Preparation method of difunctional liver cancer targeted nanobubble carrying NET-1siRNA

A dual-function, microbubble technology, applied in the field of biomedical engineering, can solve the problems of poor tumor tissue specificity and inability to guarantee siRNA concentration, and achieve the effects of improving gene transfection rate, inhibiting the growth of liver cancer cells, and improving transfection rate.

Active Publication Date: 2015-04-22
HARBIN MEDICAL UNIVERSITY
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0008] However, the specificity of nano-microbubbles to tumor tissue is generally poor, and it cannot guarantee a high siRNA concentration at the target site, improving the targeting of gene therapy

Method used

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  • Preparation method of difunctional liver cancer targeted nanobubble carrying NET-1siRNA
  • Preparation method of difunctional liver cancer targeted nanobubble carrying NET-1siRNA
  • Preparation method of difunctional liver cancer targeted nanobubble carrying NET-1siRNA

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 design, synthesis, screening effective NET-1siRNA

[0040] According to the gene sequence of human NET-1 mRNA, according to the basic design principles of siRNA, select 3 target sequences, design 3 pairs of NET-1 siRNA, and design an irrelevant control sequence: negative control siRNA (Table 1, provided by Shanghai Gemma Pharmaceutical Technology Co., Ltd. synthesis). Three pairs of NET-1 and negative control siRNA were labeled with FAM fluorescence and biotin, freeze-dried and stored at -20°C.

[0041] Table 1 Sequences of three NET-1 siRNAs and negative controls

[0042]

Embodiment 2

[0043] Example 2 Preparation of Biotinylated Nanobubbles (Nanobubble)

[0044] 10 mg of lipid mixture (mass ratio, DSPC: DSPE-PEG2000: DSPE-PEG2000-biotin=9:0.5:0.5) was placed in a round bottom flask, and a mixed solution of chloroform and methanol was added dropwise (volume ratio, chloroform:methanol= 2:1), until the lipid mixture was completely dissolved. Place the flask in a rotary evaporator and evaporate to dryness at 40°C under vacuum;

[0045] After evaporating to dryness, add 5 mL of PBS (pH=7.4, 0.15 mol / L) buffer solution in the flask, pipette it into a centrifuge tube, seal it, and replace the internal air with C 3 f 8 ;

[0046] The amalgam oscillator was shaken for 40 seconds, and then the solution was placed under an ultrasonic breaker, in a water bath for 20 seconds, and the temperature was 55°C. Stop breaking, incubate at 55°C for 60 minutes, and then continue breaking for 2 minutes. After standing still, the microspheres in the upper layer are floated, w...

Embodiment 3

[0047] Example 3 Synthesis of biotinylated galactosylated polylysine (Bio-G-PLL)

[0048] Weigh 113.2 mg of polylysine, 480.5 mg of galactose, and 300.0 mg of sodium borohydride and dissolve them in 120 mL of water, place them in a round-bottomed flask, add a stirrer, and react at a constant temperature of 37 ° C for 24 hours. Potassium hydroxide was used to adjust the pH of the reaction solution to 8.5, and the reaction was continued at 37°C for 6 hours.

[0049] Dialysis bag pretreatment: After cutting the dialysis bag to an appropriate length (about 30cm), use NaHCO with a mass fraction of 2% 3 (M / V) and 10mmol / L EDTA (pH8.0) were boiled for 10min, then cleaned with distilled water, put in 1mmol / L EDTA (pH8.0) and boiled for 10min, cooled, stored at 4°C, guaranteed bag Submerge completely in solution. Fill the bag with clean water before use, and use it after draining.

[0050] Dialysis process: put the reaction solution into a dialysis bag, tie both ends with a thin thr...

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Abstract

The invention discloses a preparation method of a difunctional liver cancer targeted nanobubble carrying NET-1siRNA. The method comprises the following steps: (1) synthesizing 3 pairs of NET-1siRNA sequences, and biotinylating the sequences; (2) preparing a biotinylated nanobubble; (3) synthesizing biotinylated galactosylated polylysine; and (4) coupling the biotinylated NET-1siRNA, the biotinylated nanobubble and the biotinylated galactosylated polylysine through a biotin-avidin system to obtain the difunctional liver cancer targeted nanobubble carrying NET-1siRNA. The liver cancer targeted nanobubble can be used in clinical ultrasonic diagnosis as a targeted ultrasonic contrast agent; and more importantly, the NET-1siRNA carried by the liver cancer targeted nanobubble can inhibit tumor growth, so the liver cancer targeted nanobubble can be used in gene therapy of the liver cancer.

Description

technical field [0001] The invention relates to a method for preparing nano-microbubbles, in particular to a method for preparing bifunctional liver cancer-targeting nano-microbubbles carrying NET-1siRNA, and belongs to the field of biomedical engineering. Background technique [0002] Liver cancer is a major killer threatening human health, known as the "king of cancer". The incidence rate is increasing year by year. According to statistics, the annual global incidence of liver cancer ranks fifth in the incidence of malignant tumors, and the death rate ranks third. 55% of new liver cancer patients occur in China. Surgical resection is the main method of its treatment. However, liver cancer cells grow rapidly and metastasize to blood earlier, so most patients lose the opportunity for surgery. Traditional radiotherapy and chemotherapy are not ideal for its treatment effect. The liver has become an ideal target organ for gene therapy because of its strong regenerative abili...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K47/48A61K49/22A61P35/00
Inventor 程文吴博林
Owner HARBIN MEDICAL UNIVERSITY
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