CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES >
Effect of albendazole-loaded vesicles on the vitality of protoscoleces of Echinococcus granulosus
Received date: 2021-11-22
Revised date: 2022-02-09
Online published: 2022-07-06
Supported by
National Natural Science Foundation of China(81760570);National Natural Science Foundation of China(81760371);XPCC Special Fund for Science and Technology Development(2018CB017);XPCC Special Fund for Science and Technology Development(2019AB031)
Objective To investigate the effect of albendazole-loaded extracellular vesicles (drug-loaded vesicles) on the vitality of Echinococcus granulosus protoscolex in vitro. Methods The mouse hepatoma cells H22 culture suspension was assigned into low, medium and high concentration groups, to each of which albendazole was added at final concentrations of 200, 400 and 600 μmol/L, respectively. UV irradiation (UVB, 300 J/m2) was performed for 1 hour, followed by 18-20 hours of incubation. The drug-loaded vesicles were produced by ultra-high speed differential centrifugation. Vesicles without albendazole were prepared using the same method. The shape of the drug-loaded vesicles was observed with a transmission electron microscope, the particle diameter was measured with a laser particle sizer, and the optimal dose determined by liquid chromatography. The E. granulosus protoscolex was divided into 4 groups and was cultured in vitro with pure medium (blank control group), no-loading vesicles (no-loading vesicle group), drug-loaded vesicles (drug-loaded vesicle group) and albendazole (albendazole positive control group), respectively. The final concentration of albendazole in the drug-loaded vesicle group and albendazole positive control group was 13 μmol/L. The protoscolex was stained with Eosin to observe the morphology and activity at 1, 3, 5 and 7 days post-treatment. The survival rate of protoscolex was calculated. The caspase-3 expression level of E. granulosus protoscolex was detected by using caspase-3 detection kit 3, 5, 7 days post-treatment to identify the apoptosis of protoscolex. One-way ANOVA was used for comparisons between groups. Results The transmission electron microscopy showed variations in vesicle of different size with double membrane structure. The particle diameter sizer showed that the particle diameter was 200-400 nm. The liquid chromatography showed that the effective drug concentrations in low, medium and high concentration groups were (36.3 ± 2.85), (79.0 ± 2.30), (99.5 ± 4.20) μmol/L. The increment of albendazole concentration from the low concentration group to the medium concentration group was higher than that from the medium concentration group to the high concentration group. The difference between the groups was statistically significant (F = 21.43, P < 0.05). The optimal concentration of the final drug-loaded vesicles was 400 μmol/L. Eosin staining showed that on day 7 post-treatment, the protoscolex remained active, and the morphology and structure of the protoscolex were clear in the blank control group and the no-loading vesicle group. In the albendazole positive control group, the protoscolex was inactive, and unsharpness and its volume decreased. In the drug-loaded vesicle group, the structure of protoscolex was disordered, shrunken and partially died. On day 7 post-treatment, the survival rate of the protoscolex in the blank control group, no-loading vesicle group, albendazole positive control group and drug-loaded vesicle group were (91.2 ± 1.07)%, (88.9 ± 1.43)%, (64.5 ± 1.19)% and (45.3 ± 0.98)%. The survival rate curves in the drug-loaded vesicle group were lower than that of the the other groups (F = 1 021.17, P < 0.05). The apoptosis of the protoscolex results showed, on day 3 post-treatment, the caspase-3 of E. granulosus protoscolex in blank control group, no-loading vesicle group, albendazole positive control group and the drug-loaded group were (41.80 ± 3.02), (40.26 ± 2.78), (55.20 ± 3.09) and (68.15 ± 3.60) μmol/L, respectively; on day 5 post-treatment, were (43.18 ± 2.43), (43.02 ± 3.13), (52.17 ± 4.13) and (62.74 ± 3.16) μmol/L, respectively; while on day 7 post-treatment, were (52.93 ± 1.46), (53.08 ± 1.60), (57.32 ± 1.81) and (61.99 ± 1.14) μmol/L, respectively. The differences were significant between the groups on 3, 5, 7 days post-treatment (F = 51.97, 24.53, 23.82; P < 0.05) and the caspase-3 in the drug-loaded vesicle group were all higher than that in the albendazole positive control group (F = 22.36, 12.43, 14.33; P < 0.05). Conclusion Albendazole-loaded vesicle could improve the solubility of albendazole, enhancing the killing effect on E. granulosus protoscolex.
Key words： Echinococcus granulosus; Albendazole; Drug-loaded vesicle
QIAO Shi-yuan , ZHOU Xue , LIU Cheng-hao , JIANG Hui-jiao , BU Yuan-yuan , CHEN Xue-ling , WU Xiang-wei . Effect of albendazole-loaded vesicles on the vitality of protoscoleces of Echinococcus granulosus[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022 , 40(3) : 324 -329 . DOI: 10.12140/j.issn.1000-7423.2022.03.007
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