ABSTRACT
The global emergency of coronavirus disease 2019 (COVID-19) has spurred extensive worldwide efforts to develop vaccines for protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our contribution to this global endeavor involved the development of a diverse library of nanocarriers, as alternatives to lipid nanoparticles (LNPs), including nanoemulsions (NEs) and nanocapsules (NCs), with the aim of protecting and delivering messenger ribonucleic acid (mRNA) for nasal vaccination purposes. A wide range of prototypes underwent rigorous screening through a series of in vitro and in vivo experiments, encompassing assessments of cellular transfection, cytotoxicity, and intramuscular administration of a model mRNA for protein translation. As a result, two promising candidates were identified for nasal administration. One of them was a NE incorporating a combination of an ionizable lipid (C12-200) and cationic lipid (DOTAP), both intended to condense mRNA, along with DOPE, which is known to facilitate endosomal escape. This NE exhibited a size of 120 nm and a highly positive surface charge (+ 50 mV). Another candidate was an NC formulation comprising the same components and endowed with a dextran sulfate shell. This formulation showed a size of 130 nm and a moderate negative surface charge (-16 mV). Upon intranasal administration of mRNA encoding for ovalbumin (mOVA) associated with optimized versions of the said NE and NCs, a robust antigen-specific CD8 + T cell response was observed. These findings underscore the potential of NEs and polymeric NCs in advancing mRNA vaccine development for combating infectious diseases.
Subject(s)
Administration, Intranasal , COVID-19 Vaccines , Emulsions , Nanocapsules , mRNA Vaccines , Nanocapsules/chemistry , Animals , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/immunology , Mice , COVID-19/prevention & control , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Humans , SARS-CoV-2/immunology , Female , Quaternary Ammonium Compounds/chemistry , Mice, Inbred BALB C , Fatty Acids, Monounsaturated/chemistry , RNA, Messenger/administration & dosage , Drug Carriers/chemistry , Drug Carriers/administration & dosageABSTRACT
K-RAS is a highly relevant oncogene that is mutated in approximately 90% of pancreatic cancers and 20-25% of lung adenocarcinomas. The aim of this work was to develop a new anti-KRAS siRNA therapeutic strategy through the engineering of functionalized lipid nanoparticles (LNPs). To do this, first, a potent pan anti-KRAS siRNA sequence was chosen from the literature and different chemical modifications of siRNA were tested for their transfection efficacy (KRAS knockdown) and anti-proliferative effects on various cancer cell lines. Second, a selected siRNA candidate was loaded into tLyp-1 targeted and non-targeted lipid nanoparticles (LNPs). The biodistribution and antitumoral efficacy of selected siRNA-loaded LNP-prototypes were evaluated in vivo using a pancreatic cancer murine model (subcutaneous xenograft CFPAC-1 tumors). Our results show that tLyp-1-tagged targeted LNPs have an enhanced accumulation in the tumor compared to non-targeted LNPs. Moreover, a significant reduction in the pancreatic tumor growth was observed when the anti-KRAS siRNA treatment was combined with a classical chemotherapeutic agent, gemcitabine. In conclusion, our work demonstrates the benefits of using a targeting approach to improve tumor accumulation of siRNA-LNPs and its positive impact on tumor reduction.
Subject(s)
Nanoparticles , Pancreatic Neoplasms , Humans , Mice , Animals , RNA, Small Interfering/genetics , RNA, Small Interfering/therapeutic use , Tissue Distribution , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/geneticsABSTRACT
Direct nose-to-brain (N-to-B) delivery enables the rapid transport of drugs to the brain, while minimizing systemic exposure. The objective of this work was to engineer a nanocarrier intended to enhance N-to-B delivery of RNA and to explore its potential utility for the treatment of neurological disorders. Our approach involved the formation of electrostatically driven nanocomplexes between a hydrophobic derivative of octaarginine (r8), chemically conjugated with lauric acid (C12), and the RNA of interest. Subsequently, these cationic nanocomplexes were enveloped (enveloped nanocomplexes, ENCPs) with different protective polymers, i.e. polyethyleneglycol - polyglutamic acid (PEG-PGA) or hyaluronic acid (HA), intended to enhance their stability and mucodiffusion across the olfactory nasal mucosa. These rationally designed ENCPs were produced in bulk format and also using a microfluidics-based technique. This technique enabled the production of a scalable nanoformulation, exhibiting; (i) a unimodal size distribution with a tunable mean size, (ii) the capacity to highly associate (100%) and protect RNA from degradation, (iii) the ability to preserve its physicochemical properties in biorelevant media and prevent the premature RNA release. Moreover, in vitro cell culture studies showed the capacity of ENCPs to interact and be efficiently taken-up by CHO cells. Finally, in vivo experiments in a mouse model of Alzheimer's disease provided evidence of a statistically significant increase of a potentially therapeutic miRNA mimic in the hippocampus area and its further effect on two mRNA targets, following its intranasal administration. Overall, these findings stress the value of the rational design of nanocarriers towards overcoming the biological barriers associated to N-to-B RNA delivery and reveal their potential value as therapeutic strategies in Alzheimer's disease.
