An in vitro investigation of redox reactive polymer nanoparticle safety using the C3A heptocyte cell line

Polymeric nanoparticles (NPs) can be relatively quick and inexpensive to produce. This has led to rapid development of numerous polymeric NPs for drug delivery. Ensuring polymer NP safety is paramount to their successful integration into the clinic, however a thorough assessment of the safety of new nanomedicines has lagged behind the enthusiasm to exploit them. Alternatives to animal testing are desirable for drug safety screening due to ethical, financial and time considerations. Polymeric NPs are commonly coated with Poly (ethylene glycol (PEG) to introduce stealth properties (i.e. to avoid recognition by cells), to increase their circulation time in the body. This study aims to utilise in vitro models to investigate the safety profile of two PEG coated biodegradable polymer NPs; PLGA-PEG (PEG-methyl ether-block-poly(lactide-co-glycolide) and PLGA-ss-PEG, a redox reactive NP designed to selectively shed its PEG coating when in contact with tumour cells. Both NPs were loaded with the green fluorescent dye, DIO (3, 3’-Dioctadecyloxacarbocyanine Perchlorate). High throughput methods were used to assess the cytotoxicity, cellular internalisation and genotoxicity of these NPs to hepatocytes.
The cytotoxicity of these NPs was investigated in the C3A (hepatocyte) cell lines using two cell viability assays (Alamar Blue and Neutral Red) at NP concentrations ranging from 5 to 250 µg/ml, 24 h post-exposure. The uptake of these NPs (5 to 250 µg/ml) over time (10, 60 and 1440 min) was determined using a high throughput quantitative plate reader based method and verified by confocal imaging. Genotoxicity (125 and 250 µg/ml, 24 h post-exposure) was determined using the comet assay in the presence and absence of FPG which indicates whether DNA damage is mediated by an oxidant mechanism.
Cell viability was above 80%, at all concentrations of NPs tested suggesting low cytotoxicity. In C3A cells there was a time and concentration dependent increase in the uptake of NPs, with uptake greatest at 1440 min and a concentration of 250 µg/ml. PLGA-ss-PEG NPs were not genotoxic, although there is a significant level of DNA damage seen for PLGA-PEG NPs, which was mediated by an oxidant mechanism. This disparity in DNA damage may be due to physiochemical differences between the NPs such as size and surface chemistry. This study demonstrated the importance of future investigation into polymer NP toxicity and related mechanisms.