Expanding the Scope of an Amphoteric Condensed Tannin, Tanfloc, for Antibacterial Coatings.

Bacterial infections are a common mode of failure for medical implants. This study aims to develop antibacterial polyelectrolyte multilayer (PEM) coatings that contain a plant-derived condensed tannin polymer (Tanfloc, TAN) with inherent antimicrobial activity. Tanfloc is amphoteric, and herein we show that it can be used as either a polyanion or a polycation in PEMs, thereby expanding the possibility of its use in PEM coatings. PEMs are ordinarily formed using a polycation and a polyanion, in which the functional (ionic) groups of the two polymers are complexed to each other. However, using the amphoteric polymer Tanfloc with weakly basic amine and weakly acidic catechol and pyrogallol groups enables PEM formation using only one or the other of its functional groups, leaving the other functional group available to impart antibacterial activity. This work demonstrates Tanfloc-containing PEMs using multiple counter-polyelectrolytes including three polyanionic glycosaminoglycans of varying charge density, and the polycations N,N,N-trimethyl chitosan and polyethyleneimine. The layer-by-layer (LbL) assembly of PEMs was monitored using in situ Fourier-transform surface plasmon resonance (FT-SPR), confirming a stable LbL assembly. X-ray photoelectron spectroscopy (XPS) was used to evaluate surface chemistry, and atomic force microscopy (AFM) was used to determine the surface roughness. The LDH release levels from cells cultured on the Tanfloc-containing PEMs were not statistically different from those on the negative control (p > 0.05), confirming their non-cytotoxicity, while exhibiting remarkable antiadhesive and bactericidal properties against Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus), respectively. The antibacterial effects were attributed to electrostatic interactions and Tanfloc's polyphenolic nature. This work underscores the potential of Tanfloc as a versatile biomaterial for combating infections on surfaces.

Decellularized Liver Nanofibers Enhance and Stabilize the Long-Term Functions of Primary Human Hepatocytes In Vitro.

Owing to significant differences across species in liver functions, in vitro human liver models are used for screening the metabolism and toxicity of compounds, modeling diseases, and cell-based therapies. However, the extracellular matrix (ECM) scaffold used for such models often does not mimic either the complex composition or the nanofibrous topography of native liver ECM. Thus, here novel methods are developed to electrospin decellularized porcine liver ECM (PLECM) and collagen I into nano- and microfibers (≈200-1000 nm) without synthetic polymer blends. Primary human hepatocytes (PHHs) on nanofibers in monoculture or in coculture with nonparenchymal cells (3T3-J2 embryonic fibroblasts or primary human liver endothelial cells) display higher albumin secretion, urea synthesis, and cytochrome-P450 1A2, 2A6, 2C9, and 3A4 enzyme activities than on conventionally adsorbed ECM controls. PHH functions are highest on the collagen/PLECM blended nanofibers (up to 34-fold higher CYP3A4 activity relative to adsorbed ECM) for nearly 7 weeks in the presence of the fibroblasts. In conclusion, it is shown for the first time that ECM composition and topography synergize to enhance and stabilize PHH functions for several weeks in vitro. The nanofiber platform can prove useful for the above applications and to elucidate cell-ECM interactions in the human liver.

Ligand Presentation Inside Protein Crystal Nanopores: Tunable Interfacial Adhesion Noncovalently Modulates Cell Attachment.

Protein crystals with sufficiently large solvent pores can non-covalently adsorb polymers in the pores. In principle, if these polymers contain cell adhesion ligands, the polymer-laden crystals could present ligands to cells with tunable adhesion strength. Moreover, porous protein crystals can store an internal ligand reservoir, so that the surface can be replenished. In this study, we demonstrate that poly(ethylene glycol) terminated with a cyclic cell adhesion ligand peptide (PEG-RGD) can be loaded into porous protein crystals by diffusion. Through atomic force microscopy (AFM), force-distance correlations of the mechanical interactions between activated AFM tips and protein crystals were precisely measured. The activation of AFM tips allows the tips to interact with PEG-RGD that was pre-loaded in the protein crystal nanopores, mimicking how a cell might attach to and pull on the ligand through integrin receptors. The AFM experiments also simultaneously reveal the detailed morphology of the buffer-immersed nanoporous protein crystal surface. We also show that porous protein crystals (without and with loaded PEG-RGD) serve as suitable substrates for attachment and spreading of adipose-derived stem cells. This strategy can be used to design surfaces that non-covalently present multiple different ligands to cells with tunable adhesive strength for each ligand, and with an internal reservoir to replenish the precisely defined crystalline surface.

Expanding the Repertoire of Electrospinning: New and Emerging Biopolymers, Techniques, and Applications.

Electrospinning has emerged as a versatile and accessible technology for fabricating polymer fibers, particularly for biological applications. Natural polymers or biopolymers (including synthetically derivatized natural polymers) represent a promising alternative to synthetic polymers, as materials for electrospinning. Many biopolymers are obtained from abundant renewable sources, are biodegradable, and possess inherent biological functions. This review surveys recent literature reporting new fibers produced from emerging biopolymers, highlighting recent developments in the use of sulfated polymers (including carrageenans and glycosaminoglycans), tannin derivatives (condensed and hydrolyzed tannins, tannic acid), modified collagen, and extracellular matrix extracts. The proposed advantages of these biopolymer-based fibers, focusing on their biomedical applications, are also discussed to highlight the use of new and emerging biopolymers (or new modifications to well-established ones) to enhance or achieve new properties for electrospun fiber materials.

Enhanced blood coagulation and antibacterial activities of carboxymethyl-kappa-carrageenan-containing nanofibers.

Ideal wound dressings should be biocompatible, exhibit high antibacterial activity, and promote blood coagulation. To impart these imperative functions, carboxymethyl-kappa-carrageenan was incorporated into poly(vinyl alcohol) nanofibers (PVA-CMKC). The antibacterial activity of the nanofibers was evaluated. Adsorption of two important blood proteins, fibrinogen and albumin, was also assessed. The adhesion and activation of platelets, and the clotting of whole blood were evaluated to characterize the ability of the nanofibers to promote hemostasis. Adhesion and morphology of both Staphylococcus aureus and Pseudomonas aeruginosa were evaluated using fluorescence microscopy and scanning electron microscopy. CMKC-containing nanofibers demonstrated significant increases in platelet adhesion and activation, percentage of coagulation in whole blood clotting test and fibrinogen adsorption, compared to PVA nanofibers, showing blood coagulation activity. Incorporating CMKC also reduces adhesion and viability of S. aureus and P. aeruginosa bacteria after 24 h of incubation. PVA-CMKC nanofibers show potential application as dressings for wound healing applications.

Biocompatible Crosslinked Nanofibers of Poly(Vinyl Alcohol)/Carboxymethyl-Kappa-Carrageenan Produced by a Green Process.

This study presents a new type of biocompatible nanofiber based on poly(vinyl alcohol) (PVA) and carboxymethyl-kappa-carrageenan (CMKC) blends, produced with no generation of hazardous waste. The nanofibers are produced by electrospinning using PVA:CMKC blends with ratios of 1:0, 1:0.25, 1:0.4, 1:0.5, and 1:0.75 (w/w PVA:CMKC) in aqueous solution, followed by thermal crosslinking. The diameter of the fibers is in the nanometer scale and below 300 nm. Fourier transform infrared spectroscopy shows the presence of the carboxyl and sulfate groups in all the fibers with CMKC. The nanofibers from water-soluble polymers are stabilized by thermal crosslinking. The incorporation of CMKC improves cytocompatibility, biodegradability, cell growth, and cell adhesion, compared to PVA nanofibers. Furthermore, the incorporation of CMKC modulates phenotype of human adipose-derived stem cells (ADSCs). PVA/CMKC nanofibers enhance ADSC response to osteogenic differentiation signals and are therefore good candidates for application in tissue engineering to support stem cells.

Polyelectrolyte multilayers containing a tannin derivative polyphenol improve blood compatibility through interactions with platelets and serum proteins.

To develop hemocompatible surfaces, a cationic tannin derivate (TN) was used to prepare polyelectrolyte multilayers (PEMs) with the glycosaminoglycans heparin (HEP) and chondroitin sulfate (CS). The surface chemistry of the PEMs was characterized using X-ray photoelectron spectroscopy and water contact angle measurements. PEMs assembled with chitosan (CHI) and HEP or CS were used as controls. We investigate the hemocompatibility of PEMs by analyzing the adsorption of key blood serum proteins, adhesion and activation of platelets, and blood clotting kinetics. TN- and CHI-based PEMs adsorb similar amounts of albumin, whereas fibrinogen adsorption was more pronounced on TN-based PEMs, due to strong association with catechol groups. However, TN-based PEMs significantly reduce both platelet adhesion and platelet activation, while CHI-based PEMs promote platelet adhesion and activation. The whole-blood clotting kinetics assay also shows lower blood coagulation on TN-based PEMs. TN is an amphoteric, cationic, condensed tannin derivative with resonance structures. It also contains catechol groups, which are similar to those in mussel adhesive protein. These chemical features enable strong association with fibrinogen, which promotes the platelet-repelling effect. This study provides a new perspective for understanding platelet adhesion and activation on biomaterial surfaces, toward the development of new blood-compatible surfaces using a tannin derivative-based polymer.

Carboxymethyl-kappa-carrageenan: A study of biocompatibility, antioxidant and antibacterial activities.

Chemical modification of polysaccharides is an important route to enhance, develop or change polysaccharide properties. In this study, carboxymethylation of kappa-carrageenan (KC) with monochloroacetic acid was performed to achieve different degrees of substitution (DS) of carboxymethyl-kappa-carrageenan (CMKC). The degree of substitution ranged from 0.8 to 1.6 and was calculated from the 1H NMR spectra. The chemical structure of the CMKCs was further characterized by FT-IR, and 13C NMR. FT-IR confirmed the carboxymethylation. Carboxymethylation increased viscosity of KC in water and decreased viscosity of KC in synthetic human sweat. Tests with human adipose derived stem cells showed higher viability and lower cytotoxicity for CMKCs when compared to KC. CMKCs showed no hemolytic activity to human red blood cells. CMKCs have increased antioxidant activity compared to KC. In antibacterial assays, CMKCs with DS of 0.8, 1.0 and 1.2 exhibited growth inhibition against Staphylococcus aureus, Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa. CMKC with DS ranging from 1.0 to 1.2 are good candidate biomaterials for cell-contacting applications.

Enhanced hemocompatibility and antibacterial activity on titania nanotubes with tanfloc/heparin polyelectrolyte multilayers.

Biomaterial-associated thrombus formation and bacterial infection remain major challenges for blood-contacting devices. For decades, titanium-based implants have been largely used for different medical applications. However, titanium can neither suppress blood coagulation, nor prevent bacterial infections. To address these challenges, tanfloc/heparin polyelectrolyte multilayers on titania nanotubes array surfaces (NT) were developed. The surfaces were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and water contact angle measurements. To evaluate the hemocompatibility of the surfaces, fibrinogen adsorption, Factor XII activation, and platelet adhesion and activation were analyzed. The antibacterial activity was investigated against Gram-negative P. aeruginosa and Gram-positive S. aureus. Bacterial adhesion and morphology, as well as biofilm formation, were analyzed using fluorescence microscopy and SEM. The anti-thrombogenic properties of the surfaces were demonstrated by significant decreases in fibrinogen adsorption, Factor XII activation, and platelet adhesion and activation. Modifying NT with tanfloc/heparin also reduces the adhesion and proliferation of P. aeruginosa and S. aureus bacteria after 24 hr of incubation, with no biofilm formation. The modified NT surfaces with tanfloc/heparin polyelectrolyte multilayers are a promising biomaterial for use on implant surfaces because of their enhanced blood biocompatibility and antibacterial properties.