Effect of albendazole-loaded vesicles on the vitality of protoscoleces of Echinococcus granulosus

doi:10.12140/j.issn.1000-7423.2022.03.007

Abstract

Abstract:

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

CLC Number:

R383.33

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.

Figures/Tables 6

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References 28

[1]	Zhang MY,, Wu WP,, Guan YY, et al. Analysis on disease burden of hydatid disease in China[J]. Chin J Parasitol Parasit Dis, 2018, 36(1): 15-19, 25. (in Chinese)
	( 张梦媛,, 伍卫平,, 官亚宜, 等. 我国棘球蚴病疾病负担分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(1): 15-19, 25.)
[2]	Wang TP,, Cao ZG. Current status of echinococcosis control in China and the existing challenges[J]. Chin J Parasitol Parasit Dis, 2018, 36(3): 291-296. (in Chinese)
	( 汪天平,, 操治国. 中国棘球蚴病防控进展及其存在的问题[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(3): 291-296.)
[3]	Wei SH,, Wu WP,, Han S, et al. Analysis of the results of echinococcosis surveillance in China from 2016 to 2017[J]. J Pathog Biol, 2020, 15(8): 924-928. (in Chinese)
	( 魏思慧,, 伍卫平,, 韩帅, 等. 2016—2017年全国棘球蚴病监测结果分析[J]. 中国病原生物学杂志, 2020, 15(8): 924-928.)
[4]	Shang JY,, Zhang GJ,, He W, et al. Taxonomy and molecular epidemiology of Echinococcus granulosus complex causing cystic echinococcosis[J]. Chin J Parasitol Parasit Dis, 2018, 36(2): 166-173, 177. (in Chinese)
	( 尚婧晔,, 张光葭,, 何伟, 等. 细粒棘球蚴病病原分类学与分子流行病学研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(2): 166-173, 177.)
[5]	Chen J,, Wen H. Progress in diagnosis and treatment of hepatic echinococcosis[J]. J Southeast Univ Med Sci Ed, 2018, 37(5): 929-934. (in Chinese)
	陈骏,, 温浩. 肝棘球蚴病的诊断与治疗进展[J]. 东南大学学报(医学版), 2018, 37(5): 929-934.)
[6]	Zhao SY,, Zhu HH,, Wang XQ, et al. Present situation and progress of comprehensive treatments for hepatic alveolar echinococcosis[J]. Chin J Schisto Control, 2019, 31(6): 676-678. (in Chinese)
	( 赵顺云,, 朱海宏,, 王向前, 等. 肝多房棘球蚴病的综合治疗现状和进展[J]. 中国血吸虫病防治杂志, 2019, 31(6): 676-678.)
[7]	Chinese Congress of Hepatobiliary Surgeons. Expert consensus on diagnosis and treatment of hepatic hydatidosis(2015 Edition)[J]. Chin J Digest Surg, 2015, 14(4): 253-264. (in Chinese)
	中国医师协会外科医师分会包虫病外科专业委员会. 肝两型包虫病诊断与治疗专家共识(2015版)[J]. 中华消化外科杂志, 2015, 14(4): 253-264.)
[8]	Sichuan Echinococcosis Diagnosis and Treatment Expert Group. Diagnosis and treatment of liver echinococcosis in Sichuan Province[J]. Chin J Bases Clin Gen Surg, 2017, 24(7): 798-803. (in Chinese)
	( 四川省包虫病诊疗专家组. 四川省肝包虫病诊治规范[J]. 中国普外基础与临床杂志, 2017, 24(7): 798-803.)
[9]	Xiong YH,, Zheng B. The analytical research on patented technology of drugs for echinococcosis prevention and control in China[J]. Chin Health Stand Manag, 2021, 12(9): 5-9. (in Chinese)
	( 熊彦红,, 郑彬. 中国棘球蚴病防治药物专利技术分析研究[J]. 中国卫生标准管理, 2021, 12(9): 5-9.)
[10]	Wang J,, Chen JY. Research progress of extracellular vesicles[J]. Chin J Tissue Eng Res, 2017, 21(4): 621-626. (in Chinese)
	( 王琎,, 陈建英. 细胞外囊泡研究新进展[J]. 中国组织工程研究, 2017, 21(4): 621-626.)
[11]	Zhai JT,, Ma F. Advances in tumor cell-derived chemotherapeutic microparticles research[J]. Chin J Front Med Sci (Electron Version), 2020, 12(3): 27-30. (in Chinese)
	翟婧彤,, 马飞. 肿瘤细胞来源的载药囊泡研究进展[J]. 中国医学前沿杂志(电子版), 2020, 12(3): 27-30.)
[12]	Yuan FM,, Li YM,, Wang ZH. Preserving extracellular vesicles for biomedical applications: consideration of storage stability before and after isolation[J]. Drug Deliv, 2021, 28(1): 1501-1509. doi: 10.1080/10717544.2021.1951896
[13]	Chen Y. Research progress of extracellular vesicles[J]. Chin J Cell Biol, 2019, 41(2): 202-210. (in Chinese)
	( 陈扬. 细胞外囊泡研究进展[J]. 中国细胞生物学学报, 2019, 41(2): 202-210.)
[14]	Meng WR,, He CS,, Hao YY, et al. Prospects and challenges of extracellular vesicle-based drug delivery system: considering cell source[J]. Drug Deliv, 2020, 27(1): 585-598. doi: 10.1080/10717544.2020.1748758
[15]	Vader P,, Mol EA,, Pasterkamp G, et al. Extracellular vesicles for drug delivery[J]. Adv Drug Deliv Rev, 2016, 106(Pt A): 148-156.
[16]	Chen B,, Zhang Y,, Tang K. The invention relates to a storage method for tumor cell vesicle preparation[P]: China, CN109200029A. 2019-01-15. (in Chinese)
	( 陈彬,, 张一,, 唐科. 一种肿瘤细胞囊泡制剂的贮存方法[P]: 中国, CN109200029A. 2019-01-15.)
[17]	Zhao Y,, Zhang P,, Tang WB, et al. Determination of albendazole in albendazole tablets by high performance liquid chromatography[J]. Chin J Vet Drug, 2004, 38(5): 33-34, 37. (in Chinese)
	( 赵英,, 张平,, 唐文标, 等. 高效液相色谱法测定兽用阿苯达唑片的含量[J]. 中国兽药杂志, 2004, 38(5): 33-34, 37.)
[18]	Zu YZ,, Tao DY,, Fang WS, et al. Status of drug treatment and prevention of echinococcosis[J]. J Clin Med Lit, 2017, 4(27): 5344, 5346. (in Chinese)
	( 祖逸峥,, 陶栋义,, 方万胜, 等. 包虫病药物治疗与预防现状[J]. 临床医药文献电子杂志, 2017, 4(27): 5344, 5346.)
[19]	Pugholm LH,, Revenfeld ALS,, Søndergaard EKL, et al. Antibody-based assays for phenotyping of extracellular vesicles[J]. Biomed Res Int, 2015, 2015: 524817.
[20]	Li L,, Piontek K,, Ishida M, et al. Extracellular vesicles carry microRNA-195 to intrahepatic cholangiocarcinoma and improve survival in a rat model[J]. Hepatology, 2017, 65(2): 501-514. doi: 10.1002/hep.28735
[21]	Zakharova L,, Svetlova M,, Fomina AF. T cell exosomes induce cholesterol accumulation in human monocytes via phosphatidy-lserine receptor[J]. J Cell Physiol, 2007, 212(1): 174-181. pmid: 17299798
[22]	Chaput N,, Théry C. Exosomes: immune properties and potential clinical implementations[J]. Semin Immunopathol, 2011, 33(5): 419-440. doi: 10.1007/s00281-010-0233-9
[23]	Ma JW,, Zhang Y,, Tang K, et al. Reversing drug resistance of soft tumor-repopulating cells by tumor cell-derived chemotherapeutic microparticles[J]. Cell Res, 2016, 26(6): 713-727. doi: 10.1038/cr.2016.53
[24]	Gao YF,, Zhang H,, Zhou NN, et al. Methotrexate-loaded tumour-cell-derived microvesicles can relieve biliary obstruction in patients with extrahepatic cholangiocarcinoma[J]. Nat Biomed Eng, 2020, 4(7): 743-753. doi: 10.1038/s41551-020-0583-0
[25]	Wang LZ,, Liu LK,, Liu J. Advances in isolation and purification techniques for exosomes[J]. Chemistry, 2021, 84(10): 1023-1030. (in Chinese)
	( 王立志,, 刘路宽,, 刘晶. 外泌体分离与纯化技术研究进展[J]. 化学通报, 2021, 84(10): 1023-1030.)
[26]	Shan ZM,, Tao SC,, Hu CM, et al. Extraction, identification and proteomic analysis of exosomes derived from human umbilical cord mesenchymal stem cells[J]. Chin J Tissue Eng Res, 2022, 26(19): 3036-3042. (in Chinese)
	( 单政铭,, 陶述春,, 胡春梅, 等. 人脐带间充质干细胞来源外泌体的提取、鉴定和蛋白组学分析[J]. 中国组织工程研究, 2022, 26(19): 3036-3042.)
[27]	Jäger R,, Zwacka RM. The enigmatic roles of caspases in tumor development[J]. Cancers, 2010, 2(4): 1952-1979. doi: 10.3390/cancers2041952
[28]	Kang JF,, Hu HH,, Chen R, et al. Observation on the apoptosis in protoscolex of hydatid cyst[J]. Chin J Zoonoses, 2010, 26(5): 433-435, 441. (in Chinese)
	( 康金凤,, 胡汉华,, 陈蓉, 等. 棘球蚴原头节细胞凋亡的观察[J]. 中国人兽共患病学报, 2010, 26(5): 433-435, 441.)