Cationic Nanoparticles for Delivery of siRNA to Treat Chronic Inflammatory Diseases and Sex Based Differences in Nanoparticle Performance

Introduction:: In this study, we developed modified cationic nanocarriers as vehicles for the intracellular delivery of therapeutic small interfering ribonucleic acid (siRNA). RNA interference is a treatment method that utilizes siRNAs to silence production of specific proteins from strands of messenger RNA, and is promising as a treatment for many inflammatory diseases that result from aberrant production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α). The potential of siRNA applications has been limited by barriers to its intracellular cytosolic delivery. The relatively large molecular weight of siRNA hinders its cellular uptake and its negative charge is repelled by the negatively charged cell membrane. Additionally, endogenous nucleases present in the bodily fluids lead to rapid siRNA degradation, and the immune system is prone to clearance of siRNA. To overcome these barriers, our research investigates the efficacy of nanomaterials to develop biocompatible, biodegradable, non-toxic carrier systems for the intracellular delivery of siRNA. We focus on developing cationic nanoparticles for the intracellular delivery of anti-TNF-α siRNA to macrophages, which are the primary cells responsible for pro-inflammatory cytokine release during chronic inflammatory disease.

Recent studies have shown that cell sex and cell type effect nanoparticle of toxicity and uptake. Therefore, this study also investigates the importance of cell sex by comparing in vitro nanoparticle performance in primary macrophages (male vs. female). Moreover, > 70% of studies are performed in cells collected from patients of European descent, rendering our current understanding of biomedicine limited. Therefore, this study utilized cells isolated from patients of Hispanic descent.


Materials and Methods:: Nanocarriers were synthesized from various cationic and hydrophobic methacrylate monomers (diethyl amino methacrylate, dimethyl amino methacrylate, tert butyl methacrylate, hexyl methacrylate, cyclohexyl methacrylate) using atoms regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) and the nanocarrier properties were evaluated as a function of structure-property relationships. Dynamic light scattering, potentiometric titration, and MTS assays in human cell lines were used to evaluate the nanocarrier properties. siRNA loading and release was performed as a function of pH and time in citric acid buffer of pH 5.5 and pH 7.5. Nanocarrier function to deliver anti-tumor necrosis factor α (TNF-α) siRNA was measured using enzyme linked immunosorbent assay and confocal microscopy in human macrophages. siRNA loaded nanocarriers were exposed to the cells for up to 24 hours before all cell assays were performed. Gene knockdown as a function of patient sex was analyzed in cells from Hispanic donors aged 30-34 (n=3).


Results, Conclusions, and Discussions:: In this study, modified cationic nanocarriers were synthesized and characterized as vehicles for the intracellular delivery of therapeutic siRNA. The importance of siRNA loading methods was investigated by studying the impact of the pH and time over which siRNA is loaded into the nanogels, with a concentration on diffusion-based loading in the presence and absence of electrostatic interactions. Loading of negatively charged siRNA at low pH (5.5) when cationic nanocarriers were not charged resulted in diffusional based loading ranging from ~30% to ~75%, dependent on both the nanocarrier formulation and the time of loading, whereas loading at neutral pH (7.5) when cationic nanocarriers are positively charged resulted in nearly 100% loading regardless of time and formulation. siRNA release kinetics were studied using samples prepared from nanocarriers loaded under the various pH and time conditions, and it was shown that nanocarriers loaded at pH 7.5 resulted in a greater burst release than nanocarriers loaded at pH 5.5. In addition, siRNA delivery was shown to be dependent on the formulation of the nanocarrier. This research provides evidence that loading conditions of siRNA affect the release behavior.

The synthesized nanocarriers were also capable of knockdown of TNF-α in human macrophage cell lines. Sex-based differences in both cytotoxicity and gene knockdown were shown to be statistically significant.

This study concludes that controlling the loading conditions and formulation of the nanocarrier could prove advantageous for eliciting prolonged intracellular release of nucleic acids and negatively charged molecules, effectively decreasing dose frequency and contributing to more effective therapies and improved patient outcomes. In addition, these findings show that controlling the loading conditions and understanding the resulting release mechanisms for charged therapeutics can be used as a pathway for the continued optimization of cationic nanocarriers in a wide array of RNA interference-based applications. Plus, sex-based differences in nanocarrier toxicity and gene knockdown were shown, which may lead to implications for sex-specific treatments in the future, rather than the one-size fits-all paradigm that is currently used by engineers who design nanotherapies.