Redispersible nanoalum-adjuvanted hepatitis B vaccine microneedles with enhanced immunogenicity

Nanoalum has been widely investigated as an advanced aluminum-based adjuvant; however, its performance in solid vaccine formulations remains poorly understood because administration depends on reconstitution after storage and delivery. Here, a hepatitis B surface antigen (HBsAg) vaccine was formulated with nanoalum, freeze-dried, and delivered using a powder-attached microneedle array patch (P-MAP). Formulations were screened based on redispersion in PBS after lyophilization to identify a lead composition (HBsAg: Alhydrogel: PAA: Trehalose = 1:5:5:50, w/w/w/w; F12), and the effects of preparation pH (7.4 vs. 5.6) were evaluated. The optimized formulation maintained a nanoscale dispersed state after PBS reconstitution (< 250 nm), whereas PEG-substituted and PAA-free formulations showed severe aggregation (> 2 μm). When administered as liquid intradermal injections, preparation pH produced minimal differences in immune responses. In contrast, clear divergence emerged following freeze-drying and P-MAP delivery. The pH 5.6 formulation (G9) generated the highest IFN-γ ELISpot response among all P-MAP groups while maintaining strong HBsAg-specific IgG responses and an increased IgG2a/IgG1 tendency. The PAA-free formulation (G10), which exhibited poor redispersion after reconstitution, produced reduced and more variable antibody responses. Notably, formulations displaying nanoscale redispersion consistently outperformed aggregated formulations in both humoral and cellular immune responses. These findings demonstrate that post-lyophilization redispersion is a critical quality attribute for nanoalum-containing solid vaccine formulations and that formulation conditions governing redispersion can substantially influence immune outcomes following P-MAP delivery.