Prophylactic anti-tumor effects in a B cell lymphoma model with DNA vaccines delivered on polyethylenimine (PEI) functionalized PLGA microparticles

Purpose: DNA vaccines are limited by inefficient delivery to antigen-presenting cells (APCs) and lack of adjuvant effect, especially for weakly immunogenic self/tumor antigens. The study addresses this by using cationic PEI-functionalized PLGA microparticles to enhance APC activation and protective anti-tumor immunity against B cell lymphoma.

Hypothesis: If plasmid DNA encoding a chemokine-fused idiotypic tumor antigen is adsorbed onto PEI-conjugated PLGA microparticles, then the formulation will activate APCs and induce protective anti-tumor immunity in a murine B cell lymphoma model. Branched PEI may outperform linear PEI, and intramuscular delivery may provide better protection than intradermal delivery.

Aims: Synthesize and characterize branched and linear PEI-conjugated PLGA microparticles. - Compare buffering ability, DNA loading/release, cytotoxicity, and APC activation in vitro. - Evaluate prophylactic anti-tumor efficacy against A20 B cell lymphoma after intradermal or intramuscular immunization. - Compare microparticle delivery with gene gun-mediated DNA administration.

Delivery system: Platform: Cationic PLGA microparticles surface-functionalized with PEI via carbodiimide chemistry. - Polymers: PLGA 50:50 (RG502H, RG503H); branched PEI 70 kDa and 25 kDa; linear PEI 25 kDa. - Payload: Plasmid DNA MCP3-sFv20 encoding B cell idiotype antigen fused to MCP-3 chemokine. - Loading: DNA adsorption at pH 6.0; loading ~7.49 µg/mg for branched PEI70k, 6.04 µg/mg for branched PEI25k, and 4.80 µg/mg for linear PEI25k. - Route: Intradermal or intramuscular injection; gene gun was used as positive control.

Approach: In vitro: RAW264.7 murine macrophage cells for cytotoxicity (MTT) and surface marker analysis (CD80, MHC class II, F4/80). - In vivo: Female Balb/c mice, 8–10 weeks old, n = 10/group. Immunized three times at 2-week intervals with 25 µg pDNA on microparticles. Gene gun group received ~2 µg pDNA. Controls included saline, irrelevant plasmid, and blank microparticles. - Tumor challenge: A20 B cell lymphoma cells, 5 × 10⁵ cells (2.5× minimal lethal dose), injected intraperitoneally 2 weeks after last immunization. Survival followed for 80 days.

Key methods: Zeta potential, laser diffraction sizing, and SEM for particle characterization. - Acid-base titration for buffering capacity. - Mass balance and Picogreen assay for DNA loading and release. - MTT assay for cytotoxicity. - Flow cytometry for APC activation markers. - Kaplan–Meier survival and log-rank test for anti-tumor efficacy.

Key results: Particle properties: Zeta potentials: unmodified PLGA −10.4 mV; branched PEI70k +37.69 mV; branched PEI25k +37.74 mV; linear PEI25k +44.9 mV. Most particles were <10 µm. - DNA release: Linear PEI particles released ~60% of DNA within 3 weeks, versus ~28% for branched PEI25k and ~20% for branched PEI70k. - Buffering: Branched PEI70k imparted strong buffering; linear PEI did not. - Toxicity: PEI-PLGA particles showed 90–100% cell viability in RAW264.7 cells, comparable to unmodified PLGA. - APC activation: pDNA-loaded PEI-PLGA microparticles upregulated CD80 and MHC class II in RAW264.7 cells, higher than unmodified PLGA but lower than LPS. - Tumor protection: Branched PEI70k microparticles significantly improved survival versus controls and outperformed linear PEI particles. Intramuscular delivery gave better long-term survival than intradermal and performed similarly to gene gun immunization (p < 0.01 vs saline/irrelevant plasmid).

Interpretation: The authors conclude that branched PEI-conjugated PLGA microparticles are reproducible, noncytotoxic, have intrinsic adjuvant effects, and provide significant protective anti-tumor immunity in a B cell lymphoma model. They propose this surface-functionalized microparticle platform as a promising strategy for improving weakly immunogenic self-antigen DNA cancer vaccines.

Limitations: In vitro APC activation was assessed in RAW264.7 macrophage-like cells, not primary dendritic cells. - Mechanism of enhanced immunity was not fully elucidated; no gene expression analysis at injection site or draining lymph nodes. - Route comparison was not dose-matched to gene gun, which used a much lower DNA dose. - Prophylactic model only; no therapeutic treatment of established tumors. - No large-animal or human data. - In vivo toxicity, biodistribution, and long-term safety of PEI-PLGA particles were not extensively evaluated. - Survival follow-up was limited to 80 days.