Page 45 - Biennial Report 2018-20 Jun 2021

treatment with LbcinB6 modified nanoassemblies. To measure intracellular ROS, B16-F10 cells
were treated with ZnO+M9+LbcinB6 nanoassembly and after 24 hours of treatment H2DCFDA
staining was done followed by mean fluorescence intensity measurement by flow cytometry.
After 24 hours of treatment, a significant increase in ROS production was observed in case of the
nanoassembly treated cells as compared to the control nanoparticle treated cells. Further, to
analyze apoptosis, B16-F10 cells were incubated with ZnO+M9+LbcinB6 nanocomplex for 1h,
cells were treated with annexin V fluorochrome and flow cytometry was performed. Flow
cytometry assays indicated that around 30% of the population were apoptotic while the necrotic
cellular population was less than 4%, suggesting that the cytotoxic effect of the nanocomplex
involved early events of apoptosis. Peptide nanocomplexes are also being used in other
biological applications like bone regeneration, in collaboration with Delhi Technological
University.

LOCALIZED AND SYSTEMIC DELIVERY OF THERAPEUTICS FOR PREVENTION
OF COLD INDUCED INJURIES INCLUDING FROSTBITE

Frostbite is caused by exposure to extreme cold conditions, or accidental and direct exposure to
freezing agents. It is a common problem faced by military personnel posted at high altitudes.
The Defence Research and Development Organization has funded a project to Munia Ganguli to
develop therapeutic strategies for frostbrite, using proteins known to protect cells of some
invertebrates from freezing. Antifreeze encoding plasmid was chosen from the literature.
Antifreeze protein RiAFP, from beetle Rhagium inquisitor, which is reported to be one of the
most potent antifreeze proteins, was selected for delivery to skin cells. The construct was
designed and cloning of RiAFP into mammalian expression vectors was carried out. After
confirming the cloning and creating the AFP expressing plasmid, nanocomplexes were prepared
with the DNA and skin penetrating peptides. Two different cationic and amphipathic peptides
earlier designed in the laboratory that show efficient skin penetration were used for this
purpose. Chondroitin sulphate was used for stabilising the nanocomplexes. The nanocomplexes
were characterized for their size, shape, morphology and charge using Atomic Force Microscopy
and Dynamic Light Scattering. The complexes were around 100-200 nm in size with surface
positive charge. Change in cell viability, following addition of these nanocomplexes, was studied
in two skin cell lines using MTS assay. The coated nanocomplexes were found to be compatible
to the cells and showed less cell death (as compared to commercial agents). The nanocomplexes
in selected conditions also showed transfection equivalent to commercial agents in multiple skin
cell lines. When cold stress was applied to the cells with and without treatment of
nanocomplexes, it was observed that while control plasmid without antifreeze did not show
rescue of cellular morphology after cold stress, the treatment helped in the rescue.

A mouse model of frostbite was established in-house on BALB/c mice based on minor
modifications of literature protocol and standardization of the cold stress regime. The model
was developed using frozen magnets (in -80°C) and created frostbite injury on skin using
continuous 5 minutes freeze. To confirm the model, phenotypic examination was performed to

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