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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 E-mail:llw0501@163.com

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Objective

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.
Methods

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.
Results

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."

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