Biomedical applications of electrostatic layer-by-layer nano-assembly of polymers,enzymes, and nanoparticles

The introduction of electrostatic layer-by-layer (LbL) self-assembly has shown broad biomedical applications in thin film coating, micropatterning, nanobioreactors, artificial cells, and drug delivery systems. Multiple assembly polyelectrolytes and proteins are based on electrostatic interaction between oppositely charged layers. The film architecture is precisely designed and can be controlled to 1-nm precision with a range from 5 to 1000 nm. Thin films can be deposited on any surface including many widely used biomaterials. Microencapsulation of micro/nanotemplates with multilayers enabled cell surface modification, controlled drug release, hollow shell formation, and nanobioreactors. Both in vitro and in vivo studies indicate potential applications in biology, pharmaceutics, medicine, and other biomedical areas.     

Enhanced resistance of polyelectrolyte multilayer microcapsules to pepsin erosion and release properties of encapsulated indomethacin

Polyelectrolyte multilayer films were prepared through layer-by-layer (LbL) self-assembly using polysaccharide sodium alginate (ALG) and chitosan (CHI). After incubation in an enzyme pepsin solution, the multilayer film was partially destroyed as detected by the decrease in fluorescent intensity because of the enzymatic degradation of CHI. The enzymatic desorption was also observed from the microcapsule wall made of the ALG/CHI multilayer film directly deposited on indomethacin (IDM) microcrystals through LbL self-assembly. After pepsin erosion, the IDM release from the microcapsules monitored by UV absorbance was obviously accelerated because of desorption. To enhance the stability of the ALG/CHI multilayer film to the enzymatic erosion, some physical and chemical methods were established to increase film thickness or to cross-link the polysaccharides within the film. Increasing the layer number and raising the deposition temperature effectively slowed down the enzymatic desorption and release rate. Especially, increasing deposition temperature was more effective because of producing a more perfect structure in the ALG/CHI multilayer film. Cross-linking the neighboring layers of ALG and CHI with 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide in the ALG/CHI multilayer film significantly reduced the enzymatic desorption and release rate. Therefore, increasing deposition temperature and cross-linking neighboring layers are effective methods to protect the multilayer film fabricated using LbL assembly from the enzymatic erosion and to prolong the release of the encapsulated drug.     

Structural stability of polypeptide nanofilms under extreme conditions

Self-assembly of designed peptides is a promising area of biomaterials research and development. Here, polypeptide nanofilms have been prepared by electrostatic layer-by-layer self-assembly (LBL) of cysteine (Cys)-containing 32mers designed to be oppositely charged at neutral pH, and structural stability of the films has been probed by subjecting them to various extreme physical and chemical conditions. The results suggest that although electrostatic attraction plays a key role in strengthening polypeptide films, stability is inversely related to absolute net charge of the supramolecular complex. This behavior is similar to the typical behavior of small globular proteins. Film structure is very stable in organic solvent and, when dehydrated, at extreme temperatures. Such stability is in marked contrast to the behavior of proteins, which tend to denature under comparable conditions. Similar to proteins, peptide nanofilms cross-linked by disulfide (S-S) bonds are considerably stronger than films stabilized by electrostatic, van der Waals, or hydrophobic interactions alone. This effect is particularly evident at extremes of pH and at elevated temperature when the film is hydrated. These results, the great variety of possible peptide structures, the inherent biocompatibility of l-amino acids, and current applications of thin films in commercial products together suggest that polypeptide films are promising for the development of new or enhanced products in food technology, drug delivery and medical device coatings, and biomaterials.     

Thermally modulated insulin release from microgel thin films

We describe investigations of thermally triggered insulin release from poly(N-isopropylacrylamide-co-acrylic acid) microgel thin films prepared by layer-by-layer (LbL) polyelectrolyte assembly. The thermoresponsivity of these films was confirmed using light scattering techniques. Simultaneous monitoring of film collapse and insulin release kinetics shows that deswelling of the films is partially decoupled from macromolecule release and that release is mainly governed by partitioning effects. We hypothesize, however, that film thermoresponsivity plays an important role in that subjection to many thermal cycles enables the embedded peptide to solubilize and subsequently partition through film layers. Direct pulsatile and extended release studies confirm the capability of these films to release bursts of insulin over many cycles, and confirm that the magnitude of the release can be controlled based on film thickness. These insulin-impregnated films are extremely stable with the potential to release constant pulses of peptide for more than 1 month at a time.     

Versatile bioelectronic interfaces on flexible non-conductive substrates.

Bioelectronic interfaces that establish electrical communication between redox enzymes and electrodes have potential applications as biosensors, biocatalytic reactors, and biological fuel cells. These interfaces are commonly formed on gold films deposited using physical vapor deposition (PVD) or chemical vapor deposition (CVD). PVD and CVD require deposition of a primer layer, such as titanium or chromium, and require the use of expensive equipment and cannot be used on a wide range of substrates. This paper describes a versatile new bench-top method to form bioelectronic interfaces containing a gold film, electron mediator, cofactor, and dehydrogenase enzyme (secondary alcohol dehydrogenase, and sorbitol dehydrogenase) on nonconductive substrates such as polystyrene and glass. The method combines layer-by-layer deposition of polyelectrolytes, electroless metal deposition, and directed molecular self-assembly. Cyclic voltammetry, chronoamperometry, field emission X-ray dispersive spectroscopy, scanning electron microscopy, and atomic force microscopy were used to characterize the bioelectronic interfaces. Interfaces formed on flexible polystyrene slides were shown to retain their activity after bending to a radius of curvature of 18mm, confirming that the approach can be applied on cheap and flexible substrates for applications where traditional wafer-scale electronics is not suitable, such as personal or structural health monitors and rolled microtube biosensors.     

Fine tuning of physical properties of designed polypeptide multilayer films by control of pH

Adjustment of pH can alter the ensemble of three-dimensional structures of a polypeptide in solution by changing the distribution of charge and Coulombic interactions. The role of pH in layer-by-layer self-assembly (LbL) of designed 32mer peptides containing the amino acid cysteine has been investigated using a combination of physical methods. Results show that pH can have a substantial influence on the mass of adsorbed peptide, surface roughness, and film density over a range of 1.5 pH units. Peptide film thickness depends on the number of layers, as with "conventional" polyelectrolytes. Film density and morphology, however, vary more with pH than does thickness, translating into a change in density on the order of 70% over the pH range 7.4-8.9. Results of this work provide insight on the physical basis of LbL and suggest that peptides are a promising class of polyelectrolytes for the creation of designer thin films for applications in biotechnology and other areas.     

Multilayer biomimetics: reversible covalent stabilization of a nanostructured biofilm

Designed polypeptides and electrostatic layer-by-layer self-assembly form the basis of promising research in bionanotechnology and medicine on development of polyelectrolyte multilayer films (PEMs). We show that PEMs can be formed from oppositely charged 32mers containing several cysteine residues. The polypeptides in PEMs become cross-linked under mild oxidizing conditions. This mimicking of disulfide (S-S) bond stabilization of folded protein structure confers on the PEMs a marked increase in resistance to film disassembly at acidic pH. The reversibility of S-S bond stabilization of PEMs presents further advantages for controlling physical properties of films, coatings, and other applications involving PEMs.     

Self-assembly of monoglycerides in beta-lactoglobulin adsorbed films at the air-water interface. Structural, topographical, and rheological consequences

In this work, we have analyzed the structural, topographical, and surface dilatational characteristics of pure beta-lactoglobulin adsorbed films and the effect of the self-assembly of monoglycerides (monopalmitin or monoolein) in beta-lactoglobulin films at the air-water interface. Measurements were performed in a single device that incorporates a Wilhelmy-type film balance, Brewster angle microscopy, and interfacial dilatational rheology. The structural and topographical characteristics of beta-lactoglobulin adsorbed and spread films are similar. However, the surface dilatational modulus of beta-lactoglobulin films shows a complex behavior depending on film formation. The self-assembly of monoglyceride in a beta-lactoglobulin adsorbed film has an effect on the structural, topographical, and dilatational properties of the mixed films, depending on the interfacial composition and the surface pressure (pi). At low pi, a mixed film of monoglyceride and beta-lactoglobulin may exist. At high pi (after the collapse of beta-lactoglobulin), the mixed films are dominated by monoglyceride molecules. However, the small amounts of collapsed beta-lactoglobulin have a significant effect on the surface dilatational properties of the mixed films. Protein displacement by monoglyceride is higher for monopalmitin than for monoolein. However, some degree of interaction exists between proteins and monoglycerides, and these interactions are more evident in adsorbed films than in spread films.     

Coating and selective deposition of nanofilm on silicone rubber for cell adhesion and growth

A recently developed method for surface modification, layer-by-layer (LbL) assembly, has been applied to silicone, and its ability to encourage endothelial cell growth and control cell growth patterns has been examined. The surfaces studied consisted of a precursor, with alternating cationic polyethyleneimine (PEI) and anionic sodium polystyrene sulfonate (PSS) layers followed by alternating gelatin and poly-d-lysine (PDL) layers. Film growth increased linearly with the number of layers. Each PSS/PEI bilayer was 3 nm thick, and each gelatin/PDL bilayer was 5 nm thick. All layers were more hydrophilic than the unmodified silicone rubber surface, as determined from contact angle measurements. The contact angle was primarily dictated by the outermost layer. Of the coatings studied, gelatin was the most hydrophilic. A film of (PSS/PEI)₄/(gelatin/PDL)₄/ gelatin was highly favorable for cell adhesion and growth, in contrast to films of (PSS/PEI)₈ or (PSS/PEI)₈/PSS. Cell growth patterns were successfully controlled by selective deposition of microspheres on silicone rubber, using microcontact printing with a silicone stamp. Cell adhesion was confined to the region of microsphere deposition. These results demonstrate that the LbL self-assembly technique provides a general approach to coat and selectively deposit films with nanometer thickness on silicone rubber. Furthermore, they show that this method is a viable technique for controlling cellular adhesion and growth.     

Electrochemical impedance detection of DNA hybridization based on dendrimer modified electrode

Bioactive ultrathin films with the incorporation of amino-terminated G4 PAMAM dendrimers have been prepared via layer-by-layer self-assembly methods on a gold electrode and used for the DNA hybridization analysis. Surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy (EIS) are used to characterize the successful construction of the multicomponent film on the gold substrate. The dendrimer-modified surfaces improve the immobilization capacity of the probe DNA greatly, compared to the AET (2-aminoethanethiol) SAM sensor surfaces without dendrimer molecules. DNA hybridization analysis is monitored by EIS. The dendrimer-based electrochemical impedance DNA biosensor shows high sensitivity and selectivity for DNA hybridization assay. The multicomponent films also display a high stability during repeated regeneration and hybridization cycles.     

Orientational order in surface layers of pullulan films

A study was made of spontaneous surface birefringence observed in pullulan films when a tilted polarized beam is passed through a plain polymer film. Birefringence was explored as a function of the angle of beam incidence and the film thickness. The orientational order parameter was estimated for pullulan chains located in the near-surface layers of films. The results were compared with data from earlier studies of other polysaccharides.     

Layer-by-layer films made from extracellular matrix macromolecules on silicone substrates

The layer-by-layer (LbL) technique has been widely used to produce nanofilms for biomedical applications. Naturally occurring polymers such as ECM macromolecules are attractive candidates for LbL film preparation. In this study, we assessed the build-up of type I collagen (Col1)/chondroitin sulfate (CS) or Col1/Heparin (HN) on polydimethylsiloxane (PDMS) substrates. The build-up was assessed by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). Integrin-mediated cell adhesion was assessed by studying the cytoskeletal organization of mammalian primary cells (chondrocytes) seeded on different end layers and number of layers. Data generated from the QCM-D observations showed a consistent build-up of films with more adsorption in the case of Col1/HN. Col1/CS films were stable in media, whereas Col1/HN films were not. AFM analysis showed that the layers were fibrillar in structure for both systems and between 20 and 30 nm thick. The films promoted cell adhesion when compared with tissue culture plastic in serum-free media with cycloheximide. Crosslinking of the films resulted in constrained cell spreading and a ruffled morphology. Finally, beta1 integrin blocking antibodies prevented cell spreading, suggesting that cell adhesion and spreading were mediated mainly by interaction with the collagen fibrils. The ability to construct stable ECM-based films on PDMS has particular relevance in mechanobiology, microfluidics, and other biomedical applications.     

Light-induced storage in layer-by-layer films of chitosan and an azo dye

The buildup of layer-by-layer (LBL) films from chitosan and the azodye Ponceau-S (PS) was investigated under various experimental conditions, and the resulting films were used in optical storage experiments. The kinetics for the writing process in optical storage was faster for LBL films prepared at low pHs, probably because the films had a larger free volume for isomerization of the chromophores. The nanostructured nature of the LBL films also affected the crystallinity of chitosan, which was considerably decreased in this type of film as chitosan became protonated because of the electrostatic interactions between adjacent layers.     

Biomimetic nanosystems and novel composite nanobiomaterials

Biophysicochemical approaches to the solution of nanotechnology problems associated with the design of functional biomimetic nanosystems, hybrid and composite nanobiomaterials and study of their structure-function relationships. The results of studies concerned with physicochemical mechanisms of the formation of organized biomimetic nanostructures and bioinorganic nanomaterials in systems involving a bulky liquid phase and the interface (gas-liquid, solid-liquid, liquid-liquid)during the synthesis and structure formation with the participation of the components of colloid systems, inorganic nanoparticles of various composition and clusters of metals, surfactants, polyelectrolytes and their complexes are discussed. In the development of the methods for the formation of composite bioinorganic nanosystems containing inorganic nanocomponents, two major approaches were used: adsorption and incorporation into the biomolecular matrix or colloid system of presynthesized inorganic nanoparticles, as well as the synthesis of the inorganic nanophase immediately in the biomolecular system. The methods of obtaining biomaterials and nanosystems are based on the principles of biomimetics, biomineralization, self-assembly and self-organization, combination and integration of a number of synthetic and physicochemical methods (physical and chemical adsorption, Langmuir technique, the formation of polycomplexes, chemical linking, competitive interactions, and substitution of ligands in supramolecular and coordination complexes) and nanocomponents of different nature. In particular, a novel approach to the preparation of highly organized nanofilm materials was developed, which is based on the effect of self-assembly and self-organization of colloid nanoparticles during the formation of their complexes with polyfunctional biogenic ligands in the volume of the liquid phase in the absence of any surfaces and interfaces. The physical and chemical factors responsible for the formation of structurally ordered biomolecular and composite nanosystems including nano-sized components of different nature and the possibilities to control the composition, structure, and properties of resulting nanomaterials and nanosystems are discussed. The experimental methods and approaches developed may be useful in studies of structure-property relationships and basic mechanisms of structural organization and transformation at the nanoscales level in biological, artificial, and hybrid nanosystems. The problems of practical application of the synthetic methods and the corresponding nanomaterials are discussed.     

S-layer-supported lipid membranes

Many prokaryotic organisms (archaea and bacteria) are covered by a regularly ordered surface layer (S-layer) as the outermost cell wall component. S-layers are built up of a single protein or glycoprotein species and represent the simplest biological membrane developed during evolution. Pores in S-layers are of regular size and morphology, and functional groups on the protein lattice are aligned in well-defined positions and orientations. Due to the high degree of structural regularity S-layers represent unique systems for studying the structure, morphogenesis, and function of layered supramolecular assemblies. Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at air/water interfaces, on planar mono- and bilayer lipid films, on liposomes and on solid supports (e.g. silicon wafers). Detailed studies on composite S-layer/lipid structures have been performed with Langmuir films, freestanding bilayer lipid membranes, solid supported lipid membranes, and liposomes. Lipid molecules in planar films and liposomes interact via their head groups with defined domains on the S-layer lattice. Electrostatic interactions are the most prevalent forces. The hydrophobic chains of the lipid monolayers are almost unaffected by the attachment of the S-layer and no impact on the hydrophobic thickness of the membranes has been observed. Upon crystallization of a coherent S-layer lattice on planar and vesicular lipid membranes, an increase in molecular order is observed, which is reflected in a decrease of the membrane tension and an enhanced mobility of probe molecules within an S-layer-supported bilayer. Thus, the terminology 'semifluid membrane' has been introduced for describing S-layer-supported lipid membranes. The most important feature of composite S-layer/lipid membranes is an enhanced stability in comparison to unsupported membranes.     

Photoelectrochemical, photophysical and morphological studies of electrostatic layer-by-layer thin films based on poly(p-phenylenevinylene) and single-walled carbon nanotubes

The preparation of multilayer films based on poly(p-phenylenevinylene) (PPV) and carboxylic-functionalized single-walled carbon nanotubes (SWNT-COOH) by electrostatic interaction using the layer-by-layer (LbL) deposition method is reported herein. The multilayer build-up, monitored by UV-Vis and photoluminescence (PL) spectroscopies, displayed a linear behavior with the number of PPV and SWNT-COOH layers deposited that undergo deviation and spectral changes for thicker films. Film morphology was evaluated by AFM and epifluorescence microscopies showing remarkable changes after incorporation of SWNT-COOH layers. Films without SWNT show roughness and present dispersed grains; films with SWNT-COOH layers are flatter and some carbon nanotube bundles can be visualized. The photoinduced charge transfer from the conducting polymer to SWNT-COOH was analyzed by PL quenching either by the decrease of the emission intensity or by the presence of dark domains in the epifluorescence micrographs. Photoelectrochemical characterization was performed under white light and the films containing SWNT-COOH displayed photocurrent values between 2.0 μA cm(-2) and 7.5 μA cm(-2), as the amount of these materials increases in the film. No photocurrent was observed for the film without carbon nanotubes. Photocurrent generation was enhanced and became more stable when an intermediate layer of PEDOT:PSS was interposed between the active layer and the ITO electrode, indicating an improvement in hole transfer to the contacts. Our results indicate that these multilayer films are promising candidates as active layers for organic photovoltaic cells.     

Semi-synthetic nucleic acid-protein conjugates: applications in life sciences and nanobiotechnology

Semi-synthetic conjugates of nucleic acids and proteins can be generated by either covalent coupling chemistry, or else by non-covalent biomolecular recognition systems, such as receptor-ligands of complementary nucleic acids. These nucleic acid-protein conjugates are versatile molecular tools which can be applied, for instance, in the self-assembly of high-affinity reagents for immunological detection assays, the fabrication of laterally microstructured biochips containing functional biological groups, and the biomimetic 'bottom-up' synthesis of nanostructured supramolecular devices. This review summarizes the current state-of-the-art synthesis and characterization methods of artificial nucleic acid-protein conjugates, as well as applications and perspectives for future developments of such hybrid biomolecular components in life sciences and nanobiotechnology.     

Stabilization of glucose oxidase in alginate microspheres with photoreactive diazoresin nanofilm coatings

The nanoassembly and photo-crosslinking of diazo-resin (DAR) coatings on small alginate microspheres for stable enzyme entrapment is described. Multilayer nanofilms of DAR with poly(styrene sulfonate) (PSS) were used in an effort to stabilize the encapsulation of glucose oxidase enzyme for biosensor applications. The activity and physical encapsulation of the trapped enzyme were measured over 24 weeks to compare the effectiveness of nanofilm coatings and crosslinking for stabilization. Uncoated spheres exhibited rapid loss of activity, retaining only 20% of initial activity after one week, and a dramatic reduction in effective activity over 24 weeks, whereas the uncrosslinked and crosslinked {DAR/PSS}-coated spheres retained more than 50% of their initial activity after 4 weeks, which remained stable even after 24 weeks for the two and three bilayer films. Nanofilms comprising more polyelectrolyte layers maintained higher overall activity compared to films of the same composition but fewer layers, and crosslinking the films increased retention of activity over uncrosslinked films after 24 weeks. These findings demonstrate that enzyme immobilization and stabilization can be achieved by using simple modifications to the layer-by-layer self-assembly technique.     

Stepwise self-assembled poly(amidoamine) dendrimer and poly(styrenesulfonate) microcapsules as sustained delivery vehicles

Hollow microcapsules comprised of poly(styrenesulfonate) (PSS) and a fourth generation poly(amidoamine) dendrimer (4G PAMAM) were prepared by depositing PSS/4G PAMAM multilayers on melamine formaldehyde (MF) colloid particles by the layer-by-layer self-assembly technique and subsequently dissolving the templated cores. The PSS/4G PAMAM layers were unstable toward the core removal procedure (pH approximately 1), resulting in a low yield of intact hollow capsules (<10% for 3.5 microm diameter MF templates). Stretching of the multilayer film due to core swelling during MF core dissolution leads to partial or complete destruction of capsules, giving discrete PSS-4G PAMAM complexes. Yields were increased by increasing inter- and intramolecular attractive forces between the PSS chains in the capsules through electrostatic, hydrophobic, and a combination of these interactions. The yields, however, were practically unaffected by enhancing such effects between dendrimer molecules. Transmission electron microscopy and scanning force microscopy measurements show no deformation for 3.5 microm capsules stabilized through the various interactions stated above. Further, capsules were filled with low molecular weight dextran sulfate and subsequently loaded with a model, therapeutically active molecule, doxorubicin hydrochloride (DOX). Release of DOX from the capsules was also studied to highlight the drug delivery potential of the dendrimer-based microcapsules.     

A simple route to fabricate controllable and stable multilayered all-MWNTs films and their applications for the detection of NADH at low potentials

This study demonstrates a polyelectrolyte-free method to fabricate controllable and stable all-MWNTs films via a covalent layer-by-layer (LBL) deposition. Aminated MWNTs and carboxylated MWNTs were prepared by surface functionalization, allowing the incorporation of MWNTs into highly tunable thin films through the formation of covalent amide bonds. Fourier transform infrared spectroscopy (FTIR) analysis demonstrated the formation of covalent linkages between MWNTs layers. Scanning electron microscopy (SEM) and ultraviolet-visible spectroscopy (UV-vis) were used to characterize the assembly process. Electrochemical studies indicated that the all-MWNTs film possessed a remarkable electrocatalytic activity toward dihydronicotinamide adenine dinucleotide (NADH) at relatively low potentials, without the need for redox mediators. The film thickness and the amount of assembled MWNTs were readily adjusted by simply changing the number of cycles in the LBL assembly process, which also effectively tuned the electrocatalytic activity of the film toward NADH. The film constructed with four bilayers showed a high sensitivity of 223.8μAmM(-1)cm(-2) and a detection limit of 1.5μM, with a fast response of less than 3s. Furthermore, the all-MWNTs film also showed good selectivity and excellent stability for the determination of NADH.