Compression of Vectors for Small Interfering RNAs Delivery: Toward Oral Administration of siRNA Lipoplexes in Tablet Forms.

Currently, most nonviral 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; As regards nucleic acid vectors, a dry form represents an advance in terms of stability. Using an optimized lipid-based small interfering RNA-delivery system, we studied the tabletability of a liquid suspension of these vectors. 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 small-angle X-ray scattering (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 allows 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 resuspension 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.

DLin-MC3-Containing mRNA Lipid Nanoparticles Induce an Antibody Th2-Biased Immune Response Polarization in a Delivery Route-Dependent Manner in Mice.

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.

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

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.

Spheroplexes: Hybrid PLGA-cationic lipid nanoparticles, for in vitro and oral delivery of siRNA.

Vectorized small interfering RNAs (siRNAs) are widely used to induce gene silencing. Among the delivery systems used, lipid-based particles are the most effective. Our objective was the development of novel lipid-polymer hybrid nanoparticles, from lipoplexes (complexes of cationic lipid and siRNAs), and poly (lactic-co-glycolic acid) (PLGA), using a simple modified nanoprecipitation method. Due to their morphology, we called these hybrid nanoparticles Spheroplexes. We elucidated their structure using several physico-chemical techniques and showed that they are composed of a hydrophobic PLGA matrix, surrounded by a lipid envelope adopting a lamellar structure, in which the siRNA is complexed, and they retain surface characteristics identical to the starting nanoparticles, i.e. lipoplexes siRNA. We analyzed the composition of the particle population and determined the final percentage of spheroplexes within this population, 80 to 85% depending on the preparation conditions, using fluorescent markers and the ability of flow cytometry to detect nanometric particles (approximately 200 nm). Finally, we showed that spheroplexes are very stable particles and more efficient than siRNA lipoplexes for the delivery of siRNA to cultured cells. We administered spheroplexes contain siRNAs targeting TNF-α to mice with ulcerative colitis induced by dextran sulfate and our results indicate a disease regression effect with a response probably mediated by their uptake by macrophages / monocytes at the level of lamina propria of the colon. The efficacy of decreased level of TNF-α in vivo seemed to be an association of spheroplexes polymer-lipid composition and the specific siRNA. These results demonstrate that spheroplexes are a promising hybrid nanoparticle for the oral delivery of siRNA to the colon.

Modifying internal organization and surface morphology of siRNA lipoplexes by sodium alginate addition for efficient siRNA delivery.

Vectorized small interfering RNAs (siRNAs) are widely used to induce specific mRNA degradation in the intracellular compartment of eukaryotic cells. Recently, we developed efficient cationic lipid-based siRNA vectors (siRNA lipoplexes or siLex) containing sodium alginate (Nalg-siLex) with superior efficiency and stability properties than siLex. In this study, we assessed the physicochemical and some biological properties of Nalg-siLex compared to siLex. While no significant differences in size, ζ potential and siRNA compaction were detected, the addition of sodium alginate modified the particle morphology, producing smoother and heterogeneous particles characterized by transmission electron microscopy. We also noted that Nalg-siLex have surface differences observed by X-ray photoelectron spectroscopy. These differences could arise from an internal reorganization of components induced by the addition of sodium alginate, that is indicated by Small-Angle X-ray Scattering results. Moreover, Nalg-siLex did not trigger significant hepatotoxicity nor inflammatory cytokine secretion compared to siLex. Taken together these results suggest that sodium alginate played a key role by structuring and reinforcing siRNA lipoplexes, leading to more stable and efficient delivery vector.

Lipoplexes Strengthened by Anionic Polymers: Easy Preparation of Highly Effective siRNA Vectors Based on Cationic Lipids and Anionic Polymers.

RNA interference is an invaluable tool in biology to specifically silence a given gene. Synthetic duplexes of RNA oligonucleotides are widely used to induce mRNA degradation in cultured cells or in whole organisms. They have to be vectorized to reach their target site. Here, we describe the preparation of highly efficient siRNA vectors based on cationic liposomes and polyanionic polymers and their application in cultured cells to silence reporter and/or endogenous genes.