Affinity purification/immobilization of poly histidine-tagged proteins by nickel-functionalized porous chitosan membranes.

Although immobilized metal ion affinity chromatography (IMAC) is one of the most effective methods for purifying his-tagged proteins, it has limitations such as expensive commercial resins and non-specific binding of unwanted proteins to the nickel immobilized on the resin. In this study, biocompatible chitosan and porous chitosan membranes as alternative resins were synthesized for protein immobilization and purification, but finally porous chitosan membrane was selected due to its higher porosity and consequently higher nickel adsorption. Once the membrane was functionalized with nickel ions and its metal adsorption confirmed by EDS and ICP methods, it was used to immobilize and purify recombinant ß-NGF as a protein model with his-tag tail in batch-fashion. Protein binding and purification were also approved by FTIR and UV-Vis spectroscopy and SDS-PAGE technique. Our results indicated that the protein of interest could bind to the nickel-functionalized porous chitosan membrane with high efficiency at pH=7. Furthermore, for protein purification, the pH value of 6 and an imidazole concentration of 750 mM were suggested for the final elution buffer. In conclusion, nickel-functionalized porous chitosan membrane could be a suitable alternative to IMAC for low cost and specific protein immobilization and purification.

3D-printing of alginate/gelatin scaffold loading tannic acid@ZIF-8 for wound healing: In vitro and in vivo studies.

In the present study, ZIF-8 metal-organic framework (MOF) modified with Tannic acid (TA@ZIF-8) was synthesized and impregnated in alginate-gelatin (Alg-Gel) hydrogel. The Alg-Gel scaffolds containing 0, 5, and 10 % of TA@ZIF-8 were fabricated through the 3D printing method specifically denoted as Alg-Gel 0 %, Alg-Gel 5 %, and Alg-Gel 10 %. XRD, FTIR, FESEM, and EDX physically and chemically characterized the synthesized ZIF-8 and TA@ZIF-8 MOFs. Besides, Alg-Gel containing TA@ZIF-8 prepared scaffolds and their biological activity were also evaluated. SEM images verified the nano-size formation of MOFs. Improved swelling and decreased degradation rates after adding TA@ZIF-8 were also reported. Increased compression strength from 0.628 to 1.63 MPa in Alg-Gel 0 % and Alg-Gel 10 %, respectively, and a 2.19 increase in elastic modulus in Alg-Gel 10 % scaffolds were exhibited. Biological activity of scaffolds, including Live-dead and Cell adhesion, antibacterial, in-vivo, and immunohistochemistry assays, demonstrated desirable fibroblast cell proliferation and adhesion, increased bacterial growth inhibition zone, accelerated wound closure and improved expression of anti-inflammatory cytokines in Alg-Gel 10 % scaffolds. The findings of this study confirm that Alg-Gel 10 % scaffolds promote full-thickness wound healing and could be considered a potential candidate for full-thickness wound treatment purposes.

Composite Microgels for Imaging-Monitored Tracking of the Delivery of Vascular Endothelial Growth Factor to Ischemic Muscles.

Monitoring the supply of vascular endothelial growth factor (VEGF) to ischemic tissues provides information on its biodistribution and delivery to meet the requirements of therapeutic angiogenesis and tissue engineering applications. We herein report the use of microfluidically generated microgels containing VEGF-conjugated fluorescent carbon dots (CDs) (VEGF-CDs), a gelatin-phenol conjugate, and silk fibroin for imaging-monitored tracking of VEGF delivery to ischemic muscles. An in vitro release study and a bioactivity assay indicated that the VEGF-CDs were released in a sustained manner with high bioactivity. The microgels showed a high angiogenesis potential, along with a strong fluorescent signal, for the chicken chorioallantoic membrane and chick embryo. Imaging and studies of therapeutic modalities of the composite microgels indicated their effective localization in ischemic tissues and sustained VEGF release, which resulted in enhanced therapeutic angiogenesis of ischemic muscles. This work reveals the success of using VEGF-loaded composite polymer microgels for efficient and monitored VEGF delivery by intramuscular administration for ischemic disease treatment.

Carbon Dots Conjugated with Vascular Endothelial Growth Factor for Protein Tracking in Angiogenic Therapy.

One of the challenges of using growth factors for tissue regeneration is to monitor their biodistributions and delivery to injured tissues for minimally invasive detection. In the present study, tracking of human vascular endothelial growth factor (VEGF) was achieved by chemically linking it to photoluminescent carbon dots (CDs). Carbon dots were synthesized by the hydrothermal method and, subsequently, conjugated with VEGF using carbodiimide coupling. ELISA and western blot analysis revealed that VEGF-conjugated CDs preserve the binding affinity of VEGF to its antibodies. We also show that VEGF-conjugated CDs maintain the functionality of VEGF for tube formation and cell migration. The VEGF-conjugated CDs were also used for in vitro imaging of human umbilical vein endothelial cells. The results of this work suggest that cell-penetrating VEGF-conjugated CDs can be used for growth factor protein tracking in therapeutic and tissue engineering applications.

Preparation and characterisation of zein/polyphenol nanofibres for nerve tissue regeneration.

Bridging strategies are required to repair peripheral nerve injuries that result in gaps >5-8 mm. Limitations such as donor-site morbidity and size mismatches with receptor sites for autografts, together with immunological problems associated with allografts and xenografts, have created an increased interest in the field of manufactured nerve guide conduits. In this study, zein, a plant protein-based polymer, was electrospun to prepare nanofibrous mats. An important challenge with zein mats is the rapid change from fibre to film under aqueous conditions. Tannic acid (TA), which is a polyphenol, was selected to prepare a blend of zein/TA with different weight ratios to investigate its effect on the wetting resistance of nanofibres. The electrospun mats were characterised and evaluated by Fourier transform infrared spectroscopy and scanning electron microscopy (SEM). Also, degradation and mechanical properties of the mats were studied. Results showed that TA had a significant effect on the resistance to film formation in nanofibres. Moreover, the degradation and elongation at break of mats were increased with increase in TA concentration. For the investigation of the peripheral nerve regeneration potential, Schwann cells were selected for cytotoxicity evaluation by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay and cell morphology by SEM. Schwann cells had good biocompatibility with zein/TA blends (%) of 90/10 and 80/20.

Poly (sodium 4-styrene sulfonate)-modified hydroxyapatite nanoparticles in zein-based scaffold as a drug carrier for vancomycin.

In order to prepare bone tissue engineered scaffolds, zein, a natural polymer isolated from corn, was used as a starting material. Zein provides ideal properties for tissue engineered scaffolds, and the products derived from its degradation are non-toxic. To enhance the osteogenic properties of the scaffold, hydroxyapatite mineral was used in the form of nanospheres. Hydroxyapatite nanoparticles are designed to carry a drug in addition to the role they play in bone tissue engineering. The surface of the hydroxyapatite nanoparticles was modified with negatively charged poly (sodium 4-styrene sulfonate) polymer, and their surface was then loaded with positively charged Vancomycin as a model drug. The scaffolds were evaluated by structural and cellular assays. FTIR and particle zeta potential tests confirmed the presence of PSS and Vancomycin in nanoparticles. The results showed a decrease in the porosity of the scaffolds and a reduction of scaffold degradation over an eight week period by increasing the concentration of hydroxyapatite nanoparticles, compared to the pure zein sample. It was observed that increasing the concentration of nanoparticles to an optimum concentration can improve the mechanical properties of scaffolds. The drug release from the scaffolds over two weeks was increased with an increase in hydroxyapatite concentration, and cell viability assays showed >90% viability of cells in scaffolds containing hydroxyapatite nanoparticles, which were confirmed to be accumulating in a proper fashion according to cell adhesion assays.

Surface modification of polypropylene membrane by polyethylene glycol graft polymerization.

Polypropylene hollow fiber microporous membranes have been used in a wide range of applications, including blood oxygenator. The hydrophobic feature of the polypropylene surface causes membrane fouling. To minimize fouling, a modification consisting of three steps: surface activation in H2 and O2 plasma, membrane immersion in polyethylene glycol (PEG) and plasma graft polymerization was performed. The membranes were characterized by contact angle measurement, Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), tensile test, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Oxygen transfer of modified membranes was also tested. The stability of grafted PEG was measured in water and in phosphate buffer saline (PBS) at 37°C. Blood compatibility of modified surfaces was evaluated by the platelet adhesion method. Water contact angel reduction from 110° to 72° demonstrates the enhanced hydrophilicity, and XPS results verify the presence of oxygenated functional groups due to the peak existence in 286 eV as a result of PEG grafting. The results clearly indicate that plasma graft-polymerization of PEG is an effective way for antifouling improvement of polypropylene membranes. Also, the results show that oxygen transfer changes in PEG grafted membranes are not significant.