RESUMEN

Recently, mRNA-based therapeutics, including vaccines, have gained significant attention in the field of gene therapy for treating various diseases. Among the various mRNA delivery vehicles, lipid nanoparticles (LNPs) have emerged as promising vehicles for packaging and delivering mRNA with low immunogenicity. However, while mRNA delivery has several advantages, the delivery efficiency and stability of LNPs remain challenging for mRNA therapy. In this study, an ionizable helper cholesterol analog, 3ß[L-histidinamide-carbamoyl] cholesterol (Hchol) lipid is developed and incorporated into LNPs instead of cholesterol to enhance the LNP potency. The pKa values of the Hchol-LNPs are ≈6.03 and 6.61 in MC3- and SM102-based lipid formulations. Notably, the Hchol-LNPs significantly improve the delivery efficiency by enhancing the endosomal escape of mRNA. Additionally, the Hchol-LNPs are more effective in a red blood cell hemolysis at pH 5.5, indicating a synergistic effect of the protonated imidazole groups of Hchol and cholesterol on endosomal membrane destabilization. Furthermore, mRNA delivery is substantially enhanced in mice treated with Hchol-LNPs. Importantly, LNP-encapsulated SARS-CoV-2 spike mRNA vaccinations induce potent antigen-specific antibodies against SARS-CoV-2. Overall, incorporating Hchol into LNP formulations enables efficient endosomal escape and stability, leading to an mRNA delivery vehicle with a higher delivery efficiency.

Asunto(s)

Colesterol , Nanopartículas , ARN Mensajero , SARS-CoV-2 , Animales , Colesterol/química , Colesterol/análogos & derivados , Nanopartículas/química , Ratones , ARN Mensajero/genética , Humanos , Histidina/química , Histidina/análogos & derivados , Lípidos/química , COVID-19 , Vacunas contra la COVID-19/química , Endosomas/metabolismo , Femenino , Hemólisis/efectos de los fármacos , Ratones Endogámicos BALB C , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Liposomas

RESUMEN

Metal chelators are used for various industrial and medical purposes based on their physicochemical properties and biological activities. In biological systems, copper ions bind to certain enzymes as cofactors to confer catalytic activity or bind to specific proteins for safe storage and transport. However, unbound free copper ions can catalyze the production of reactive oxygen species (ROS), causing oxidative stress and cell death. The present study aims to identify amino acids with copper chelation activities that might mitigate oxidative stress and toxicity in skin cells exposed to copper ions. A total of 20 free amino acids and 20 amidated amino acids were compared for their copper chelation activities in vitro and the cytoprotective effects in cultured HaCaT keratinocytes exposed to CuSO4. Among the free amino acids, cysteine showed the highest copper chelation activity, followed by histidine and glutamic acid. Among the amidated amino acids, cysteinamide showed the highest copper chelation activity, followed by histidinamide and aspartic acid. CuSO4 (0.4-1.0 mM) caused cell death in a concentration-dependent manner. Among the free and amidated amino acids (1.0 mM), only histidine and histidinamide prevented the HaCaT cell death induced by CuSO4 (1.0 mM). Cysteine and cysteinamide had no cytoprotective effects despite their potent copper-chelating activities. EDTA and GHK-Cu, which were used as reference compounds, had no cytoprotective effects either. Histidine and histidinamide suppressed the CuSO4-induced ROS production, glutathione oxidation, lipid peroxidation, and protein carbonylation in HaCaT cells, whereas cysteine and cysteinamide had no such effects. Bovine serum albumin (BSA) showed copper-chelating activity at 0.5-1.0 mM (34-68 mg mL-1). Histidine, histidinamide, and BSA at 0.5-1.0 mM enhanced the viability of cells exposed to CuCl2 or CuSO4 (0.5 mM or 1.0 mM) whereas cysteine and cysteinamide had no such effects. The results of this study suggest that histidine and histidinamide have more advantageous properties than cysteine and cysteinamide in terms of alleviating copper ion-induced toxic effects in the skin.