LipoParticles: Lipid-Coated PLA Nanoparticles Enhanced In Vitro mRNA Transfection Compared to Liposomes

Abstract

The approval of two mRNA vaccines as urgent prophylactic treatments against Covid-19 made them a realistic alternative to conventional vaccination methods. However, naked mRNA is rapidly degraded by the body and cannot effectively penetrate cells. Vectors capable of addressing these issues while allowing endosomal escape are therefore needed. To date, the most widely used vectors for this purpose have been lipid-based vectors. Thus, we have designed an innovative vector called LipoParticles (LP) consisting of poly(lactic) acid (PLA) nanoparticles coated with a 15/85 mol/mol DSPC/DOTAP lipid membrane. An in vitro investigation was carried out to examine whether the incorporation of a solid core offered added value compared to liposomes alone. To that end, a formulation strategy that we have named particulate layer-by-layer (pLbL) was used. This method permitted the adsorption of nucleic acids on the surface of LP (mainly by means of electrostatic interactions through the addition of LAH4-L1 peptide), allowing both cellular penetration and endosomal escape. After a thorough characterization of size, size distribution, and surface charge- and a complexation assessment of each vector-their transfection capacity and cytotoxicity (on antigenic presenting cells, namely DC2.4, and epithelial HeLa cells) were compared. LP have been shown to be significantly better transfecting agents than liposomes through pLbL formulation on both HeLa and DC 2.4 cells. These data illustrate the added value of a solid particulate core inside a lipid membrane, which is expected to rigidify the final assemblies and makes them less prone to early loss of mRNA. In addition, this assembly promoted not only efficient delivery of mRNA, but also of plasmid DNA, making it a versatile nucleic acid carrier that could be used for various vaccine applications. Finally, if the addition of the LAH4-L1 peptide systematically leads to toxicity of the pLbL formulation on DC 2.4 cells, the optimization of the nucleic acid/LAH4-L1 peptide mass ratio becomes an interesting strategy-essentially reducing the peptide intake to limit its cytotoxicity while maintaining a relevant transfection efficiency.

Keywords: LipoParticles; cell-penetrating peptides; delivery systems; liposomes; mRNA vaccines; nanoparticles; nucleic acids; transfection.

Figure 1

Schematic representation of cationic liposomes (left), an anionic poly(lactic) acid (PLA) nanoparticle (middle), and a resulting LipoParticle (right). Note that the scale and charge proportions are not respected.

Figure 2

Schematic representation of the particulate layer-by-layer formulation strategy used to successively adsorb mRNA and LAH4-L1 on either a liposome (top) or a LipoParticle (bottom). Note that the scale proportions are not respected.

Figure 3

Agarose gel electrophoresis analysis of mRNA-based formulations (A) without and (B) with mRNA desorption treatment. For desorption treatment, samples were incubated at room temperature for 30 min and then treated with heparin (to desorb mRNAs and peptides) and proteinase K (to degrade peptides) at 56 °C for 15 min.

Figure 4

In vitro evaluation at +24 h (top) of transfection efficiency through measurement of luminescence intensity (Bright-Glo luciferase assay) and (bottom) cell viability (PrestoBlue assay) obtained after transfection of 90 ng eq. of Fluc mRNA, formulated either in liposomes or in LP using pLbL strategy (ratio mRNA/LAH4-L1 = 1:20 w/w) on HeLa (A,C) and DC 2.4 (B,D) cells. Lipofectamine 2000^TM was used as positive control. Data are presented as mean ± SD and statistically analyzed using one-way ANOVA followed by Tukey’s multiple comparison test (not significant (ns): p > 0.01, **: p < 0.001, ***: p < 0.0001 and ****: p < 0.00001).

Figure 5

In vitro assessment of (top images) the cytotoxicity (normal phase mode) and (bottom images) transfection efficiency (fluorescent mode) of pLbL formulations using microscopy. Images taken 24 h after transfection of 90 ng eq. of eGFP mRNA, formulated either in liposomes or in LP using pLbL strategy (ratio mRNA/LAH4-L1 = 1:20 w/w) on DC 2.4 ((A), (a–h)) and HeLa ((B), (a’–h’)) cells. Lipofectamine 2000^TM was used as positive control. Scale bar: 100µm. (a,a’) Cells alone (b,b’) LAH4-L1 peptide (c,c’) Liposomes, (d,d’) LP (e,e’) Naked mRNA (f,f’) Lipofectamine 2000^TM (g,g’) pLbL Liposomes mRNA (h,h’) pLbL LP mRNA.

Figure 6

In vitro evaluation at +24 h (top) of transfection efficiency through the measurement of luminescence intensity (Bright-Glo luciferase assay) and (bottom) cell viability (PrestoBlue assay) obtained after transfection of 90 ng eq. of either Fluc mRNA or luciferase-pcDNA3.1, formulated in LP using pLbL strategy (ratio nucleic acids/LAH4-L1 = 1:20 w/w) on HeLa (A,C) and DC 2.4 (B,D) cells. Lipofectamine 2000^TM was used as positive control. Data are presented as mean ± SD and statistically analyzed using one-way ANOVA followed by Tukey’s multiple comparison test (ns: p > 0.01, **: p <0.001, ***: p < 0.0001 and ****: p < 0.00001).

Figure 7

Evaluation of the influence of nucleic acids/ LAH4-L1 w/w ratio during pLbL formulation. Analysis of (top) the transfection efficiency by measuring luminescence intensity (Bright-Glo luciferase assay) and (bottom) cell viability (PrestoBlue assay) obtained after transfection of 90 ng eq. of either Fluc mRNA or luciferase-pcDNA3.1 formulated in LP using the pLbL strategy. Nucleic acids/LAH4-L1 w/w ratio ranged from 1:2 to 1:20. Transfection was performed both on HeLa (A,C) and DC 2.4 (B,D) cells. Lipofectamine 2000^TM was used as positive control. Data are presented as mean ± SD and statistically analyzed using one-way ANOVA followed by Tukey’s multiple comparison test (ns: p > 0.01, *: p < 0.01, **: p < 0.001 and ***: p < 0.0001).