Development of chitosan-based chitosan nanoparticles for intranasal vaccination and dissolving microneedles for intradermal vaccination of Japanese encephalitis

Abstract

In this study, chitosan-based nanoparticles (CS-NPs) for intranasal (IN) immunization and chitosan-based MNs patch (CS-MNs patch) for intradermal (ID) immunization, non-invasive alternative approaches for Japanese encephalitis (JE) vaccination were developed to improve the immune responses of the live attenuated Japanese encephalitis-chimeric virus vaccine (JE-CV), a commercial JE vaccine, compared with traditional subcutaneous (SC) immunization. This work, therefore, demonstrates the formulations, characterization, and efficacies of using JE-CV-loaded CS-NPs for IN immunization and JE-CV-loaded sCS-MNs patches for ID immunization. For IN immunization study, positive charges CS-NPs at the mass ratio of vaccine antigen: NPs of 6:1 with an incubation time of 1 h at 4°C were successfully developed with high loading efficiency (LE) >85% and loading capacity (LC) >1.2 × 104 PFU/mg. FITC-BSA-loaded NPs exhibited superior mucoadhesive and uptake properties on ex vivo nasal swine mucosa, compared to antigen alone without NPs. Mice immunized with JE-CV at 1 × 104 PFU (full dose vaccine) via IN immunization stimulated systemic and mucosal immune responses in mice, while SC immunization only stimulated systemic immune responses but lack of mucosal immune responses. IN immunization elicited PRNT50 at seroprotection level (PRNT50 titers ≥ 10, as recommended by WHO) similar to SC immunization with JE-CV solution. In the case of IFN-γ, a major cell-mediated immune response for preventing flavivirus amplification, the results revealed that IN immunization of JE-CV in mice with CS-NPs produced more IFN-γ than that of the JE-CV solution. Moreover, IN immunization did not induce adverse events when observed body weight change in mice. In the case of ID immunization, the blended polymer of NaHCO3:CS:polyvinyl alcohol (PVA) (0.25:1:4) was used for the CS-MNs patch fabrication and successfully developed with an elegant appearance, able to resist sufficient compression forces with good ability for skin insertion. In addition, JE-CV was loaded into a CS-MNs patch to obtain the optimal mass ratio of 1:4. The JE-CV-loaded CS-MNs patch was then successfully prepared using a cellulose membrane as an MN base to achieve a separating CS-based MNs patch (sCS-MNs patch). The morphology, mechanical strength, and penetration properties of the JE-CV loaded sCS-MNs patch were evaluated and showed desirable results with the detachable property. The percentages of LE and LC of the JE-CV-loaded sCS-MNs patch were approximately 79% and 1.2 × 104 PFU/g, respectively. For safety, after applying MNs, the skin appeared the microholes and naturally resealed and completely recovered at 48 h without skin infection after the MNs patches removal. ID immunization in mice with a half-dose of JE-CV-loaded sCS-MNs patch (5 × 103 PFU/mouse) effectively elicited PRNT50 and cell-mediated immune responses (CMIR) similar to full dose vaccination via SC. Therefore, immunization with JE-CV-loaded CS-NPs via IN and JE-CV-loaded sCS-MNs patch via ID could be promising approaches for effective immunization.

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