Abstract
The development of new gene-editing technologies has fostered the need for efficient and safe vectors capable of encapsulating large nucleic acids. In this work we evaluate the synthesis of large-size plasmid-loaded PLGA nanoparticles by double emulsion (considering batch ultrasound and microfluidics-assisted methodologies) and magnetic stirring-based nanoprecipitation synthesis methods. For this purpose, we characterized the nanoparticles and compared the results between the different synthesis processes in terms of encapsulation efficiency, morphology, particle size, polydispersity, zeta potential and structural integrity of loaded pDNA. Our results demonstrate particular sensibility of large pDNA for shear and mechanical stress degradation during double emulsion, the nanoprecipitation method being the only one that preserved plasmid integrity. However, plasmid-loaded PLGA nanoparticles synthesized by nanoprecipitation did not show cell expression in vitro, possibly due to the slow release profile observed in our experimental conditions. Strong electrostatic interactions between the large plasmid and the cationic PLGA used for this synthesis may underlie this release kinetics. Overall, none of the methods evaluated satisfied all the requirements for an efficient non-viral vector when applied to large-size plasmid encapsulation. Further optimization or alternative synthesis methods are thus in current need to adapt PLGA nanoparticles as delivery vectors for gene editing therapeutic technologies.
Highlights
PLGA, aqueous buffers with hydrophilic pacity, cargo integrity preservation and release are of particular relevance, which will be cargo (i.e.,throughout Plasmid DNA (pDNA)) andthis surfactants promote analyzed work for to each of the emulsion synthesis formation, procedures.stability, and size control
Supercoiled conformafluidics-assisted w/o/w and nanoprecipitation) rely on different physical principles, they tion possesses the highest transfection capacity and is considered to be transcriptionally all share the use of organic solutions containing dissolved PLGA, aqueous buffers with active, while other isoforms result in a significant loss of functionality
As measured from TEM images, in this order, our results showed that microfluidic technology resulted in larger nanoparticles (212 ± 87 and 258 ± 140 nm for sodium cholate and polyvinyl alcohol (PVA) syntheses, respectively) as compared to ultrasound-assisted w/o/w and nanoprecipitation techniques, the latter two being within the desired 70–200 nm size range for this study
Summary
With the development of gene therapies, and especially the CRISPR/Cas system, the use of plasmids as therapeutic tools is emerging. Major drawbacks of this novel approach are the instability of nucleic acids, which are rapidly degraded by the endonucleases within the body [1], and the inefficient internalization of exogenous naked plasmids by cells. RNA and CRISPR-associated endonuclease) are often delivered into cells encoded on largesize plasmid vectors (9–19 kb) [2]. Due to their large size, these vectors are notably difficult 4.0/).
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