New class of ultrathin,highly cell-adhesion-resistant polyelectrolyte multilayers with micropatterning capabilities

Hydrogen-bonded multilayers comprised of polyacrylamide (PAAm) and a weak polyelectrolyte, such as poly(acrylic acid) (PAA) or poly(methacrylic acid) (PMA), were investigated for their surface-cell interactions. The assembled films were lightly cross-linked thermally or photochemically in order to render them stable in a physiological environment. Both PAA/PAAm and PMA/PAAm multilayers were found to exhibit a high resistance to the adhesion (cytophobicity) of mammalian fibroblasts, even with only a single bilayer coating. Protein adsorption to the multilayers, as revealed by surface plasmon resonance measurements, was greatly reduced for fibronectin and serum-containing medium. In situ swelling experiments indicate that the H-bonded multilayers are hydrogellike coatings capable of a high level of swelling in buffered solution. Utilizing the H-bonding nature of these multilayers, we were able to micropattern the films to create more complex cell-resistant/-adhesive surfaces. The long-term stability of the cell-resistant multilayers was found to be exceptionally good even under conditions (pH 7.4, buffered solution) where a high degree of swelling takes place. No degradation of the micropatterned films was observed over a period of a month, during which time the multilayer coatings remained highly resistant to cell-adhesion.     

Patterning of diverse mammalian cell types in serum free medium with photoablation

Integration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer‐by‐layer self‐assembly technique and photolithography offer a simple, versatile, and silicon compatible approach that overcomes chemical surface patterning limitations, such as short‐term stability and low‐protein adsorption resistance. In this study, direct photolithographic patterning of two types of multilayers, PAA (poly acrylic acid)/PAAm (poly acryl amide) and PAA/PAH (poly allyl amine hydrochloride), were developed to pattern mammalian neuronal, skeletal, and cardiac muscle cells. For all studied cell types, PAA/PAAm multilayers behaved as a cytophobic surface, completely preventing cell attachment. In contrast, PAA/PAH multilayers have shown a cell‐selective behavior, promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108‐15 cells) to a greater extent, while providing little attachment for neonatal rat cardiac and skeletal muscle cells (C2C12 cell line). PAA/PAAm multilayer cellular patterns have also shown a remarkable protein adsorption resistance. Protein adsorption protocols commonly used for surface treatment in cell culture did not compromise the cell attachment inhibiting feature of the PAA/PAAm multilayer patterns. The combination of polyelectrolyte multilayer patterns with different adsorbed proteins could expand the applicability of this technology to cell types that require specific proteins either on the surface or in the medium for attachment or differentiation, and could not be patterned using the traditional methods. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Hydrogen bonding versus ion pairing in polyelectrolyte multilayers with homopolynucleotides

Homopolynucleotides--poly(adenylic acid), poly(A), and poly(uridylic acid), poly(U)--were assembled, layer-by-layer, into thin films with poly(ethylenimine), PEI. Various combinations and sequences of polynucleotide and PEI were used to highlight contributions of electrostatic versus hydrogen bonding as driving forces for multilayer build-up. Assembly of alternating poly(A) and poly(U) failed to yield growing films, due to excessively strong interactions between these complimentary strands. The surface morphology of multilayers depended on the deposition order and whether films had been annealed by salt. Films assembled from preformed A/U duplexes (having high persistence lengths) were very smooth. Individual adsorption steps, followed by optical waveguide light-mode spectroscopy, showed that only complementary polynucleotides adsorb by H-bonding to the surface of a growing multilayer. In contrast to behavior usually observed for polyelectrolyte multilayer build-up, the films decreased in thickness with increasing salt concentration.     

Cytotoxicity of free versus multilayered polyelectrolytes

The cytotoxicity of polyelectrolytes commonly employed for layer-by-layer deposition of polyelectrolyte multilayers (PEMUs) was assessed using rat smooth muscle A7r5 and human osteosarcoma U-2 OS cells. Cell growth, viability, and metabolic assays were used to compare the responses of both cell lines to poly(acrylic acid), PAA, and poly(allylamine hydrochloride), PAH, in solution at concentrations up to 10 mM and to varying thicknesses of (PAA/PAH) PEMUs. Cytotoxicity correlated with increasing concentration of solution polyelectrolytes for both cell types and was greater for the positively charged PAH than for the negatively charged PAA. While metabolism and proliferation of both cell types was slower on PEMUs than on tissue culture plastic, little evidence for direct toxicity on cells was observed. In fact, evidence for more extensive adhesion and cytoskeletal organization was observed with PAH-terminated PEMUs. Differences in cell activity and viability on different thickness PEMU surfaces resulted primarily from differences in attachment for these adhesion-dependent cell lines.     

Effect of the polycation nature on the structure of layer-by-layer electrostatically self-assembled multilayers of polyphenol oxidase

Self-assembled multilayers comprised of alternate layers of polyphenol oxidase (PPO) and poly(allylamine) (PAH) or PPO and poly(diallyldimethylamine) (PDDA), deposited on a 3-mercaptopropanesulfonic acid (MPS)-modified gold surface, were studied "in-situ" (under water) by means of ellipsometry and quartz crystal microbalance (QCM), and "ex-situ" (in open air) by ellipsometry and fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS). Optical ellipsometric properties of (PAH)(n)(PPO)(n) and (PDDA)(n)(PPO)(n) multilayers were obtained at two wavelengths, employing an isotropic single-layer model with the substrate parameters measured after thiol adsorption. Film thickness as well as ellipsometric mass values based on the de Feijter equation were also evaluated. The quartz crystal impedance analysis showed that self-assembled multilayers behaved as acoustically thin films, and therefore, the shifts observed in the film inductive impedance parameter were interpreted in terms of gravimetric mass. The enzyme mass up-take in each adsorption step was determined on PAH or on PDDA topmost layer. A comparative study between the ellipsometric thickness and acoustic mass values allowed us to obtain average values of "apparent" densities of (2.1 +/- 0.1) and (2.4 +/- 0.1) g cm(-3) for (PAH)(n)(PPO)(n) and (PDDA)(n)(PPO)(n) multilayers, respectively. The content of water included in the open polymer-enzyme structure was evaluated by comparison of QCM and ellipsometric mass values. FT-IRRAS spectra of each layer in (PAH)(n)(PPO)(n) and (PDDA)(n)(PPO)(n) films were recorded, and the intensity ratio of the amide bands was evaluated to obtain information about layer-by-layer enzyme conformation. An enzyme/polycation distribution model for (PAH)(n)(PPO)(n)and (PDDA)(n)(PPO)(n) multilayer structures is presented on the basis of combined ellipsometric, QCM, and FT-IRRAS results.     

Multilayer DNA/poly(allylamine hydrochloride) microcapsules: assembly and mechanical properties

We report the preparation, characterization, and mechanical properties of DNA/poly(allylamine hydrochloride) (PAH) multilayer microcapsules. The DNA/PAH multilayers were first constructed on a planar support to examine their layer-by-layer buildup. Surface plasmon resonance spectroscopy (SPR) showed a nonlinear growth of the assembly upon each bilayer deposited independently on a concentration of salt. A weak increase in the film thickness with the DNA concentration was, however, detected. A post-treatment of the multilayers in the salt solutions has shown a thinning of the film. The optimal conditions of the planar film growth were used to deposit the same multilayers on the surface of colloidal templates and to study their roughness and morphology with the atomic force microscope (AFM) imaging. When an outer layer is formed by DNA, we observe large domains of oriented parallel DNA loops, while an outer layer formed by PAH shows highly porous morphology. The dissolution of colloidal templates led to a formation of highly porous DNA/PAH microcapsules. We probe their mechanical properties by measuring force-deformation curves with the AFM-related setup. The experiment suggests that the DNA/PAH capsules are softer than capsules made from the flexible polyelectrolytes studied before. The softening is due to both higher permeability and smaller Young's modulus of the shell material. The Young's modulus of the DNA/PAH shells increases after post-treatment in salt solutions of relatively low concentration.     

Substrata mechanical stiffness can regulate adhesion of viable bacteria

The competing mechanisms that regulate adhesion of bacteria to surfaces and subsequent biofilm formation remain unclear, though nearly all studies have focused on the role of physical and chemical properties of the material surface. Given the large monetary and health costs of medical-device colonization and hospital-acquired infections due to bacteria, there is considerable interest in better understanding of material properties that can limit bacterial adhesion and viability. Here we employ weak polyelectrolyte multilayer (PEM) thin films comprised of poly(allylamine) hydrochloride (PAH) and poly(acrylic acid) (PAA), assembled over a range of conditions, to explore the physicochemical and mechanical characteristics of material surfaces controlling adhesion of Staphylococcus epidermidis bacteria and subsequent colony growth. Although it is increasingly appreciated that eukaryotic cells possess subcellular structures and biomolecular pathways to sense and respond to local chemomechanical environments, much less is known about mechanoselective adhesion of prokaryotes such as these bacteria. We find that adhesion of viable S. epidermidis correlates positively with the stiffness of these polymeric substrata, independently of the roughness, interaction energy, and charge density of these materials. Quantitatively similar trends observed for wild-type and actin analogue mutant Escherichia coli suggest that these results are not confined to only specific bacterial strains, shapes, or cell envelope types. These results indicate the plausibility of mechanoselective adhesion mechanisms in prokaryotes and suggest that mechanical stiffness of substrata materials represents an additional parameter that can regulate adhesion of and subsequent colonization by viable bacteria.     

Glycated polyelectrolyte multilayer films: differential adhesion of primary versus tumor cells

Glycated polymers have already been widely employed for cell transfection studies, as cells possess specific lectins. However, up to now, these glycated polymers have barely been investigated for their cell adhesive properties, save macrophages. In this work, we use polyelectrolyte multilayer films made of poly(L-lysine) and poly(L-glutamic) acid as polymeric substrates to investigate the role of sugar molecules (e.g., mannose and lactose) on the adhesion of primary cells as compared to that of a tumor cell line. The glycated polymeric films were compared to ungrafted and chemically cross-linked films, which are known to present opposite adhesive properties. A differential adhesion could be evidenced on mannose grafted films: primary chondrocytes adhere and proliferate well on these films, whereas chondrosarcoma cells do not grow well. Although present, the effect of lactose on cell adhesion was much less important. This adhesion, mediated by glycated polymers, appears to be specific. These results show that it is possible to use glycated polyelectrolytes not only as nonviral vectors but also as cell adhesive substrates.     

Protamine assembled in multilayers on colloidal particles can be exchanged and released

Biocomposite thin films assembled on colloidal particles by means of layer-by-layer adsorption have been suggested as drug carriers and diagnostic devices. Protamine (PRM)/dextransulfate (DXS) and protamine/bovine serum albumine (BSA) multilayers were fabricated on colloidal silica and subsequently investigated by means of fluorescence activated cell sorting (FACS) and microelectrophoresis. Fluorescein labeled polyelectrolytes were embedded at different positions in the multilayers as a marker for layer growth. FACS showed that PRM and DXS formed regular growing stable multilayers, yet adsorbed PRM can be nevertheless exchanged with PRM in solution during layer formation and also after the multilayer formation has been completed. Up to 90% of the PRM pool was available for exchange. PRM together with BSA as demonstrated by SFM did not form multilayers under the applied conditions although the zeta-potential, commonly used as an indicator for stepwise adsorption, observed characteristic alternations. The capability of bound PRM to exchange with PRM in solution is attributed to its relatively small size. The demonstrated exchange may have importance in designing multilayers with smart release features. Furthermore, FACS proved to be a rather suitable means to quantify the aggregation behavior during coating and washing. Singulets, doublets, triplets, and aggregates of higher order could be clearly resolved. The aggregation of particles coated with PRM/DXS layers was higher than that of silica particles coated with PAH/PSS layers. In the first case about 50% of all recorded events are attributed to aggregats, while the PAH/PSS coating produced only about 10% aggregates.     

Polyelectrolyte multilayer films as substrates for photoreceptor cells

Reconstruction of extracellular matrix substrates for delivery of functional photoreceptors is crucial in pathologies such as retinal degeneration and age-related macular degeneration. In this study, we assembled polyelectrolyte films using the layer-by-layer deposition method. The buildup of three different films composed of poly(L-lysine)/chondroitin sulfate (PLL/CSA), poly(L-lysine)/poly(styrenesulfonate) (PLL/PSS), or poly(L-lysine)/hyaluronic acid (PLL/HA) was followed by means of quartz crystal microbalance measurements, optical waveguide light mode spectroscopy, confocal microscopy, and atomic force microscopy. The exponential growth regime and the diffusion of PLL chains from the bulk through the PLL/CSA, PLL/PSS, and PLL/HA films was examined. Evaluation of photoreceptor cell viability was optimal on one layer of PLL (PLL(1)), followed by 10 bilayers of PLL/HA [(PLL/HA)(10)] and 10 bilayers of PLL/CSA [(PLL/CSA)(10)]. The number of bilayers and the type of terminating layer also had a significant influence on the number of photoreceptor cells attached. Functionalized polyelectrolyte multilayer films were obtained by adsorbing basic fibroblastic factor (bFGF) or the insoluble fraction of interphotoreceptor matrix (IPM) on or within polyelectrolyte multilayers. bFGF and IPM adsorption on top of the (PLL/CSA)(10)/PLL polyelectrolyte films increased the number of photoreceptor cells attached and maintained the differentiation of rod and cone cells.     

Amperometric glucose biosensor based on layer-by-layer assembly of multilayer films composed of chitosan, gold nanoparticles and glucose oxidase modified Pt electrode

A new strategy for fabricating glucose biosensor was presented by layer-by-layer assembled chitosan (CS)/gold nanoparticles (GNp)/glucose oxidase (GOD) multilayer films modified Pt electrode. First, a cleaned Pt electrode was immersed in poly(allylamine) (PAA), and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/PAA/Pt). Second, the GOD/GNp/PAA/Pt electrode was immersed in CS, and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/CS/GOD/GNp/PAA/Pt). Third, different layers of multilayer films modified Pt electrodes were assembled by repeating the second process. Film assembling and characterization were studied by quart crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The results confirmed that the assembling process of multilayer films was simple to operate, the immobilized GOD displayed an excellent catalytic property to glucose, and GNp in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. The amperometric response of the biosensors uniformly increased from one to six layers of multilayer films, and then reached saturation after the seven layers. Among the resulting biosensors, the biosensor based on the six layers of multilayer films was best. It showed a wide linear range of 0.5-16 mM, with a detection limit of 7.0 microM estimated at a signal-to-noise ratio of 3, fast response time (within 8s). Moreover, it exhibited good reproducibility, long-term stability and interference free. This method can be used for constructing other thin films, which is a universal immobilization method for biosensor fabrication.     

Addition of biological functionality to poly(epsilon-caprolactone) films

Biodegradable polyesters such as poly(epsilon-caprolactone) (PCL) have a number of biomedical applications; however, their usage is often limited by a lack of biological functionality. In this paper, a PCL-based polymer containing pendent groups activated by 4-nitrophenyl chloroformate (NPC) and reactive toward primary amines has been cast into thin films. The reactivity of the films toward poly(l-lysine) and the cell adhesion peptide, GRGDS, was assessed, and their cell adhesive capabilities were characterized. ATR-FTIR analysis found that NPC functional groups were present on the surface of the cast film, and the synthesis, conjugation, and visualization of a fluorescent molecule on these films further demonstrated the success of this functionalization methodology. The immersion of these films into a solution of either poly(l-lysine) (PLL) or GRGDS in PBS (pH 7.4) and subsequent 3T3 fibroblast adhesion studies demonstrated significant improvement in cell adhesion and spreading over films cast from unmodified PCL. This investigation has shown that this novel NPC-containing polymer can be utilized in many applications where increased cellular adhesion is required, or the coupling of specific molecules to polymer surfaces is of interest.     

Novel agmatine-containing poly(amidoamine) hydrogels as scaffolds for tissue engineering

Novel biocompatible and biodegradable amphoteric poly(amidoamine) (PAA) hydrogels were designed for applications as scaffolds for tissue engineering. These hydrogels (PAA-AG1 and PAA-AG2) were obtained by polyaddition of 2,2-bisacrylamidoacetic acid with 2-methylpiperazine and 4-aminobutyl guanidine, a bioactive molecule with a known ability to induce adhesion to cell membranes. They contain carboxylic functions in their main chain and interchain connections deriving from two different cross-linking agents: for PAA-AG1, a multifunctional primary amine, that is, 1,10-decanediamine; for PAA-AG2, a purposely synthesized PAA (PAA-NH(2)) containing pendant NH(2). Both PAA-AG1 and PAA-AG2 proved noncytotoxic and adhesive to cell membranes, as ascertained by means of cytotoxicity and proliferation tests carried out on fibroblast cell lines. Good apparent mechanical strength was also observed in the case of PAA-AG2, cross-linked with the PAA-NH(2). Both PAA-AG1 and PAA-AG2 underwent degradation tests under controlled conditions simulating the biological environments, that is, Dulbecco medium at pH 7.4 and 37 degrees C. They completely dissolved within 10 and about 40 days, respectively. In both cases, the degradation products were completely noncytotoxic. All the results of this paper point to the conclusion that agmatine-based PAA hydrogels are excellent substrates for cell proliferation.     

Fibronectin and cell attachment to cell and protein resistant polyelectrolyte surfaces

Culture of A7r5 smooth muscle cells on a polyelectrolyte multilayer film (PEMU) can influence various cell properties, including adhesion, motility, and cytoskeletal organization, that are modulated by the extracellular matrix (ECM) in vivo. ECM contribution to cell behavior on PEMUs was investigated by determining the amount of fibronectin (FN) bound to charged and hydrophobic PEMUs by optical waveguide lightmode spectroscopy and immunofluorescence microscopy. FN bound best to poly(allylamine hydrochloride) (PAH)-terminated and Nafion-terminated PEMUs. FN bound poorly to PEMUs terminated with a copolymer of poly(acrylic acid) (PAA) and 3-[2-(acrylamido)-ethyl dimethylammonio] propane sulfonate (PAA-co-AEDAPS). Cells adhered and spread well on the Nafion-terminated PEMU surfaces. In contrast, cells spread less and migrated more on both FN-coated and uncoated PAH-terminated PEMU surfaces. Both cells and FN interacted much better with Nafion than with PAA-co-PAEDAPS in a micropatterned PEMU. These results indicate that A7r5 cell adhesion, spreading, and motility on PEMUs can be independent of FN binding to the surfaces.     

Disulfide cross-linked polymer capsules: en route to biodeconstructible systems

Hydrogen-bonded multilayer thin films were constructed using poly(vinylpyrrolidone) and poly(methacrylic acid) functionalized with cysteamine. The resulting films included thiol moieties that were cross-linked to render the films stable at physiological pH. Film buildup was followed using quartz crystal microgravimetry, which was also used to demonstrate the improved stability imparted by reacting the thiol moieties to form disulfide bonds. Films without disulfide bonds were readily deconstructed at physiological pH, while those with disulfide bonds were swollen upon exposure to this pH (7) but remained intact. Addition of a common thiol-disulfide exchange reagent, dithiothreitol (DTT) at pH 7 led to disassembly of the multilayer films. The films were also prepared on colloidal substrates (as demonstrated using confocal microscopy) and were used to retain a model drug (fluorescently labeled transferrin) and release this molecule when triggered by the addition of DTT. This approach has potential for the in vivo applications of hollow capsules, as thiol-disulfide exchange leading to deconstruction of the capsules can occur with the assistance of intracellular proteins.     

Environmental responsiveness of polygalacturonic acid-based multilayers to variation of pH

The effect of pH on the stability of layer-by-layer deposited polygalacturonic acid (PGalA)-based multilayer films prepared with the polycations poly-L-lysine, chitosan, and lysozyme is studied. The response was characterized using a quartz crystal microbalance, dual polarization interferometry, and Fourier transform infrared spectroscopy which probe multilayer thickness, density, polymer mass (composition and speciation), and hydration. All multilayers showed irreversible changes in response to pH change becoming thinner due to the partial disassembly. Preferential loss of the polycation (50-80% w/w) and relative small losses of PGaLA (10-35% w/w) occurred. The charge density on the polycation has a strong influence on the response to the acid cycle. Most of the disassembly takes place at the pH lower that pK(a) of PGaLA, indicating that this factor was crucial in determining the stability of the films. The pH challenge also revealed a polycation-dependent shift to acid pH in the PGaLA pK(a).     

Ultrathin antibiotic walled microcapsules

Ultrathin microcapsules comprised of anionic polyelectrolytes (PE) and a polycationic aminoglycoside (AmG) antibiotic drug were prepared by depositing PE/AmG multilayers on zinc oxide (ZnO) colloid particles using the layer-by-layer self-assembly technique and subsequently dissolving the ZnO templated cores. The polyelectrolytes, dextran sulfate sodium (DxS) and poly(styrenesulfonate) (PSS), were selected owing to their different backbone structure. An aminoglycoside, tobramycin sulfate (TbS), was used for studying DxS/TbS or PSS/TbS multilayer films. The multilayer growth on ZnO cores was characterized by alternating zeta potential values that were different for the DxS/TbS and PSS/TbS multilayers due to the PE chemistry and its interaction with Zn(2+) ions. Transmission and scanning electron microscopy provide evidence of PE/TbS multilayer coating on ZnO core particles. The slow acid-decomposition of the ZnO cores using weak organic acids and the presence of sufficient quantity of Zn(2+) in the dispersion were required to produce antibiotic multilayer capsules. There was no difference in the morphological characteristics of the two types of capsules; although, the yield for [PSS/TbS](5) capsules was significantly higher than for [DxS/TbS](5) capsules which was related to the physicochemical properties of DxS/TbS/Zn(2+) and PSS/TbS/Zn(2+) complexes forming the capsule wall. The TbS quantity in the multilayer films was determined using a quartz crystal microbalance and high performance liquid chromatography techniques which showed less TbS loading in both, capsules and multilayers on planar gold substrate, than the theoretical DxS:TbS or PSS:TbS stoichiometric ratio. The decomposition of the [PE/TbS](6) multilayers was fastest in physiological buffer followed by mannitol and water. The decomposition rate of the [PSS/TbS](6) multilayers was slower than [DxS/TbS](6) monolayers. The incomplete decomposition of DxS/TbS under saline conditions suggests the major role of hydrogen bonding for stability of DxS/TbS multilayers. A combination of hydrogen bonding and hydrophobic interaction between phenyl rings in PSS was responsible for PSS/TbS multilayer stability. In vivo studies in rabbits highlight the safety and sustained drug delivery potential of the PE/AmG microcapsules. The antibiotic walled ultrathin capsules presented here are suitable for sustained ophthalmic antibiotic delivery.     

Multifunctional poly(ethylene glycol) semi-interpenetrating polymer networks as highly selective adhesive substrates for bioadhesive peptide grafting

Novel artificial extracellular matrices were synthesized in the form of semi-interpenetrating polymer networks containing copolymers of poly(ethylene glycol) and acrylic acid (PEG-co-AA) grafted with synthetic bioadhesive peptides onto exposed carboxylic acid moieties. These substrates were very resistant to cell adhesion, but when they were grafted with adhesive peptides they were highly biospecific in their ability to support cell adhesion. Extensive preadsorption of adhesive proteins or peptides did not render these materials cell adhesive; yet covalent grafting of adhesive peptides did render these materials highly cell adhesive even in the absence of serum proteins. Polymer networks containing immobilized PEG-co-AA were grafted with peptides at densities of 475 +/- 40 pmol/cm(2). Polymer networks containing immobilized PEG-co-AA N-terminally grafted with GRGDS supported cell adhesion efficiencies of 42 +/- 4% 4 h after seeding and became confluent after 12 h. These cells displayed cell spreading and cytoskeletal grafted with inactive control peptides (GRDGS, GRGES, or no peptide) supported cell adhesion efficiencies of 0 +/- 0%, even when challenged with high seeding densities (to 100,000 cell/cm(2)) over 14 days. These polymer networks are suitable substrates to investigate in vitro cell-surface interactions in the presence of serum proteins without nonspecific protein adsorption adhesion signals other than those immobilized for study.     

Inherent charge-shifting polyelectrolyte multilayer blends: a facile route for tunable protein release from surfaces

Recent research has highlighted degradable multilayer films that enable the programmed release of different therapeutics. Multilayers constructed by the layer-by-layer (LbL) deposition that can undergo disassembly have been demonstrated to be of considerable interest, particularly for biomedical surface coatings due to their versatility and mild aqueous processing conditions, enabling the inclusion of biologic drugs with high activity. In this study, we examine the controlled release of a protein using a different mechanism for film disassembly, the gradual dissociation of film interactions under release conditions. Poly(β-amino ester)s and poly(L-lysine) (PLL) were used as the positively charged multilayer components coassembled with a model negatively charged antigen protein, ovalbumin (Ova). The release of the protein from these multilayer films is dominated by the slow shift in the charge of components under physiological pH conditions rather than by hydrolytic degradative release. The time scale of release can be varied over almost 2 orders of magnitude by varying the ratio of the two polyamines in the deposition solution. The highly versatile and tunable properties of these films form a basis for designing controlled and sequential delivery of drug coatings using a variety of polyions.     

pH-responsive properties of multilayered poly(L-lysine)/hyaluronic acid surfaces

Multilayer films have been prepared by the sequential electrostatic adsorption of poly(L-lysine) and hyaluronic acid onto charged silicon surfaces from dilute aqueous solutions under various pH conditions. Microelectrophoresis was used to examine the local acid-base equilibria of the polyelectrolytes within the films as a function of the total number of layers in the film and the assembly solution pH. The acid-base dissociation constants were observed to deviate significantly from dilute solution values upon adsorption, to be layer dependent only within the first 3-4 layers, and to show sensitivity to the assembly solution pH. As a result, some of the physicochemical properties of the films have also been found to exhibit pH-responsive behavior. For example, the thickest films result when at least one of the polyelectrolytes is only partially dissociated in solution. As well, the pH-dependent degree of dissociation of the surface functional groups can be used to vary the contact angle of a water droplet by as much as 25 degrees and the coefficient of friction by up to an order of magnitude. In addition, the extent to which PLL/HA films can be made to swell in solution can be varied by a factor of 7 depending on the assembly solution and swelling solution pH. The anomalies found in the dissociation constants account for the trends in these pH-dependent properties. Here, we demonstrate that knowledge of the acid-base dissociation behavior in multilayer films is key to understanding and controlling the physical properties of the films, particularly surface friction and wettability, which are fundamentally important factors for many biomaterials applications. For example, we outline a mechanism whereby biopolymer thin films can be electrostatically adsorbed under highly charged "sticky" conditions and then quickly transformed into stable low-friction films simply by altering the pH environment.