Drug liposome capable of efficient and highly specific killing of P53 gene mutation type of breast cancer cells

A technology of breast cancer cells and liposomes, which is applied in the field of medicine and biology, can solve the problems of poor drug efficacy, recurrence, and metastasis, and achieve the effects of exact drug efficacy, high gene expression, and high efficiency

Inactive Publication Date: 2012-10-10
SUN YAT SEN UNIV CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The curative effect of breast cancer is good, but there are still some patients who will relapse...

Method used

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  • Drug liposome capable of efficient and highly specific killing of P53 gene mutation type of breast cancer cells
  • Drug liposome capable of efficient and highly specific killing of P53 gene mutation type of breast cancer cells
  • Drug liposome capable of efficient and highly specific killing of P53 gene mutation type of breast cancer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Construction of T-VISA-miR34a therapeutic vector

[0020] (1) pCRII-TOPO-hTERT plasmid cloning

[0021] 1. The DNA of breast cancer MCF-7 cells (purchased from ATCC, USA) was extracted by conventional methods.

[0022] 2. Using the DNA as a template, design PCR primers for amplifying hTERT, hTERT PCR upstream (Forward) and downstream (Reverse) primers:

[0023] Forward: 5′-ata tct aga ggc ccc tcc ctc ggg tta ccc cac agc-3′(XbaI)

[0024] Reverse: 5′-ata gat ctt atg cgg ccg ccc acg tgc gca gca gga cgc agc gc-3′.

[0025] 3. Using breast cancer MCF-7 cell DNA as a template, hTERT PCR primers (Forward: 5′-ata tct aga ggc ccc tcc ctc ggg tta ccc cac agc-3′; Reverse: 5′-ata gat ctt atg cgg ccg ccc acg tgc gca gca gga cgc agc gc-3′), Pfu DNA polymerase, dNTP and PCR reaction solution, amplified to obtain hTERT promoter (-416 to+1) product, its sequence is shown in SEQ ID NO.2. The PCR reaction system: 5 μl of 10×PCR buffer, 1 μl of upstream (Forward) and downst...

Embodiment 2

[0082] Embodiment 2: the preparation of liposome:

[0083] Lipids were taken out from the refrigerator (DOTAP was stored at -20°C, cholesterol was stored at -4°C) and returned to room temperature. Two rotary evaporators were heated in a water bath to 30°C and 50°C respectively. Weigh 68.75mg of cholesterol and put it into a 1000ml round bottom flask. Into a round bottom flask was added 100 mg DOTAP and 25 mg Chloroform. Swirl the flask to mix well. Rotate the round-bottom flask in a water bath at 30°C for 2 minutes to make it evenly mixed and form a film on the wall of the flask. Turn on the vacuum aspirator, 30min at 30°C. Add 8.9ml of preheated 5% glucose solution to dissolve the dried film, and rotate rapidly at 105 rpm for 45min at 50°C. Then lower the temperature to 35 °C and rotate for 10 min. Seal the flask with plastic wrap (or paraffin), and store it overnight at room temperature, away from light. Measure the volume, add double distilled water to 8.9ml. The fl...

Embodiment 3

[0084] Embodiment 3: Preparation of T-VISA-miR34a liposome

[0085] T-VISA-miR34a treatment carrier is dissolved in 5% glucose solution, so that its final concentration is 1ug / ul, and the liposome of embodiment 2 of storage concentration (20mM) is diluted to working concentration (8mM) with 5% glucose solution simultaneously . Add 1μg DNA / ul T-VISA-miR34a therapeutic carrier slowly into 8mM liposome (therapeutic carrier: liposome = 20ug: 50ul), let it stand at room temperature for 20 minutes, and react to form drug T-VISA-miR34a Liposomes.

[0086] Then test, biological characteristic detection and quality control such as endotoxin, aseptic packaging. Finished product testing, packaging, storage at 4-8°C; put at room temperature for 20 minutes before use, can be used directly for in vitro research, can also be used for in vivo research, and can also be used after dilution with 5% glucose solution.

[0087] 2. Effect experiment:

[0088] 1. Biological characteristics of T-V...

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Abstract

The invention discloses a drug T-VISA-miR34a liposome capable of efficient and highly specific killing of P53 gene mutation type of breast cancer cells. A T-VISA-miR34a treatment carrier capable of efficient and highly specific killing of P53 gene mutation type of breast cancer cells has a nucleotide sequence shown as a SEQ ID NO.1. The drug T-VISA-miR34a liposome capable of efficient and highly specific killing of P53 gene mutation type of breast cancer cells includes a liposome and a T-VISA-miR34a treatment carrier encapsulated by the liposome; and the T-VISA-miR34a treatment carrier has a nucleotide sequence shown as a SEQ ID NO.1. Encapsulated and delivered by the liposome, the T-VISA-miR34a treatment carriers of the invention enrich in the breast cancer site, and can efficiently target breast cancer BCL-2 and CD44 targets, completely or incompletely pair with 3 ' UTR, degrade mRNA of the target gene or inhibit its translation and lead to apoptosis of breast cancer cells without killing the normal cells. The drug can be delivered systemically, has advantages of high gene expression amount, long time, high efficiency, exact efficacy, low toxicity and side effect, no toxicity on kidney or liver, and has broad prospects in the treatment of P53 mutation type breast cancer.

Description

Technical field: [0001] The invention belongs to the field of medicine and biology, and in particular relates to a T-VISA-miR34a treatment carrier capable of efficiently and specifically killing P53 gene mutant breast cancer cells and a drug T-VISA-miR34a liposome. Background technique: [0002] Breast cancer is the most common malignant tumor in women, and its incidence rate is increasing year by year, seriously endangering human health. The curative effect of breast cancer is good, but there are still some patients who will relapse and metastasize after comprehensive treatment, and the curative effect on existing drugs is very poor. microRNAs (miRNAs) are a class of small non-coding RNAs discovered in recent years with a length of 18-24 nucleotides. It mainly degrades target gene mRNA or inhibits its translation through complete or incomplete pairing with the target gene 3'UTR, thereby participating in the regulation of individual development, cell apoptosis, proliferatio...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K9/127A61P35/00A61K31/513
Inventor 谢小明李来胜谢新华郭姣丽伍民庆孔亚楠韦尉东肖祥胜
Owner SUN YAT SEN UNIV CANCER CENT
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