Controlled release of PEI/DNA complexes from mannose-bearing chitosan microspheres as a potent delivery system to enhance immune response to HBV DNA vaccine

Purpose: DNA vaccines can induce antibody and CTL responses, but clinical translation is limited by weak potency and the need for high or repeated doses. The study addresses this by targeting DNA to antigen-presenting cells (APCs) and improving endosomal/lysosomal release, using mannose-bearing chitosan microspheres loaded with PEI/DNA complexes for an HBV DNA vaccine.

Hypothesis: If PEI/DNA complexes are entrapped in mannose-bearing chitosan microspheres, then after intramuscular injection the particles will target mannose receptor–expressing APCs such as dendritic cells and macrophages, release intact PEI/DNA in a controlled manner, enhance gene expression, and induce stronger HBV-specific humoral and cellular immune responses than naked DNA or unmodified chitosan microspheres.

Aims: Synthesize and characterize mannose-bearing chitosan (m-chitosan) with improved water solubility and lysozyme-mediated biodegradability. - Prepare m-chitosan microspheres entrapping PEI/DNA complexes and characterize size, zeta potential, encapsulation efficiency, and in vitro release. - Evaluate in vitro transfection and cytotoxicity in macrophage-like cells, and in vivo reporter gene expression after intramuscular injection. - Assess HBV-specific humoral and cellular immune responses after immunization with the microsphere-formulated DNA vaccine.

Delivery system: Platform: Mannose-bearing chitosan microspheres formed by complex coacervation, encapsulating ternary PEI/DNA complexes. - Polymer chemistry: Chitosan modified with mannose/mannobiose via reductive amination using NaBH₃CN, attaching mannose to primary amines. - Payload: Plasmid DNA encoding HBV S antigen (HBV S/V1012) for immunization; pCMV Luc plasmid for reporter gene expression. - Carrier components: PEI 25 kDa condensed DNA at N/P = 6; microspheres prepared with sodium sulfate as desolvating agent. - Targeting ligand: Mannose groups for recognition by mannose receptors on immature dendritic cells and macrophages. - Route: Intramuscular injection.

Approach: In vitro: RAW264.7 murine macrophage/monocyte cells for transfection and uptake studies; COS-7 cells for MTT cytotoxicity assay. - In vivo gene expression: Female BALB/c mice (n = 6/group) injected i.m. with naked pCMV Luc DNA, u-chitosan microspheres, or m-chitosan microspheres containing 50 µg pCMV Luc DNA; muscles harvested at days 1, 7, and 14. - Immunization: Female BALB/c mice (n = 8/group) immunized at weeks 0, 2, 4, and 6 with m-chitosan microspheres, control DNA, or PBS at different doses. - Immune readouts: Serum anti-HBsAg IgG by ELISA; CTL activity by LDH-release assay; IFN-γ-producing splenocytes by ELISPOT.

Key methods: Dynamic light scattering (DLS) for size and zeta potential. - TEM and AFM for microsphere morphology. - UV absorbance at 260 nm for DNA loading, encapsulation efficiency, and in vitro release. - Luciferase reporter assay for in vitro and in vivo gene expression. - MTT assay for cytotoxicity. - ELISA for HBsAg-specific serum IgG. - LDH-release assay for HBsAg-specific CTL activity. - IFN-γ ELISPOT for antigen-specific T-cell responses.

Key results: Microsphere characterization: m-chitosan microspheres were 326.3 ± 31.9 nm, with 89 ± 2.6% DNA encapsulation efficiency and zeta potential 10.19 ± 3.23 mV. u-chitosan microspheres were 415.2 ± 44.2 nm, 94 ± 5.4% encapsulation, and 19.44 ± 2.78 mV. - Release: No initial burst; by day 7, 22.4% of PEI/DNA was released from m-chitosan microspheres versus 5.1% from u-chitosan microspheres. m-Chitosan degraded faster in lysozyme. - In vitro transfection/toxicity: m-Chitosan microspheres gave about one order of magnitude higher luciferase expression than u-chitosan microspheres, though 150–300-fold lower than PEI/DNA alone. m- and u-chitosan microspheres showed no cytotoxicity in RAW264.7 cells; PEI alone reduced viability to 30–50% at 5–25 µg. - In vivo gene expression: m-Chitosan microspheres produced higher luciferase expression than u-chitosan microspheres and naked DNA at days 7 and 14. u-Chitosan was 1.5–2.5-fold lower than m-chitosan. - Humoral response: m-Chitosan microspheres were effective at 1 µg DNA, whereas naked DNA required 10 µg, indicating >10-fold increase in potency; antibody responses were also accelerated by about 2 weeks. - Cellular response: m-Chitosan microspheres induced CTL responses at 100 ng DNA versus 100 µg for naked DNA, indicating 100–1000-fold potency increase. IFN-γ ELISPOT showed 1 µg m-chitosan microspheres matched 100 µg naked DNA, about 100-fold increase.

Interpretation: The authors attribute the enhanced immunogenicity to controlled DNA release, endosomal escape via PEI, and mannose receptor–mediated targeting of APCs. They conclude that this approach may reduce the DNA dose required for vaccination, improving safety, and that the technology holds promise for human DNA vaccine delivery, with supportive subsequent studies in guinea pigs and rhesus macaques.

Limitations: Efficacy and immunogenicity were demonstrated in mice only; no HBV challenge or therapeutic chronic-infection model was used. - In vivo mannose receptor targeting is inferred from in vitro mannose competition and APC biology, not directly blocked or visualized in vivo. - Follow-up was relatively short (immune responses up to ~7 weeks; gene expression up to 14 days). - Large-animal and human validation are not included in this paper; the authors only mention subsequent studies. - PEI is used in the formulation, and although encapsulated microspheres showed low cytotoxicity, long-term safety of the full system is not established here.