Journal of Controlled Release1999ResearchNon-viral Gene Delivery

Encapsulation of plasmid DNA in biodegradable poly(D,L-lactic-co-glycolic acid) microspheres as a novel approach for immunogene delivery

Daqing Wang, Deborah R. Robinson, Glen S. Kwon, John Samuel29 September 2026DOI 10.1016/S0168-3659(98)00093-8

Summary

Naked plasmid DNA vaccination is largely limited to intramuscular, cutaneous, and intradermal routes because naked DNA is ineffective at crossing mucosal barriers and is rapidly degraded by nucleases. Delivery systems that protect plasmid DNA and target it to antigen-presenting cells are needed for mucosal DNA vaccines and broader immunogene delivery. Encapsulation efficiency increased with PLGA molecular mass: 22.5% at 6,000 Da, 30.0% at 12,500 Da, 50.7% at 30,000 Da, and 53.3% at 50,000 Da. DNA loading ranged from 0.62 to 1.41 µg/mg PLGA. - Structural and.

Keywords

DNAGene deliveryTransfectionPLGAPolymericMacrophagesNanocarriers

Purpose: Naked plasmid DNA vaccination is largely limited to intramuscular, cutaneous, and intradermal routes because naked DNA is ineffective at crossing mucosal barriers and is rapidly degraded by nucleases. Delivery systems that protect plasmid DNA and target it to antigen-presenting cells are needed for mucosal DNA vaccines and broader immunogene delivery.

Hypothesis: If plasmid DNA is encapsulated in biodegradable PLGA microspheres by a double-emulsion method, then the DNA will retain structural and functional integrity, be protected from nuclease digestion, release in a controlled manner dependent on PLGA molecular mass, and be phagocytosed by macrophages. Co-encapsulated monophosphoryl lipid A (MPLA) will further increase the rate of phagocytosis.

Aims: Primary aim: Prepare and characterize PLGA microspheres containing an 8.1 kb plasmid DNA (pcDNA3/zeo/nlacZ) for immunogene delivery.

Secondary aim 1: Determine encapsulation efficiency, DNA loading, particle size, and morphology across PLGA molecular masses (6,000–60,000 Da).

Secondary aim 2: Assess structural and functional integrity of extracted plasmid DNA by restriction digestion and in vitro transfection.

Secondary aim 3: Evaluate protection of encapsulated DNA from DNase I and measure in vitro release kinetics.

Secondary aim 4: Assess phagocytosis of DNA-loaded microspheres by J774A-1 macrophages and the effect of co-encapsulated MPLA.

Delivery system:

Component: Polymer; Description: PLGA (50:50 lactide:glycolide), molecular mass range 6,000–60,000 Da

Component: Formulation Type; Description: Microspheres prepared by water-oil-water (w/o/w) emulsion solvent evaporation

Component: Payload; Description: Plasmid pcDNA3/zeo/nlacZ, 8.1 kb, encoding E. coli β-galactosidase

Component: Co-Payload; Description: Monophosphoryl lipid A (MPLA), 200 µg, dissolved in organic phase

Component: Surfactant / Stabilizer; Description: Polyvinyl alcohol (PVA), 9% w/v

Component: Organic Solvent; Description: Chloroform

Component: Particle Size; Description: 0.4–2 µm diameter; spherical and compact

Component: DNA Loading; Description: 0.62 ± 0.06 to 1.41 ± 0.11 µg DNA/mg PLGA depending on polymer molecular mass

Component: Encapsulation Efficiency; Description: 22.5% (6k), 30.0% (12.5k), 50.7% (30k), 53.3% (50k); ~47% (60k)

Component: Targeting Ligand; Description: None; passive targeting to phagocytic antigen-presenting cells

Approach: In vitro cell lines: COS-1 (African green monkey kidney) for transfection; J774A-1 (murine macrophage) for phagocytosis; 410.4 (mouse mammary epithelial) as non-phagocytic control. - No in vivo studies were performed. - Groups / variables: PLGA molecular masses 6,000, 12,500, 30,000, 50,000, and 60,000 Da; DNA-loaded vs. empty microspheres; with or without MPLA; naked DNA and empty-microsphere controls. - DNase I protection: Incubation for 1, 4, 8, and 16 h. - Release study: PBS pH 7.4 at 37°C over 30 days. - Transfection: COS-1 cells transfected with DOTAP-complexed plasmid DNA extracted from microspheres or control naked DNA; β-galactosidase activity measured 48 h post-transfection.

Key methods:

Technique: Scanning electron microscopy (SEM); Purpose: Particle size and morphology

Technique: Chloroform–water extraction + UV absorbance (260 nm); Purpose: Quantify encapsulated plasmid DNA

Technique: Agarose gel electrophoresis + Pvu II restriction digestion; Purpose: Assess structural integrity of extracted DNA

Technique: COS-1 transfection + X-gal / β-galactosidase activity assay; Purpose: Assess functional integrity of extracted DNA

Technique: DNase I digestion assay; Purpose: Evaluate protection of encapsulated DNA from nuclease

Technique: PicoGreen dsDNA quantification; Purpose: Measure in vitro plasmid DNA release

Technique: Phase-contrast microscopy; Purpose: Count phagocytosed microspheres per J774A-1 cell

Key results: Encapsulation efficiency increased with PLGA molecular mass: 22.5% at 6,000 Da, 30.0% at 12,500 Da, 50.7% at 30,000 Da, and 53.3% at 50,000 Da. DNA loading ranged from 0.62 to 1.41 µg/mg PLGA. - Structural and functional integrity retained: Extracted plasmid DNA showed the same restriction digestion pattern as control DNA. Transfection of COS-1 cells gave β-galactosidase activity of 199.4 ± 18.2 mU/mg cell protein for encapsulated DNA vs. 216.7 ± 19.7 mU/mg for unencapsulated control DNA (P = 0.327). - Protection from DNase I: Encapsulated DNA remained intact after up to 16 h of DNase I exposure, whereas naked DNA or DNA mixed with empty microspheres was completely digested within 1 h. - Release depended on PLGA molecular mass: Approximately 20% release over 30 days from 6,000 Da PLGA, ~8% from 12,500 Da, and little or no release from PLGA >30,000 Da. - Phagocytosis: J774A-1 macrophages phagocytosed PLGA microspheres; DNA loading did not alter uptake, and PLGA molecular mass did not significantly affect phagocytosis. Co-encapsulated MPLA markedly increased the rate of phagocytosis.

Interpretation: The authors conclude that PLGA microspheres can encapsulate plasmid DNA without compromising structural or functional integrity, protect it from nuclease degradation, and release it at controlled rates depending on polymer molecular mass. They propose PLGA microspheres as a promising platform for joint delivery of genes and immunomodulators (e.g., MPLA) to antigen-presenting cells, with particular relevance for mucosal DNA vaccines and immunogene delivery.

Limitations: No in vivo immunogenicity, protective efficacy, or toxicity data; authors state immune-response studies are in progress. - Transfection was performed with plasmid DNA extracted from microspheres, not with intact DNA-loaded microspheres delivered to cells. - Microsphere size (0.4–2 µm) may be suboptimal for certain routes or systemic delivery. - No active targeting ligand; delivery relies on phagocytosis by APCs. - Plasmid release was slow and incomplete, especially for high-molecular-mass PLGA, which could limit gene expression. - Sonication increased the relaxed-to-supercoiled DNA ratio; supercoiled DNA is more efficient for gene expression, so encapsulation conditions require further optimization. - No zeta potential, colloidal stability, or in vivo nuclease-protection data were reported.

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Encapsulation of plasmid DNA in biodegradable poly(D,L-lactic-co-glycolic acid) microspheres as a novel approach for immunogene delivery

Summary

Keywords

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