Comparison of Physicochemical Properties of LipoParticles as mRNA Carrier Prepared by Automated Microfluidic System and Bulk Method

Abstract

Polymeric and/or lipid platforms are promising tools for nucleic acid delivery into cells. We previously reported a lipid-polymer nanocarrier, named LipoParticles, consisting of polylactic acid nanoparticles surrounded by cationic lipids, and allowing the addition of mRNA and cationic LAH4-1 peptide at their surface. Although this mRNA platform has shown promising results in vitro in terms of mRNA delivery and translation, the bulk method used to prepare LipoParticles relies on a multistep and time-consuming procedure. Here, we developed an automated process using a microfluidic system to prepare LipoParticles, and we compared it to the bulk method in terms of morphology, physicochemical properties, and ability to vectorize and deliver mRNA in vitro. LipoParticles prepared by microfluidic presented a smaller size and more regular spherical shape than bulk method ones. In addition, we showed that the total lipid content in LipoParticles was dependent on the method of preparation, influencing their ability to complex mRNA. LipoParticles decorated with two mRNA/LAHA-L1 ratios (1/20, 1/5) could efficiently transfect mouse DC2.4 cells except for the automated 1/5 assay. Moreover, the 1/5 mRNA/LAHA-L1 ratio drastically reduced cell toxicity observed in 1/20 ratio assays. Altogether, this study showed that homogeneous LipoParticles can be produced by microfluidics, which represents a promising platform to transport functional mRNA into cells.

Keywords: LipoParticles; biodegradable polymer; bulk method; hybrid nanoparticle; liposomes; mRNA transfection; microfluidics.

Figure 1

Preparation of LipoParticles (A) using the standard bulk method or (B) through an automated mixing using microfluidic system.

Figure 2

TEM images of PLA-NP (A–C), LP (E–G), and LP_auto (I–K) stained with 1% (w/v in water) tungsten silicate solution. White arrows indicate lipid layers on LipoParticles (G,K), while black asterisks indicate eccentric lipid vesicles attached to LP (F). Size particle analysis was performed on 1 µm scaled images to evaluate the size distribution frequency of nanoparticles (graphs D,H,L). Scale bars are indicated on each micrograph.

Figure 3

Agarose gel electrophoresis assays of pLbL formulations (A) without and (B) with mRNA desorption treatment. The concentration of Fluc mRNA was 10 µg/mL.

Figure 4

Determination of the amount of mRNA that can be adsorbed onto (A) LP and (B) LP_auto using agarose gel electrophoresis assays. To this aim, formulations containing increasing concentrations of mRNA were prepared and directly deposited on 1% agarose gel. The black box represents the mRNA concentration used in the pLbL formulations.

Figure 5

In vitro evaluation of (left) transfection efficiency using Bright-Glo luciferase assay and (right) cell viability (PrestoBlue assay) of pLbL formulations. Transfections were performed on DC2.4 cells. Measurements were always performed 24 h post transfection. Data are presented as the mean ± SD (not significant (ns): p > 0.05, ***: p < 0.001, ****: p < 0.0001).