Tarun Agarwal

Calcium alginate-carboxymethyl cellulose beads for colon-targeted drug delivery

The present study delineates preparation, characterization and application of calcium alginate (CA)-carboxymethyl cellulose (CMC) beads for colon-specific oral drug delivery. Here, we exploited pH responsive swelling, mucoadhesivity and colonic microflora-catered biodegradability of the formulations for colon-specific drug delivery. The CA-CMC beads were prepared by ionic gelation method and its physicochemical characterization was done by SEM, XRD, EDAX, DSC and texture analyzer. The swelling and mucoadhesivity of the beads was found higher at the simulated colonic environment. Variation was more prominent in compositions with lower CMC concentrations. CA-CMC formulations degraded slowly in simulated colonic fluid, however the degradation rate increased drastically in the presence of colonic microflora. In vitro release study of anticancer drug 5-fluorouracil (5-FU) showed a release (>90%) in the presence of colonic enzymes. A critical analysis of drug release profile along with FRAP (fluorescence recovery after photobleaching) study revealed that the presence of CMC in the formulation retarded the release rate of 5-FU. 5-FU-loaded formulations were tested against colon adenocarcinoma cells (HT-29). Cytotoxicity data, nuclear condensation-fragmentation and apoptosis analysis (by flow cytometry) together confirmed the therapeutic potential of the CA-CMC formulations. In conclusion, CA-CMC beads can be used for colon-specific drug delivery.

Guar gum and sesame oil based novel bigels for controlled drug delivery

Bigels are novel semi-solid formulations which have been drawing attention of many research scientists due to their numerous advantages over the conventional gels. The objective of this study was to develop and characterize novel bigels by mixing guar gum hydrogel and sorbitan monostearate-sesame oil based organogel for controlled drug delivery applications. The confocal microscopy suggested the existence of both aqueous and oil phases together as bigel. Micro-scale deformation (viscometric) analysis in conjugation with macro-scale deformation studies suggested shear-thinning and viscoelastic nature of the bigels. Thermal study suggested an increase in thermal stability with the increase in organogel proportion in the bigels. The developed bigels were biocompatible in nature. The in vitro drug release study showed that the release of ciprofloxacin (lipophilic drug) increased with a decrease in the organogel content. Further analysis showed that the drug release from all the bigels followed zero order diffusion kinetics which is desirable for a controlled release system. The drug loaded gels showed good antimicrobial efficiency against Bacillus subtilis. In conclusion, the developed bigels may be tried as matrices for topical drug delivery.

Cobalt doped proangiogenic hydroxyapatite for bone tissue engineering application

The present study delineates the synthesis and characterization of cobalt doped proangiogenic-osteogenic hydroxyapatite. Hydroxyapatite samples, doped with varying concentrations of bivalent cobalt (Co(2+)) were prepared by the ammoniacal precipitation method and the extent of doping was measured by ICP-OES. The crystalline structure of the doped hydroxyapatite samples was confirmed by XRD and FTIR studies. Analysis pertaining to the effect of doped hydroxyapatite on cell cycle progression and proliferation of MG-63 cells revealed that the doping of cobalt supported the cell viability and proliferation up to a threshold limit. Furthermore, such level of doping also induced differentiation of the bone cells, which was evident from the higher expression of differentiation markers (Runx2 and Osterix) and better nodule formation (SEM study). Western blot analysis in conjugation with ELISA study confirmed that the doped HAp samples significantly increased the expression of HIF-1α and VEGF in MG-63 cells. The analysis described here confirms the proangiogenic-osteogenic properties of the cobalt doped hydroxyapatite and indicates its potential application in bone tissue engineering.

Gelatin/Carboxymethyl chitosan based scaffolds for dermal tissue engineering applications.

The present study delineates the preparation, characterization and application of gelatin-carboxymethyl chitosan scaffolds for dermal tissue engineering. The effect of carboxymethyl chitosan and gelatin ratio was evaluated for variations in their physico-chemical-biological characteristics and drug release kinetics. The scaffolds were prepared by freeze drying method and characterized by SEM and FTIR. The study revealed that the scaffolds were highly porous with pore size ranging between 90 and 170μm, had high water uptake (400-1100%) and water retention capacity (>300%). The collagenase mediated degradation of the scaffolds was dependent on the amount of gelatin present in the formulation. A slight yet significant variation in their biological characteristics was also observed. All the formulations supported adhesion, spreading, growth and proliferation of 3T3 mouse fibroblasts. The cells seeded on the scaffolds also demonstrated expression of collagen type I, HIF1α and VEGF, providing a clue regarding their growth and proliferation along with potential to support angiogenesis during wound healing. In addition, the scaffolds showed sustained ampicillin and bovine serum albumin release, confirming their suitability as a therapeutic delivery vehicle during wound healing. All together, the results suggest that gelatin-carboxymethyl chitosan based scaffolds could be a suitable matrix for dermal tissue engineering applications.

Development of soy lecithin based novel self-assembled emulsion hydrogels.

The current study reports the development and characterization of soy lecithin based novel self-assembled emulsion hydrogels. Sesame oil was used as the representative oil phase. Emulsion gels were formed when the concentration of soy lecithin was >40% w/w. Metronidazole was used as the model drug for the drug release and the antimicrobial tests. Microscopic study showed the apolar dispersed phase in an aqueous continuum phase, suggesting the formation of emulsion hydrogels. FTIR study indicated the formation of intermolecular hydrogen bonding, whereas, the XRD study indicated predominantly amorphous nature of the emulsion gels. Composition dependent mechanical and drug release properties of the emulsion gels were observed. In-depth analyses of the mechanical studies were done using Ostwald-de Waele power-law, Kohlrausch and Weichert models, whereas, the drug release profiles were modeled using Korsmeyer-Peppas and Peppas-Sahlin models. The mechanical analyses indicated viscoelastic nature of the emulsion gels. The release of the drug from the emulsion gels was diffusion mediated. The drug loaded emulsion gels showed good antimicrobial activity. The biocompatibility test using HaCaT cells (human keratinocytes) suggested biocompatibility of the emulsion gels.

Nickel doped nanohydroxyapatite: vascular endothelial growth factor inducing biomaterial for bone tissue engineering

Biomaterial induced activation of vascular endothelial growth factor (VEGF) pathway for angiogenesis is now gaining recognition as an effective option for tissue engineering. In this context, bivalent nickel (Ni2+) ion doped nano-hydroxyapatites (nHAp) were synthesized by wet chemical method, characterized and evaluated for their osteoconductive and proangiogenic properties. Electron microscopy (FESEM and TEM) along with ICP-OES analysis ensured formation of Ni2+ doped nanoparticles (average ferret diameter 15–17 nm). The ‘apatite identity’ of the nanoparticles was confirmed by XRD and FT-IR. Analysis revealed that Ni2+ doping caused no significant distortion in the crystal structure but decreased the crystallinity of nHAp considerably. Similarly, no major variation in the surface area (BET analysis), the zeta potential and the protein adsorption was observed among the samples. Biological characterization showed that Ni2+ doping influenced the cell viability, proliferation and differentiation of bone cells (MG-63) in a concentration dependent way. ELISA and RT-PCR study revealed that Ni2+ doped nHAp induced cellular VEGF expression many fold in comparison to control. Profiling of hypoxia inducible factor 1 alpha (HIF-1α) expression by immuno-cytochemistry and RT-PCR implied its involvement in cellular VEGF production. In conclusion, Ni2+ doped nHAp may serve as proangiogenic–osteogenic biomaterial for bone tissue engineering.

Molecular modeling and docking study to elucidate novel chikungunya virus nsP2 protease inhibitors

Chikungunya is one of the tropical viral infections that severely affect the Asian and African countries. Absence of any suitable drugs or vaccines against Chikungunya virus till date makes it essential to identify and develop novel leads for the same. Recently, nsP2 cysteine protease has been classified as a crucial drug target to combat infections caused by Alphaviruses including Chikungunya virus due to its involvement viral replication. Here in, we investigated the structural aspects of the nsP2 protease through homology modeling based on nsP2 protease from Venezuelan equine encephalitis virus. Further, the ligands were virtually screened based on various pharmacological, ADME/Tox filters and subjected to docking with the modeled Chikungunya nsP2 protease using AutoDock4.2. The interaction profiling of ligand with the protein was carried out using LigPlot+. The results demonstrated that the ligand with PubChem Id (CID_5808891) possessed highest binding affinity towards Chikungunya nsP2 protease with a good interaction profile with the active site residues. We hereby propose that these compounds could inhibit the nsP2 protease by binding to its active site. Moreover, they may provide structural scaffold for the design of novel leads with better efficacy and specificity for the nsP2 protease.

Gum tragacanth–alginate beads as proangiogenic–osteogenic cell encapsulation systems for bone tissue engineering

There is a dearth of biologically active matrices for the encapsulation of bone cells. Here, we hypothesize that the use of gum tragacanth (GT) with alginate might improve the biological properties of calcium alginate (CA) beads, a common cell encapsulation system. We show that the incorporation of GT in the bead-composition significantly improves the molecular transport, swelling and degradation properties of the CA bead. Although no significant molecular interaction between GT and CA was found, a decrease in the concentration of calcium with an increase in GT concentration was noticed. We show that the presence of GT in the bead-composition resulted in improved viability, proliferation, and differentiation of encapsulated bone cells. We further demonstrate that bone cell loaded CA-GT beads are capable of inducing angiogenesis. In conclusion, we prove that CA-GT beads are more osteo-conductive and proangiogenic in comparison to pure CA beads.

PAMAM (generation 4) incorporated gelatin 3D matrix as an improved dermal substitute for skin tissue engineering

The study explored the prospects of PAMAM (generation 4) applicability in gelatin based scaffolds for skin tissue engineering. The effect of PAMAM on physico-chemical and biological characteristics of gelatin scaffolds was evaluated. Gelatin scaffolds (with/without PAMAM) were prepared by lyophilization, chemically crosslinked by glutaraldehyde and characterized for their morphology (pore size), chemical features (bond nature), water adsorption, biodegradation and biological compatibility. The study demonstrated that addition of PAMAM did not significantly alter the pore size distribution or porosity of the scaffolds. However, water adsorption potential and collagenase mediated degradation significantly enhanced over period of the study. Both the scaffolds (with/without PAMAM) were highly biocompatible and hemocompatible. PAMAM (G4) blended scaffolds showed relatively higher cellular adhesion and proliferation of both keratinocytes and fibroblasts with an improved gene expression profile of native collagen type I of fibroblasts. Moreover, expression of angiogenesis inducing genes, HIF1α and VEGF were also higher in PAMAM blended gelatin matrix. Also, PAMAM incorporated gelatin matrix showed a slower rate of drug release which confirms its suitability for therapeutic delivery during wound healing. These results clearly suggest that blending PAMAM (G4) into the matrix could provide an additional support to scaffold assisted wound healing.

Biophysical changes of ATP binding pocket may explain loss of kinase activity in mutant DAPK3 in cancer: A molecular dynamic simulation analysis.

DAPK3 belongs to family of DAPK (death-associated protein kinases) and is involved in the regulation of progression of the cell cycle, cell proliferation, apoptosis and autophagy. It is considered as a tumor suppressor kinase, suggesting the loss of its function in case of certain specific mutations. The T112M, D161N and P216S mutations in DAPK3 have been observed in cancer patients. These DAPK3 mutants have been associated with very low kinase activity, which results in the cellular progression towards cancer. However, a clear understanding of the structural and biophysical variations that occur in DAPK3 with these mutations, resulting in the decreased kinase activity has yet not been deciphered. We performed a molecular dynamic simulation study to investigate such structural variations. Our results revealed that mutations caused a significant structural variation in DAPK3, majorly concentrated in the flexible loops that form part of the ATP binding pocket. Interestingly, D161N and P216S mutations collapsed the ATP binding pocket through flexible loops invasion, hindering ATP binding which resulted in very low kinase activity. On the contrary, T112M mutant DAPK3 reduces ATP binding potential through outward distortion of flexible loops. In addition, the mutant lacked characteristic features of the active protein kinase including proper interaction between HR/FD and DFG motifs, well structured hydrophobic spine and Lys42-Glu64 salt bridge interaction. These observations could possibly explain the underlying mechanism associated with the loss of kinase activity with T112M, D161N and P216S mutation in DAPK3.