Chitosan microspheres as delivery vesicle for effective mucosal immunity to Brucella abortus malate dehydrogenase (MDH) = Chitosan microspheres as delivery vesicle for effective mucosal immunity to Brucella abortus malate dehydrogenase (MDH)

다국어 초록 (Multilingual Abstract)

Introduction: Brucellosis is a world widespread zoonotic disease that is transmitted from domestic animals to humans, effective and safe vaccine development have been required. In previous study, stimulation of recombinant proteins in THP-1 cells revealed malate dehydrogenase (MDH) as a high pro-inflammatory cytokine inducer. Also, it has been reported to be immunogenic and to be related to the bacterial pathogenesis. Brucellosis can be transmitted via aerosol exposure; hence the induction of protective immunity at respiratory mucosal surfaces is usually an expected attribute in the field of development of new vaccines. Therefore, this study produced delivery vesicle with immunostimulatory activities using chitosan, which is known to enhance mucosal absorption for effective B.abortus MDH delivery to the respiratory tract.
Materials and Methods: After protein expression and purification, size and specificity was confirmed by SDS-PAGE and Western blotting with an anti-His antibody. Chitosan microspheres (CMs) were prepared based on the ionic gelation of chitosan with sodium tripolyphosphate anions. Prepared CMs were dispersed in 1ml of protein solution (4mg/ml, pH 7.4 PBS) and loading efficiency was measured by quantifying the unloaded protein in the supernatant in each time. Transmissible electron microscope (TEM) and dynamic light scattering spectrophotometer (DLS) was using to characterize morphology and size distribution.
Results: SDS-PAGE profiles indicated purified TF and MDH was 52kDa and 85.71kDa sized, respectively. The recombinant proteins expressed with pCold TF vector were confirmed by western blot using anti-histidine antibody. The loading efficiency of TF and MDH into CMs was 61.23±3.58 and 50.97±0.82 %. TEM results showing that morphologies of CMs were spherical shapes. Average microsphere sizes of CMs, TF-loaded CMs, and MDH-loaded PCMs measured by DLS were 0.32±0.09, 1.87±0.46 and 0.66±0.16Om, respectively, with the increased particle sizes of CMs after the loading of TF and MDH. Release of TF and MDH from loaded CMs was released at 27% and 39% up to 80 hours respectively.
Conclusions: Chitosan microspheres were produced and characterized as delivery carriers for effective mucosal immunity. Immune stimulatory activities will be analyzed in THP-1 cells and mice intranasally immunized with the CMs in further studies.
Acknowledgements: This work was supported by KHIDI (No. HI16C2130), the BK21 PLUS program and the RIVS, Seoul Nat’l University, Republic of Korea.