Multifunctional polyelectrolyte multilayer films: combining mechanical resistance, biodegradability, and bioactivity

Cross-linked polyelectrolyte multilayer films (CL PEM) have an increased rigidity and are mechanically more resistant than native (e.g., uncrosslinked) films. However, they are still biodegradable, which make them interesting candidates for biomedical applications. In this study, CL PEM films have been explored for their multifunctional properties as (i) mechanically resistant, (ii) biodegradable, and (iii) bioactive films. Toward this end, we investigated drug loading into CL chitosan/hyaluronan (CHI/HA) and poly(L-lysine)/hyaluronan (PLL/HA) films by simple diffusion of the drugs. Sodium diclofenac and paclitaxel were chosen as model drugs and were successfully loaded into the films. The effect of varying the number of layers in the (CHI/HA) films as well as the cross-linker concentration on diclofenac loading were studied. Diclofenac was released from the film in about 10 h. Paclitaxel was also found to diffuse within CL films. Its activity was maintained after loading in the CL films, and cellular viability could be reduced by about 55% over 3 days. Such a simple approach may be applied to other types of cross-linked films and to other drugs. These results prove that it is possible to design multifunctional multilayer films that combine mechanical resistance, biodegradability, and bioactivity properties into a single PEM architecture.     

Incorporation of photoreceptor membrane into a multilamellar film.

Multilamellar arrays of photoreceptor membrane up to 50 micrometer thick have been produced using a new method. Rhodopsin chromophore orientation in the films was studied using optical linear dichroism. The rhodopsin appears to be structurally intact and capable of photobleaching and regeneration. The production of biologically active liquid-crystal films offers a promising new approach to the study of biomembranes.     

Ultrathin hydrogel films for rapid optical biosensing

Novel biosensors have been designed by reporting an analyte-induced (de)swelling of a stimuli-responsive hydrogel (usually in a form of thin film) with a suitable optical transducer. These simple, inexpensive hydrogel biosensors are highly desirable, however, their practical applications have been hindered, largely because of their slow response. Here we show that quick response hydrogel sensors can be designed from ultrathin hydrogel films. By the adoption of layer-by-layer assembly, a simple but versatile approach, glucose-sensitive hydrogel films with thickness on submicrometer or micrometer scale, which is 2 orders of magnitude thinner than films used in ordinary hydrogel sensors, can be facilely fabricated. The hydrogel films can not only respond to the variation in glucose concentration, but also report the event via the shift of Fabry-Perot fringes using the thin film itself as Fabry-Perot cavity. The response is linear and reversible. More importantly, the response is quite fast, making it possible to be used for continuous glucose monitoring.     

Reactive polymer multilayers fabricated by covalent layer-by-layer assembly: 1,4-conjugate addition-based approaches to the design of functional biointerfaces

We report on conjugate addition-based approaches to the covalent layer-by-layer assembly of thin films and the post-fabrication functionalization of biointerfaces. Our approach is based on a recently reported approach to the "reactive" assembly of covalently cross-linked polymer multilayers driven by the 1,4-conjugate addition of amine functionality in poly(ethyleneimine) (PEI) to the acrylate groups in a small-molecule pentacrylate species (5-Ac). This process results in films containing degradable β-amino ester cross-links and residual acrylate and amine functionality that can be used as reactive handles for the subsequent immobilization of new functionality. Layer-by-layer growth of films fabricated on silicon substrates occurred in a supra-linear manner to yield films ≈ 750 nm thick after the deposition of 80 PEI/5-Ac layers. Characterization by atomic force microscopy (AFM) suggested a mechanism of growth that involves the reactive deposition of nanometer-scale aggregates of PEI and 5-Ac during assembly. Infrared (IR) spectroscopy studies revealed covalent assembly to occur by 1,4-conjugate addition without formation of amide functionality. Additional experiments demonstrated that acrylate-containing films could be postfunctionalized via conjugate addition reactions with small-molecule amines that influence important biointerfacial properties, including water contact angles and the ability of film-coated surfaces to prevent or promote the attachment of cells in vitro. For example, whereas conjugation of the hydrophobic molecule decylamine resulted in films that supported cell adhesion and growth, films treated with the carbohydrate-based motif D-glucamine resisted cell attachment and growth almost completely for up to 7 days in serum-containing media. We demonstrate that this conjugate addition-based approach also provides a means of immobilizing functionality through labile ester linkages that can be used to promote the long-term, surface-mediated release of conjugated species and promote gradual changes in interfacial properties upon incubation in physiological media (e.g., over a period of at least 1 month). These covalently cross-linked films are relatively stable in biological media for prolonged periods, but they begin to physically disintegrate after ≈ 30 days, suggesting opportunities to use this covalent layer-by-layer approach to design functional biointerfaces that ultimately erode or degrade to facilitate elimination.     

STATISTICAL ASPECTS OF THE INDEPENDENT JOINT ACTION OF POISONS, PARTICULARLY INSECTICIDES

Methods are described for discovering whether a mixture of two poisons is as toxic as predicted on the hypothesis of independent joint action. These include X²tests and a procedure for finding the maximum likelihood estimate of the coefficient of correlation in resistance to the poisons. The methods are illustrated using data from insecticidal tests.
In the insecticidal tests, flour beetles, Tribolium castaneum Herbst, were sprayed with, or exposed to films of, different insecticides in solution in Shell oil P 31. The insecticides were pyrethrins, D.D.T., and B.H.C., singly and in pairs. The statistical analysis of the results showed that pyrethrins and D.D.T. could have acted independently both as films and as direct sprays; D.D.T. and B.H.C. could have acted independently as films but not as direct sprays; and B.H.C. and pyrethrins could not have acted independently either as films or as direct sprays.
These findings are discussed, and it is concluded that independent action should be regarded as a special case of a more general type of joint action for which the term dissimilar is proposed. A general method of approach is suggested for the conception and development of hypotheses of the joint action of poisons.     

An electrochemical investigation of ligand-binding abilities of film-entrapped myoglobin

Film-entrapped myoglobin exhibits well-defined electrochemistry which, upon ligand binding, displays a titratable redox potential shift. This effect has been observed to be highly dependent on the charged state of involved films. We have demonstrated that this approach may act as a model system for studies of molecular recognition between proteins and ligands.     

Covalent attachment of synthetic DNA to self-assembled monolayer films.

The covalent attachment of thiol-modified DNA oligomers; to self-assembled monolayer silane films on fused silica and oxidized silicon substrates is described. A heterobifunctional crosslinking molecule bearing both thiol- and amino-reactive moieties was used to tether a DNA oligomer (modified at its terminus with a thiol group) to an aminosilane film formed on silica surfaces. A variety of aminosilanes, crosslinkers and treatment conditions have been tested to identify optimal conditions for DNA immobilization using this approach. The DNA films which result have been characterized using UV spectroscopy, water contact angle measurement, radiolabeling and hybridization methods.     

Folic acid modified gelatine coated quantum dots as potential reagents for in vitro cancer diagnostics

Background

Highly hydrophobic surfaces can have very low surface energy and such low surface energy biological interfaces can be obtained using fluorinated coatings on surfaces. Deposition of biocompatible organic films on solid-state surfaces is attained with techniques like plasma polymerization, biomineralization and chemical vapor deposition. All these require special equipment or harsh chemicals. This paper presents a simple vapor-phase approach to directly coat solid-state surfaces with biocompatible films without any harsh chemical or plasma treatment. Hydrophilic and hydrophobic monomers were used for reaction and deposition of nanolayer films. The monomers were characterized and showed a very consistent coating of 3D micropore structures.

Results

The coating showed nano-textured surface morphology which can aid cell growth and provide rich molecular functionalization. The surface properties of the obtained film were regulated by varying monomer concentrations, reaction time and the vacuum pressure in a simple reaction chamber. Films were characterized by contact angle analysis for surface energy and with profilometer to measure the thickness. Fourier Transform Infrared Spectroscopy (FTIR) analysis revealed the chemical composition of the coated films. Variations in the FTIR results with respect to different concentrations of monomers showed the chemical composition of the resulting films.

Conclusion

The presented approach of vapor-phase coating of solid-state structures is important and applicable in many areas of bio-nano interface development. The exposure of coatings to the solutions of different pH showed the stability of the coatings in chemical surroundings. The organic nanocoating of films can be used in bio-implants and many medical devices.     

Nanocomposite films based on xylan-rich hemicelluloses and cellulose nanofibers with enhanced mechanical properties

Interest in xylan-rich hemicelluloses (XH) film is growing, and efforts have been made to prepare XH films with improved mechanical properties. This work described an effective approach to produce nanocomposite films with enhanced mechanical properties by incorporation of cellulose nanofibers (CNFs) into XH. Aqueous dispersions of XH (64-75 wt %), sorbitol (16-25 wt %), and CNF (0-20 wt %) were cast at a temperature of 23 °C and 50% relative humidity. The surface morphology of the films was revealed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The thermal properties and crystal structure of the films were evaluated by thermal analysis (TG) and X-ray diffraction (XRD). The surface of XH films with and without CNF was composed primarily of nanonodules, and CNFs were embedded in the XH matrix. Freeze-dried XH powder was amorphous, whereas the films with and without CNF showed a distinct peak at around 2θ = 18°, which suggested that XH molecules aggregated or reordered in the casting solution or during water evaporation. Furthermore, the nanocomposite films had improved thermal stability. XH film with 25 wt % plasticizer (sorbitol, based on dry XH weight) showed poor mechanical properties, whereas incorporation of CNF (5-20 wt %, based on the total dry mixture) into the film resulted in enhanced mechanical properties due to the high aspect ratio and mechanical strength of CNF and strong interactions between CNF and XH matrix. This effective method makes it possible to produce hemicellulose-based biomaterials of high quality.     

Vibrational circular dichroism of carbohydrate films formed from aqueous solutions

Vibrational circular dichroism (VCD) spectra in the entire 2000-900 cm(-1) region have been recorded, for the first time, for films of carbohydrates prepared from aqueous solutions. Eight different carbohydrates, alpha-D-glucopyranosyl-(1-->4)-D-glucose, cyclomaltohexaose, alpha-D-glucopyranosyl alpha-D-glucopyranoside, beta-D-glucopyranosyl-(1-->6)-D-glucose, beta-D-glucopyranosyl-(1-->4)-D-glucose, D-glucose, and both enantiomers of 6-deoxygalactose and of allose, were investigated. The VCD spectra obtained for films are found to be identical to the corresponding spectra obtained for aqueous solutions of carbohydrates. These measurements demonstrate several advantages of significant importance. The strong infrared absorption of water has prevented, in the past, the pursuit for routine applications of VCD in determining the structures of carbohydrates in aqueous solutions. This limitation is not present for film studies because water solvent is removed in the process of preparing the films. Also, strong infrared absorption of water at 1650 cm(-1) requires the use of very short-pathlength (6 microm) cells for measurements on aqueous solutions. This requirement and concomitant inconveniences (such as laborious assembling of a demountable liquid cell or purchasing an expensive variable pathlength liquid cell) have been eliminated for film measurements. The removal of interfering water absorption in film studies resulted in higher light throughput and better signal-to-noise ratios for VCD measurements. Another point of significance is that the amount of carbohydrate sample required for VCD measurements on films is approximately one to two orders of magnitude smaller than that required for corresponding VCD measurements on aqueous solutions. Since carbohydrate samples can now be studied as films, VCD spectroscopy becomes much more broadly applicable for carbohydrates than previously believed. The present work, in combination with other film measurements in our laboratory, indicate that VCD studies on films can be used more generally, providing a convenient and powerful approach for probing structural information for biologically important compounds.     

Invertase-lipid biocomposite films: preparation, characterization, and enzymatic activity

The formation of biocomposite films of the industrially important enzyme invertase and fatty lipids under enzyme-friendly conditions is described. The approach involves a simple beaker-based diffusion protocol wherein invertase diffuses into the cationic lipid octadecylamine during immersion of the lipid film in the enzyme solution. Entrapment of invertase in the octadecylamine film is highly pH-dependent, underlining the role of attractive electrostatic interactions between the enzyme and the lipid in the biocomposite film formation. The kinetics of formation of the enzyme-lipid biocomposites has been studied by quartz crystal microgravimetry (QCM) measurements. The stability of the enzyme in the lipid matrix was confirmed by fluorescence spectroscopy and biocatalytic activity measurements. The biocatalytic activity of the invertase-lipid biocomposite films was comparable to that of the free enzyme in solution and showed marginally higher temperature stability. Particularly exciting was the excellent reuse characteristics of the biocomposite films, indicating potential industrial application of these films.     

In vitro study of the proteolytic degradation of Antheraea pernyi silk fibroin

In this study, Antheraea pernyi silk fibroin (Ap-SF) films were incubated with Protease Type XXI from Streptomyces griseus, at 37 degrees C, to investigate the degradation behavior in an in vitro model system. The enzyme-resistant fractions of Ap-SF films and the soluble peptides formed by proteolytic degradation were collected at specified times, from 1 to 17 days, and analyzed by high performance liquid chromatography, differential scanning calorimetry, FT-Raman, and FT-IR spectroscopy. Proteolysis resulted in extensive weight loss and progressive fragmentation of films, especially at long degradation times. A range of soluble peptides was formed by proteolysis. By high performance-size exclusion chromatography it was found that their average molecular weight changed with the time of incubation. The chemical analysis of the enzyme-resistant fraction of Ap-SF films at different times of degradation indicated that the proteolytic attack preferentially occurred in the less ordered Gly rich sequences and that the contribution of the Ala rich crystalline regions to the composition of biodegraded films became progressively larger. Accordingly, DSC and spectroscopic results showed an enhancement of the crystalline character of the biodegraded films. From the behavior of the most important thermal transitions, it was deduced that the alpha-helix domains probably represent the most enzyme-resistant fraction. The in vitro approach used in the present study seems to be a valid tool for studying the rate and mechanism of degradation of Ap-SF films and of other biopolymers of potential biomedical utility.     

Growth of thin processable films of poly(pyrrole) using phosphomolybdate clusters

Facile control over the chemical polymerization of pyrrole from solutions of pyrrole-phosphomolybdic acid has allowed processibility of conducting poly(pyrrole). This approach has allowed thin conducting films of poly(pyrrole) to be deposited in a single step, and in contrast to electrochemical polymerization methods, is not limited to electrically conducting substrates. For example, 40–100 nm thick poly(pyrrole) films with conducivities in the range of 15 to 30 S cm⁻¹ have been spin coated onto insulating glass substrates. Insight into the mechanism of polymerization has been obtained with cyclic voltammetry and UV-Vis spectroscopy.     

Enthalpy changes associated with protein binding to thin films

Molecularly imprinted thin films consisting of proteins embedded in polymerised aminophenyl boronic acid have been made on glass supports. The protein contents of the films have been optimised to achieve a maximum energy of interaction between the film and the native template. The fabrication of the films and the subsequent removal from their surfaces of the imprint proteins has been shown to be a facile and easily reproduced process. The enthalpy changes associated with the rebinding of the films with their original templates (lysozyme and cytochrome c) and with non-native templates has been examined by micro-calorimetry. The results demonstrate that thin films can be successfully imprinted as shown by the significant reduction in the enthalpy (DeltaH) observed when the films were rebound with proteins other than the original templates. Additionally, it was shown that after binding, non-template proteins could be removed by washing and a greater enthalpy again observed when the films were rebound with the native protein compared to that which had been found with the non-native protein.     

Touring the Dream Factory: The Dream-Film Connection in The Wizard of Oz and A Nightmare on Elm Street

This essay explores the complex interplay of dreams and film, using an analysis of The Wizard of Oz (1939) and A Nightmare on Elm Street (1984) to illustrate how an interdisciplinary approach can provide fruitful insights into how dreams influence films, and films influence dreams. The essay suggests that reflecting on the dreams-film connection can deepen our understanding of the cultural dimensions of human development, particularly in the context of modern American culture. By using the methodological resources of psychology, sociology, history, film criticism, and theology, the essay argues more generally that the interdisciplinary analysis of films offers significant new possibilities for the development of dream studies. Kelly Bulkeley, Ph.D.     

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

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

Membrane-Membrane contact: Involvement of interfacial instability in the generation of discrete contacts

The classical approach to understanding the closeness of approach of two membranes has developed from consideration of the net effect of an attractive van der Waals force and a repulsive electrostatic force. The repulsive role of hydration forces and stereorepulsion glycocalyx forces have been recently recognized and an analysis of the effect of crosslinking molecules has been developed. Implicit in these approaches is the idea of an intercellular water layer of uniform thickness which narrows but retains a uniform thickness as the cells move towards an equilibrium separation distance. Most recently an attempt has been made to develop a physical chemical approach to contact which accommodates the widespread occurrence of localized spatially separated point contacts between interacting cells and membranes. It is based on ideas drawn from analysis of the conditions required to destabilize thin liquid films so that thickness fluctuations develop spontaneously and grow as interfacial instabilities to give spatially periodic contact. Examples of plasma membrane behaviour which are consistent with the interfacial instability approach are discussed and experiments involving polycation, polyethylene glycol, dextran and lectin adhesion and agglutination of erythrocytes are reviewed.     

Analysis of Vaginal Microbicide Film Hydration Kinetics by Quantitative Imaging Refractometry

We have developed a quantitative imaging refractometry technique, based on holographic phase microscopy, as a tool for investigating microscopic structural changes in water-soluble polymeric materials. Here we apply the approach to analyze the structural degradation of vaginal topical microbicide films due to water uptake. We implemented transmission imaging of 1-mm diameter film samples loaded into a flow chamber with a 1.5×2 mm field of view. After water was flooded into the chamber, interference images were captured and analyzed to obtain high resolution maps of the local refractive index and subsequently the volume fraction and mass density of film material at each spatial location. Here, we compare the hydration dynamics of a panel of films with varying thicknesses and polymer compositions, demonstrating that quantitative imaging refractometry can be an effective tool for evaluating and characterizing the performance of candidate microbicide film designs for anti-HIV drug delivery.     

Inexpensive color evaluation of dye-based pressure-sensitive films for plantar studies

Dye-based pressure-sensitive films are advantageous in plantar pressure studies due to their ease of use, cost-effectiveness, and ability to produce measurements within the shoe. To circumvent the use of proprietary equipment and software to relate the dye-stained film to load, an alternative approach of using a conventional flatbed scanner and generic image processing software is attempted here instead. The technique revealed high linear increasing and decreasing trends for the respective red and blue normalized intensities (R(2) > 0.95) and low standard deviation in all readings (<0.06) overall. By subtracting the blue from the red normalized intensity, it was discovered that the measurement sensitivity could be doubled. The results here confirm the viability of using a conventional flatbed scanner and generic image processing software to relate the dye-stained pressure films to load. The adoption of this approach promises substantial cost savings.     

Fungal biosorption of silver particles on 20th-century photographic documents

Microbial deterioration is a common problem in photographic collections and is considered a major cause of loss of documents. However, few studies so far have been addressed to biological damage on these materials. Several species of naturally occurring fungi can cause infections on the gelatin-silver emulsion of both positive and negative photographic material, producing defacement and loss of mechanical and aesthetical properties of the objects. In this study a particular phenomenon, spontaneously caused by fungi on 20th-century photographic films and positive supports, was documented by means of variable pressure scanning electron microscopy (VP-SEM) combined with electronic dispersion spectroscopy (EDS). This technique allowed the observation of entire, unaltered films without metallisation thus with a not invasive approach. The ability of some fungi to alter the distribution of silver crystals in the gelatin emulsion was described thanks to a backscattered electrons detector that showed differences in the atomic number of the visualised objects, giving rise to an appreciable contrast in case of different chemical composition.