pH-Dependent Phase Behavior and Stability of Cationic Lipid-mRNA Nanoparticles.

Lipid nanoparticles (LNPs) containing mRNA can deliver genetic material to cells for use as vaccines or protein replacement therapies. We characterized the effect of solution pH on cationic LNPs containing green fluorescent protein (EGFP) mRNA and their transfection efficiency. We compared the structural and colloidal properties of mRNA LNPs with LNPs not containing mRNA and mRNA free in solution. We used a combination of biophysical technique to build a picture of the structure of the lipids and mRNA across pH and temperature in the form of an empirical phase diagram (EPD). A combination of Fourier-transform infrared (FTIR) spectroscopy and differential scanning calorimetry was used to investigate lipid phase behavior. The mRNA-LNPs transition from an inverse hexagonal phase at pH values below the pKa of the cationic lipid to a lamellar phase above the pKa. At higher temperatures the mRNA-LNPs also transitioned from an inverse hexagonal phase to a lamellar phase indicating the inverse hexagonal phase is more thermodynamically favorable. Based on circular dichroism, the mRNA within the LNP has more A form structure at pH values below the lipid pKa than above it. Optical density, zeta potential and dynamic light scattering measurements were used to probe the colloidal stability of the mRNA-LNPs. The particles were larger and more prone to aggregation below the pKa. A stability study was performed to relate the biophysical characteristics to the storage of the particles in solution at 4 and 25 °C. mRNA-LNPs had the highest transfection efficiency and stability at pH values below the pKa. However, there was a trade-off between the stability and aggregation propensity since at very low pH the particles were most prone to aggregation. We performed kinetic experiments to show that the time scale of the pH-dependent phase behavior is slow (6 hour transition) and the transition from lamellar to inverse hexagonal phases is irreversible. This suggests that the lamellar phase is less stable and kinetically trapped. Our findings deepen our structural understanding of mRNA-LNPs and will aid the development of related formulations.

A scalable and robust cationic lipid/polymer hybrid nanoparticle platform for mRNA delivery.

mRNA based gene therapies hold the potential to treat multiple diseases with significant advantages over DNA based therapies, including rapid protein expression and minimized risk of mutagenesis. However, successful delivery of mRNA remains challenging, and clinical translation of mRNA therapeutics has been limited. This study investigated the use of a lipid/polymer hybrid (LPH) nanocarrier for mRNA, designed to address key delivery challenges and shuttle mRNA to targeted tissues. LPH nanocarriers were synthesized using a scalable microfluidic process with a variety of material compositions and mRNA loading strategies. Results show that a combination of permanently ionized and transiently, pH-dependent ionizable cationic lipids had a synergistic effect upon on mRNA gene translation, when compared to each lipid independently. Upon intravenous administration, particles with adsorbed mRNA outperformed particles with encapsulated mRNA for protein expression in the lungs and the spleen despite significant LPH nanoparticle localization to the liver. In contrast, encapsulated particles had higher localized expression when injected intramuscularly with protein expression detectable out to 12 days post injection. Intramuscular administration of particles with OVA mRNA resulted in robust humoral immune response with encapsulated outperforming adsorbed particles in terms of antibody titers at 28 days. These results demonstrate LPH nanocarriers have great potential as a vehicle for mRNA delivery and expression in tissues and that tissue expression and longevity can be influenced by LPH composition and route of administration.

Synthesis, characterization and in vivo efficacy of PEGylated insulin for oral delivery with complexation hydrogels.

PURPOSE: This work evaluated the feasibility of combining insulin PEGylation with pH responsive hydrogels for oral insulin delivery. METHODS: A mono-substituted PEG-insulin conjugate was synthesized and purified. The site of conjugation was determined by MALDI-TOF MS. Uptake and release of PEGylated insulin was performed in complexation hydrogels to simulate oral dosing. The bioactivity of the conjugate and PK/PD profile was measured in vivo in rats. RESULTS: PEGylation was confirmed to be specifically located at the amino terminus of the B-chain of insulin. Higher loading efficiency was achieved with PEGylated insulin than regular human insulin in pH responsive hydrogels. The release of PEGylated insulin was lower than that of human insulin at all pH levels considered. Full retention of bioactivity of the PEG-insulin conjugate was confirmed by intravenous dosing while subcutaneous dosing exhibited a relative hypoglycemic effect 127.8% that of human insulin. CONCLUSIONS: Polyethylene glycol conjugated specifically to the amino terminus of the B-chain of insulin maintained the bioactivity of the protein and significantly extended the duration of the hypoglycemic effect. Used in combination with pH responsive hydrogels, PEGylated insulin has significant potential for oral delivery.