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

International Journal of Industrial Chemistry https://doi.org/10.1007/s40090-019-0185-4 RESEARCH Cytotoxicity and in vitro evaluation of whey protein‑based hydrogels for diabetes mellitus treatment S. J. Owonubi1 · E. Mukwevho2 · B. A. Aderibigbe3 · Neerish Revaprasadu1 · E. R. Sadiku4 Received: 18 July 2018 / Accepted: 23 May 2019 © The Author(s) 2019 Abstract 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 confirmed 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, differentiated and treated with the drug-encapsulated hydrogels to detect the effect on the adipose tissue cells by quantitative real-time polymerase chain reaction (qPCR) identifying gene expression levels by utilizing gene markers specific for diabetes and obesity: cpt-1, glut-4, acc1, pgc-1, mef2a and nrf-1 with comparison to positive control metformin. The cytoxicity studies confirmed non-toxic nature of the hydrogels; identified dosage of drugs encapsulated were effective 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 identified positive effects employing combination therapy, but further studies are required to ascertain the actual effect 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 significantly different from positive control metformin treatments, but the combination therapy investigation showed more promise. Keywords Hydrogel biocomposite · Type II diabetes · Drug release · Adipocytes · Metformin · Gene markers Introduction * S. J. Owonubi * E. Mukwevho 1 Department of Chemistry, University of Zululand, KwaDlangezwa, KwaZulu Natal, South Africa 2 Department of Biochemistry, North-West University, Private Bag X2046, Mmabatho 2735, South Africa 3 Department of Chemistry, University of Fort Hare, Alice Campus, Alice, Eastern Cape, South Africa 4 Polymer Division, Institute of NanoEngineering Research (INER) and Department of Chemical, Metallurgical and Materials Engineering, Tshwane University of Technology, Staatsartillerie Rd, Pretoria West Campus, Pretoria, South Africa In recent times, drug delivery systems are made of natural components, or synthetic materials, such as: carbon nanotubes, silica nanostructures, synthetic hydroxyapatite beads and polyelectrolyte microcapsules gaining significant importance due to their potential benefits in numerous cell-based therapies, tissue engineering techniques, lenses manufacture, cancer therapy, diabetic therapy and drug delivery [1–7]. Hydrogels are of particular interest for drug delivery applications due to their ability to address targeted drug delivery, in addition to their good biocompatibility, tunable network structure needed to control the diffusion of drugs and their ability to imbibe drugs within their mesh network structure [8–10]. Graphene oxide (GO) has gained popularity for its biomedical advancements [11, 12]. The π structure of 13 Vol.:(0123456789) International Journal of Industrial Chemistry graphene enables its interaction with hydrophobic drugs and these features make graphene materials ideal drug carriers, especially for hydrophobic drugs [13–16]. As a result of challenges with targeting causative substances using single drug molecules, combination of different drugs has been administered optimally and at different periods of the treatment [17–19]. Until recently, limited researches on dual drug delivery systems have so far provided positive results [5, 20–22]. Obesity is the accumulation of excess body fat and a prelude to type II diabetes mellitus (T2DM), a number of cardiovascular conditions: stroke, hypertension and certain forms of cancer [23]. Diabetes has become an ever increasing problem of pandemic proportions globally, with 425 million people estimated to be living presently with the condition, it is envisaged to increase by 55% by the year 2035 [24]. Diabetes is a heterogeneous endocrine disorder that is associated with hyperglycemia and there are two main types known, namely: type I diabetes mellitus and T2DM. Literature has shown that excessive lipid levels and their oxidation appear to play a major role in the development of insulin resistance and non-insulin-dependent diabetes (T2DM) [25, 26]. It has also been established that there is a link between the development of insulin resistance and pathogenesis of T2DM [27]. Physical inactivity, tissue inflammation, endoplasmic reticulum stress (ER-stress) in β-cells, oxidative stress, tissue lipid accumulation, aging, obesity and β-cell dysfunction are some of the factors known to be linked to insulin resistance which can progress to T2DM [28]. Oxidative stress and/or pro-inflammatory mediators are/is the most significant factor(s) [29]. The production of excess endogenous oxidative species causes oxidative stress, which can manipulate signaling pathways and damage cells [30, 31]. Therefore, the inhibition of oxidation stress aids in the management of insulin resistance and diabetes [32]. Insulin resistance and T2DM can be induced by pro-inflammatory mediators through stimulation of metabolic and/or various transcriptional mediated molecular pathways and oxidative stress [27]. Some studies have focused on oxidative stress mediators, notably pro-inflammatory cytokines such as adipocytokines, chemokines, tumor necrosis factor-alpha (TNFα), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β) epigenetic factors, glucolipotoxicity, various transcriptional and metabolic pathways for the management of T2DM [33–37]. Metabolic pathways involving the regulation of lipids and carbohydrates depend on mitochondrial genes; similarly, glucose and fatty acids modulate the expression levels of a number of genes encoding metabolic enzymes in variety of cell types [38]. Thus, the insulin signaling pathway can be influenced and investigated by so many mediators and intermediates, such as: adenosine monophosphate-activated protein kinase (AMPK), c (...truncated)