Encapsulation of Large-Size Plasmids in PLGA Nanoparticles for Gene Editing: Comparison of Three Different Synthesis Methods

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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.
Funding Information
  • Instituto de Salud Carlos III (PI17/00949)
  • European Regional Development Fund ("Una manera de hacer Europa")
  • Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED612-CB18/05/00037)
  • H2020 Marie Skłodowska-Curie Actions (Fellowship 752349)
  • Consolidated Groups from Gobierno de Aragón (LAGENBIO)
  • Ministerio de Ciencia, Innovación y Universidades (Programa Retos Investigación, Proyect REF: RTI2018-099019-A-I00)
  • Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (Iniciativa Ingenio 2010, Consolider Program)

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