Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA

Abstract

Lipid nanoparticles (LNP) are the leading systems for in vivo delivery of small interfering RNA (siRNA) for therapeutic applications. Formulation of LNP siRNA systems requires rapid mixing of solutions containing cationic lipid with solutions containing siRNA. Current formulation procedures employ macroscopic mixing processes to produce systems 70-nm diameter or larger that have variable siRNA encapsulation efficiency, homogeneity, and reproducibility. Here, we show that microfluidic mixing techniques, which permit millisecond mixing at the nanoliter scale, can reproducibly generate limit size LNP siRNA systems 20 nm and larger with essentially complete encapsulation of siRNA over a wide range of conditions with polydispersity indexes as low as 0.02. Optimized LNP siRNA systems produced by microfluidic mixing achieved 50% target gene silencing in hepatocytes at a dose level of 10 µg/kg siRNA in mice. We anticipate that microfluidic mixing, a precisely controlled and readily scalable technique, will become the preferred method for formulation of LNP siRNA delivery systems.

The Niemann-Pick C1 Inhibitor NP3.47 Enhances Gene Silencing Potency of Lipid Nanoparticles Containing siRNA

“…22 The third area of interest concerns whether enhanced gene silencing potency observed in vitro in the presence of NP3.47 can be extended to in vivo situations. As noted elsewhere the gene silencing potency of LNP-siRNA systems for hepatocyte targets is extremely high, requiring doses of as little as 10 μg/kg body weight to achieve 50% gene silencing; 3,4 however, for other tissues dose levels of 1 mg/kg body weight or more are required, [23][24][25] limiting clinical applications. The addition of agents such as NP3.47 to enhance potency would appear an attractive option, however the problems associated with the likely toxicities of In this regard we have recently shown 26 that a hydrophobic prodrug derivative of dexamethasone, when associated with LNP systems is a 20-fold or more potent for suppressing immunostimulatory effects of encapsulated RNA or DNA as compared with free dexamethasone.…”

“…2 These systems employ optimized ionizable cationic lipids and satisfy key issues for the development of clinically relevant siRNA-loaded LNP (LNP-siRNA) including efficient siRNA encapsulation, high gene silencing potencies and therapeutic indices (for liver targets) as well as scalable manufacturing processes. [3][4][5] However, there remains a critical need to improve the potency of LNP-siRNA systems in order to extend applications of this gene silencing technology to tissues other than the liver.…”

Harnessing RNAi-based nanomedicines for therapeutic gene silencing in B-cell malignancies

“…These LNPs were produced by a scalable production process that increases siRNA encapsulation and transfection potency 20). Similar LNPs demonstrated potent siRNA activity in hepatocytes (14), which are relatively easy to transfect in vivo.…”

“…We next constructed LNPs encapsulating siRNAs using a microfluidic mixing system as previously described (17,18,20) (Fig. 3A).…”

siRNA Lipid Nanoparticle Potently Silences Clusterin and Delays Progression When Combined with Androgen Receptor Cotargeting in Enzalutamide-Resistant Prostate Cancer

“…The lipid composition of all LNPs containing siRNA (LNP-siRNA) was cationic lipid/DSPC/ cholesterol/PEG-DMG (50/10/38.5/1.5; mol%) for in vitro, while cationic lipid/DSPC/cholesterol/PEG-DSG (50/10/37.5/ 2.5; mol%) or (50/10/35/5; mol%) for in vivo. LNP-siRNAs were prepared using a microfluidic mixing apparatus as previously described (30,32).…”