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Preparation method of polycationic liposome/calcium phosphate nanoparticle drug delivery vector

A technology of polycation and drug delivery carrier, applied in the field of gene drug carrier preparation, can solve the problems of reduced cell uptake, particle aggregation and precipitation, low transfection efficiency, etc., achieves broad application prospects, overcomes easy aggregation and precipitation, and improves stability Effect

Inactive Publication Date: 2013-05-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, like most non-viral vector-gene complexes, calcium phosphate is mainly combined with genes through electrostatic interaction, which is prone to particle aggregation and precipitation, and has poor stability, resulting in reduced cell uptake. Using calcium phosphate alone as a gene carrier, Its transfection efficiency is usually lower than that of liposome vectors

Method used

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  • Preparation method of polycationic liposome/calcium phosphate nanoparticle drug delivery vector
  • Preparation method of polycationic liposome/calcium phosphate nanoparticle drug delivery vector
  • Preparation method of polycationic liposome/calcium phosphate nanoparticle drug delivery vector

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Embodiment 1

[0028] (1) Take 1ml of 75 mmol / L calcium chloride aqueous solution, add 0.1mg / ml oligonucleotide (ODN, 5'-CTCAGTTAGGGTTAG-3', 0.3nmol phosphate) solution 1ml, 0.4ml (4mg) 1% poloxamer 188, gently shaken and incubated for 30 minutes, as mixture A. Take another 2 ml of 6 mmol / L disodium hydrogen phosphate aqueous solution, add 0.5 ml of 25 mmol / L sodium citrate aqueous solution, and mix with 0.6 ml (6 mg) 1% poloxamer 188, as the mixed solution B. Add mixed solution B to mixed solution A dropwise, and stir at room temperature (25°C) for 30 min to obtain 5.5 ml of calcium phosphate nanoparticle solution (ODN mass: 0.1 mg). (2) Take soybean lecithin 10mg, cholesterol 5mg, polyethyleneimine-cholesterol (PEI-Chol) 1mg (PEI molecular weight 800 Da, amino molarity 20nmol) dissolved in 10ml chloroform, vacuum rotary evaporation to dry chloroform, add 10ml Deionized water was hydrated for 10 minutes under ultrasonic conditions in a water bath, and then further ultrasonic treatment was ...

Embodiment 2

[0031] (1) Take 1ml of 50 mmol / L calcium chloride aqueous solution, add 0.1mg / ml green fluorescent protein particle (pEGFP-N1, GenBank Accession #U55762, 4.7kb, molar mass of phosphate radical is 0.3nmol) solution 1ml, 0.4ml ( 4mg) 1% poloxamer 188, shake gently and incubate for 30 minutes as mixture A. Take another 2ml of 25mmol / L disodium hydrogen phosphate aqueous solution, add 2ml of 25mmol / L sodium citrate aqueous solution, and mix with 0.8ml (8mg) 1% poloxamer 188 as the mixed solution B. Add the mixed solution B to the mixed solution A dropwise, and stir at room temperature for 30 min to obtain a calcium phosphate nanoparticle solution (the mass of pEGFP-N1 plasmid is 0.1 mg). (2) Take polyethyleneimine-cholesterol 5mg (PEI molecular weight 800 Da, amino molarity 100nmol), DOPE 2.5mg dissolved in 10ml chloroform, vacuum rotary evaporation to dry the chloroform, add 5ml deionized water, in water bath ultrasonic condition Hydrate for 10 minutes, and then use an ultrasoni...

Embodiment 3

[0034] (1) Mix 2.52ml of polyethylene glycol octylphenyl ether (Triton X-1), 1.68ml of n-hexanol, and 2.8ml of cyclohexane to form 7ml of an oil phase ternary system. Mix 100 μL of 250 mmol / L calcium chloride aqueous solution, 100 μL of 25 mmol / L disodium hydrogen phosphate aqueous solution, 500 μL of 0.1 mg / ml pEGFP-N1 plasmid (the molar mass of phosphate radical is 0.15 nmol) and 100 μL of 15 mmol / L sodium citrate aqueous solution Mix to make a mixture. Add the mixed solution drop by drop into the ternary system under stirring, stir at room temperature for 0.5 hours, dialyze the mixed solution with a dialysis bag (molecular weight cut-off 14000 Da) for 72 hours to remove the organic phase, the dialysate is absolute ethanol, take the retained solution and Disperse in 5ml of deionized water to obtain the calcium phosphate nanoparticle solution (the mass of pEGFP-N1 plasmid is 0.05mg). (2) Take PEI-Chol 5mg (PEI molecular weight 800 Da, amino molarity 100nmol), DOPE 2.5mg diss...

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Abstract

The invention discloses a preparation method of a gene drug delivery vector with polycationic liposome coating calcium phosphate nanoparticles. The calcium phosphate nanoparticles are prepared with an improved coprecipitation method or a reverse microemulsion method, and the stability of the calcium phosphate nanoparticles is improved; and an amphiphilic compound polyethyleneimine-cholesterol, phosphatide and / or cholesterin are / is taken as liposome membrane material(s), and incubation is performed by adopting both the lipidosome and the calcium phosphate nanoparticles, so that the gene drug delivery vector is obtained. According to the preparation method, the stability of the calcium phosphate nanoparticles is improved through improvements of a calcium phosphate nanoparticle prescription and a preparation process; the trend that the calcium phosphate nanoparticles can gather and precipitate easily is further overcome by a lipidosome coating technology; and polycations on the surface of the lipidosome increase the uptake of a cell for the vector and improve the endosomal escape capability. The prepared compound gene drug delivery vector improves the cellular uptake and transfection efficiencies on the basis of safely and economically reservation of the calcium phosphate vector, and has a broad application prospect.

Description

(1) Technical field [0001] The present invention relates to the field of preparation of a gene drug carrier, in particular to a method for preparing a gene drug delivery carrier prepared by polycationic liposomes encapsulating calcium phosphate nanoparticles, that is, a method for preparing a polycationic liposome / calcium phosphate nanoparticle drug delivery carrier . (2) Background technology [0002] Gene therapy provides good prospects for the treatment of hereditary diseases, infectious diseases and cancer, and the development of a safe and effective gene delivery system will become one of the most important factors for the success of gene therapy. As a research hotspot, the non-viral vector has a good application prospect due to its simple preparation, unlimited gene size and no specific immune response in vivo. Among them, cationic liposomes and cationic polymers are the two most widely studied non-viral vectors because they can form complexes with genes through elect...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K47/34A61K47/32A61K47/02A61K47/10
Inventor 陈金亮章建军孙晓译高建青梁文权
Owner ZHEJIANG UNIV
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