Poster W14 - Inhibiting Skin Cancer Progression in Hyperglycemia By Mesenchymal Stem Cell derived Exosomes based Nanotherapeutics

Introduction: Skin cancers are the most common form of malignancy and has emerged as a significant public health concern due to rising rates and intricate treatment approaches. Hyperglycemia or high blood glucose has shown to contribute to increasing skin cancer cell proliferation, inducing mutation, augmenting invasion and migration, and activating cancer related pathways due to immunosuppression. Mesenchymal stem cells have shown promising potential as an anticancer treatment due to their anti-tumorigenicity, low immunogenicity. Recent studies showed secreted paracrine factors from MSCs such as exosomes can mimic the quality of parent MSCs with higher safety and administration advantages. Exosomes have also shown to alleviate hyperglycemic condition. In this study, we investigated the therapeutic effect of curcumin loaded exosomes in skin cancer in high glucose condition. Curcumin has shown to have anti-neoplastic activity and triggers process of apoptosis in cancer cells. We hypothesize, by encapsulating curcumin in MSC exosomes will improve the capability of exosome in inhibiting skin cancer cell progression. Currently we are investigating the therapeutic effect of the exosome based nano formulation in skin cancer inhibition in hyperglycemic condition.

Materials and

Methods: 80% confluent MSCs were cultured in serum free media and conditioned media was collected after 24h and 48h and pooled together. The pooled conditioned media was filtered using Amicon Ultra Centrifugal Filters and exosomes were isolated with ExoQuick TC PLUS kit. Total protein concentration of the exosomes was observed using BCA assay. Curcumin was loaded inside exosome using saponin with optimization. Curcumin with exosomes were incubated with 0.2% saponin at RT for 20 mins in a shaker at 300 rpm. The curcumin loaded exosomes were purified and unbound curcumin was removed by filtering using centrifugal filters of 3kDA at 17,000 g for 30 mins at RT. The loaded exosome pellet was taken and mixed with RIPA buffer and fluorescence value was observed at 433/534 Ex/Em to observe the loading efficiency of the drug. Size of the formulation was quantified using DLS. A431 cancer cell line was cultured in DMEM with 4.5 g/l glucose supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. Toxicity effect of the formulation was observed in A431 cells using Alamar blue assay at Ex/Em 570/590. Cells were treated with 1.25 µM or 2.5 µM exosome-curcumin dose. Cells were also treated with either 1.25 µM or 2.5 µM curcumin dose and corresponding exosomes dose of 10 µg/ml or 20 µg/ml, respectively. Cells treated with only DMEM acted as control. Statistical analysis was conducted using One way ANOVA . * p < 0.05, or ** p < 0.01, or *** p < 0.001, ****p < 0.0001.

Results, Conclusions, and Discussions:

Results:
Exosomes isolated from 5ml of conditioned media showed exosomal protein concentration around 206.45 µg/ml ± 55.51 ug/ml. Then curcumin was loaded inside exosome by incubating with saponin and loading efficiency of curcumin was observed from calibration curve of varying concentrations of curcumin vs fluorescence intensity (Figure 1). From calibration curve equation, curcumin loading efficiency inside exosomes was observed around 5.6% ± 0.6%. Further exosome and exosome-curcumin formulation was characterized by the size. The average size of the exosomes was found to be 95.0 ± 3.0 d.nm, and exosomes-curcumin formulation was 149.8 ± 7.3 d.nm. To observe the efficacy of the exosome-curcumin nanoformulation as a therapeutic approach in skin cancer, A431 cells were incubated with 1.25 µM or 2.5 µM curcumin, or exosome-curcumin formulation. As observed from Figure 2, cells treated with 1.25 µM curcumin and corresponding exosome showed no significant increase in toxicity compared to control. However, when cells were treated with 1.25 µM exosome-curcumin formulation, 23.4% increase in toxicity was observed compared to control, (p-value < 0.0001). However, for 2.5 µM exosome-curcumin formulation, 70.02%, 65.98%, and 64.95% increase in toxicity was observed compared to control, 2.5 µM curcumin dose, or corresponding exosome dose, respectively (p-value < 0.0001). Observing the promising initial results of the exosome-curcumin nanoformulation in skin cancer inhibition, exosome nanoformulation shows promising potential of skin cancer therapy in hyperglycemic condition. Further optimization of curcumin encapsulation inside exosomes will be investigated and its potential in diabetic skin cancer therapy and mechanistic behavior will be observed.

Conclusion:
In summary, MSC-derived exosomes could promote enhanced delivery of drug in skin cancer treatment in hyperglycemic condition. Our research findings suggest that exosomes could be considered as a delivery vehicle of drug for skin cancer and enhance toxicity in the cells. Optimization of exosome nanoformulation production and consecutive effect in hyperglycemic skin cancer will be presented.

Acknowledgements (Optional): We would like to thank University of Michigan-Dearborn Department of Mechanical Engineering for funding.