Engineering a “PEG-g-PEI/DNA nanoparticle-in-PLGA microsphere” hybrid controlled release system to enhance immunogenicity of DNA vaccine

Purpose: DNA vaccines require high doses because naked DNA is poorly delivered to antigen-presenting cells (APCs), and conventional PLGA microsphere encapsulation by W/O/W emulsion can damage DNA, lower supercoiled DNA content, and give low encapsulation efficiency. A controlled-release system is needed that protects DNA during formulation and enhances immunogenicity at lower doses.

Hypothesis: If PEG-g-PEI/DNA polyplexes are lyophilized/micronized and encapsulated into PLGA microspheres via a solid-in-oil-in-water (S/O/W) emulsion, then DNA will remain structurally and functionally stable, encapsulation efficiency will improve, release will be pH-sensitive and sustained, and the resulting nanoparticle-in-microsphere (NIM) system will passively target phagocytic APCs and enhance HIV DNA vaccine humoral and cellular immune responses at low doses.

Aims: Synthesize and characterize PEG-g-PEI copolymer and PEG-g-PEI/DNA polyplexes. - Prepare NIMs by S/O/W encapsulation and characterize size, zeta potential, encapsulation efficiency, and release kinetics. - Evaluate DNA stability, in vitro transfection, cytotoxicity, and macrophage uptake. - Assess in vivo gene expression and HIV-specific humoral and cellular immune responses after intramuscular immunization with a DNA prime/MVA boost regimen.

Delivery system: Platform: “Nanoparticle-in-microsphere” (NIM) hybrid controlled-release system. - Inner core: PEG-g-PEI/DNA polyplexes. PEG-g-PEI was synthesized by linking branched PEI (25 kDa) and mPEG (5 kDa) via hexamethylene diisocyanate to form urethane bonds; average ~6.2 PEG blocks per PEI. - Payload: HIV-1 D-GPEi plasmid (13,113 bp) encoding Gag, Pol, and Env; pCMV Luc plasmid used for reporter gene studies. - Outer shell: PLGA (RG505) microspheres prepared by S/O/W emulsion with PVA as stabilizer. - Targeting strategy: Passive targeting to phagocytic APCs by size exclusion; mean NIM size ~1.59 µm. - Key formulation feature: Lyophilized PEG-g-PEI/DNA polyplexes were dispersed in organic solvent, avoiding the aqueous/organic interface that damages DNA in W/O/W methods.

Approach: In vitro cell lines: COS-7 cells for transfection and cytotoxicity; RAW264.7 murine macrophages for phagocytosis and cytotoxicity; L929 fibroblasts as non-phagocytic control. - In vivo gene expression: Female BALB/c mice (n = 5/group) injected intramuscularly with saline, 50 µg naked pCMV Luc DNA, PEG-g-PEI/DNA polyplex, or NIMs containing 50 µg pCMV Luc DNA. Muscle harvested at days 1, 7, and 14. - Immunization: Female BALB/c mice (n = 5 per group) vaccinated intramuscularly with NIMs containing 10, 1, or 0.1 µg HIV DNA; naked DNA at 100, 10, 1, or 0.1 µg; or saline. Primed at weeks 0 and 4, boosted at week 8 with recombinant MVA (1 × 10⁷ pfu). Blood collected at weeks 2, 6, and 9; CTL assay 5 days after boost.

Key methods: DLS for polyplex and NIM size/zeta potential. - SEM and TEM for microsphere morphology and core-shell structure. - UV absorbance at 260 nm and Hoechst assay for DNA loading/encapsulation efficiency. - Circular dichroism and agarose gel electrophoresis for DNA conformational and structural stability. - Luciferase reporter assay for in vitro and in vivo gene expression. - MTT assay for cytotoxicity. - Fluorescence microscopy for macrophage uptake. - Western blotting for anti-HIV p24 antibody responses. - LDH-release assay for CTL activity. - IFN-γ ELISPOT for HIV-1 p7g-specific CD8⁺ T cells.

Key results: Polyplex characterization: PEG-g-PEI/DNA polyplexes at N/P = 20 had a size of 74.1 ± 21.2 nm and zeta potential of 11.47 ± 0.78 mV; transfection peaked at N/P ≥ 20, and cell viability was >90% at N/P ≤ 20. - NIM encapsulation: S/O/W NIMs achieved 87.5 ± 4.9% DNA encapsulation efficiency, compared with 32.5 ± 4.8% for W/O/W NIMs. Mean NIM size was 1590 ± 914 nm and zeta potential was −19.24 ± 5.24 mV. - Release: Initial burst was <15%; at pH 7.4 release was sustained near zero-order, while at pH 4.5 more than 70% of DNA was released within 2 days, indicating pH-sensitive lysosomal release. - DNA stability and function: DNA released from NIMs retained supercoiled structure and showed luciferase expression similar to freshly prepared polyplexes. - Uptake and toxicity: NIMs were internalized by RAW264.7 macrophages but not L929 fibroblasts, and showed no significant cytotoxicity against RAW264.7 cells. - In vivo gene expression: NIMs gave higher luciferase expression than naked DNA at all timepoints and a longer expression profile than PEG-g-PEI/DNA polyplexes at day 14. - Immune response: NIM formulation was effective at 1 µg DNA, whereas naked DNA required 100 µg, indicating ~100-fold dose reduction. At 10 µg DNA, NIMs produced a 15-fold increase in anti-p24 antibody response versus 100 µg naked DNA. CTL lysis at E/T 100:1 was 10.1% for 100 µg naked DNA, versus 49.6% for 10 µg NIMs and 14.2% for 1 µg NIMs. IFN-γ ELISPOT responses from 1 µg NIMs were comparable to 100 µg naked DNA (~100-fold increase).

Interpretation: The authors claim the NIM system integrates the advantages of nanoscale polyplexes and controlled-release microspheres. The S/O/W method protects DNA from shear and aqueous-organic interface damage, improves encapsulation, passively targets APCs by size, and accelerates intracellular DNA release. This enhanced immunogenicity at low DNA doses may improve DNA vaccine safety and reduce cost, supporting further clinical evaluation.

Limitations: No viral challenge or protection study was performed; only immune correlates were measured. - Efficacy was shown in mice only; large-animal data are not included in this paper, although the authors mention subsequent guinea pig and rhesus macaque studies. - In vivo APC targeting is inferred from in vitro phagocytosis and size exclusion, not directly demonstrated by receptor blocking or in vivo imaging. - Follow-up was relatively short (up to week 9 for antibodies; day 14 for gene expression). - S/O/W NIMs did not show significantly higher or longer gene expression than W/O/W NIMs in vivo, despite improved encapsulation. - Long-term safety, repeated-dose toxicity, and PLGA degradation effects were not extensively evaluated.