MRNA-based vaccines have made a leap forward since the SARS-CoV-2 pandemic and are currently used to develop anti-infectious therapies. If the selection of a delivery system and an optimized mRNA sequence are two key factors to reach in vivo efficacy, the optimal administration route for those vaccines remains unclear. We investigated the influence of lipid components and immunization route regarding the intensity and quality of humoral immune responses in mice. The immunogenicity of HIV-p55Gag encoded mRNA encapsulated into D-Lin-MC3-DMA or GenVoy-ionizable lipid-based LNPs was compared after intramuscular or subcutaneous routes. Three sequential mRNA vaccines were administrated followed by a heterologous boost composed of p24-HIV protein antigen. Despite equivalent IgG kinetic profiles of general humoral responses, IgG1/IgG2a ratio analysis showed a Th2/Th1 balance toward a Th1-biased cellular immune response when both LNPs were administrated via the intramuscular route. Surprisingly, a Th2-biased antibody immunity was observed when DLin-containing vaccine was injected subcutaneously. A protein-based vaccine boost appeared to reverse this balance to a cellular-biased response correlated to an increase in antibody avidity. Our finding suggests that the intrinsic adjuvant effect of ionizable lipids appears to be dependent on the delivery route used, which could be relevant to reach potent and long-lasting immunity after mRNA-based immunization.

Nanomedicine as a therapeutic approach for pregnancy-related diseases could offer improved treatments for the mother while avoiding side effects for the fetus. In this study, we evaluated the potential of liposomes as carriers for small interfering RNAs to placental cells. Three neutral formulations carrying rhodamine-labelled siRNAs were evaluated on an in vitro model, i.e., human primary villous cytotrophoblasts. siRNA internalization rate from lipoplexes were compared to the one in the presence of the lipofectamine reagent and assessed by confocal microscopy. Results showed cellular internalization of nucleic acid with all three formulations, based on two cationic lipids, either DMAPAP or CSL-3. Moreover, incubation with DMAPAP+AA provided a rate of labelled cells as high as with lipofectamine (53 ± 15% and 44 ± 12%, respectively) while being more biocompatible. The proportion of cells which internalized siRNA were similar when using DMAPAP/DDSTU (16 ± 5%) and CSL-3 (22 ± 5%). This work highlights that liposomes could be a promising approach for gene therapy dedicated to pregnant patients.

Currently, most non-viral nucleic acid vectors are in the form of colloidal suspensions administered primarily parenterally. This type of formulation and the mode of administration impose strong constraints such as the size of the administered vectors or the production of sterile preparations. The tablet form provides access to easy oral administration, well accepted by patients, it is also an advance for the stability of the nucleic acids vectors, since it is a dry form. Using an optimized lipid-based small interfering RNA delivery system, we studied the tabletability of a liquid suspension of these vector. We optimized the conditions of freeze-drying by choosing excipients and process, allowing for the conservation of both the gene silencing efficacy of the formulated siRNAs and the supramolecular structure of the lipid particulate system. Gene silencing efficacy was assayed on luciferase-expressing cells and the structure of the siRNA vector in freeze-dried and tablet forms was examined using SAXS Synchrotron radiation. The freeze-dried powders were then mixed with excipients necessary for the good progress of the compression by allowing for a regular supply of the matrix and the reduction of friction. The compression was carried out using a rotary press simulator that allow for complete monitoring of the compression conditions. After compression, formulated siRNAs retained more than 60% of their gene silencing efficacy. Within the tablets, a specific SAXS signal was detectable and the lamellar and cubic phases of the initial liquid suspension were restored after re-suspension of siRNA vectors by disintegration of the tablets. These results show that the bilayer lipid structures of the particles were preserved despite the mechanical constraints imposed by the compression. If such a result could be expected after the freeze-drying step, it was never shown, to our knowledge, that siRNA delivery systems could retain their efficacy and structure after mechanical stress such as compression. This opens promising perspectives to oral administration of siRNA as an alternative to parenteral administration.

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.