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

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.

PLGA fine nanoparticle for drug delivery systems: experimental and simulation

“…The great limitation remains for size of nanocapsules which must be finer than 100 nm to be desirable for injection and diffusible through cancer cells [31]. Certainly the synthesised capsule has to be stable in blood, where nanoparticles are susceptible to precipitation with high risk for body direct injection [32]. In this paper, some methods of stability will be reported, beside.…”

Multi‐modal cell structure formation of poly (lactic‐co‐glycolic acid)/superparamagnetic iron oxide nanoparticles composite scaffolds by supercritical CO2 varying‐temperature foaming

“…Before the foaming process, 1 g PLGA raw materials were dissolved in dichloromethane using ultrasonic dispersion for 60 min with a concentration of 10% (g·ml −1 , w/v) 32–35 . Then, 5% (relative to the mass of PLGA) SPIONs were added to the solution, and the mixture was stirred for 4 h. After dichloromethane was completely volatilized, the mixed samples were put into a vacuum drying oven for 48 h. The dried mixed samples were shattered by shredding machines, and the collected PLGA/SPIONs tiny particles were pressed to a cylindrical sheet with a fixed sheet diameter of 15 mm by a tablet press machine (769YP‐24B, Nuoleixinda, China) under 12 MPa for 2 min.…”

“…Before the foaming process, 1 g PLGA raw materials were dissolved in dichloromethane using ultrasonic dispersion for 60 min with a concentration of 10% (gÁml À1 , w/v). [32][33][34][35] Then, 5% (relative to the mass of PLGA) SPIONs were added to the solution, and the mixture was stirred for 4 h. After dichloromethane was completely volatilized, the mixed samples were put into a vacuum drying oven for The experimental process was carried out via the varyingtemperature mode, as shown in Figure 2. Briefly, the sample was placed in the high-pressure vessel and then the vessel was injected with cooled CO 2 for pressurization.…”

Biomimetic cell membrane‐coated poly(lactic‐co‐glycolic acid) nanoparticles for biomedical applications

“…Drug loading, encapsulation, and release are significantly affected when one of emulsion‐based technique is switched to another one (O/W, W/O, and W/O/W) 282 . Because conventional methods suffer from limitations, such as small batch and large batch production can alter the formulation characteristics, new emulsification technologies, such as microfluidics‐based and membrane emulsification have been developed 283 . The production and storage costs of NPs also need to be considered when patient's own cells are used.…”

“…283 The production and storage costs of NPs also need to be considered when patient's own cells are used. Therefore, continuous screening of these NPs for better biocompatibility, higher specificity, better method of preparation and wider applications should constitute a major future direction of research and development.…”