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

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.

Oral delivery of siRNA lipid nanoparticles: Fate in the GI tract.

Oral delivery, a patient-friendly means of drug delivery, is preferred for local administration of intestinal therapeutics. Lipidoid nanoparticles, which have been previously shown to deliver siRNA to intestinal epithelial cells, have potential to treat intestinal disease. It is unknown, however, whether the oral delivery of these particles is possible. To better understand the fate of lipid nanoparticles in the gastrointestinal (GI) tract, we studied delivery under deconstructed stomach and intestinal conditions in vitro. Lipid nanoparticles remained potent and stable following exposure to solutions with pH values as low as 1.2. Efficacy decreased following exposure to "fed", but not "fasting" concentrations of pepsin and bile salts. The presence of mucin on Caco-2 cells also reduced potency, although this effect was mitigated slightly by increasing the percentage of PEG in the lipid nanoparticle. Mouse biodistribution studies indicated that siRNA-loaded nanoparticles were retained in the GI tract for at least 8 hours. Although gene silencing was not initially observed following oral LNP delivery, confocal microscopy confirmed that nanoparticles entered the epithelial cells of the mouse small intestine and colon. Together, these data suggest that orally-delivered LNPs should be protected in the stomach and upper intestine to promote siRNA delivery to intestinal epithelial cells.

Achieving long-term stability of lipid nanoparticles: examining the effect of pH, temperature, and lyophilization.

The broadest clinical application of siRNA therapeutics will be facilitated by drug-loaded delivery systems that maintain stability and potency for long times under ambient conditions. In the present study, we seek to better understand the stability and effect of storage conditions on lipidoid nanoparticles (LNPs), which have been previously shown by our group and others to potently deliver RNA to various cell and organ targets both in vitro and in vivo. Specifically, this study evaluates the influence of pH, temperature, and lyophilization on LNP efficacy in HeLa cells. When stored under aqueous conditions, we found that refrigeration (2°C) kept LNPs the most stable over 150 days compared to storage in the -20°C freezer or at room temperature. Because the pH of the storage buffer was not found to influence stability, it is suggested that the LNPs be stored under physiologically appropriate conditions (pH 7) for ease of use. Although aggregation and loss of efficacy were observed when LNPs were subjected to freeze-thaw cycles, their stability was retained with the use of the cryoprotectants, trehalose, and sucrose. Initially, lyophilization of the LNPs followed by reconstitution in aqueous buffer also led to reductions in efficacy, most likely due to aggregation upon reconstitution. Although the addition of ethanol to the reconstitution buffer restored efficacy, this approach is not ideal, as LNP solutions would require dialysis prior to use. Fortunately, we found that the addition of trehalose or sucrose to LNP solutions prior to lyophilization facilitated room temperature storage and reconstitution in aqueous buffer without diminishing delivery potency.