CN102697732A - Preparation method of gene and hydrophobic drug co-supported PEG (polyethyleneglycol) nanoparticles - Google Patents
Preparation method of gene and hydrophobic drug co-supported PEG (polyethyleneglycol) nanoparticles Download PDFInfo
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- CN102697732A CN102697732A CN2012101534971A CN201210153497A CN102697732A CN 102697732 A CN102697732 A CN 102697732A CN 2012101534971 A CN2012101534971 A CN 2012101534971A CN 201210153497 A CN201210153497 A CN 201210153497A CN 102697732 A CN102697732 A CN 102697732A
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Abstract
The invention discloses a preparation method of gene and hydrophobic drug co-supported PEG (polyethyleneglycol) nanoparticles. The method comprises the following steps of: 1) preparing beta-cyclodextrin modified polycation solution; 2) preparing ferrocene modified polyethylene glycol solution; 3) mixing solutions in step 1) and step 2), standing after ultrasound is carried out; 4) preparing a hydrophobic drug solution; 5) dripping the hydrophobic drug solution prepared in step 4) into the solution in step 3), stirring away from light, dialyzing and lyophilizing to obtain inclusion powder; 6) preparing an inclusion solution; 7) preparing a DNA (deoxyribonucleic acid) solution; and 8) adding the solution in step 6) to isovolumetric solution in step 7), performing eddy mixing and standing to obtain the gene and hydrophobic drug co-supported PEG nanoparticles. The gene and hydrophobic drug co-supported PEG nanoparticles prepared by virtue of a host-guest effect; and the preparation method is simple and easy in operation, so that the stability of the gene and hydrophobic drug co-supported PEG nanoparticles in physiological salt solution can be remarkably improved.
Description
Technical field
The present invention relates to a kind of gene and dewatering medicament load P EGization nanometer particle process method altogether.It has combined gene therapy and chemotherapy, belongs to the method for preparing and the technology of the new and effective non-virus gene delivery system of field of gene.
Background technology
Cancer (malignant tumor) has become the important diseases that influences human health owing to lack effective treatment means, and the annual expense number that directly is used for oncotherapy is in 10,000,000,000.One of the most obstinate fort that cancer becomes that scientist do not capture as yet also is one of the most challenging problem of contemporary science research.Chemotherapy is one of important means of malignant tumor Comprehensive Treatment.But because its drug dose is big, lack specificity, make and when suppressing cancerous tissue, the normal human cell is also caused serious toxic and side effects, the patient is easy to generate multi-drug resistant during treating.Therefore, be to reduce the toxic and side effects of anticarcinogen to the human normal cell, the pharmaceutical carrier of seeking safety and stability to chemotherapeutic further develop most important.
Gene therapy is that people's healthy gene or medicative gene are imported human body cell through certain way, with defective or the performance therapeutical effect of correcting gene, thus the biomedical new technique of treatment disease.Its key is to select suitable genophore and method of gene introduction, makes genes of interest can in target cell, obtain safe, efficient, controlled and stable expression.Polycation becomes one of main developing direction of gene therapy vector owing to have characteristics such as preparation is simple, immunogenicity is low, year gene capacity is big.But this type polycation/DNA assembly is assembled in vivo easily, and is cleared out of in the body by reticuloendothelial system, reduces the gene carrier and ties up to intravital circulation time and influence gene transfection efficient.The gene that preparation Polyethylene Glycol (PEG) is changed transmits system and can effectively address the above problem.
Existing discovering; Be transported to therapeutic gene and cancer therapy drug in the tumor cell simultaneously; Make up the genomic medicine complex carrier of highly effective and safe, reach gene therapy and chemotherapeutic synergism, realize effective transmission of gene and cancer therapy drug; Effectively solve the multidrug resistance of cancerous cell, have important science and using value.Therefore introduce the PEG segment through the Subjective and Objective effect of beta-schardinger dextrin-and ferrocene; Inclusion used load medicine through beta-schardinger dextrin-and dewatering medicament; Can prepare gene and dewatering medicament load P EGization nanoparticle altogether, new method for preparing is provided for adopting chemicals and gene therapeutic alliance malignant tumor.
Summary of the invention
The purpose of this invention is to provide a kind of gene and dewatering medicament load P EGization nanometer particle process method altogether.The step that gene and dewatering medicament are total to load P EGization nanometer particle process method is following:
1) configuration concentration is the beta-schardinger dextrin-modification said polycation solution of 0.5～4 mg/mL;
2) configuration concentration is the ferrocene modification polyglycol solution of 0.5～4 mg/mL;
3) with step 1) and step 2) the solution mixing, the mol ratio that makes beta-schardinger dextrin-and ferrocene is 4:1～2:1, leaves standstill behind ultrasonic 30 min;
4) configuration concentration is the dewatering medicament solution of 2 mg/mL;
5) the dewatering medicament drips of solution with the step 4) preparation is added in the step 3) solution; The mol ratio that makes beta-schardinger dextrin-and dewatering medicament is 1:1～1:4; Lucifuge stirs 24 h; Dialysis 8～24h, lyophilizing obtains beta-schardinger dextrin-and modifies polycation/ferrocene modification Polyethylene Glycol/dewatering medicament clathrate powder;
6) configuration concentration is beta-schardinger dextrin-modification polycation/ferrocene modification Polyethylene Glycol/dewatering medicament inclusion complex in solution of 0.05～1 mg/mL;
7) configuration concentration is the dna solution of 75～100 μ g/mL;
8) step 6) solution is joined in the equal-volume step 7) solution, whirlpool mixes and leaves standstill 30 min, obtains gene and dewatering medicament load P EGization nanoparticle altogether.
Described beta-schardinger dextrin-is modified polycation and comprised: beta-schardinger dextrin-modifying polyethyleneimine, beta-schardinger dextrin-are modified polylysine or beta-schardinger dextrin-beautify chitosan.Described dewatering medicament comprises: amycin or paclitaxel.
The present invention prepares the PEGization nanoparticle of gene and the common load of dewatering medicament through the Subjective and Objective effect; The PEG segment is introduced in Subjective and Objective effect through beta-schardinger dextrin-and ferrocene; Through the inclusion used load medicine of beta-schardinger dextrin-and dewatering medicament, new method for preparing is provided for adopting chemicals and gene therapeutic alliance malignant tumor.Method for preparing is simple to operation, and the useful load of the degree of PEGization, medicine and gene is prone to regulate, and has significantly improved gene and the dewatering medicament stability of load nano particle in physiological solt solution altogether.
Description of drawings
Fig. 1 is common load P EGization nanoparticle of gene and dewatering medicament and PEI-g-CD/DOX/ DNA assembly are placed different time in physiological solt solution a change of size;
Fig. 2 is that gene and dewatering medicament are total to the transmission electron microscope photo of load P EGization nanoparticle after physiological solt solution is placed 1h;
Fig. 3 is the Zeta-potential value that gene and dewatering medicament are total to load P EGization nanoparticle and PEI-g-CD/DNA assembly and PEI-g-CD/DOX/ DNA assembly;
Fig. 4 is the vitro drug release curve that gene and dewatering medicament are total to load P EGization nanoparticle.
The specific embodiment
The step that a kind of gene and dewatering medicament are total to load P EGization nanometer particle process method is following:
1) configuration concentration is the beta-schardinger dextrin-modification said polycation solution of 0.5～4 mg/mL;
2) configuration concentration is the ferrocene modification polyglycol solution of 0.5～4 mg/mL;
3) with step 1) and step 2) the solution mixing, the mol ratio that makes beta-schardinger dextrin-and ferrocene is 4:1～2:1, leaves standstill behind ultrasonic 30 min;
4) configuration concentration is the dewatering medicament solution of 2 mg/mL;
5) the dewatering medicament drips of solution with the step 4) preparation is added in the step 3) solution; The mol ratio that makes beta-schardinger dextrin-and dewatering medicament is 1:1～1:4; Lucifuge stirs 24 h; Dialysis 8～24h, lyophilizing obtains beta-schardinger dextrin-and modifies polycation/ferrocene modification Polyethylene Glycol/dewatering medicament clathrate powder;
6) configuration concentration is beta-schardinger dextrin-modification polycation/ferrocene modification Polyethylene Glycol/dewatering medicament inclusion complex in solution of 0.05～1 mg/mL;
7) configuration concentration is the dna solution of 75～100 μ g/mL;
8) step 6) solution is joined in the equal-volume step 7) solution, whirlpool mixes and leaves standstill 30 min, obtains gene and dewatering medicament load P EGization nanoparticle altogether.
Described beta-schardinger dextrin-is modified polycation and comprised: beta-schardinger dextrin-modifying polyethyleneimine, beta-schardinger dextrin-are modified polylysine or beta-schardinger dextrin-beautify chitosan.Described dewatering medicament comprises: amycin or paclitaxel.
Embodiment 1:
Configuration concentration is that beta-schardinger dextrin-modifying polyethyleneimine (PEI-g-CD) solution of 4 mg/mL and ferrocene that concentration is 4 mg/mL are modified the Polyethylene Glycol (solution of PEG-Fc).Is the mixed of 2:1 with two kinds of solution by the mol ratio of β-CD among the PEI-g-CD and PEG-Fc, set aside for use behind ultrasonic 30 min.2 mg/mL amycin (DOX) dimethyl sulphoxide solutions are added drop-wise in the above-mentioned mixed solution; Make that the mol ratio of CD and DOX is the ratio of 1:1 among the PEI-g-CD, lucifuge stirs 24 h, 8 h that dialyse again ~ 24 h; Lyophilizing obtains PEI-g-CD/PEG-Fc/DOX clathrate powder.PEI-g-CD/PEG-Fc/DOX clathrate powder by above-mentioned preparation; Adopt ultraviolet-visible spectrophotometer (UV) to measure the drug loading and the envelop rate of amycin (DOX); Recording drug loading is 9%; Envelop rate is 57%, and effectively enclose dewatering medicament DOX of beta-schardinger dextrin-modifying polyethyleneimine is described.
Configuration concentration is the PEI-g-CD/PEG-Fc/DOX solution of 0.5 mg/mL and p53 (antioncogene) solution that concentration is 75 μ g/mL.Get above-mentioned PEI-g-CD/PEG-Fc/DOX solution and mix with 250 μ L dna solution equal-volume whirlpools and leave standstill 30 min, be prepared into gene and dewatering medicament load P EGization nanoparticle altogether.Gene and dewatering medicament by above-mentioned preparation are total to load P EGization nanoparticle, utilize dynamic light scattering (DLS) and transmission electron microscope (TEM) the research stability of load nano particle under physiological salt concentration altogether, and the result sees Fig. 1, Fig. 2.Fig. 1 is for being total to load nano particle and PEI-g-CD/DOX/ DNA assembly are placed the particle diameter of different time in physiological solt solution situation of change, and Fig. 2 is for being total to the transmission electron microscope photo of load P EGization nanoparticle after physiological solt solution is placed 1h.Experimental result shows the less gathering in physiological solt solution of common load P EGization nanoparticle, and stability better.Adopt the Zeta-potential analyser to study the gene of above-mentioned preparation and the surface potential that dewatering medicament is total to load P EGization nanoparticle, because segmental shielding action of PEG and volume excluding effect, the Zeta-potential of assembly significantly reduces, and sees Fig. 3.
Embodiment 2:
Configuration concentration is that the beta-schardinger dextrin-of 2 mg/mL is modified the ferrocene modification Polyethylene Glycol that polylysine (PLL-g-CD) solution and concentration the are 2 mg/mL (solution of PEG-Fc).Is the mixed of 3:1 with two kinds of solution by the mol ratio of β-CD among the PLL-g-CD and PEG-Fc, set aside for use behind ultrasonic 30 min.2 mg/mL paclitaxel (PTX) chloroformic solutions are added drop-wise in the above-mentioned mixed solution; Make that the mol ratio of CD and PTX is the ratio of 1:2 among the PLL-g-CD, lucifuge stirs 24 h, 8 h that dialyse again ~ 24 h; Lyophilizing obtains PLL-g-CD/PEG-Fc/PTX clathrate powder.Configuration concentration is the PLL-g-CD/PEG-Fc/PTX solution of 0.5 mg/mL and the p16 that concentration is 75 μ g/mL (many TIF 1 genes) solution.Get above-mentioned PLL-g-CD/PEG-Fc/PTX solution and mix with 250 μ L dna solution equal-volume whirlpools and leave standstill 30 min, be prepared into gene and dewatering medicament load P EGization nanoparticle altogether.
Embodiment 3:
Configuration concentration is that beta-schardinger dextrin-beautify chitosan (CS-g-CD) solution of 0.5 mg/mL and ferrocene that concentration is 1 mg/mL are modified the Polyethylene Glycol (solution of PEG-Fc).Is the mixed of 4:1 with two kinds of solution by the mol ratio of β-CD among the CS-g-CD and PEG-Fc, set aside for use behind ultrasonic 30 min.2 mg/mL paclitaxel (PTX) chloroformic solutions are added drop-wise in the above-mentioned mixed solution, make that the mol ratio of CD and PTX is the ratio of 1:4 among the CS-g-CD, lucifuge stirs 24 h, 8 h that dialyse again ~ 24 h, and lyophilizing obtains CS-g-CD/PEG-Fc/PTX clathrate powder.Configuration concentration is that CS-g-CD/PEG-Fc/PTX solution and the concentration of 0.5 mg/mL is the p53 solution of 75 μ g/mL.Get above-mentioned CS-g-CD/PEG-Fc/PTX solution and mix with 250 μ LDNA solution equal-volume whirlpools and leave standstill 30 min, be prepared into gene and dewatering medicament load P EGization nanoparticle altogether.
Embodiment 4:
Configuration concentration is that beta-schardinger dextrin-modifying polyethyleneimine (PEI-g-CD) solution of 1 mg/mL and ferrocene that concentration is 0.5 mg/mL are modified the Polyethylene Glycol (solution of PEG-Fc).Is the mixed of 3:1 with two kinds of solution by the mol ratio of β-CD among the PEI-g-CD and PEG-Fc, set aside for use behind ultrasonic 30 min.2 mg/mL amycin (DOX) dimethyl sulphoxide solutions are added drop-wise in the above-mentioned mixed solution; Make that the mol ratio of CD and DOX is the ratio of 1:3 among the PEI-g-CD, lucifuge stirs 24 h, 8 h that dialyse again ~ 24 h; Lyophilizing obtains PEI-g-CD/PEG-Fc/DOX clathrate powder.Configuration concentration is the PEI-g-CD/PEG-Fc/DOX solution of 1 mg/mL and pORF-hTRAIL (tumor necrosin relative death inducing ligand plasmid) solution that concentration is 100 μ g/mL.Get above-mentioned PEI-g-CD/PEG-Fc/DOX solution and mix with 3.75 mL dna solution equal-volume whirlpools and leave standstill 30 min, be prepared into gene and dewatering medicament load P EGization nanoparticle altogether.
Gene and dewatering medicament by above-mentioned preparation are total to load P EGization nanoparticle, and utilization ultraviolet-visible spectrophotometer (UV) is measured the release profiles of amycin (DOX).The common load nano particle solution of 5 mL embodiment, 4 preparations is joined in the bag filter that molecular cut off is 3500 Da, bag filter is placed 10 mL PBS solution, then in 37 ℃ of waters bath with thermostatic control, shaking table concussion dialysis down.At set intervals, take out 1 mL dialysis solution, and add the fresh PBS solution of 1 mL.Calculate the burst size of DOX through the ultraviolet absorption value that detects DOX in the releasing solution, the result is as shown in Figure 4.
Embodiment 5:
Configuration concentration is that beta-schardinger dextrin-modifying polyethyleneimine (PEI-g-CD) solution of 1 mg/mL and ferrocene that concentration is 2 mg/mL are modified the Polyethylene Glycol (solution of PEG-Fc).Is the mixed of 2:1 with two kinds of solution by the mol ratio of β-CD among the PEI-g-CD and PEG-Fc, set aside for use behind ultrasonic 30 min.2 mg/mL amycin (DOX) dimethyl sulphoxide solutions are added drop-wise in the above-mentioned mixed solution; Make that the mol ratio of CD and DOX is the ratio of 1:4 among the PEI-g-CD, lucifuge stirs 24 h, 8 h that dialyse again ~ 24 h; Lyophilizing obtains PEI-g-CD/PEG-Fc/DOX clathrate powder.By the PEI-g-CD/PEG-Fc/DOX clathrate powder of above-mentioned preparation, adopt ultraviolet-visible spectrophotometer (UV) to measure the drug loading and the envelop rate of amycin (DOX), recording drug loading is 5.6%, envelop rate is 48%.
Configuration concentration is that PEI-g-CD/PEG-Fc/DOX solution and the concentration of 0.05 mg/mL is the dna solution that contains p53 (antioncogene) of 100 μ g/mL.Get above-mentioned PEI-g-CD/PEG-Fc/DOX solution and mix with 24 μ L dna solution equal-volume whirlpools and leave standstill 30 min, be prepared into gene and dewatering medicament load P EGization nanoparticle altogether.
Configuration concentration be PEI-g-CD/PEG-Fc/DOX solution and the concentration of 0.05 mg/mL be 100 μ g/mL contain the pEGFP dna solution of (the GFP egfp grain does not contain antioncogene).Get above-mentioned PEI-g-CD/PEG-Fc/DOX solution and mix with 24 μ L dna solution equal-volume whirlpools and leave standstill 30 min, be prepared into gene and dewatering medicament load P EGization nanoparticle altogether.
Gene and dewatering medicament by above-mentioned preparation are total to load P EGization nanoparticle, adopt mtt assay, utilize ELIASA to carry out the cytotoxicity dynamic experiment.Place human liver cancer cell HepG2 cell to hatch 48 h, 72 h, 96 h, 144 h respectively the gene that contains two kinds of different plasmids and the common load P EGization nanoparticle of dewatering medicament of the embodiment of the invention 5 preparations; In cell culture fluid, add 5 mg/mL tetrazolium salts (MTT) solution then; Continue to cultivate 4 h, inhale that to go original fluid, every hole to add 200 μ LDMSO dissolving crystallized; Measure every hole OD value in 570 nm places with ELIASA at last, and calculate the cytoactive under the variable concentrations.Can know that by the result the common load P EGization nanoparticle that contains the p53 antioncogene is not bigger than not containing the common load P EGization nanoparticle toxicity of antioncogene, and p53 antioncogene and amycin combined effect are described, kills the HepG2 cell gradually.
Claims (3)
1. gene and dewatering medicament load P EGization nanometer particle process method altogether is characterized in that its step is following:
1) configuration concentration is the beta-schardinger dextrin-modification said polycation solution of 0.5～4 mg/mL;
2) configuration concentration is the ferrocene modification polyglycol solution of 0.5～4 mg/mL;
3) with step 1) and step 2) the solution mixing, the mol ratio that makes beta-schardinger dextrin-and ferrocene is 4:1～2:1, leaves standstill behind ultrasonic 30 min;
4) configuration concentration is the dewatering medicament solution of 2 mg/mL;
5) the dewatering medicament drips of solution with the step 4) preparation is added in the step 3) solution; The mol ratio that makes beta-schardinger dextrin-and dewatering medicament is 1:1～1:4; Lucifuge stirs 24 h; Dialysis 8～24h, lyophilizing obtains beta-schardinger dextrin-and modifies polycation/ferrocene modification Polyethylene Glycol/dewatering medicament clathrate powder;
6) configuration concentration is beta-schardinger dextrin-modification polycation/ferrocene modification Polyethylene Glycol/dewatering medicament inclusion complex in solution of 0.05～1 mg/mL;
7) configuration concentration is the dna solution of 75～100 μ g/mL;
8) step 6) solution is joined in the equal-volume step 7) solution, whirlpool mixes and leaves standstill 30 min, obtains gene and dewatering medicament load P EGization nanoparticle altogether.
2. a kind of gene according to claim 1 and dewatering medicament be load P EGization nanometer particle process method altogether, and it is characterized in that described beta-schardinger dextrin-modification polycation comprises: beta-schardinger dextrin-modifying polyethyleneimine, beta-schardinger dextrin-are modified polylysine or beta-schardinger dextrin-beautify chitosan.
3. a kind of gene according to claim 1 and dewatering medicament be load P EGization nanometer particle process method altogether, it is characterized in that described dewatering medicament comprises: amycin or paclitaxel.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104069507A (en) * | 2014-06-11 | 2014-10-01 | 广州吉家庄生物科技有限公司 | High-strength supramolecular hydrogel, as well as preparation method and application thereof |
| CN104530441A (en) * | 2015-01-19 | 2015-04-22 | 中山大学 | Amylase modified product for co-delivery of genes and antitumor drugs as well as preparation method and application thereof |
| CN106197718A (en) * | 2016-08-31 | 2016-12-07 | 北京埃德万斯离子束技术研究所股份有限公司 | A kind of film temperature sensor and preparation method |
| CN114377144A (en) * | 2022-01-28 | 2022-04-22 | 南通大学 | PH/active oxygen dual-responsive supramolecular polypeptide prodrug nanoparticle |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101716346A (en) * | 2009-12-16 | 2010-06-02 | 中山大学 | Supramolecular hydrogel gene vector material, and preparation method and application thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101716346A (en) * | 2009-12-16 | 2010-06-02 | 中山大学 | Supramolecular hydrogel gene vector material, and preparation method and application thereof |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104069507A (en) * | 2014-06-11 | 2014-10-01 | 广州吉家庄生物科技有限公司 | High-strength supramolecular hydrogel, as well as preparation method and application thereof |
| CN104069507B (en) * | 2014-06-11 | 2017-04-12 | 广州吉家庄生物科技有限公司 | High-strength supramolecular hydrogel, as well as preparation method and application thereof |
| CN104530441A (en) * | 2015-01-19 | 2015-04-22 | 中山大学 | Amylase modified product for co-delivery of genes and antitumor drugs as well as preparation method and application thereof |
| CN104530441B (en) * | 2015-01-19 | 2017-08-25 | 中山大学 | A kind of amylose modifier transmitted altogether for gene and antineoplastic and preparation method and application |
| CN106197718A (en) * | 2016-08-31 | 2016-12-07 | 北京埃德万斯离子束技术研究所股份有限公司 | A kind of film temperature sensor and preparation method |
| CN106197718B (en) * | 2016-08-31 | 2019-08-27 | 北京埃德万斯离子束技术研究所股份有限公司 | A kind of film temperature sensor and preparation method |
| CN114377144A (en) * | 2022-01-28 | 2022-04-22 | 南通大学 | PH/active oxygen dual-responsive supramolecular polypeptide prodrug nanoparticle |
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Application publication date: 20121003 |