Rapid Discovery of Potent siRNA-Containing Lipid Nanoparticles Enabled by Controlled Microfluidic Formulation

Abstract

The discovery of potent new materials for in vivo delivery of nucleic acids depends upon successful formulation of the active molecules into a dosage form suitable for the physiological environment. Because of the inefficiencies of current formulation methods, materials are usually first evaluated for in vitro delivery efficacy as simple ionic complexes with the nucleic acids (lipoplexes). The predictive value of such assays, however, has never been systematically studied. Here, for the first time, by developing a microfluidic method that allowed the rapid preparation of high-quality siRNA-containing lipid nanoparticles (LNPs) for a large number of materials, we have shown that gene silencing assays employing lipoplexes result in a high rate of false negatives (~90%) that can largely be avoided through formulation. Seven novel materials with in vivo gene silencing potencies of >90% at a dose of 1.0 mg/kg in mice were discovered. This method will facilitate the discovery of next-generation reagents for LNP-mediated nucleic acid delivery.

Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates

“…Lipid derivatives from Fig. 2 were formulated with cholesterol, DSPC, PEG-lipid, and siRNA via a microfluidic-based mixing technology (25). Formulations that were unstable in solution or had no siRNA entrapment were not evaluated in vivo (SI Appendix, Table S1).…”

Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric nanoparticle infusion delays glioblastoma progression

“…Several obstacles with respect to tissue specificity and sustainable potency must be overcome to achieve therapeutic outcomes. Protection against naturally occurring nuclease cleavage, enhanced cellular uptake, and endosomal escape with charge modification and the reduction of kidney clearance by increasing size are key milestones of successful in vivo delivery of siRNAs, and our solution of using a unique class of lipopolymeric nanoconstructs to complex therapeutic siRNA duplexes accomplishes all indices in one fell swoop (14,15,22,42). Overcoming these obstacles helps reduce off-target effects and minimizes toxicity to liver and immune cells in vivo; however, the endothelia-targeting properties of these NPs indicate that systemic delivery of candidate siRNAs is not ideal for targeting brain tumors, even though tumor-induced angiogenesis is prevalent in GBM (43) and cerebral blood flow volume is one of the largest parts of the total cardiac output.…”

“…siRNA was dissolved in pH 3 10 mM citrate solution (Teknova) in a separate syringe. The two syringes were connected to a syringe pump, and the fluid was pushed through a microfluidic device as previously described (22). The resulting NPs were dialyzed in 1× PBS and were sterilized using a 0.22-μm polyethersulfone syringe filter (Genesee Scientific).…”

“…A class of lipopolymeric nanoparticles (LPNPs) has been formulated to maximize systemic delivery to vasculature-rich organs for antitumor RNAi therapies, and the combination of ionizable, low-molecular-weight lipopolymers custom synthesized to optimize cell entry facilitates tumor cell targeting when delivered directly (14)(15)(16)(17)(18)(19)(20)(21)(22). The polymers were synthesized by Significance Glioblastoma is a deadly brain tumor with no cure.…”

Multiphase microfluidic synthesis of micro- and nanostructures for pharmaceutical applications

“…Furthermore, a VFF approach using a large microchannel aspect ratio (channel depth to channel width), up to 100:1, was developed for producing liposomes at a throughput of nearly 100 mg/h (Hood and DeVoe, 2015), which makes the microfluidic technology promising for fabricating liposomes for practical applications. Liposomes have been developed as nanocarriers by loading various functional cargoes for pharmaceutical applications, including drugs (Hood et al, 2014;Kastner et al, 2015) and genes (Balbino et al, 2013;Belliveau et al, 2012;Chen et al, 2012). The cargo can be loaded into liposomes either by active or passive loading.…”