Temperature-dependent and time-dependent effects of hyperthermia mediated by dextran-coated La0.7Sr0.3MnO3: in vitro studies

Reihaneh Haghniaz, Rinku D Umrani, Kishore M Paknikar Centre for Nanobioscience, Agharkar Research Institute, Pune, Maharashtra, India Background: The purpose of this study was to investigate the therapeutic efficacy of dextran-coated (Dex) La0.7Sr0.3MnO3 (LSMO) nanoparticles-mediated hyperthermia at different temperatures (43°C, 45°C, and 47°C) based on cell killing potential and induction of heat shock proteins in a murine melanoma cell (B16F1) line. Methods: LSMO nanoparticles were synthesized by a citrate-gel method and coated with dextran. B16F1 cells were exposed to the Dex-LSMO nanoparticles and heated using a radiofrequency generator. After heating, the morphology and topology of the cells were investigated by optical microscopy and atomic force microscopy. At 0 hours and 24 hours post heating, cells were harvested and viability was analyzed by the Trypan blue dye exclusion method. Apoptosis and DNA fragmentation were assessed by terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay and agarose gel electrophoresis, respectively. An enzyme-linked immunosorbent assay was used to quantify heat shock protein levels. Results: Our data indicate that cell death and induction of heat shock proteins in melanoma cells increased in a time-dependent and temperature-dependent manner, particularly at temperatures higher than 43°C. The mode of cell death was found to be apoptotic, as evident by DNA fragmentation and TUNEL signal. A minimum temperature of 45°C was required to irreversibly alter cell morphology, significantly reduce cell viability, and result in 98% apoptosis. Repeated cycles of hyperthermia could induce higher levels of heat shock proteins (more favorable for antitumor activity) when compared with a single cycle. Conclusion: Our findings indicate a potential use for Dex-LSMO-mediated hyperthermia in the treatment of melanoma and other types of cancer. Keywords: hyperthermia, Dex-LSMO nanoparticles, heat shock proteins, melanoma, apoptosi

Hyperthermia mediated by dextran-coated La0.7Sr0.3MnO3 nanoparticles: in vivo studies

Reihaneh Haghniaz, Rinku D Umrani, Kishore M Paknikar Centre for Nanobioscience, Agharkar Research Institute, Pune, India Purpose: The aim of this study was to evaluate radiofrequency-induced dextran-coated lanthanum strontium manganese oxide nanoparticles-mediated hyperthermia to be used for tumor regression in mice.Materials and methods: Nanoparticles were injected intra-tumorally in melanoma-bearing C57BL/6J mice and were subjected to radiofrequency treatment.Results: Hyperthermia treatment significantly inhibited tumor growth (~84%), increased survival (~50%), and reduced tumor proliferation in mice. Histopathological examination demonstrated immense cell death in treated tumors. DNA fragmentation, increased terminal deoxynucleotidyl transferase-dUTP nick end labeling signal, and elevated levels of caspase-3 and caspase-6 suggested apoptotic cell death. Enhanced catalase activity suggested reactive oxygen species-mediated cell death. Enhanced expression of heat shock proteins 70 and 90 in treated tumors suggested the possible development of “antitumor immunity”.Conclusion: The dextran-coated lanthanum strontium manganese oxide-mediated hyperthermia can be used for the treatment of cancer. Keywords: lanthanum strontium manganese oxide nanoparticles, melanoma, tumor regression, survival, heat shock proteins, MR

Carbon nanospheres mediated delivery of nuclear matrix protein SMAR1 to direct experimental autoimmune encephalomyelitis in mice

Sijo V Chemmannur,1,* Prasad Bhagat,2,* Bhalchandra Mirlekar,1 Kishore M Paknikar,2 Samit Chattopadhyay1,3 1Disease and Chromatin Biology Laboratory, National Center for Cell Science, Pune University Campus, Pune, Maharashtra, India; 2Center for Nanobioscience, Agharkar Research Institute, Pune, Maharashtra, India; 3Indian Institute of Chemical Biology, Kolkata, India *These authors have contributed equally to this work Abstract: Owing to the suppression of immune responses and associated side effects, steroid based treatments for inflammatory encephalitis disease can be detrimental. Here, we demonstrate a novel carbon nanosphere (CNP) based treatment regime for encephalomyelitis in mice by exploiting the functional property of the nuclear matrix binding protein SMAR1. A truncated part of SMAR1 ie, the DNA binding domain was conjugated with hydrothermally synthesized CNPs. When administered intravenously, the conjugate suppressed experimental animal encephalomyelitis in T cell specific conditional SMAR1 knockout mice (SMAR-/-). Further, CNP-SMAR1 conjugate delayed the onset of the disease and reduced the demyelination significantly. There was a significant decrease in the production of IL-17 after re-stimulation with MOG. Altogether, our findings suggest a potential carbon nanomaterial based therapeutic intervention to combat Th17 mediated autoimmune diseases including experimental autoimmune encephalomyelitis. Keywords: carbon nanospheres, EAE, IL-17, SMAR1, Th1

In vitro and in vivo studies of a novel bacterial cellulose-based acellular bilayer nanocomposite scaffold for the repair of osteochondral defects

Jyoti V Kumbhar,1 Sachin H Jadhav,2 Dhananjay S Bodas,1 Amruta Barhanpurkar-Naik,3 Mohan R Wani,3 Kishore M Paknikar,1 Jyutika M Rajwade1 1Nanobioscience, 2Animal Sciences Division, Agharkar Research Institute, 3National Centre for Cell Science, Savitribai Phule Pune University, Pune, India Abstract: Bacterial cellulose (BC) is a naturally occurring nanofibrous biomaterial which exhibits unique physical properties and is amenable to chemical modifications. To explore whether this versatile material can be used in the treatment of osteochondral defects (OCD), we developed and characterized novel BC-based nanocomposite scaffolds, for example, BC-hydroxyapatite (BC-HA) and BC-glycosaminoglycans (BC-GAG) that mimic bone and cartilage, respectively. In vitro biocompatibility of BC-HA and BC-GAG scaffolds was established using osteosarcoma cells, human articular chondrocytes, and human adipose-derived mesenchymal stem cells. On subcutaneous implantation, the scaffolds allowed tissue ingrowth and induced no adverse immunological reactions suggesting excellent in vivo biocompatibility. Implantation of acellular bilayered scaffolds in OCD created in rat knees induced progressive regeneration of cartilage tissue, deposition of extracellular matrix, and regeneration of subchondral bone by the host cells. The results of micro-CT revealed that bone mineral density and ratio of bone volume to tissue volume were significantly higher in animals receiving bilayered scaffold as compared to the control animals. To the best of our knowledge, this study proves for the first time, the functional performance of acellular BC-based bilayered scaffolds. Thus, this strategy has great potential for clinical translation and can be used in repair of OCD. Keywords: bacterial cellulose-hydroxyapatite, bacterial cellulose-glycosaminoglycan, osteochondral defect, stem cell differentiation, acellular bilayer scaffold&nbsp