High content analysis of cytotoxic effects of pDMAEMA on human intestinal epithelial and monocyte cultures − Rakyat Biologi

HCA analysis allows the combination of fluorescent probes to be used concurrently to investigate different parameters of cytotoxicity [9-13]. Separate fluorescence signals can be detected simultaneously using HCA and dyes that have overlapping fluorescence may be differentiated if their cellular locations do not overlap [35]. By incorporating three to four fluorescent dyes, up to eight markers of cytotoxicity have been successfully investigated concurrently [10]. These include nuclear morphology, plasma membrane integrity, mitochondrial function, intracellular calcium and cell proliferation [10, 11]. Use of HCA in the development of novel antimicrobial agents may increase the speed by which these molecules are assessed and processed to the clinical trials stage of development. To date, in vitro assays including MTT and lactate dehydrogenase have been commonly used to assess cytotoxicity of pDMAEMA [3, 36, 37, 38]. Although useful indicators, they do not give much information about the processes of cytotoxicity nor early stage events. Other fluorescent-based tests using flow cytometry and confocal microscopy may provide additional information about the gradual process of cytotoxicity induced by a polymer, nor their mechanism. They are also time consuming, costly and labour intensive. HCA overcomes these problems by providing a high throughput process that allows testing of numerous cytotoxicity markers concurrently.

Data from MTT analysis has suggested variable cytotoxic effects of pDMAEMA in different cell types [3]. Therefore, the cytotoxic-inducing aspects of this polymer were investigated by HCA. NA was found to increase in U937 cells and decrease in Caco-2 cells upon exposure to pDMAEMA. An increase in NA may be associated with nuclear swelling as a result of premature senescence [39-41]. Cellular shrinkage and condensation of the chromatin are markers of apoptosis [42]. The decrease in NA in Caco-2 cells may be an indication of apoptosis and the swelling of the U937 nuclei may indicate necrosis. The increased [Ca²⁺]i observed in Caco-2 cells after treatment with pDMAEMA may also be an indicator of apoptosis [43]. In U937 cells, [Ca²⁺]i decreased upon treatment with pDMAEMA. This may be related to the increased plasma membrane permeability (PMP), which may cause dye release from the cell thereby masking an increase in calcium. In addition, the nuclear intensity (NI) of both cell types increased with increasing concentrations of pDMAEMA. This may also be due to an increase in the PMP allowing more dye into the cell. pDMAEMA appeared to induce apoptosis in Caco-2 cells but not in U937 cells, and it has previously been hypothesised to be inducing cell death in these cells through a necrotic process as detected by flow cytometry [38]. Our data supports this conclusion, although caution should be taken when discussing the cytotoxicity of this polymer. Jones et al. [38] found a late-stage decrease in MMP in U937 cells. They hypothesized that, due to the fact that disruption of MMP is an early indicator of apoptosis and also a late stage event, the reduction in MMP was an indicator of necrosis. The data presented here supports this conclusion, as the concentrations that induced a decrease in MMP in U937 cells were similar to those that produced other cytotoxic effects. This indicates a late-stage necrotic event. However, in Caco-2, the MMP was decreased at concentrations significantly lower than those that induced other cytotoxicity effects, including PMP and this is evidence further supporting a process of apoptosis in Caco-2.

Hormesis is a process produced by some drugs where a biphasic effect is seen at high versus low concentrations of drugs [11, 44]. This process is characterised by low concentration stimulation and high concentration inhibition and has been found to occur with parameters such as MMP and CN [11]. In U937, both parameters were increased at low pDMAEMA concentrations. At higher concentrations however, there was a decrease in MMP, but no change in CN. Hormesis may be a reaction of the cells to sub-cytotoxic concentrations of drug whereby the cells try to overcompensate for its presence by increasing their biochemical protective mechanisms [45]. In Caco-2 cells, hormesis was also evident with CN increasing at low concentrations of pDMAEMA, then decreasing at high concentrations. For MMP, a multiphasic reaction to pDMAEMA was seen in Caco-2 cells. At all time points there was a decrease in MMP at lower concentrations, followed by recovery, then a decrease again as the concentration of pDMAEMA increases. The reason this occurs is unclear, but it may be that a dual mechanism of action is occurring, i.e., at low concentrations pDMAEMA induces an apoptotic process of cell death, while at high concentrations a necrotic process is induced.

Modifications of pDMAEMA have also been found to reduce cytotoxicity. These include PEGylation [6], reduction of terminal thiol groups [46] and complex formation with n-carboxyethylchitosan [47]. Complexation of pDMAEMA with DNA also improves transfection efficiency, as did copolymer formation with methyl methacrylate [48]. It is known that altering the MW of pDMAEMA impacts on cytotoxicity: low MW pDMAEMA (43 kDa) is considered less toxic than high MW pDMAEMA (915 kDa) [49]. The MW of the pDMAEMA used here was also quite low (12.8 kDa). Deciphering these relationships using HCA will be the subject of future investigations of pDMAEMA polymers of different MW, in co-polymer formats and in complexes with DNA and siRNA.

pDMAEMA itself has not been tested by oral administration in vivo. However, methyl methacrylate, a component of pDMAEMA, has been found to be non-toxic by oral administration in vivo in rats [50]. It was rapidly hydrolysed by serum carboxylesterases resulting in rapid degradation of methacrylic acid to less toxic compounds. Due to the variation in effect of pDMAEMA on different cell lines, its effect on whole tissue was assessed. Against rat colonic tissue, pDMAEMA did not cause any histopathology, while against rat ileal tissue there was a degree of sloughing of cells at high concentrations. Fluorescent hostasol-conjugated pDMAEMA has previously been used to visualise interactions of the polymer with rat intestinal tissue [2]. In stomach, caecum and colon tissue, pDMAEMA was retained in the surface mucus layer of the tissue, while in ileum, it penetrated into the intestinal crypts. This varying distribution may explain the reason for the increased histological damage found here in ileal versus colonic tissue. It is possible that the small amount of damage seen with pDMAEMA in vitro on rat intestinal ileal tissue may be diminished in vivo. Absorption enhancers have typically been found to be cytotoxic in vitro, while they produce reversible damage in vivo, possibly due to the presence of mucus, or to the large capacity for the epithelium to repair itself [51].