| Objective: The aim of this research is to prepare antiFcεRIα Fab conjugated celastrol-loaded micelles(antiFcεRIα Fab-NMs-celastrol), analyze the targeting, cytotoxicity and apoptosis-inducing effect of micelles on mast cells, and explore the in vivo biodistribution and anti-allergic effect in mouse models of allergic asthma and passive cutaneous reaction.Methods:(1)Celastrol-loaded(PEO-block-PPO-block-PEO, Pluronic) polymeric nanomicelles(celastrol-NMs) were prepared using thin-film hydration method. The antiFcεRIα Fab Fragment was then conjugated to carboxyl groups on drug-loaded micelles via EDC amidation reaction. The characteristics of antiFcεRIα Fab-conjugated celastrol-loded micelles including particle size, morphology, drug loading percentage and encapsulation efficiency were characterized.(2) The in vitro effect of antiFcεRIα Fab-NMs-celastrol was investigated on human basophil cell line KU812 cells which expressed FcεRIα and immature human mast cell line HMC-1 cells which did not express FcεRIα on its surface. In the cellular uptake assay, antiFcεRIα Fab-NMs-coumarin 6 was prepared and the targeting effect of micelles was detected after cells incubated with antiFcεRIα Fab-NMs-coumarin 6 using flow cytometry and confocal microscopy. To explore the cytotoxicity and apoptotic effect of micelles to mast cells, the cell proliferation inhibitory rate, apoptotic cells number, and expressing of apoptotic molecular were detected using CCK-8 assay, Annexin V-FITC/PI staining and western blot respectively.(3) Furthermore, antiFcεRIα Fab-conjugated Di R-loaded micelles were synthesized and in vivo distribution of micelles was explored in mouse model of allergic asthma. To explore the anti-allergic effect in mouse model of allergic asthma, mice was executed after treatment with micelles. The OVA-s Ig E in serum, histamine and Th1/Th2 cytokines in BALF were detected using ELISA and Luminex, respectively. Then WBC and eosnophils were also counted. Also, the pathological change in lung tissue was observed. In addition, the anti-allergic effect was investigated passive cutaneous reaction, and the extravasation of Evans blue was detected.Results:(1)The antiFcεRIα Fab-conjugated celastrol-loaded micelles were successfully synthesized, which revealed uniform particle size(93.43 ± 12.93 nm) with high loading percentage(22.8±1.0% w/w) and high encapsulation efficiency(98.9±3.3% w/w). The image of micelles showed oval and rod like.(2) The antiFcεRIα Fab-conjugated micelles demonstrated enhanced cellular uptake by KU812 cells than non-conjugated micelles. In vitro cytotoxicity and apoptosis assay showed that the cell growth inhibition ratio, apoptotic cell number, caspase 3 activity and cleaved PARP expression was much higher in antiFcεRIα Fab-NMs-celastrol-treated group than that for celastroland celastrol-NMs-treated group.(3)In mouse model of allergic asthma, treatment with antiFcεRIα Fab-conjugated micelles increased lung accumulation of micelles, and significantly reduced OVA-s Ig E, histamine and Th2 cytokines(IL-4, IL-5, IL-13 and TNF-α) levels, eosinophils infiltration and mucus production. In addition, in mouse model of passive cutaneous anaphylaxis, antiFcεRIα Fab-conjugated celastrol-loaded micelles treatment significantly decreased extravasated evans blue in the ear. The in vivo anti-allergic effect of antiFcεRIα Fab-NMs-celastrol was stronger than that of celastrol and celastrol-NMs.Conclusion: These results indicate that antiFcεRIα Fab-conjugated celastrol-loaded micelles were successfully synthesized with good characteristics. The micelles can target and selectively kill mast cells and basophils which express FcεRIα, and showed inhibitory effect to allergic inflammation in mouse model of allergic asthma and passive cutaneous reaction. Thus the micelles may be efficient reagents for the treatment of allergic disorders and mast cell related diseases. |
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