Effect of the nanoformulation of siRNA-lipid assemblies on their cellular uptake and immune stimulation

Abstract

Two lipid-based nanoformulations have been used to date in clinical studies: lipoplexes and lipid nanoparticles (LNPs). In this study, we prepared small interfering RNA (siRNA)-loaded carriers using lipid components of the same composition to form molecular assemblies of differing structures, and evaluated the impact of structure on cellular uptake and immune stimulation. Lipoplexes are electrostatic complexes formed by mixing preformed cationic lipid liposomes with anionic siRNA in an aqueous environment, whereas LNPs are nanoparticles embedding siRNA prepared by mixing an alcoholic lipid solution with an aqueous siRNA solution in one step. Although the physicochemical properties of lipoplexes and LNPs were similar except for small increases in apparent size of lipoplexes and zeta potential of LNPs, siRNA uptake efficiency of LNPs was significantly higher than that of lipoplexes. Furthermore, in the case of LNPs, both siRNA and lipid were effectively incorporated into cells in a co-assembled state; however, in the case of lipoplexes, the amount of siRNA internalized into cells was small in comparison with lipid. siRNAs in lipoplexes were thought to be more likely to localize on the particle surface and thereby undergo dissociation into the medium. Inflammatory cytokine responses also appeared to differ between lipoplexes and LNPs. For tumor necrosis factor-α, release was mainly caused by siRNA. On the other hand, the release of interleukin-1β was mainly due to the cationic nature of particles. LNPs released lower amounts of tumor necrosis factor-α and interleukin-1β than lipoplexes and were thus considered to be better tolerated with respect to cytokine release. In conclusion, siRNA-loaded nanoformulations effect their cellular uptake and immune stimulation in a manner that depends on the structure of the molecular assembly; therefore, nanoformulations should be optimized before extending studies into the in vivo environment.

Inside out: optimization of lipid nanoparticle formulations for exterior complexation and in vivo delivery of saRNA

“…To our knowledge, this proof-of-concept study is the first systematic comparison of LNPs with saRNA on the interior or exterior of the particles. In order to be able to compare the formulations, we employed a single N/P ratio of 12:1, which can be further optimized for each formulation [37, 38]. It would be particularly useful to characterize the exact distribution of saRNA for each formulation, i.e., whether the saRNA is completely encapsulated or a portion is still accessible on the surface.…”

RNA interference-based therapies for the control of atherosclerosis risk factors

“…Lipoplexes have not been clinically tested yet, but there is one FDA approved drug utilizing synthetic lipid nanoparticles (LNPs), for hereditary transthyretin amyloidosis. LNPs and lipoplexes share many similar features, but while lipoplexes are slightly bigger in size and formed by binding existing liposomes with nucleic acids, LNPs are rather formed in one step by embedding the nucleic acid with the lipid solution [54]. Currently, LNPs are also being used in two COVID-19 mRNA vaccines used worldwide, and there is an increasing interest in their use as tissue specific therapeutic vehicles for various diseases [55].…”

Amino acid-based liposomal assemblies: Intracellular plasmid DNA delivery nanoparticles

“…Furthermore, encapsulation strategy helps to protect plasmid DNA from the attack of endogenous nucleases [44,110]. Recently, Kobuta et al have reported that encapsulated siRNA within lipid nanoparticles (LNPs) also showed higher stability and cell uptake efficiency as well as low cytotoxicity including reduced release of cytokines (i.e., TNF-α and IL-1β) [111]. However, the encapsulation of plasmid DNA in the aqueous core of liposome is a very tedious process and the encapsulation efficiency is also very low.…”