Surface modification of poly(L-lactic acid) with biomolecules to promote endothelialization

Rapid endothelialization is important for biodegradable blood-contacting devices not only to prevent thrombosis but also to prevent degradation debris from entering the bloodstream and causing further complications. Here the authors report a three-step surface modification method, by which biomolecules, such as gelatin and chitosan, are covalently immobilized on the surface of plasma-treated poly(L-lactic acid) (PLLA) via -COOH groups introduced by acrylic acid grafting polymerization. Surface characterization techniques, including x-ray photoelectron spectroscopy, contact angle measurement, and colorimetric methods for surface density of functional groups, proved the feasibility and stability of this surface modification method. Surface wettability was increased by biomolecules immobilization. The -COOH surface density was measured to be 4.17±0.15?μmol/cm(2), the and amount of gelatin immobilized was 4.8?μg/cm(2). Human umbilical vein endothelial cell was used during in vitro study at seeding density of 10(4)?cells/cm(2). PLLA-gAA-gelatin surface was found to enhance cell adhesion, spreading, focal adhesion formation, and proliferation significantly. Chitosan-modified PLLA shows marginally improvement in cell adhesion and proliferation. Endothelialization was achieved within 7 days on both modified PLLA surfaces. In conclusion, this work demonstrates the feasibility of the surface modification method, and its ability to promote complete endothelialization for cardiovascular applications.     

Sustained release of complexed DNA from films: Study of bioactivity and intracellular tracking

Sustained DNA delivery from polymeric films provides a means for localized and prolonged gene therapy. However, in the case of bioactive molecules such as plasmid DNA (pDNA), there are limitations on the achievable release profiles as well as on the maintenance of bioactivity over time. In this report, the authors have investigated the bioactivity of the released DNA (naked and complexed with lipofectamine) from polymeric films using in vitro cell transfection of COS-7 cell lines. The polymeric system consists of a biodegradable semicrystalline polymer such as poly(ε-caprolactone) (PCL) with or without blended gelatin. Sustained release of lipoplexes and of pDNA is shown over several days. However, lipoplexes released from pure PCL films show no transfection on day 18, whereas lipoplexes released from PCL-gelatin films continue to transfect cells on day 18 of release. Confocal studies were used to determine the reasons for this difference in transfection efficiency, and it is proposed that association of the lipoplex with gelatin confers protection from degradation in the cytoplasm. The results also showed that the bioactivity of released lipoplexes was superior to that of the naked pDNA. For both naked pDNA and the lipoplexes, the presence of gelatin helped to maintain the bioactivity over several days.     

Immobilization of Mucor miehei esterase on core-shell magnetic beads via adsorption and covalent binding: Application in esters synthesis

In this work, poly(hydroxyethyl methacrylate/glycidylmethacrylate), p(HPMA-GMA), magnetic beads were prepared via suspension polymerization, and grafted with poly(methacrylic acid), p(MMAc) via redox polymerization. Esterase from M. miehei was immobilized onto core-shell magnetic beads with two different methods (i.e., adsorption and covalent binding). For adsorption, the effects of pH and initial esterase concentration on the immobilization efficiency via adsorption have been studied. The maximum adsorption capacity of the magnetic beads for esterase was found to be 27.6 mg/g magnetic beads at pH 7.0. The covalent immobilization of esterase via carbodiimide coupling on mp(HEMA-GMA)-g-p(MAAc) beads was found to be the more effective than the direct immobilization of the enzyme on the mp(HEMA-GMA) beads. The covalently immobilized esterase on the mp(HEMA-GMA)-g-p(MAAc) beads exhibited higher activity compared to the adsorbed esterase on the same magnetic beads. Tributyrin hydrolysis properties of the free and both immobilized esterase preparations were determined under different experimental conditions. The operational, thermal and storage stabilities of the immobilized esterase preparations were increased compared with the free esterase. Finally, esterification reactions have been performed to produce citronellyl-esters (namely: acetate, butyrate and propionate) in n-hexane medium using covalently immobilized esterase. The immobilized esterase was effectively reused in successive batch runs in n-hexane medium for citronellyl-butyrate synthesis, and only 23 % activity was lost after 6 cycles.     

Chitosan Activated with Genipin: A Nontoxic Natural Carrier for Tannase Immobilization and Its Application in Enhancing Biological Activities of Tea Extract

In this work, a non-toxic chitosan-based carrier was constructed via genipin activation and applied for the immobilization of tannase. The immobilization carriers and immobilized tannase were characterized using Fourier transform infrared spectroscopy and thermogravimetric analysis. Activation conditions (genipin concentration, activation temperature, activation pH and activation time) and immobilizations conditions (enzyme amount, immobilization time, immobilization temperature, immobilization pH, and shaking speed) were optimized. The activity and activity recovery rate of the immobilized tannase prepared using optimal activation and immobilization conditions reached 29.2 U/g and 53.6%, respectively. The immobilized tannase exhibited better environmental adaptability and stability. The immobilized tannase retained 20.1% of the initial activity after 12 cycles and retained 81.12% of residual activity after 30 days storage. The catechins composition analysis of tea extract indicated that the concentration of non-ester-type catechins, EGC and EC, were increased by 1758% and 807% after enzymatic treatment. Biological activity studies of tea extract revealed that tea extract treated with the immobilized tannase possessed higher antioxidant activity, higher inhibitory effect on α-amylase, and lower inhibitory effect on α-glucosidase. Our results demonstrate that chitosan activated with genipin could be an effective non-toxic carrier for tannase immobilization and enhancing biological activities of tea extract.     

Covalent immobilization of urease to modified ethyl cellulose

Graft polymerization technology is a good way to modify polymers. New functional groups are added to polymer structure using graft polymerization. Enzyme immobilization could be done from these added functional groups. Ethyl cellulose was selected as a support for enzyme immobilization and no many studies has been conducted about it. It is insoluble in water and suitable for reuse. In this study, methacrylic acid was graft polymerized to ethyl cellulose using benzophenone. In graft polymerization studies, optimum amounts of methacrylic acid and benzophenone were determined as 60 mmol and 0.6 g, respectively. Percentage of graft polymerization was maximum in toluene as solvent and optimum graft polymerization time was found as 3 hours. The graft polymerization percentage was 225.7 % at optimum conditions. This value was very good for UV-induced graft polymerization technique. FT-IR spectra of ethyl cellulose and methacrylic acid graft polymerized ethyl cellulose showed that graft polymerization was carried out successfully. -COOH groups were added to ethyl cellulose structure after graft polymerization. Then, urease was immobilized to methacrylic acid graft polymerized ethyl cellulose using 1-ethyl-3-(3-dimetylaminopropyl)-carbodiimide as the condensing agent which accelerates the reaction between -COOH from methacrylic acid graft polymerized ethyl cellulose and -NH2 from urease. Optimization studies were also performed for the immobilization of urease. Optimized values for urease immobilization; optimum amount of 1-ethyl-3-(3-dimetylaminopropyl)-carbodiimide was found as 5 mg, temperature was determined as 37 °C, 2 hours were selected as optimum time, pH and amount of urease were found to be pH 7 and 10 mg, respectively. Remained activity of immobilized urease was 1.74 % before optimization studies. After optimization of immobilization studies, this ratio has increased to 29.85 %. The immobilized urease activity was increased 17.2 times.     

Poly(methyl methacrylate-glycidiyl methacrylate) film with immobilized iminodiacetic acid and Cu(II) Ion: For protein adsorption

Poly(methyl methacrylate-glycidiyl methacrylate) p(MMA-GMA) film was fabricated by UV initiated photo-polymerization. Iminodiacetic acid ligand (IDA) was covalently immobilized to the p(MMA-GMA) film via ring opening reaction of epoxy groups. Cu(II) ion was chelated with the immobilized ligand. The morphology and properties of the films were characterized with SEM, FTIR, AFM, water content, the specific surface area, and contact angle. HSA (human serum albumin) was used as a model protein to describe the adsorption propency of the support. The information obtained in this research will serve for scaling up the process in industrial applications. The HSA adsorption capacity of the metal chelated film was found to be 2.7 mg/cm² film. The p(MMA-GMA)-IDA-Cu(II) films exhibited a high adsorption capacity and fast adsorption rate compared to Cu(II) ion free counterpart.     

Evaluating the potential of wine-making residues and corn cobs as support materials for cell immobilization for ethanol production

Three wine-making residues (grape seeds, skins and stems), and corn cobs were evaluated as support material for immobilization of Saccharomyces cerevisiae and the ethanol production by the immobilized cells was assessed. The main objective of this study was to find an abundant and low cost material suitable for the cells immobilization and able to be used in a next step of wine production by immobilized yeast cells. The four natural materials were used as support in two different forms: untreated, and treated by a sequence of acid and basic reactions. Untreated grape skin and corn cobs provided the highest cell immobilization results (25.1 and 22.2 mg cells/g support, respectively). The maximum ethanol production yield (about 0.50 g/g) was also obtained when the cells were immobilized in these untreated materials. It was also found that the support materials released nutrients to the medium, which favored the yeast development and the ethanol production. The use of immobilized cells systems under agitated conditions gave ethanol yields similar to those obtained by the static fermentations, but the immobilized cell concentration was significantly lower. In brief, static fermentation with cells immobilized on grape skins or corn cobs appear to be an interesting alternative for use on wine-making. The use of grape skins, particularly, which is a by-product of the wine elaboration, could be of larger interest to obtain an integrated wine production process with by-product reuse.     

Preparation of zero-valent Co/N-CNFs as an immobilized thin film onto graphite disc for methanol electrooxidation

In this study, we developed new method to prepare one-pot current collector/electrode plate to be utilized in the fuel cell technology. In situ preparation of zero-valent N-doped Co carbon nanofibers (Co/N-CNFs) as an immobilized thin film onto graphite disc was achieved by electrospinning technique, followed by calcination at 1100 oC in argon atmosphere. This catalyst was used for methanol electrooxidation in alkaline media. The effects of nitrogen doping and immobilization on the activity and stability of the prepared catalysts were studied using cyclic voltammetry and chronoamperometry, respectively. SEM, TEM, EDX, XRD, and TGA were used to characterize the morphology and composition of Co/N-CNFs catalysts. The results showed that the electrocatalytic activity as well as the stability of Co/N-CNFs towards methanol electrooxidation in alkaline media were significantly affected by both nitrogen doping and immobilization of the catalyst on the graphite disc. Moreover, the methanol concentration has also affected the electrocatalytic activity of Co/N-CNFs-supported immobilized onto graphite disc and Co/N-CNFs-unsupported graphite disc.     

A promising approach for bio-finishing of cotton using immobilized acid-cellulase

In this research, cellulases were immobilized on Eudragit S-100 to minimize the tensile strength loss of cotton fabric caused by the enzymatic hydrolysis. About 76 % of the enzyme activity and 81 % of the amount of protein were recovered after the immobilization process, and the immobilized cellulase exhibited good reuse ability. The immobilized cellulase had the better adsorptive performance on cotton than the free cellulase. In addition, the results revealed that the catalytic efficiency of the immobilized cellulase on cotton was degradation, perhaps because the diffusion of the enlarged cellulase molecules is significantly inhibited in the interior of the cotton fiber. Moreover, the cotton fabric treated with the immobilized cellulase showed less weight and strength losses. SEM pictures further indicated that the cotton fabric treated with the immobilized cellulase suffered less damage.     

Manufacturing polymethyl methacrylate nanofibers as a support for enzyme immobilization

Nanofibers have a great potential for enzyme immobilization application due to their large surface area to volume ratio besides their porous structure. In this work, we produce polymethyl methacrylate (PMMA) nanofibers via electrospinning method in dimethylformamide (DMF) as solvent. Thereafter, we employ a chemical method on final PMMA nanofiberous web to covalently immobilize acetylcholinesterase (AChE) enzyme on membrane surface. Morphology and tensile properties of nanofibers are studied as first steps of characterization to make sure of obtaining a properly stable membrane for enzyme carrying application. Thereafter, the stability and activity of immobilized enzymes as two main characteristic parameters are tested and reported for different applications such as biosensor manufacturing.     

Surface-modified nanofibrous biomaterial bridge for the enhancement and control of neurite outgrowth

Biomaterial bridges constructed from electrospun fibers offer a promising alternative to traditional nerve tissue regeneration substrates. Aligned and unaligned polycaprolactone (PCL) electrospun fibers were prepared and functionalized with the extracellular matrix proteins collagen and laminin using covalent and physical adsorption attachment chemistries. The effect of the protein modified and native PCL nanofiber scaffolds on cell proliferation, neurite outgrowth rate, and orientation was examined with neuronlike PC12 cells. All protein modified scaffolds showed enhanced cellular adhesion and neurite outgrowth compared to unmodified PCL scaffolds. Neurite orientation was found to be in near perfect alignment with the fiber axis for cells grown on aligned fibers, with difference angles of less than 7° from the fiber axis, regardless of the surface chemistry. The bioavailability of PCL fibers with covalently attached laminin was found to be identical to that of PCL fibers with physically adsorbed laminin, indicating that the covalent chemistry did not change the protein conformation into a less active form and the covalent attachment of protein is a suitable method for enhancing the biocompatibility of tissue engineering scaffolds.     

Preparation of amidoxime-modified polyacrylonitrile nanofibers immobilized with laccase for dye degradation

A novel approach to preparing multifunctional composite nanofibrous membrane was developed. Polyacrylonitrile (PAN) nanofibrous membrane was fabricated by electrospinning and then the nitrile groups in PAN copolymer was chemically modified to obtain amidoxime modified PAN (AOPAN) nanofiber membrane which was further used as a functional support for laccase immobilization. During the process of reactive dye degradation catalyzed by the AOPAN nanofiber membrane immobilized with laccase, metal ion adsorption occurred at the same time. The chemical modification was confirmed by Fourier transform spectroscopy (FTIR). Scanning electron microscope (SEM) was employed to investigate the surface morphologies of the electrospun nanofibers before and after laccase immobilization. The effects of environmental factors on laccase activity were studied in detail. It was found that the optimum pH and temperature for the activity of immobilized laccase was 3.5 and 50 °C. The relative activity retention of the immobilized laccase decreased dramatically during the initial four repeated uses. After 20 days’ storage, the activity retention of immobilized laccase was still high above 60 %. It has also proved that laccase immobilized on AOPAN nanofiber membrane performed well in dye degradation and metal ion adsorption.     

Thrombin immobilization to Poly(Methacrylicacid) graft polymerized PET and PAN fabrics

Poly(ethylene terephthalate) and poly(acrylonitrile) fabrics are the most produced synthetic fabrics in the world. Their production and usage increase at medical textile. There is no functional group in their structure for enzyme immobilization. Hence, they are not inclined to react. However, functional groups could be added to their structure using graft polymerization. In this study, methacrylic acid was graft polymerized to poly(ethylene terephthalate) and poly(acylonitrile) fabrics. Characterization of modified fabrics was carried out and thrombin was immobilized to poly(methacrylic acid) graft polymerized poly(ethylene terephthalate) and poly(acylonitrile) fabrics using 1-Ethyl-3-(3-dimetylaminopropyl)-carbodiimide. Optimization studies were also performed for the immobilization of thrombin. Thrombin immobilized poly(methacrylic acid) graft polymerized poly(ethylene terephtalate) and poly(acrylonitrile) fabrics were reduced recalcification time 30 % and 25 %, respectively. It is the first time, an enzyme was immobilized to fabric and its in vitro applications were performed. Thrombin has not been immobilized to synthetic fabric, yet.     

The Role of Biodegradable Engineered Nanofiber Scaffolds Seeded with Hair Follicle Stem Cells for Tissue Engineering

Background

The aim of this study was to fabricate the poly caprolactone (PCL) aligned nanofiber scaffold and to evaluate the survival, adhesion, proliferation, and differentiation of rat hair follicle stem cells (HFSC) in the graft material using electrospun PCL nanofiber scaffold for tissue engineering applications.

Methods

The bulge region of rat whisker was isolated and cultured in DMEM: nutrient mixture F-12 supplemented with epidermal growth factor. The morphological and biological features of cultured bulge cells were observed by light microscopy using immunocytochemistry methods. Electrospinning was used for production of PCL nanofiber scaffolds. Scanning electron microscopy (SEM), 3-(4, 5-di-methylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and histology analysis were used to investigate the cell morphology, viability, attachment and infiltration of the HFSC on the PCL nanofiber scaffolds.

Results

The results of the MTT assay showed cell viability and cell proliferation of the HFSC on PCL nanofiber scaffolds. SEM microscopy images indicated that HFSC are attached, proliferated and spread on PCL nanofiber scaffolds. Also, immunocytochemical analysis showed cell infiltration and cell differentiation on the scaffolds.

Conclusion

The results of this study reveal that PCL nanofiber scaffolds are suitable for cell culture, proliferation, differentiation and attachment. Furthermore, HFSC are attached and proliferated on PCL nanofiber scaffolds.Key Words: Nanofiber, Electrospinning, Stem cells, Tissue engineering     

Fabrication of Biocompatible PLGA/PCL/PANI Nanofibrous Scaffolds with Electrical Excitability

Application of electrospun nanofibrous scaffolds has received immense attention in tissue engineering. Fabrication of scaffolds with appropriate electrical properties plays a key role in neural tissue engineering. Since fibers orientation in the scaffolds affects the growth and proliferation of the cells, this study aimed to prepare aligned electrospun conductive nanofibers by mixing 1 %, 10 % and 18 % (w/v) doped polyaniline (PANI) with polycaprolactone (PCL)/poly lactic-coglycolic acid (PLGA) (25/75) solution through the electrospinning process. The fibers diameter, hydrophilicity and conductivity were measured. In addition, the shape and proliferation of the nerve cells seeded on fibers were evaluated by MTT cytotoxicity assay and scanning electron microscopy. The results revealed that the conductive nanofibrous scaffolds were appropriate substrates for the attachment and proliferation of nerve cells. The electrical stimulation enhanced neurite outgrowth compared to those PLGA/PCL/PANI scaffolds that were not subjected to electrical stimulation. As polyaniline ratio increases, electric stimulation through nanofibrous PLGA/PCL/PANI scaffolds results in cell proliferation enhancement. However, a raise more than 10 % in polyaniline will result in cell toxicity. It was concluded that conductive scaffolds with appropriate ratio of PANI along with electrical stimulation have potential applications in treatment of spinal cord injuries.     

Removal of reactive orange 16 by immobilized Coprinus plicatilis in the batch shaking bioreactor

Coprinus plicatilis, white rot fungi, capable of removing Reactive Orange 16 (Remazol Brillant Orange 3R) was immobilized in calcium alginate gel beads and used for decolorization of RO16 in aqueous in the Batch Shaking Bioreactor. The optimum conditions for immobilization of the microorganism, such as alginate and calcium ion concentration, initial cell amout, hardening time and bead size, were determined with a view to improving the RO16 removal rate. The characteristics of RO16 decolorization by immobilized fungal cells were investigated. The repeated use of immobilized cells for RO16 decolorization was performed and the results revealed that the bioactivity of immobilized cells was stable over 120 hours in the repeated batch cultivation RO16 removal. At the end of decolorization process, the metabolites of the dye decolorization produced by immobilized C. plicatilis, were analyzed via GC-MS. It was concluded that the removal of the dye by immobilized C. plicatilis was achieved.     

Thrombin immobilization to enzymatic modified PET and PAN fabrics and their applications

Enzymatic modification of synthetic materials has immense potential both of the functionalization of polymeric materials, such as poly(acrylonitrile) or polyesters, and the production of polymers for special applications, such as medical devices and enzyme immobilization. In this study, poly(ethyleneterephtalate) and poly(acrylonitrile) fabrics were modified with commercial laccase and nitrilase, respectively. Contact angles of enzymatic modified and unmodified fabrics were measured and it was found contact angles of enzymatic modified fabrics were less than those of unmodified fabrics. Attenuated-Total-Reflection-Fourier-Transform infrared spectroscopy showed that carboxylic acid groups occurred on fabrics after enzymatic modifications. Surfaces of modified and unmodified fabrics were investigated using scanning electron microscopy. Surfaces of unmodified fabrics were smooth but surfaces of modified fabrics were rugged and cracked. Thrombin was immobilized in modified fabrics by using 1-Ethyl-3-(3-dimetylaminopropyl)-carbodiimide. Optimization studies were also performed for the immobilization of thrombin. After prepared material was tested to stop bleeding in vitro conditions and it was found that thrombin immobilized poly(ethyleneterephtalate) and poly(acrylonitrile) fabrics had a reduced recalcification time to 51 % and 89 %, respectively. Thrombin immobilized poly(ethyleneterephtalate) fabric was also tested in in vivo conditions by using Cavia porcellus and it was observed that this material caused bleeding to stop at a ratio of 24.6 %. The results were statistically significant.     

Sericin-fixed filk fiber as an immobilization support of enzyme

In this study, we attempted to evaluate a novel use of sericin-fixed silk fiber (SFx) as an immobilization support of enzyme. Sericin was fixed on the silk fiber using glutaraldehyde as a fixation reagent. After 6 hours of fixation, the degree of fixation increases linearly with linear decrease of the amount of bound α-chymotrypsin (CT). This suggests that the increase of the degree of fixation is due to the further crosslinking of free aldehyde groups on the surface of sericin-fixed silk fiber (SFx). Even though perfect fixation was not achieved, sericin did not dissolve seriously and could be removed by further washing. The specific activity did not differ significantly after 6 hours of fixation. The activity of immobilized CT on SFx decreased to its half after 6 hours of incubation at 50°C. However, it retained 78% of initial activity even after 1 hour of treatment with 100% ethanol. As a result, the SFx could be used as an immobilization support of enzyme in non-aqueous media at ambient temperature.     

Designing double-layered nanofibrous membranes as a wound dressing material

New generation wound dressings require the criteria that both bioactive and conventional wound dressing materials can recompense the fundamental properties like defense of wound from microbial invasion, dehydration during the wound care duration and mimic the healing process. In this study, functional double-layered nanofibrous composite membranes were fabricated via electrospinning method. The matrices consist of a sheet of ampicillin loaded poly(2-hydroxylethyl methacrylate/polyacrylic acid (pHEMA/pAA) nanofibers on the upper side (first layer: pH sensitive antibacterial barrier) and a sheet of poly(ε-caprolactone) (PCL)/gelatin nanofibers (second layer: bioactive part). Ampicillin was successfully incorporated to double-layered matrices which greatly changed the mechanical properties, biodegradability and water uptake ratios (up to 4 fold higher values). The success of the antimicrobial activity of ampicillin on Staphylococcus aureus and Escherichia coli was indicated by the inhibition zone test. pH sensitivity was confirmed by the swelling and ampicillin release studies by shifting pH value to basic environment. Thus, double-layered pHEMA-pAA nanofibers suggest as a potential wound dressing material for its pH sensitive drug delivery ability and its bioactive part.