and a
second anion or mixture of anions is released after a delay or lag
period of at least about 20 days.
[0061] In another aspect, the present invention encompasses a
method of releasing two or more anions into a selected environment
wherein at least two of the anions are released from the assembly
with separate, distinct or both separate and distinct release
profiles. In specific embodiments, the environment is in vivo. In
other specific embodiments, the environment is in vitro. Such a
method includes steps of contacting the selected environment with a
polyelectrolyte assembly of this invention comprising two or more
anions. In such method, one or more anions are released by
disruption of one or more bilayers containing such one or more
anions by decreasing the cationic charge on one or more cationic
polymers of formula I, formula II or both.
[0062] In a more specific embodiment, the present invention
encompasses a method of delivering two or more nucleic acids into a
selected environment comprising one or more cells wherein at least
two of the nucleic acids are released from the assembly with
separate, distinct or both release profiles and thus are delivered
to the one or more cells with separate, distinct or both delivery
profiles. Such a method includes a step of contacting a cell with a
polyelectrolyte assembly of the invention which comprised two or
more nucleic acids. In such method, one or more nucleic acids are
released by disruption of one or more bilayers containing such one
or more anions by decreasing the cationic charge on one or more
cationic polymers of formula I, formula II or both. Methods of
delivery of nucleic acid according to the invention can be carried
out in the presence of cell culture medium or, alternatively and
more preferably, in the context of a medical device implanted in a
living tissue.
[0063] In another aspect, the invention is directed to a method of
providing an implantable medical device capable of delivery of two
or more anions wherein at least two of the anions are released from
the device with separate, distinct or both separate and distinct
release profiles. In particular, the two or more anions are two or
more nucleic acids. In particular, the anions are released into
living tissue or cells. In particular, the anions are delivered for
uptake into one or more cells. In particular, the anions are
nucleic acids and the nucleic acids are delivered to one or more
cells in contact with or located at an implantation site of the
respective device.
[0064] The method includes steps of layer-by-layer depositing of
anions and cationic polymer of formula I, formula II or both on a
surface of an implantable medical device to provide a
polyelectrolyte assembly coating at least a portion of the
implantable medical device. In a specific embodiment, the
polyelectrolyte assembly includes at least two different nucleic
acid/cationic polymer bilayers.
[0065] The polyelectrolyte assemblies, devices and methods of the
present invention are particularly advantageous in that they allow
for release of two or more anions with selected release profiles
and/or delivery to tissue or cells of two or more anions with
selected release profiles. In a specific embodiment, a device can
be coated with a film comprising two or more nucleic acid
sequences, which delivers two or more nucleic acids to cells or
tissues with separate, distinct or both temporal profiles to
provide for transfection of cells of a subject in situ providing
for the production of therapeutic agents that facilitate a certain
therapeutic activity including, for example, the acceptance of the
device by the subject through the reduction of inflammation
associated with implant placement.
[0066] Additional aspects and embodiments of the invention will be
apparent upon review of the following non-limiting description,
drawings and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0067] FIG. 1 is a schematic illustration of a `charge-shifting`
polymer synthesized by the conjugate addition of methyl acrylate to
poly(allylamine). Gradual hydrolysis of ester-functionalized side
chains introduces carboxylate functionality and reduces the net
charge of the polymer. Relative changes in net charge shown are
exemplary and provided for illustrative purposes only (see text).
Bottom: Polymer 2 is cationic and can be used to fabricate
DNA-containing polyelectrolyte multilayers. Time-dependent changes
in the net charge of the polymer result in changes in the nature of
the ionic interactions in the multilayers and promote film erosion
and the release of DNA.
[0068] FIG. 2 is a graph showing the kinetics of side chain ester
hydrolysis for polymers 2a (.DELTA.), 2b (.tangle-solidup.), 2c
(.quadrature.) and 2d (.box-solid.) in deuterated phosphate-buffer
(pH=7.4) at 37.degree. C., as determined by .sup.1H NMR
spectroscopy.
[0069] FIG. 3 is a plot of film thickness versus the number of
polymer/DNA bilayers deposited for films fabricated from plasmid
DNA and either PAH ( ), polymer 2a (.DELTA.), 2b
(.tangle-solidup.), 2c (.quadrature.), or 2d (.box-solid.) on
planar silicon substrates.
[0070] FIG. 4A is a plot of film erosion vs time for polymer/DNA
films eight bilayers thick fabricated from DNA and either PAH ( ),
2a (.DELTA.), 2b (.tangle-solidup.), 2c (.quadrature.), 2d
(.box-solid.), or polymer 3 (.smallcircle.) upon incubation in PBS
at 37.degree. C. Decreases in film thickness were determined by
ellipsometry and are expressed as a percentage of the original
thickness at each time point.
[0071] FIG. 4B is a plot of absorbance at 260 nm vs time showing
release of DNA from films in part A above, fabricated from DNA and
either PAH ( ), 2a (.DELTA.), 2b (.tangle-solidup.), 2c
(.quadrature.), 2d (.box-solid.), or polymer 3 (.smallcircle.).
Markers represent absorbance values recorded for the incubation
buffer; error bars in most cases are smaller than the symbols
used.
[0072] FIG. 5 is a plot of the percentage of DNA released vs time
for the release of fluorescently labeled DNA from films having the
general structure
(2c/pEGFP-Cy5).sub.4(2c/SPS).sub.2(2a/pDsRed-Cy3).sub.4 incubated
in PBS at 37.degree. C. Data points correspond to amounts of
pDsRed-Cy3 (.DELTA.) and pEGFP-Cy5 (.quadrature.) in solution
determined from solution fluorescence measurements.
[0073] FIG. 6 shows representative fluorescence microscopy images
showing relative levels of EGFP (green channel) and RFP (red
channel) expressed in COS-7 cells. Cells were transfected with
samples of DNA released from a film having the structure
(2c/pEGFP).sub.4(2c/SPS).sub.2(2a/pDsRed).sub.4; cells were
transfected by combining released DNA with Lipofectamine 2000 as a
transfection agent. The presence, absence, or relative levels of
EGFP and RFP observed correspond qualitatively to relative levels
of each plasmid released and collected over each of the following
time periods: 0-0.5 hrs, 0.5-3 hrs, 3-6 hrs, 6-24 hrs, 24-120
hrs.
[0074] FIG. 7 is a graph showing Kinetics of side chain ester
hydrolysis for polymer 5 in phosphate buffer (500 mM, pH=7.2) at
37.degree. C., as determined by .sup.1H NMR spectroscopy.
[0075] FIG. 8 is a plot of ellipsometric thickness versus the
number of polymer 5/DNA bilayers deposited on a silicon substrate.
Symbols represent average values and error for multiple independent
measurements made on three different films.
[0076] FIG. 9A is a plot of absorbance at 260 nm versus time
showing the release of DNA from films fabricated from polymer 5
(solid diamonds) and polymer 6 (solid squares). Symbols represent
the average and error of absorbance values recorded for the
incubation buffer.
[0077] FIG. 9B is a plot of change in film thickness versus time
for films fabricated from polymer 5 (solid diamonds) and polymer 6
(solid squares).
[0078] FIGS. 10A, 10B and 10C are schematic illustrations of
exemplary polyelectrolyte assemblies of this invention.
DETAILED DESCRIPTION OF THE INVENTION
[0079] The invention provides an approach for fabrication of
multilayer films, i.e. a polyelectrolyte assembly, using
`charge-shifting` polymers of formula I or formula II or a
combination of such polymers of formula I and formula II. In
contrast to the use of degradable cationic polymers, it is possible
to disrupt ionic interactions in polyelectrolyte assemblies of this
invention in physiologically relevant media using cationic polymers
designed to undergo gradual reductions in net charge upon exposure
to aqueous media.
[0080] In an aspect, in which two or more different cationic
polymers of formula I or cationic polymers of each of formula I and
II are combined, the polyelectrolyte assemblies (multilayers or
films) of this invention allow release of one or more anions from
the assembly exhibiting separate, distinct or both separate and
distinct release profiles. The invention is useful for the release
of anions from such an assembly wherein at least a portion of the
anions are released with a profile that is distinct and/or separate
compared to the release of another portion of the anions in the
assembly. The first portion and the second portion of anions may be
the same anions or different anions. In a specific embodiment, the
anions are one or more nucleic acids. In a specific embodiment, the
one or more nucleic acids encode one or more polypeptides and are
capable of expressing the encoded polypeptide on release from the
assembly and introduction of the one or more nucleic acids into
tissues or cells.
[0081] In an aspect, in which two or more different cationic
polymers of formula I or cationic polymers of each of formula I and
II are combined, the polyelectrolyte assemblies (multilayers or
films) of this invention allows release of at least two different
anions with separate, distinct or both separate and distinct
release profiles. The invention is particularly useful for the
release of two or more different anions from such an assembly
wherein at least one of the different anions is released from the
assembly with distinct and/or separate release profiles compared to
at least one other anion in the assembly. The first and the second
anions may be the same anions or different anions. In a specific
embodiment the first and second anions are two different nucleic
acids.
[0082] In another aspect, in which bilayers are formed from one or
more cationic polymers of formula II, the polyelectrolyte
assemblies (multilayers or films) of this invention allow long-term
release of one or more anions from the assembly over weeks or
months in contrast to short-term release over hours or days. More
specifically, this aspect of the invention is useful for the
release of one or more anions from the assembly after a delay or
lag period of 20 days or more. The invention is particularly useful
for long-term release of one or more nucleic acids from the
assembly over weeks or months. More specifically, the invention is
useful for the release of one or more nucleic acids from the
assembly after a delay or lag period of 20 days or more.
[0083] In another aspect, in which bilayers are formed from one or
more cationic polymers of formula I and one or more cationic
polymers of formula II, the polyelectrolyte assemblies (multilayers
or films) of this invention allows a combined short-term release of
a portion of the anions in the assembly over hours or days and a
long-term release of another portion of the anions in the assembly
over weeks or months. The first portion of anions that are released
over a short term (typically 10 days or less) may comprise one
anion or a mixture of more than one anions. The second portion of
anions that are released long-term (typically 20 days or more) may
comprise one anion or a mixture of more than one anion. The first
portion of anions may be the same or different from the second
portion of anions. For example, a first anion may be released
short-term and a different anion may be released long-term.
Alternatively, the same anion that was released shot-term may be
released long-term after a delay or lag period. More specifically,
this aspect of the invention is useful for the release of one or
more anions from the assembly for a period up to 10 days and for
release of one or more anions from the assembly after a delay or
lag period of 20 days or more. The invention is particularly useful
for controlled short-term and long-term release of one or more
nucleic acids.
[0084] Two anions exhibit "distinct" release profiles if the
relative amount of the two anions released is not constant as a
function of time. Two anions exhibit "separate" release profiles if
a portion of one of the anions is released when there is no release
of the second anion. In a specific embodiment, two anions can
exhibit "selective separate" release if one of the anions is
predominantly (50% by weight or more in the polyelectrolyte
assembly) released prior to the release of the second anion. In a
specific embodiment, two anions can exhibit "sequential" release if
one of the anions is substantially (90% by weight or more in the
polyelectrolyte assembly) released prior to the release of the
second anion. In a specific embodiment, two anions can exhibit
"distinct sequential" release if one of the anions is approximately
completely released (99% by weight or more in the polyelectrolyte
assembly) prior to the initiation of release of the second anion.
It will be understood that two or more different anions can exhibit
distinct and/or separate and/or selective separate and/or
sequential release and/or distinct sequential release profiles on
release from an appropriate polyelectrolyte assembly of this
invention.
[0085] It will, however, also be understood that when only a single
anion is present in the polyelectrolyte assembly, a portion of the
anion in a given assembly can exhibit release that is distinct
and/or separate and/or selective separate and/or sequential and/or
distinct sequential compared to another portion of the same anion
in that assembly. Differences in the release profile of a single
anion can be assessed by tagging or labeling sub-portions of the
anion to be released, for example, employing isotopic or
radiolabeling.
[0086] In view of the descriptions herein regarding anion release
profiles from representative cationic polymers of formulas I and
II, one of ordinary skill in the art can prepare polyelectrolyte
assemblies to achieve desired distinct, separate, distinct and
separate, long-term or combinations of long-term and short-term
release profiles by combining anion/cationic polymer bilayers
formed from selected polymers of formulas I and II. The choice of
cationic polymer is generally made based on the structure of the
polymer as described particularly in the examples herein.
[0087] In specific embodiments, the invention provides multilayer
polyelectrolyte films comprising two or more different
anion/cationic bilayers which provide for distinct and/or separate
and/or selective separate and/or sequential release and/or distinct
sequential release profiles of anions therein.
[0088] The present invention relates to multilayer polyelectrolyte
films which are formed from at least two different cationic charge
dynamic polymers, also called charge shifting polymers, selected
from cationic polymers having formula I:
##STR00003##
where: n, m, l, k and i are zero or integers where n+m+l+k+i=N, the
total number of repeat units in the polymer; A.sub.1-3, B.sub.1-3,
C.sub.1-3, D.sub.1-3 and E.sub.1-3 are linkers which may be the
same or each may be different; Z.sub.1-Z.sub.5 are covalent bonds
or degradable bonds; R.sub.1-4 and R.sub.11-19, independently, can
be hydrogen, or alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic,
aryl, or heteroaryl groups with the exception that R.sub.1-4 are
not esters; and R.sub.5-R.sub.10 are linkers or covalent bonds
which may be the same or each may be different. Variables in
formula I are further described above.
[0089] In specific embodiments, cationic polymers of formula IA are
useful in this invention:
##STR00004##
where variables are as defined above. The number of repeating units
in the polymer N is n+m+. In specific embodiments, (m+)/N is 0.01
to 0.1, 0.05 to 0.2, 0.1 to 0.25, 0.25 to 0.50, 0.25 to 0.75, or
0.75 to 1.
[0090] In specific embodiments, cationic polymers of formula IB are
useful in this invention:
##STR00005##
where each r is an integer ranging from 1-10 and other variables
are as defined above. The number of repeating units in the polymer
N is n+m+. In specific embodiments, (m+)/N is 0.01 to 0.1, 0.05 to
0.2, 0.1 to 0.25, 0.25 to 0.50, 0.25 to 0.75, or 0.75 to 1. In
specific embodiments, each r is 1, 2 or 3. In specific embodiments,
all r have the same value.
[0091] In specific embodiments, cationic polymers of formula IC are
useful in this invention:
##STR00006##
where variables are as defined above. The number of repeating units
in the polymer N is n+m+. In specific embodiments, (m+l)/N is 0.01
to 0.1, 0.05 to 0.2, 0.1 to 0.25, 0.25 to 0.50, 0.25 to 0.75, or
0.75 to 1. In specific embodiments, each r independently is 1, 2 or
3 and each p independently is 1, 2 or 3. In specific embodiments,
all r have the same value. In specific embodiments, all p have the
same value.
[0092] In specific embodiments, cationic polymers of formula ID are
useful in this invention:
##STR00007##
where variables are as defined above. The number of repeating units
in the polymer N is m++k+i. In specific embodiments, (m+)/N is 0.01
to 0.1, 0.05 to 0.2, 0.1 to 0.25, 0.25 to 0.50, 0.25 to 0.75, or
0.75 to 1. In specific embodiments, k is 0, k is less than 0.01 or
k is less than 0.1. In specific embodiments, each r is 1, 2 or 3.
In specific embodiments, all r are the same. In specific
embodiments, R.sub.3 and R.sub.4 are hydrogens.
[0093] In specific embodiments, cationic polymer of formula IE are
useful in this invention:
##STR00008##
where variables are as defined above. The number of repeating units
in the polymer N is n+m+. In specific embodiments, (m+)/N is 0.01
to 0.1, 0.05 to 0.2, 0.1 to 0.25, 0.25 to 0.50, 0.25 to 0.75, or
0.75 to 1. In specific embodiments, each r independently is 1, 2 or
3 and each p independently is 1, 2 or 3. In specific embodiments,
all r have the same value. In specific embodiments, all p have the
same value. In specific embodiments, R.sub.3 and R.sub.4 are
hydrogens.
[0094] In specific embodiments of formulas IA-IE all of
R.sub.11-R.sub.19 are alkyl groups having 1-8, 1-6 or 1-3 carbon
atoms.
[0095] In specific embodiments, polyelectrolyte assemblies of this
invention comprise two or more different cationic polymers of any
of formulas IA-IE where each different cationic polymer (m+)/N is a
different value.
[0096] In specific embodiments, polyelectrolyte assemblies of this
invention comprise a first bilayer comprising a cationic polymer of
any of formulas I, or IA-IE where (m+)/N is 0.01 to 0.50, 0.01 to
0.1, 0.05 to 0.2, 0.1 to 0.25, or 0.25 to 0.50 and a second bilayer
comprising a cationic polymer of any of formulas I, or IA-IE where
(m+)/N is 0.50 to 1.0, 0.50 to 0.75 or 0.75 to 1, wherein the first
and second cationic polymers have different values of (m+)/N.
[0097] In specific embodiments, polyelectrolyte assemblies of this
invention comprise a first bilayer comprising a cationic polymer of
any of formulas I, or IA-IE where (m+)/N is 0.01 to 0.25, 0.01 to
0.1, 0.05 to 0.2, or 0.1 to 0.25, and a second bilayer comprising a
cationic polymer of any of formulas I, or IA-IE where (m+l)/N is
0.50 to 1.0, 0.50 to 0.75 or 0.75 to 1.0.
[0098] In specific embodiments, polyelectrolyte assemblies of this
invention comprise a first bilayer comprising a cationic polymer of
any of formulas I, or IA-IE where (m+)/N is 0.01 to 0.25, a second
bilayer comprising a cationic polymer of any of formulas i, or
IA-IE where (m+)/N is 0.50 to 0.75 and a third bilayer comprising a
cationic polymer of any of formulas I, or IA-IE where (m+)/N is
0.80 to 1.0.
[0099] In specific embodiments, polyelectrolyte assemblies of this
invention comprise one more intermediate bilayers each comprising a
cationic polymer of any of formulas I or IA-IE.
[0100] In specific embodiments, polyelectrolyte assemblies of this
invention comprise one or more intermediate bilayers each
comprising a cationic polymer of any of formulas I, or IA-IE where
(m+)/N is 0.01 to 0.25, 0.01 to 0.1, 0.05 to 0.2, 0.1 to 0.25, or
0.25 to 0.5. In specific embodiments, polyelectrolyte assemblies of
this invention comprise one or more intermediate bilayers each
comprising a cationic polymer of any of formulas I, or IA-IE where
(m+)/N is 0.50 to 1.0, 0.50 to 0.75 or 0.75 to 1.0.
[0101] In specific embodiments, cationic polymers of formula II
include those of formula IIA, IIB, IIC, IID, IIE or IIF which are
all useful in this invention:
##STR00009## ##STR00010##
where variable are as defined above. In specific embodiments of
formulas IA-IF, all R.sup.3 are alkyl groups and particularly are
C1-C3 alkyl groups. In other embodiments, all R.sup.3 are methyl.
In other embodiments of formulas IA-IF, all R.sup.1 are hydrogen or
alkyl. In other embodiments, all R.sup.1 are C1-C3 alkyl. In other
embodiments, all R.sup.1 are methyl. In specific embodiments, all x
are the same. In other embodiments all y are the same. In specific
embodiments, each y is 2. In specific embodiments, each x is 2, 3
or 4.
[0102] In specific embodiments of formulas IIA-IIF, m is zero. In
other embodiments of formulas IIA-IIF, n is zero. In specific
embodiments of formulas IIA-IIF, N=5 to 100,000. More specifically,
N can range from 20 to 100,000, from 100 to 100,000, from 1,000 to
100,000 or from 10,000 to 100,000. In specific embodiments of
formulas IIA-IIF, (n)/(m+n) ranges from 0.1 to 1, including 0.05 to
0.25, 0.25 to 0.50, 0.25 to 0.75, 0.75 to 1, 0.85 to 1, 0.90 to 1
or 0.95 to 1. In specific embodiments of formulas IIA-IIF,
(n)/(n+m) is 0.50 to 1. In other specific embodiments of formulas
IIA-IIF, (n)/(n+m) is 0.01 to 0.5.
[0103] In specific embodiments of formulas IIA-IIF, 10% or more of
the groups bonded as side groups to the polymer are ester groups.
In specific embodiments, 25% or more of the groups bonded as side
groups to the polymer are ester groups. In specific embodiments,
50% or more of the groups bonded as side groups to the polymer are
ester groups. In specific embodiments, 75% or more of the groups
bonded as side groups to the polymer are ester groups. In specific
embodiments, 90% or more of the groups bonded as side groups to the
polymer are ester groups.
[0104] In other specific embodiments of formulas IIA-IIF, 10%-25%
of the groups bonded as side groups to the polymer are ester
groups. In specific embodiments, 25%-50% of the groups bonded as
side groups to the polymer are ester groups. In specific
embodiments, 50%-75% of the groups bonded as side groups to the
polymer are ester groups. In specific embodiments, 75%-90% of the
groups bonded as side groups to the polymer are ester groups. In
specific embodiments, 90%-100% of the groups bonded as side groups
to the polymer are ester groups.
[0105] In specific embodiments, polyelectrolyte assemblies of this
invention comprise one or two or more different cationic polymers
of any of formulas II or IIA-IIF. In specific embodiments,
polyelectrolyte assemblies of this invention comprise two or more
different cationic polymers of any of formulas II or IIA-IIF, where
each different cationic polymer has (n)/(n+m) that is
different.
[0106] In specific embodiments, polyelectrolyte assemblies of this
invention comprise a first bilayer comprising a polycation of any
of formulas II, or IIA-IIF where (n)/(n=m) is 0.1 to 1, 0.25 to 1,
0.5 to 1, or 0.75 to 1 and a second bilayer comprising a polycation
of any of formulas II, or IIA-IIF where (n)/(n=m) is 0.01 to 0.25,
0.1 to 0.25, 0.01 to 0.1 or 0.05 to 0.25.
[0107] In specific embodiments, polyelectrolyte assemblies of this
invention comprise at least a first bilayer comprising a polycation
of any of formulas I, or IA-IE and at least a second bilayer
comprising a polycation of any of formulas II, or IIA-IIF.
[0108] In specific embodiments, polyelectrolyte assemblies of this
invention comprise at least a first bilayer comprising a polycation
of any of formulas I, or IA-IE and at least a second bilayer
comprising a polycation of any of formulas II, or IIA-IIF wherein m
is zero or (n)/(n+m) is 0.5 to 1.
[0109] In specific embodiments, polyelectrolyte assemblies of this
invention comprise at least a first bilayer comprising a polycation
of any of formulas I, or IA-IE wherein and at least a second
bilayer comprising a polycation of any of formulas II, or IIA-IIF
wherein m is zero or (n)/(n+m) is 0.5 to 1.
[0110] In specific embodiments, polyelectrolyte assemblies of this
invention comprise a first bilayer comprising a polycation of any
of formulas I, or IA-IE where (m+l)/N is 0.01 to 0.25, or a first
bilayer comprising a polycation of any of formulas I, or IA-IE
where (m+l)/N is 0.50 to 0.75 or a first bilayer comprising a
polycation of any of formulas I, or IA-IE where (m+l)/N is 0.80 to
1.0 in combination with a second bilayer of a polycation of any of
formulas II or IIA-IIF where m is 0 or (n)/(n+m) is 0.5 to 1.
[0111] In specific embodiments, polyelectrolyte assemblies of this
invention comprise a first bilayer comprising a polycation of any
of formulas I, or IA-IE where (m+)/N is 0.01 to 0.25, a second
bilayer comprising a polycation of any of formulas I, or IA-IE
where (m+)/N is 0.50 to 1 in combination with a third bilayer of a
polycation of any of formulas II or IIA-IIF where m is 0 or
(n)/(n+m) is 0.5 to 1.
[0112] In specific embodiments, polyelectrolyte assemblies of this
invention comprise a first bilayer comprising a polycation of any
of formulas I, or IA-IE where (m+l)/N is 0.50 to 1, a second
bilayer comprising a polycation of any of formulas I, or IA-IE
where (m+)/N is 0.80 to 1.0 in combination with a third bilayer of
a polycation of any of formulas II or IIA-IIF where m is 0 or
(n)/(n+m) is 0.5 to 1.
[0113] In specific embodiments, polyelectrolyte assemblies of this
invention comprise a first bilayer comprising a polycation of any
of formulas I, or IA-IE where (m+)/N is 0.80 to 1.0 and a second
bilayer comprising a polycation of any of formulas II or IIA-IIF
where m is 0 or (n)/(n+m) is 0.5 to 1.
[0114] In specific embodiments, polyelectrolyte assemblies of this
invention comprise a first bilayer comprising a polycation of any
of formulas I, or IA-IE where n, m, k and i are all zero and a
second bilayer comprising a polycation of any of formulas II or
IIA-IIF where m is 0.
[0115] In specific embodiments, polyelectrolyte assemblies of this
invention further comprise one more intermediate bilayers each
comprising a cationic polymer of any of formulas II or IIA-IIF.
[0116] In specific embodiments, polyelectrolyte assemblies of this
invention further comprise one or more intermediate bilayers each
comprising a cationic polymer of any of formulas I, or IIA-IIF
where m is 0, or where (n)/(n+m) ranges from 0.5 to 1.0. In
specific embodiments, polyelectrolyte assemblies of this invention
comprise one or more intermediate bilayers each comprising a
cationic polymer of any of formulas II, or IIA-IIF where (n)/(n+m)
is 0.1 to 0.5.
[0117] In specific embodiments, polyelectrolyte assemblies of this
invention further comprise one or more intermediate bilayers each
comprising a cationic polymer other than a polymer of formulas I or
II, but which is a degradable polymer and which in particular is a
cationic polymer the polymer backbone of which is hydrolytically or
enzymatically degradable.
[0118] The number of bilayers in polyelectrolyte assemblies of this
invention is not particularly limited. The number of bilayers can
for example be 1-10, 1-20, 1-100, 1-500, or 1-1000. In specific
embodiments, 75% to 100% of the bilayers in the polyelectrolyte
assembly can be those formed by one or more polymers of formula II.
In other specific embodiments, 75% to 100% of the bilayers in the
polyelectrolyte assembly can be those formed by one or more
polymers of formula I. In other specific embodiments, 50% to 90% of
the bilayers in the polyelectrolyte assembly can be those formed by
one or more polymers of formula II, and 10% to 50% of the bilayers
in the polyelectrolyte assembly can be those formed by a one or
more polymers of formula I. In other specific embodiments, 50% to
90% of the bilayers in the polyelectrolyte assembly can be those
formed by one or more polymers of formula I, and 10% to 50% of the
bilayers in the polyelectrolyte assembly can be those formed by a
one or more polymers of formula II. In other specific embodiments,
50% to 99% of the bilayers in the polyelectrolyte assembly can be
those formed by one or more polymers of formula II, and 1% to 50%
of the bilayers in the polyelectrolyte assembly can be those formed
by a one or more polymers of formula I.
[0119] In other specific embodiments, 50% to 99% of the bilayers in
the polyelectrolyte assembly can be those formed by one or more
polymers of formula I, and 1% to 50% of the bilayers in the
polyelectrolyte assembly can be those formed by a one or more
polymers of formula II.
[0120] FIGS. 10A-C illustrate exemplary polyelectrolyte assemblies
of this invention comprising one or more cationic polymers of
formulas I, IA, IB, IC, ID, IE, II, IIA, IIB, IIC, IID, IIE, IIF or
combinations thereof.
[0121] In the assembly (10) of FIG. 1A, a plurality of sequential
first bilayers (2) and a plurality of second sequential bilayers
(3) are optionally separated by one or more than one intermediate
bilayer (4). The assembly optionally comprises one or more base
bilayers (5) formed on the substrate (9). The assembly optionally
comprises one or more top protective bilayers (6). The number of
first bilayers and second bilayers can be the same or different.
There can be more first bilayers than second bilayers or there can
be fewer first bilayers than second bilayers. It will be apparent
that the assembly of FIG. 10A can contain additional pluralities of
bilayers which may be pluralities of the first or second bilayers
or a plurality of one or more different bilayers, e.g., a third,
fourth, fifith or other bilayer. Each such plurality of bilayers is
optionally separated by one or more intermediate bilayers.
Assemblies as in FIG. 10A can, for example, comprise a total of
four or more different bilayers. In specific embodiments,
assemblies of FIG. 10A comprise 1000, 500 or 200 or fewer bilayers
in addition to any base bilayers. In specific embodiments,
assemblies of FIG. 10A comprise 100, 50 or 20 or fewer bilayers in
addition to any base bilayers. In specific embodiments, assemblies
of FIG. 10A comprise 10 or fewer bilayers in addition to any base
bilayers.
[0122] The assembly (10) of FIG. 10B, comprises a plurality of
sequential first bilayers (2a), a second plurality of first
bilayers (2b) and a plurality of second sequential bilayers (3).
Each plurality of bilayers is optionally separated by one or more
than one intermediate bilayers (4a and 4b). The assembly optionally
comprises one or more base bilayers (5) formed on the substrate.
(9). The assembly optionally comprises one or more top protective
bilayers (6). The assembly can contain additional pluralities of
first bilayers and/or additional pluralities of second bilayers.
The number of first bilayers and second bilayers in the assembly
can be the same or different. There can be more first bilayers than
second bilayers or there can be fewer first bilayers than second
bilayers. It will be apparent that the assembly of FIG. 10B can
contain additional pluralities of bilayers which may be pluralities
of the first or second bilayers or a plurality of one or more
different bilayers, e.g., a third, fourth, fifth or other bilayer.
Each such plurality of bilayers is optionally separated by one or
more intermediate bilayers. The number of bilayers in the first
plurality of first bilayers and the number of bilayers in any
additional pluralities of first bilayers can be the same or
different. The number of bilayers in the first plurality of second
bilayers and the number of bilayers in any additional pluralities
of second bilayers can be the same or different. Assemblies as in
FIG. 10B can, for example, comprise a total of 6 or more bilayers.
In specific embodiments, assemblies of FIG. 10B comprise 1000, 500,
200 or fewer bilayers in addition to any base bilayers. In specific
embodiments, assemblies of FIG. 10B comprise 100, 50, 20 or fewer
bilayers in addition to any base bilayers. In specific embodiments,
assemblies of FIG. 10B comprise 10 or fewer bilayers in addition to
any base bilayers. In an assembly of FIG. 10B it will be recognized
that the order of the pluralities of bilayers in the assembly can
be changed. For example, first and second pluralities of first
bilayers can be layered sequentially, separated by one or more
intermediate bilayers, and first and second pluralities of second
bilayers can be layered sequentially separated by one or more
intermediate bilayers.
[0123] The assembly (10) of FIG. 1C comprises one or a plurality of
(two or more) sequential first bilayers (2), one or a plurality of
second bilayers (3) and one or a plurality of third sequential
bilayers (7). Each plurality of bilayers is optionally separated by
one or more than one intermediate bilayers (not shown). The
assembly optionally comprises one or more base bilayers (5) formed
on the substrate (9). The assembly optionally comprises one or more
top protective bilayers (6). The number of first bilayers, second
bilayers and third bilayers in the assembly can be the same or
different. There can be more first bilayers than second bilayers or
there can be fewer first bilayers than second bilayers. It will be
apparent that the assembly of FIG. 10C can contain additional
single bilayers or pluralities of bilayers which may be first,
second, third or additional bilayers, e.g., a fourth, fifth or
other bilayer. Each such plurality of bilayers is optionally
separated by one or more intermediate bilayers. Assemblies as in
FIG. 10C can comprise a total of three or more bilayers. In
specific embodiments, assemblies of FIG. 10C comprise 1000, 500,
200 or fewer bilayers in addition to any base bilayers. In specific
embodiments, assemblies of FIG. 10C comprise 100, 50, 20 or fewer
bilayers in addition to any base bilayers. In specific embodiments,
assemblies of FIG. 10C comprise 10 or fewer bilayers in addition to
any base bilayers.
[0124] Bilayers in any of FIGS. 1A-C are different if they comprise
a different polycation composition or a different anion
composition. Different bilayers include those formed using
different cationic polymers or different mixtures of cationic
polymers. Different bilayers include those comprising different
anions or different mixtures of anions. In a specific embodiment,
different bilayers can contain the same anion or mixture of anions
which are present in the different bilayers at different
concentrations or amounts.
[0125] In a specific embodiment, a polyelectrolyte assembly as in
FIG. 10A is designed for short-term release of a first anion and
long-term release of a second anion. The anions may be first and
second nucleic acids. In the assembly, upper bilayers (2) contain
the anion to be released short-term (hours or days or 10 days or
less) and lower bilayers (3) contain the anion to be released
long-term (20 days or more, weeks, or months). Bilayers 2 are
preferably formed with one or more cationic polymers of formula I
wherein (m+l)/N is 0.5 or more and bilayers 3 are preferably formed
with a cationic polymer of formula II, particularly where (n)/(n+m)
is 0.5 or more and more specifically where m is 0. The assembly
optionally further comprises one or more base, top or intermediate
layers between the short-term and long-term release bilayers. In
specific embodiments, the first bilayers 2 and second bilayers 3
are sequential and contain 1-1000 (or 1-500, 1-200, 1-200, 1-50,
1-20 or 1-10) bilayers and are optionally separated by 1-100 (or
1-50, 1-20 or 1-10) intermediate bilayers. In specific embodiments,
the assembly comprises 1-100 (or 1-50, 1-20, or 1-10) base bilayers
and/or 1-100 (or 1-50, 1-20, or 1-10) top protective bilayers.
[0126] In specific embodiments, the invention provides
polyelectrolyte assemblies comprising 1-1000 (or 1-500, 1-200,
1-100, 1-50 or 1-20) first bilayers formed from a first cationic
polymer of any of formulas I, or IA-IE where (m+l)/N is 0.01 to
0.50, 0.01 to 0.1, 0.05 to 0.2, 0.1 to 0.25, or 0.25 to 0.50 and
1-1000 (or 1-500, 1-200, 1-100, 1-50 or 1-20) second bilayers
formed from a second polycation of any of formulas IA-IE where
(m+)/N is 0.50 to 1.0, 0.50 to 0.75 or 0.75 to 1, wherein the first
and second polycations have different values of (m+l)/N. In
specific embodiments, the first bilayers and second bilayers are
sequential and are optionally separated by 1-100 (or 1-50, 1-20 or
1-10) intermediate bilayers. In specific embodiments, the assembly
comprises 1-100 (or 1-50, 1-20, or 1-10) base bilayers and/or 1-100
(or 1-50, 1-20, or 1-10) top protective bilayers. In specific
embodiments, the first and second bilayers comprise different
anions or different mixtures of anions. In specific embodiments,
the first and second bilayers comprise different nucleic acids or
different mixtures of nucleic acids. In specific embodiments, the
first and second bilayers comprise different nucleic acids carried
on one or more vectors. In specific embodiments, the first and
second bilayers comprise different nucleic acids carried on one or
more expression vectors. In specific embodiments, the different
nucleic acids have different sequences. In specific embodiments,
the first and second bilayers comprise different nucleic acids
which encode one or more polypeptides. In specific embodiments, the
first and second bilayers comprise different nucleic acids each of
which encodes a different one or more polypeptides. In specific
embodiments, the assembly comprises 1-1000 (or 1-500, 1-200, 1-100,
1-50, 1-20 or 1-10) first bilayers and 1-1000 (or 1-500, 1-200,
1-100, 1-50, 1-20 or 1-10) second bilayers. In specific
embodiments, the assembly comprises 1-10 first bilayers and 1-10
second bilayers. In specific embodiments, the assembly comprises
1-10 first bilayers and 1-10 second bilayers separated by 1-10
intermediate bilayers.
[0127] Dynamic charge state cationic polymers are polymers designed
to have cationic charge densities that decrease by removal of
removable functional groups from the polymers. In specific
embodiments, the removable functional group is a hydrolysable
group, such as a pendant ester which is converted on hydrolysis to
a pendant --COO.sup.- (anionic group). For some polymers herein,
the ester bond will generally be readily hydrolysable, whereas the
amide bond is not readily hydrolysable.
[0128] The polymers of the present invention may have any desired
molecular weight, such as from 1,000 to 100,000 grams/mole, or from
about 2,000 to 50,000 grams/mole. The dynamic charge state cationic
polymers of this invention can be associated with a ligand
facilitating the delivery of the polymer to a specific target, such
as a target cell.
[0129] The cationic polymers of this invention can also be part of
a copolymer, which can be composed of any other polymers, for
example a polymer such as PEG or PEO which are commonly used to
give stability toward protein adsorption. The cationic polymers of
the invention are generally cationic, but different functional
groups attached to the polymer can render the polymer zwitterionic.
To impart a cationic charge to the polymer, the attached functional
groups can be positively charged. The cationic polymers of the
invention may also be capable of buffering changes in pH which
results from the make-up of the polymer backbone and/or the
attached functional groups. Thus, the invention further relates to
polyelectrolyte assemblies which comprise one or more copolymer
comprising a cationic polymer of formula I or formula II.
[0130] Certain cationic polymers of this invention carry positive
charge on polymer side chains. Dependent upon the specific
structure of the cationic polymer, hydrolysis of side chain groups,
such as esters, results in the formation of negatively charged
species on the side chains and an overall decrease in positive
charge of the polymer. In specific embodiments, the polymer
backbones of the cationic polymers of this invention do not carry
charge. In specific embodiments, the polymer backbones of the
cationic polymers of this invention are not hydrolytically or
enzymatically degradable.
[0131] The present dynamic charge state cationic polymers may be
non-immunogenic, non-toxic or both non-immunogenic and non-toxic.
In the present polymers, the polymeric backbone can be degradable
or nondegradable. The present polymers do not require that the
degradation of the backbone occur at the same time as the shift in
cationic charge. One skilled in the art will recognize that the
measure of degradability will be commensurate with the
environmental conditions and desired properties for any particular
application for the present polymers.
[0132] As one non-limiting example, for biomedical uses of the
present polymers, the present invention contemplates polymers that
degrade in a desired time frame (from an hour to a week to a month
to a year) under physiological conditions typically found in the
body or in a cell or cell compartment [e.g., pH ranges from about
5.0 (endosomal/lysosomal) to 7.4 (extracellular and cytosol), a
temperature of about 37.degree. C. and an ionic strength of a
typical physiological solution (generally around 130-150 mM NaCl,
for example)]. In the present invention, the degradability of the
polymer can be measured by a variety of methods, including, but not
limited to, GPC (gel permeation chromatography).
[0133] The present invention also provides cationic polymers
complexed with one or more anionic molecules thereby forming an
interpolyelectrolyte complex.
[0134] Suitable anions of the invention may be naturally occurring,
synthetic, or both. In some embodiments, suitable examples of
anions include nucleic acids, such as RNA, DNA, and analogs
thereof. In other embodiments, the anion is a synthetic polyanion.
In still other embodiments, the anions of the invention are nucleic
acids, such as RNA, DNA, or analogs thereof, and a synthetic
polyanion. When the anion is a nucleic acid, the nucleic acid can
have the sequence of a nucleic acid molecule of interest or its
complement. As such, the nucleic acid can encode for a protein or a
functional fragment thereof or be useful in antisense treatment or
RNA interference. In some embodiments, the nucleic acid is a
plasmid. In other embodiments, the anionic molecule or agent may be
a therapeutic molecule, diagnostic molecule, peptide, or
carbohydrate, for example a macromolecular carbohydrate such as
heparin.
[0135] The following are terms used in the present application:
The term "alkyl" as used herein refers to saturated, straight- or
branched-chain hydrocarbon radicals derived from a hydrocarbon
moiety containing between one and thirty (more typically between
1-22) carbon atoms by removal of a single hydrogen atom. In some
embodiments, alkyl groups have from 1 to 12, from 1 to 8 carbon
atoms, from 1 to 6 or 1 to 3 carbon atoms. Examples of alkyl
radicals include, but are not limited to, methyl, ethyl, propyl,
isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl,
n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl. A "cycloalkyl"
group is a cyclic alkyl group typically containing from 3 to 8 ring
members such as, but not limited to, a cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl group.
[0136] The term "alkoxy" as used herein refers to an alkyl group,
as previously defined, attached to the parent molecular moiety
through an oxygen atom. Examples include, but are not limited to,
methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy,
neopentoxy, and n-hexoxy groups.
[0137] The term "alkenyl" denotes a monovalent group derived from a
hydrocarbon moiety having at least one carbon-carbon double bond by
the removal of a single hydrogen atom. Alkenyl groups typically can
have 1-22 carbon atoms and include, for example, ethenyl, propenyl,
butenyl, l-methyl-2-buten-l-yl, and the like. Alkenyl groups
include those having from 2-12 carbon atoms, those having 2-8, and
those having 2-6 carbon atoms.
[0138] The term "alkynyl" as used herein refers to a monovalent
group derived form a hydrocarbon having at least one carbon-carbon
triple bond by the removal of a single hydrogen atom. Alkynyl
groups can typically have 1-22 carbon atoms. Representative alkynyl
groups include ethynyl, 2-propynyl (propargyl), l-propynyl, and the
like. Alkynyl groups include those having from 2-12 carbon atoms,
those having 2-8, and those having 2-6 carbon atoms.
[0139] Alkyl, alkenyl and alkynyl groups can be optionally
substituted with groups including alkoxy, thioalkoxy, amino,
alkylamino, dialkylamino, trialkylamino, acylamino, cyano, hydroxy,
halo, mercapto, nitro, carboxyaldehyde, carboxy, alkoxycarbonyl,
and carboxamide groups.
[0140] The term "aryl" as used herein refers to carbocyclic ring
systems having at least one aromatic ring including, but not
limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, and
indenyl groups, and the like. Aryl groups can be unsubstituted or
substituted with substituents selected from the group consisting of
branched and unbranched alkyl, alkenyl, alkynyl, haloalkyl, alkoxy,
thioalkoxy, amino, alkylamino, dialkylamino, trialkylamino,
acylamino, cyano, hydroxy, halo, mercapto, nitro, carboxyaldehyde,
carboxy, alkoxycarbonyl, and carboxamide. In addition, substituted
aryl groups include tetrafluorophenyl and pentafluorophenyl.
[0141] The term carbocyclic is used generally herein to refer to
groups containing one or more carbon rings. The groups may be
aromatic or aryl groups. Rings may contain 3-10 carbon atoms and
one, two or three double bonds or a triple bond. These groups may
include single rings of 3 to 8 atoms in size and bi- and tri-cyclic
ring systems which may include aromatic six membered aryl or
aromatic groups fused to a non-aromatic ring.
[0142] The terms "heterocyclic" and "heterocyclyl", are used
broadly herein to refer to an aromatic, partially unsaturated or
fully saturated 3- to 10-membered ring system, which includes
single rings of 3 to 8 atoms in size and bi- and tri-cyclic ring
systems which may include aromatic six membered aryl or aromatic
heterocyclic groups fused to a non-aromatic ring. These
heterocyclic and heterocyclyl rings and groups include those having
from one to three heteroatoms independently selected from oxygen,
sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms
may optionally be oxidized and the nitrogen heteroatom may
optionally be quaternary.
[0143] The terms "aromatic heterocyclic" or "heteroaryl" as used
herein, refer to a cyclic aromatic radical having from five to 12
ring atoms of which one ring atom is selected from sulfur, oxygen,
and nitrogen; zero, one, or two ring atoms are additional
heteroatoms independently selected from sulfur, oxygen, and
nitrogen; and the remaining ring atoms are carbon, the radical
being joined to the rest of the molecule via any of the ring atoms.
The term includes heteroaromatic rings fused to aryl ring or to
carbocylic rings. Examples of such aromatic heterocyclyl groups
include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl,
pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl,
thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, and
isoquinolinyl groups, and the like.
[0144] Specific heterocyclic and aromatic heterocyclic groups that
may be included in the compounds of the invention include:
3-methyl-4-(3-methylphenyl)piperazine, 3-methylpiperidine,
4-(bis-(4-fluorophenyl)methyl)piperazine,
4-(diphenylmethyl)piperazine, 4-(ethoxycarbonyl)piperazine,
4-(ethoxycarbonylmethyl)piperazine, 4-(phenylmethyl)piperazine,
4-(1-phenylethyl)piperazine,
4-(1,1-dimethylethoxycarbonyl)piperazine,
4-(2-(bis-(2-propenyl)amino)ethyl)piperazine,
4-(2-(diethylamino)ethyl)piperazine, 4-(2-chlorophenyl)piperazine,
4-(2-cyanophenyl)piperazine, 4-(2-ethoxyphenyl)piperazine,
4-(2-ethylphenyl)piperazine, 4-(2-fluorophenyl)piperazine,
4-(2-hydroxyethyl)piperazine, 4-(2-methoxyethyl)piperazine,
4-(2-methoxyphenyl)piperazine, 4-(2-methylphenyl)piperazine,
4-(2-methylthiophenyl)piperazine, 4-(2-nitrophenyl)piperazine,
4-(2-nitrophenyl)piperazine, 4-(2-phenylethyl)piperazine,
4-(2-pyridyl)piperazine, 4-(2-pyrimidinyl)piperazine,
4-(2,3-dimethylphenyl)piperazine, 4-(2,4-difluorophenyl)piperazine,
4-(2,4-dimethoxyphenyl)piperazine,
4-(2,4-dimethylphenyl)piperazine, 4-(2,5-dimethylphenyl)piperazine,
4-(2,6-dimethylphenyl)piperazine, 4-(3-chlorophenyl)piperazine,
4-(3-methylphenyl)piperazine,
4-(3-trifluoromethylphenyl)piperazine,
4-(3,4-dichlorophenyl)piperazine, 4-3,4-dimethoxyphenyl)piperazine,
4-(3,4-dimethylphenyl)piperazine,
4-(3,4-methylenedioxyphenyl)piperazine,
4-(3,4,5-trimethoxyphenyl)piperazine,
4-(3,5-dichlorophenyl)piperazine,
4-(3,5-dimethoxyphenyl)piperazine,
4-(4-(phenylmethoxy)phenyl)piperazine,
4-(4-(3,1-dimethylethyl)phenylmethyl)piperazine,
4-(4-chloro-trifluoromethylphenyl)piperazine,
4-(4-chlorophenyl)-3-methylpiperazine,
4-(4-chlorophenyl)piperazine, 4-(4-chlorophenyl)piperazine,
4-(4-chlorophenylmethyl)piperazine, 4-(4-fluorophenyl)piperazine,
4-(4-methoxyphenyl)piperazine, 4-(4-methylphenyl)piperazine,
4-(4-nitrophenyl)piperazine, 4-(4-trifluoromethylphenyl)piperazine,
4-cyclohexylpiperazine, 4-ethylpiperazine,
4-hydroxy-4-(4-chlorophenyl)methylpiperidine,
4-hydroxy-4-phenylpiperidine, 4-hydroxypyrrolidine,
4-methylpiperazine, 4-phenylpiperazine, 4-piperidinylpiperazine,
4-(2-furanyl)carbonyl)piperazine,
4-((1,3-dioxolan-5-yl)methyl)piperazine-,
6-fluoro-1,2,3,4-tetrahydro-2-methylquinoline,
1,4-diazacylcloheptane, 2,3-dihydroindolyl, 3,3-dimethylpiperidine,
4,4-ethylenedioxypiperidine, 1,2,3,4-tetrahydroisoquinoline,
1,2,3,4-tetrahydroquinoline, azacyclooctane, decahydroquinoline,
piperazine, piperidine, pyrrolidine, thiomorpholine, and
triazole.
[0145] The term "hydrocarbon", as used herein, refers to any
chemical group comprising hydrogen and carbon. The hydrocarbon may
be substituted or unsubstituted. The hydrocarbon may be
unsaturated, saturated, branched, unbranched, cyclic, polycyclic,
or heterocyclic. Illustrative hydrocarbons include, for example,
methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, allyl, vinyl,
n-butyl, tert-butyl, ethynyl, cyclohexyl, methoxy, diethylamino,
and the like. As would be known to one skilled in this art, all
valencies must be satisfied in making any substitutions.
[0146] The terms "substituted", whether preceded by the term
"optionally" or not, and "substituent", as used herein, refer to
the ability, as appreciated by one skilled in this art, to change
one functional group for another functional group provided that the
valency of all atoms is maintained. When more than one position in
any given structure may be substituted with more than one
substituent selected from a specified group, the substituent may be
either the same or different at every position. The substituents
may also be further substituted (e.g., an aryl group substituent
may be further substituted. For example, a non limiting example is
an aryl group that may be further substituted with, for example, a
fluorine group at one or more position.
[0147] As used herein, "biodegradable" compounds are those that,
when introduced into cells, are broken down by the cellular
machinery or by hydrolysis into components that the cells can
either reuse or dispose of, in some cases without significant toxic
effect on the cells (e.g., fewer than about 20% of the cells are
killed when the components are added to cells in vitro). The
components preferably do not induce inflammation or other adverse
effects in vivo. In certain embodiments, the chemical reactions
relied upon to break down the biodegradable compounds are
uncatalyzed.
[0148] A "labile bond" is a covalent bond that is capable of being
selectively broken. That is, a labile bond may be broken in the
presence of other covalent bonds without the breakage of other
covalent bonds. For example, a disulfide bond is capable of being
broken in the presence of thiols without cleavage of any other
bonds, such as carbon-carbon, carbon-oxygen, carbon-sulfur,
carbon-nitrogen bonds, which may also be present in the molecule.
"Labile" also means cleavable.
[0149] A "labile linkage" is a chemical compound that contains a
labile bond and provides a link or spacer between two other groups.
The groups that are linked may be chosen from compounds such as
biologically active compounds, membrane active compounds, compounds
that inhibit membrane activity, functional reactive groups,
monomers, and cell targeting signals. The spacer group may contain
chemical moieties chosen from a group that includes alkanes,
alkenes, esters, ethers, glycerol, amide, saccharides,
polysaccharides, and heteroatoms such as oxygen, sulfur, or
nitrogen. The spacer may be electronically neutral, may bear a
positive or negative charge, or may bear both positive and negative
charges with an overall charge of neutral, positive or
negative.
[0150] In general, the "effective amount" of an active agent refers
to the amount necessary to elicit the desired biological response.
As will be appreciated by those of ordinary skill in this art, the
effective amount of an agent or device may vary depending on such
factors as the desired biological endpoint, the agent to be
delivered, the composition of the encapsulating matrix, the target
tissue, etc. The polyelectrolyte assemblies of this invention can
be employed to deliver an effective amount of one or more active
agents which are anions and particularly which are nucleic
acids.
[0151] As used herein, "peptide", means peptides of any length and
includes proteins. The terms "polypeptide" and "oligopeptide" are
used herein without any particular intended size limitation, unless
a particular size is otherwise stated. The only limitation to the
peptide or protein drug which may be utilized is one of
functionality. The terms "protein" and "peptide" may be used
interchangeably. Peptide may refer to an individual peptide or a
collection of peptides. peptides preferably 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/.about.da-dgrplUnnatstruct.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 an peptide 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. In some embodiments,
the modifications of the peptide lead to a more stable peptide
(e.g., greater half-life in vivo). These modifications may include
cyclization of the peptide, the incorporation of D-amino acids,
etc. Anions of this invention include anionic polypeptides,
proteins and/or peptides.
[0152] As used herein, "administering", and similar terms means
delivering the composition to the individual being treated. In some
instances the composition is capable of being circulated
systemically where the composition binds to a target cell and is
taken up by endocytosis. In specific embodiments of this invention,
polyelectrolyte assemblies can be employed to administer or deliver
two or more anions to an individual.
[0153] The present methods may be carried out by performing any of
the steps described herein, either alone or in various
combinations. The present compounds may also have any or all of the
components described herein. One skilled in the art will recognize
that all embodiments of the present invention are capable of use
with all other embodiments of the invention described herein.
Additionally, one skilled in the art will realize that the present
invention also encompasses variations of the present methods and
compositions that specifically exclude one or more of the steps,
components or groups described herein.
[0154] As will be understood by one skilled in the art, for any and
all purposes, particularly in terms of providing a written
description, all ranges disclosed herein also encompass any and all
possible subranges and combinations of subranges thereof. Any
listed range can be easily recognized as sufficiently describing
and enabling the same range being broken down into at least equal
halves, thirds, quarters, fifths, tenths, etc. As a non-limiting
example, each range discussed herein can be readily broken down
into a lower third, middle third and upper third, etc. As will also
be understood by one skilled in the art all language such as "up
to," "at least," "greater than," "less than," "more than" and the
like include the number recited and refer to ranges which can be
subsequently broken down into subranges as discussed above. In the
same manner, all ratios disclosed herein also include all subratios
falling within the broader ratio.
[0155] Accordingly, for all purposes, the present invention
encompasses not only the main group, but also the main group absent
one or more of the group members. The present invention also
envisages the explicit exclusion of one or more of any of the group
members in the claimed invention.
[0156] When a group of substituents is disclosed herein, it is
understood that all individual members of that group and all
subgroups, including any isomers, enantiomers, and diastereomers of
the group members, are disclosed separately. When a Markush group
or other grouping is used herein, all individual members of the
group and all combinations and subcombinations possible of the
group are intended to be individually included in the disclosure. A
number of specific groups of variable definitions have been
described herein. It is intended that all combinations and
subcombinations of the specific groups of variable definitions are
individually included in this disclosure. Accordingly, for all
purposes, the present invention encompasses not only the main
group, but also the main group absent one or more of the group
members. The present invention also envisages the explicit
exclusion of one or more of any of the group members in the claimed
invention.
[0157] Compounds described herein may exist in one or more isomeric
forms, e.g., structural or optical isomers. When a compound is
described herein such that a particular isomer, enantiomer or
diastereomer of the compound is not specified, for example, in a
formula or in a chemical name, that description is intended to
include each isomers and enantiomer (e.g., cis/trans isomers, R/S
enantiomers) of the compound described individual or in any
combination. Additionally, unless otherwise specified, all isotopic
variants of compounds disclosed herein are intended to be
encompassed by the disclosure. For example, it will be understood
that any one or more hydrogens in a molecule disclosed can be
replaced with deuterium or tritium. Isotopic variants of a molecule
are generally useful as standards in assays for the molecule and in
chemical and biological research related to the molecule or its
use. Isotopic variants, including those carrying radioisotopes, may
also be useful in diagnostic assays and in therapeutics. Methods
for making such isotopic variants are known in the art. Specific
names of compounds are intended to be exemplary, as it is known
that one of ordinary skill in the art can name the same compounds
differently.
[0158] Molecules disclosed herein may contain one or more ionizable
groups [groups from which a proton can be removed (e.g., --COOH) or
added (e.g., amines) or which can be quaternized (e.g., amines)].
All possible ionic forms of such molecules and salts thereof are
intended to be included individually in the disclosure herein. With
regard to salts of the compounds herein, one of ordinary skill in
the art can select from among a wide variety of available
counterions those that are appropriate for preparation of salts of
this invention for a given application. In specific applications,
the selection of a given anion or cation for preparation of a salt
may result in increased or decreased solubility of that salt. Every
formulation or combination of components described or exemplified
herein can be used to practice the invention, unless otherwise
stated.
[0159] Whenever a range is given in the specification, for example,
a temperature range, a time range, or a composition or
concentration range, all intermediate ranges and subranges, as well
as all individual values included in the ranges given are intended
to be included in the disclosure. It will be understood that any
subranges or individual values in a range or subrange that are
included in the description herein can be excluded from the claims
herein.
[0160] All patents and publications mentioned in the specification
are indicative of the levels of skill of those skilled in the art
to which the invention pertains. References cited herein are
incorporated by reference herein in their entirety to indicate the
state of the art as of their publication or filing date and it is
intended that this information can be employed herein, if needed,
to exclude specific embodiments that are in the prior art. For
example, when composition of matter are claimed, it should be
understood that compounds known and available in the art prior to
Applicant's invention, including compounds for which an enabling
disclosure is provided in the references cited herein, are not
intended to be included in the composition of matter claims
herein.
[0161] As used herein, "comprising" is synonymous with "including,"
"containing," or "characterized by," and is inclusive or open-ended
and does not exclude additional, unrecited elements or method
steps. As used herein, "consisting of" excludes any element, step,
or ingredient not specified in the claim element. As used herein,
"consisting essentially of" does not exclude materials or steps
that do not materially affect the basic and novel characteristics
of the claim. The broad term comprising is intended to encompass
the narrower consisting essentially of and the even narrower
consisting of. Thus, in any recitation herein of a phrase
"comprising one or more claim element" (e.g., "comprising A and B),
the phrase is intended to encompass the narrower, for example,
"consisting essentially of A and B" and "consisting of A and B."
Thus, the broader word "comprising" is intended to provide specific
support in each use herein for either "consisting essentially of"
or "consisting of." The invention illustratively described herein
suitably may be practiced in the absence of any element or
elements, limitation or limitations which is not specifically
disclosed herein.
[0162] One of ordinary skill in the art will appreciate that
starting materials, catalysts, reagents, synthetic methods,
purification methods, analytical methods, and assay methods, other
than those specifically exemplified can be employed in the practice
of the invention without resort to undue experimentation. All
art-known functional equivalents, of any such materials and methods
are intended to be included in this invention. The terms and
expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that
in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are
possible within the scope of the invention claimed. Thus, it should
be understood that although the present invention has been
specifically disclosed by examples, preferred embodiments and
optional features, modification and variation of the concepts
herein disclosed may be resorted to by those skilled in the art,
and that such modifications and variations are considered to be
within the scope of this invention as defined by the appended
claims.
[0163] All references cited herein are hereby incorporated by
reference to the extent that there is no inconsistency with the
disclosure of this specification. Some references provided herein
are incorporated by reference to provide details concerning sources
of starting materials; alternative starting materials, reagents,
methods of synthesis, purification methods, and methods of
analysis; as well as additional uses of the invention.
[0164] Unless otherwise specified, "a" or "an" means "one or
more".
THE EXAMPLES
Example 1
Multilayered Films Assembled from Charge-Shifting Cationic Polymers
Providing Separate and/or Distinct Release Profiles of DNA
Constructs
[0165] In general, approaches to the design of `charge-shifting`
polymers have taken one of two basic routes: (i) the attachment of
amine-functional side chains to polymer backbones through cleavable
linkages, or (ii) the conjugate addition of ester-functionalized
`charge-shifting` side chains to the backbones of cationic
polymers. [X. H. Liu, J. W. Yang, A. D. Miller, E. A. Nack, D. M.
Lynn, Macromolecules 2005, 38, 7907.]
[0166] Polymer 1 is exemplary of this second design approach (ii);
gradual reductions in the net charge of this polymer can be made to
occur upon hydrolysis of ester-functionalized side chains and the
introduction of anionic charge (Eq 1; full protonation of amine
functionality is shown for illustrative purposes)..sup.[24]
##STR00011##
[0167] This polymer can promote both self-assembly and
time-dependent disassembly with DNA in solution in ways that can be
understood in terms of side chain hydrolysis and subsequent changes
in the net charges of the polymer. This approach also permits
tunable control over the nature of electrostatic interactions with
DNA by control over the number of charge-shifting side chains added
to the polymer.
[0168] This example demonstrates that this approach to the
disruption of ionic interactions in polyelectrolyte assemblies can
be exploited to exert control over the time-dependent stability of
polyelectrolyte multilayers in aqueous environments. Recently, it
has been reported that `charge-shifting` cationic polymers designed
using polymers having amine-functional side chains attached through
hydrolyzable linkages can be used to fabricate multilayers. [J. T.
Zhang, D. M. Lynn, Adv Mater 2007, 19, 4218; B. G. De Geest, R. E.
Vandenbroucke, A. M. Guenther, G. B. Sukhorukov, W. E. Hennink, N.
N. Sanders, J. Demeester, S. C. De Smedt, Adv Mater 2006, 18,
1005.] De Geest et al. in particular reported the use of this
approach to fabricate multilayered microcapsules designed for
intracellular delivery.
[0169] In the context of designing films that provide tunable
control over film disassembly, the approach used to design polymer
1 can provide practical advantages relative to the approaches noted
above (which require the synthesis of specialized monomers) because
this approach is (i) modular and (ii) it can be used to introduce
`charge-shifting` side chains to a broad range of commercially
available polyamines. [Liu et al., 2005, supra]
[0170] The addition of ester-functionalized `charge-shifting` side
chains to poly(allylamine hydrochloride) (PAH) (polymer 2) can be
used to provide control over the erosion of DNA-containing films
and design multilayered films that orchestrate the release of
multiple different DNA constructs with separate and distinct
release profiles.
##STR00012##
[0171] The methyl ester-functionalized polymer 2 was synthesized by
the conjugate addition of PAH to methyl acrylate using a procedure
similar to that described previously for the synthesis of polymer
1. [Liu et al., 2005, supra] Treatment of PAH with an excess of
methyl acrylate resulted in the exhaustive functionalization of PAH
(i.e., polymer 2; n=m=0), as determined by .sup.1H NMR
spectroscopy. To investigate the influence of polymer structure on
film growth and behavior, we synthesized four derivatives of
polymer 2 having approximately 100%, 75%, 50%, and 25% substitution
(referred to hereafter as polymers 2a, 2b, 2c, and 2d) by varying
the amount of methyl acrylate added. PAH contains primary amine
functionality that can participate in up to two conjugate addition
reactions with methyl acrylate. As a result, the structures of
polymers 2b, 2c, and 2d (each substituted at <100%) consist of
mixtures of repeat units that are either exhaustively alkylated,
partially alkylated, or non-alkylated (as shown in FIG. 1). The
extents of substitution of polymers 2b-d are reported here as
percentages relative to the number of side chains that would be
present in an exhaustively substituted polymer (e.g., polymer 2a,
n=m=0).
[0172] The side chain methyl esters of polymers 2a-d hydrolyze to
unmask anionic carboxylate functionality when these materials are
incubated in physiologically relevant media (e.g., FIG. 1, top).
Characterization of ester hydrolysis in deuterated
phosphate-buffered saline (PBS, pH=7.4) at 37.degree. C. revealed
differences in the rates of hydrolysis for these four materials
(e.g., half-lives ranging from .about.4.5 days for polymer 2a to
.about.1 day or less for polymers 2b-d; see FIG. 2).
Characterization of the resulting acid-functionalized materials by
FTIR spectroscopy demonstrated that side chain hydrolysis occurred
without the formation of amide crosslinks between the amine
functionality and ester functionality in these materials.
[0173] A series of experiments was conducted to determine whether
ester-functionalized polymers 2a-d could be used to fabricate
polyelectrolyte multilayers using a plasmid DNA construct
(pEGFP-N1) encoding enhanced green fluorescent protein (EGFP) and
an alternate dipping procedure similar to that used in our past
studies to fabricate films using hydrolytically degradable cationic
polymers. [J. Zhang, L. S. Chua, D. M. Lynn, Langmuir 2004, 20,
8015; C. M. Jewell, J. Zhang, N. J. Fredin, D. M. Lynn, J Control
Release 2005, 106, 214; C. M. Jewell, J. Zhang, N. J. Fredin, M. R.
Wolff, T. A. Hacker, D. M. Lynn, Biomacromolecules 2006, 7,
2483.]
[0174] For these and all other experiments described below, films
were fabricated on planar silicon substrates to permit
characterization of film growth and erosion using ellipsometry.
[0175] FIG. 3 shows a plot of optical film thickness versus the
number of polyamine/DNA layers (referred to hereafter as
`bilayers`) deposited for films fabricated using either polymers
2a-d or unsubstituted PAH. Inspection of these data reveals that
the optical thicknesses of all films increased in a manner that was
linear or roughly linear with respect to the number of polymer/DNA
bilayers deposited. Further inspection, however, reveals large
differences in rates of film growth and final film thicknesses. For
example, films 8 bilayers thick fabricated using polymers 2a and 2b
were .about.110 nm thick, whereas films fabricated using polymers
2c and 2d were only .about.45 nm thick after the deposition of 8
bilayers. The thicknesses of films fabricated using less
substituted polymers 2c and 2d (which should have a higher
percentage of unsubstituted amine functionality and, thus, greater
PAH character) are, in general, closer in thickness to films
fabricated using PAH (.about.28 nm). These observations, when
combined, indicate that alkylation of the primary amines of PAH may
influence the ability of these polymers to form electrostatic
interactions with DNA (for example, by creating more sterically
hindered secondary or tertiary amines) or lead to differences in
the ionization or solution conformations of the polymers in ways
that influence the thickness of each adsorbed layer.
[0176] The stability of films fabricated from polymers 2a-d were
characterized in physiologically relevant media to determine
whether differences in the structures of these ester-functionalized
polymers could be exploited to provide control over rates of film
erosion and the release of DNA (e.g., FIG. 1, bottom). FIG. 4A
shows a plot of decreases in film thickness for DNA-containing
films fabricated using either PAH or polymers 2a-d upon incubation
in PBS at 37.degree. C. Inspection of the data in FIG. 4A reveals
that the thickness of films fabricated using unsubstituted PAH
(closed circles) does not decrease significantly for up to 250
hours. These results demonstrate that DNA-containing multilayers
fabricated from PAH are stable for at least 10 days under these
conditions, and provide a baseline from which to characterize
time-dependent changes in the stability of films fabricated from
polymers 2a-d.
[0177] The data in FIG. 4A reveal large differences in the
stabilities of films fabricated from polymers 2a-d that correlate
to differences in the amount of ester-functionalized side chains
incorporated into these materials. Films fabricated from
100%-substituted polymer 2a (open triangles) decreased in film
thickness very rapidly, and essentially completely, within the
first hour of incubation in PBS. The thicknesses of films
fabricated from 75%-substituted polymer 2b also decreased rapidly,
although not as completely, during the first hour (e.g., an
.about.80% decrease within the first hour), with the remainder of
the film eroding more slowly over an additional two day period.
[0178] These results demonstrate that films fabricated from these
two polymers are unstable and erode rapidly upon incubation in PBS.
The small differences in erosion profile noted above correlate, in
general, with differences in the number of side chains incorporated
into these polymers. Film erosion occurs sufficiently rapidly in
these cases, however, that it is difficult to interpret this
behavior solely in terms of side chain hydrolysis or the potential
`charge-shifting` nature of these ester-functionalized materials.
For example, rapid decreases in film thickness are also consistent
with film dissolution processes that could occur upon the immersion
of these ionically crosslinked assemblies in solutions of high
ionic strength (e.g., PBS).
[0179] To probe the nature of the film disassembly processes
further, we conducted an additional series of experiments using
films fabricated from DNA and amide-substituted polymer 3.
##STR00013##
[0180] Polymer 3 is an analog of polymer 2a with
dimethylamide-functionalized side chains (synthesized by the
conjugate addition of N,N-dimethylacrylamide to PAH; see below)
that do not hydrolyze readily under the conditions used here.
Inspection of the data in FIG. 4A reveals that films fabricated
using polymer 3 (open circles) are stable and do not decrease in
thickness for up to 10 days under these conditions. These data,
when combined with those above, provide support for the view that
the ester functionality in polymer 2 plays an important role in
governing the stability (or instability) of these materials in
PBS.
[0181] The remaining data in FIG. 4A correspond to films fabricated
from polymers 2c and 2d and demonstrate further that rates of film
erosion are influenced significantly by the number of ester side
chains incorporated into the polymer. Films fabricated from
25%-substituted polymer 2d (closed squares) were stable and did not
decrease in thickness substantially for over 10 days when incubated
in PBS buffer. However, films fabricated using 50%-substituted
polymer 2c (open squares) decreased in thickness gradually over a
period of 10 days under these conditions.
[0182] The physical erosion of these films also results in the
surface-mediated release of plasmid DNA into solution. FIG. 4B
shows a plot of solution absorbance (at 260 nm, the absorbance
maximum of DNA) versus time measured during the film erosion
experiments described above. The differences in the DNA release
profiles shown in FIG. 4B are consistent with the differences in
the erosion profiles shown in FIG. 4A, and demonstrate that it is
possible to control the rates at which DNA is released from
film-coated surfaces by changing the structure of the polyamines
used to fabricate the films. The differences in the final solution
absorbance values arising from films fabricated from polymers 2a
and 2b and films fabricated from less-substituted polymer 2c
correlate directly to differences in the amounts of DNA in these
films, and correlate to differences in the initial thicknesses of
these films (see FIG. 3). These differences in film erosion and DNA
release profiles can be exploited to design films with
architectures that permit control over the release of two DNA
constructs with separate and distinct release profiles.
[0183] Several recent reports have demonstrated that layer-by-layer
methods of assembly can be used to fabricate polyelectrolyte
multilayers composed of multiple different layers of multiple
different polyelectrolytes. [S N. Jessel, M. Oulad-Abdelghani, F.
Meyer, P. Lavalle, Y. Haikel, P. Schaaf, J. C. Voegel, Proc Natl
Acad Sci USA 2006, 103, 8618; T. Dubas, T. R. Farhat, J. B.
Schlenoff, J Am Chem Soc 2001, 123, 5368. J. Cho, F. Caruso,
Macromolecules 2003, 36, 2845. A. J. Nolte, M. F. Rubner, R. E.
Cohen, Langmuir 2004, 20, 3304. K. C. Wood, H. F. Chuang, R. D.
Batten, D. M. Lynn, P. T. Hammond, Proc Natl Acad Sci USA 2006,
103, 10207.]
[0184] Jessel et al. demonstrated recently that this general
approach could be used to fabricate DNA-containing multilayers that
provide control over the order in which two different DNA
constructs were expressed by attached cells (e.g., by depositing
two different plasmid DNA constructs at different depths within an
enzymatically degradable film). Zhang et al. also demonstrated that
hydrolytically degradable polyamines could be used to fabricate
films that provide control over the release of two plasmid
constructs into solution. [J. T. Zhang, S. I. Montanez, C. M.
Jewell, D. M. Lynn, Langmuir 2007, 23, 11139.]
[0185] This approach permitted measures of control over the
relative orders with which two plasmid constructs were released
(e.g., by controlling the relative orders with which they were
incorporated into the films), but it was not possible to fabricate
films that provided large differences in individual release
profiles. For example, it was not possible to fabricate films
capable of regulating the release of two different DNA constructs
with separate and mutually exclusive release profiles (that is,
films for which one DNA construct could be released largely before
the onset of the release of a second DNA construct). We have
demonstrated this type of control for release of DNA in exemplary
multilayers formed employing polymers 2a and 2c.
[0186] Films were fabricated using both the pEGFP-N1 plasmid
described above and a second plasmid construct (pDsRed-N1) encoding
red fluorescent protein (RFP). In the experiments described below,
we used films fabricated from polymers 2a and 2c and either (i)
plasmid DNA fluorescently labeled with Cy5 or Cy3 fluorescent dyes
(denoted pEGFP-Cy5 and pDsRed-Cy3; used to permit characterization
of the release profiles of each plasmid independently using
fluorimetry), or (ii) unlabeled plasmid DNA (to permit
characterization of gene expression in cell-based assays). Films
used in these experiments were fabricated layer-by-layer to contain
four bottommost layers containing polymer 2c (which released DNA
slowly in the above experiments) and four topmost layers containing
polymer 2a (which released DNA rapidly in the above
experiments).
[0187] Additionally two bilayers fabricated from polymer 2c and
sodium poly(styrene sulfonate) (SPS) were deposited as intermediate
layers between the plasmid-containing layers of these films. Films
having this general structure are denoted hereafter in the
following manner:
(2c/plasmid.sub.1).sub.4(2c/SPS).sub.2(2a/plasmid.sub.2).sub.4 (see
also the schematic illustration in FIG. 6).
[0188] FIG. 5 shows the results of an experiment conducted using a
film having the structure
(2c/pEGFP-Cy5).sub.4(2c/SPS).sub.2(2a/pDsRed-Cy3).sub.4. Inspection
of these data reveals that the pDsRed-Cy3 plasmid, deposited in the
topmost layers of the film, is released rapidly and completely
within the first 30 min of incubation in PBS (open triangles). In
contrast, the pEGFP-Cy5 plasmid, deposited in the bottommost layers
of the film, is released more slowly over a period of 48 hours
(open squares). The relative order in which these two plasmids are
released is consistent with the order in which they were deposited,
and the relative rates at which they are released are consistent
with the behaviors of polymer 2a (rapid release) and polymer 2c
(slow release) observed in the experiments described above.
Reversing the order in which the two different plasmids were
deposited during fabrication [i.e., using films having the general
structure (2c/pDsRed-Cy3).sub.4(2c/SPS).sub.2(2a/pEGFP-Cy5).sub.4]
resulted in a reversal of the order in which the DNA constructs
were released.
[0189] Additional consideration of the data in FIG. 5 reveals that
the release profiles for each DNA construct are distinct and almost
completely non-overlapping (e.g., only .about.15% of the pEGFP-Cy5
plasmid is released during the time required for all of the
pDsRed-Cy3 plasmid to be released). These results are believed to
arise from the large differences in the release profiles that can
be achieved using polymers 2a and 2c.
[0190] These results also indicate that a relatively low level of
physical interpenetration may exist among the layers in the topmost
and bottommost portions of these films. Additional delay in the
onset of the release of the plasmid located in the bottommost
layers of these films can be obtained by manipulating the number or
structure of the intermediate layers deposited between the
DNA-containing layers..sup.[17,34]
[0191] We conducted another set of experiments using films having
the structure (2c/pEGFP).sub.4(2c/SPS).sub.2(2a/pDsRed).sub.4 (that
is, films identical to those described above, but fabricated using
plasmid that was not fluorescently labeled) to characterize the
functional integrity of released DNA and determine whether the
differences in the release profiles shown in FIG. 5 could also be
observed as differences in EGFP and RFP expression profiles in
cells. FIG. 6 shows a series of fluorescence micrographs of COS-7
cells 48 hours after treatment with samples of plasmid DNA
collected at five predetermined time points during the erosion of
these films.
[0192] Inspection of the data in the left column of FIG. 6 (red
fluorescence channel) reveals high levels of red fluorescence in
cells treated with samples of DNA collected after 30 min of
incubation. Further inspection reveals little red fluorescence in
cells treated with DNA collected at subsequent time points. These
data demonstrate that the pDsRed plasmid released from these films
is released in a form that remains transcriptionally active, and
they provide an additional indication that essentially all of the
pDsRed located in the topmost layers of the film is released within
the first 30 min of incubation. The right column of FIG. 6 (green
fluorescence channel) shows images of the same cells shown in the
left column. These images demonstrate that significant levels of
EGFP expression are not observed in cells treated with samples of
DNA collected at 30 min, but that the number of cells expressing
EGFP increases throughout the remainder of the experiment. These
temporal differences in the expression of EGFP and RFP are
consistent with the results shown in FIG. 5 and demonstrate that it
is possible to exploit the structures and properties of polymer 2
to design films that permit control over the release of two
different DNA constructs with release profiles that are distinct
and essentially non-overlapping.
[0193] This work provides an approach to the fabrication of
ultrathin polyelectrolyte multilayers that provides temporal
control over the release of two different DNA constructs from
surfaces. The addition of ester-functionalized side chains to
poly(allylamine) provides control over the stability of
DNA-containing multilayers in aqueous environments. By control over
the number of ester-functionalized side chains added to the
polymer, it is possible to design films that release DNA rapidly,
slowly, or that are stable and do not release DNA upon incubation
in physiologically relevant media. These differences in film
erosion can be exploited to design multilayers with architectures
that provide control over the release of two or more different
plasmid constructs with distinct and largely non-overlapping
release profiles. Such control has been difficult to achieve using
conventional methods for the incorporation of DNA into thin films
and coatings. We and others have demonstrated in past reports that
polyelectrolyte multilayers fabricated from DNA can be used to
promote localized and surface-mediated cell transfection..sup.[23]
In this context, the approach reported here contributes to the
development of thin films and coatings capable of regulating the
localized release of well-defined quantities of multiple different
DNA constructs (or other agents) of interest in a broad range of
fundamental and applied contexts
[0194] Materials. Test grade n-type silicon wafers were purchased
from Si-Tech, Inc. (Topsfield, Mass.). Poly(allylamine
hydrochloride) (PAH, MW.apprxeq.60,000) was obtained from Alfa
Aesar Organics (Ward Hill, Pa.). Sodium poly(styrene sulfonate)
(SPS, MW=70,000), methyl acrylate, and N,N-dimethylacrylamide were
obtained from Aldrich Chemical Co. (Milwaukee, Wis.). Plasmid DNA
[pEGFP-N1 or pDsRed2-N1 (4.7 kb, >95% supercoiled)] was
purchased from Elim Biopharmaceuticals, Inc. (San Francisco,
Calif.). Cy3 and Cy5 Label-IT nucleic acid labeling kits were
purchased from Mirus (Madison, Wis.). All commercial materials were
used as received without further purification unless otherwise
noted. Deionized water (18 M.OMEGA.) was used for washing steps and
to prepare all polymer solutions. PBS buffer was prepared by
diluting commercially available concentrate (EM Science) and
adjusting the pH to 7.4 with 1.0 M HCl or NaOH. All buffers and
polymer solutions were filtered through a 0.2-.mu.m membrane
syringe filter prior to use unless otherwise noted.
[0195] General Considerations. .sup.1H and .sup.13C nuclear
magnetic resonance (NMR) spectra were recorded on Bruker AC+ 300
(300.135 MHz) and Varian UNITY 500 (499.896 MHz) spectrometers.
Chemical shift values are given in ppm and are referenced with
respect to residual protons from solvent. Attenuated total
reflectance infrared spectroscopy data were collected on a Bruker
TENSOR 27 FTIR instrument (Billerica, Mass.) outfitted with an ATR
transmission cell from PIKE Technologies (Madison, Wis.). Silicon
substrates (e.g., 0.5.times.2.0 cm.sup.2) used for the fabrication
of multilayered films were cleaned with methylene chloride,
ethanol, methanol, and deionized water, and dried under a stream of
filtered compressed air. Surfaces were then activated by etching
with oxygen plasma for 5 min (Plasma Etch, Carson City, Nev.) prior
to film deposition. The optical thicknesses of films deposited on
silicon substrates were determined using a Gaertner LSE
ellipsometer (632.8 nm, incident angle=70.degree.). Data were
processed using the Gaertner Ellipsometer Measurement Program.
Relative thicknesses were calculated assuming an average refractive
index of 1.577 for the multilayered films. Thicknesses were
determined in at least five different standardized locations on
each substrate and are presented as an average (with standard
deviation) for each film. All films were dried under a stream of
filtered compressed air prior to measurement.
[0196] UV-visible absorbance values for phosphate-buffered saline
(PBS) solutions used to determine film release kinetics were
recorded on a Beckman Coulter DU520 UV-vis spectrophotometer
(Fullerton, Calif.). Absorbance values were recorded at a
wavelength of 260 nm (the absorbance maximum of DNA). Fluorescence
measurements of solutions used to erode multilayered films
fabricated from DNA labeled with Cy3 and Cy5 fluorescent dyes were
made using a Fluoromax-3 fluorimeter (Jobin Yvon, Edison, N.J.).
Fluorescence microscopy images used to evaluate the expression of
enhanced green fluorescent protein (EGFP) or red fluorescent
protein (RFP) in cell transfection experiments were recorded using
an Olympus IX70 microscope and were analyzed using the Metavue
version 4.6 software package (Universal Imaging Corporation). Image
acquisition settings were identical for all samples, using an
exposure time of 200 ms, a gain of +0.25, and a binning of two.
Data were stored in single channel, 12-bit TIF format. Additional
image processing was limited to false coloring and scaling.
[0197] Synthesis of Ester-Functionalized PAH (Polymer 2). The
conjugate addition of PAH to methyl acrylate was performed using a
protocol similar to that reported previously for the synthesis of
ester-functionalized linear poly(ethylene imine) (LPEI) [24]. PAH
(550 mg) was dissolved in methanol (.about.5 wt % in methanol) and
1.1 mL of a sodium methoxide solution (35 wt % in methanol) was
added. The resulting reaction mixture was stirred for 4 hr at
45.degree. C., precipitated NaCl was removed by filtration, and
methyl acrylate was added. The amount of methyl acrylate added was
varied (e.g., from 0.5 to 2.2 equivalents relative to the molar
amount of amine functionality in PAH) to achieve desired mole
percent substitutions. Reaction mixtures were stirred at room
temperature for two hours (for polymers synthesized at low
acrylate/amine ratios) or up to 48 hours (for polymers synthesized
at higher acrylate/amine ratios). Reactions requiring longer
reaction times were monitored to prevent the formation of amide
crosslinks resulting from potential reactions between amines and
the ester functionality of methyl acrylate using attenuated total
reflectance infrared spectroscopy. One equivalent of HCl was added
to the reaction mixture, and the resulting reaction product was
concentrated by rotary evaporation. The final product was dissolved
in a mixture of dichloromethane and methanol (v/v=9:1) and
precipitated into hexanes. The isolated material was dried under
vacuum to yield the desired product as a white solid in near
quantitative yield. Representative .sup.1H NMR data for a polymer
with 100% substitution: (D.sub.2O) .delta. (ppm)=1.6 (br, 2H); 2.1
(br, 1H); 2.8-3.3 (br, 8H); 3.5 (br, 2H), 3.72 (s, 6H).
[0198] Synthesis of Amide-Functionalized PAH. The conjugate
addition of PAH to N,N-dimethylacrylamide was performed using a
protocol similar to that reported previously for the synthesis of
amide-functionalized LPEI [24] and conducted in analogy to the
synthesis of polymer 2 above. Representative .sup.1H NMR data for a
polymer with 100% substitution: (D.sub.2O) .delta. (ppm)=1.6 (br,
2H); 2.1 (br, 1H); 2.8-3.3 (br m, 20H); 3.4 (br, 2H).
[0199] Characterization of Side Chain Ester Hydrolysis. .sup.1H NMR
experiments used to characterize the kinetics of ester hydrolysis
for ester-functionalized PAH in physiologically relevant media were
conducted in the following general manner. Ester-functionalized
polymer (.about.10 mg) was dissolved in deuterated PBS buffer (0.6
mL, pH.about.7.4), 3-(trimethylsilyl)-1-propanesulfonic acid sodium
salt (.about.3 mg) was added as an internal standard, and the
resulting solution was placed in a glass NMR tube. The NMR tube was
placed in a 37.degree. C. incubator and removed periodically for
analysis by .sup.1H NMR spectroscopy. The disappearance of the
methyl ester resonance at 3.72 ppm was monitored and integrated
versus the trimethylsilyl protons of the internal standard.
[0200] Preparation of Polyelectrolyte Solutions. Solutions of
Cationic Polymers used for dipping (10 mM with respect to the MW of
the polymer repeat unit) were prepared in 18 M.OMEGA. water and pH
was adjusted to .about.5 using 1N NaOH. Solutions of SPS (20 mM
with respect to the MW of the polymer repeat unit) were prepared in
18 M.OMEGA. water. DNA solutions (1 mg/mL) used for the deposition
of polymer/DNA layers were prepared in sodium acetate buffer (100
mM, pH=5) and were not filtered prior to use.
[0201] Fabrication of Multilayered Films. Multilayered films were
fabricated on planar silicon substrates using an alternating
dipping procedure according to the following general protocol: (1)
Substrates were submerged in a solution of polycation for 5 min,
(2) substrates were removed and immersed in an initial water bath
for 1 min followed by a second water bath for 1 min, (3) substrates
were submerged in a solution of polyanion for 5 min, and (4)
substrates were rinsed in the manner described above. This cycle
was repeated until the desired number of polycation/polyanion layer
pairs (typically eight) had been deposited. For experiments
designed to characterize film growth profiles by ellipsometry,
films were dried after every two cycles of the above procedure
using filtered compressed air prior to measurement. Films to be
used in erosion and release experiments were either used
immediately after fabrication or dried under a stream of filtered
compressed air and stored in a vacuum desiccator until use. All
films were fabricated at ambient room temperature.
[0202] Characterization of Film Erosion and Release Kinetics.
Experiments designed to investigate film erosion and release
kinetics were performed in the following general manner:
Film-coated substrates were placed in a plastic UV-transparent
cuvette and 1.0 mL of PBS (pH=7.4, 137 mM NaCl) was added to cover
the film-coated portion of the substrate. The samples were
incubated at 37.degree. C. and removed at predetermined intervals
for characterization by ellipsometry. Films were rinsed under
deionized water and dried under a stream of filtered compressed air
prior to measurement. Values of optical film thickness were
determined in at least four different predetermined locations on
the substrate by ellipsometry and the samples were returned
immediately to the buffer solution. For experiments designed to
monitor the concentration of DNA in solution, UV absorbance
readings were made using the solution used to incubate the sample
(at 260 nm, the absorbance maximum of DNA). For experiments in
which fluorescently labeled DNA was used, changes in the
concentration of DNA in solution were monitored by fluorimetry. For
release experiments designed to produce samples of DNA suitable for
use in cell transfection experiments, erosion experiments were
conducted as described above with the following exceptions: at each
predetermined time interval substrates were removed from the
buffer, placed into a new cuvete containing fresh PBS, and the
original DNA-containing solution was stored for use in transfection
experiments.
[0203] Cell Transfection Assays. COS-7 cells were grown in 96-well
plates at an initial seeding density of 12,000 cells/well in 200 mL
of growth medium (90% Dulbecco's modified Eagle's medium, 10% fetal
bovine serum, penicillin 100 units/mL, streptomycin 100 mg/mL).
Cells were grown for 24 h, at which time 50 mL of a Lipofectamine
2000 (Invitrogen, Carlsbad, Calif.) and plasmid mixture was added
directly to the cells according to the general protocol provided by
the manufacturer. The Lipofectamine 2000/plasmid transfection
milieu was prepared by mixing 25 mL of the plasmid solution
collected at each time point during release experiments (arbitrary
concentrations but constant volumes) with 25 mL of diluted
Lipofectamine 2000 reagent (24 mL stock diluted into 976 mL of
water). Fluorescence images were taken after 48 h using an Olympus
IX70 microscope and analyzed using the Metavue version 4.6 software
package (Universal Imaging Corporation).
Example 2
Preparation of Polyelectrolyte Multilayers for Extended Long-Term
Release of Nucleic Acid
[0204] Side-chain functionalized polymer 5 was synthesized by the
reaction of 3-dimethylamino-1-propanol with poly
(2-vinyl-4,4-dimethyl azlactone) (4, Mn .about.50,000; Scheme 1).
This general approach permits conjugation of tertiary
amine-functionalized side chains to a poly(acrylamide) backbone
through a hydrolysable ester bond. Polymer 5 is a weak
polyelectrolyte; it is soluble in aqueous media and behaves as a
cationic polymer by virtue of protonation of pendant tertiary
amines. As illustrated in Scheme 1 hydrolysis of the ester bonds in
the side chains of polymer 5 leads to gradual loss of amine
functionality and the introduction of anionic carboxylate
functionality. Thus, polymer 5 is capable of transforming gradually
from a polymer that is completely positively charged to a polymer
that is completely negatively charged upon complete side chain
hydrolysis.
##STR00014##
.sup.1H NMR spectroscopy was used to characterize the loss of ester
functionality in solutions of polymer 5 as a function of time upon
incubation in phosphate buffer (pH=7.2; 37.degree. C.). The results
of these experiments demonstrate that side chain hydrolysis occurs
slowly in physiologically relevant media (t1/2 .about.200 days; see
FIG. 7). Incomplete or partial hydrolysis of the side chains in
polymer 5 would lead to a polymer containing both cationic and
anionic side chains and that, in general, the overall net charge of
these polymers would depend upon additional environmental factors
such as pH and ionic strength.
[0205] Polymer 5 was used to fabricate multilayered films using a
plasmid DNA construct (pEGFP-N1) encoding enhanced green
fluorescent protein (EGFP). All films used in these initial studies
were deposited on planar silicon substrates to permit
characterization of film thicknesses and growth profiles using
ellipsometry FIG. 8 shows a plot of the optical thickness of films
versus the number of polymer 5/DNA layers (hereafter referred to as
`bilayers`) deposited. Film thickness increased in a nonlinear
manner for the first three bilayers and then, subsequently, as a
linear function of the number of bilayers deposited, resulting in
films .about.100 nm thick after the deposition of 8 bilayers.
[0206] On the basis of these optical measurements, the average
thickness of a polymer 2/DNA bilayer in these films was .about.12.5
nm. Characterization of these films by atomic force microscopy
(AFM) revealed the surfaces of these assemblies to be rough (RRMS
.about.47 nm; data not shown). The thicknesses and surface
morphologies of these films are similar to those reported in past
studies for the assembly of multilayered films using plasmid DNA
and a variety of other cationic polymers. [J. Blacklock, H. Handa,
D. Soundara Manickam, G. Mao, A. Mukhopadhyay, D. Oupicky,
Biomaterials 2007, 28, 117; J. Chen, S. Huang, W. Lin, R. Zhuo,
Small 2007, 3, 636; N. J. Fredin, J. Zhang, D. M. Lynn, Langmuir
2005, 21, 5803.]
[0207] Additional experiments were performed to determine whether
assemblies fabricated from polymer 5 and plasmid DNA could erode
and release DNA when incubated in aqueous media. FIG. 9A (closed
diamonds) shows a plot of solution absorbance (at 260 nm, the
absorbance maximum of DNA) as a function of time for a polymer
5/DNA film .about.80 nm thick incubated in PBS at 37.degree. C.
[0208] These data demonstrate that DNA is released into solution
over a period of 90 days. On the basis of these absorbance data,
the amount of DNA incorporated into a film .about.80 nm thick was
estimated to be .about.4.8 .mu.g/cm.sup.2. Further inspection of
this release profile reveals the presence of a lag phase of
.about.25 days prior to the release of measurable amounts of DNA
into solution. This behaviour contrasts significantly to that of
polyamine/DNA films fabricated from hydrolytically or enzymatically
degradable polyamines, for which DNA is generally observed to be
released immediately upon exposure of film to aqueous environments
or enzymes (and often with an initial burst of DNA release). [J.
Zhang, L. S. Chua, D. M. Lynn, Langmuir 2004, 20, 8015; K. F. Ren,
J. Ji, J. C. Shen, Biomaterials 2006, 27, 1152; C. M. Jewell, J.
Zhang, N. J. Fredin, M. R. Wolff, T. A. Hacker, D. M. Lynn,
Biomacromolecules 2006, 7, 2483.] The presence of a lag phase in
this current system provides insight into possible molecular level
processes that may contribute to the extended release profiles of
these materials.
[0209] FIG. 9B (closed diamonds) shows a plot of film thickness
versus time corresponding to the erosion profile for the film shown
in FIG. 9A. Film thickness does not decrease significantly over the
first .about.25 days. This period of apparent film stability
corresponds closely to the lag phase in the DNA release profile
shown in FIG. 9A. Film thickness decreases in a nearly linear
manner upon further incubation, corresponding to the period of time
over which DNA is observed to be released into solution.
Characterization of the surfaces of these films during erosion by
AFM revealed changes in surface morphologies from films that were
initially rough (RRMS .about.40 nm; as described above) to surfaces
that were smooth and uniform (RRMS .about.3 nm) over a period of
.about.20 days. This behaviour varies considerably from the
behaviour of multilayered films fabricated using plasmid DNA and
hydrolytically degradable poly(.beta.-amino ester)s, which undergo
dramatic changes in nanometer-scale surface structure upon
incubation in PBS. [N. J. Fredin, J. Zhang, D. M. Lynn, Langmuir
2005, 21, 5803; N. J. Fredin, J. Zhang, D. M. Lynn, Langmuir 2007,
23, 2273.]
[0210] These results demonstrate that polymer 5 can be used to
fabricate ultrathin films that erode and release plasmid DNA over
long periods of time. This behavior is believed to result from the
gradual hydrolysis of the side chains in polymer 5 which is
supported, in part, by the solution-phase side-chain hydrolysis
experiments discussed above.
[0211] Polymer 6 was synthesized by the reaction of
3-dimethylamino-1-propylamine with polymer 4. Polymer 6 has a
molecular weight, polydispersity, and chemical structure that is
identical to that of polymer 5, with the exception that the
tertiary amine functionality of the side chain is linked to the
backbone of the polymer through an amide linkage that does not
hydrolyze readily in physiologically relevant media.
##STR00015##
Amide-functionalized polymer 6 was used to fabricate DNA containing
films on silicon substrates using a procedure identical to that
described above for polymer 5. The growth profiles of polymer 6/DNA
films were similar that of polymer 5/DNA films (data not shown).
However, striking differences were observed in stability of films
fabricated from polymers 5 and 6 when films were incubated in
PBS.
[0212] As shown in FIGS. 9A and 9B (closed squares), films
fabricated from polymer 6 did not decrease in thickness or release
measurable amounts of DNA into solution for periods of up to 90
days. The results of these experiments demonstrate that replacement
of the ester functionality in polymer 5 with amide functionality
leads to assemblies that do not erode or release DNA under
otherwise identical conditions. These results provide strong
support that the erosion and release of DNA from films fabricated
from polymer 5 results from the hydrolysis of the side chains of
polymer 5, and not from other factors (such as changes in pH or
ionic strength) that could arise during the incubation of these
assemblies.
[0213] The hydrolysis of the side chains of polymer 5 should result
in a change in the net charge of the polymer and, as a result, a
change in the nature of electrostatic interactions within an
ionically crosslinked film. The results indicate that such changes
in the strength of these ionic interactions are sufficient to
disrupt these films and promote the release of DNA. The erosion and
release of DNA from films fabricated from polymer 5 occurs over
periods of time .about.55 times longer than films fabricated from
hydrolytically or enzymatically degradable polymers (under
otherwise similar conditions). [J. Zhang, L. S. Chua, D. M. Lynn,
Langmuir 2004, 20, 8015; K. F. Ren, J. Ji, J. C. Shen, Biomaterials
2006, 27, 1152; C. M. Jewell, J. Zhang, N. J. Fredin, M. R. Wolff,
T. A. Hacker, D. M. Lynn, Biomacromolecules 2006, 7, 2483.]
[0214] One possible explanation for these differences is that the
hydrolysis of the side chains in polymer 5 occurs slowly in PBS (as
noted above). However, we also considered fundamental differences
in the structures of these cationic polymers that could lead to
such large differences in film behaviour. For example, for films
fabricated using hydrolytically or enzymatically degradable
cationic polymers, mechanisms of film erosion and DNA release
involve polymer chain backbone scission. In assemblies fabricated
from these degradable polymers, the hydrolysis of a single bond in
the backbone of a polymer chain can result in a dramatic change in
the molecular weight of the polymer and, as a result, a significant
reduction of the stability of a film. By contrast, the backbone of
polymer 5 is not degradable--the hydrolysis of a single ester bond
in the side chain reduces the net charge of a polymer chain by two,
but the polymer chain itself is not cleaved. As such, films
fabricated from polymer 5 would likely remain stable in
physiological media longer than films fabricated from degradable
polyamines, and erode or release DNA only after a threshold number
of side chains esters are cleaved. This view is supported by the
observation of lag phases in the release and erosion profiles shown
in FIGS. 9A and 9B.
[0215] Additionally, a consideration important with respect to the
application of these materials to promote localized or
surface-mediated cell transfection is the structural and functional
integrity of the plasmid DNA that is released. Cell transfection
experiments using samples of DNA collected at various times during
the erosion of a polymer 5/DNA film and a commercially available
cationic lipid transfection agent. Fluorescence micrographs of
COS-7 cells were obtained 48 h after treatment with samples of DNA
collected over three periods ranging from .about.16 to 27 days, 48
to 59 days, or 70 to 80 days. These micrographs demonstrated that
the DNA released over these extended time periods remained capable
of mediating high levels of expression of EGFP in mammalian cells.
The structural integrity of the DNA released over these time
periods was also examined using agarose gel electrophoresis. These
experiments demonstrated that a significant fraction of DNA was
released as supercoiled DNA (e.g., from 30% to 50%), with the
remainder being released in an open circular topology. These
results contrast significantly with those of past studies of the
release of DNA from multilayered assemblies fabricated from
degradable poly(.beta.-amino ester)s, for which DNA is released
almost entirely in an open circular form. [J. Zhang, L. S. Chua, D.
M. Lynn, Langmuir 2004, 20, 8015.]
[0216] Characterization of solutions of released DNA by dynamic
light scattering demonstrated the presence of aggregates ranging in
size from .about.100 to 600 nm. The zeta potentials of these
aggregates were measured to be negative (-11.3 mV). However, these
values were less negative than zeta potentials measured for
solutions of naked plasmid DNA (-29.2 mV). These results indicated
that the DNA released from polymer 5/DNA films may be released in a
form that is at least partially associated with polymer 5.
[0217] Materials. Poly(2-vinyl-4,4-dimethylazlactone) (polymer 4,
Mn=49,800, PDI=4.3) is prepared by art known methods.
3-Dimethylamino-1-propanol, 3-dimethylamino-1-propylamine, and 1,8
diazabicyclo[5.4.0]undec-7-ene (DBU) were obtained from Acros
Organics. Sodium acetate buffer was purchased from Aldrich Chemical
Company (Milwaukee, Wis.). Test grade n-type silicon wafers were
purchased from Si-Tech, Inc. (Topsfield, Mass.). Phosphate-buffered
saline (PBS) was prepared by dilution of commercially available
concentrate (EM science, Gibbstown, N.J.). Plasmid DNA [pEGFP-N1
(4.7 kb), >95% supercoiled] was obtained from Elim
Biopharmaceuticals, Inc. (San Francisco, Calif.). All materials
were used as received without further purification unless noted
otherwise. Deionized water (18 M.OMEGA.) was used for washing steps
and to prepare all buffer and polymer solutions. Compressed air
used to dry films and coated substrates was filtered through a 0.4
.mu.m membrane syringe filter. General Considerations. .sup.1H NMR
spectra were recorded on a Bruker AC+ 300 spectrometer. Chemical
shift values are reported in ppm and are referenced to residual
protons from solvent. Silicon substrates (e.g., 0.5.times.2.0 cm)
used for the fabrication of multilayered films were cleaned with
acetone, ethanol, methanol, and deionized water, and dried under a
stream of filtered compressed air. Surfaces were then activated by
etching with an oxygen plasma for 5 minutes (Plasma Etch, Carson
City, Nev.) prior to film deposition. The optical thicknesses of
films deposited on silicon substrates were determined using
air-dried films and a Gaertner LSE ellipsometer (632.8 nm, incident
angle=70.degree.). Data were processed using the Gaertne
Ellipsometer Measurement Program. Relative thicknesses were
calculated assuming an average refractive index of 1.58 for the
multilayered films. Thicknesses were determined in at least four
different standardized locations on each substrate and are
presented as an average (with standard deviation) of independent
measurements made on three separate films. UV-visible absorbance
values for PBS solutions used to determine film release kinetics
were recorded on a Beckman Coulter DU520 UV/vis Spectrophotometer
(Fullerton, Calif.). Film topography and surface roughness of
air-dried films were obtained from height data imaged under air in
tapping mode on a Nanoscope Multimode atomic force microscope
(Digital Instruments, Santa Barbara, Calif.). Silicon cantilevers
with a spring constant of 40 N/m and a radius of curvature of less
than 10 nm were used (model NSC15/Al BS, MikroMasch USA, Inc.,
Portland, Oreg.). For each sample, at least two different scans
were obtained at randomly chosen points near the center of the film
at each time point. Height data were flattened using a 2nd-order
fit. Root-mean squared surface roughness (Rrms) was calculated over
the scan area using the Nanoscope.RTM. IIIa software package
(Digital Instruments, Santa Barbara, Calif.).
[0218] Synthesis of Polymer 5 and 6. Polymers 5 and 6 were prepared
by reacting poly(2-vinyl-4,4'-dimethylazlactone) (4) with hydroxyl
or primary amine functionalized nucleophiles. [23 Jewell and Lynn
2008] For the synthesis of polymer 5: polymer 4 (2 mmol),
3-dimethylamino-1-propanol (3 mmol), and DBU (0.2 mmol) were
weighed into a vial and dissolved in THF (2.0 mL). The reaction
mixture was sealed, heated to 60.degree. C., and stirred for 24
hrs. The resulting reaction products were concentrated in vacuo and
precipitated into a hexane and acetone mixture (1:1, v/v)
containing 2 mmol of HCl. The precipitate was then dissolved in
methanol and reprecipitated twice more. The final product was dried
under vacuum to yield off-white flakes. .sup.1H NMR data for
polymer 5: (D.sub.2O, 300.135 MHz) .delta. (ppm)=4.23 (br t, 2H);
3.15 (br, t, 2H); 2.80 (br m, 7H); 2.10 (br m, 2H); 1.50 (br m,
8H).
[0219] For the synthesis of polymer 6: polymer 4 (0.7 mmol) and
3-dimethylamino-1-propylamine (1.1 mmol) were weighed into a vial
and dissolved in THF (2.0 mL) and the mixture was subsequently
heated to 50.degree. C. in an oil bath. After 7 hours, the
resulting reaction products were concentrated in vacuo, dissolved
in methanol, and precipitated into a hexane and acetone mixture
(1:1, v/v) containing 1.5 mmol of HCl. The precipitate was isolated
by centrifugation, dissolved in methanol, and reprecipitated twice
more. The final product was dried under vacuum to yield light
yellow flakes. .sup.1H NMR data for polymer 6: (D.sub.2O, 300.135
MHz) .delta. (ppm)=3.20 (br m, 5H); 2.89 (s, 6H); 1.95 (br m, 4H);
1.52 (br m, 6H).
[0220] Characterization of Kinetics of Ester Hydrolysis. .sup.1H
NMR experiments designed to characterize the loss of ester
functionality in polymer 5 in aqueous solution were performed in
the following manner. Polymer 2 (10 mg) was dissolved in deuterated
phosphate buffer (1.0 mL, 0.5 M, pH=7.2).
3-(trimethylsilyl)-1-propanesulfonic acid sodium salt (2 mg) was
added as an internal standard, and this solution was transferred to
a glass NMR tube. The NMR tube was incubated at 37.degree. C. and
removed periodically for analysis by .sup.1H NMR spectroscopy. The
change of the resonance corresponding to the methylene protons
adjacent to the ester functionality (at 4.2 ppm) to a resonance at
3.7 ppm after hydrolysis was monitored, and the extent of
hydrolysis was determined by integrating these signals versus the
trimethylsilyl protons of the internal standard.
[0221] Fabrication of Multilayered Films. Solutions of polymers 5
and 6 (5 mM with respect to the molecular weight of polymer repeat
units) and DNA (1 mg/ml) used for dipping were prepared in sodium
acetate buffer (100 mM, pH=5.1). Multilayered films were fabricated
on planar silicon substrates manually using an alternating dipping
procedure according to the following general protocol: 1)
Substrates were submerged in a solution of polyamine for 5 minutes,
2) substrates were removed and immersed in an initial water bath
for 1 minute followed by a second water bath for 1 minute, 3)
substrates were submerged in a solution of DNA for 5 minutes, and
4) substrates were rinsed in the manner described above. This cycle
was repeated until the desired number of polyamine/DNA bilayers was
reached. Films were either used immediately or dried under a stream
of filtered, compressed air and stored in a vacuum dessicator until
use. All films were fabricated at ambient room temperature.
[0222] Characterization of Film Erosion and Release Kinetics.
Experiments designed to investigate film erosion and DNA release
kinetics were performed in the following general manner:
Film-coated substrates were placed in a plastic UV-transparent
cuvette and 1.0 mL of phosphate buffered saline (PBS, pH=7.4, 137
mM NaCl) was added to cover the film-coated portion of the
substrate. The samples were incubated at 37.degree. C. and removed
at predetermined intervals to be characterized by ellipsometry or
atomic force microscopy (AFM). Films were rinsed under deionized
water and dried under a stream of filtered compressed air prior to
measurement. Values were determined in at least four different
predetermined locations on the substrate by ellipsometry and the
sample was returned immediately to the buffer solution. For
experiments designed to monitor the concentration of DNA in the
solution, a UV absorbance reading at 260 nm was made on the
solution used to incubate the sample.
[0223] For plasmid release experiments designed to produce samples
for cell transfection experiments, erosion experiments were
conducted as above with the following exceptions: at each
predetermined time interval substrates were removed from the
incubation buffer, placed into a new cuvette containing fresh PBS,
and the original plasmid-containing solution was stored for
analysis.
[0224] Agarose Gel Electrophoresis Assays. Samples of plasmid DNA
collected from film erosion experiments were evaluated by loading
30 .mu.L of plasmid solution into 1% agarose gels (HEPES, 20 mM,
pH=7.2, 108V, 45 min). Samples were loaded on the gel with 2 .mu.L
of a loading buffer consisting of 50/50 glycerol water (v/v). DNA
bands were visualized by ethidium bromide staining, and relative
intensities of bands corresponding to supercoiled and open circular
DNA were determined using Image J. Assignment of bands was aided by
restriction enzyme digestion of recovered DNA samples by digestion
by Not I and by digestion by NotI and BamHI.
[0225] Cell Transfection Assays. COS-7 cells were grown in 96-well
plates at an initial seeding density of 15,000 cells/well in 200
.mu.L of growth medium (90% Dulbecco's modified Eagle's medium, 10%
fetal bovine serum, penicillin 100 units/mL, streptomycin 100
.mu.g/mL). Cells were grown for 24 hours, at which time the 50
.mu.l of a Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) and
plasmid mixture was added directly to the cells according to the
general protocol provided by the manufacturer. The Lipofectamine
2000/plasmid transfection milieu was prepared by mixing 25 .mu.l of
the plasmid solution collected at each time point during release
experiments (arbitrary concentrations but constant volumes) with 25
.mu.l of diluted Lipofectamine 2000 reagent (25 .mu.L stock diluted
into 975 .mu.L of water). Fluorescence microscopy images were
acquired after 48 hours using an Olympus IX70 microscope and
analyzed using the Metavue version 4.6 software package (Universal
Imaging Corporation).
[0226] While certain specific embodiments have been illustrated and
described, it should be understood that changes and modifications
can be made therein in accordance with ordinary skill in the art
without departing from the invention in its broader aspects as
defined in the following claims.
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