Page 56 - Annual report 2021-22

Annual Report 2021-22 |

One of the interests in the lab is to develop anti-freeze formulations for delivery and validate them in
a mouse model of frostbite since this may serve as a preventive intervention in vivo. Munia Ganguli’s
group has previously developed a combination of Poly (vinyl alcohol) (PVA) and Dimethyl Sulfoxide
(DMSO) which gave cellular protection from cold stress and applied this combination in vivo in the
frostbite mice model standardized previously in the laboratory. The combination called fSynAFP when
applied on mouse skin showed no irritation and absence of any erythema or edema. Skin histology 39
showed that there was no perturbation to skin layers when this combination was applied under
physiological conditions. Even an application regime of three consecutive applications 24 hours apart
did not visibly perturb the mice skin for over 48 hours of follow-up from the last application. In vivo
experiments were then done with pre-treatment of PVA-DMSO combination on mice skin, prior to
subjecting it to a 2 min acute cold exposure. The control group was exposed only to cold without any
pre-application of chemicals. In the experimental group, the wound was minor with significantly less
wound surface area as compared to the control group. The wound in the treated mice was healed
when followed up at Day 10, while that in control mice did not show any re-epithelialization. The
histological sections of treated mice showed less infiltration of inflammatory cells and no damage
beyond stratum corneum, while the sections of cold control mice showed an injury up to the dermis
of the mice with an increased infiltration of inflammatory cells.

On checking the histological sections and performing immunostaining experiments for checking skin
integrity markers, there was no perturbation to any layer of skin, which was evident from an intact
epidermis, which showed expression of loricrin as well as keratin 14 markers in its respective layers.
In collaboration with Shantanu Sengupta, the mechanism of action of these agents was explored by
performing whole-cell quantitative proteomics to check for the protein profile when subjected to an
acute cold temperature. Comparison of the cold-stressed cells, those given the treatment with SynAFP
before cold stress and untreated control indicated a pool of 107 proteins that were differentially
expressed on cold stress but remained unaltered with SynAFP pretreatment. These proteins belonged
to pathways like cell redox homeostasis, TCA and glycolysis and cell-cell adhesion regulation. A
dysregulation of proteins involved in cell redox homeostasis along with an overexpression of
apoptosis-inducing factor (Aifm) in -20 °C cells, indicates that cells succumb to cold stress via the
intrinsic pathway of apoptosis.

SynAFP appears to be maintaining the redox homeostasis and therefore significantly decreases the
effect of cold stress. They also observed significant upregulation of the TCA cycle proteins under cold
stress which might be an indicator of accumulation of TCA intermediates on cold exposure. SynAFP
was able to prevent the overexpression of these proteins, thus protecting cells from catastrophic
effects of cold on the energy and metabolic processes of the cells under sustained sub-zero conditions.

Cytotoxic effects of nano-assemblies

Munia Ganguli’s group has also analyzed the cytotoxic effect of nanoassemblies created by surface
decorating ZnO nanoparticles with apoptotic peptide LbcinB6 on both B16-F10 and HaCaT skin cells.
They observed significant cancer cell killing by ZnO-B6 nanoparticle system as compared to normal
keratinocyte cells. They found that specific apoptosis was induced in cancer cells, but not in normal
keratinocytes.

They also conjugated mitochondria outer membrane targeting Amphipathic Tail Anchoring Peptide
(ATAP) to ZnO nanoparticles using an MMP linker and attached a cell penetrating peptide on the
nanoparticle surface. Cellular viability of both B16-F10 and HaCaT cells were analyzed by MTT assay
24 and 48 hours post treatment. They observed higher loss of cellular viability in cancerous B16-F10
cells compared to HaCaT cells. They observed more than 30% apoptotic cell population using Annexin-