Poster X20 - Development of Mesoscale Lipid Nanoparticles for Targeted Nucleic Acid Delivery to Renal Epithelial Cells

Introduction: The increasing prevalence of acute and chronic kidney diseases poses a major problem in healthcare. Due to limited treatment options, there is a need for novel therapeutics that would efficiently accumulate in the kidneys and exert a sufficient therapeutic effect. Selective targeting of the proximal tubular epithelial cells in the kidneys has been demonstrated with nanoparticles in the mesoscale size range (300 – 400 nm). This drug delivery platform has the potential to improve therapeutic efficacy through increased accumulation of kidney therapeutics at the disease site, as well as reduced off-target effects. Moreover, nanocarriers enable delivery of biological molecules, which hold promise as next-generation treatments. With the development of nucleic acid therapeutics, lipid nanoparticles have proven to be efficient carriers for easily degradable biological cargo. However, kidney targeting of mesoscale nanoparticles has thus far only been demonstrated with polymeric carriers. Hence, the purpose of this work was to develop a lipid-based mesoscale nanoparticle formulation, which can later be applied for targeted delivery of nucleic acids for kidney disease treatment.

Materials and

Methods: Mesoscale lipid nanoparticles (MLNP) with siRNA cargo have been formulated with an ionizable lipid, a phospholipid, cholesterol, and a PEGylated lipid. Nanoparticles were prepared using the nanoprecipitation method. Briefly, varied amounts of lipids were dissolved in ethanol and added dropwise to the aqueous phase containing siRNA, NaCl, and sodium acetate buffer (pH=5.2) under magnetic stirring. After nanoparticle formation, the buffer was changed to PBS by overnight dialysis to neutralize the ionizable lipid charge. Modifications of the formulation composition and process parameters have been applied to optimize the nanoparticle size. MLNP size and polydispersity index were characterized via dynamic light scattering and zeta potential via electrophoretic light scattering. siRNA encapsulation efficiency was measured using a fluorescence-based RNA-quantitation assay. MTT assay has been applied to test the MLNP cytotoxicity to renal epithelial cells. 786-O cells with LPS-induced inflammatory response have been treated with MLNPs encapsulating IL-6 siRNA to test the efficacy of siRNA-induced cytokine silencing. To test in vivo siRNA delivery, healthy C57/BI/6J mice were injected IV with 50 mg/kg Cdkn1a siRNA MLNPs. Organs have been collected 24 h post-injection for qPCR analysis of Cdkn1a mRNA levels.

Results, Conclusions, and Discussions: Five formulation modifications have been tested to obtain siRNA-loaded lipid nanoparticles with a size in the mesoscale range (Fig. 1). While all modifications resulted in an increase of nanoparticle size, MLNP with a size >300 nm have been produced through a combination of increased total lipid concentration, modification of lipid molar ratio, increased ion concentration, and modification of mixing parameters. To achieve the stable PEGylation required for kidney targeting, the lipid composition of MLNPs has also been optimized. MTT assay has demonstrated that treatment with the optimized MLNP formulation encapsulating non-targeting siRNA and IL-6 siRNA has no effect on the viability of renal epithelial cells. In vitro testing has shown a silencing effect of siRNA encapsulating MLNPs on the target inflammatory cytokine. MLNP treatment has been well-tolerated in healthy mice.
In the current work, we have developed a formulation of mesoscale lipid nanoparticles that has been optimized for targeted nucleic acid delivery to the renal epithelial cells. The MLNP formulation has demonstrated preliminary indications of safety and efficacy, and additional in vitro and in vivo studies will be carried out to apply this drug delivery system for kidney disease treatment.

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