Search JIM Advanced Search

Journal of Chinese Integrative Medicine ›› 2009, Vol. 7 ›› Issue (9): 836-841.doi: 10.3736/jcim20090908

• Original Experimental Research • Previous Articles     Next Articles

Effects of intratumoral injection of microspheres containing cobra venom cytotoxin on transplanted human hepatoma in nude mice

Yan Wang , Li-wu Lin,Zhi-kui Chen, En-sheng Xue, Xiao-dong Lin, Li-yun Yu, Zhen-hu Lin   

  1. Department of Ultrasonics, Institute of Ultrasonic Medicine, Union Hospital, Fujian Medical University, Fuzhou 350001, Fujian Province, China
  • Received:2009-06-18 Accepted:2009-07-22 Online:2009-09-20 Published:2009-09-15
  • Contact: Li-wu Lin


To evaluate the safety and efficacy of intratumoral injection of polylactic-co-glycolic acid (PLGA) microspheres containing cobra venom cytotoxin in nude mice with transplanted human hepatoma.

Cytotoxic activity of cytotoxin from cobra venom was determined by using methyl thiazolyl tetrazolium method in vitro. Microspheres containing cobra venom cytotoxin were prepared with a double emulsion-solvent evaporation method. Forty BALB/c nude mice were inoculated subcutaneously in right flank with hepatoma BEL-7404 cells. Thirty-two mice whose tumor size reached about 1.0 cm in diameter, were randomly assigned into normal saline group, blank microsphers group, cytotoxin group and cytotoxin-PLGA group. Nude mice were intratumorally injected with normal saline, blank microspheres, cytotoxin or cytotoxin-PLGA microspheres respectively. Internal echo characteristics and blood flow of tumors were observed by high-frequency ultrasound every week after treatment. Twenty-six days after treatment, the tumors were removed to calculate the inhibition rate of tumor growth. The tumor, heart, liver and kidney tissues were obtained for histopathological examination.

The cytotoxin separated and purified from crude cobra venom caused intense cytotoxic effects to the BEL-7404 cells in vitro. The diameter of PLGA microspheres containing cobra venom cytotoxin was about (34.45±9.85) μm. Encapsulation rate was up to (78.13±8.92)%, and cumulative amount of cobra venom cytotoxin released from the PLGA microspheres in vitro during 30 days was up to 84.3%. After intratumoral injection, tumor volumes and weights in the cytotoxin-PLGA group were lower than those in the normal saline group, with a tumor growth inhibition rate of 52.36%. Observed under a light microscope, most tumor tissues were necrotic. No obvious morphological change could be seen on the liver, kidney and heart tissues.
Conclusion: The above findings indicate that intratumoral injection of cytotoxin-PLGA microspheres has strong antitumor effect and can obviously lessen systemic toxicity, which may provide an effective and feasible method for hepatocellular carcinoma treatment.

Key words: Cobra venom cytotoxin, Liver neoplasms, Drug therapy, Delayed-action preparation, Mice, Nude

Table 1

Inhibition rate of BEL-7404 cell proliferation cultured with cobra venom CTX ($\bar{x}$±s, %)"

Concentration (μg/mL) n Inhibition rate
12 h 24 h 48 h
Control 3 0 0 0
0.5 3 16.71±2.27* 24.36±2.54 29.42±1.88
1 3 21.85±1.67* 37.82±1.15 44.36±2.35
2 3 33.82±2.15* 52.37±1.75 60.14±1.36
3 3 47.39±2.03* 64.43±2.27 76.32±2.33
4 3 69.46±1.57* 82.34±0.89 90.58±1.18

Figure 1

Shape of cobra venom CTX-PLGA microspheres observed by ESEM (A: ×300; B: ×1 000)"

Figure 2

Release curve of CTX from cobra venom CTX-PLGA microspheres in vitro"

Table 2

Changes of tumor volume of nude mice in different groups monitored by ultrasonography ($\bar{x}$±s, mm3)"

Group n Tumor volume
Before treatment 8 days
after treatment
14 days
after treatment
20 days
after treatment
26 days
after treatment
Normal saline 8 256.3±24.7 636.8±94.1 1 616.6±203.6 3 237.9±382.8 5 768.9±442.8
Blank microspheres 8 249.5±23.3 617.2±88.5 1 572.8±217.2 3 229.5±316.3 5 447.5±426.3
CTX 8 261.3±30.2 459.3±78.4* 842.3±205.2* 1 937.7±257.1* 3 637.5±293.2*
CTX-PLGA 8 251.5±28.4 379.2±58.1** 603.2±139.3** 915.6±251.2** 1 219.6±263.7**

Figure 3

Ultrasonogram of tumor tissue of nude mice in different groups after treatment A, B: Normal saline group; C, D: Blank microspheres group; E, F: CTX group; G, H: CTX-PLGA group."

Table 3

Body and tumor weights and inhibition rates of nude mice bearing BEL-7404 liver cancer in different groups after 26-day treatment"

Group n Body weight ($\bar{x}$±s, g) Tumor weight ($\bar{x}$±s, g) Inhibition rate (%)
Normal saline 8 27.90±1.43 2.75±0.52
Blank microspheres 8 28.05±0.79 2.51±0.39 8.73
CTX 8 28.29±1.29 2.02±0.46* 26.55
CTX-PLGA 8 28.74±0.70 1.31±0.31** 52.36

Figure 4

Histopathologic changes of tumor tissue of nude mice in different groups observed by light microscopy (HE staining, ×400) A: Normal saline group; B: Blank microspheres group; C: CTX group; D: CTX-PLGA group."

[1] Abu-Sinna G, Esmat AY, Al-Zahaby AA, Soliman NA, Ibrahim TM . Fractionation and characterization of Cerastes cerastes cerastes snake venom and the antitumor action of its lethal and non-lethal fractions[J]. Toxicon, 2003,42(2):207-215
doi: 10.1016/S0041-0101(03)00138-7
[2] Feofanov AV, Sharonov GV, Dubinnyi MA, Astapova MV, Kudelina IA, Dubovskii PV, Rodionov DI, Utkin YN, Arseniev AS . Comparative study of structure and activity of cytotoxins from venom of the cobras Naja oxiana, Naja kaouthia, and Naja haje[J]. Biochemistry (Mosc), 2004,69(10):1148-1157
doi: 10.1023/B:BIRY.0000046890.46901.7e
[3] Oron U, Chaim-Matyas A, Ovadia M . Histopathological changes in WEHI-3B leukemia cells following intoxication by cytotoxin P4 from Naja nigricollis nigricollis venom[J]. Toxicon, 1992,30(9):1122-1126
doi: 10.1016/0041-0101(92)90058-D
[4] Chen ZK, Lin LW, Lin Q, Zhuang QQ, He YM, Xue ES . Preparation and characterization of poly(lactide-co-glycolide acid) microspheres containing cytotoxin from Naja Naja Atra venom[J]. Zhongguo Sheng Hua Yao Wu Za Zhi, 2008,29(3):161-164
陈志奎, 林礼务, 林琦, 庄权权, 何以敉, 薛恩生 . 眼镜蛇毒细胞毒素缓释微球制备及体外性质研究[J]. 中国生化药物杂志, 2008,29(3):161-164
[5] Sapin A, Garcion E, Clavreul A, Laqarce F, Benoit JP, Menei P . Development of new polymer-based particulate systerms for anti-glioma vaccination[J]. Int J Pharm, 2006,309(1-2):1-5
doi: 10.1016/j.ijpharm.2005.10.046 pmid: 16386390
[6] DeFail AJ, Chu CR, Izzo N, Marra KG . Controlled release of bioactive TGF-beta 1 from microspheres embedded within biodegradable hydrogels[J]. Biomaterials, 2006,27(8):1579-1585
doi: 10.1016/j.biomaterials.2005.08.013
[7] Sun L, Wang Y, Liu ML, Xu MD . Recent developments on PLA/PLGA microspheres and its application[J]. Yao Wu Fen Xi Za Zhi, 2005,25(11):1409-1415
孙磊, 王岩, 刘明理, 许鸣镝 . 聚乳酸/乳酸-乙醇酸共聚物微球制剂研究的新进展[J]. 药物分析杂志, 2005,25(11):1409-1415
[8] Huang WD, Long DH, Gu HG . Explore of preparation and formulation parameters on BSA-PLGA microspheres[J]. Jie Pou Xue Yan Jiu, 2008,30(4):241-244
黄婉丹, 龙大宏, 谷海刚 . BSA-PLGA缓释微球的制备及优化条件的探索[J]. 解剖学研究, 2008,30(4):241-244
[9] Wang PP, Frazier J, Brem H . Local drug delivery to the brain[J]. Adv Drug Deliv Rev, 2002,54(7):987-1013
doi: 10.1016/S0169-409X(02)00054-6 pmid: 12384318
[10] Li Q, Fan ZZ, Li XQ, Liu XH, Sun J, Gu W, Heng P, Gao H . Effects of interventional therapy with norcantharidin microsphere on hepatoma in rats and its mechanism[J]. J Chin Integr Med, 2006,4(4):378-383
李琦, 范忠泽, 李先茜, 刘晓华, 孙珏, 顾伟 , Heng P, 高虹.去甲斑蝥素微球介入治疗大鼠肝癌疗效及其机制研究[J]. 中西医结合学报, 2006,4(4):378-383
[11] Chen ZK, Lin LW, Yang FD, Weng XH, Cai HJ, Xue ES, Wang Y . Effects of interstitial chemotherapy using thermosensitive gel-coated ricin on hepatoma H22-bearing mice[J]. J Chin Integr Med, 2009,7(5):447-452
doi: 10.3736/jcim20090509
陈志奎, 林礼务, 杨发端, 翁秀华, 蔡华晶, 薛恩生, 王艳 . 蓖麻毒素温敏型凝胶间质化疗对H22肝癌荷瘤小鼠的影响[J]. 中西医结合学报, 2009,7(5):447-452
doi: 10.3736/jcim20090509
[12] Shive MS, Anderson JM . Biodegradation and biocompatibility of PLA and PLGA microspheres[J]. Adv Drug Deliv Rev, 1997,28(1):5-24
doi: 10.1016/S0169-409X(97)00048-3
[13] Allen TM, Cullis PR . Drug delivery systems: entering the mainstream[J]. Science, 2004,303(5665):1818-1822
doi: 10.1126/science.1095833
[14] Lesniak MS, Upadhyay U, Goodwin R, Tyler B, Brem H . Local delivery of doxorubicin for the treatment of malignant brain tumors in rats. Anticancer Res[J]., 2005,25(6B):3825-3831
doi: 10.3171/jns.2002.97.5.1173 pmid: 16312042
[1] Victor Abou Nehmi Filho, Gilson Masahiro Murata, Ruan Carlos Macêdo de Moraes, Gabriely Cristina Alves Lima, Danielle Araujo de Miranda, Katrin Radloff, Raquel Galvão Figuerêdo Costa, Joyce de Cassia Rosa de Jesus, Jéssica Alves De Freitas, Nayara Izabel Viana, Ruan Pimenta, Katia Ramos Moreira Leite, José Pinhata Otoch, Ana Flávia Marçal Pessoa. A novel supplement with yeast β-glucan, prebiotic, minerals and Silybum marianum synergistically modulates metabolic and inflammatory pathways and improves steatosis in obese mice. Journal of Integrative Medicine, 2021, 19(5): 439-450.
[2] Hao Gou, Ruo-chen Huang, Yong-hua Su, Wei Li. Design of dual targeting immunomicelles loaded with bufalin and study of their anti-tumor effect on liver cancer. Journal of Integrative Medicine, 2021, 19(5): 408-417.
[3] Yi Ran Hu, San Li Xing, Chuan Chen, Ding Zhu Shen, Jiu Lin Chen. Tiaoxin Recipe, a Chinese herbal formula, inhibits microRNA-34a expression in the APPswe/PS1ΔE9 mouse model of Alzheimer's disease. Journal of Integrative Medicine, 2019, 17(6): 404-409.
[4] Xuan Liu, Li-juan Xiu, Jian-peng Jiao, Jing Zhao, Ying Zhao, Ye Lu, Jun Shi, Yong-jin Li, Min Ye, Yu-fang Gu, Xiao-wei Wang, Jing-yu Xu, Ci-an Zhang, Yong-ying Liu, Yun Luo, Xiao-qiang Yue. Traditional Chinese medicine integrated with chemotherapy for stage IV non-surgical gastric cancer: A retrospective clinical analysis. Journal of Integrative Medicine, 2017, 15(6): 469-475.
[5] Bhesh Raj Sharma, Dong Wook Kim, Dong Young Rhyu. Korean Chungtaejeon tea extract attenuates weight gain in C57BL/6J-Lep ob/ob mice and regulates adipogenesis and lipolysis in 3T3-L1 adipocytes. Journal of Integrative Medicine, 2017, 15(1): 56-63.
[6] Shi-li Jiang, Xu-dong Hu, Ping Liu. Immunomodulation and liver protection of Yinchenhao decoction against concanavalin A-induced chronic liver injury in mice. Journal of Integrative Medicine, 2015, 13(4): 262-268.
[7] Xuanbin Wang, Ning Wang, Fan Cheung, Lixing Lao, Charlie Li, Yibin Feng. Chinese medicines for prevention and treatment of human hepatocellular carcinoma: Current progress on pharmacological actions and mechanisms. Journal of Integrative Medicine, 2015, 13(3): 142-164.
[8] Elizabeth Abidemi Balogun, Sylvia Orume Malomo, Joseph Oluwatope Adebayo, Ahmed Adebayo Ishola, Ayodele Olufemi Soladoye, Lawrence Aderemi Olatunji, Olatunji Matthew Kolawole, Stephen Olubunmi Oguntoye, Abiola Samuel Babatunde, Oluwole Busayo Akinola . In vivo antimalarial activity and toxicological effects of methanolic extract of Cocos nucifera (Dwarf red variety) husk fibre. Journal of Integrative Medicine, 2014, 12(6): 504-511.
[9] Md. Asadujjaman, Ahmed Ullah Mishuk, Md. Aslam Hossain, Utpal Kumar Karmakar. Medicinal potential of Passiflora foetida L. plant extracts: Biological and pharmacological activities. Journal of Integrative Medicine, 2014, 12(2): 121-126.
[10] Marjan Nassiri-Asl, Taghi Naserpour Farivar, Esmail Abbasi, Hamid Reza Sadeghnia, Mehdi Sheikhi, Mina Lotfizadeh, Parisa Bazahang. Effects of rutin on oxidative stress in mice with kainic acid-induced seizure. Journal of Chinese Integrative Medicine, 2013, 11(5): 337-342.
[11] Md. Khirul Islam, Imran Mahmud, Sanjib Saha, Asit Baron Sarker, Himangsu Mondal, A.S.M. Monjur-Al-Hossain, Md. Anisuzzman. Preliminary pharmacological evaluation of Alocasia indica Schott tuber. Journal of Chinese Integrative Medicine, 2013, 11(5): 343-351.
[12] Sanjib Saha, Faroque Hossain, Md. Anisuzzman, Md. Khirul Islam. Pharmacological evaluation of Musa seminifera Lour. Fruit. Journal of Chinese Integrative Medicine, 2013, 11(4): 253-261.
[13] Sunil A. Nirmal, Jayashri M. Ingale, Shashikant R. Pattan, Sanjay B. Bhawar. Amaranthus roxburghianus root extract in combination with piperine as a potential treatment of ulcerative colitis in mice. Journal of Chinese Integrative Medicine, 2013, 11(3): 206-212.
[14] Lin Wang, Guan-liang Wang, Jia-han Liu, Di Li, De-zeng Zhu, Liang-neng Wu . Effects of ursolic acid in ameliorating insulin resistance in liver of KKAy mice via peroxisome proliferator-activated receptors α and γ. Journal of Chinese Integrative Medicine, 2012, 10(7): 793-799.
[15] Mei-jiao Mao, Jun-ping Hu, Cong Wang, Yi-yi Zhang, Ping Liu. Effects of Chinese herbal medicine Guanxinkang on expression of PPARγ-LXRα-ABCA1 pathway in ApoE-knockout mice with atherosclerosis. Journal of Chinese Integrative Medicine, 2012, 10(7): 814-820.
Full text



[1] Wei-xiong Liang. Problems-solving strategies in clinical treatment guideline for traditional Chinese medicine and integrative medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 1-4
[2] Zhao-guo Li. Discussion on English translation of commonly used sentences in traditional Chinese medicine: part one. Journal of Chinese Integrative Medicine, 2008, 6(1): 107-110
[3] Jun Hu, Jian-ping Liu. Non-invasive physical treatments for chronic/recurrent headache. Journal of Chinese Integrative Medicine, 2008, 6(1): 31
[4] Xue-mei Liu, Qi-fu Huang, Yun-ling Zhang, Jin-li Lou, Hong-sheng Liu, Hong Zheng. Effects of Tribulus terrestris L. saponion on apoptosis of cortical neurons induced by hypoxia-reoxygenation in rats. Journal of Chinese Integrative Medicine, 2008, 6(1): 45-50
[5] . Uniform requirements for manuscripts submitted to biomedical journals: Writing and editing for biomedical publication (Chinese version, part two). Journal of Chinese Integrative Medicine, 2010, 8(11): 1001-1005
[6] Daniel Weber, Janelle M Wheat, Geoffrey M Currie. Inflammation and cancer: Tumor initiation, progression and metastasis,and Chinese botanical medicines. Journal of Chinese Integrative Medicine, 2010, 8(11): 1006-1013
[7] Hong Liu , Guo-liang Zhang, Li Shen , Zhen Zeng, Bao-luo Zhou, Cheng-hai Liu, Guang Nie . Application and evaluation of a pseudotyped virus assay for screening herbs for anti-H5Nl avian influenza virus. Journal of Chinese Integrative Medicine, 2010, 8(11): 1036-1040
[8] Zhao-guo Li . A discussion of English translation of 1995 and 1997 Chinese National Standards of Traditional Chinese Medical Terminologies for Clinical Diagnosis and Treatment. Journal of Chinese Integrative Medicine, 2010, 8(11): 1090-1096
[9] Rui Jin, Bing Zhang. A complexity analysis of Chinese herbal property theory: the multiple formations of herbal property (Part 1). Journal of Chinese Integrative Medicine, 2012, 10(11): 1198-1205
[10] Hui-min Liu, Xian-bo Wang, Yu-juan Chang, Li-li Gu. Systematic review and meta-analysis of randomized controlled trials of integrative medicine therapy for treatment of chronic severe hepatitis. Journal of Chinese Integrative Medicine, 2012, 10(11): 1211-1228