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Toad venom targeted liposome and preparation method and application thereof

A technology for targeting liposomes and toadstool liposomes, applied in the directions of liposome delivery, antibodies, pharmaceutical formulations, etc., can solve the problems of low expression amount and no CA9 given, and achieves simple preparation process and good stability. , the effect of reducing drug toxicity

Inactive Publication Date: 2016-05-11
CHANGSHU JINDA TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In general, CA9 is only expressed in very few normal tissues, such as stomach, bile duct, pancreas, small intestine and vas deferens epithelium, and the expression amount is very small, while in colon cancer, bladder cancer, kidney cancer, cervical cancer, brain glue Carbonic anhydrase CA9 can be highly expressed in tumors and gastric cancers, and most of the prior art is used for tumor diagnosis. Although it is described in the prior art that CA9 can be used for treatment, it does not give specific instructions on how to use CA9 to treat treat

Method used

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  • Toad venom targeted liposome and preparation method and application thereof
  • Toad venom targeted liposome and preparation method and application thereof
  • Toad venom targeted liposome and preparation method and application thereof

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Embodiment 1

[0039] The preparation method of embodiment 1 Bufona targeting liposome

[0040] 1. Preparation of liposomes encapsulating venomous venom

[0041] 1) Take distearoylphosphatidylcholine (DSPC) 60mg, cholesterol (Chol) 10mg, distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) 9mg, distearate phosphatidylethanolamine- Dissolve 1.5mg of polyethylene glycol 2000-carboxyl cross-linked product (DSPE-PEG2000-COOH) in 15ml of chloroform (A solution); dissolve venom in methanol (B solution), mix A solution and B solution at a ratio of 1:1 , using film dispersion method, 60 ° C, 40r / min, distilled to remove chloroform and methanol.

[0042] 2) The lipid film was hydrated in 10 mM Tris-isotonic saline buffer (10 mM Tris, 137 mM NaCl, 25° C., pH 7.4). The final concentration of lipid was controlled at 5-20mg / ml.

[0043] 3) The mixture was then kept at 80°C (above the transition temperature of all lipids) for 60 min to solidify the liposome structure. During cur...

Embodiment 2

[0050] The characterization of embodiment 2 venom targeting liposomes

[0051] 1. Determination of Encapsulation Efficiency

[0052] Use AvantiJ-E centrifuge (JA-20, 17400*g, 6°C, 20min) to centrifuge 150 μL of newly prepared venom targeting liposomes through a MicroconY-10 centrifugal filter device (Millipore) with a molecular weight cut-off of 10KD . The concentration of Bufoni in the centrifuged solution was determined spectrophotometrically at 296nm. This concentration represents the concentration of venom in the continuous phase of the liposomes (non-encapsulated venom). Spectrophotometry is also used to determine the total concentration of venom in the dispersed and continuous phases of liposomes. Absolute ethanol was added to the liposome suspension to completely rupture the liposomes and completely dissolve the encapsulated venom in the solution. Absorbance was measured at a wavelength of 296 nm by a UV-VIS Varian Cary 50 spectrometer equipped with a thermostatic q...

Embodiment 3

[0064] Example 3, the effect of toad venom targeted liposomes on the survival rate of tumor cells (MTT colorimetric method)

[0065] HCT116 cells in the logarithmic growth phase were digested with trypsin and diluted with culture medium to make 3×10 4 / ml cell suspension, seeded in 96-well culture plate, 100μl per well. The experiment consisted of an experimental group, a negative control group and a blank control group. Incubate for 24 hours after inoculation, add 25 μL of samples from each group, repeat 5 wells for each, culture at 37°C 5% CO2 for 0h, 4h, 8h, 16h, and 24h respectively, add MTT5mg / ml 20μl to each well, and continue in a 37°C incubator Incubate for 4h. Discard the supernatant of the 96-well plate, add 120 μL of dimethyl sulfoxide (DMSO) to each well, shake the shaker at low speed for 10 min to fully dissolve, and measure the absorbance (OD) value on a microplate reader with a detection wavelength of 492 nm. Cell survival rate%=[(OD value of anticancer drug ...

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Abstract

The present invention discloses a cancer-treating toad venom targeted liposome and a preparation method and application thereof. The toad venom targeted liposome includes a targeted site carbonic anhydrase CA9 antibody, a liposome and toad venom. The liposome covers the toad venom, and then is connected with the carbonic anhydrase CA9 antibody, so that the toad venom targeted liposome can specifically act on CA9 overexpressing cancer cells for targeted killing of the cancer cells. Results of studies on characteristics of the prepared toad venom targeted liposome, tumor cell MTT experiments and in-vivo pharmacodynamic experiments show that the toad venom targeted liposome is relatively stable, difficult to gather, good in targeting property and high in therapeutic index. The toad venom targeted liposome is useful in the treatment of colon cancer, bladder cancer, renal cancer, cervical cancer, brain glioma and gastric cancer.

Description

technical field [0001] The invention belongs to the field of pharmaceutical preparations, and relates to targeting liposomes, in particular to venom liposomes modified with carbonic anhydrase CA9 antibody, a construction method thereof and an anti-tumor application thereof. Background technique [0002] Toad (Bufotoxin) is the dry secretion of the ear glands and skin glands of toads (Toad, commonly known as toad). Anti-inflammatory and anti-tumor effects, is an important traditional Chinese medicine. Chansu is mostly available in pills and powders. The well-known Chinese patent medicines Liushen Pill, Houzheng Pill, and Meihuadianyuedan all contain Chansu. Liquid, toad analgesic ointment, compound toad pills, compound toad ointment, toad analgesic babu ointment. However, toad venom is a type 2 toxic traditional Chinese medicine, and the maximum dosage is 0.015-0.03g. The bufo toxin contained in it stimulates the vagus nerve center or end, and directly acts on the myocardiu...

Claims

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Application Information

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IPC IPC(8): A61K39/44A61K47/48A61K9/127A61P35/00A61K35/56
Inventor 杨智钧王锦锦徐健卞兆祥吕爱平
Owner CHANGSHU JINDA TECH CO LTD
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