Cytotoxicity and in vitro evaluation of whey protein‑based hydrogels for diabetes mellitus treatment

Obesity is the accumulation of excess body fat and the hallmark of type II diabetes mellitus, characterized by hyperglycemia. Glycemic control is very critical to reduce long-term vascular complications resulting from the progressive nature of hyperglycemia. In previous studies, thermally reduced graphene oxide (rGO)-based hydrogel biocomposites were prepared and in vitro drug release studies confrmed their potential as a biodegradable-targeted drug delivery system. Thus, the in vitro biological evaluation of these rGO-based hydrogels was investigated. The hydrogels were encapsulated with chloroquine diphosphate (CQ) and proguanil (P) drugs to investigate potential of combination therapy. The non-toxic nature of the hydrogels was investigated by the use of the MTT assay against 3T3-L1 and c2c12 cell lines. 3T3-L1 pre-adipocytes were grown, diferentiated and treated with the drug-encapsulated hydrogels to detect the efect on the adipose tissue cells by quantitative real-time polymerase chain reaction (qPCR) identifying gene expression levels by utilizing gene markers specifc for diabetes and obesity: cpt-1, glut-4, acc1, pgc-1, mef2a and nrf-1 with comparison to positive control metformin. The cytoxicity studies confrmed non-toxic nature of the hydrogels; identifed dosage of drugs encapsulated were efective within investigated treatment time and qPCR revealed an upregulation of CPT-1, GLUT-4, PGC-1, MEF2A and NRF-1 marker genes, but a downregulation of ACC-1 marker gene. The results from the expression of investigated genes suggest the anti-obesity potential of drugs released from the hydrogels. There were identifed positive efects employing combination therapy, but further studies are required to ascertain the actual efect of the drugs in combination, by further varying the ratios of drugs (instead of the presented 1:1 ratio) employed. Statistically, the results from the individual drug release treatments were not signifcantly diferent from positive control metformin treatments, but the combination therapy investigation showed more promise.