A PLGA Nanoparticle-based Vaccine For Clostridium Perfringens Epsilon Toxin Dissolving Microneedle Vaccine

The toxicity of Clostridium perfringens epsilon toxin（ETX）is only surpassed by botulinum toxin and tetanus toxin,and can cause enterotoxemia,necrotic enteritis,and neurological diseases in mammals.Treatment with drugs alone is difficult to achieve efficacy after ETX poisoning,and vaccination is an important method for preventing such diseases.This study aimed to develop a new type of microneedle patch based on PLGA nanoparticles as a vaccine adjuvant for delivering the r ETX^Y196E-C vaccine to enhance the immune response.The specific research contents are as follows:（1）Preparation of PLGA nanoparticles.Poly（lactic-co-glycolic acid）（PLGA）,a FDA-approved safe copolymer,was successfully used to prepare nanoparticles（NPs）that were modified with a cationic surfactant,dimethyldioctadecylammonium bromide（DDAB）,to allow antigen adsorption.The morphology,particle size,zeta potential,cytotoxicity,and amount of DDAB added were investigated.The results showed that PLGA NPs with smooth surfaces,sizes of 100∽200 nm,good dispersion,positive surface charge,and good safety were successfully prepared and able to efficiently adsorb the detoxified mutant protein r ETX^Y196E-C.（2）Preparation of PLGA-DDAB dissolving microneedle（d MN）vaccine.Gelatin and sucrose were chosen as the MN matrix to prepare d MN vaccines.The morphology,mechanical properties,mouse skin penetration,and in vivo release time were investigated.The results showed that the PLGA-DDAB d MN vaccine had intact patch tips,good mechanical properties,and the PLGA NPs carrying the antigen were concentrated at the tips for precise delivery.Optical coherence tomography（OCT）showed that the PLGA-DDAB d MN could be smoothly inserted into pig skin with thumb pressure and completely dissolved in the skin within 10∽15 minutes.In vivo release time studies in mice have shown that PLGA is able to form a short-term reservoir of antigen at the injection site with some slow release.（3）The immunogenicity and protective effects of PLGA-DDABd MN vaccine were evaluated.PLGA NPs enhanced both cellular and humoral immune responses,with a specific Ig G antibody titer of 10⁵after triple immunization,increased Ig G2a/Ig G1 ratio,and effectively activated the expression of cytokines such as IL-2,IL-4,IL-6 and interferonγin T cells.MN enhanced the immune response and produced more neutralizing antibodies.In vivo neutralization experiments showed that MN immunization had a better protective effect compared to subcutaneous immunization,and in vitro neutralization experiments showed that MN immunization produced significantly better protection for mice with MDCK cells than the subcutaneous immunization group.In addition,both immunisation modalities produced 100%protection when used in combination with PLGA NPs.In summary,our d MN vaccine patch based on PLGA NPs has the advantages of MN vaccine patches such as quick and easy vaccination,painless and good thermal stability,as well as the features of PLGA NPs to enhance the immune effect,and the preparation method is simple,safe and effective,and easy for subsequent mass production.It provides an effective means to prevent ETX poisoning,reduce livestock losses due to ETX poisoning,and protect against biosecurity threats posed by ETX as a bioterror agent,and is expected to be a new vaccine delivery system extended to other vaccines or drugs.