An Orthogonal Array Optimization of Lipid-like Nanoparticles for mRNA Delivery in Vivo


Authors: Bin Li, Xiao Luo, Binbin Deng, Junfeng Wang, David W. McComb, Yimin Shi, Karin M. L. Gaensler, Xu Tan, Amy L. Dunn, Bryce A. Kerlin, and Yizhou Dong

Journal: Nano Letters

DOI: 10.1021/acs.nanolett.5b03528

Publication - Summary

September 03, 2015

Summary:

Although messenger RNA (mRNA) therapeutics are useful for transiently expressing antibodies and proteins, antigen-specific immune responses make the systemic delivery of mRNA challenging. Lipid-like nanoparticles (LLNs) have been shown to efficiently deliver short interfering RNA (siRNA), so it is possible that LLNs may also be effective for mRNA delivery due to the physicochemical similarity between siRNA and mRNA. The Dong research group at Ohio State University investigated a series of LLNs to evaluate their potential for mRNA delivery. They used NanoAssemblr™ technology to produce a LLN that improved mRNA delivery efficiency over 350 times more than their original formulations, showing how LLNs can be modified to increase their efficiency as mRNA delivery tools in future biological and therapeutic applications.

Previous work by the authors indicated that an appropriate starting LLN design consisted of a phenyl ring connected to three amino lipid chains by three amide linkers, so they synthesized seven variants of N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (TT) derived LLNs with different length amino lipid chains (TT2-TT8) based on this template. The particle size, zeta potential, and entrapment efficiency of the resulting LLNs were measured and the LLNs were found to be between 99 and 178 nm in size, with polydispersity indexes below 0.2, mostly positive charges, and mRNA entrapment efficiencies between 15% and 82% for firefly luciferase (FLuc) mRNA. Cytotoxicity was also evaluated and was found to be minimal to moderate for all LLNs. TT3 was chosen as a lead material as it showed the highest FLuc expression of all TT LLNs.

A correlation analysis between transfection efficiency and particle size, surface charge, entrapment efficiency, and cell viability found a positive correlation between transfection and entrapment efficiencies, but no correlation with the other variables. Additionally, LLNs formulated with 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were found to be the most efficient for mRNA delivery of the three helper lipids tested. To further optimize the formulation of TT3 LLNs, an orthogonal experimental design was used to streamline the evaluation of the effects of varying the formulation ratios of each of the four LLN components: TT3, DOPE, cholesterol (Chol), and 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG2000). After two rounds of orthogonal optimization, LLNs with molar ratios of TT3/DOPE/Cholesterol/DMG-PEG2000 of 20/30/40/0 (Hi-TT3) were predicted to have the highest transfection efficiency, which was then confirmed by evaluating FLuc expression levels.

Hi-TT3 LLNs increased transfection efficiency over 350 times more than the original TT3 LLNs and over 65 times more than C12-200-DSPC LLNs, a lead material reported previously. Unfortunately, it was noticed that Hi-TT3 LLNs were not stable without DMG-PEG2000, which increases LLN stability, but is negatively correlated with delivery efficiency and particle size. To increase particle stability without significantly compromising delivery efficiency, LLNs with molar ratios of TT3/DOPE/Cholesterol/DMG-PEG2000 of 20/30/40/0.75 (O-TT3) were formulated and found to be stable for a minimum of two weeks. Based on these results, O-TT3 LLNs were selected for in vivo studies.

In vivo, O-TT3 was shown to effectively deliver FLuc mRNA to the liver and spleen, but not to the kidneys, lungs, or heart. Based on this biodistribution, O-TT3 LLNs were then used to encapsulate mRNA for human factor IX (hFIX), a blood clotting protein normally produced by the liver, the deficiency of which causes hemophilia B. O-TT3 hFIX LLNs were found to efficiently deliver hFIX mRNA in wild-type mice and restore FIX activity in FIX-knockout mice in a dose dependent manner, which indicated that LLNs can be modified to be efficient mRNA delivery systems for therapeutic applications.

This paper shows how LLNs can be rationally designed to increase their mRNA delivery efficiency and that increasing mRNA entrapment efficiency is important for increasing transfection efficiency. Additionally, it validated that PEGylation improves particle stability, but hinders transfection efficiency. Most importantly, it showed that LLNs can be used to efficiently deliver therapeutically relevant mRNAs in vivo to fully restore normal protein expression in deficient mouse models. Taken together, these results indicate that modified LLNs show considerable promise in the delivery of mRNA for biological and therapeutic applications in the future.

Abstract:

 

Systemic delivery of mRNA-based therapeutics remains a challenging issue for preclinical and clinical studies. Here, we describe new lipid-like nanoparticles (TT-LLNs) developed through an orthogonal array design, which demonstrates improved delivery efficiency of mRNA encoding luciferase in vitro by over 350-fold with significantly reduced experimental workload. One optimized TT3 LLN, termed O-TT3 LLNs, was able to restore the human factor IX (hFIX) level to normal physiological values in FIX-knockout mice. Consequently, these mRNA based nanomaterials merit further development for therapeutic applications.

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