Kishor Sarkar

pH sensitive N-succinyl chitosan grafted polyacrylamide hydrogel for oral insulin delivery.

pH sensitive PAA/S-chitosan hydrogel was prepared using ammonium persulfate (APS) as an initiator and methylenebisacrylamide (MBA) as a crosslinker for oral insulin delivery. The synthesized copolymer was characterized by Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) study; morphology was observed under scanning electron microscope (SEM). The PAA/S-chitosan with ∼ 38% of insulin loading efficiency (LE) and ∼ 76% of insulin encapsulation efficiency (EE), showed excellent pH sensitivity, retaining ∼ 26% of encapsulated insulin in acidic stomach pH 1.2 and releasing of ∼ 98% of insulin in the intestine (pH 7.4), providing a prolonged attachment with the intestinal tissue. The oral administration of insulin loaded PAA/S-chitosan hydrogel was successful in lowering the blood glucose level of diabetic mice. The bioavailability of insulin was ∼ 4.43%. Furthermore, no lethality or toxicity was documented after its peroral administration. Thus, PAA/S-chitosan hydrogel could serve as a promising oral insulin carrier in future.

Oral insulin delivery by self-assembled chitosan nanoparticles: in vitro and in vivo studies in diabetic animal model.

We have developed self-assembled chitosan/insulin nanoparticles for successful oral insulin delivery. The main purpose of our study is to prepare chitosan/insulin nanoparticles by self-assembly method, to characterize them and to evaluate their efficiency in vivo diabetic model. The size and morphology of the nanoparticles were analyzed by dynamic light scattering (DLS), atomic force microscopy (AFM) and scanning electron microscopy (SEM). The average particle size ranged from 200 to 550 nm, with almost spherical or sub spherical shape. An average insulin encapsulation within the nanoparticles was ~85%. In vitro release study showed that the nanoparticles were also efficient in retaining good amount of insulin in simulated gastric condition, while significant amount of insulin release was noticed in simulated intestinal condition. The oral administrations of chitosan/insulin nanoparticles were effective in lowering the blood glucose level of alloxan-induced diabetic mice. Thus, self-assembled chitosan/insulin nanoparticles show promising effects as potential insulin carrier system in animal models.

Preparation of low molecular weight N-maleated chitosan-graft-PAMAM copolymer for enhanced DNA complexation

Low molecular weight N-maleated chitosan-graft-PAMAM (polyamidoamine) copolymer was prepared through N-maleated chitosan (NMC) by Michael type addition reaction to enhance its solubility in water as well as its cationic character for enhancement of DNA complexation. FTIR, (1)H NMR, XRD and GPC were used to characterize the graft copolymers. The copolymer showed better DNA complexation ability at low N/P ratio than that of chitosan due to increased surface charge density by the incorporation of PAMAM molecule on to chitosan backbone. The copolymer can effectively protect the DNA toward anionic surfactant. In vitro release study showed efficient DNA release occurred at physiological pH (pH 7.4). In vitro cell cytotoxicity test indicated toward less cytotoxicity of NMC-graft-PAMAM copolymers compared to that of 25 kDa PEI. Thus, the synthesized NMC-graft-PAMAM copolymers have great potential of finding application in drug and gene delivery.

Preparation of low toxic fluorescent chitosan-graft-polyethyleneimine copolymer for gene carrier

Fluorescent chitosan-graft-polyethyleneimine (PEI) copolymer was prepared by incorporating PEI molecule onto chitosan backbone through naphthalimide moiety by simple substitution reaction. 4-Bromo-1,8-naphthalic anhydride was used as fluorescent probe due to its strong fluorescence and good photo-stability property and the presence of a fine tunable bromide functional group in the naphthalimide ring, in this work. The copolymer was characterized by FTIR, elemental analysis and XRD. The fluorescence property of the copolymer was determined by UV-vis spectrometer and spectrofluorometer. The effects of pH and temperature on fluorescence property of the copolymer were also studied. The graft copolymer with degree of substitution 37.6 of PEI onto chitosan showed better complexation ability with DNA at comparatively low N/P (nitrogen to phosphate ratio) ratio 1.0 compared to that of chitosan (N/P ratio 2.0). The cytotoxicity of PEI largely decreased after grafting with chitosan and all the copolymers showed above 50% cell viability even at high polymer concentration (300 μg/mL). Therefore, the prepared fluorescent chitosan-graft-PEI copolymer may be used as a biological marker as well as drug or gene carrier with low toxicity.

Efficient oral insulin delivery by dendronized chitosan: in vitro and in vivo studies

The development of efficient and bio-safe polymeric carriers for oral insulin delivery is a major thrust in biomedical research. In this paper, dendronized chitosan (DCTS) is prepared using a Michael-type addition reaction by grafting polyamidoamine (PAMAM) onto chitosan to improve its water solubility, pH sensitivity, and insulin encapsulation efficiency for enhanced bioavailability of the oral insulin. The self-assembled dendronized chitosan nanoparticles are prepared using a mild coacervation method, in which almost sub-spherically shaped nanoparticles of 85–150 nm size are produced, with an insulin encapsulation of approx. 95%. In vitro release study confirms a pH-sensitive and self-sustained release of insulin, where the oral administration of these nanoparticles exhibits a pronounced hypoglycemic effect in diabetic mice, producing a relative bioavailability of ∼9.19%. As no systemic toxicity is observed with its peroral delivery, these DCTS nanoparticles can effectively serve as a promising device in the efficient administration of oral insulin.

PAMAM conjugated chitosan through naphthalimide moiety for enhanced gene transfection efficiency

Development of efficient and safe gene carrier is the main hurdle for successful gene therapy till date. Poor water solubility and low transfection efficiency of chitosan are the main drawbacks to be efficient gene carrier for successful gene therapy. In this work, PAMAM conjugated chitosan was prepared through naphthalimide moiety by simple substitution reaction. The synthesis of the chitosan conjugates was confirmed by FTIR, (1)H NMR and XRD analyses. The conjugates showed enhanced DNA binding capability compared to that of unmodified chitosan. Moreover, the conjugates showed minimal cytotoxicity compared to that of polyethyleneimine (PEI, 25 kDa) and also showed good blood compatibility with negligible haemolysis. The transfection efficiency of the conjugate was significantly increased compared to that of unmodified chitosan and it also surpassed the transfection efficiency by PEI. Therefore, PAMAM conjugated chitosan can be used safely as alternate efficient gene delivery vector in gene therapy.

Recyclable Crosslinked O-Carboxymethyl Chitosan for Removal of Cationic Dye from Aqueous Solutions

Carboxymethyl chitosan have been investigated for many biomedical applications as well as for the removal of metal ion and cationic dye from aqueous solution. But, carboxymethyl chitosan is soluble in water and therefore, it is difficult to reuse. The aim of the work was to prepare cross-linked O-carboxymethyl chitosan (OCMCTS) with different degree of substitution for the removal of Crystal Violet (CV) cationic dye from aqueous solution. The influence of the parameters such as initial pH of the dye solution, initial dye concentration, adsorption temperature, degree of substitution of OCMCTS and adsorption time on the adsorption capacity was studied using batch method. The results showed that the adsorption capacity of modified CTS increased from 28.49 mg/g to 239.54 mg/g. The kinetic study of OCMCTS showed that it follows the pseudo-second-order kinetic rather than pseudo-first-order kinetic. The adsorption equilibrium showed that the experimental data could be best fitted to the Langmuir equation. The desorbed OCMCTS can be reused to absorb the cationic dyes. Therefore, cross-linked OCMCTS may be favorable adsorbent and could be employed as low-cost alternatives for the removal of cationic dyes in wastewater treatment.

Biofunctionalized surface-modified silver nanoparticles for gene delivery

Silver nanoparticles (AgNPs) find use in different biomedical applications including wound healing and cancer. We propose that the efficacy of the nanoparticles can be further augmented by using these particles for gene delivery applications. The objective of this work was to engineer biofunctionalized stable AgNPs with good DNA binding ability for efficient transfection and minimal toxicity. Herein, we report on the one-pot facile green synthesis of polyethylene glycol (PEG) stabilized chitosan-g-polyacrylamide modified AgNPs. The size of the PEG stabilized AgNPs was 38 ± 4 nm with a tighter size distribution compared to the unstabilized nanoparticles which showed bimodal distribution of particle sizes of 68 ± 5 nm and 7 ± 4 nm. To enhance the efficiency of gene transfection, the Arg-Gly-Asp-Ser (RGDS) peptide was immobilized on the silver nanoparticles. The transfection efficiency of AgNPs increased significantly after immobilization of the RGDS peptide reaching up to 42 ± 4% and 30 ± 3% in HeLa and A549 cells, respectively, and significantly higher than 34 ± 3% and 23 ± 2%, respectively, with the use of polyethyleneimine (25 kDa). These nanoparticles were found to induce minimal cellular toxicity. Differences in cellular uptake mechanisms with RGDS immobilization resulting in improved efficiency are elucidated. This study presents biofunctionalized AgNPs for potential use as efficient nonviral carriers for gene delivery with minimal cytotoxicity toward augmenting the therapeutic efficacy of AgNPs used in different biomedical products.

Blood compatible N-maleyl chitosan-graft-PAMAM copolymer for enhanced gene transfection

To improve transfection efficiency, we prepared N-maleyl chitosan-graft-polyamidoamine (NMCTS-graft-PAMAM) copolymer. Self-assembled NMCTS-graft-PAMAM/pDNA complexes were prepared by complex coacervation method at different N/P (nitrogen to phosphate ratio) ratios. The copolymer effectively formed complexes with pDNA at lower N/P ratio (N/P ratio 1.0) than that of unmodified chitosan (N/P ratio 2.0) and the complexes were spherical with particle size of 100-150 nm. The copolymer showed significant protection of DNA from nuclease attack with lower toxicity against HeLa cell. The copolymer also showed no noticeable hemolytic effects up to 10mg/mL indicating no detectable disturbance of the red blood cell membranes. The transfection efficiency of the copolymer was increased significantly compared to that of chitosan and reached up to 36±2% at N/P ratio 7.0 which was higher than that of PEI (30±3% at N/P ratio 10). Therefore, the copolymer may be a strong alternative candidate as effective nonviral vector.

Removal of Anionic Dye in Acid Solution by Self Crosslinked Insoluble Dendronized Chitosan

Insoluble Dendronized Chitosan (DCTS) was prepared to improve the adsorption capacity of chitosan (CTS) as well as to lower its solubility at lower pH for efficient removal of acid dye, Acid Blue 9 (AB 9) from aqueous solutions. Dendronized chitosan was prepared by grafting ‘dendrimer-like’ Polyamidoamine (PAMAM) onto CTS surface using Michael addition reaction followed by amidation reaction. The obtained CTS derivative became insoluble at any pH medium due to self inter- and intra-molecular cross linking during the reaction without any external crosslinker. The adsorption capacity of the CTS derivative was studied using batch method with respect to various parameters like, initial pH of the dye solution, initial dye concentration, adsorption temperature and adsorption time. The batch study showed that the adsorption capacity of CTS derivative increased many times than that of chitosan. From the adsorption kinetic study, it was found that the adsorption of dye molecule on the adsorbent surface obeyed pseudosecond- order kinetic instead of generally reported pseudo-first-order kinetic. Dsorbent surface obeyed pseudo-second-order kinetic instead of generally reported pseudo-first-order kinetic. The adsorption equilibrium showed that the Langmuir equation represented best fit of the experimental data than that of Freundlich equation. The desorbed DCTS could be reused for the adsorption of the acid dyes. The results in this study showed that DCTS may be an attractive candidate for removing anionic dyes from the wastewater.