Lipid Nanoparticle Formulations for Enhanced Co-delivery of siRNA and mRNA

Abstract

Although mRNA and siRNA have significant therapeutic potential, their simultaneous delivery has not been previously explored. To facilitate the treatment of diseases associated with aberrant gene upregulation and downregulation, we sought to co-formulate siRNA and mRNA in a single lipidoid nanoparticle (LNP) formulation. We accommodated the distinct molecular characteristics of mRNA and siRNA in a formulation consisting of an ionizable and biodegradable amine-containing lipidoid, cholesterol, DSPC, DOPE, and PEG-lipid. Surprisingly, the co-formulation of siRNA and mRNA in the same LNP enhanced the efficacy of both drugs in vitro and in vivo. Compared to LNPs encapsulating siRNA only, co-formulated LNPs improved Factor VII gene silencing in mice from 44 to 87% at an siRNA dose of 0.03 mg/kg. Co-formulation also improved mRNA delivery, as a 0.5 mg/kg dose of mRNA co-formulated with siRNA induced three times the luciferase protein expression compared to when siRNA was not included. As not all gene therapy applications require both RNA drugs, we sought to extend the benefit of co-formulated LNPs to formulations encapsulating only a single type of RNA. We accomplished this by substituting the "helper" RNA with a negatively charged polymer, polystyrenesulfonate (PSS). LNPs containing PSS mediated the same level of protein silencing or expression as standard LNPs using 2-3-fold less RNA. For example, LNPs formulated with and without PSS induced 50% Factor VII silencing at siRNA doses of 0.01 and 0.03 mg/kg, respectively. Together, these studies demonstrate potent co-delivery of siRNA and mRNA and show that inclusion of a negatively charged "helper polymer" enhances the efficacy of LNP delivery systems.

Cancer‐Targeting Nanoparticles for Combinatorial Nucleic Acid Delivery

“…Larger nucleic acids, such as plasmid DNA and mRNA, provide a high density of negative charge which can stabilize lipid or polymer carriers. Therefore, combination therapy with larger nucleic acid molecules can enhance the delivery and efficacy of smaller molecules, even if they act on distinct targets . However, the relative amount of these stabilizing nucleic acids should be optimized, as very stable carriers with strong electrostatic interactions may not efficiently release their cargos.…”

“…[32,33] Similarly, cationic lipids have been used recently to coencapsulate multiple types of nucleic acids into lipidoid nanoparticles (LNPs). [34] Liposomes contain both hydrophilic and hydrophobic moieties which can bind small molecule therapeutic agents for codelivery with nucleic acids. Artificial vesicles like liposomes have an aqueous core that can be used to encapsulate the hydrophilic nucleic acids, [35][36][37] and other materials, like polyesters, can physically entrap nucleic acids via emulsification.…”

Lipid Polymer Hybrid Nanomaterials for mRNA Delivery

“…7, 17 Recently, lipid polymer hybrid nanoparticles (LPNs), by integrating the complementary properties of lipid and polymeric nanomaterials, are emerging as a class of nanomaterials for RNA delivery. 2, 11, 31, 37 Although various combinations of lipids and polymers were applied in establishing LPNs for siRNA delivery, little is known about the effects of different polymers on lipid based nanomaterials for mRNA delivery.…”

Lipid nanoparticles silence tumor necrosis factor α to improve wound healing in diabetic mice

“…[49][50][51] Nanoparticles formulated with lipidoid, a type of ionizable lipid-like material, have been shown to potently deliver siRNA in vivo to several cell types, including hepatocytes, epithelial cells, and difficult to transfect cell lines, like immune cells. 38,39,[52][53][54] To transfect wound bed cells, lipidoid nanoparticles must remain in the wound tissue without being degraded by enzymes or becoming stuck in wound debris. Confocal images show that they remained in the tissue, most likely conforming to wound bed topology, and avoided degradation long enough to be taken up by cells ( Figure 2).…”