Chitosan Composites Containing Boron-Dipyrromethene Derivatives for Biomedical Applications.

The work is devoted to preparing and characterizing the properties of photosensitive composites, based on chitosan proposed for photodynamic therapy. Chitosan films with a 5% addition of two BODIPY dyes were prepared by solution casting. These dyes are dipyrromethene boron derivatives with N-alkyl phthalimide substituent, differing in the presence of iodine atoms in positions 2 and 6 of the BODIPY core. The spectral properties of the obtained materials have been studied by infrared and UV-vis absorption spectroscopy and fluorescence, both in solutions and in a solid state. Surface properties were investigated using the contact angle measurement. The morphology of the sample has been characterized by Scanning Electron and Atomic Force Microscopy. Particular attention was paid to studying the protein absorption and kinetics of the dye release from the chitosan. Adding BODIPY to the chitosan matrix leads to a slight increase in hydrophilicity, higher structure heterogeneity, and roughness, than pure chitosan. The presence of iodine atoms in the BODIPY structure caused the bathochromic effect, but the emission quantum yield decreased in the composites. It has been found that BODIPY-doped chitosan interacts better with human serum albumin and acidic α-glycoprotein than unmodified chitosan. The release rate of dyes from films immersed in methanol depends on the iodine present in the structure.

Water Infused Surface Protection as an Active Mechanism for Fibrin Sheath Prevention in Central Venous Catheters.

Protein adhesion in central venous catheters (CVCs) leads to fibrin sheath formation, the precursor to thrombotic and biofilm-related CVC failures. Advances in material properties and surface coatings do not completely prevent fibrin sheath formation and post-formation treatment options are limited and expensive. We propose water infused surface protection (WISP), an active method for prevention of fibrin sheath formation on CVCs, which creates a blood-free boundary layer on the inner surface of the CVC, limiting blood contact with the CVC lumen wall. A hollow fiber membrane (HFM) in a benchtop device served as a CVC testing model to demonstrate the WISP concept. Porcine blood was pumped through the HFM while phosphate buffered saline (PBS) was infused through the HFM wall, creating the WISP boundary layer. Protein adherences on model CVC surfaces were measured and imaged. Analytical and finite volume lubrication models were used to justify the assumption of a blood-free boundary layer. We found a 92.2% reduction in average adherent protein density when WISP is used, compared with our model CVC without WISP flow. Lubrication models matched our experimental pressure drop measurements suggesting that a blood-free boundary layer was created. The WISP technique also provides a novel strategy for drug administration for biofilm treatment. Reduction in adherent protein indicates a restriction on long-term fibrin sheath and biofilm formation making WISP a promising technology which improves a wide range of vascular access treatments.

Single-molecule resolution of protein structure and interfacial dynamics on biomaterial surfaces.

A method was developed to monitor dynamic changes in protein structure and interfacial behavior on surfaces by single-molecule Förster resonance energy transfer. This method entails the incorporation of unnatural amino acids to site-specifically label proteins with single-molecule Förster resonance energy transfer probes for high-throughput dynamic fluorescence tracking microscopy on surfaces. Structural changes in the enzyme organophosphorus hydrolase (OPH) were monitored upon adsorption to fused silica (FS) surfaces in the presence of BSA on a molecule-by-molecule basis. Analysis of >30,000 individual trajectories enabled the observation of heterogeneities in the kinetics of surface-induced OPH unfolding with unprecedented resolution. In particular, two distinct pathways were observed: a majority population (∼ 85%) unfolded with a characteristic time scale of 0.10 s, and the remainder unfolded more slowly with a time scale of 0.7 s. Importantly, even after unfolding, OPH readily desorbed from FS surfaces, challenging the common notion that surface-induced unfolding leads to irreversible protein binding. This suggests that protein fouling of surfaces is a highly dynamic process because of subtle differences in the adsorption/desorption rates of folded and unfolded species. Moreover, such observations imply that surfaces may act as a source of unfolded (i.e., aggregation-prone) protein back into solution. Continuing study of other proteins and surfaces will examine whether these conclusions are general or specific to OPH in contact with FS. Ultimately, this method, which is widely applicable to virtually any protein, provides the framework to develop surfaces and surface modifications with improved biocompatibility.

Bio-inspired Adaptive and Responsive Protein-based Materials.

In nature, the inherent adaptability and responsiveness of proteins play a crucial role in the survival and reproduction of organisms, enabling them to adjust to ever-changing environments. A comprehensive understanding of protein structure and function is essential for unraveling the complex biological adaptive processes, providing new insights for the design of protein-based materials in advanced fields. Recently, materials derived from proteins with specific properties and functions have been engineered. These protein-based materials, distinguished by their engineered adaptability and responsiveness, range from the nanoscale to the macroscale through meticulous control of protein structure. First, the review introduces the natural adaptability and responsiveness of proteins in organisms, encompassing biological adhesion and the responses of organisms to light, magnetic fields, and temperature. Next, it discusses the achievements in protein-engineered adaptability and adhesion through protein assembly and nanotechnology, emphasizing precise control over protein bioactivity. Finally, the review briefly addresses the application of protein engineering techniques and the self-assembly capabilities of proteins to achieve responsiveness in protein-based materials to humidity, light, magnetism, temperature, and other factors. We hope this review will foster a multidimensional understanding of protein adaptability and responsiveness, thereby advancing the interdisciplinary integration of biomedical science, materials science, and biotechnology.

Passivation protein-adhesion platform promoting stent reendothelialization using two-electron-assisted oxidation of polyphenols.

Superhydrophilic surfaces play an important role in nature. Inspired by this, scientists have designed various superhydrophilic materials that are widely used in the field of biomaterials, such as PEG molecular brushes and zwitterionic materials. However, superhydrophilic coatings with only anti-fouling properties do not satisfy the requirements for rapid reendothelialization of cardiovascular stent surfaces. Herein, a novel polyphenol superhydrophilic surface with passivated protein-adsorption properties was developed using two-electron oxidation of dopamine and polyphenols. This coating has a multiscale effects: 1) macroscopically: anti-fouling properties of superhydrophilic; 2) microscopically: protein adhesion properties of active groups (quinone-, amino-, hydroxyphenyl groups and aromatic ring). Polyphenols not only enhance the ability of coating to passivate protein-adsorption, but also make the coating have polyphenol-related biological functions. Therefore, the polyphenol and passivated protein-adsorption platform together maintain the stability of the scaffold microenvironment. This, in turn, provides favorable conditions for the growth of endothelial cells on the scaffold surface. In vivo implantation of the coated stents into the abdominal aorta resulted in uniform and dense endothelial cells covering the surface of the neointima. Moreover, new endothelial cells secreted large amounts of functional endothelial nitric oxide synthase like healthy endothelial cells. These results indicate that the polyphenol superhydrophilic coating potentially resists intra-stent restenosis and promotes surface reendothelialization. Hence, polyphenol superhydrophilic coatings with passivated protein-adsorption properties constructed by two-electron-assisted oxidation are a highly effective and versatile surface-modification strategy for implantable cardiovascular devices.

Self-assembly of a barnacle cement protein into intertwined amyloid fibres and determination of their adhesive and viscoelastic properties.

The stalked barnacle Pollicipes pollicipes uses a multi-protein cement to adhere to highly varied substrates in marine environments. We investigated the morphology and adhesiveness of a component 19 kDa protein in barnacle cement gland- and seawater-like conditions, using transmission electron microscopy and state-of-the art scanning probe techniques. The protein formed amyloid fibres after 5 days in gland-like but not seawater conditions. After 7-11 days, the fibres self-assembled under gland-like conditions into large intertwined fibrils of up to 10 µm in length and 200 nm in height, with a distinctive twisting of fibrils evident after 11 days. Atomic force microscopy (AFM)-nanodynamic mechanical analysis of the protein in wet conditions determined E' (elasticity), E'' (viscosity) and tan δ values of 2.8 MPa, 1.2 MPa and 0.37, respectively, indicating that the protein is a soft and viscoelastic material, while the adhesiveness of the unassembled protein and assembled fibres, measured using peak force quantitative nanomechanical mapping, was comparable to that of the commercial adhesive Cell-Tak™. The study provides a comprehensive insight into the nanomechanical and viscoelastic properties of the barnacle cement protein and its self-assembled fibres under native-like conditions and may have application in the design of amyloid fibril-based biomaterials or bioadhesives.

The Effect of Germanium-Loaded Hydroxyapatite Biomaterials on Bone Marrow Mesenchymal Stem Cells Growth.

Hydroxyapatite (HA) is a hard mineral component of mineralized tissues, mainly composed of calcium and phosphate. Due to its bioavailability, HA is potentially used for the repair and regeneration of mineralized tissues. For this purpose, the properties of HA are significantly improved by adding natural and synthetic materials. In this sense, the germanium (Ge) mineral was loaded in HA biomaterial by cold isostatic pressure for the first time and characterization and biocompatibility using bone marrow mesenchymal stem cells (BM-MSCs) were investigated. The addition of Ge at 5% improved the solubility (3.32%), stiffness (18.34 MPa), water holding (31.27%) and biodegradation (21.87%) properties of HA, compared to control. Compared to all composite biomaterials, the drug-releasing behavior of HA-3% Ge was higher at pH 1 and 3 and the maximum drug release was obtained at pH 7 and 9 with HA-5% Ge biomaterials. Among the different mediums tested, the DMEM-medium showed a higher drug release rate, especially at 60 min. HA-Ge biomaterials showed better protein adhesion and apatite layer formation, which ultimately proves the compatibility in BM-MSCs culture. Except for higher concentrations of HA (5 and 10 mg/mL), the different concentrations of Ge and HA and wells coated with 1% of HA-1% Ge had higher BM-MSCs growth than control. All these findings concluded that the fabricated HA biomaterials loaded with Ge could be the potential biomaterial for culturing mammalian cells towards mineralized tissue repair and regeneration.

Manipulating cell behavior on a bacterial macro-polymer poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) via tuning the S-doped graphene ratio.

Graphene is a material with various application potentials Graphene is a unique material with superiorities and has been applied in various fields for different purposes. Although studies on the utility of graphene oxide in the biomedical field are available, no evaluation has yet been done regarding the utility of sulfur doped (S-doped) graphene. The study focuses on the effect of blending the poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) membrane with sulfur heteroatom doped graphene and the evaluation of biological responses to S-doped graphene/PHBHHx. PHBHHx membranes were blended with 1%, 0.5%, 0.1% (w/v) S-doped graphene. The morphological (SEM and Microscopy), chemical (FTIR and Raman spectroscopy), and surface area (BET) characterizations of S-doped graphene/PHBHHx membranes were performed. The presence of S groups on the surface was determined with the EDS results. Besides, the swelling profile and biodegradation tendency of the membranes were evaluated. The differentiation of protein adhesion, cell viability, cell adhesion, and cell proliferation by the increasing content of S-doped graphene was examined. The contact angle analysis revealed that modification of PHBHHx with S-doped Graphene reduced the free surface energy of PHBHHx membranes. Blending with S-doped Graphene has decreased the polarity of the PHBHHx membrane. The protein adsorption on the PHBHHx membrane was determined as 10.12 ± 0.247 mg/ml. Protein absorption on 1%, 0.5% and 0.1% S-doped graphene/PHBHHx membranes were determined as 11.34 ± 0.551 mg/ml, 9.91 ± 0.294 mg/ml and 9.48 ± 0.093 mg/ml, respectively. The cell attachment to the surface decreased with the increasing amount of S-doped graphene, however, PHBHHx membranes with graphene did not affect cytotoxicity. S-doped graphene blended PHBHHx membrane seems like a suitable patch for biomedical treatments as a hydrophobic membrane where less cell adhesion and proliferation are required like the prevention of peritoneal adhesion.

Fabrication of nanofiltration membrane based on non-biofouling PVP/lecithin nanofibers reinforced with microcrystalline cellulose via needle and needle-less electrospinning techniques.

In this work, polyvinylpyrrolidone nanofibers were electrospun incorporated with lecithin, zero-charge natural segment, as non-biofouling nanofiltration membrane with tunable porous structures. Optimum conditions were studied to obtain nano-pore size capable of nano-scaled objects reduction using needle and needleless electrospinning apparatuses. Fiber diameters were in proportional relationship with PVP concentrations to range from 1.2 um to 34 nm at 10 to 5% wt/v PVP respectively. Microcrystalline cellulose (MCC) was added and PVP fibers were photo-crosslinked to enhance the mechanical strength. Mechanical properties of electrospun fibers were enforced up to 279% in the presence of microcrystalline cellulose while increased by 125% when exposed to photo-crosslinking for 8 h by UV-light radiation. UV-crosslinking has significantly improved the hydrophobicity of the final mat to report contact angle bigger than 90° at 16 h. Protein adhesion test was conducted to indicate the capability of the electrospun membrane to bypass the blood-plasma products. Zero protein adhesion was recorded by adding only 2% wt/v of lecithin.

Small changes in PDMMLA structure influence the adsorption behavior of ECM proteins and syndecan-4 on PDMMLA derivative surfaces: Experimental validation by tensiometric surface force measurements.

Cardiovascular diseases are the leading cause of death around the world according to the World Health Organization. In-stent restenosis is an inflammatory response of the immune system to endovascular stent implantation in atherosclerotic patients. Biocompatible and biodegradable polymers are of great interest in this field in order to limit the side effects of stent treatments. Poly([R,S]-3,3-dimethylmalic acid) (PDMMLA) is a new biodegradable statistical polyester which presents promising properties as a stent coating. In this work, we studied by dynamic tensiometry, the adhesion of extracellular matrix proteins (bovine serum albumin, fibronectin, fibrinogen, and vitronectin) and plasma membrane proteoglycan (syndecan-4) on three PDMMLA derivatives with different hydrophilicity levels. The results show that proteins have different adhesion profiles and affinity on these surfaces. They show similar behavior on the most hydrophilic surface, making hydrophilic, ionic and hydrogen type bonds. Then we compared each protein's individual profile to that of a mixture of all studied proteins. The comparison shows that vitronectin and syndecan-4 are the quantitatively dominating proteins adsorbed by specific interactions. Based on the results from previous studies, this work allowed us to identify the most important PDMMLA surface as a promising biomaterial for bioactive stent-coating.

Interfacial Coating Method for Amine-Rich Surfaces using Poly(ethylene glycol) Diacrylate Applied to Bioprosthetic Valve Tissue Models.

Bioprosthetic heart valve implants are beset by calcification and failure due to the interactions between the body and the transplant. Hydrogels can be used as biological blank slates that may help to shield implants from these interactions; however, traditional light-based hydrogel polymerization is impeded by tissue opacity and topography. Therefore, new methods must be created to bind hydrogel to implant tissues. To address these complications, a two-step surface-coating method for bioprosthetic valves was developed. A previously developed bioprosthetic valve model (VM) was used to investigate and optimize the coating method. Generally, this coating is achieved by first reacting surface amine groups with an NHS-PEG-acrylate while also allowing glucose to absorb into the bulk. Then, glucose oxidase, poly(ethylene glycol) diacrylate (PEGDA), and iron ions are added to the system to initiate free-radical polymerization that bonds the PEGDA hydrogel to the acrylates sites on the surface. Results showed a thin (∼8 µm), continuous coating on VM samples that is capable of repelling protein adhesion (2% surface fouling versus 20% on uncoated samples) and does not significantly affect the surface mechanical properties. Based on this success, the coating method was translated to glutaraldehyde-fixed valve tissue samples. Results showed noncontinuous but evident coating on the surface, which was further improved by adjusting the coating solution. These results demonstrate the feasibility of the proposed two-step surface coating method for modifying the surface of bioprosthetic valve replacements.

Development of surface functionalization strategies for 3D-printed polystyrene constructs.

There is a growing interest in 3D printing to fabricate culture substrates; however, the surface properties of the scaffold remain pertinent to elicit targeted and expected cell responses. Traditional 2D polystyrene (PS) culture systems typically require surface functionalization (oxidation) to facilitate and encourage cell adhesion. Determining the surface properties which enhance protein adhesion from media and cellular extracellular matrix (ECM) production remains the first step to translating 2D PS systems to a 3D culture surface. Here we show that the presence of carbonyl groups to PS surfaces correlated well with successful adhesion of ECM proteins and sustaining ECM production of deposited human mesenchymal stem cells, if the surface has a water contact angle between 50° and 55°. Translation of these findings to custom-fabricated 3D PS scaffolds reveals carbonyl groups continued to enhance spreading and growth in 3D culture. Cumulatively, these data present a method for 3D printing PS and the design considerations required for understanding cell-material interactions. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2566-2578, 2019.

Surface Assembly Configurations and Packing Preferences of Fibrinogen Mediated by the Periodicity and Alignment Control of Block Copolymer Nanodomains.

The ability to control the specific adsorption and packing behaviors of biomedically important proteins by effectively guiding their preferred surface adsorption configuration and packing orientation on polymeric surfaces may have utility in many applications such as biomaterials, medical implants, and tissue engineering. Herein, we investigate the distinct adhesion configurations of fibrinogen (Fg) proteins and the different organization behaviors between single Fg molecules that are mediated by the changes in the periodicity and alignment of chemically alternating nanodomains in thin films of polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA) block copolymer (BCP). Specifically, the adsorption characteristics of individual Fg molecules were unambiguously resolved on four different PS-b-PMMA templates of dsa PS-b-PMMA, sm PS-b-PMMA, com PS-b-PMMA, and PS-r-PMMA. By direct visualization through high resolution imaging, the distinct adsorption and packing configurations of both isolated and interacting Fg molecules were determined as a function of the BCP template-specific nanodomain periodicity, domain alignment (random versus fully aligned), and protein concentration. The three dominant Fg adsorption configurations, SP∥, SP⊥, and TP, were observed and their occurrence ratios were ascertained on each PS-b-PMMA template. During surface packing, the orientation of the protein backbone was largely governed by the periodicity and alignment of the underlying PS-b-PMMA nanodomains whose specific direction was explicitly resolved relative to the polymeric nanodomain axis. The use of PS-b-PMMA with a periodicity much smaller than (and comparable to) the length of Fg led to a Fg scaffold with the protein backbone aligned parallel (and perpendicular) to the nanodomain major axis. In addition, we have successfully created fully Fg-decorated BCP constructs analogous to two-dimensional Fg crystals in which aligned protein molecules are arranged either side-on or end-on, depending on the BCP template. Our results demonstrate that the geometry and orientation of the protein can be effectively guided during Fg self-assembly by controlling the physical dimensions and orientations of the underlying BCP templates. Finally, the biofunctionality of the BCP surface-bound Fg was assessed and the Fg/BCP construct was successfully used in the Ca-P nanoparticle nucleation/growth and microglia cell activation.

Effect of tissue scaffold topography on protein structure monitored by fluorescence spectroscopy.

The impact of surface topography on the structure of proteins upon adhesion was assessed through non-invasive fluorescence monitoring. This study aimed at obtaining a better understanding about the role of protein structural status on cell-scaffold interactions. The changes induced upon adsorption of two model proteins with different geometries, trypsin (globular conformation) and fibrinogen (rod-shaped conformation) on poly-l-lactic acid (PLLA) scaffolds with different surface topographies, flat, fibrous and surfaces with aligned nanogrooves, were assessed by fluorescence spectroscopy monitoring, using tryptophan as structural probe. Hence, the maximum emission blue shift and the increase of fluorescence anisotropy observed after adsorption of globular and rod-like shaped proteins on surfaces with parallel nanogrooves were ascribed to more intense protein-surface interactions. Furthermore, the decrease of fluorescence anisotropy observed upon adsorption of proteins to scaffolds with fibrous morphology was more significant for rod-shaped proteins. This effect was associated to the ability of these proteins to adjust to curved surfaces. The additional unfolding of proteins induced upon adsorption on scaffolds with a fibrous morphology may be the reason for better cell attachment there, promoting an easier access of cell receptors to initially hidden protein regions (e.g. RGDS sequence), which are known to have a determinant role in cell attaching processes.

Control of supramolecular chirality of nanofibers and its effect on protein adhesion.

Chiral nanostructure, such as the double helix of DNA and α-helix of protein, plays an important role in biochemistry and material sciences. In the organism system, the biological entities always exhibit homochirality and show preference toward one specific enantiomer. How the opposite enantiomers will affect the chirality of the supramolecular nanostructures and their interactions with the biological molecules remains an important issue. In this study, two gelators bearing amphiphilic l-glutamide and d- or l-pantolactone (abbreviated as DPLG and LPLG) were designed, and their self-assembly behavior and interactions with proteins were investigated. It was found that both of the gelators could form gels in the mixed solvent of ethanol and water, and the corresponding gels were characterized with UV-vis spectroscopy, circular dichroism, Fourier transform infrared spectroscopy, X-ray diffraction, and atomic force microscopy. Although both gels formed nanofiber structures and showed many similar properties, their supramolecular chiralities were opposite, which was determined by the chirality of the terminal group. The chirality of the nanofibrous structure is found to influence the protein adhesion significantly. Quartz crystal microbalance technique was used to investigate the adsorption of human serum albumin on the nanofibrous structures. It was revealed that supramolecular nanostructure of DPLG exhibited stronger adhesive ability than that of LPLG, while there is no clear difference at a molecular level. This suggested that slightly different interactions between d and l substances with the biological molecules could be amplified when they formed chiral nanostructures. Molecular dynamic simulations were performed to verify the interaction between the two gelators and protein molecules. A possible model was proposed to explain the interaction between the nanofibers and the proteins.