Target quaternary ammonium salt cationic polymer lipid gene carrier, preparation method and application thereof

Abstract

The invention discloses a target quaternary ammonium salt cationic polymer lipid gene carrier, a preparation method and an application thereof. The target quaternary ammonium salt cationic polymer lipid genetic carrier is characterized in that: a polymeric quaternary ammonium salt and lipid are adopted for preparing a quaternary ammonium salt cationic polymer lipid genetic carrier according to a mass ratio, wherein the mass ratio of the polymeric quaternary ammonium salt to the lipid is 0.05-20:1; then a assembly method or a modification method is adopted for modifying to prepare a folic acid or EGFR antibody modified cationic polymer lipid gene carrier. Results of gene transfection experiments show that: gene transfection efficiencies of the target quaternary ammonium salt cationic polymer lipid gene carrier in 293T cells and NIH-3T3 cells are the same as the gene transfection efficiencies of positive control lipofectamine of lipofectamine<TM>2000 in the 293T cells and the NIH-3T3 cells; the gene transfection efficiencies of the EGFR antibody modified cationic polymer lipid genetic carrier in liver cancer Huh-7 cells and breast cancer MCF-7 cells are higher than the gene transfection efficiencies of the lipofectamine<TM>2000 in the liver cancer Huh-7 cells and the breast cancer MCF-7 cells. The cationic polymer lipid genetic carrier system provided by the present invention has good biocompatibility and low cytotoxicity, and can be as an excellent non-viral gene delivery carrier.

Description

technical field

[0001] The invention relates to a drug and gene carrier constructed by a modified high molecular polymer, a preparation method and an application thereof, and further to a targeted quaternary ammonium salt cationic high molecular lipid gene carrier, a preparation method and an application, belonging to functional Liposome preparation and application technology. Background technique

[0002] Gene therapy is mainly to introduce exogenous gene DNA or RNA fragments into target cells or tissues to correct or compensate gene defects or inhibit abnormally expressed genes, so as to achieve the purpose of treatment, while gene delivery system refers to the delivery of target genes, genes Fragments or short-chain interfering RNAs are introduced into target cells to achieve therapeutic purposes. The gene delivery system is the most critical bottleneck of gene therapy for malignant tumors, and it is also the core technical problem that restricts gene therapy for maligna...

Examples

Embodiment 1

[0050] This example discusses the effect of different ratios of polyethyleneimine tris(octadecyl)ammonium chloride (PQA) and cholesterol on the particle size of cationic polymer liposomes when the reverse evaporation method is adopted.

[0051] PQA (the weight-average molecular weight is 20,000) and cholesterol in different proportions are co-dissolved in dichloromethane, and mixed to obtain solution I; prepare deionized aqueous solution II, wherein the ratio of solution I and solution II is 1:2; After the two solutions were mixed, they were fully ultrasonically emulsified, and dichloromethane was distilled off under reduced pressure on a rotary evaporator to obtain a polymer liposome solution. As can be seen from Table 2, adjusting the mass ratio of PQA and cholesterol can obtain polymer liposomes with different particle sizes, and the particle size distribution of the cationic polymer liposomes in aqueous solution is also relatively uniform. in figure 1 It is a TEM photo of...

Embodiment 2

[0055] This example mainly provides an example of using other lipid components to prepare cationic polymer liposomes.

[0056] Weigh carboxymethyl chitosan-based tri(octadecyl)ammonium chloride (quaternary ammonium salt substitution degree is 90.0%, weight average molecular weight is 1000) 15.0mg, DOPE 12.0mg is dissolved in 3.0ml dichloromethane , oscillate evenly to obtain solution I, put it into an eggplant-shaped bottle, distill under reduced pressure on a rotary evaporator, and pass nitrogen gas from time to time until the dichloromethane is completely volatilized, then dry it in vacuum at room temperature for 24 hours, and then use 3.0 mg of water-soluble 5.0ml PBS (pH = 7.4) buffer solution II of magnetic particles was ultrasonically hydrated for 10min (thin film method); or the solution I solution II was blended and emulsified, ultrasonically 10min, after forming a stable emulsion, put it into an eggplant-shaped bottle, and Distill under reduced pressure on a rotary ev...

Embodiment 3

[0059] This example is a method for preparing PEG-modified cationic polymer liposomes. PEG-modified positive

[0060] This example is a method for preparing folic acid-modified cationic polymer liposomes. Take folic acid-modified cationic polymer liposome FA-PQA (folate-modified polylysine-based tri(octadecyl)ammonium chloride) / PQA / cholesterol as an example: first prepare FA-PQA, Then assemble, the specific experimental process is as follows:

[0061] (1) Preparation of FA-PQA:

[0062] Dissolve 2.5g of folic acid FA in 50mL of DMSO solution, add 1.5ml of triethylamine to it, dissolve 1.3g of NHS with a certain amount of DMSO, add it to the above reaction system and react in the dark for more than 12 hours. The product was filtered to obtain the NHS-FA active as a yellow solid. Take 1.0 g of NHS-FA active substance and dissolve it in 25 mL of DMSO, and add a certain amount of PQA. use Na 2 HPO 4 Adjust the pH of the reaction system to 10 with NaOH buffer solution, and r...

technical field

[0001] The invention relates to a drug and gene carrier constructed by a modified high molecular polymer, a preparation method and an application thereof, and further to a targeted quaternary ammonium salt cationic high molecular lipid gene carrier, a preparation method and an application, belonging to functional Liposome preparation and application technology. Background technique

[0002] Gene therapy is mainly to introduce exogenous gene DNA or RNA fragments into target cells or tissues to correct or compensate gene defects or inhibit abnormally expressed genes, so as to achieve the purpose of treatment, while gene delivery system refers to the delivery of target genes, genes Fragments or short-chain interfering RNAs are introduced into target cells to achieve therapeutic purposes. The gene delivery system is the most critical bottleneck of gene therapy for malignant tumors, and it is also the core technical problem that restricts gene therapy for maligna...

Examples

Embodiment 1

[0050] This example discusses the effect of different ratios of polyethyleneimine tris(octadecyl)ammonium chloride (PQA) and cholesterol on the particle size of cationic polymer liposomes when the reverse evaporation method is adopted.

[0051] PQA (the weight-average molecular weight is 20,000) and cholesterol in different proportions are co-dissolved in dichloromethane, and mixed to obtain solution I; prepare deionized aqueous solution II, wherein the ratio of solution I and solution II is 1:2; After the two solutions were mixed, they were fully ultrasonically emulsified, and dichloromethane was distilled off under reduced pressure on a rotary evaporator to obtain a polymer liposome solution. As can be seen from Table 2, adjusting the mass ratio of PQA and cholesterol can obtain polymer liposomes with different particle sizes, and the particle size distribution of the cationic polymer liposomes in aqueous solution is also relatively uniform. in figure 1 It is a TEM photo of...

Embodiment 2

[0055] This example mainly provides an example of using other lipid components to prepare cationic polymer liposomes.

[0056] Weigh carboxymethyl chitosan-based tri(octadecyl)ammonium chloride (quaternary ammonium salt substitution degree is 90.0%, weight average molecular weight is 1000) 15.0mg, DOPE 12.0mg is dissolved in 3.0ml dichloromethane , oscillate evenly to obtain solution I, put it into an eggplant-shaped bottle, distill under reduced pressure on a rotary evaporator, and pass nitrogen gas from time to time until the dichloromethane is completely volatilized, then dry it in vacuum at room temperature for 24 hours, and then use 3.0 mg of water-soluble 5.0ml PBS (pH = 7.4) buffer solution II of magnetic particles was ultrasonically hydrated for 10min (thin film method); or the solution I solution II was blended and emulsified, ultrasonically 10min, after forming a stable emulsion, put it into an eggplant-shaped bottle, and Distill under reduced pressure on a rotary ev...

Embodiment 3

[0059] This example is a method for preparing PEG-modified cationic polymer liposomes. PEG-modified positive

[0060] This example is a method for preparing folic acid-modified cationic polymer liposomes. Take folic acid-modified cationic polymer liposome FA-PQA (folate-modified polylysine-based tri(octadecyl)ammonium chloride) / PQA / cholesterol as an example: first prepare FA-PQA, Then assemble, the specific experimental process is as follows:

[0061] (1) Preparation of FA-PQA:

[0062] Dissolve 2.5g of folic acid FA in 50mL of DMSO solution, add 1.5ml of triethylamine to it, dissolve 1.3g of NHS with a certain amount of DMSO, add it to the above reaction system and react in the dark for more than 12 hours. The product was filtered to obtain the NHS-FA active as a yellow solid. Take 1.0 g of NHS-FA active substance and dissolve it in 25 mL of DMSO, and add a certain amount of PQA. use Na 2 HPO 4 Adjust the pH of the reaction system to 10 with NaOH buffer solution, and r...

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