Nanoparticle-Driven Adipose Thermogenesis: A Novel Strategy to Reduce Frostbite Wound Severity

Introduction: : Cold induced injuries such as hypothermia are significant concerns for people who engage in mountaineering, skiing, and other outdoor activities in cold weather, especially for our military personnel and researchers who spend extensive amounts of time in the arctic. Frostbite is one of the most common cold-related injuries and requires immediate medical attention. Prolonged exposure to below freezing temperatures causes local hypothermia, ice crystal formation in tissue, and reduced blood flow and oxygen deprivation which can result in significant tissue injuries. We hypothesize that enhancing the thermogenic activity of dermal and subcutaneous adipose tissue can reduce the severity of frostbite injuries and promote wound healing. To test this, we developed a nanotherapeutic that increases thermogenic activity in white adipose tissue. This nanomedicine is composed of lipid-coated mesoporous silica nanoparticles loaded with forskolin. In this study, we propose that localized stimulation of thermogenic activity in subcutaneous white adipose tissue (sWAT) will prevent, halt, and treat cold-induced tissue damage by increasing local tissue temperature and supporting the wound healing process.

Materials and

Methods: : Dendritic mesoporous silica nanoparticles (MSNs) were synthesized and loaded with forskolin (FSK), then fused with a liposomal film via electrostatic interactions to form lipid-coated mesoporous silica nanoparticles (LCMSNs-FSK). The nanoparticles were characterized for size, zeta potential, and drug loading efficiency. In vitro, cytotoxicity, temporal cellular uptake, and thermogenic modulation were evaluated using differentiated human adipose stem cells.

Part One – Local Skin Temperature Modulation: To assess the effect of LCMSNs-FSK on local skin temperature, 16-week-old male C57BL/6J mice were injected with 20 mg/kg of LCMSNs-FSK in the interscapular region, three hours before exposure to a cold chamber at 6°C. (This time point was chosen based on prior observation of peak UCP1 expression at six hours post-treatment.) Rectal temperatures were recorded every 30 minutes for three hours. Surface temperature was captured using an infrared digital thermographic camera positioned 70 cm from the animals.

Part Two – Frostbite Injury Prevention and Treatment: To evaluate the therapeutic potential of LCMSNs-FSK for frostbite injuries, mice received the same treatment as in Part One. A simulated frostbite injury was induced using a metal rod cooled in liquid nitrogen, applied tightly to the skin for one minute. All procedures adhered to UTSA LARC protocols for ethical animal treatment. On day four post-injury, mice were euthanized, and tissues were harvested for histological and gene expression analysis to assess inflammation, adipocyte thermogenesis, and wound healing (n = 3).

Results, Conclusions, and Discussions:: During the cold chamber experiment, we observed a gradual increase in local skin temperature in mice treated with LCMSN-FSK, a change not seen in PBS-treated controls. This temperature shift was calculated by subtracting the average temperature within a region of interest (ROI) at various timepoints from the initial baseline temperature (Figure 1A). A comparison of the maximum temperature change revealed a consistent increase over the experimental time course in the LCMSN-FSK group, supporting its thermogenic effect (Figure 1B). This treatment not only helped maintain local temperature but also enhanced localized heat output.

In Part Two of the experiment, although no significant difference in frostbite wound size was detected on Day 4 (data not shown), RT-PCR analysis revealed significant changes in gene expression related to inflammation, thermogenesis, and wound healing in both the dermal and subcutaneous layers (Figures 2 and 3). In the dermis, both pro-inflammatory genes (Tnfα, Il-6) and anti-inflammatory genes (Arg1, Il-10) were significantly upregulated (p < 0.05 and p < 0.001, respectively), suggesting a balanced and potentially resolving inflammatory response. Ucp1 expression was significantly increased, and Adipoq (adiponectin) was reduced, indicating enhanced thermogenic activity in dermal adipocytes. Although no significant changes were observed in Cd31 and Vegf α , there was a notable increase in Col3a1, suggesting enhanced early wound healing processes.

In the subcutaneous adipose tissue, pro-inflammatory genes were significantly downregulated while Arg1 was significantly upregulated, further supporting an anti-inflammatory shift with LCMSN-FSK treatment. Additionally, Ucp1 and Atgl (a marker of triglyceride catabolism) were upregulated, indicating elevated thermogenic and metabolic activity. Increased expression of Vegfα and a trend toward elevated Cd31 also suggest enhanced vascular remodeling.

In summary, LCMSN-FSK effectively increased local skin temperature in cold-exposed mice and showed potential to modulate inflammation, thermogenesis, and early wound healing responses following cold-induced injury.

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