Cytotoxic, Apoptotic and Genotoxic Effects of Lipid-Based and Polymeric Nano Micelles, an In Vitro Evaluation

Abstract

Self-assembly systems (SAS) mainly consist of micelles, and liposomes are the classes of Nano Drug Delivery Systems with superior properties compared to traditional therapeutics in targeting cancer tumors. All commercially available nano-formulations of chemotherapeutics currently consist of SAS. According to our knowledge, a specific toxicity comparison based on material differences has not yet been performed. The purpose of this study was to evaluate and compare the toxicity of two SAS consisting of Sterically Stabilized Micelles (SSM) made of a lipid-based amphiphilic distearoyl-sn-glycero-phosphatidylethanolamine-polyethylene glycol (PEG)-2000 and a polymeric micelle (PM) consisting of Y-shape amphiphilic block copolymer, synthesized using poly ε-caprolactone and PEG. The mechanism of cytotoxicity and genotoxicity of micelles on L-929 healthy mouse fibroblast cells was assessed using Sulforhodamine-B, WST-1, Acridine Orange/Ethidium Bromide and alkaline single-cell gel electrophoresis assays. Results showed that SSM in conc. of 40 mg/mL shows very low cytotoxicity at the end of 24, 48 and 72 h. The DNA damage caused by SSM was much lower than PM while the latter one showed significant toxicity by causing apoptosis with the ED50 value of 3 mg/mL. While the DNA damage caused by SSM was ignorable, some DNA chain breaks were detected on cells treated with PM.

Keywords: Nano Drug Delivery System; cytotoxicity; genotoxicity; lipid-based micelles; polymeric micelle; targeted cancer therapy.

Figure 1

Synthesis of Y-shaped amphiphilic PEG-PCL₂ in order to prepare polymeric micelles. The figure is adopted from our previously published data [13].

Figure 2

Particle size distribution of SSM and PM; (a) % Volume (b) % Number (c) % Intensity of SSM and (d) % Volume (e) % Number (f) % Intensity of PM.

Figure 3

Cytotoxic effect of (a) SSM and (b) PM at 24, 48 and 72 h on L-929 cell lines determined by SRB assay. Data are representative of tree independent trials and are expressed as the mean ± SD. Significant differences between cell viabilities at 24, 48 and 72 h were indicated by * p < 0.05.

Figure 4

Cytotoxic effect of (a) SSM and (b) PM at 24, 48 and 72 h on L-929 cell lines determined by WST-1. Data are representative of tree independent trials and are expressed as the mean ± SD. Significant differences between cell viabilities at 24, 48 and 72 h were indicated by * p < 0.05.

Figure 5

Genotoxic effect of 4 mg/mL of SSM and 1 mg/mL of PM on L-929 cells compared to non-treated ones after 24, 48 and 72 h incubation. Comet formation pattern showed that SSM induces a much lower DNA damage on these cells than PM after 48 and 72 h of incubation.

Figure 6

The comet assay results of SSM and PM on L-929 cell lines. Values are shown as Mean ± SD, which, are three separate experiments, performed in triplicate. Data are representative of tree independent trials and are expressed as the mean ± SD. Significant differences between DNA damage at 24, 48 and 72 h were indicated by p < 0.05 as: A: Significant differences between DNA damage of control and related Nano-micelles at indicated incubation periods, B: Significant differences between DNA damage of 24 h incubated cells and 48 h incubated cells, C: Significant differences between DNA damage of 24 h incubated cells and 72 h incubated cells, D: Significant differences between DNA damage of 48 h incubated cells and 72 h incubated cells with related Nano-micelles.

Figure 7

Different morphological patterns of cell death induced by 4 mg/mL of SSM and 1 mg/mL of PM on L-929 cells compared to non-treated ones after 24, 48 and 72 h incubation. Green live cells show normal morphology; green early apoptotic cells show nuclear margination and chromatin condensation. Late orange apoptotic cells showed fragmented chromatin and apoptotic bodies. Necrotic cells stain uniformly red.

Figure 8

Apoptotic and necrotic activity of SSM and PM on L-929 cell lines. Cells were treated with different concentrations of SSM and PM for 24 (up, left, a), 48 (up, right, b) and 72 h (below middle, c), AO/EB double staining and measured by fluorimetery. There were positive correlations between cell viability ratios obtained from cytotoxicity assays and cell death (apoptosis + necrosis) ratios obtained from AO/EB assay. Significant differences between cell death ratios at 24, 48 and 72 h were indicated by p < 0.05 as: A: Significant differences between cell death at control and cells treated with related Nano-micelles B: Significant differences between 1 mg/mL and 2 mg/mL SSM, C: Significant differences between 1 mg/mL and 4 mg/mL SSM, D: Significant differences between 2 mg/mL and 4 mg/mL SSM, E: Significant differences between 0.25 mg/mL and 0.5 mg/mL PM, F: Significant differences between 0.25 mg/mL and 1 mg/mL PM, G: Significant differences between 0.25 mg/mL and 1 mg/mL PM.