Munia Ganguli, a biochemist at the Institute of Genomics and Integrative Biology, has dedicated her career to developing methods for delivering genes and other drugs to various organs. While presenting her research on skin delivery platforms at a meeting, a fellow scientist asked if she had considered applications in preventing damage to the skin from freezing temperatures. “We had no idea about frostbite at that point or cold-induced injuries,” Ganguli said. “So, this was kind of serendipitous that we moved into this area.”

Ganguli has since explored several biomolecular approaches to equip the skin with antifreeze-like protection. Her team recently developed a concoction of synthetic cryopreservative chemicals that helps to prevent cold-induced injury to cells and mice. Ganguli hopes to provide a preventative solution to frostbite, mitigating pain, swelling, and tissue death in people routinely exposed to frigid conditions.
What is your motivation for studying preventative approaches to frostbite?
Researchers have investigated heat stress extensively, but studies on the effects of cold and how to address them are not as prevalent in the scientific literature. We don’t have a good option for preventing cold-induced injuries, but sometimes, people have no choice but to be exposed to severe temperatures. In the civilian population, homeless people and those who work in the cold are regularly exposed to very low temperatures. And in the military, people endure brutally cold conditions for long hours.
For the people in these scenarios, preventative approaches are very important. Short encounters with extremely low temperatures can quickly wreak havoc, so once they’ve been exposed to the cold, there is a small window of time to stop the damage. Current treatments such as rewarming, vasodilators that increase blood flow, and thrombolytics that break up blood clots may be helpful, but they are given after cold exposure when some damage has already set in (1). And in remote settings, people may not have access to these therapeutic interventions. Having an option that can be applied in advance could prevent the injury from happening in the first place.
What types of approaches have you explored?
We have investigated antifreeze proteins, which combat the ice crystals that form inside and outside the cell due to severe cold, causing cellular damage. These proteins bind to the ice crystals, reduce the freezing point of water, and inhibit recrystallization of the ice. Antifreeze proteins are naturally expressed in some organisms that survive in low temperatures. In one key study, researchers developed a transgenic mouse expressing an antifreeze glycoprotein present in ticks, conferring protection against the cold (2). In humans, the protein needs to be expressed transiently, and ensuring sufficient expression and delivery of the DNA are challenges with this approach. We developed agents to facilitate the delivery of antifreeze proteins in the form of plasmids, which are too large and negatively charged to penetrate the skin by themselves. However, we still could not transfect many cells and did not observe an effect on cold resistance at a relevant temperature range.
We then considered whether it might be more feasible to work with existing chemicals used for cryopreservation, such as dimethyl sulfoxide (DMSO) and polyvinyl alcohol (PVA). We sought to identify a combination of molecules that would prevent intracellular and extracellular ice crystal formation and maintain cell viability at both low temperatures and room temperature. In order to use the mixture preventatively, someone would apply it at room temperature before being exposed to the cold, so we needed to ensure that it was nontoxic during this stage. We screened a large number of combinations and identified a ratio of DMSO to PVA that is well tolerated at room temperature and protects cells from the cold (3).
How did you determine that this mixture protects cells from the cold?
When we treated cells with the cryopreservative composition (called SynAFP) and then incubated them at -20°C for one hour, we observed significantly higher viability compared to untreated cells. We found that cold exposure induced changes in cellular morphology, including damage to the cytoskeleton and cell membrane, and that pretreatment with SynAFP prevented these effects. Cold can also trigger cell cycle arrest, but we found that the cryopreservative chemicals restored a normal cell cycle progression. Additionally, we analyzed the proteome and found that while cold-stressed cells showed overexpression of proteins associated with oxidative stress, SynAFP-treated cells did not exhibit cold-induced proteomic changes. These experiments demonstrate that in the presence of SynAFP, the cells not only survive, but retain their inherent properties.
You then tested SynAFP in mice. How did you develop an animal model of cold injury and what did you observe?
We used chilled magnets to apply cold stress to the dorsal skin surfaces on the backs of the mice and standardized how long we needed to administer the treatment to produce a consistent cold injury. We analyzed the skin tissue histologically and found that these mice had significant damage to the epidermis. Pretreating the mice with SynAFP prevented this damage, yielding an intact epidermis. When we examined the mice phenotypically, we saw that the SynAFP-treated mice showed a much smaller cold-induced wound than the untreated mice. We also formulated SynAFP as an aloe vera-based cream and saw that it maintained similar protection against cold injury.
There is so much to explore, and we are at the tip of the literal iceberg.
- Munia Ganguli, Institute of Genomics and Integrative Biology
We obtained these results when we applied the cold stress 15 minutes after SynAFP but did not observe the same degree of protection with longer gap times. We are interested in seeing if multiple applications of the cream can provide a longer lasting effect without producing toxicity toward the skin. Promisingly, we found that applying SynAFP to the mice multiple times did not compromise skin integrity.
What do you see as the future of this budding field of cold protection?
We need multiple sustainable options. While there are challenges with using antifreeze proteins, there are also immense possibilities. Researchers are investigating how specific antifreeze proteins work and developing synthetic analogues of these proteins, and I think both antifreeze proteins and their mimics will be important. On the other hand, there are many more molecules available to cryopreserve cells that are well-suited for applications in cold injury and frostbite prevention. There is so much to explore, and we are at the tip of the literal iceberg.
This interview has been condensed and edited for clarity.
References
- Gupta, A., Soni, R., & Ganguli, M. Frostbite — manifestation and mitigation. Burns Open 5, 96-103 (2021).
- Heisig, M. et al. Frostbite protection in mice expressing an antifreeze glycoprotein. PLoS ONE 10, e0116562 (2015).
- Gupta, A. et al. A combination of synthetic molecules acts as antifreeze for the protection of skin against cold-induced injuries. ACS Appl Bio Mater 5, 252-264 (2022).