Toxicological analysis of Lipid-Based Nanoparticles: A Comparative Study

Abstract

Lipid-based nanoparticles (LNPs) are frontliners in nanomedicine, as their use as mRNA carriers and in drug delivery has become prominent over the years. Despite their increasing application in medicine, there are limited studies on their potential toxicity or on any adverse effects that could be associated with them. Also, growing concerns about their toxicity have moderated their clinical translation. This study elucidates the potential toxicological behavior of three lipid-based nanoparticles. Lipid based nanoparticles are divided into two major groups based on the charge of their head group. These include Cationic Lipid nanoparticles (cLNPs) which are positively charged and Ionizable Lipid Nanoparticles (iLNPs) which are neutral, but they take on the charge of the environment they are. Both systems are emerging as promising tools for the delivery of therapeutic drugs, including nucleic acids. In our toxicity studies of Lipid-based nanoparticles, firstly, we carried out comparative in vitro analysis and animal studies using cLNPs (Liposomes and jetMESSENGER) and iLNPs (Nanostructure Lipid Carriers – NLCs). Secondly, we employed RNA sequencing and transcriptomics analysis. RNA seq analysis was done due to inconsistent outcomes often observed with the results from in vitro studies, suggesting the cruciality of more studies on the molecular level. In vitro toxic effects of all three LNPs were evaluated based on oxidative stress, cell viability, cell proliferation, programmed cell death and cell morphology. We treated Chinese hamster ovary (CHO) cells, a mammalian cell line, with the LNPs to measure the production of reactive oxygen species (ROS). Cell viability was ascertained with trypan blue exclusion assay. Flow cytometry was used to analyze cell apoptosis and necrosis. Moreover, the cell proliferation assay, Alamar blue assay, was used to study the metabolic activity of LNP treated cells by using resazurin, a non-fluorescent dye to measure NADH production. Being an acute toxicity study, two exposure periods, 4 h and 24 h, were considered. For animal studies, serum from blood samples that were collected from mice after 24 h intramuscular and intravenous injection with NLCs and NLC with eGFP-mRNA encapsulation. Specific enzymes, i.e. aspartate transaminase –AST and alanine transaminase –ALT, were used as biomarkers, because increase in their level could be indicative of specific organ or cell damage. Results from our in vitro study showed that cells treated with the LNPs compared to the control induced some level of cytotoxicity, although there was no significant increase in ROS level after 24 h. Meanwhile, animal assessment showed no significant toxicity when LNPs were intravenously administered compared to intramuscular administration. In the RNA-Seq study, RNA from human lung fibroblasts (WI-38) that were treated with LNP-eGF-mRNA for 24 h, was sequenced. The mRNA transcripts of exposed cells were compared to transcripts from untreated and differentially expressed genes generated with bioinformatics tools. Exposure of WI-38 cells to LNP-mRNA led to specific changes in the expression of genes associated with immune response, inflammation, cell response to oxidative stress, and apoptosis. Pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) related to the immune system response, inflammation and carcinogenesis were significantly upregulated. Overall, our findings suggest that though LNP-based nanoparticles are valuable tools in medicine, some levels of toxicity can be attributed to their interaction with biological systems. Our findings will aid the evaluation of potential risks associated with LNPs use such as oxidative stress, increased immune and inflammatory responses, reduced cell metabolic activity.