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Novel cationic graft copolymer, and preparation method and application of multiple composite non-viral gene vector

A technology of graft copolymer and gene carrier, applied in the fields of biotechnology and pharmaceutical preparations, can solve the problems of high toxicity, coagulation of blood cells, low transfection efficiency, etc., and achieves low cytotoxicity, broad application prospects and high transfection efficiency Effect

Inactive Publication Date: 2014-03-26
CHINA PHARM UNIV
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Problems solved by technology

[0007] In view of the high cytotoxicity of the cationic polymer PEI, the low transfection efficiency and the coagulation of blood cells during the transfection process, a new cationic graft polymer was synthesized, and the lipophilic material polycaprolactone (PCL) was selected to connect the hydrophilic end. Polyethyleneimine (LPEI) makes cationic LPEI cover the surface of nanoparticles, makes the nanoparticles positively charged, constructs plasmid DNA complexes, and then uses hyaluronic acid (HA) to wrap gene complexes to construct multiple complexes, thereby improving Its cell transfection efficiency and tumor active targeting are expected to have potential application value in clinical gene therapy

Method used

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  • Novel cationic graft copolymer, and preparation method and application of multiple composite non-viral gene vector
  • Novel cationic graft copolymer, and preparation method and application of multiple composite non-viral gene vector
  • Novel cationic graft copolymer, and preparation method and application of multiple composite non-viral gene vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Synthesis of cationic polymer PCL-g-LPEI

[0034] Using the ring-opening copolymerization method, ε-caprolactone (106.7mL, 1.0mol) and purified water (1mL, 55.6mmol) were injected into the three-necked flask, and the catalyst Sn(Oct) was added. 2 (1.8mL, 5mmol), react at 120°C for 2h, cool to 25°C, add appropriate amount of chloroform to dissolve, add to excess methanol for precipitation, filter the precipitate, and vacuum dry at 40°C for 4h to obtain polycaprolactone (PCL).

[0035] By infrared FT-IR and proton nuclear magnetic resonance spectroscopy 1 The structure of PCL is characterized by H NMR, and the spectrum is resolved as follows:

[0036] FT-IRIR(KBr): 3426.2(v OH (COOH)), 2946.2 (v as CH2 ), 2866.4(V s CH2 ), 1739.1 (v s C=O (COOH)), 1471.6(β CH2 ), 1419.8(β OH (COOH)), 1398.1(β oH (CH 2 OH)), 1295.4(v C-O-C ).

[0037] 1 H-NMR(300MHz, CDCl 3 ): δ1.35~1.41(H1, CH 2 ), δ1.62~1.68(H2, CH 2 ), δ2.28~2.31(H3, CH 2 ), δ4.05~4.07(H4, CH 2 ).

[0038] The...

Embodiment 2

[0044] Example 2: Synthesis of cationic polymer PCL-g-LPEI

[0045] Using the ring-opening copolymerization method, ε-caprolactone (106.7mL, 1.0mol) and purified water (1mL, 55.6mmol) were injected into the three-necked flask, and the catalyst Sn(Oct) was added. 2 (1.8mL, 5mmol), react at 120°C for 2h, cool to room temperature, add appropriate amount of chloroform to dissolve, add to methanol for precipitation, filter the precipitate, and dry under vacuum at 40°C to obtain polycaprolactone (PCL).

[0046] The yield of PCL was 58.3% and the molecular weight was 2392 Da.

[0047] Through the amide reaction, dissolve PCL (12.0g, 5mmol) in an appropriate amount of DMSO, add DCC (1.0g, 5mmol), NHS (0.6g, 5mmol), and react under nitrogen protection at 25°C for 12h, add LPEI (1.1g, 25mmol) , Stirring, dissolving, reacting at 80°C for 48h, filtering the solution, dialysis in deionized water for 72h (dialysis bag cut-off molecular weight 8000), freeze-drying to obtain PCL-g-LPEI.

[0048] The ...

Embodiment 3

[0049] Example 3: Synthesis of cationic polymer PCL-g-LPEI

[0050] Through the ring-opening copolymerization reaction, ε-caprolactone (106.7mL, 1.0mol) and purified water (0.9mL, 50.0mmol) were injected into the three-necked flask, and the catalyst Sn(Oct) was added. 2 (1.8mL, 5mmol), react at 120°C for 4h, cool to room temperature, add appropriate amount of chloroform to dissolve, add dropwise to excess methanol for precipitation, filter the precipitate, and dry under vacuum at 40°C for 4h to obtain polycaprolactone (PCL).

[0051] The yield of PCL is 51.8% and the molecular weight is 2996Da

[0052] Using amide reaction, dissolve PCL (15.0g, 5mmol) in an appropriate amount of DMSO, add DCC (1.0g, 5mmol), NHS (0.6g, 5mmol), and react under nitrogen protection at 25℃ for 12h, add LPEI (2.2g, 25mmol) , Stirring, dissolving, reacting at 80°C for 48h, filtering the solution, dialysis in deionized water for 72h (dialysis bag cut-off molecular weight 8000), freeze-drying to obtain PCL-g-...

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Abstract

The invention relates to a novel cationic graft copolymer, and a preparation method and an application of a multiple composite non-viral gene vector. The novel cationic graft copolymer PCL-g-LPEI (polycaprolactone-g-linear polyethylenimine) is synthesized by ring-opening copolymerization and amidation reaction. The multiple composite non-viral gene vector comprises: (A) a plasmid DNA (deoxyribonucleic acid)-supported PCL-g-LPEI cationic compound serving as a kernel of a multiple gene compound; (B) a tumor-targeting ligand HA (hyaluronic acid) wrapping the cationic compound under the interaction of positive and negative charges to form a shell of the multiple gene compound. A multiple composite gene constructed by the non-viral gene vector has the advantages of low cytotoxicity, high blood compatibility, high transfection efficiency, tumor targeting effects and the like, and is expected to be applied to clinical gene therapy.

Description

technical field [0001] The invention belongs to the fields of biotechnology and pharmaceutical preparations, and relates to a preparation method of a novel cationic graft copolymer and a multi-composite non-viral gene carrier. Specifically relate to the synthesis of novel cationic graft copolymer PCL-g-LPEI; The present invention also relates to the preparation method of constructing PCL-g-LPEI / DNA compound and carrying hyaluronic acid (HA) multiple gene complex, as low toxicity and high efficiency Gene carriers are used in gene therapy. Background technique [0002] In recent years, new gene therapy methods provide good development prospects for cancer treatment, genetic diseases and so on. The development of safe, effective, low cytotoxicity and tumor-targeted non-viral gene delivery system will become an important factor for the success of gene therapy. Gene carrier is the key difficulty of current gene therapy, mainly including viral vector and non-viral vector. Altho...

Claims

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Application Information

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IPC IPC(8): C08G81/00C08G73/04C08G63/91C08G63/08C12N15/63A61K47/34A61K48/00A61P35/00
Inventor 李娟尹少平王晶杨勇
Owner CHINA PHARM UNIV
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