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Nanomaterials2021ResearchNon-viral Gene Delivery

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

Tresa Lopez-Royo, Victor Sebastian, Laura Moreno-Martinez, Laura Uson, Cristina Yus, Teresa Alejo, Pilar Zaragoza, Rosario Osta, Manuel Arruebo, Raquel ManzanoDOI 10.3390/nano11102723

Summary

CRISPR/Cas gene-editing components are often encoded on large plasmids (9–19 kb), which are difficult to encapsulate and transfect. Most PLGA nanoparticle (NP) gene-delivery studies use small nucleic acids (siRNA) or small plasmids (<6 kb), and the structural integrity and functional performance of large plasmids in PLGA NPs remain poorly characterized. There is a need to compare synthesis methods for encapsulating large plasmids without. pDNA stability: Batch ultrasound double emulsion completely degraded the 9.4 kb plasmid (standard) or left only 8.80% supercoiled (modified). Microfluidics-assisted double emulsion avoided degradation but produced only.

Keywords

PLGANanoparticlesGene editingMicrofluidicsTransfectionDNACRISPR
Purpose: CRISPR/Cas gene-editing components are often encoded on large plasmids (9–19 kb), which are difficult to encapsulate and transfect. Most PLGA nanoparticle (NP) gene-delivery studies use small nucleic acids (siRNA) or small plasmids (<6 kb), and the structural integrity and functional performance of large plasmids in PLGA NPs remain poorly characterized. There is a need to compare synthesis methods for encapsulating large plasmids without degrading the DNA or losing transfection capacity.
Hypothesis: If large plasmid DNA is encapsulated in PLGA NPs using milder synthesis methods — microfluidics-assisted double emulsion or nanoprecipitation — then plasmid structural integrity, encapsulation efficiency, and downstream cell expression will be better preserved than with conventional batch ultrasound-assisted double emulsion, which exposes DNA to high shear and mechanical stress.
Aims: Primary aim: Compare three PLGA NP synthesis methods — double emulsion (batch ultrasound and microfluidics-assisted) and magnetic-stirring nanoprecipitation — for encapsulation of a large 9.4 kb plasmid.
  • Secondary aim 1: Characterize NPs for morphology, size, polydispersity, zeta potential, NP yield, and pDNA encapsulation efficiency.
  • Secondary aim 2: Assess structural integrity of encapsulated pDNA (supercoiled, linear, relaxed, nicked open-circular isoforms) and pDNA release profile.
  • Secondary aim 3: Evaluate in vitro cytotoxicity, cellular internalization, and plasmid expression in NSC-34 motor neuron-like cells.
Delivery system:

Component: Polymer; Description: PLGA-COOH (Resomer RG 503H) or PLGA-NH₂ (50:50, 20 kDa)

Component: NP Type; Description: Double emulsion (w/o/w) or nanoprecipitation

Component: Targeting Ligand; Description: None

Component: Payload; Description: pRFP plasmid, 9431 bp (9.4 kb), encoding red fluorescent protein

Component: Surfactants / Stabilizers; Description: Sodium cholate, PVA, Pluronic F-127, PEG (modified ultrasound method)

Component: Fluorescent Tracer; Description: TIPS pentacene (for nanoprecipitation uptake tracking)

Component: Formulations Tested; Description: Standard ultrasound-assisted w/o/w; modified ultrasound-assisted w/o/w; microfluidics-assisted w/o/w with sodium cholate; microfluidics-assisted w/o/w with PVA; nanoprecipitation with PLGA-COOH; nanoprecipitation with PLGA-NH₂

Component: Size Range; Description: Nanoprecipitation: 88 ± 21 nm (PLGA-COOH), 93 ± 29 nm (PLGA-NH₂); ultrasound: 116 ± 24 nm, 123 ± 67 nm; microfluidics: 212 ± 87 nm, 258 ± 140 nm

Component: Zeta Potential; Description: −33 to −57 mV (all formulations negatively charged)

Component: Encapsulation Efficiency; Description: 0% (nanoprecipitation PLGA-COOH), 24.90% (microfluidics sodium cholate), 33.16 ± 20.81% (standard ultrasound), 50.91 ± 23.27% (microfluidics PVA), 56.84 ± 16.09% (nanoprecipitation PLGA-NH₂); modified ultrasound not determined due to PEG interference

Component: pDNA Integrity; Description: Standard ultrasound: 100% degraded; modified ultrasound: 73.64% degraded, 8.80% supercoiled, 17.86% nicked open-circular; microfluidics sodium cholate: 64.80% linear, 35.20% nicked open-circular; microfluidics PVA: 45.01% linear, 54.99% nicked open-circular; nanoprecipitation PLGA-NH₂: 41.97% supercoiled, 58.03% relaxed

Approach: In vitro model: NSC-34 motor neuron-like cells. - No in vivo model was used. - Cytotoxicity: Resazurin assay at NP concentrations 0.01, 0.05, 0.1, 0.2, and 0.4 mg/mL for 24 h. - Cellular uptake: Fluorescence quantification and confocal microscopy at 4, 8, 12, 24, and 48 h using pDNA-loaded NPs synthesized by nanoprecipitation with PLGA-NH₂ at 0.2 mg/mL. - Transfection: pDNA-loaded NPs corresponding to 2 µg encapsulated plasmid incubated for 72 h; lipofectamine/pDNA as positive control; RFP expression assessed by immunofluorescence. - Controls: Untreated cells, empty PLGA NPs, induced cell death positive control, and no-cell background controls.
Key methods:

Technique: Agarose gel electrophoresis; Purpose: Assess pDNA conformation (supercoiled, linear, relaxed, nicked open-circular) and structural integrity

Technique: Qubit dsDNA high-sensitivity assay; Purpose: Quantify pDNA encapsulation efficiency and release

Technique: TEM and SEM; Purpose: Determine NP morphology and size

Technique: Dynamic light scattering / phase analysis light scattering; Purpose: Measure zeta potential

Technique: Resazurin assay; Purpose: Assess in vitro cytotoxicity

Technique: Fluorescence spectroscopy; Purpose: Quantify cellular internalization of PLGA NPs and pDNA per cell

Technique: Confocal microscopy; Purpose: Visualize intracellular NP localization over time

Technique: Immunofluorescence; Purpose: Detect RFP expression as transfection readout

Key results: pDNA stability: Batch ultrasound double emulsion completely degraded the 9.4 kb plasmid (standard) or left only 8.80% supercoiled (modified). Microfluidics-assisted double emulsion avoided degradation but produced only linear and nicked open-circular isoforms, with 0% supercoiled DNA. Nanoprecipitation with PLGA-NH₂ preserved 41.97% supercoiled pDNA, the highest functional isoform retention. - Encapsulation efficiency: Nanoprecipitation with PLGA-NH₂ achieved 56.84 ± 16.09%; microfluidics with PVA achieved 50.91 ± 23.27%; standard ultrasound achieved 33.16 ± 20.81%; microfluidics with sodium cholate achieved 24.90%; nanoprecipitation with PLGA-COOH achieved 0%. - Cellular uptake: NSC-34 cells internalized 0.127 ± 0.029 ng PLGA NPs/cell at 24 h and 0.137 ± 0.016 ng/cell at 48 h, corresponding to 2.282 pg pDNA/cell at 24 h and 2.469 pg/cell at 48 h (>235,000 plasmid copies/cell). - Cytotoxicity: Empty and pDNA-loaded PLGA NPs were non-cytotoxic across 0.01–0.4 mg/mL. - Transfection: No RFP expression was detected from any pDNA-loaded PLGA NP formulation. The most promising formulation (nanoprecipitation with PLGA-NH₂) released only ~2.2% pDNA at 4°C and ~4% at 37°C in water over 24 h, likely due to strong electrostatic binding between the large anionic plasmid and cationic PLGA-NH₂.
Interpretation: The authors conclude that none of the evaluated methods fully satisfied the requirements for an efficient non-viral vector for large plasmids. Double emulsion — both ultrasound- and microfluidics-assisted — damaged or altered plasmid structure, while nanoprecipitation with PLGA-NH₂ preserved the highest fraction of supercoiled pDNA and achieved the best encapsulation efficiency, but suffered from poor cargo release and no detectable cell expression. The authors state that further optimization or alternative synthesis methods are needed before PLGA NPs can serve as delivery vectors for CRISPR/Cas gene-editing plasmids.
Limitations: No in vivo validation; all work is in vitro. - No successful plasmid expression or functional gene-editing readout was achieved. - Only one cell line (NSC-34) and one large plasmid (pRFP, 9.4 kb) were tested. - pDNA release in water was very low (~2–4% in 24 h); release improved in PBS but was still incomplete. - Nanoprecipitation with PLGA-COOH failed to encapsulate pDNA. - The study did not test targeting ligands or surface modifications to improve uptake or release. - Plasmid degradation at 37°C in PLGA-NH₂ formulations was observed, possibly due to depurination in the presence of primary amines; this may confound release measurements. - Microfluidics-assisted NPs had higher polydispersity than expected, possibly due to DCM use. - No direct comparison with viral vectors or commercial transfection reagents beyond lipofectamine positive control.

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