Drug eluting coatings that can direct the host tissue response to implanted medical devices have the potential to ameliorate both the medical and financial burden of complications from implantation. However, because many drugs useful in this arena are biologic in nature, a paucity of delivery strategies for biologics, including growth factors, currently limits the control that can be exerted on the implantation environment. Layer-by-Layer (LbL) polyelectrolyte multilayer films are highly attractive as ultrathin biologic reservoirs, due to conformal coating of difficult geometries, aqueous processing likely to preserve fragile protein function, and tenability of incorporation and release profiles. Herein, we describe the first LbL films capable of microgram-scale release of the biologic Bone Morphogenetic Protein 2 (BMP-2), which is capable of directing the host tissue response to create bone from native progenitor cells. Ten micrograms of BMP-2 are released over a period of two weeks in vitro; less than 1% is released in the first 3 hours (compared with commercial collagen matrices which can release up to 60% of BMP-2, too quickly to induce differentiation). BMP-2 released from LbL films retains its ability to induce bone differentiation in MC3T3 E1S4 preosteoblasts, as measured by induction of alkaline phosphatase and stains for calcium (via Alizarin Red) and calcium matrix (via Von Kossa). In vivo, BMP-2 film coated scaffolds were compared with film coated scaffolds lacking BMP-2. BMP-2 coatings implanted intramuscularly were able to initiate host progenitor cells to differentiate into bone, which matured and expanded from four to nine weeks as measured by MicroCT and histology. Such LbL films represent new steps towards controlling and tuning host response to implanted medical devices, which may ultimately increase the success of implanted devices, provide alternative new approaches toward bone wound healing, and lay the foundation for development of a multi-therapeutic release coating.
We report here the synthesis and characterization of polydiacetylene (PDA) films and nanotubes using layer-by-layer (LBL) chemistry. 10,12-Docosadiyndioic acid (DCDA) monomer was self-assembled on flat surfaces and inside of nanoporous alumina templates. UV irradiation of DCDA provided polymerized-DCDA (PDCDA) films and nanotubes. We have used zirconium-carboxylate interlayer chemistry to synthesize PDCDA multilayers on flat surfaces and in nanoporous template. PDCDA multilayers were characterized using optical (UV–vis, fluorescence, ellipsometry, FTIR) spectroscopies, ionic current–voltage (I–V) analysis, and scanning electron microscopy. Ellipsometry, FTIR, electronic absorption and emission spectroscopies showed a uniform DCDA deposition at each deposition cycle. Our optical spectroscopic analysis indicates that carboxylate-zirconium interlinking chemistry is robust. To explain the disorganization in the alkyl portion of PDCDA multilayer films, we propose carboxylate-zirconium interlinkages act as “locks” in between PDCDA layers which restrict the movement of alkyl portion in the films. Because of this locking, the induced-stresses in the polymer chains can not be efficiently relieved. Our ionic resistance data from I–V analysis correlate well with calculated resistance at smaller number of PDCDA layers but significantly deviated for thicker PDCDA nanotubes. These differences were attributed to ion-blocking because some of the PDCDA nanotubes were totally closed and the nonohmic and permselective ionic behaviors when the diameter of the pores approaches the double-layer thickness of the solution inside of the nanotubes.
Layer-by-layer assembly (LBL) can create advanced composites with exceptional properties unavailable by other means, but the laborious deposition process and multiple dipping cycles hamper their utilization in microtechnologies and electronics. Multiple rinse steps provide both structural control and thermodynamic stability to LBL multilayers but they significantly limit their practical applications and contribute significantly to the processing time and waste. Here we demonstrate that by employing inkjet technology one can deliver the necessary quantities of LBL components required for film build-up without excess, eliminating the need for repetitive rinsing steps. This feature differentiates this approach from all other recognized LBL modalities. Using a model system of negatively charged gold nanoparticles and positively charged poly(diallyldimethylammonium) chloride, the material stability, nanoscale control over thickness and particle coverage offered by the inkjet LBL technique are shown to be equal or better than the multilayers made with traditional dipping cycles. The opportunity for fast deposition of complex metallic patterns using a simple inkjet printer was also shown. The additive nature of LBL deposition based on the formation of insoluble nanoparticle-polyelectrolyte complexes of various compositions provides an excellent opportunity for versatile, multi-component, and non-contact patterning for the simple production of stratified patterns that are much needed in advanced devices.
layer-by-layer deposition; inkjet; patterning; nanoparticles; nanocomposites; flexible electronics
This work presents the use of nanoporous anodic aluminium oxide [AAO] for reflective interferometric sensing of volatile sulphur compounds and hydrogen sulphide [H2S] gas. Detection is based on changes of the interference signal from AAO porous layer as a result of specific adsorption of gas molecules with sulphur functional groups on a gold-coated surface. A nanoporous AAO sensing platform with optimised pore diameters (30 nm) and length (4 µm) was fabricated using a two-step anodization process in 0.3 M oxalic, followed by coating with a thin gold film (8 nm). The AAO is assembled in a specially designed microfluidic chip supported with a miniature fibre optic system that is able to measure changes of reflective interference signal (Fabry-Perrot fringes). When the sensor is exposed to a small concentration of H2S gas, the interference signal showed a concentration-dependent wavelength shifting of the Fabry-Perot interference fringe spectrum, as a result of the adsorption of H2S molecules on the Au surface and changes in the refractive index of the AAO. A practical biomedical application of reflectometric interference spectroscopy [RIfS] Au-AAO sensor for malodour measurement was successfully shown. The RIfS method based on a nanoporous AAO platform is simple, easy to miniaturise, inexpensive and has great potential for development of gas sensing devices for a range of medical and environmental applications.
nanoporous alumina; reflectometric interference spectroscopy; volatile sulphur compounds; hydrogen sulphide sensor; oral malodour
We describe protein- and oligonucleotide-loaded layer-by-layer (LbL)-assembled multiplayer films incorporating a hydrolytically degradable polymer for transcutaneous drug or vaccine delivery. Films were constructed based on electrostatic interactions between a cationic poly(β-amino ester) (denoted Poly-1) with a model protein antigen, ovalbumin (ova), and/or immunostimulatory CpG (Cytosine–phosphate diester—Guanine rich) DNA oligonucleotide adjuvant molecules. Linear growth of nanoscale Poly-1/ova bilayers was observed. Dried ova protein-loaded films rapidly deconstructed when rehydrated in saline solutions, releasing ova as non-aggregated/non-degraded protein, suggesting that the structure of biomolecules integrated into these multilayer films are preserved during release. Using confocal fluorescence microscopy and an in vivo murine ear skin model, we demonstrated delivery of ova from LbL films into barrier-disrupted skin, uptake of the protein by skin-resident antigen-presenting cells (Langerhans cells), and transport of the antigen to the skin-draining lymph nodes. Dual incorporation of ova and CpG oligonucleotides into the nanolayers of LbL films enabled dual release of the antigen and adjuvant with distinct kinetics for each component; ova was rapidly released while CpG was released in a relatively sustained manner. Applied as skin patches, these films delivered ova and CpG to Langerhans Cells in the skin. To our knowledge, this is the first demonstration of LbL films applied for the delivery of biomolecules into skin. This approach provides a new route for storage of vaccines and other immunotherapeutics in a solid-state thin film for subsequent delivery into the immunologically-rich milieu of the skin.
layer-by-layer; transcutaneous delivery; vaccine; polymer assembly; biodegradable
A study of reflective interference spectroscopy [RIfS] properties of nanoporous anodic aluminium oxide [AAO] with the aim to develop a reliable substrate for label-free optical biosensing is presented. The influence of structural parameters of AAO including pore diameters, inter-pore distance, pore length, and surface modification by deposition of Au, Ag, Cr, Pt, Ni, and TiO2 on the RIfS signal (Fabry-Perot fringe) was explored. AAO with controlled pore dimensions was prepared by electrochemical anodization of aluminium using 0.3 M oxalic acid at different voltages (30 to 70 V) and anodization times (10 to 60 min). Results show the strong influence of pore structures and surface modifications on the interference signal and indicate the importance of optimisation of AAO pore structures for RIfS sensing. The pore length/pore diameter aspect ratio of AAO was identified as a suitable parameter to tune interferometric properties of AAO. Finally, the application of AAO with optimised pore structures for sensing of a surface binding reaction of alkanethiols (mercaptoundecanoic acid) on gold surface is demonstrated.
nanoporous alumina; reflective interference spectroscopy; interference spectrum; optical label-free biosensing
We present the hydrolytically degradable polymeric multilayer films that can co-deliver multiple therapeutics of differing chemical characteristics (charged biomacromolecules and neutral hydrophobic small molecules) from a surface. This multi-agent-delivery (MAD) nanolayer system integrates the hydrolytically degradable poly(β-amino ester) as a structural component to control the degradation of the multilayers to release active therapeutic macromolecules, as well as hydrophobic drugs imbedded within amphiphilic block copolymer micellar carriers within layer-by-layer (LbL) films, which would otherwise be difficult to include within the multilayers. By varying the anionic therapeutic agents (heparin and dextran sulfate) within the multilayer, we examine how different structural components can be used to control the release kinetics of multiple therapeutics from MAD nanolayers. Controlled release profiles and the in vitro efficacy of the MAD nanolayers in suppressing the growth of human smooth muscle cell lines were evaluated. The dual delivery of a charged macromolecular heparin and a small hydrophobic drug, paclitaxel, is found to be synergistic and beneficial toward effective therapeutic activity. Furthermore, we compared the classical dipping method we employed here with an automated spray-LbL technique. Spray-LbL significantly facilitates film processing time while preserving the characteristic release profiles of the MAD nanolayers. With the highly versatile and tunable nature of LbL assembly, we anticipate that MAD nanolayers can provide a unique platform for delivering multiple therapeutics from macromolecular to small molecules with distinct release profiles for applications in biological and biomedical surface coatings.
Layer-by-layer assembly; Drug delivery; Degradable polymer; Multicomponent assembly; Polymer micelle; Surface coating
Hyaluronic acid (HA), an anionic polysaccharide, is one of the major components of the natural extracellular matrix (ECM). Although HA has been widely used for tissue engineering applications, it does not support cell attachment and spreading and needs chemical modification to support cellular adhesion. Here, we present a simple approach to functionalize photocrosslinked HA hydrogels by deposition of poly(L-lysine) (PLL) and HA multilayer films made by the layer-by-layer (LbL) technique. PLL/HA multilayer film formation was assessed by using fluorescence microscopy, contact angle measurements, cationic dye loading and confocal microscopy. The effect of polyelectrolyte multilayer film formation on the physicochemical and mechanical properties of hydrogels revealed polyelectrolyte diffusion inside the hydrogel pores, increased hydrophobicity of the surface, reduced equilibrium swelling, and reduced compressive moduli of the modified hydrogels. Furthermore, NIH-3T3 fibroblasts seeded on the surface showed improved cell attachment and spreading on the multilayer functionalized hydrogels. Thus, modification of HA hydrogel surfaces with multilayer films affected their physicochemical properties and improved cell adhesion and spreading on these surfaces. This new hydrogel/PEM composite system may offer possibilities for various biomedical and tissue engineering applications, including growth factor delivery and co-culture systems.
Hydrogels; Hyaluronic acid; Photocrosslinked; Surface functionalization; Layer-by-layer; Polyelectrolyte diffusion; Cell adhesion
Electrostatically driven layer-by-layer (LbL) assembly is a simple and robust method for producing structurally tailored thin film biomaterials, of thickness ca. 10 nanometers, containing biofunctional ligands. We investigate the LbL formation of multilayer films composed of polymers of biological origin (poly(L-lysine) (PLL) and dextran sulfate (DS)), the adsorption of fibronectin (Fn) - a matrix protein known to promote cell adhesion - onto these films, and the subsequent spreading behavior of human umbilical vein endothelial cells (HUVEC). We employ optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microgravimetry with dissipation (QCMD) to characterize multilayer assembly in situ, and find adsorbed Fn mass on PLL terminated films to exceed that on DS terminated films by 40%, correlating with the positive charge and lower degree of hydration of PLL terminated films. The extent and initial rate of Fn adsorption to both PLL and DS terminated films exceed those onto the bare substrate, indicating the important role of electrostatic complexation between negatively charged protein and positively charged PLL at or near the film surface. We use phase contrast optical microscopy to investigate the time dependent morphological changes of HUVEC as a function of layer number, charge of terminal layer, and the presence of Fn. We observe HUVEC to attach, spread, and lose circularity on all surfaces. (Positively charged) PLL terminated films exhibit a greater extent of cell spreading than do (negatively charged) DS terminated films, and spreading is enhanced while circularity loss is suppressed by the presence of adsorbed Fn. The number of layers plays a significant role only for DS terminated films with Fn, where spreading on a bilayer greatly exceeds that on a multilayer, and PLL terminated films without Fn, where initial spreading is significantly higher on a monolayer. We observe initial cell spreading to be followed by retraction (i.e. decreased cell area and circularity with time) for films without Fn, and for DS terminated films with Fn. Overall, the Fn coated PLL monolayer and the Fn coated PLL terminated multilayer are the best performing films in promoting cell spreading. We conclude the presence of Fn to be an important factor (more so than film charge or layer number) in controlling the interaction between multilayer films and living cells, and thus to represent a promising strategy toward in vivo applications such as tissue engineering.
fibronectin; poly(L-lysine); dextran sulfate; protein adsorption; endothelial cell; layer-by-layer
This paper demonstrates the generation of systemically deliverable layer-by-layer (LbL) nanoparticles for cancer applications. LbL-based nanoparticles designed to navigate the body and deliver therapeutics in a programmable fashion are promising new and alternative systems for drug delivery; but there have been very few demonstrations of their systemic delivery in vivo due to a lack of knowledge in building LbL nanofilms that mimic traditional nanoparticle design to optimize delivery. The key to the successful application of these nanocarriers in vivo requires a systematic analysis of the influence of film architecture and adsorbed polyelectrolyte outer layer on their pharmacokinetics, which has thus far not been examined for this new approach to nanoparticle delivery. Herein, we have taken the first steps in stabilizing and controlling the systemic distribution of multilayer nanoparticles. Our findings highlight the unique character of LbL systems: the electrostatically assembled nanoparticles gain increased stability in vivo with larger numbers of deposited layers, and the final layer adsorbed generates a critical surface cascade, which dictates the surface chemistry and biological properties of the nanoparticle. This outer polyelectrolyte layer dramatically affects not only the degree of nonspecific particle uptake, but also the nanoparticle biodistribution. For hyaluronic acid (HA) outer layers, a long blood elimination half-life (~9 h) and low accumulation (~ 10–15 % recovered fluorescence/g) in the liver were observed, illustrating that these systems can be designed to be highly appropriate for clinical translation.
Layer-by-layer; Nanoparticles; Drug Delivery; Biomaterials
Nanoporous anodic aluminum oxide (AAO) has been explored for various applications due to its regular cell arrangement and relatively easy fabrication processes. However, conventional two-step anodization based on self-organization only allows the fabrication of a few discrete cell sizes and formation of small domains of hexagonally packed pores. Recent efforts to pre-pattern aluminum followed with anodization significantly improve the regularity and available pore geometries in AAO, while systematic study of the anodization condition, especially the impact of acid composition on pore formation guided by nanoindentation is still lacking. In this work, we pre-patterned aluminium thin films using ordered monolayers of silica beads and formed porous AAO in a single-step anodization in phosphoric acid. Controllable cell sizes ranging from 280 nm to 760 nm were obtained, matching the diameters of the silica nanobead molds used. This range of cell size is significantly greater than what has been reported for AAO formed in phosphoric acid in the literature. In addition, the relationships between the acid concentration, cell size, pore size, anodization voltage and film growth rate were studied quantitatively. The results are consistent with the theory of oxide formation through an electrochemical reaction. Not only does this study provide useful operational conditions of nanoindentation induced anodization in phosphoric acid, it also generates significant information for fundamental understanding of AAO formation.
Anodic aluminum oxide; Phosphoric acid; Anodization; Imprinting; Silica nanobeads
Background and the purpose of the study
Layer-by-layer (LbL) deposition of polyelectrolytes (PEs) has received a great attention in the area of drug delivery due to its simplicity and versatility. This research was aimed to develop multilayered microcapsules through LbL deposition of chitosan (CHI) and sodium alginate (NaALG) and utilize them as vehicle for controlled delivery of isoniazid (INH).
CaCO3 particles, prepared by colloidal crystallization of CaCl2 and Na2CO3 solutions, were used as micro-templates for LbL deposition of CHI and NaALG. Subsequent to the deposition, templates were decomposed to obtain hollow microcapsules. Prepared microcapsules were subjected to physicochemical evaluations, drug release and stability studies.
Results and major conclusion
Though CaCO3 particles possessed a rough and irregular surface, prepared hollow microcapsules were spherical in shape, having smooth surface and regular thickness. Following deposition of each layer, alternating values of zeta potential were observed, indicating the formation of multilayered films. Microcapsules with 5 bilayers, i.e. (CHI/NaALG)5 provided 39% entrapment efficiency and exhibited a controlled release behavior, lasting up to 24 hrs. An improvement in drug release rate and stability profile of the formulation was observed by increasing the number of deposition steps and performing the crosslinking of polyelectrolytes. This study showed that the prepared formulation could promisingly be utilized as controlled delivery vehicle for INH.
Layer-by-layer; Polyelectrolyte; Multilayers microcapsules; Controlled delivery
A universal nitric oxide (NO) generating surface is assembled via Layer-by-Layer (LbL) deposition of sodium alginate (Alg) and organoselenium modified polyethyleneimine (SePEI) on quartz and polymeric substrates. The immobilized SePEI species is capable of catalytically decomposing S-nitrosothiol species (RSNO) to NO in the presence of thiol reducing agents (e.g., glutathione, cysteine, etc.). The stepwise buildup of the multilayer films is monitored by UV-Vis spectroscopy, SEM and surface contact angle measurements. X-ray photoelectron spectroscopy is used to study the stoichiometry between the polyanion and polycation, and also the presence of Se in the catalytic LbL film. A reductive annealing process is necessary to improve the stability of freshly coated multilayer films via chain rearrangement. Chemiluminescence measurements illustrate the ability of the LbL films to generate NO from S-nitrosoglutathione (GSNO) in the presence of S-glutathione (GSH). Enhanced NO fluxes can be achieved by increasing the number of catalytic (SePEI/Alg) bilayers coated on the substrates. Nitric oxide generation is observed even after prolonged contact with sheep whole blood. Preliminary applications of this LbL on silicone rubber tubings and polyurethane catheters reveal similar NO generation behavior from these biomedical grade polymeric substrates.
Electrochemical deposition of crosslinked oxo-cyanoruthenate, Ru-O/CN-O, from a mixture of RuCl3 and K4Ru(CN)6 is known to yield a film on glassy carbon that promotes oxidations by a combination of electron and oxygen transfer. Layer-by-layer (LbL) deposition of this species and of a film formed by cycling of the electrode potential in a ZrO2 solution systematically increases the number of catalytically active sites of the Ru-O/CN-O on the electrode. The evaluation of the electrocatalytic activity was by cyclic voltammetric oxidation of cysteine at pH 2. Plots of the anodic peak current vs. the square root of scan rate were indicative of linear diffusion control of this oxidation, even in the absence of ZrO2, but the slopes of these linear plots increased with bilayer number, n, of (ZrO2 | Ru-O/CN-O)n. The latter observation is hypothesized to be due to an increased number of active sites for a given geometric electrode area, but proof required further study. To optimize utilization of the catalyst and to provide a size-exclusion characteristic to the electrode, the study was extended to LbL deposition of the composite in 50-nm pores of an organically modified silica film deposited by electrochemically assisted sol-gel processing using surface-bound poly(styrene sulfonate) nanospheres as a templating agent.
Electrocatalysis; Layer-by-layer assembly; Sol-gel processing; Cysteine oxidation
A promising strategy to accelerate joint implant integration and reduce recovery time and failure rates is to deliver a combination of certain growth factors to the integration site. There is a need to control the quantity of growth factors delivered at different times during the healing process to maximize efficacy. Polyelectrolyte multilayer (PEM) films, built using the layer-by-layer (LbL) technique, are attractive for releasing controlled amounts of potent growth factors over a sustained period. Here, we present PEM films that sequester physiological amounts of osteogenic rhBMP-2 (recombinant human bone morphogenetic protein - 2) and angiogenic rhVEGF165 (recombinant human vascular endothelial growth factor) in different ratios in a degradable [poly(β-amino ester)/polyanion/growth factor/ polyanion] LbL tetralayer repeat architecture where the biologic load scaled linearly with the number of tetralayers. No burst release of either growth factor was observed as the films degraded. The release of rhBMP-2 was sustained over a period of 2 weeks, while rhVEGF165 eluted from the film over the first 8 days. Both growth factors retained their efficacy, as quantified with relevant in vitro assays. rhBMP-2 initiated a dose dependent differentiation cascade in MC3T3-E1S4 pre-osteoblasts while rhVEGF165 upregulated HUVEC proliferation, and accelerated closure of a scratch in HUVEC cell cultures in a dose dependent manner. In vivo, the mineral density of ectopic bone formed de novo by rhBMP-2/rhVEGF165 PEM films was approximately 33% higher than when only rhBMP-2 was introduced, with a higher trabecular thickness, which would indicate a decrease in the risk of osteoporotic fracture. Bone formed throughout the scaffold when both growth factors were released, which suggests more complete remodeling due to an increased local vascular network. This study demonstrates a promising approach to delivering precise doses of multiple growth factors for a variety of implant applications where control over spatial and temporal release profile of the biologic is desired.
Controlled drug release; BMP; VEGF; bone; hip replacement prosthesis; layer-by-layer; polyelectrolyte multilayer; dose response
By making use of an e-beam deposition system, the [Co(2 Å)/Pd(10 Å)]15 multilayers were prepared on a Si(100) substrate and anodized aluminum oxide [AAO] templates with average pore diameters of around 185, 95, and 40 nm. The mechanism of magnetization reversal of the Co/Pd multilayers was investigated. Wall motion was observed on the Co/Pd multilayers grown on the Si substrate. A combination of wall motion and domain rotation was found in the sample grown on the AAO template with a 185-nm pore diameter. For the samples grown on the AAO templates with pore diameters of around 95 and 40 nm, the reversal mechanism was dominated by domain rotation. The rotational reversal was mainly contributed from the underlying nanoporous AAO templates that provided an additional pinning effect.
PACS: 75.30.Gw, magnetic anisotropy; 78.67.Rb, nanoporous materials; 75.60.Jk, magnetization reversal mechanisms.
Co/Pd; porous anodized aluminum oxide; magnetization reversal
Titanium surface modification is crucial to improving its bioactivity, mainly its bone binding ability in bone implant materials. In order to functionalize titanium with small interfering RNA (siRNA) for sustained gene silencing in nearby cells, the layer-by-layer (LbL) approach was applied using sodium hyaluronate and chitosan/siRNA (CS/siRNA) nanoparticles as polyanion and polycation, respectively, to build up the multilayered film on smooth titanium surfaces. The CS/siRNA nanoparticle characterization was analyzed first. Dynamic contact angle, atomic force microscopy, and scanning electron microscopy were used to monitor the layer accumulation. siRNA loaded in the film was quantitated and the release profile of film in phosphate-buffered saline was studied. In vitro knockdown effect and cytotoxicity evaluation of the film were investigated using H1299 human lung carcinoma cells expressing green fluorescent protein (GFP). The transfection of human osteoblast-like cell MG63 and H1299 were performed and the osteogenic differentiation of MG63 on LbL film was analyzed. The CS/siRNA nanoparticles exhibited nice size distribution. During formation of the film, the surface wettability, topography, and roughness were alternately changed, indicating successful adsorption of the individual layers. The scanning electron microscope images clearly demonstrated the hybrid structure between CS/siRNA nanoparticles and sodium hyaluronate polymer. The cumulated load of siRNA increased linearly with the bilayer number and, more importantly, a gradual release of the film allowed the siRNA to be maintained on the titanium surface over approximately 1 week. In vitro transfection revealed that the LbL film-associated siRNA could consistently suppress GFP expression in H1299 without showing significant cytotoxicity. The LbL film loading with osteogenic siRNA could dramatically increase the osteogenic differentiation in MG63. In conclusion, LbL technology can potentially modify titanium surfaces with specific gene-regulatory siRNAs to enhance biofunction.
sustained gene silencing; osteogenic differentiation; chitosan; small interfering RNA; titanium
The layer-by-layer (LbL) assembly of polyelectrolyte pairs on temperature and pH-sensitive cross-linked poly(N-isopropylacrylamide)-co-(methacrylic acid), poly(NIPAAm-co-MAA), microgels enabled a fine tuning of the gel swelling and responsive behavior according to the mobility of the assembled polyelectrolyte (PE) pair and the composition of the outermost layer. Microbeads with well-defined morphology were initially prepared by synthesis in supercritical carbon dioxide. Upon LbL assembly of polyelectrolytes, interactions between the multilayers and the soft porous microgel led to differences in swelling and thermoresponsive behavior. For the weak PE pairs, namely poly(L-lysine) / poly(L-glutamic acid) and poly(allylamine hydrochloride) / poly(acrylic acid), polycation-terminated microgels were less swollen and more thermoresponsive than native microgel; while polyanion-terminated microgels were more swollen and not significantly responsive to temperature, in a quasi-reversible process with consecutive PE assembly. For the strong PE pair, poly(diallyldimethylammonium chloride) / poly(sodium styrene sulfonate), the differences among polycation and polyanion-terminated microgels are not sustained after the first PE bilayer due to extensive ionic cross-linking between the polyelectrolytes. The tendencies across the explored systems became less noteworthy in solutions with larger ionic strength due to overall charge shielding of the polyelectrolytes and microgel. ATR FT-IR studies correlated the swelling and responsive behavior after LbL assembly on the microgels with the extent of H-bonding and alternating charge distribution within the gel. Thus, the proposed LbL strategy may be a simple and flexible way to engineer smart microgels in terms of size, surface chemistry, overall charge and permeability.
Layer-by-layer (LbL) films have multiple features which make them attractive for drug delivery including the possibility of sequential delivery of growth factors, however, to date, proof of concept has been lacking for protein delivery from such films. Here, LbL polyelectrolyte films constructed with lysozyme (a model protein) and a hydrolytically degradable and biocompatible synthetic polycation are shown to be capable of release attractive for the localized delivery of therapeutic proteins from implanted medical devices. Milligram/cm2 scale release with power law or linear profile can be achieved over 3 weeks to 3 months at room temperature. The release rate at 37°C increases in a way that is compatible with a surface erosion mechanism of release from 100 days to 5 days. This time scale of release can be tuned by changing the degradability of the synthetic polycation, and an increase to 34 days of release at 37°C is seen by increasing the hydrophobicity of this degradable polyester. The enzyme released from these films retains 80–100% functionality, underscoring the mild processing conditions that are apt to preserve fragile protein function. These results uncover many possibilities for incorporation of therapeutic proteins to modulate the interaction between implanted surfaces and the cells they contact.
Multilayer nanofilms, formed by the layer-by-layer (LbL) adsorption of positively and negatively charged polyelectrolytes, are promising substrates for tissue engineering. We investigate here the attachmemt and function of hepatic cells on multilayer films in terms of film composition, terminal layer, rigidity, charge, and presence of biofunctional species. Human hepatocellular carcinoma cells (HepG2), adult rat hepatocytes (ARH), and human fetal hepatoblasts (HFHb) are studied on films composed of the polysaccharides chitosan (CHI) and alginate (ALG), the polypeptides poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA), and the synthetic polymers poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS). The influence of chemical cross-linking following LbL assembly is also investigated. We find HepG2 to reach confluence after seven days of culture on only 2 of 18 candidate multilayer systems: (PAH-PSS)n (i.e. n PAH-PSS bilayers) and cross-linked (PLL-ALG)n-PLL. These two systems, as well as cross-linked (PLL-PGA)n-PLL, support attachment and function (in terms of albumin production) of ARH, provided collagen is adsorbed to the top of the film. (PAH-PSS)n, cross-linked (PLL-ALG)n, and cross-linked (PLL-PGA)n-PLL films all support attachment, layer confluence, and function of HFHb, with the latter film promoting the greatest level of function at 8 days. Overall, film composition, terminal layer, and rigidity are key variables in promoting attachment and function of hepatic cells, while film charge and biofunctionality are somewhat less important. These studies reveal optimal candidate multilayer biomaterials for human liver tissue engineering applications.
layer-by-layer; multilayer film; nanofilm biomaterial; hepatocyte; liver
An inorganic short chain polymer, poly(sodium phosphate), PSP, together with poly(allylamine hydrochloride), PAH, is used to fabricate layer-by-layer (LbL) films. The thickness, roughness, contact angle, and optical transmittance of these films are studied depending on three parameters: the precursor solution concentrations (10-3 and 10-4 M), the number of bilayers deposited (20, 40, 60, 80, and 100 bilayers), and the specific technique used for the LbL fabrication (dipping or spraying). In most cases of this experimental study, the roughness of the nanofilms increases with the number of bilayers. This contradicts the basic observations made in standard LbL assemblies where the roughness decreases for thicker coatings. In fact, a wide range of thickness and roughness was achieved by means of adjusting the three parameters mentioned above. For instance, a roughness of 1.23 or 205 nm root mean square was measured for 100 bilayer coatings. Contact angles close to 0 were observed. Moreover, high optical transmittance is also reported, above 90%, for 80 bilayer films fabricated with the 10-4 M solutions. Therefore, these multilayer structures can be used to obtain transparent superhydrophilic surfaces.
Layer-by-layer; Dipping and spray deposition; Inorganic polymer; Hydrophilic film; Functionalized surfaces
In this work, we report an electrochemical surface plasmon resonance/waveguide (EC-SPR/waveguide) glucose biosensor, which could detect enzymatic reactions in a conducting polymer/glucose oxidase (GOx) multilayer thin film. In order to achieve a controlled enzyme electrode and waveguide mode, GOx (negatively charged) was immobilized with a water-soluble conducting N-alkylaminated polypyrrole (positively charged) using the layer-by-layer (LbL) electrostatic self-assembly technique. The electrochemical and optical signals were simultaneously obtained from the composite LbL enzyme electrode upon addition of glucose as mediated by the electroactivity and electrochromic property of the polypyrrole layers. The signal enhancement in the EC-SPR detection is obtained by monitoring the doping-dedoping events on the polypyrrole. The real time optical signal could be distinguished between the change in the dielectric constant of the enzyme layer and other non-enzymatic reaction events such as adsorption of glucose and change of refractive index of solution. This was possible by a correlation of both the SPR mode, m=0, and m=1 mode of the waveguide in an SPR/waveguide spectroscopy experiment.
surface plasmon; glucose; biosensor; conducting polymer; enzyme; waveguide
Complex coacervates prepared from poly-Aspartic acid (polyAsp) and poly-L-Histidine (polyHis) were investigated as models of the metastable protein phases used in the formation of biological structures such as squid beak. When mixed, polyHis and polyAsp form coacervates whereas poly-L-Glutamic acid (polyGlu) forms precipitates with polyHis. Layer-by-layer (LbL) structures of polyHis-polyAsp on gold substrates were compared with those of precipitate-forming polyHis-polyGlu by monitoring with iSPR and QCM-D. PolyHis-polyAsp LbL was found to be stiffer than polyHis-polyGlu LbL with most water evicted from the structure but with sufficient interfacial water remaining for molecular rearrangement to occur. This thin layer is believed to be fluid and like preformed coacervate films, capable of spreading over both hydrophilic ethylene glycol as well as hydrophobic monolayers. These results suggest that coacervate-forming polyelectrolytes deserve consideration for potential LbL applications and point to LbL as an important process by which biological materials form.
Complex coacervates; biomaterials; iSPR; QCM; polyelectrolytes; Layer-by-layer
A simple approach to the mechanical modulation of layer-by-layer (LbL) films is through manipulation of the film assembly. Here, we report results based on altering the salt concentration during film assembly and its effect on film rigidity. Based on changes in film rigidity, cell adhesion characteristics and transfection activity were investigated in vitro. LbL films consisting of reducible hyperbranched poly(amide amine) (RHB) have been implemented along with DNA for investigating fibroblast adhesion on [RHB/DNA]n/2 films with varying rigidities. The rigidity was varied by changing the ionic concentration of the deposition solution between 0.01 M NaCl and 1.0 M NaCl. Molecular force probe (MFP) measurements were performed to measure the apparent Young’s modulus, EAPP, of the films in situ. Cell adhesion and stress-fiber characteristics were investigated using total internal reflectance microscopy (TIRF-M). The average cell peripheral area, fiber density and average fiber length during 5 days of cell growth on films with either low (below 2.0 MPa) or high (above 2.0 MPa) film elastic modulus were investigated. Transfection studies were performed using gfpDNA and SEAP-DNA to investigate if changes in cell adhesion affect transfection activity. Furthermore, cell proliferation and cytotoxicity studies were used to investigate cellular viability over a week. The results have shown that surface modification of bioreducible LbL films of controlled thickness and roughness promotes cellular adhesion, stress-fiber growth and increased transfection activity without the need for an additional adhesive protein pre-coating of the surface or chemical cross-linking of the film.
molecular force probe; MFP; cell adhesion; transfection; TIRF-M; stress fiber orientation; layer-by-layer; LbL; mechanical properties; thin films; elastic modulus; film rigidity
Polyelectrolyte multilayers built via the layer-by-layer (LbL) method has been one of the most promising systems in the field of materials science. Layered structures can be constructed by the adsorption of various polyelectrolyte species onto the surface of a solid or liquid material by means of electrostatic interaction. The thickness of the adsorbed layers can be tuned precisely in the nanometer range. Stable, semiconducting thin films are interesting research subjects. We use a conducting polymer, poly(p-phenylene vinylene) (PPV), in the preparation of a stable thin film via the LbL method. Cyclic voltammetry and electrochemical impedance spectroscopy have been used to characterize the ionic conductivity of the PPV multilayer films. The ionic conductivity of the films has been found to be dependent on the polymerization temperature. The film conductivity can be fitted to a modified Randle’s circuit. The circuit equivalent calculations are performed to provide the diffusion coefficient values.
electrochemical impedance spectroscopy; cyclic voltammetry; layer-by-layer deposition; poly(p-phenylene vinylene); modified Randle’s circuit