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Preparation method of bionic siRNA nano complex with anticancer activity

A nanocomposite, anticancer activity technology, applied in the field of biomimetic siRNA nanocomposite preparation, can solve the problem of difficult to penetrate into the internal cells of tumors, etc.

Pending Publication Date: 2022-02-01
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inventor investigated the literature and found that the main reason for the difference in in vivo and in vitro effects is that nanocarriers are difficult to effectively accumulate, infiltrate and retain in tumor tissues, and it is extremely difficult to penetrate deep into tumor cells, and the efficiency of nanocarriers reaching and accumulating in tumor sites is less than 1%!

Method used

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  • Preparation method of bionic siRNA nano complex with anticancer activity
  • Preparation method of bionic siRNA nano complex with anticancer activity
  • Preparation method of bionic siRNA nano complex with anticancer activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Synthetic PLGA 50k -PEI 25k polymer

[0049] 3 g of PLGA (50 kD) was dissolved in 30 ml of DMSO, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide (EDC) and N-hydroxy succinimide (both) (both) The mass ratio was 5: 1), and stirred until it was completely dissolved for two hours, then 0.6 g of Pei (25 kD) was added dropwise, at room temperature for 48 h, dialysis (MW = 65kd) three days, centrifuge, pick it into PLGA 50k -PEI 25k sample. Nuclear magnetic resonance techniques were used for obtained samples ( 1 H NMR) is characterized to obtain a nuclear magnetic resonance spectrum (with figure 1 . from figure 1 The peaks prepared at 5.2 ppm are assigned by H, and after the above test analysis, the peak at 5.2 ppm is at home.

Embodiment 2

[0050] Example 2: Extramicomacemic reticulum (EM) extracting tumor cells

[0051] Prepare 1 × 10 8 Human breast cancer MCF-7 cells, add 2.5 mL of the homogenate separation liquid having an enzyme inhibitor, and then placed in the ice homogenate for 10 min, use the cell scraper to scrape, collect the suspension at 4 ° C, 1000g centrifugation 10min, discarded precipitation; absorb the supernatant to another centripette, 12000 g from centrifugation for 15 minutes, discard the precipitate, collect the supernatant (light yellow clear and transparent liquid); transfer the supernatant round bottom flask, Add 7.5 times volume of pre-cooling CaCl drop 2 (8 mM) solution (turbid liquid), the ice continued for 15 min and then centrifuged at 8000 g of 10 min, and the deposition is a rich microparticles (endoplasmic reticulum, referred to as EM); discard the supernatant, separation The liquid resuspendent, and the scroll is dispersed to obtain an endoplasmic reticulum. 3 μl of solution was adde...

Embodiment 3

[0052] Example 3: Preparation and characterization of inner reticular modified PLGA-PEI / siRNA nano-composite

[0053] PLGA 50k -PEI 25k Dichloromethane solution, 37 ° C decompression rotation evaporation remove organic solvent, 60 ° C water bath to add 5% glucose solution, water bath ultrasonic hydration, PLGA 50k -PEI 25k The final concentration is 10 mg / ml to get PLGA 50k -PEI 25k Cationic nanoparticles (PP NPS) were dissolved in 500 μl of 5% glucose solution at a final concentration of 0.064 mg / ml (4 μM). 25 μl siRNA (4 μM) was drawn, and a different concentration of PP NPS solution was added dropwise, mixed uniform, incubated for 15 min at 37 ° C to obtain a nanocomposite of siRNA (PP / siRNA NPS).

[0054] Determination of particle size distribution and potential using the Marvin Parthenometer (DLS) to get attached image 3 . like image 3 , Under different ratios, along with PLGA 50k -PEI 25k Elevation, the better the SiRNA's packaging effect.

[0055] Further, 50 μl of ...

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Abstract

The invention relates to a preparation method of a bionic siRNA nano complex with anticancer activity. The preparation method comprises the following steps: (1) preparing a PLGA-PEI polymer by a one-step amidation method; (2) extracting endoplasmic omentum of tumor cells by a calcium chloride precipitation method; (3) preparing PLGA-PEI cation nanoparticles through a film dispersion method and ultrasonic assistance; and (4) loading a siRNA drug, and externally modifying the endoplasmic reticulum membrane to obtain the EPP / siRNA nano complex. According to the invention, the preparation method of the bionic siRNA nano complex with anticancer activity aims to solve the key scientific problems of enhancing the accumulation infiltration capacity of nanoparticles at a tumor site and improving the antitumor curative effect; and by means of active protein for regulating cell vesicle transport in endoplasmic reticulum membrane, the nano complex is endowed with unique transcellular endocytosis capability, the purposes of enhancing accumulation of tumor sites across vascular endothelial cells and enhancing deep penetration of tumor tissues across tumor cells are achieved, and stronger gene silencing and anti-tumor effects are achieved.

Description

Technical field [0001] The present invention belongs to the technical field of nano-drug carrier materials, and in particular to a method of preparing a biochemical siRNA nanocomposite having anti-cancer activity. Background technique [0002] siRNA can specifically lysis target mRNA, down-regulate protein expression levels associated with disease gene, exhibited powerful genetic potential in the treatment of tumors. However, its unique pharmaceutical defects mainly include ribozyme degradation, membrane permeability, short-half-life shortage degradation damage, and directly use free siRNA to achieve genetic silencing effects inside and outside the body, and severely restrict its clinical application. [0003] The existing research workers have developed various types of carrier delivery systems for solving the application problem of siRNA, especially non-viral gene vectors based on cationic materials, such as lipids (such as DOTAP, DLINDMA, etc.), polymers. Polymer (such as poly...

Claims

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Application Information

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IPC IPC(8): A61K47/69A61K31/7088A61K9/51A61K47/46A61P35/00B82Y5/00B82Y30/00B82Y40/00
CPCA61K31/7088A61K47/6937A61K47/6901A61K9/5068A61K9/0019A61P35/00B82Y5/00B82Y30/00B82Y40/00
Inventor 邱崇夏斐郭秋岩史巧莉
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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