Wound healing strategies based on nanoparticles incorporated in hydrogel wound patches.

The intricate, tightly controlled mechanism of wound healing that is a vital physiological mechanism is essential to maintaining the skin's natural barrier function. Numerous studies have focused on wound healing as it is a massive burden on the healthcare system. Wound repair is a complicated process with various cell types and microenvironment conditions. In wound healing studies, novel therapeutic approaches have been proposed to deliver an effective treatment. Nanoparticle-based materials are preferred due to their antibacterial activity, biocompatibility, and increased mechanical strength in wound healing. They can be divided into six main groups: metal NPs, ceramic NPs, polymer NPs, self-assembled NPs, composite NPs, and nanoparticle-loaded hydrogels. Each group shows several advantages and disadvantages, and which material will be used depends on the type, depth, and area of the wound. Better wound care/healing techniques are now possible, thanks to the development of wound healing strategies based on these materials, which mimic the extracellular matrix (ECM) microenvironment of the wound. Bearing this in mind, here we reviewed current studies on which NPs have been used in wound healing and how this strategy has become a key biotechnological procedure to treat skin infections and wounds.

Local epidermal growth factor delivery using nanopillared chitosan-gelatin films for melanogenesis and wound healing.

Epidermal growth factor (EGF) is required for various regulations of skin tissue including wound healing; however, it has limited stability due to the physicochemical conditions of the wound milieu. The lack of functional EGF within the wound can cause permanent tissue defects and therefore, current wound patch designs involve EGF-releasing components. Consequently, the focus of such systems is to improve the wound healing mechanism, with minimal attention on melanogenesis of the scar tissue. The present study investigates in vitro/in vivo wound healing and melanogenesis potential of the EGF-doped films comprised of arrays of chitosan:gelatin nanopillars (nano C:G films) prepared by using nanoporous anodic alumina molds. The potential of EGF-doped films in wound healing was examined with individual and coculture systems of fibroblasts and melanocytes to mimic the wound conditions. The outcomes demonstrated that compared to the control groups, the combination of EGF doping and nanotopography consistently provided the highest levels of melanogenic activity-related genes, melanin contents as well as EGFR expressions for both melanocyte-only and coculture setups. Proteomic, genomic and histological analysis of the excisional wound model further demonstrated that if EGF was present within the nanostructured films, the performance of these substrates in terms of wound closure, collagen thickness as well as melanin deposition was considerably improved. Furthermore, when compared with the control saline treatment and healthy mice groups, significant differences for such parameters were obtained for the nano C:G films, irrespective of their EGF contents. Overall, the results indicate that EGF-doped nano C:G films are good candidates as wound patches that not only provide desirable healing characteristics but also cause improved melanogenic outputs.

Bioinspired Collagen/Gelatin Nanopillared Films as a Potential Implant Coating Material.

Collagen-based Sharpey's fibers are naturally located between alveolar bone and tooth, and they have critical roles in a well-functioning tooth such as mechanical stability, facile differentiation, and disease protection. The success of Sharpey's fibers in these important roles is due to their unique location, vertical alignment with respect to tooth surface, as well as their micronanofiber architecture. Inspired by these structures, herein, we introduce the use of nanoporous anodic aluminum oxide molds in a drop-casting setup to fabricate biopolymeric films possessing arrays of uniform Collagen:Gelatin (Col:Gel) nanopillars. Obtained structures have diameters of ∼90 nm and heights of ∼300 nm, yielding significantly higher surface roughness values compared to their flat counterparts. More importantly, the nanostructures were parallel to each other but perpendicular to the underlying film surface imitating the natural collagenous structures of Sharpey's fibers regarding nanoscale morphology, geometrical orientation, as well as biochemical content. Viability testing showed that the nanopillared Col:Gel films have high cell viabilities (over 90%), and they display significantly improved attachment (ca. ∼ 2 times) and mineralization for Saos-2 cells when compared to flat Col:Gel films and Tissue Culture Polystyrene (TCPS) controls, plausibly due to their largely increased surface roughness and area. Hence, such Sharpey's fiber-inspired bioactive nanopillared Col:Gel films can be used as a dental implant coating material or tissue engineering platform with enhanced cellular and osteogenic properties.

Nanopillared Chitosan/Gelatin Films: A Biomimetic Approach for Improved Osteogenesis.

Biomimicry strategies, inspired from natural organization of living organisms, are being widely used in the design of nanobiomaterials. Particularly, nonlithographic techniques have shown immense potential in the facile fabrication of nanostructured surfaces at large-scale production. Orthopedic biomaterials or coatings possessing extracellular matrix-like nanoscale features induce desirable interactions between the bone tissue and implant surface, also known as osseointegration. In this study, nanopillared chitosan/gelatin (C/G) films were fabricated using nanoporous anodic alumina molds, and their antibacterial properties as well as osteogenesis potential were analyzed by comparing to the flat C/G films and tissue culture polystyrene as controls. In vitro analysis of the expression of RUNX2, osteopontion, and osteocalcin genes for mesenchymal stem cells as well as osteoblast-like Saos-2 cells was found to be increased for the cells grown on nano C/G films, indicating early-stage osteogenic differentiation. Moreover, the mineralization tests (quantitative calcium analysis and alizarin red staining) showed that nanotopography significantly enhanced the mineralization capacity of both cell lines. This work may provide a new perspective of biomimetic surface topography fabrication for orthopedic implant coatings with superior osteogenic differentiation capacity and fast bone regeneration potential.

Protein-releasing conductive anodized alumina membranes for nerve-interface materials.

Nanoporous anodized alumina membranes (AAMs) have numerous biomedical applications spanning from biosensors to controlled drug delivery and implant coatings. Although the use of AAM as an alternative bone implant surface has been successful, its potential as a neural implant coating remains unclear. Here, we introduce conductive and nerve growth factor-releasing AAM substrates that not only provide the native nanoporous morphology for cell adhesion, but also induce neural differentiation. We recently reported the fabrication of such conductive membranes by coating AAMs with a thin C layer. In this study, we investigated the influence of electrical stimulus, surface topography, and chemistry on cell adhesion, neurite extension, and density by using PC 12 pheochromocytoma cells in a custom-made glass microwell setup. The conductive AAMs showed enhanced neurite extension and generation with the electrical stimulus, but cell adhesion on these substrates was poorer compared to the naked AAMs. The latter nanoporous material presents chemical and topographical features for superior neuronal cell adhesion, but, more importantly, when loaded with nerve growth factor, it can provide neurite extension similar to an electrically stimulated CAAM counterpart.

Anemone-like nanostructures for non-lithographic, reproducible, large-area, and ultra-sensitive SERS substrates.

The melt-infiltration technique enables the fabrication of complex nanostructures for a wide range of applications in optics, electronics, biomaterials, and catalysis. Here, anemone-like nanostructures are produced for the first time under the surface/interface principles of melt-infiltration as a non-lithographic method. Functionalized anodized aluminum oxide (AAO) membranes are used as templates to provide large-area production of nanostructures, and polycarbonate (PC) films are used as active phase materials. In order to understand formation dynamics of anemone-like structures finite element method (FEM) simulations are performed and it is found that wetting behaviour of the polymer is responsible for the formation of cavities at the caps of the structures. These nanostructures are examined in the surface-enhanced-Raman-spectroscopy (SERS) experiment and they exhibit great potential in this field. Reproducible SERS signals are detected with relative standard deviations (RSDs) of 7.2-12.6% for about 10,000 individual spots. SERS measurements are demonstrated at low concentrations of Rhodamine 6G (R6G), even at the picomolar level, with an enhancement factor of ∼10(11). This high enhancement factor is ascribed to the significant electric field enhancement at the cavities of nanostructures and nanogaps between them, which is supported by finite difference time-domain (FDTD) simulations. These novel nanostructured films can be further optimized to be used in chemical and plasmonic sensors and as a single molecule SERS detection platform.

One-dimensional surface-imprinted polymeric nanotubes for specific biorecognition by initiated chemical vapor deposition (iCVD).

Molecular imprinting is a powerful, generic, and cost-effective technique; however, challenges still remain related to the fabrication and development of these systems involving nonhomogeneous binding sites, insufficient template removing, incompatibility with aqueous media, low rebinding capacity, and slow mass transfer. The vapor-phase deposition of polymers is a unique technique because of the conformal nature of coating and offers new possibilities in a number of applications including sensors, microfluidics, coating, and bioaffinity platforms. Herein, we demonstrated a simple but versatile concept to generate one-dimensional surface-imprinted polymeric nanotubes within anodic aluminum oxide (AAO) membranes based on initiated chemical vapor deposition (iCVD) technique for biorecognition of immunoglobulin G (IgG). It is reported that the fabricated surface-imprinted nanotubes showed high binding capacity and significant specific recognition ability toward target molecules compared with the nonimprinted forms. Given its simplicity and universality, the iCVD method can offer new possibilities in the field of molecular imprinting.

Nanoporous polymeric nanofibers based on selectively etched PS-b-PDMS block copolymers.

One-dimensional nanoporous polymeric nanofibers have been fabricated within an anodic aluminum oxide (AAO) membrane by a facile approach based on selective etching of poly(dimethylsiloxane) (PDMS) domains in polystyrene-block-poly(dimethylsiloxane) (PS-b-PDMS) block copolymers that had been formed within the AAO template. It was observed that prior to etching, the well-ordered PS-b-PDMS nanofibers are solid and do not have any porosity. The postetched PS nanofibers, on the other hand, had a highly porous structure having about 20-50 nm pore size. The nanoporous polymeric fibers were also employed as a drug carrier for the native, continuous, and pulsatile drug release using Rhodamine B (RB) as a model drug. These studies showed that enhanced drug release and tunable drug dosage can be achieved by using ultrasound irradiation.

Surface-induced self-assembly of dipeptides onto nanotextured surfaces.

There is an increasing interest for the utilization of biomolecules for fabricating novel nanostructures due to their ability for specific molecular recognition, biocompatibility, and ease of availability. Among these molecules, diphenylalanine (Phe-Phe) dipeptide is considered as one of the simplest molecules that can generate a family of self-assembly based nanostructures. The properties of the substrate surface, on which the self-assembly process of these peptides occurs, play a critical role. Herein, we demonstrated the influence of surface texture and functionality on the self-assembly of Phe-Phe dipeptides using smooth silicon surfaces, anodized aluminum oxide (AAO) membranes, and poly(chloro-p-xylylene) (PPX) films having columnar and helical morphologies. We found that helical PPX films, AAO, and silicon surfaces induce similar self-assembly processes and the surface hydrophobicity has a direct influence for the final dipeptide structure whether being in an aggregated tubular form or creating a thin film that covers the substrate surface. Moreover, the dye staining data indicates that the surface charge properties and hence the mechanism of the self-assembly process are different for tubular structures as opposed to the peptidic film. We believe that our results may contribute to the control of surface-induced self-assembly of peptide molecules and this control can potentially allow the fabrication of novel peptide based materials with desired morphologies and unique functionalities for different technological applications.

Novel antifouling oligo(ethylene glycol) methacrylate particles via surfactant-free emulsion polymerization.

The use of particle formulations with antifouling surface properties attracts increasing interest in several biotechnological applications. Majority of these studies utilize a poly(ethylene glycol) coating to render the corresponding surface nonrecognizable to biological macromolecules. Herein, we report a simple way to prepare novel antifouling colloids composed of oligo(ethylene glycol) backbones via surfactant-free emulsion polymerization. Monodisperse cross-linked poly(ethylene glycol) ethyl ether methacrylate particles were characterized by dynamic light scattering and transmission electron microscopy. The effects of monomer, cross-linker and initiator on particle characteristics were investigated. More importantly, a prominent blockage of bovine serum albumin adsorption was obtained for the poly(ethylene glycol)-based sub-micron (~200 nm) particles when compared with similar-sized poly(methyl methacrylate) counterparts.

Size controlled synthesis of sub-100 nm monodisperse poly(methylmethacrylate) nanoparticles using surfactant-free emulsion polymerization.

Surfactant-free emulsion polymerization (SFEP) is a well-known technique for the production of polymeric nanoparticles that does not require post-synthetic cleaning steps. Obtaining hydrophobic particles at sub-100 nm scale, however, is quite challenging with this polymerization method. Here, we demonstrate a single step synthetic approach that yields poly(methylmethacrylate) (PMMA) nanoparticles with controlled sub-100 nm size and relatively high resultant solid content. Dynamic light scattering (DLS) was used for the particle characterization. Spherical and uniformly sized nanoparticles were confirmed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Acetone was used as a cosolvent in order to obtain monodisperse sub-100 nm diameter particles. Stable PMMA nanoparticle dispersions were obtained for all formulations where the persulfate initiator causes the negative charges on the particle surface. The effects of acetone, monomer and initiator concentration were studied to optimize average particle hydrodynamic diameter and polydispersity index of the final particles. Non-crosslinked monodisperse PMMA nanoparticles (polydispersity index less than 0.05) with diameters from 32 nm to 72 nm were synthesized by using this method.

Resistive-pulse detection of short dsDNAs using a chemically functionalized conical nanopore sensor.

AIMS: To develop nanopore resistive-pulse sensors for the detection of short (50 base-pair [bp] and 100 bp) DNAs. MATERIALS & METHODS: Conically shaped nanopores were chemical etched into polyethylene terphthalate membranes. The as-etched membrane had anionic carboxylate sites on the pore walls. Neutral and hydrophilic ethanolamine functional groups were attached to these carboxylate sites using well-established EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) chemistry. RESULTS & DISCUSSION: The ethanolamine-functionalized pores were used to detect 50 and 100 bp DNAs via the resistive-pulse method. The resistive-pulse signature produced by the 50 bp DNA could be distinguished from that of the 100 bp DNA with these sensors. CONCLUSIONS: Attachment of ethanolamine to the carboxylate groups on the pore wall lowered the anionic charge density on the wall. This mitigated the problem of electrostatic rejection of the anionic DNAs from the pore and enabled the detection of these DNA analytes.

Antibody-functionalized nano test tubes target breast cancer cells.

AIM: To develop nano test tubes that will deliver a biomedical payload to a specific cell type. METHODS: The template-synthesis method was used to prepare silica nano test tubes. An antibody that is specific for breast cancer cells was attached to the outer tube surfaces. A fluorophore was attached to the inner surfaces of the nano test tubes. The tubes were incubated with the breast cancer cells and the extent of attachment to the cell surfaces was investigated by fluorescence microscopy. RESULTS: Tubes modified on their outer surfaces with the target antibody showed enhanced attachment to breast-cancer cells, relative to tubes modified on their outer surfaces with a species and isotype-matched control antibody. CONCLUSIONS: This work is a first step toward demonstrating that nano test tubes can be used as cell-specific delivery vehicles.

Biofunctionalization and capping of template synthesized nanotubes.

Using alumina templates both nanotubes (open on both ends) and nano test tubes (open on only one end) have been synthesized from many different materials and these have great potential as delivery vehicles for biomedical applications. This review focuses on our recent results directed towards developing "smart" nanotubes for biomolecule delivery applications. While intensive efforts have focused on spherical nanoparticles that are easier to make, cylindrical particles or nanotubes offer many advantages. First, the tunable alumina template allows one to dictate both the pore diameter and length of the nanotube. In addition, template synthesized nanotubes can be differentially functionalized on their inner and outer surfaces. This review highlights these advantages in the contexts of drug extraction and antibody-antigen interactions, the synthesis of protein nanotubes, and recent advances in covalently capped ("corked") nanotubes designed to prevent premature payload leakage. Though diverse applications for nanotubes have already been discovered, many new and exciting paths await exploration.

Corking nano test tubes by chemical self-assembly.

There is tremendous current interest in using nanoparticles to deliver biomolecules and macromolecules (e.g., drugs and DNA) to specific sites in living systems. Release of the biomedical payload from the nanoparticle can be accomplished by chemical or enzymatic degradation of the nanoparticle or of the link between the payload and the nanoparticle. We are exploring an alternative payload-release strategy that builds on our work on template-synthesized nano test tubes. These are hollow nanotubes that are closed on one end and open on the other, and the dimensions can be controlled at will. If these nano test tubes could be filled with a payload and then the open end corked with a chemically labile cap, they might function as a universal delivery vehicle. We show here that silica nano test tubes can be covalently corked by chemical self-assembly of nanoparticles to the tubes. We also show that the nanoparticle corks remain attached to the mouths of the nano test tubes after liberation from the alumina template. For this proof-of-principle study, we used simple imine linkages to attach the corks to the test tubes. Schiff's bases are thermodynamically unstable in the presence of water; however, the multiple points of contact between the nano test tubes and nanoparticles allow the assembled structure to be metastable under our experimental conditions. Other chemical linkages-either more or less stable-may be more appropriate for other applications, and these are currently under development.

Template synthesized nanotubes for biomedical delivery applications.

This review details the advances made in alumina template-synthesized nanotubes and nano test tubes as delivery vehicles for biomedical applications. Most current research has focused on spherical nanoparticles because they are easier to make; however, cylindrical particles or nanotubes offer many advantages over spherical particles. One advantage is that the template is tunable, which means the pore diameter and template thickness can be controlled, resulting in larger payload capacities for nanotubes. Another advantage is that template synthesized nanotubes can be differentially functionalized on their inner and outer surfaces. Inner and outer surface nanotube modification for use in drug extraction, antibody-antigen interactions and magnetization is discussed. Recent advances made in covalent capping ('corking') nanotubes to prevent premature payload leakage are also covered. Although many applications for nanotubes have already been discovered, many new and exciting paths await exploration.