(534c) siRNA Delivery from Cationic Nanocarriers Prepared By Diffusion-Assisted Loading in the Presence and Absence of Electrostatic Interactions

In this study, we use 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 (mRNA), 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 in the clinic has been limited by barriers to its intracellular cytosolic delivery. The relatively large molecular weight (~13 kDa) of siRNA hinders its cellular uptake and its negative charge is repelled by the negatively charged cell membrane. Additionally, endogenous nucleases present in the blood stream and in other bodily fluids lead to rapid siRNA degradation, and the immune system is prone to clearance of siRNA [1-3]. To overcome these barriers, recent research investigates the efficacy of nanomaterials to develop biocompatible, biodegradable, non-toxic, nano-sized carrier systems for the intracellularly delivery of siRNA [4]. In particular, polymer-based systems offer advantages for siRNA delivery when compared with viral vectors: tailorable properties, ease of production, and safety [5,6]. Of particular interest are cationic materials, as they can ionically complex with the negatively charged siRNA and get taken up by the cell through the negatively charged cell membrane [7].

Our lab has previously used cationic monomers 2-(diethylamino)ethyl methacrylate (DEAEMA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) to synthesize crosslinked nanoscale hydrogels (nanocarriers). We synthesize these nanocarriers by activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) with a hydrophobic comonomer, such as tert butyl methacrylate (tBMA) and a poly(ethylene glycol) (PEG) graft which reduce cytotoxicity associated with the cationic monomer and allow for adjustment of pH-responsiveness and swell-ability of the nanogels.

In our previous studies, siRNA was loaded into DEAEMA at low pH values where the DEAEMA is positively charged, leading to a loading mechanism that is controlled by ionic complexation to the negatively charged siRNA [8,9]. However, this leads to a large burst release when the pH is increased to neutral values above the pK_a of the DEAEMA particles and the ionic complex is lost [10]. Therefore in this work, after developing nanocarrier formulations with appropriate pK_a, size, swellability, and cytocompatibility, we investigate the importance of siRNA loading methods by studying the impact of the pH and time over which siRNA is loaded into the nanogels. We concentrate on diffusion-based loading in the presence and absence of electrostatic interactions. siRNA release kinetics were studied using samples prepared from nanocarriers loaded by both mechanisms. In addition, siRNA delivery was evaluated for two formulations. While previous studies were conducted with samples prepared by siRNA loading at low pH values, this research provides evidence that loading conditions of siRNA affect the release behavior. Nanocarriers were also shown to be capable of successful gene knockdown in both male and female cells.

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:

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. The different loading results and proposed loading mechanisms are shown in Figure 1. 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.

Conclusions:

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.

References:

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