Debasish Mishra

Biofunctionalized, phosphonate-grafted, ultrasmall iron oxide nanoparticles for combined targeted cancer therapy and multimodal imaging.

A novel, inexpensive biofunctionalization approach is adopted to develop a multimodal and theranostic nanoagent, which combines cancer-targeted magnetic resonance/optical imaging and pH-sensitive drug release into one system. This multifunctional nanosystem, based on an ultrasmall superparamagnetic iron oxide (USPIO) nanocore, is modified with a hydrophilic, biocompatible, and biodegradable coating of N-phosphonomethyl iminodiacetic acid (PMIDA). Using appropriate spacers, functional molecules, such as rhodamine B isothiocyanate, folic acid, and methotrexate, are coupled to the amine-derivatized USPIO-PMIDA support with the aim of endowing simultaneous targeting, imaging, and intracellular drug-delivering capability. For the first time, phosphonic acid chemistry is successfully exploited to develop a stealth, multifunctional nanoprobe that can selectively target, detect, and kill cancer cells overexpressing the folate receptor, while allowing real-time monitoring of tumor response to drug treatment through dual-modal fluorescence and magnetic resonance imaging.

Bio-functionalization of magnetite nanoparticles using an aminophosphonic acid coupling agent: new, ultradispersed, iron-oxide folate nanoconjugates for cancer-specific targeting

The present study describes a systematic approach towards the design and development of novel, bio-functionalized, magneto-fluorescent nanoparticles for cancer-specific targeting. Biocompatible, hydrophilic, magneto-fluorescent nanoparticles with surface-pendant amine, carboxyl or aldehyde groups, to be later used for bio-conjugation, were designed using an aminophosphonic acid coupling agent. These magneto-fluorescent nanoparticles were further functionalized with folic acid, using diverse conjugation strategies. A series of new iron-oxide folate nanoconjugates with excellent aqueous dispersion stability and reasonably good hydrodynamic sizes under a wide range of physiological conditions were developed. These ultradispersed nanosystems were analyzed for their physicochemical properties and cancer-cell targeting ability, facilitated by surface modification with folic acid. The nanoparticle size, charge, surface chemistry, magnetic properties and colloidal stability were extensively studied using a variety of complementary techniques. Confocal microscopy, performed with folate receptor positive human cervical HeLa cancer cells, established that these non-cytotoxic iron-oxide folate nanoconjugates were effectively internalized by the target cells through receptor-mediated endocytosis. Cell-uptake behaviors of nanoparticles, studied using magnetically activated cell sorting (MACS), clearly demonstrated that cells over-expressing the human folate receptor internalized a higher level of these nanoparticle-folate conjugates than negative control cells.

Chemical Synthesis, Characterization, and Biocompatibility Study of Hydroxyapatite/Chitosan Phosphate Nanocomposite for Bone Tissue Engineering Applications

A novel bioanalogue hydroxyapatite (HAp)/chitosan phosphate (CSP) nanocomposite has been synthesized by a solution-based chemical methodology with varying HAp contents from 10 to 60% (w/w). The interfacial bonding interaction between HAp and CSP has been investigated through Fourier transform infrared absorption spectra (FTIR) and x-ray diffraction (XRD). The surface morphology of the composite and the homogeneous dispersion of nanoparticles in the polymer matrix have been investigated through scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. The mechanical properties of the composite are found to be improved significantly with increase in nanoparticle contents. Cytotoxicity test using murine L929 fibroblast confirms that the nanocomposite is cytocompatible. Primary murine osteoblast cell culture study proves that the nanocomposite is osteocompatible and highly in vitro osteogenic. The use of CSP promotes the homogeneous distribution of particles in the polymer matrix through its pendant phosphate groups along with particle-polymer interfacial interactions. The prepared HAp/CSP nanocomposite with uniform microstructure may be used in bone tissue engineering applications.

“Clickable”, Trifunctional Magnetite Nanoparticles and Their Chemoselective Biofunctionalization

A multifunctional iron oxide based nanoformulation for combined cancer-targeted therapy and multimodal imaging has been meticulously designed and synthesized using a chemoselective ligation approach. Novel superparamagnetic magnetite nanoparticles simultaneously functionalized with amine, carboxyl, and azide groups were fabricated through a sequence of stoichiometrically controllable partial succinylation and Cu (II) catalyzed diazo transfer on the reactive amine termini of 2-aminoethylphosphonate grafted magnetite nanoparticles (MNPs). Functional moieties associated with MNP surface were chemoselectively conjugated with rhodamine B isothiocyanate (RITC), propargyl folate (FA), and paclitaxel (PTX) via tandem nucleophic addition of amine to isothithiocyanates, Cu (I) catalyzed azide--alkyne click chemistry and carbodiimide-promoted esterification. An extensive in vitro study established that the bioactives chemoselectively appended to the magnetite core bequeathed multifunctionality to the nanoparticles without any loss of activity of the functional molecules. Multifunctional nanoparticles, developed in the course of the study, could selectively target and induce apoptosis to folate-receptor (FR) overexpressing cancer cells with enhanced efficacy as compared to the free drug. In addition, the dual optical and magnetic properties of the synthesized nanoparticles aided in the real-time tracking of their intracellular pathways also as apoptotic events through dual fluorescence and MR-based imaging.

PLGA Microspheres Incorporated Gelatin Scaffold: Microspheres Modulate Scaffold Properties

Freeze drying is one of the popular methods of fabrication for poly(lactide-co-glycolide) (PLGA) microspheres incorporated polymer scaffolds. However, the consequence of microspheres incorporation on physical and biological properties of scaffold has not been studied yet. In this study, attempt has been made to characterize the effect of PLGA microsphere incorporation on the physical properties of freeze-dried gelatin scaffold and its influence on cytocompatibility. Scaffolds loaded with varying amount of PLGA microspheres (10%, 1%, 0.1% w/w) were subjected to microarchitecture analysis, swelling, porosity, mechanical properties, biodegradation, cell adhesion, and cell proliferation studies. Results revealed that an increase in percentage loading of microspheres reduced the pore size and uniformity of the pore structure. Moreover, loading of PLGA microspheres up to 1% w/w significantly increased porosity, swelling, and mechanical properties of the scaffold but variations were not proportional for 10% w/w loading. Results also showed that PLGA microspheres have no significant effect on cell adhesion but influenced the growth kinetics.

Nanocomposites of bio-based hyperbranched polyurethane/funtionalized MWCNT as non-immunogenic, osteoconductive, biodegradable and biocompatible scaffolds in bone tissue engineering

This study focused on the design of novel mechanically tough, biocompatible, osteoconductive and biodegradable scaffolds based on sunflower oil modified hyperbranched polyurethane (HBPU)/functionalized multi-walled carbon nanotube (f-MWCNT) nanocomposites (NCs), and the response of an animal model on their post-implantation. The NC was prepared by an in situ polymerization technique with different wt% of f-MWCNTs. The tensile strength of the NCs was enhanced to 36.98-47.6 MPa from 23.93 MPa (HBPU) and toughness from 12 767 to 18 427-19 440 due to the addition and efficient dispersion of the f-MWCNTs in the HBPU matrix. The post-60 days in vitro biodegraded NC retained sufficient strength (39 ± 1.65 MPa). The increase in wt% of f-MWCNTs had a significant effect on tailoring the physico-mechanical properties of the polymer. The hematological, histological and immunological indices of toxicity suggested the safety potential of the prepared systems within the tested animal model. Moreover, the cytokines (viz. IL-6 and TNF-α) detection, MTT assay and anti-hemolytic assay boosted the non-toxic behavior of the systems. The NC with interconnected pores size (200-330 μm) showed better proliferation and adherence of osteoblast (MG63) cells compared to the HBPU and the results were comparable with the control. Thus the findings confirmed the non-toxicity of f-MWCNTs in association with the polymer and thereby endorsed the NC as a potential biomimetic scaffold for bone tissue engineering.

Abrus abrin derived peptides induce apoptosis by targeting mitochondria in HeLa cells

In our previous study, Abrus abrin derived peptide fraction (ABP) with molecular weight in range of 600–1500 Da was shown to have potent antitumor activity in Daltons lymphoma (DL) tumor bearing mice. The purpose of this study was to elucidate the mechanism of mitochondrial apoptosis induced by the peptide fraction. ABP was found to have selective antiproliferative activity (10 ng–100 ng/ml) on several tumor cell lines in vitro without having any cytotoxic effect on normal cell lines with a dose of 1000 ng/ml. Analysis of the growth inhibitory mechanism in HeLa cells revealed DNA fragmentation with appearance of the sub G0/G1 peak indicative of apoptosis. Further investigation results showed that the apoptotic machinery of HeLa induced by ABP was associated with the release of reactive oxygen species, a drop in mitochondrial transmembrane potential, upregulation of Bax, downregulation of Bcl‐2, and activation of caspase‐3. The peptide fraction was found to target mitochondria of HeLa cells as observed by confocal microscopy. This peptide fraction offers a source of mitochondria penetrating peptides which might have therapeutic induction of apoptosis in cancer cells.

Wound pH-Responsive Sustained Release of Therapeutics from a Poly(NIPAAm-co-AAc) Hydrogel

Wound pH strongly influences residence time and activity of various growth factors during wound healing. Hence, a pH-responsive sustained release growth factor delivery system could be beneficial for effective treatment of wound. In this context, an effort was made to evaluate the potential of a poly(N-isopropylacrylamide-co-acrylic acid) hydrogel as pH-sensitive sustained release system for wound-pH-dependent therapeutics delivery. The polymer was synthesized via radical copolymerization and influence of pH on lower critical solution temperature (LCST), microarchitechture and swelling of the hydrogel was evaluated. Results showed a pH-dependent variation in the physical properties of the hydrogel within the wound pH range. Fluorescence recovery after photobleaching (FRAP) analysis endorsed a pH dependent restricted diffusion of the BSA in the hydrogel. Later, release of bovine serum albumin (BSA), vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) (each 5%, w/v) from the hydrogel within the range of wound pH (pH 6.7–7.9) were examined. Analysis showed non-Fickian release of therapeutics from the hydrogel with a significant variation in release rate and cumulative release with the increase in pH. Retention of the bioactivity of the released EGF was confirmed by studying murine dermal fibroblast cell proliferation in vitro. Finally, a growth factor (EGF or VEGF)-loaded hydrogel was applied on a murine excisional wound model and showed augmentation of wound healing in comparison to conventional sustained release growth factor therapy.

Caprine (Goat) Collagen: A Potential Biomaterial for Skin Tissue Engineering

Collagens presently used in tissue engineering are primarily of bovine or porcine origin. However, a risk of a spongiform encephalopathy epidemic has limited the use of collagen from these sources. Keeping the aforementioned perspective in mind, we explored the possibility of using domestic goat available in the subcontinent as a potential source of collagen for tissue-engineering application. This article delineates the isolation, physico-chemical characterization, biocompatibility study and wound healing application of acid soluble caprine (goat) tendon collagen (GTC). Physico-chemical characterization of 1% acetic acid extracted GTC was done by SDS-PAGE, amino-acid composition analysis, FT-IR and CD spectroscopy. Results revealed that GTC was comprised of type-I collagen. Biocompatibility study showed that GTC augmented cell adhesion, cell cycle progression and proliferation. Immuno-cytochemical analysis in conjugation with traction force microscopy further confirmed a superior focal adhesion complex mediated cell–substrate interaction in GTC. Finally, in vivo study in mice model revealed that GTC has low immunogenicity and it augments healing process significantly. Throughout the study, calf skin collagen (CSC) was used as standard for comparative evaluation. In conclusion, it can be said that GTC may find its application as biomaterial in skin tissue engineering.

On-chip lectin microarray for glycoprofiling of different gastritis types and gastric cancer

An on-chip lectin microarray based glycomic approach is employed to identify glyco markers for different gastritis and gastric cancer. Changes in protein glycosylation have impact on biological function and carcinogenesis. These altered glycosylation patterns in serum proteins and membrane proteins of tumor cells can be unique markers of cancer progression and hence have been exploited to diagnose various stages of cancer through lectin microarray technology. In the present work, we aimed to study the alteration of glycan structure itself in different stages of gastritis and gastric cancer thoroughly. In order to perform the study from both serum and tissue glycoproteins in an efficient and high-throughput manner, we indigenously developed and employed lectin microarray integrated on a microfluidic lab-on-a-chip platform. We analyzed serum and gastric biopsy samples from 8 normal, 15 chronic Type-B gastritis, 10 chronic Type-C gastritis, and 6 gastric adenocarcinoma patients and found that the glycoprofile obtained from tissue samples was more distinctive than that of the sera samples. We were able to establish signature glycoprofile for the three disease groups, that were absent in healthy normal individuals. In addition, our findings elucidated certain novel signature glycan expression in chronic gastritis and gastric cancer. In silico analysis showed that glycoprofile of chronic gastritis and gastric adenocarcinoma formed close clusters, confirming the previously hypothesized linkage between them. This signature can be explored further as gastric cancer marker to develop novel analytical tools and obtain in-depth understanding of the disease prognosis.