Surendra Kumar Verma

Selenocysteine vs Cysteine: Tuning the Derivatization on Benzenesulfonyl Moiety of a Triazole Linked Dansyl Connected Glycoconjugate for Selective Recognition of Selenocysteine and the Applicability of the Conjugate in Buffer, in Serum, on Silica Gel, and in HepG2 Cells

A dansyl derivatized triazole linked glucopyranosyl conjugate ((NO2)L) has been synthesized and characterized and was used in the present study. The conjugate (NO2)L releases a fluorescent product upon reaction by Cys-SeH in aqueous PBS buffer by exhibiting a ∼210-fold fluorescence enhancement even in the presence of 20 other amino acids with a minimum detection limit of (1.5 ± 0.2) × 10(-7) M. The selectivity of the Cys-SeH to (NO2)L was further proven by extending the fluorescence study to different other selenium compounds. The role of para-nitrobenzenesulfonyl (pNBS) center in (NO2)L in the selective recognition of Cys-SeH was confirmed when the fluorescence emission studies were carried out using five different derivatizations possessing two NO2, five fluoro, two fluoro, one fluoro, and no fluoro groups. The nucleophilic substitution reaction of Cys-SeH on (NO2)L has been clearly demonstrated on the basis of (1)H NMR, ESI-MS, and absorption spectroscopy, and the heat changes were monitored by isothermal titration calorimetry. The application potential of (NO2)L has been demonstrated by studying its selectivity toward Cys-SeH in aqueous PBS buffer, in bovine serum, and on the silica gel surface that lead to minimum detection limits of (25 ± 2), (80 ± 5), and (168 ± 16) ppb, respectively. The biological applicability of (NO2)L for Cys-SeH was further demonstrated in HepG2 cells by fluorescence microscopy. Thus, (NO2)L is aqueous soluble and a biologically acceptable probe for Cys-SeH.

Graphene oxide nanosheets and d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) doping improves biocompatibility and ultrafiltration in polyethersulfone hollow fiber membranes

Novel graphene oxide (G)-and d-α-Tocopheryl polyethylene glycol 1000 succinate (T)-doped polyethersulfone (P) hollow fiber membranes (GTP HFMs) were efficiently prepared. GTP HFMs were found to be a desirable biocompatible substrate for attachment and proliferation of human embryonic kidney-293 (HEK-293) cells. Significantly high porosity (94.58±1.1%), low contact angle (61.1±2.5°), low hemolysis (0.58% in batch mode and 0.64% in continuous mode), low terminal complement complex activation (SC5b-9 marker level ∼6.73ng/mL), prolonged blood coagulation time, and low platelet adhesion were measured for GTP HFMs indicating the superior suitability of GTP HFMs for blood-contact applications. Further, SEM and confocal laser microscopy studies showed the significantly high HEK-293 cells attachment and proliferation on GTP HFMs which was corroborated by results of glucose consumption analysis and MTT cell proliferation assay. High ultrafiltration coefficient (110±3mL/m2/h/mmHg), and albumin solute rejection (94.87±0.5%) were also measured for GTP HFMs. Thus, these results clearly indicated that the synergistic effect of additives improved the biocompatibility and ultrafiltration in GTP HFMs. The developed GTP HFMs can potentially be used for simultaneous/sequential cells attachment and proliferation, and ultrafiltration applications such as the bioartificial kidney.

Improved hemodialysis with hemocompatible polyethersulfone hollow fiber membranes: In vitro performance

We show that addition of nanozeolite (NZ) and vitamin E D-α-Tocopherol polyethylene glycol succinate (TPGS or T) considerably improves the performance of polyethersulfone (PES or P) hollow fiber membrane (HFM) for hemodialysis. Nanocomposite HFMs were manufactured using PES as a polymer, TPGS as an additive and NZ as a filler to give a composite membrane called PT-NZ. HFMs were spun by dry-wet spinning principle based on liquid-liquid phase separation. TPGS and NZ were successfully incorporated in HFMs, as confirmed by EDX elemental mapping. The resultant PT-NZ HFMs had improved hemocompatibility: lower percent hemolysis (0.28% in batch mode and 0.32% in continuous mode), lower platelet adhesion, higher coagulation time and lower protein adsorption (16.34 µg/cm2 ), compared with P, PT, and commercial (F60S) HFMs. The ultrafiltration coefficient of PT-NZ HFM-based module (274.59 mL/m2 /h/mmHg) was ∼1.5-times higher than that of F60S membranes (151.67 mL/m2 /h/mmHg), and the solute rejection of both the membranes was comparable. The toxin clearance performance of lab-scale PT-NZ HFM-based hemodialyzer with uremic toxin spiked goat blood was remarkably higher (five times) than that of F60S. Hence, the synthesized PT-NZ HFMs are a potentially attractive membrane material for hemodialysis application, particularly due to decreased treatment time and minimal side reactions.

Graphene oxide-doping improves the biocompatibility and separation performance of polyethersulfone hollow fiber membranes for bioartificial kidney application

HYPOTHESIS Graphene oxide (GO)-doping in polyethersulfone hollow fiber membranes (PES HFMs) improves the biocompatibility and separation performance for bioartificial kidney (BAK) application. EXPERIMENTS GO was doped in PES HFMs. The physicochemical characterization of the developed HFMs was carried out. The biocompatibility tests including hemocompatibility and cytotoxicity tests, and separation experiments including uremic toxins clearance were performed. FINDINGS GO-doping resulted in low hemolysis (0.37 ± 0.15%), prolonged coagulation times, and low SC5b-9 marker level (6.84 ± 1.7 ng/mL), i.e., significantly improved hemocompatibility of GP HFMs. The monolayer attachment and improved proliferation of kidney cells on the outer surface of GP HFMs were achieved. GO-doping significantly enhanced the separation performance, i.e., high pure water permeability (154 ± 3 mL/m2/h/mmHg) was measured, and similar solute rejection profile as that of the commercial dialyzer membranes was recorded. The clearance of urea, creatinine and phosphorous from the simulated blood was measured to be almost 1.6 to 3.3 times higher than that measured for the commercial membranes. Thus, these results indicated that the GO-doping remarkably improved the performance of the developed GP HFMs thereby making them a potential membrane material for the BAK application.

Porosity and compatibility of novel polysulfone-/vitamin E-TPGS-grafted composite membrane

Polysulfone (Psf) hollow fiber membranes are widely used for hemodialysis. Despite its popularity as a biomaterial, the hydrophobicity of the polymer is a major concern for blood contact applications. Various blends of the polymer with hydrophilic ligands have been reported in the literature, to achieve desired surface property. To increase hydrophilicity, we report here a hydrophilic polysulfone polymer by covalently coupling vitamin E tocopheryl polyethylene glycol 1000 succinate (Vit E-TPGS or TPGS) and Psf. 1H NMR confirmed grafting of TPGS to Psf. With a high degree of TPGS substitution, the Psf-g-TPGS was completely hydrophilic, which resulted in no fiber formation alone. Hence, composite membranes were prepared by mixing plain Psf and Psf-g-TPGS in 1.8:1 ratio, which also resulted in a hydrophilic character. This can be tuned toward slightly hydrophilic with a higher ratio. The porosity and biocompatibility of the Psf-g-TPGS (M-III, PTC) were compared against those of unmodified Psf membrane (M-I, Psf) and unmodified Psf-TPGS blend membrane (M-II, PTB). The in vitro cellular compatibility was tested in hepatocarcinoma cell line (HepG2); the cells grown on membrane surface were examined by SEM. Results confirmed that the modified membrane is hydrophilic, is non-toxic, and may have improved efficiency in hemodialysis. Hemocompatibility of M-III, PTC had a slightly better performance over M-I; M-I showed better performance in the cellular attachment, which shows the promising role of the grafted hydrophilic polymer for related biocompatibility applications.

Functionally coated polyethersulfone hollow fiber membranes: A substrate for enhanced HepG2/C3A functions

Hollow fiber membrane (HFM) based liver assist systems are a life-saving bridge for patients until a donor organ is available for transplantation or until liver regeneration. However, liver cell attachment and functional maintenance on HFM surface is a major challenge in bio-artificial liver (BAL) support systems. In the present study, novel glutaraldehyde (GTA)-crosslinked gelatin (gel)-coated polyethersulfone (X-gel-PT) HFMs were manufactured using triple orifice spinneret by the dry-wet spinning method. HFMs were characterized for morphology, outer surface roughness, hydrophilicity, tensile strength, thermal stability, BET surface area and pore volume measurements, permeability and rejection. Fourier transform infrared spectroscopy, and transmission electron microscopy confirmed the GTA-crosslinked gel-coating in the X-gel-PT HFMs, which provided the desirable extracellular matrix-like environment to the HepG2/C3A cells. The results of in-vitro hemocompatibility tests showed the better suitability of the developed HFMs for the blood-contact application. X-gel-PT HFMs showed significantly better cellular attachment and proliferation of HepG2/C3A cells on day 3 and 6, as shown by scanning electron and confocal microscopy. Significantly high urea synthesis and albumin secretion seen indicated the improved functional and metabolic activity of HepG2/C3A cells. Thus, the developed X-gel-PT HFMs is a suitable substrate for the hepatocyte culture, mass culture, and development of BAL support system.

Islet encapsulated implantable composite hollow fiber membrane based device: A bioartificial pancreas

Islets from xeno-sources and islet like clusters derived from autologus stem cells have emerged as alternatives to cadaveric pancreas used for treatment of type 1 diabetes. However, the immuno-isolation of these islets from the host immune system suffers from the issue of biocompatibility and hypoxia. To overcome the issues of immunobarrier biocompatibility, we developed a Polysulfone (Psf)/TPGS composite hollow fiber membrane (HFM) using a hollow fiber spinning pilot plant specially developed for this purpose. Important structural variables such as fiber material, dope composition, dimensions, surface characteristics etc., were precisely engineered and tuned for bioartificial pancreas application. The HFMs were characterized for their morphology, molecular diffusion, selectivity and protein absorption. The optimized Polysulfone(Psf)/TPGS composite HFMs, which contained TPGS, exhibited uniformed structure with low insulin adsorption and high permeability of insulin. The HFM was further studied for the encapsulation and in-vitro growth with porcine and differentiated islets isolated from human umbilical cord Whartons jelly. To prove their efficacy under in-vivo conditions, the Polysulfone(Psf)/TPGS composite HFMs were encapsulated with either of these isolated cells (porcine islets or islet like cell clusters derived from mesenchymal stem cells isolated from human umbilical cord Whartons jelly) and they were transplanted in experimental STZ induced diabetic mice. The results showed restoration of normoglycemia for 30days, indicating their ability to respond efficiently to high glucose without immune-rejection. Thus, these results indicate that Polysulfone (Psf)/TPGS composite HFMs can be used as an implantable, immune-competent bioartificial pancreas as a therapy for type 1 diabetes.

Hydrophilic ZIF-8 decorated GO nanosheets improve biocompatibility and separation performance of polyethersulfone hollow fiber membranes: A potential membrane material for bioartificial liver application

The hydrophobic nature of zeolitic imidazole framework-8 (ZIF-8) nanoparticles restricts their use as additives in hollow fiber membranes (HFMs) for biomedical applications. In this study, hydrophilic ZIF-8 decorated graphene oxide nanosheets (ZGs) were synthesized and used as additives (0-1 wt%) in polyethersulfone (P) HFMs with the aim of improving the biocompatibility and separation performance so as to make the ZGP HFMs suitable for bioartificial liver (BAL) application. Elemental mapping and Fourier transform infrared studies confirmed the efficacious incorporation of ZG nanohybrids in the ZGP HFMs, which resulted in their improved hydrophilicity. The remarkably improved biocompatibility was experimentally demonstrated for the ZGP HFMs, which also were antioxidative and hemocompatible. There was a significantly high attachment and proliferation of HepG2 cells on these HFMs, and they showed remarkably high urea synthesis and albumin secretion. Further, the ZGP HFMs showed high ultrafiltration coefficient (392.2 ± 26.5 mL/h/m2/mm Hg), high flux recovery ratio (84.3%), low flux reduction (15.7%), and desirable molecular weight cutoff (125-135 kDa). Thus, these results experimentally demonstrated that the hydrophilic ZG nanohybrids improve the desirable properties of ZGP HFMs making them a potential biocompatible material for biomedical applications including BAL application.

Extracellular matrix-coated polyethersulfone-TPGS hollow fiber membranes showing improved biocompatibility and uremic toxins removal for bioartificial kidney application

In this study, L-3, 4-dihydroxyphenylalanine and human collagen type IV were coated over the outer surface of the custom-made hollow fiber membranes (HFMs) with the objective of simultaneously improving biocompatibility leading to proliferation of human embryonic kidney cells-293 (HEK-293) and improving separation of uremic toxins, thereby making them suitable for bioartificial kidney application. Physicochemical characterization showed the development of coated HFMs, resulting in low hemolysis (0.25 ± 0.10%), low SC5b-9 marker level (7.95 ± 1.50 ng/mL), prolonged blood coagulation time, and minimal platelet adhesion, which indicated their improved human blood compatibility. Scanning electron microscopy and confocal laser scanning microscopy showed significantly improved attachment and proliferation of HEK-293 cells on the outer surface of the coated HFMs, which was supported by the results of glucose consumption and MTT cell proliferation assay. The solute rejection profile of these coated HFMs was compared favorably with that of the commercial dialyzer membranes. These coated HFMs showed a remarkable 1.6-3.2 fold improvement in reduction ratio of uremic toxins as compared to standard dialyzer membranes. These results clearly demonstrated that these extracellular matrix-coated HFMs can be a potential biocompatible substrate for the attachment and proliferation of HEK-293 cells and removal of uremic toxins from the simulated blood, which may find future application for bioartificial renal assist device.

Lactobionic acid-functionalized polyethersulfone hollow fiber membranes promote HepG2 attachment and function

Surface modification of polyethersulfone hollow fibers, which are important in bio-artificial liver, is increasingly used to improve biocompatibility and promote the adhesion and proliferation of hepatocytes resulting in improved cell functionality. Hepatocytes are anchorage-dependent cells, and membrane surface modification enhances the hepatic cell adhesion and proliferation. Specific interaction of the asialoglycoprotein receptor on hepatocyte cell surfaces with a galactose moiety enhances the attachment of the cells on a biocompatible substrate. In this study, the outer surface of the polyethersulfone (P) hollow fiber membranes (HFMs) was chemically modified by covalent coupling with lactobionic acid (LBA). The energy dispersive X-ray spectrometry elemental mapping, attenuated total reflectance-Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy confirmed the LBA-coupling on the outer surface of P-LBA HFMs. Hemocompatibility study indicated the suitability of the modified membranes with human blood. These membranes showed remarkably improved biocompatibility with human primary mesenchymal stem cells and HepG2 cells. Characteristic multi-cellular spheroids of HepG2 cells were observed under scanning electron and confocal microscopy. HepG2 cell functional activity was measured by quantifying the urea synthesis, albumin secretion and glucose consumption in the culture media, which indicated the improved HepG2 functions. These experimental results clearly suggest the potentiality of these LBA-modified P HFMs as a suitable biocompatible substrate for promoting HepG2 attachment and function leading to their application in bioreactors and bio-artificial liver devices.