Polypeptide multilayer film co-delivers oppositely-charged drug molecules in sustained manners

The current state-of-the-art for drug-carrying biomedical devices is mostly limited to those that release a single drug. Yet there are many situations in which more than one therapeutic agent is needed. Also, most polyelectrolyte multilayer films intended for drug delivery are loaded with active molecules only during multilayer film preparation. In this paper, we present the integration of capsules as vehicles within polypeptide multilayer films for sustained release of multiple oppositely charged drug molecules using layer-by-layer nanoassembly technology. Calcium carbonate (CaCO(3)) particles were impregnated with polyelectrolytes, shelled with polyelectrolyte multilayers, and then assembled onto polypeptide multilayer films using glutaraldehyde. Capsule-integrated polypeptide multilayer films were obtained after decomposition of CaCO(3) templates. Two oppositely charged drugs were loaded into capsules within polypeptide multilayer films postpreparation based on electrostatic interactions between the drugs and the polyelectrolytes impregnated within capsules. We determined that the developed innovative capsule-integrated polypeptide multilayer films could be used to load multiple drugs of very different properties (e.g., opposite charges) any time postpreparation (e.g., minutes before surgical implantation inside an operating room), and such capsule-integrated films allowed simultaneous delivery of two oppositely charged drug molecules and a sustained (up to two weeks or longer) and sequential release was achieved.     

Reversibility of structural changes of polypeptides in multilayer nanofilms

Structural properties of different polypeptide multilayer nanofilms fabricated at neutral pH have been analyzed by UV spectroscopy, circular dichroism spectroscopy (CD), and Fourier-transform infrared spectroscopy (FTIR). The various peptides studied exhibit a strong tendency to adopt a beta sheet conformation in the films. Changes in film structure on dehydration are completely reversed on rewetting. The time scale of reversibility is, however, substantially shorter for the polymer backbone than the side chains, as in protein folding.     

层层自组装纳米粒作为非病毒基因载体

基因治疗的效果严重依赖于基因载体。与传统包封技术相比,在自组装技术基础上发展起来的以DNA为聚阴离子,与荷正电的高分子材料在溶液中形成纳米粒的方法,已成为目前最重要的非病毒基因载体制备手段,具有良好的应用前景。采用层层自组装(layer-by-layer assembly,LbL)技术可提高基因装载率,其优势还在于纳米粒表面性质的可控性:在温和的条件下实现多种材料在载体表面的固定,实现载体多功能化等。本文将对近年来国内外有关层层自组装纳米粒作为非病毒基因载体的研究进展以及本课题组在此方向的研究进行简要综述。     

Layer-by-layer assembly of multilayer films composed of avidin and biotin-labeled antibody for immunosensing

Protein multilayers composed of avidin and biotin-labeled antibody (bio-Ab) were prepared on gold surface by layer-by-layer assembly technology using the high specific binding constant (K(a): approximately 10(15) M(-1)) between avidin and biotin. The assembly process of the multilayer films was monitored by using real-time BIA technique based on surface plasmon resonance (SPR). The multilayer films were also characterized by electrochemical impedance spectroscopy (EIS) and reflection absorption Fourier transform infrared spectroscopy (FTIR). The results indicate that the growth of the multilayer is uniform. From response of SPR for each layer, the stoichiometry S for the interaction between avidin and bio-Ab is calculated to be 0.37 in the multilayer whereas 0.82 in the first layer. The protein mass concentration for each layer was also obtained. The schematic figure for the multilayer assembly was proposed according to the layer mass concentration and S value. The utility of the mutilayer films for immunosensing has been investigated via their subsequent interaction with hIgG. The binding ability of the multilayer increased for one to three layers of antibody, and then reach saturation after the fourth layer. These layer-by-layer constructed antibody multilayers enhance the binding ability than covalently immobilized monolayer antibody. This technology can be also used for construction of other thin films for immunosensing and biosensor.     

Orientation and lateral mobility of cytochrome c on the surface of ultrathin lipid multilayer films.

We have previously shown that cytochrome c can be electrostatically bound to an ultrathin multilayer film having a negatively charged hydrophilic surface; furthermore, x-ray diffraction and absorption spectroscopy techniques indicated that the cytochrome c was bound to the surface of these ultrathin multilayer films as a molecular monolayer. The ultrathin fatty acid multilayers were formed on alkylated glass, using the Langmuir-Blodgett method. In this study, optical linear dichroism was used to determine the average orientation of the heme group within cytochrome c relative to the multilayer surface plane. The cytochrome c was either electrostatically or covalently bound to the surface of an ultrathin multilayer film. Horse heart cytochrome c was electrostatically bound to the hydrophilic surface of fatty acid multilayer films having an odd number of monolayers. Ultrathin multilayer films having an even number of monolayers would not bind cytochrome c, as expected for such hydrophobic surfaces. Yeast cytochrome c was covalently bound to the surface of a multilayer film having an even number of fatty acid monolayers plus a surface monolayer of thioethyl stearate. After washing extensively with buffer, the multilayer films with either electrostatically or covalently bound cytochrome c were analyzed for bound protein by optical absorption spectroscopy; the orientation of the cytochrome c heme was then investigated via optical linear dichroism. Polarized optical absorption spectra were measured from 450 to 600 nm at angles of 0 degrees, 30 degrees, and 45 degrees between the incident light beam and the normal to the surface plane of the multilayer. The dichroic ratio for the heme alpha-band at 550 nm as a function of incidence angle indicated that the heme of the electrostatically-bound monolayer of cytochrome c lies, on average, nearly parallel to the surface plane of the ultrathin multilayer. Similar results were obtained for the covalently-bound yeast cytochrome c. Furthermore, fluorescence recovery after photobleaching (FRAP) was used to characterize the lateral mobility of the electrostatically bound cytochrome c over the monolayer plane. The optical linear dichroism and these initial FRAP studies have indicated that cytochrome c electrostatically bound to a lipid surface maintains a well-defined orientation relative to the membrane surface while exhibiting measurable, but highly restricted, lateral motion in the plane of the surface.     

Stability and fusion of lipid layers on polyelectrolyte multilayer supports studied by colloidal force spectroscopy

The interaction between lipid layers supported by polyelectrolyte multilayer cushions has been studied by means of colloidal force spectroscopy. In a typical experiment, a colloidal probe engineered with a layer-by-layer film and a lipid bilayer on top is approached to a planar surface coated in a symmetrical way. Kinks of a few nanometres in width appear when lipid layers are pressed together—reflecting either fusion processes between lipid layers or membranes, or the penetration of polymer blobs into or through the lipid layers. Retracting curves show a stepwise shape, which results from lipid tether formation or from polymer stretching, the latter suggesting that polyelectrolyte multilayers make contact as a result of penetration or lipid fusion. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Controlled drug release from porous polyelectrolyte multilayers

Microporous and nanoporous polyelectrolyte multilayer films have been explored as ultrathin coatings for controlled drug release. Ketoprofen and cytochalasin D were successfully loaded into nanoporous films and showed zero-order release kinetics over a period of many days. In addition to homogeneous porous multilayers, heterostructures comprising porous regions stacked alternately with nonporous regions were assembled. The heterostructures behaved as dielectric mirrors, which made it possible to optically monitor the loading process. The effects of varying the number of layers in porous and nonporous regions as well as the pore size on the drug release properties were studied. Nonporous regions in the film had no effect on the release rate or duration of release. The amount of drug released could be tuned by varying the number of layers in the porous regions of films, and the release rate depended on the pore size in the films. Microporous multilayers exhibited a Fickian diffusion of drug that was approximately twice as fast as the corresponding nanoporous films. Finally, cell culture experiments with WT NR6 fibroblasts confirmed that cytochalasin D retained its ability to inhibit mitosis after release from the multilayers.     

A modified squeeze-out mechanism for generating high surface pressures with pulmonary surfactant

The exact mechanism by which pulmonary surfactant films reach the very low surface tensions required to stabilize the alveoli at end expiration remains uncertain. We utilized the nanoscale sensitivity of atomic force microscopy (AFM) to examine phospholipid (PL) phase transition and multilayer formation for two Langmuir-Blodgett (LB) systems: a simple 3 PL surfactant-like mixture and the more complex bovine lipid extract surfactant (BLES). AFM height images demonstrated that both systems develop two types of liquid condensed (LC) domains (micro- and nano-sized) within a liquid expanded phase (LE). The 3 PL mixture failed to form significant multilayers at high surface pressure (π while BLES forms an extensive network of multilayer structures containing up to three bilayers. A close examination of the progression of multilayer formation reveals that multilayers start to form at the edge of the solid-like LC domains and also in the fluid-like LE phase. We used the elemental analysis capability of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to show that multilayer structures are enriched in unsaturated PLs while the saturated PLs are concentrated in the remaining interfacial monolayer. This supports a modified squeeze-out model where film compression results in the hydrophobic surfactant protein-dependent formation of unsaturated PL-rich multilayers which remain functionally associated with a monolayer enriched in disaturated PL species. This allows the surface film to attain low surface tensions during compression and maintain values near equilibrium during expansion.     

The location of cytochrome c on the surface of ultrathin lipid multilayer films using x-ray diffraction.

X-ray diffraction and spectroscopic techniques were used to characterize ultrathin fatty acid multilayers having a bound surface layer of cytochrome c. Three to six monolayers of arachidic acid were deposited onto an alkylated glass surface, using the Langmuir-Blodgett method. These fatty acid multilayer films were stored either in a 1 mM NaHCO3 pH 7.5 solution or a buffered 10 microM cytochrome c solution, pH 7.5. After washing extensively with buffer, these multilayer films were assayed for bound cytochrome c by optical spectroscopy. It was found that the cytochrome c bound only to the odd-numbered monolayer films (which have hydrophilic surfaces). The theoretical number of cytochrome c molecules bound to the ultrathin multilayer films having three or five monolayers was calculated as N = 1.2 x 10(13)/cm2 (assuming a hexagonally close-packed monolayer of protein), which would produce an optical density of 0.002 at a wavelength of 550 nm; for a three or five monolayer ultrathin film that was incubated with cytochrome c, OD550 approximately equal to 0.002. The protein was released from the film when treated with greater than 100 mM KCl solution, as would be expected for an electrostatic interaction. Meridional x-ray diffraction data were collected from the arachidic acid films with and without a bound cytochrome c layer. A box refinement technique, previously shown to be effective in deriving the profile structures of nonperiodic ultrathin films, was used to determine the multilayer electron density profiles. The electron density profiles and their autocorrelation functions showed that bound cytochrome c resulted in an additional electron dense feature on the multilayer surface, consistent with a bound cytochrome c monolayer. The position of the bound protein relative to the multilayer surface was independent of the number of fatty acid monolayers in the multilayer. Future studies will use these methods to investigate the structures of membrane protein complexes bound directly to the surface of multilayer films.     

Manipulating the salt and thermal stability of DNA multilayer films via oligonucleotide length

DNA films are promising materials for diverse applications, including sensing, diagnostics, and drug/gene delivery. However, the ability to tune the stability of DNA films remains a crucial aspect for such applications. Herein, we examine the role of oligonucleotide length on the formation, and salt and thermal stability, of DNA multilayer films using oligonucleotides of homopolymeric diblocks (polyAG and polyTC), with each block (A, G, T, or C) ranging from 5 to 30 bases (10-, 20-, 30-, 40-, and 60-mer). Using a combination of quartz crystal microgravimetry, dual polarization interferometry, and flow cytometry, we demonstrate that at least 10 bases per hybridizing block in the DNA diblocks (that is, 20-mer) are required for successful hybridization and, hence, DNA multilayer film formation. Films assembled using longer oligonucleotide blocks were more stable in low salt conditions, with the DNA multilayer films assembled from the 60-mer oligonucleotides remaining intact in solutions of about 25 mM NaCl. A systematic increase in film melting temperature ( T m) was observed for the DNA multilayer films (assembled on colloids) with increasing oligonucleotide length, ranging from 38.5 degrees C for the 20-mer films to 53 degrees C for the 60-mer films. Further, an alternating trend in T m of the DNA multilayer films was observed with layer number (AG or TC); DNA multilayer films terminated with an AG layer exhibited a higher T m (44-49 degrees C) than films with an outermost TC layer (ca. 38 degrees C), suggesting a rearrangement of the film structure upon hybridization of the outermost layer. This work shows that the stability of DNA multilayer films can be tuned by varying the length of the oligonucleotide building blocks, thus providing a versatile means to tailor the salt and thermal stability of DNA films, which is necessary for the application of such films.     

Degradability of polysaccharides multilayer films in the oral environment: an in vitro and in vivo study

Biomedical devices and modified biomaterial surfaces constitute an expanding research domain in the dental field. However, such oral applications have to face a very particular environment containing specific physiological conditions and specific enzymes. To evaluate their suitability in the development of novel oral applications, the degradability of polyelectrolyte multilayer films made of the natural polysaccharides chitosan and hyaluronan (CHI/HA) was investigated in vitro and in vivo in a rat mouth model. The films were either native or cross-linked using a water-soluble carbodiimide (EDC) in combination with N-hydroxysulfosuccinimide. The in vitro degradation of the films by different enzymes present in the oral environment, such as lysozyme and amylase, was followed by quartz crystal microbalance measurements and confocal laser scanning microscopy observations after being film labeled with CHI(FITC). Whereas native films were subjected to degradation by all the enzymes, cross-linked films were more resistant to enzymatic degradation. Films were also put in contact with whole saliva, which induced a slow degradation of the native films over an 18 h period. The in vivo degradation of the films deposited on polymer disks and sutured in the rat mouth was followed over a 3 day period. Whereas film degradation is fast for native films, it is much slower for the cross-linked ones. More than 60% of these films remained on the disks after 3 days in the mouth. Taken together, these results suggest that the multilayer films made of natural polysaccharides are of high potential interest for oral applications, especially as drug release systems, offering various degradation rates and consequent release characteristics.     

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.     

Effect of pectin charge density on formation of multilayer films with chitosan

The effect of pectin charge density on the formation of multilayer films with chitosan (PEC/CHI) is studied by means of electro-optics. Pectins of low (21%) and high (71%) degrees of esterification, which are inversely proportional to the pectin charge density, are used to form films on colloidal beta-FeOOH particles at pH 4.0 when the CHI is fully ionized. We find that, after deposition of the first 3-4 layers, the film thickness increases linearly with the number of adsorbed layers. However, the increase in the film thickness is larger when the film is terminated with CHI. Irregular increase of the film thickness is more marked for the PEC with higher density of charge. Oscillation in the electrical polarizability of the film-coated particles with the number of deposited layers is also registered in the PEC/CHI films. The charge balance of the multilayers, calculated from electrical polarizability of the film-coated particles, is positive, with larger excess of positive charge within the film constructed from CHI and less charged PEC. This is attributed to the ability of CHI to diffuse into the film at each deposition step. Despite the CHI diffusion, the film thickness increases linearly due to the dissolution of unstable PEC/CHI complexes from the film surface.     

Structural investigation of the covalent and electrostatic binding of yeast cytochrome c to the surface of various ultrathin lipid multilayers using x-ray diffraction.

X-Ray diffraction was used to characterize the profile structures of ultrathin lipid multilayers having a bound surface layer of cytochrome c. The lipid multilayers were formed on an alkylated glass surface, using the Langmuir-Blodgett method. The ultrathin lipid multilayers of this study were: five monolayers of arachidic acid, four monolayers of arachidic acid with a surface monolayer of dimyristoyl phosphatidylserine, and four monolayers of arachidic acid acid with a surface monolayer of thioethyl stearate. Both the phosphatidylserine and the thioethyl stearate surfaces were found previously to covalently bind yeast cytochrome c, while the arachidic acid surface electrostatically binds yeast cytochrome c. Meridional x-ray diffraction data were collected from these lipid multilayer films with and without a bound yeast cytochrome c surface layer. A box refinement technique, previously shown to be effective in deriving the profile structures of ultrathin multilayer lipid films with and without electrostatically bound cytochrome c, was used to determine the multilayer electron density profiles. The surface monolayer of bound cytochrome c was readily apparent upon comparison of the multilayer electron density profiles for the various pairs of ultrathin multilayer films plus/minus cytochrome c for all cases. In addition, cytochrome c binding to the multilayer surface significantly perturbs the underlying lipid monolayers.     

Rational design of cytophilic and cytophobic polyelectrolyte multilayer thin films

Nanostructured polyelectrolyte multilayer thin films electrostatically assembled alternately from such polymers as poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were investigated for their in vitro cell interactions. Not surprisingly, NR6WT cells, a highly adhesive murine fibroblast cell line, attached to many different multilayer combinations tested. However, PAH/PAA multilayers constructed at pH deposition conditions of 2.0/2.0 were completely bioinert. Analogous cell interactions were observed with PAH/poly(methacrylic acid) (PAH/PMA), PAH/sulfonated poly(styrene) (PAH/SPS), and poly(diallyldimethylammonium chloride)/SPS (PDAC/SPS) systems, thereby suggesting a general trend in the fibroblasts' response to multilayers. Specifically, highly ionically stitched films attracted cells, whereas weakly ionically cross-linked multilayers, which swell substantially in physiological conditions to present richly hydrated surfaces, resisted fibroblast attachment. Thus, by manipulating the multilayer pH or ionic strength assembly conditions or both, which in turn dictate the molecular architecture of the thin films, one may powerfully direct a single multilayer combination to be either cell adhesive or cell resistant.     

Structured thin films as functional components within biosensors

This review provides an introduction to the field of thin films formed by Langmuir-Blodgett or self-assembly techniques and discusses applications in the field of biosensors. The review commences with an overview of thin films and methods of construction. Methods covered will include Langmuir-Blodgett film formation, formation of self-assembled monolayers such as gold-thiol monolayers and the formation of multilayers by the self-assembly of polyelectrolytes. The structure and forces governing the formation of the materials will also be discussed. The next section focussed on methods for interrogating these films to determine their selectivity and activity. Interrogation methods to be covered will include electrochemical measurements, optical measurements, quartz crystal microbalance, surface plasmon resonance and other techniques. The final section is dedicated to the functionality of these films, incorporation of biomolecules within these films and their effect on film structure. Species for incorporation will include antibodies, enzymes, proteins and DNA. Discussions on the location, availability, activity and stability of the included species are included. The review finishes with a short consideration of future research possibilities and applications of these films.     

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.     

Biomolecule-based antibacterial coating on a stainless steel surface: multilayer film build-up optimization and stability study

The goal of this paper was to establish the durability profile of antibacterial multilayer thin films under storage and usage conditions. Thin films were built on stainless steel (SS) by means of a layer-by-layer process alternating a negatively charged polyelectrolyte, polyacrylic acid, with a cationic antibacterial peptide, nisin. SS coupons coated with the antibacterial film were challenged under environmental and usage conditions likely to be encountered in real-world applications. The change in antibacterial activity elicited by the challenge was used as an indicator of multilayer film resistance. Antibacterial SS samples could be stored for several weeks at 4°C in ambient air and antibacterial films were resistant to dipping and mild wiping in water and neutral detergent. The multilayer coating showed some weaknesses, however, that need to be addressed.     

Biomolecule-based antibacterial coating on a stainless steel surface: multilayer film build-up optimization and stability study

The goal of this paper was to establish the durability profile of antibacterial multilayer thin films under storage and usage conditions. Thin films were built on stainless steel (SS) by means of a layer-by-layer process alternating a negatively charged polyelectrolyte, polyacrylic acid, with a cationic antibacterial peptide, nisin. SS coupons coated with the antibacterial film were challenged under environmental and usage conditions likely to be encountered in real-world applications. The change in antibacterial activity elicited by the challenge was used as an indicator of multilayer film resistance. Antibacterial SS samples could be stored for several weeks at 4°C in ambient air and antibacterial films were resistant to dipping and mild wiping in water and neutral detergent. The multilayer coating showed some weaknesses, however, that need to be addressed.     

Molecular "wiring" glucose oxidase in supramolecular architecture

Supramolecular organized multilayers were constructed by multiwalled carbon nanotubes modified with ferrocene-derivatized poly(allylamine) redox polymer and glucose oxidase by electrostatic self-assembly. From the analysis of voltammetric signals and fluorescence results, a linear increment of the coverage of enzyme per bilayer was estimated, which demonstrated that the multilayer is constructed in a spatially ordered manner. The cyclic voltammograms obtained from the indium tin oxide (ITO) electrodes coated by the (Fc-PAH@CNT/GOx)n multilayers revealed that bioelectrocatalytic response is directly correlated to the number of deposited bilayers; that is, the sensitivity is tunable by controlling the number of bilayers associated with ITO electrodes. The incorporation of redox-polymer-functionalized carbon nanotubes (CNT) into enzyme films resulted in a 6-10-fold increase in the glucose electrocatalytic current; the bimolecular rate constant of FADH2 oxidation (wiring efficiency) was increased up to 12-fold. Impedance spectroscopy data have yielded the electron diffusion coefficient (De) of this nanostructure to be over 10(-8) cm2 s(-1), which is typically higher than those systems without CNT by at least a factor of 10, indicating that electron transport in the new supramolecular architecture was enhanced by communication of the redox active site of enzyme, redox polymer, and CNT.