EP2029357A1 - Methods of making decomposable thin films of polyelectrolytes and uses thereof - Google Patents
Methods of making decomposable thin films of polyelectrolytes and uses thereofInfo
- Publication number
- EP2029357A1 EP2029357A1 EP07762359A EP07762359A EP2029357A1 EP 2029357 A1 EP2029357 A1 EP 2029357A1 EP 07762359 A EP07762359 A EP 07762359A EP 07762359 A EP07762359 A EP 07762359A EP 2029357 A1 EP2029357 A1 EP 2029357A1
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- EP
- European Patent Office
- Prior art keywords
- thin film
- polyelectrolyte
- poly
- polymer
- entity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/68—Polyesters containing atoms other than carbon, hydrogen and oxygen
- C08G63/685—Polyesters containing atoms other than carbon, hydrogen and oxygen containing nitrogen
- C08G63/6854—Polyesters containing atoms other than carbon, hydrogen and oxygen containing nitrogen derived from polycarboxylic acids and polyhydroxy compounds
Definitions
- LBL layer-by- layer
- the LBL approach is based on electrostatic attractions between polyelectrolytes and oppositely charged surfaces.
- a negatively charged substrate is first dipped in a polycation solution.
- Electrostatic attractions result in deposition of the polycation and a resulting reversal of surface charge (see Figure 1, step 1).
- the positively charged substrate is then submerged in a polyanion solution, resulting in deposition of the polyanion and restoration of the negative charge on the surface (see Figure 1, step 2).
- Repetition of these steps leads to the buildup of layers of alternating oppositely charged polyelectrolytes on the substrate surface.
- other factors and secondary interactions such as hydrophobicity, salt interactions, solvent quality, polymer concentrations, and deposition time may affect the multi-layer growth of the film (for a review of these factors, see Dubas and Schlenoff, Macromolecules 32:8153, 1999, the contents of which are incorporated herein by reference).
- the array of materials available for LBL assembly is broad, including synthetic polyelectrolytes, conducting polymers, dyes, and metal colloids, as well as a variety of biological species such as proteins, viruses, and DNA.
- Applications as diverse as conductive and light-emitting films, biologically-active surfaces, selective membranes, patterned films, and hollow multi-layer structures underscore the potential of the LBL technique (for a review of applications, see Hammond, Curr. Opin. Coll. Interface Sci. 3:32, 1998, the contents of which are incorporated herein by reference).
- multi-layer thin films designed to release incorporated or encapsulated compounds there remains a need in the art for thin film controlled release systems that function under physiological conditions.
- the invention is a decomposable thin film.
- the thin film includes a plurality of polyelectrolyte bilayers including a first polyelectrolyte layer having a first charge and a second polyelectrolyte layer having a second charge. At least a first portion of the polyelectrolyte bilayers include an entity selected from a biomolecule, a small molecule, and a bioactive agent. Decomposition of the thin film is characterized by sequential removal of at least a portion of the polyelectrolyte layers having the first charge and degradation of polyelectrolyte layers having the second charge and by release of the entity from the corresponding bilayer.
- the decomposable thin film further includes at least one polyelectrolyte bilaver through which the entity does not readily diffuse.
- the first polyelectrolyte layer and the second polyelectrolyte layer may be covalently cross-linked to one another within at least one of the polyelectrolyte bilayers.
- a plurality of the polyelectrolyte bilayers may be cross-linked.
- the thin film may include alternating polycationic and polyanionic layers and decomposition of the thin film may be characterized by hydro lytic degradation of at least a portion of the polycationic layers, polyanionic layers, or both.
- At least a portion of the first polyelectrolyte layers, the second polyelectrolyte layers, or both, may include a polymer including an anionic group, a cationic group, or both.
- the group may be incorporated into the backbone of the polymer, covalently attached to the backbone of the polymer, or covalently attached to a pendent group of the polymer. Degradation may be hydro lytic, thermal, enzymatic, photo lytic, or some combination of these.
- the at least one covalently cross-linked polyelectrolyte bilayer may prevent diffusion of the entity within the thin film.
- the invention is a method of encapsulating an entity within a thin film.
- the method includes disposing the entity within a thin film including a plurality of polyelectrolyte bilayer including a first polyelectrolyte layer having a first charge and a second polyelectrolyte layer having a second charge.
- the decomposable thin film includes at least one polyelectrolyte bilayer through which the entity does not readily diffuse.
- the method may further include covalently cross-linking at least one of the polyelectrolyte bilayers.
- the invention is a method of releasing an entity from a thin film.
- the method includes providing a decomposable thin film including a plurality polyelectrolyte bilayers including a first polyelectrolyte layer having a first charge and a second polyelectrolyte layer having a second charge.
- the entity is associated with at least one of the bilayers and the decomposable thin film includes at least one polyelectrolyte bilayer through which the entity does not readily diffuse.
- the method further includes placing the thin film in a medium in which at least a portion of the thin film decomposes via the substantially sequential removal of at least a portion of the polyelectrolyte layers having a first charge and degradation of polyelectrolyte layers having the second charge.
- the method may further include covalently cross-linking at least one of the polyelectrolyte bilayers.
- Animal refers to humans as well as non- human animals, including, for example, mammals, birds, reptiles, amphibians, and fish.
- the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig).
- An animal may be a transgenic animal.
- Associated with When two entities are “associated with” one another as described herein, they are linked by a direct or indirect covalent or non-covalent interaction.
- exemplary non-covalent interactions include hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc.
- Biomolecules refers to molecules (e.g., proteins, amino acids, peptides, polynucleotides, nucleotides, carbohydrates, sugars, lipids, nucleoproteins, glycoproteins, lipoproteins, steroids, etc.) whether naturally- occurring or artificially created (e.g. , by synthetic or recombinant methods) that are commonly found in cells and tissues.
- molecules e.g., proteins, amino acids, peptides, polynucleotides, nucleotides, carbohydrates, sugars, lipids, nucleoproteins, glycoproteins, lipoproteins, steroids, etc.
- biomolecules include, but are not limited to, enzymes, receptors, neurotransmitters, hormones, cytokines, cell response modifiers such as growth factors and chemotactic factors, antibodies, vaccines, haptens, toxins, interferons, ribozymes, anti-sense agents, plasmids, DNA, and RNA.
- Biocompatible The term “biocompatible”, as used herein is intended to describe materials that do not elicit a substantial detrimental response in vivo.
- Biodegradable As used herein, “biodegradable” polymers are polymers that degrade fully under physiological or endosomal conditions. In preferred embodiments, the polymers and biodegradation byproducts are biocompatible. Biodegradable polymers are not necessarily hydrolytically degradable and may require enzymatic action to fully degrade. "Degradation”: The phrase “degradation”, as used herein, relates to the cleavage of a covalent polymer backbone. Full degradation of a polymer breaks the polymer down to monomeric species.
- Endosomal conditions The phrase “endosomal conditions”, as used herein, relates to the range of chemical (e.g., pH, ionic strength) and biochemical (e.g., enzyme concentrations) conditions likely to be encountered within endosomal vesicles.
- chemical e.g., pH, ionic strength
- biochemical e.g., enzyme concentrations
- endosomal pH ranges from about 5.0 to 6.5.
- Hydrolytically degradable polymers are polymers that degrade fully in the sole presence of water. In preferred embodiments, the polymers and hydro lytic degradation byproducts are biocompatible. As used herein, the term “non-hydrolytically degradable” refers to polymers that do not fully degrade in the sole presence of water.
- physiological conditions relate to the range of chemical (e.g., pH, ionic strength) and biochemical (e.g., enzyme concentrations) conditions likely to be encountered in the intracellular and extracellular fluids of tissues.
- chemical e.g., pH, ionic strength
- biochemical e.g., enzyme concentrations
- Polyelectrolyte or “polyion”: The terms “polyelectrolyte” or “polyion”, as used herein, refer to a polymer which under some set of conditions (e.g., physiological conditions) has a net positive or negative charge. Polycations have a net positive charge and polyanions have a net negative charge. The net charge of a given polyelectrolyte or polyion may depend on the surrounding chemical conditions, e.g., on the pH.
- Polynucleotide refers to a polymer of nucleotides.
- a polynucleotide comprises at least three nucleotides. DNAs and RNAs are polynucleotides.
- the polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxy cytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified
- Polypeptide “peptide”, or “protein”: According to the present invention, a “polypeptide”, “peptide”, or “protein” comprises a string of at least three amino acids linked together by peptide bonds.
- the terms “polypeptide”, “peptide”, and “protein”, may be used interchangeably.
- Peptide may refer to an individual peptide or a collection of peptides.
- Inventive 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/ -dadgrp/Unnatstruct.gif, which displays structures of non-natural amino acids that have been successfully incorporated into functional ion channels) and/or amino acid analogs as are known in the art may alternatively be employed.
- non-natural amino acids i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain; see, for example, http://www.cco.caltech.edu/ -dadgrp/Unnatstruct.gif, which displays structures of non-natural amino acids that have been successfully incorporated into functional ion channels
- amino acid analogs as are known in the art may alternatively be employed.
- one or more of the amino acids in an inventive 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.
- 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.
- 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. None of the modifications should substantially interfere with the desired biological activity of the peptide.
- Polysaccharide “carbohydrate” or “oligosaccharide”: The terms “polysaccharide”, “carbohydrate”, or “oligosaccharide” refer to a polymer of sugars. The terms “polysaccharide”, “carbohydrate”, and “oligosaccharide”, may be used interchangeably. Typically, a polysaccharide comprises at least three sugars.
- the polymer may include natural sugars (e.g., glucose, fructose, galactose, mannose, arabinose, ribose, and xylose) and/or modified sugars (e.g., 2'-fluororibose, T- deoxyribose, and hexose).
- natural sugars e.g., glucose, fructose, galactose, mannose, arabinose, ribose, and xylose
- modified sugars e.g., 2'-fluororibose, T- deoxyribose, and hexose
- Small molecule As used herein, the term “small molecule” is used to refer to molecules, whether naturally-occurring or artificially created (e.g., via chemical synthesis), that have a relatively low molecular weight. Typically, small molecules are monomeric and have a molecular weight of less than about 1500 g/mol. Preferred small molecules are biologically active in that they produce a local or systemic effect in animals, preferably mammals, more preferably humans.
- the small molecule is a drug. Preferably, though not necessarily, the drug is one that has already been deemed safe and effective for use by the appropriate governmental agency or body. For example, drugs for human use listed by the FDA under 21 C.F.R. ⁇ 330.5, 331 through 361, and 440 through 460; drugs for veterinary use listed by the FDA under 21 C.F.R. ⁇ 500 through 589, incorporated herein by reference, are all considered acceptable for use in accordance with the present invention.
- Bioactive agents As used herein, “bioactive agents” is used to refer to compounds or entities that alter, inhibit, activate, or otherwise affect biological or chemical events.
- bioactive agents may include, but are not limited to, anti-AIDS substances, anti-cancer substances, antibiotics, immunosuppressants, anti-viral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinson substances, anti-spasmodics and muscle contractants including channel blockers, miotics and anti-cholinergics, anti-glaucoma compounds, anti-parasite and/or anti-protozoal compounds, modulators of cell-extracellular matrix interactions including cell growth inhibitors and anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNA or protein synthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal and non-steroidal anti-inflammatory agents
- SPS poly(styrene sulfonate)
- PAA poly(acrylic acid)
- LPEI linear polyethylene imine
- PDAC poly(diallyl dimethyl ammonium chloride)
- PAH poly(allylamine hydrochloride)
- PAZO is the azobenzene functionalized polymer poly ⁇ l-[4-(3-carboxy-4- hydroxyphenylazo) benzensulfonamido]-l,2 -ethanediyl ⁇ .
- Figure 1 is a schematic illustrating the construction of a thin film via layer-by- layer deposition of polyelectrolytes on a charged substrate;
- Figure 2 is a schematic illustrating the construction and decomposition of a thin film according to one embodiment of the invention by (A) layer-by-layer deposition of alternating polyanionic and polycationic layers and (B) degradation of polycationic layers and release of polyanionic components;
- Figure 3 depicts the chemical structure of exemplary hydro lyrically degradable polycations for the fabrication of a decomposable thin film
- Figure 4 depicts the chemical structure of exemplary non-degradable polyanions and polycations for the fabrication of a thin film according to one embodiment of this invention
- Figure 5 is a schematic illustrating entities (shown as black triangles), e.g., biomolecules or small molecules that are non-covalently associated with polycations of a thin film according to one embodiment of the invention
- Figure 6 is a schematic illustrating entities (shown as black triangles), e.g., biomolecules or small molecules that are covalently associated with polycations of a thin film according to one embodiment of the invention
- Figure 7 depicts the chemical structure of PAZO, an azobenzene functionalized photochromic polyanion;
- Figure 8 is a schematic illustrating the deposition of a decomposable thin film on a particulate template and construction of a decomposable hollow thin film microcapsule by dissolution of the template
- Figure 9 is a schematic illustrating the construction of a tunnel-like microstructure according to one embodiment of the invention by (A) deposition of an ionic or polar self- assembled monolayer (SAM), e.g., by micro-contact printing, (B) layer-by-layer deposition of a decomposable thin film, (C) flooding of the substrate with a non- degradable material, and (D) decomposition of the thin film
- Figure 10 depicts the chemical structure of Polymer 1 (Poly 1), a hydro lyrically degradable poly( ⁇ -amino ester);
- Figure 11 is a prof ⁇ lometry scan often bilayers of Poly 1/SPS deposited on ten precursor bilayers of LPEI/SPS;
- Figure 12 illustrates the thickness change as bilayers of Poly 1/SPS are deposited on ten precursor bilayers of LPEI/SPS;
- Figure 13 is a reflective FTIR scan often bilayers of Poly 1/SPS deposited on ten precursor bilayers of LPEI/SPS on a gold substrate (1734 cm "1 carboxyl stretch indicated);
- Figure 14 illustrates the decomposition in PBS buffer at pH 7.4 (at 37°C) of a thin film comprising Poly 1/SPS bilayers deposited on precursor bilayers of LPEI/SPS;
- Figure 15 illustrates the decomposition in TAE buffer at pH 8.3 (at 37°C) of a thin film comprising Poly 1/SPS bilayers deposited on precursor bilayers of LPEI/SPS;
- Figure 16 illustrates the thickness of six different thin films comprising Poly
- Figure 17 illustrates the decomposition in PBS buffer at pH 7.4 (at 37°C) of a thin film comprising Poly 1/DNA bilayers deposited on precursor bilayers of LPEI/SPS;
- Figure 18 is a schematic depicting exemplary strategies to construct physical barriers to control interlayer diffusion in multi-component films according to an exemplary embodiment
- Figure 19 is a graph showing how dextran sulfate (base layer, triangle) and heparin (surface layer, diamond)-loaded layers separated by a single, cross-linked layer of (PAH/PAA) according to an exemplary embodiment exhibit sequential release.
- A Fraction of mass released versus degradation time.
- B Fractional release rate versus time.
- Figure 20 is a graph illustrating that heparin (base layer, diamond) and dextran sulfate (surface layer, triangle)-loaded layers according to an exemplary embodiment, without dividing layers, sustain simultaneous release.
- A Fraction of mass released versus degradation time (error bars are small).
- B Fractional release rate versus time;
- Figure 21 is a graph illustrating that heparin (base layer, diamond) and dextran sulfate (surface layer, triangle)-loaded layers according to an exemplary embodiment, separated by (Polyl/SPS)20 degradable dividing layers, sustain simultaneous release.
- A Fraction of mass released versus degradation time (error bars are small).
- B Fractional release rate versus time;
- Figure 22 is a graph illustrating that heparin (base layer, diamond) and dextran sulfate (surface layer, triangle)-loaded layers, separated by (PDAC/SPS)50 non- degradable dividing layers according to an exemplary embodiment, sustain simultaneous release.
- A Fraction of mass released versus degradation time (error bars are small).
- B Fractional release rate versus time.
- Figure 23 is a graph illustrating that a single cross-linked according to an exemplary embodiment layer of (PAH/PAA) does not significantly delay the release of heparin. Release of heparin-loaded films coated with a single layer of (PAH/PAA) cross- linked for 45 min at 215° C (filled diamond) is compared with untreated heparin-loaded films (open diamonds);
- Figure 24 is a graph illustrating that dextran sulfate (base layer, triangle) and heparin (surface layer, diamond)-loaded layers according to an exemplary embodiment, without dividing layers, sustain simultaneous release.
- A Fraction of mass released versus degradation time (error bars are small).
- B Fractional release rate versus time;
- Figure 25 is a graph illustrating that dextran sulfate (base layer, triangle) and heparin (surface layer, diamond)-loaded layers according to an exemplary embodiment, separated by 50 (Polyl/SPS) degradable dividing layers, sustain simultaneous release.
- A Fraction of mass released versus degradation time (error bars are small).
- B Fractional release rate versus time;
- Figure 26 is a graph illustrating that dextran sulfate (base layer, triangle) and heparin (surface layer, diamond)-loaded layers according to an exemplary embodiment, separated by 50 (PDAC/SPS) non-degradable dividing layers, sustain simultaneous release.
- A Fraction of mass released versus degradation time.
- B Fractional release rate versus time;
- Figure 27 is a graph illustrating the normalized initial average release rate ( ⁇ g/h- cm 2 ) from base films according to an exemplary embodiment containing (A) dextran sulfate and (B) heparin coated with no separation layers (control), or with a single layer of (PAH/PAA) cross-linked at 215° C for variable times, non-degradable (PDAC/SPS), or degradable (Polyl/SPS).
- Initial average release rates were calculated from the average slope of the linear portion of the mass released versus time curve during the first 5O h (dextran sulfate) or 10 h (heparin) of degradation; Figure 28.
- a plot of FTIR absorbance versus number of thin film bilayers demonstrates layer-by-layer assembly of (polymer 1 /heparin) (diamond) and (polymer 1/dextran sulfate) (triangle) films exhibiting exponential and linear growth, respectively.
- Figure 29 illustrates degradation (square) and drug release (triangle) from single component films according to an exemplary embodiment (A) (polymer l/HEP)20. (B) (polymer l/DS)20; Figure 30 illustrates release from 20 (diamond), 50 (square), and 80 (triangle) bilayer films according to an exemplary embodiment containing (A) heparin (exponential) and (B) dextran sulfate (linear) with time (surface area normalized; error bars are small); and
- Figure 31 provides a schematic illustration a drug delivery film and release profiles for two drugs, drug 1 and drug 2, from a layer-by-layer thin film.
- the present invention provides a method for the gradual and controlled release of one or more entities from decomposable thin films.
- the decomposition is characterized by the substantially sequential degradation of at least a portion of the polyelectrolyte layers that make up the thin films.
- the degradation may be at least partially hydro lytic, at least partially enzymatic, at least partially thermal, and/or at least partially photolytic.
- the thin films are about 1 nm and about 100 ⁇ m thick, for example, between about 1 nm and about 100 nm thick, between about 100 nm and about 1 ⁇ m thick, between about 1 ⁇ m and about 10 ⁇ m thick, or between about 10 ⁇ m and about 100 ⁇ m thick.
- the released entities are structural polyelectrolyte components of the inventive films.
- One such embodiment of the invention is illustrated in Figure 2.
- the thin film is deposited on a substrate via layer-by-layer assembly as depicted in Figure 1.
- the thin film includes a plurality of alternating polyanionic and polycationic layers.
- the polycationic layers include a degradable polycation.
- the thin film is exposed to a degrading medium (e.g., intracellular fluid), whereupon the polycationic layers degrade and the polyanionic layers delaminate sequentially from the surface toward the substrate.
- the component polyanions of the exposed polyanionic layers are thus gradually and controllably released from the surface of the thin film.
- the released polyanions are biomolecules, for example, DNA molecules.
- the polycations and their degradation byproducts are preferably biocompatible. It will be appreciated that the roles of the two layers of the thin film can be reversed.
- the polyanionic layers include a degradable polyanion and the polycationic layers may include, for example, a polycationic protein.
- both the polycationic and polyanionic layers may both include degradable polyelectrolytes.
- a variety of materials can be used as substrates of the present invention such as, but not limited to, metals, e.g., gold, silver, platinum, and aluminum; metal-coated materials; metal oxides; plastics; ceramics; silicon; glasses; mica; graphite; hydrogels; and polymers such as polyamides, polyphosphazenes, polypropylfumarates, polyethers, polyacetals, polycyanoacrylates, polyurethanes, polycarbonates, polyanhydrides, polyorthoesters, polyhydroxyacids, polyacrylates, ethylene vinyl acetate polymers and other cellulose acetates, polystyrenes, poly( vinyl chloride), poly(vinyl fluoride), poly( vinyl imidazole), poly( vinyl alcohol), poly(ethylene terephthalate), polyesters, polyureas, polypropylene, polymethacrylate, polyethylene, poly(ethylene oxide)s and chlorosulphonated polyolefms; and combinations thereof.
- materials with an inherently charged surface are particularly attractive substrates for LBL assembly of a thin film.
- a range of methods are known in the art that can be used to charge the surface of a material, including but not limited to plasma processing, corona processing, flame processing, and chemical processing, e.g., etching, micro-contact printing, and chemical modification.
- plastics can be used as substrates, particularly if they have been chemically modified to present polar or charged functional groups on the surface.
- substrates can be primed with specific polyelectrolyte bilayers such as, but not limited to, LPEI/SPS, PDAC/SPS, PAH/SPS, LPEI/PAA, PDAC/PAA, and PAH/PAA bilayers, that form readily on weakly charged surfaces and occasionally on neutral surfaces.
- primer layers provide a uniform surface layer for further LBL assembly and are therefore particularly well suited to applications that require the deposition of a uniform thin film on a substrate that includes a range of materials on its surface, e.g., an implant or a complex tissue engineering construct.
- Any degradable polyelectrolyte can be used in a thin film of the present invention, including, but not limited to, hydrolytically degradable, biodegradable, thermally degradable, and photolytically degradable polyelectrolytes.
- Hydrolytically degradable polymers known in the art include for example, certain polyesters, polyanhydrides, polyorthoesters, polyphosphazenes, and polyphosphoesters.
- Biodegradable polymers known in the art include, for example, certain polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, poly(amino acids), polyacetals, polyethers, biodegradable polycyanoacrylates, biodegradable polyurethanes and polysaccharides.
- biodegradable polymers that may be used in the present invention include but are not limited to polylysine, poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), poly(lactide-co- caprolactone) (PLC), and poly(glycolide-co-caprolactone) (PGC).
- PLA poly(lactic acid)
- PGA poly(glycolic acid)
- PCL poly(caprolactone)
- PLA poly(lactide-co-glycolide)
- PLA poly(lactide-co-caprolactone)
- PLC poly(glycolide-co-caprolactone)
- PLC poly(glycolide-co-caprolactone)
- the anionic polyelectrolytes may be degradable polymers with anionic groups distributed along the polymer backbone.
- the anionic groups which may include carboxylate, sulfonate, sulphate, phosphate, nitrate, or other negatively charged or ionizable groupings, may be disposed upon groups pendant from the backbone or may be incorporated in the backbone itself.
- the cationic polyelectrolytes may be degradable polymers with cationic groups distributed along the polymer backbone.
- the cationic groups which may include protonated amine, quaternary ammonium or phosphonium derived functions or other positively charged or ionizable groups, may be disposed in side groups pendant from the backbone, may be attached to the backbone directly, or can be incorporated in the backbone itself.
- polyesters examples include poly(L-lactide-co-L-lysine) (Barrera et al. J. Am. Chem. Soc. 115:11010-11011, 1993; incorporated herein by reference), poly(serine ester) (Zhou et al. Macromolecules 23:3399-3406, 1990; which is incorporated herein by reference), poly(4-hydroxy-L-proline ester) (Putnam et al. Macromolecules 32:3658-3662, 1999.; Lim et al. J. Am. Chem. Soc. 121 :5633-5639, 1999; each of which is incorporated herein by reference), and more recently, poly[ ⁇ -(4- aminobutyl)-L-glycolic acid].
- poly( ⁇ -amino ester)s prepared from the conjugate addition of primary or secondary amines to diacrylates, are suitable for use with the invention.
- poly( ⁇ -amino ester)s have one or more tertiary amines in the backbone of the polymer, preferably one or two per repeating backbone unit.
- a co-polymer may be used in which one of the components is a poly( ⁇ -amino ester).
- Poly( ⁇ -amino ester)s are described in USSN 09/969,431, filed October 2, 2001, entitled “Biodegradable poly( ⁇ -amino esters) and uses thereof and Lynn et al., J. Am. Chem. Soc. 122:10761- 10768, 2000, the entire contents of both of which are incorporated herein by reference.
- Exemplary poly( ⁇ -amino ester)s are shown in Figure 3.
- Exemplary R groups include hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohvdroxvl.
- alkvlthioether amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiol groups.
- Exemplary linker groups A and B include carbon chains of 1 to 30 carbon atoms, heteroatom-containing carbon chains of 1 to 30 atoms, and carbon chains and heteroatom-containing carbon chains with at least one substituent selected from the group consisting of branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiol groups.
- the polymer may include, for example, between 5 and 10,000 repeat units.
- zwitterionic polyelectrolytes may be used. Such polyelectrolytes may have both anionic and cationic groups incorporated into the backbone or covalently attached to the backbone as part of a pendant group. Such polymers may be neutrally charged at one pH, positively charged at another pH, and negatively charged at a third pH.
- a film may be deposited by LBL deposition using dip coating in solutions of a first pH at which one layer is anionic and a second layer is cationic. If the film is put into a solution having a second different pH, then the first layer may be rendered cationic while the second layer is rendered anionic, thereby changing the charges on those layers.
- the LBL assembly of films may involve a series of dip coating steps in which the substrate is dipped in alternating polycationic and polyanionic solutions (see Figure 1). Additionally or alternatively, it will be appreciated that deposition of alternating polycationic and polyanionic layers may also be achieved by spray coating, brush coating, roll coating, spin casting, or combinations of any of these techniques.
- the composition of the polyanionic and polycationic layers can be fine-tuned to adjust the degradation rate of each layer within the film.
- the degradation rate of hydro lyrically degradable polyelectrolyte layers can be decreased by associating hydrophobic polymers such as hydrocarbons and lipids with one or more of the layers.
- the polyelectrolyte layers may be rendered more hydrophilic to increase their hydrolytic degradation rate.
- the degradation rate of a given layer can be adjusted by including a mixture of polyelectrolytes that degrade at different rates or under different conditions.
- the polyanionic and/or polycationic layers may include a mixture of degradable and non-degradable polyelectrolytes.
- non-degradable polyelectrolyte can be used with the present invention.
- exemplary non-degradable polyelectrolytes that could be used in thin films are shown in Figure 4 and include poly(styrene sulfonate) (SPS), poly(acrylic acid) (PAA), linear poly(ethylene imine) (LPEI), poly(diallyldimethyl ammonium chloride) (PDAC), and poly(allylamine hydrochloride) (PAH).
- SPS poly(styrene sulfonate)
- PAA poly(acrylic acid)
- LPEI linear poly(ethylene imine)
- PDAC poly(diallyldimethyl ammonium chloride)
- PAH poly(allylamine hydrochloride)
- the degradation rate may be fine-tuned by associating or mixing non-biodegradable, yet biocompatible polymers (polyionic or non- polyionic) with one or more of the polyanionic and/or polycationic layers.
- Suitable nonbiodegradable, yet biocompatible polymers are well known in the art and include polystyrenes, certain polyesters, non-biodegradable polyurethanes, polyureas, poly(ethylene vinyl acetate), polypropylene, polymethacrylate, polyethylene, polycarbonates, and poly(ethylene oxide)s.
- the composition of individual layers may be varied to tailor the degradation rate of various portions of the film.
- the upper layers of the film, closer to the surface may be adjusted to degrade faster than the layers of the film closer to the substrate, or vice versa.
- the degradation rate within the film may be varied cyclically (e.g., for periodic release).
- the upper layers of the film, closer to the surface may be adjusted to degrade under a first set of conditions (e.g., endosomal conditions) while the layers of the film that are closer to the substrate are adjusted to degrade under a second set of conditions (e.g., physiological conditions).
- the various layers of the film may be modified to control the diffusion of materials within the film.
- the released entity may be one that does not readily diffuse through the layers of the film.
- bilayers may be covalently cross-linked to prevent diffusion of materials across the layers of the film.
- a bilayer of two polymers of opposite charge may be cross- linked thermally or by other mechanisms.
- Thermal cross-linking may be achieved by heating the film for a particular period of time.
- Chemical cross-linking may be achieved by exposing a film to UV light.
- polymers having double bonds in or pendant to the backbone may be employed in the thin film and cross-linked after deposition.
- reactive groups such as carboxyl, thiol, amine, hydroxyl, or halogen may be exploited to covalently cross-link films.
- These groups may be made more reactive by methods known to those of skill in the art, for example, using carbodiimides or other groups such as isocyanates, 3-[(2-aminoethyl)dithio]propionic acid, and succinimidyl 4-[N-maleimidomethyl]cyclohexane-l-carboxylate (SMCC), that provide additional reactivity and good leaving groups.
- SCC succinimidyl 4-[N-maleimidomethyl]cyclohexane-l-carboxylate
- Additional groups that are suitable for cross-linking will depend on the composition of the various layers, as will be understood by those of skill in the art. A variety of cross-linking agents are available from Pierce Biotechnologies, Rockford, IL.
- a range of strategies were employed to control the relative positions of multiple, labeled species within a single film by constructing physical barriers to separate the two components.
- films were constructed containing 20-40 base layers of polymer 1/HEP, followed by a set of "barrier" layers including polymer 1/SPS (degradable), PDAC/SPS (non-degradable), thermally cross-linked PAH/PAA, or nothing at all, followed by 20-40 surface layers of polymer 1/DS ( Figure 18).
- a similar set of films was also constructed, but with the order of the labeled components reversed (e.g., DS base layers and HEP surface layers).
- FIG. 27 A illustrates the effect of barrier layers on the average release rate from the exemplary two-component systems described above.
- the average release rate (taken as the average slope of the initial, linear portion of the release curve) of systems including of an underlying layer of linearly-growing DS can be broadly controlled using both multiple layers of a nondegradable system PDAC/SPS or as little as a single layer of cross-linked PAH/PAA.
- the degree of cross-linking e.g., cross-linking time, temperature, number of cross-linked layers
- the release rate can be dramatically altered. For example, cross- linking times of greater than 1.5 h at 215° C, as well as barriers containing more than five cross-linked layers, resulted in one to two order of magnitude decreases in release rate (data not shown).
- Figures 19 and 27 reveal numerous principles for the manipulation of diffusion and release from multi-component, hydrolytically degradable LbL films.
- the released entity need not serve as a structural component of the film (see Figures 19 and 27).
- polyelectrolytes may be associated or mixed with polymeric or non-polymeric moieties to regulate the degradation rate.
- neutral, zwitterionic, or charged biomolecules, small molecules, or bioactive agents may be associated or mixed with a polycation or polyanion and incorporated into a layer.
- the charged atoms on a zwitterionic molecule may facilitate electrostatic interactions with both the polyanionic and polycationic layers.
- a zwitterionic biomolecule, small molecule, or bioactive agent may be combined in solution with the polyelectrolytes for one of the layers or placed in a separate solution to form a "sandwich" between two layers. When the thin film degrades, the biomolecule, small molecule, or bioactive agent will be released.
- a biomolecule, small molecule, or bioactive agent may be associated with a polyelectrolyte under conditions which facilitate a strong interaction between the molecule and the polyelectrolyte, while the medium in which the biomolecule, small molecule, or bioactive agent is released is one which competes with the polyelectrolyte for the biomolecule, small molecule, or bioactive agent, thereby decreasing the strength of the interaction with the polyelectrolyte.
- composition of the various layers may be adjusted to release different entities as the thin film degrades.
- a thin film may be designed to release a chemotactic factor tailored to attract cells to an implant site for a specified number of layers, followed by a growth factor tailored to stimulate a desired metabolic or proliferative activity in cells now at the implant site.
- inventive thin films can, for example, be investigated using a variety of known techniques, including ellipsometry, dynamic light scattering (DLS), zeta-potential analysis, quartz crystal microbalance (QCM), and atomic force microscopy (AFM).
- the QCM method is particularly attractive since it can be used with rough films and allows continuous monitoring without removal of the thin films from the degradation milieu.
- AFM can be also used to monitor changes in the multi-layer surface morphology as a function of degradation.
- a model chromic compound e.g., the commercially available photochromic polyanion PAZO (see Figure 7)
- Radioisotopes may also be used to label components of the films, following which the activity of a solution containing the released material may be measured using techniques known to those of skill in the art.
- the thin film may also be used to create a degradable substrate for cell seeding and culture. Some cells, for example, chondrocytes, proliferate better when deposited on a substrate to which they can attach. However, to use these cells in other applications, they may need to be separated from the substrate. Cells may be deposited on the surface of a multi-layer thin film and maintained in vitro.
- Integrins and cell adhesion sequences may be included in the top layer or layers of the film to facilitate cell adhesion. Integrins are part of a large family of cell adhesion receptors which are involved in cell- extracellular matrix and cell-cell interactions.
- the RGD sequence present in proteins such as fibronectin, has been shown to be active in promoting cell adhesion and proliferation (see Massia et al., J. Cell. Biol. 114:1089, 1991).
- the thin films may include electroactive polymers.
- conductive polymers may enhance the proliferation and metabolism of cells deposited thereon (see USPN 6,095,148, issued Aug. 1, 2000, and 6,190,893, issued Feb. 20, 2001).
- the voltage may be an externally applied voltage.
- a voltage may be applied by native tissue, for example, nerve.
- Bone is piezoelectric, and physiologic loading will generate a potential across a film implanted therein.
- Exemplary electroactive polymers include, but are not limited to, polypyrrole, poly(p-phenylene), poly(p-phenylene vinylene), polythiophene, polyaniline, polyporphyrin, polyheme, and derivatives thereof.
- polymers may be derivatized.
- hydrocarbon groups, methoxy, cyano, phenyl, alkoxy, amino, and halides may be added to aromatic groups in the polymer, and except for halides (which would lead to the production of poly(phenylene acetylene)), to the non-aromatic carbons.
- halides which would lead to the production of poly(phenylene acetylene)
- the resulting derivative should be biocompatible.
- the invention can employ a wide range of cell types and is not limited to any specific cell type.
- cell types include but are not limited to bone or cartilage forming cells such as chondrocytes and fibroblasts, other connective tissue cells, epithelial cells, endothelial cells, blood vessel cells, cancer cells, organ cells such as hepatocytes, islet cells, kidney cells, intestinal cells, and lymphocytes, smooth muscle cells, skeletal muscle cells, heart muscle cells, nerve cells, and stem cells such as human embryonic stem cells or mesenchymal stem cells.
- bone or cartilage forming cells such as chondrocytes and fibroblasts, other connective tissue cells, epithelial cells, endothelial cells, blood vessel cells, cancer cells, organ cells such as hepatocytes, islet cells, kidney cells, intestinal cells, and lymphocytes, smooth muscle cells, skeletal muscle cells, heart muscle cells, nerve cells, and stem cells such as human embryonic stem cells or mesenchymal stem cells.
- the thin film may encapsulate a decomposable substrate (e.g., a drug nano- or micro-crystal). Additionally or alternatively, the thin film may be exploited to regulate diffusion of the substrate into the surrounding medium. In certain embodiments, particularly for drug delivery, it may be desirable to target an encapsulated substrate to a particular cell or tissue. A variety of agents that can direct an encapsulated substrate to particular cells are known in the art (see, for example, Cotten et al. , Methods Enzym. 217:618, 1993).
- LDLs low-density lipoproteins
- transferrin asiaglycoproteins
- gpl20 envelope protein of the human immunodeficiency virus HIV
- toxins antibodies, and carbohydrates.
- Certain exemplary encapsulated substrates include one or more targeting agents that are associated with polyelectrolyte components of the inventive thin film and/or with the entity to be released.
- the substrate geometry may be manipulated to deposit films having a variety of shapes.
- films may be deposited on particles, tubes, or spheres to facilitate a more uniform release distribution.
- Films may be deposited on strands such as sutures to release factors such as analgesics or antibiotics at a surgical site.
- these films may be deposited onto capillary networks or tissue engineering constructs.
- a thin film deposited on a three-dimensional tissue engineering construct may be used to attract cells to a newly implanted construct and then to promote specific metabolic or proliferative activity.
- the methods of the invention may also be used to create three-dimensional microstructures.
- the thin film may be deposited on a substrate that can be dissolved to leave a hollow shell of the thin film (see Figure 8).
- multilayers may be deposited having regions that are more and less degradable. Degradation of the degradable portions leaves a three-dimensional microstructure (see Figure 9).
- a first step the surface of a substrate is divided into regions in which LBL deposition of an inventive thin film is more or less favorable (see Figure 9, step A).
- a pattern of self-assembled monolayers (SAMs) is deposited on a substrate surface by microcontact printing (see, for example, U.S. Patent No.
- the substrate surface is neutral and the exposed surface of the deposited SAMs is polar or ionic (i.e., charged).
- polar or ionic head groups are known in the art of self- assembled monolayers.
- a uniform coating of a polymer is deposited on a substrate, and that coating is transformed into a patterned layer by means of photolithography.
- the substrate surface is selectively exposed to plasmas, various forms of electromagnetic radiation, or to electron beams.
- the substrate may possess the desired surface characteristics by virtue of its inherent composition.
- the substrate may be a composite in which different regions of the surface have differing compositions, and thus different affinities for the polyelectrolyte to be deposited.
- step B polyelectrolyte layers of alternating charge are deposited by LBL on receptive regions of the surface (see Figure 9, step B) as described for a homogeneous surface above and selective regions in Jiang and Hammond, Langmuir, 16:8501, 2000; Clark et al., Supramolecular Science 4:141, 1997; and Hammond and
- Poly 1 has also been shown to form electrostatic complexes with polyanions such as DNA in solution, we hypothesized that it would readily absorb to negatively-charged surfaces and model polyanions such as poly(styrene sulfonate) (SPS) and poly(acrylic acid) (PAA) commonly used for LBL assembly.
- SPS poly(styrene sulfonate)
- PAA poly(acrylic acid)
- SPS sodium 4-styrenesulfonate
- LPEI linear poly(ethylene imine), "LPEI”, MW ⁇ 25,000, poly(dimethyldiallylammonium chloride) "PDAC”, MW ⁇ 240,000) and poly(acrylic acid), "PAA”, MW ⁇ 90,000 were obtained from Polysciences, Warrington, PA.
- Poly 1 (see Figure 10, MW ⁇ 10,000), was synthesized as described in Lynn et al., J. Am. Chem. Soc. 122:10761-10768, 2000, the contents of which are hereby incorporated by reference.
- Polyelectrolytes were used without further purification or filtration, with the exception of Poly 1.
- Poly 1 solutions were filtered using a 0.45 ⁇ m membrane syringe filter prior to use. All polymer solutions used for deposition were pH adjusted to 5.1 with a concentration of 5 mM for Poly 1 and 20 mM for all other polyelectrolytes (calculations based on monomer unit). Poly 1 was dissolved in a 100 mM sodium acetate buffer. Other solutions were prepared with deionized water and the pH was adjusted using sodium hydroxide and hydrochloric acid.
- the slide stainer was programmed to submerge the substrates in the polycation solution for five minutes and then to rinse the substrates in two successive deionized water baths. The first rinse was of one minute and the following of two minutes and thirty seconds. The substrates were then submerged five more minutes in the polyanion solution and then rinsed in the same manner. After one bilayer was deposited, the substrates were ultra sonicated for four minutes and thirty seconds. Without being bound by any particular theory, this ultra sonication step is believed to improve the surface's topography. The program was then cycled to obtain the desirable bilayers of film. After deposition, films were dried with nitrogen to remove visible drops of solution from the surface before ellipsometry or prof ⁇ lometry was performed.
- Multilayers incorporating poly 1 were removed from aqueous water baths immediately after final layers were deposited, dried under a stream of dry nitrogen, placed in a vacuum dessicator and dried overnight to minimize degradation due to incorporated water.
- Root mean square roughness was determined using a Tencor Corporation KLA Model PlO Surface Prof ⁇ lometer with a 2um stylus.
- Reflective FTIR spectra were recorded using a Nicolet Magna-IR 550 Series II Spectrometer.
- Substrates to be used for reflective FTIR analysis were coated with a thin layer of gold using a thermal evaporator.
- the substrates were submerged in buffered solutions at room temperature or 37°C and the decomposition followed by ellipsometry or UV-visible spectroscopy at desired time intervals. The thickness of each sample was determined by ellipsometry at nine different predetermined locations on the substrate surface (measured in triplicate), and the sample was returned to the buffer solution.
- Poly 1/SPS films were grown on gold substrates for analysis by reflective FTIR. The observation of a strong peak between 1725 and 1750 cm “1 corresponding to the carboxyl stretch of Poly 1 confirms the presence of Poly 1 in the films (see Figure 13).
- the Poly 1/SPS system was selected for the FTIR, rather than the Poly 1/PAA systems, to prevent the carboxyl group of the PAA from obscuring the CO stretch in Poly 1.
- Decomposition of Poly 1/SPS and Poly 1/PAA films Once procedures for the construction of films using Poly 1 were optimized, experiments to investigate film decomposition were performed.
- Film erosion rates were also dependent on the structures of the incorporated polyanions. For example, while 100 nm thick Poly 1/SPS films eroded completely over a period of 40 hours at pH 7.4 ( Figure 2), 600 nm films formed from Poly 1 and PAA degraded completely over a period of 9 hours under identical conditions. This behavior is consistent with the pH/dissolution profile observed for other weak polyacid multilayer systems, in which the increased ionization of PAA at elevated pH contributes to repulsive electrostatic interactions (Sukhishvili, S.A., et al, S. Macromolecules, 35:301-310, 2002; Sukhishvili, S.A., et al., J. Am. Chem.
- films containing thick layers of PAA often possess a lower overall effective crosslink density than more compact films constructed from strong polyelectrolytes such as SPS (Shiratori, S.S., 2000; Lvov, Y., et al, Langmuir, 9:481-486, 1993); the less crosslinked morphology may support more rapid permeation of water and breakdown of the polymer layers.
- Preliminary AFM analysis of partially eroded films is also consistent with this gradual erosion process - surface roughness values for partially eroded films (RMS roughness - 6.9 nm) were less than the thickness of an average bilayer (10 nm) and surfaces were consistent over 1 ⁇ m (Hammond, P. T., 1998) portions of the film.
- RMS roughness - 6.9 nm surface roughness values for partially eroded films
- the different decomposition rates provide a potential means for controlling the exact decomposition rates of films by customization with these two or any other polyanions.
- Controlled release of DNA from Poly 1 /DNA films Preliminary experiments designed to explore the application of these decomposable thin films to the controlled release of polyanions were done by adsorbing polyanionic dyes and calf thymus DNA into the decomposable polymeric films. Adsorption of DNA by electrostatic layer-by- layer deposition was achieved by the negative charge of the phosphate-sugar group that composes the helix. Poly 1/DNA films were deposited on silicon substrates previously prepared with ten precursor bilayers of LPEI/SPS. These films were measured by ellipsometry and film thickness ranged from 800 A to 1000 A (see Figure 16). Poly 1/DNA films decomposed over a 50-hour period in PBS buffer pH 7.4 at 37 0 C (see Figure 17). Build up and release properties of single component films
- Radiolabeled and corresponding unlabeled polymers were chosen with similar molecular weights and polydispersities in order to mimic the behavior of the unlabeled species as closely as possible. All materials and solvents were used as received without further purification.
- a Harrick PDC-32G plasma cleaner was used to etch silicon substrates (3 cm x 2 cm) following rinsing with methanol and deionized water and drying under a stream of dry nitrogen. Layer-by-layer thin films were deposited using an automated Carl Zeiss HMS Series Programmable Slide Stainer. Absorbances from growing films were measured using Fourier Transform Infrared Spectroscopy (FTIR) using a Nicolet Magna IR 550 Series II Spectrometer.
- FTIR Fourier Transform Infrared Spectroscopy
- Zinc selenide substrates used for transmission FTIR analysis were prepared using the same method employed for silicon substrates. Ellipsometric measurements for film thickness were conducted using a Gaertner Variable Angle Ellipsometer (6328 nm, 70° incident angle) and Gaertner Ellipsometer Measurement Program (GEMP) Version 1.2 software interface. The release of radiolabeled polymers was quantified using a Tri-carb liquid scintillation counter
- IX PBS buffer pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4
- Films used in this study were constructed on either silicon (for ellipsometry and degradation studies) or zinc selenide (for transmission mode FTIR) planar substrates.
- degradable, polymer 1- based films were constructed directly on top often bilayer, nondegradable base films containing linear poly(ethylenimine) (LPEI) and sulfonated poly(styrene) (SPS) to ensure uniform adhesion to the substrate. Following deposition, films were removed from rinsing baths and dried thoroughly under a stream of dry nitrogen to avoid premature degradation.
- LPEI linear poly(ethylenimine)
- SPS sulfonated poly(styrene)
- drug release experiments were performed by immersing each film in 50 mL IX PBS buffer in a 200 mL screw top vial.
- Figure 28A is a plot of the transmission mode FTIR absorbance recorded from films containing either (polymer 1/HEP) or (polymer 1/DS). Specifically, the absorbances from sulfonic acid groups at 1035 cm “1 (heparin) and 1017 cm “1 (dextran sulfate) were measured after the deposition of indicated bilayers. All measurements were taken from the same spot on the surface of the film in transmission mode. The inset shows the film thickness versus number of deposited bilayers for a (polymer 1/DS) film.
- heparin-based films exhibit an exponential increase in absorbance with increasing numbers of adsorbed layers.
- Exponentially growing films which often include hydrophilic polyelectrolytes or biologically-derived materials (i.e., peptides and polysaccharides), are poorly organized, blended architectures characterized by the complete "in” and “out” diffusion of adsorbing species throughout the growing film during the film's assembly process (Picart, 2002; Elbert, et al. (1999) Langmuir 15, 5355-5362; Picart, et al. (2001) Langmuir 17, 7414-7424; Lavalle, et al. (2002) Macromolecules 35, 4458-4465).
- Figure 28B depicts the chemical structures of the repeat units of polymer 1, HEP, and DS.
- Polymer 1 is a cationic, degradable poly ( ⁇ amino ester) synthesized by the conjugate addition-step polymerization of a diamine and a diacrylate; it represents one member of a library of over 2350 degradable poly ( ⁇ amino esters) recently synthesized and screened for their abilities to deliver DNA to cells in culture (Anderson, et al., (2003) Angew. Chem. Int. Ed. 42, 3153-3158).
- Both model drug compounds, HEP and DS are polysaccharides that possess similar structural attributes, including strong (sulfonic) acid groups on each repeat unit and relatively low molecular weights.
- Figures 29A and 29B show degradation and release from 20 bilayer (polymer 1/HEP) and (polymer 1/DS) systems, respectively, following immersion in PBS buffer at pH 7.4. Complete degradation and consequent release from (polymer 1/HEP) systems occurred within 20 hours. Film thickness was observed to decrease linearly following a brief swelling period of 0.5-2.0 h on first exposure to aqueous solution (Wood, 2005). DS-based films exhibited similar degradation and release behavior, though with kinetics approximately five-fold slower than their HEP-based counterparts.
- the initial release observed in both cases within the first few hours of degradation likely reflects passive release from the surface, as the outermost layer of each film includes the labeled compound.
- film thickness was observed to decrease linearly with time; further, the apparent roughness of the film surface, taken from the standard deviation in film thickness measured at 10 predetermined spots on the surface, was observed to remain constant, or even decrease, with time.
- the vastly different kinetics of degradation and release exhibited by these two systems may reflect differences in film organization, wherein the diffusive character of HEP may contribute to loose gradient films with larger quantities of HEP in the top layers, in comparison to their relatively more stratified, and more densely ion-crosslinked DS counterparts that have a constant distribution of DS throughout the film.
- Figure 31 provides a schematic illustrating a drug delivery film and release profiles for two drugs, drug 1 and drug 2.
- a first drug (drug 1) is mixed with a cationic or anionic polymer (polymer 1), where a cationic polymer is illustrated in Figure 31, and deposited to form a first layer (3102).
- a second drug (drug 2) is mixed with second polymer and a second layer formed (3104).
- the second polymer e.g., could be the same as the first polymer (polymer 1) or different.
- FIG. 31 is a illustrative plot of the mass release of drug 1 (3106) and drug 2 (3107) by ,e.g., hydroplytic degradation of layer 2 and layer 1. It is to be understood that by a combination of drugs, polymers and other layers and additives that complex drug release profiles and/or schedules can be provided.
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