Abstract
The development of safe and effective nucleic acid delivery systems remains a challenge, with solid lipid nanoparticle (SLN)-based vectors as one of the most studied systems. In this work, different SLNs were developed, by combination of cationic and ionizable lipids, for delivery of mRNA and pDNA. The influence of formulation factors on transfection efficacy, protein expression and intracellular disposition of the nucleic acid was evaluated in human retinal pigment epithelial cells (ARPE-19) and human embryonic kidney cells (HEK-293). A long-term stability study of the vectors was also performed. The mRNA formulations induced a higher percentage of transfected cells than those containing pDNA, mainly in ARPE-19 cells; however, the pDNA formulations induced a greater protein production per cell in this cell line. Protein production was conditioned by energy-dependent or independent entry mechanisms, depending on the cell line, SLN composition and kind of nucleic acid delivered. Vectors containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as unique cationic lipid showed better stability after seven months, which improved with the addition of a polysaccharide to the vectors. Transfection efficacy and long-term stability of mRNA vectors were more influenced by formulation-related factors than those containing pDNA; in particular, the SLNs containing only DOTAP were the most promising formulations for nucleic acid delivery.
Highlights
With the development of RNA-based products, the potential of therapeutic targets has expanded significantly
Only the solid lipid nanoparticle (SLN) prepared with DOTAP as the only cationic lipid (SLN1) and those containing a mixture of DOTAP and DODAP (SLN2) showed physicochemical features adequate to be used as nucleic acid delivery systems
MRNA vectors were able to transfect a larger number of cells, plasmid DNA (pDNA)-mediated transfection in ARPE-19 cells was the most effective in terms of the amount of protein synthetized per cell
Summary
With the development of RNA-based products, the potential of therapeutic targets has expanded significantly. The United States Food and Drug Administration (FDA) has authorized a number of RNA drugs targeted to various human diseases. These RNA drugs include aptamer RNAs (e.g., pegaptanib), antisense oligonucleotides (ASOs) or antisense RNAs (asRNAs) (e.g., mipomersen, eteplirsen, nusinersen, inotersen and golodirsen), and short interfering RNAs (siRNAs) (e.g., patisiran and givosiran) [1]. MRNAs are a promising tool for use in the expanding area of genome editing [4,5]. All of these therapeutic applications have been addressed through the administration of plasmid DNA (pDNA). From a delivery point of view, mRNA must only reach the cytoplasm to interact with the cellular translation machinery, whereas pDNA requires entry into the nucleus, which is one of the most limiting steps for transfection
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