Artificial oil body carrier for targeted therapy of mastocarcinoma as well as preparation method and application thereof

A technology of targeted therapy and oil body, which is applied in the fields of genetic engineering and medical biology, can solve problems such as pollution, increased manufacturing costs, and difficult absorption, and achieve the effects of inhibiting tumor growth, increasing drug concentration, and excellent biological effects

Inactive Publication Date: 2011-06-22
陈顺基 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Currently known carriers include polymeric particles, micells, liposomes, and viral carriers. Unstable in blood or body fluids, etc., there are many limitations in practical application
In addition, when this type of carrier is combined with the above-mentioned substances that can identify cancer cells, complex and time-consuming chemical synthesis steps are usually required, which not only increases the manufacturing cost, but also causes environmental pollution

Method used

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  • Artificial oil body carrier for targeted therapy of mastocarcinoma as well as preparation method and application thereof
  • Artificial oil body carrier for targeted therapy of mastocarcinoma as well as preparation method and application thereof
  • Artificial oil body carrier for targeted therapy of mastocarcinoma as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of oil body carrier

[0052] The recombinant strain BL21(DE3) / pJO1-oleZH2 was cultured at 37°C, and the initial bacterial density was OD 550 The value is 0.08. Inoculate into fresh LB medium containing ampicillin, when the bacterial density is OD 550 When the value is 0.3, induce for 4 hours at 37°C, collect the bacteria, refrigerate and centrifuge at 4000 rpm, 4°C for 10 min, wash once with 0.1 M pH7.5 sodium phosphate buffer, collect the bacteria, and wash with 0.1M pH7.5 sodium Phosphate buffer concentrated into bacterial density OD 550 Bacteria with a value of 10.

[0053] Bacterial density OD 550 The bacterial solution with a value of 10 was sonicated on ice with an ultrasonic cell disruptor (power: 3.5%, time: 2 minutes, run: 0.5 seconds, rest: 0.5 seconds). After sonication, centrifuge at 13,000 rpm and 4°C for 10 min, and collect the supernatant and precipitate water-insoluble protein after centrifugation. Take 30μl of supernatant and...

Embodiment 2

[0056] Example 2 Fluorescent substance coated with oil body carrier and AFM test of oil body

[0057] Take oleZH2 protein, add 0.01M pH7.5 sodium phosphate buffer, and then add nile red (brand: SIGMA, concentration: 0.5μg / ml) or Yellow GGK (brand: Huaide Biochemical Co., Ltd., concentration : 1 μg / ml) of sesame oil, ultrasonically mixed on ice with an ultrasonic cell grinder (power: 3.0%, time: 20 seconds, run: 0.5 seconds, rest: 0.5 seconds), and centrifuged at 13000 rpm, 4°C for 10 min , take out the upper layer of white oil cake, add 0.01M pH7.5 sodium phosphate buffer, break it up with an ultrasonic cell pulverizer, and perform the same steps three times to construct a fluorescent oil body carrier. Dilute with 0.01M pH7.5 sodium phosphate buffer, and observe with a fluorescence microscope. Yellow GGK is excited at 488 nm, and the oil body carrier can be seen to emit green fluorescence. Nile red is excited at 454-458 nm, and the oil body carrier can be seen to emit red flu...

Embodiment 3

[0059] Example 3 Test of oil body carrier under different preparation conditions

[0060] Different ratios of oil and protein build the oil body carrier. Mix the oil and oleZH2 protein at a ratio of 10:1, 2:1, 1:1, 1:5, and 1:10 respectively, take the oleZH2 protein, add 0.01M pH7.5 sodium phosphate buffer, and then add sesame oil (Table 1 ), use an ultrasonic cell grinder to ultrasonically mix on ice (power: 3.0%, time: 20 seconds, run: 0.5 seconds, rest: 0.5 seconds), centrifuge at 13,000 rpm, 4°C for 10 min, and take out the white layer above Add 1 ml of 0.01M pH7.5 sodium phosphate buffer to the oil cake, break it up with an ultrasonic cell pulverizer, and carry out the same steps three times to construct the oil body carrier.

[0061] Oil body vehicles were constructed under different pH conditions. Fix the ratio of oil and fusion protein, take oleZH2 protein, add sodium phosphate buffer of various pH (pH6.5, pH7.0, pH7.5, pH8.0, pH9.0), then add sesame oil, and use ult...

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Abstract

The invention discloses an artificial oil body carrier for the targeted therapy of mastocarcinoma as well as a preparation method and an application thereof. With the preparation method, a pJO1-oleZH2 plasmid is converted to enter the host cell of escherichia coli BL21 (DE3), oleZH2 proteins are produced by induction, and oil and the oleZH2 proteins are mixed to prepare an artificial oil body carrier. Invitro and invivo experiments confirm that the artificial oil body carrier disclosed by the invention can be effectively applied to target therapy, and the oil body carried covered with fluorescent substances can reflect HER2/neu positive cancer cells so as to specifically detect the mastocarcinoma cell excessively expressing HER2/neu. The artificial oil body carrier has the advantages of simple preparation process, extra small environmental pollution, good stability in blood cells or serum, small volume and the like, is cheap and efficient, is convenient to operate and is easy to transport. The artificial oil body carrier can be used for covering anti-mastocarcinoma drugs of different types of oil solubility, can effectively kill mastocarcinoma cells after being coated on drugs and can carry out specific detection and target therapy for mastocarcinoma cells.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and medical biology, and relates to a method for expressing oleZH2 protein using Escherichia coli, a method for preparing an artificial oil body carrier and the application of the carrier in specific detection and target treatment of breast cancer. Background technique [0002] Cancer, also known as malignant tumor, according to current understanding, its cause is abnormal division of cancer cells and metastasis throughout the body, resulting in abnormal physiological function and difficult to cure, it has become the number one cause of human death in the world in recent years. Since 2003, breast cancer has become the most common cancer among women in Taiwan, and 50% of breast cancer patients in the world have been diagnosed as HER2 / neu positive. These patients lack clinical symptoms and are difficult to detect at an early stage. Therefore, there is an urgent need for early diagnosis tools for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/44A61K47/42A61K31/4745C12N15/62G01N33/574A61P35/00
CPCA61K38/00A61K31/475A61K47/44A61P35/00Y02A50/30
Inventor 朱宝美陈顺基
Owner 陈顺基
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