CN102805873A - Gene drug co-carrier system using cation liposome to establish gold nano-spherical shell and preparation method - Google Patents
Gene drug co-carrier system using cation liposome to establish gold nano-spherical shell and preparation method Download PDFInfo
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Abstract
The invention relates to a gene drug co-carrier system using cation liposome to establish a gold nano-spherical shell and a preparation method. The method comprises: using phosphatides such as DPPC, MPPC and DPPE-PEG and the derivatives thereof to form the cation liposome via membrane extrusion; carrying the hydrophilic drug hydrochloric acid adriacin into the liposome in the forming process; forming the drug-carried cation liposome; adding chlorauric acid solution into cation liposome solution; adsorbing on the surface of the cation liposome; adding ascorbic acid solution for reduction; forming the gold nano-spherical shell structure; reducing sulfhydryl out of the single chain of green fluorescent protein silent gene with a disulfide bond, grafting onto the surface of the gold nano-spherical shell; and forming the gene drug co-carrier system. The gold nano-spherical shell has low toxicity and unique optical properties. The gene drug co-carrier system using cation liposome to establish the gold nano-spherical shell can provide carrier for the co-treatment of drugs and genes, can select light to illuminate the gold nano-spherical shell, and utilize light-heat conversion property to realize the co-release of the drugs and genes.
Description
Technical field
The present invention relates to the cationic-liposome is that template forms the gold nano spherical shell structure, is carrier with the gold nano spherical shell structure, and the common carrier that makes up medicine and gene is, contained medicine is cancer therapy drugs such as amycin, and contained gene is siRNA etc.
Background technology
Cationic-liposome relatively is widely used in pharmaceutical carrier and genophore, but liposome is metastable structure, and stable inadequately in body fluid, circulation time is shorter in vivo, can modify cationic-liposome, improves its stability.
Gold nano-material has the unique physicochemical characteristics of comparison, and the gold nano spherical shell structure is because of surface plasma body resonant vibration effect, the absworption peak in the near infrared region.Researcher is existing to utilize liposome to be template, at the surface deposition gold, forms nano-hollow sphere structuredly, and utilizes liposome to carry cancer therapy drugs such as doxorubicin hydrochloride, with pulsed laser irradiation gold nano spherical shell, can reach the purpose of control drug release.
The gold nano spherical shell also is widely used in the carrier of gene, utilizes sulfydryl and golden reaction, cation property copolymer is modified at the surface of gold nano hollow ball; Utilize the Electrostatic Absorption effect; Gene is adsorbed on gold nano hollow ball surface, thereby makes up genophore, the gene one terminal modified sulfydryl of going up that perhaps will deliver; Utilize sulfydryl and golden combination again, reach the purpose of delivering gene.
Research shows that in tumor therapeutic procedure, gene and medication combined treatment have good therapeutic effect, and the common carrier that makes up a gene and medicine is tied to form and is medicine and important content of gene therapeutic alliance.
The method that a lot of now structure medicine genes carry altogether is that medicine and the chemical method of gene utilization are connected together, the process more complicated of the chemical reaction that carries out, and reaction impurity later is difficult for removal; And gene is very unstable; Operating process, careless slightly, can cause the decomposition of gene; The researcher that also has has been realized carrying altogether of medicine and gene; But after carrier carries medicine and gene entering cell; Medicine and gene can not be discharged in the Cytoplasm; And medicine and gene must can discharge in cell and just can work, and the common carrier that therefore need want the method for a simple and fast to make up a gene and medicine is, and can be good at realizing the release of medicine and gene.
The common carrier that this invention utilizes the gold nano hollow ball to make up gene and medicine is to be a reasonable method; The toxicity of gold nano spherical shell structure own is little; And have the good optical performance, research shows the pulsed laser irradiation of near infrared region; Spherical shell structure is broken, thereby can discharge medicine and gene.
Summary of the invention
The objective of the invention is to utilize with the cation is the gold nano spherical shell structure that template forms, and makes up the common carrier system of a genomic medicine, for the common delivery of gene and medicine provides an excellent carrier.
Principle of the present invention is to utilize the cationic liposome to carry out medicine carrying, in liposome solutions, adds gold chloride and Reducing agent, at surface deposition one deck gold nano hollow ball shell of liposome.The one terminal modified sulfydryl of going up of the target gene that will deliver utilize the sulfydryl and the combination of gold that gene is connected on the surface of gold nano spherical shell again, thereby the common carrier that forms a medicine and gene is.
Method for preparing of the present invention is following:
A. dipalmitoyl phosphatidyl choline and single palm fibre are plucked phosphatidyl choline and between 1:1 and 1:0, feed intake, add and be modified with 1, the Macrogol 2000 of two (diphenylphosphine) ethane of 2-according to weight ratio; 1; The quality of the Macrogol 2000 of two (diphenylphosphine) ethane of 2-accounts for 5% of gross mass, and above-mentioned substance is dissolved in dibastic sodium phosphate-sodium dihydrogen phosphate buffer, makes that phospholipid concentration concentration is 40-60nM; Add doxorubicin hydrochloride, make that doxorubicin hydrochloride concentration is 5-10nM.Circulating frozen melts, and extrudes through the polycarbonate membrane of 100nm, forms the cationic-liposome of medicine carrying; Get the liposome solutions 1mL of above-mentioned preparation, adding 7-18uL concentration is the 100nM chlorauric acid solution, adds the ascorbic acid solution that 10.5uL-27uL concentration is 500nM again, forms the gold nano spherical shell structure;
B. the strand siRNA (siRNA) of 50nmol is reduced in the buffer solution of pH=3.8 and obtain being with sulfydryl strand siRNA; Join in the 1ml gold nano spherical shell solution; Under the 6000rpm condition centrifugal 10 minutes; In 30mM hydroxyethyl piperazine second sulfacid 100mM, pH=7.5 SAS, heavily disperse three times, form the gold nano spherical shell that the medicine gene carries altogether.
The volume of gold chloride and ascorbic acid adds different, and the golden thickness of the shell of formation is different, and absworption peak is also different.
The contained gene of the present invention is the gene of band sulfydryl; Can the gene that have disulfide bond be reduced, 5 '-HO-(CH2) 6-SS-(CH2) 6-spacer18-ACCCUGAAGUU-CAUCUGCACCACdCdG-(C-H2) 6-NH2 → SH-(CH2) 6-spacer18-ACCCUGAAGUU-CAUCUGCACCACdCdG-(C-H2) 6-NH
2The strand siRNA of 50nmol reduced in the buffer solution of pH=3.8 obtain being with sulfydryl strand siRNA; Join in the 1ml gold nano spherical shell solution; Under the 6000rpm condition centrifugal 10 minutes; At 30mM hydroxyethyl piperazine second sulfacid (HEPES), heavily disperse three times in the 100mM sodium acetate (pH=7.5), form the gold nano spherical shell that the medicine gene carries altogether;
Also can directly have the gene of band sulfydryl made to order; For example SH-miR-21 is dissolved in the gene of being with sulfydryl in the TE buffer solution, and concentration is 2umol/L; According to gold nano spherical shell liquor capacity than in the scope of 64:1 and 2:1, mixing the gold nano spherical shell that formation medicine gene carries altogether.
Advantage of the present invention is to utilize sophisticated liposome medicine-carried system to form nontoxic gold nano hollow ball, with the common carrier system of gold nano hollow ball gene that has been fundamental construction and medicine, for the common delivery of gene and medicine provides method
Figure of description
Fig. 1: with the cationic-liposome is the TEM photo of the gold nano spherical shell structure of template formation;
Fig. 2: medicine gene carrier altogether is a sketch map
The specific embodiment
Through example the present invention is further set forth below.
Embodiment 1:
A. the preparation of cationic-liposome: DPPC; MPPC, DPPE-PEG2000 is dissolved in (pH=7.4) in the PBS solution according to the ratio of weight ratio 40:40:10, forms the lipid solution of 60nM; Add doxorubicin hydrochloride; The concentration of the doxorubicin hydrochloride that makes is 5nM, with the circulating frozen freeze thaw method system liposome of solution according to standard, in the polycarbonate membrane through 100nm, extrudes the formation liposome.The liposome for preparing is dialysed in PBS solution, remove unnecessary doxorubicin hydrochloride.
B. the formation of gold nanoshell: get the liposome solutions of the above-mentioned preparation of 1mL, add the chlorauric acid solution of 18uL 100nM and the ascorbic acid solution of 27uL 500nM, the gold nano spherical shell absworption peak of formation is 1064nm; Again in PBS solution, the sample that makes is dialysed 12h.
C.siRNA is connected with the gold nano spherical shell: the siRNA that chooses is the green fluorescent protein silent gene, and the nucleotides sequence of positive-sense strand is classified 5 '-HO-(CH2) 6-SS-(CH2) 6-spacer18-ACCCUGAAGUU-CAUCUGCACCACdCdG-(C-H2) 6-NH2 as, and oligonucleotide at first carries out the strong stability effect; The 50nmol oligonucleotide is hatched 30min in the buffer solution of 60 ℃ pH=3.8; Be dissolved in RNA in the 50uL water, and Tris (2-carboxyethyl) phosphineHCl of adding pH=7.0 (TCEP, 0.5M); Behind the 10min; Add chloroform extraction, the water intaking layer obtains the RNA of sulfhydrylation.(3uM in 5mM TCEP pH=7.00) adds among the gold nano spherical shell solution 1mL for preparing among the step b, and is ultrasonic, placement overnight under 4 ℃ of conditions with the RNA of new reductive band sulfydryl.Under the 6000rpm condition centrifugal 10 minutes,, heavily disperse three times in the 100mM sodium acetate (pH=7.5) at 30mM HEPES.Obtain the complex of strand siRNA and gold nano spherical shell, thereby the common carrier that has made up medicine and gene is.
Embodiment 2:
A. the preparation of cationic-liposome: DPPC, MPPC, the DPPE-PEG2000 weight ratio is 90:0:10; Add PBS solution; Add doxorubicin hydrochloride, make that the concentration of doxorubicin hydrochloride is 10nM, all the other implementation steps are identical with the preparation process of cationic liposome among the embodiment 1.
B. the formation of gold nanoshell: chlorauric acid solution is different with the concentration that ascorbic acid solution adds, and the golden thickness of the shell of formation is all different with particle diameter, and the absworption peak that the plasma resonance effect produces is also different.The gold nano spherical shell absworption peak that forms among the embodiment 1 is 1064nm, in this example, adds 7uL chlorauric acid solution and 10.5uL ascorbic acid solution, and forming gold nano spherical shell absworption peak is 655nm.The sample that the makes 12h that in PBS solution, dialyses again.
C.siRNA is connected with the gold nano spherical shell: the siRNA that chooses is the green fluorescent protein silent gene, and the nucleotides sequence of positive-sense strand is classified 5 '-HO-(CH2) 6-SS-(CH2) 6-spacer18-ACCCUGAAGUU-CAUCUGCACCACdCdG-(C-H2) 6-NH2 as, and oligonucleotide at first carries out the strong stability effect; The 50nmol oligonucleotide is hatched 30min in the buffer solution of 60 ℃ pH=3.8; Be dissolved in RNA in the 50uL water, and Tris (2-carboxyethyl) phosphineHCl of adding pH=7.0 (TCEP, 0.5M); Behind the 10min; Add chloroform extraction, the water intaking layer obtains the RNA of sulfhydrylation.(3uM in 5mM TCEP pH=7.00) adds among the gold nano spherical shell solution 1mL for preparing among the step b, and is ultrasonic, placement overnight under 4 ℃ of conditions with the RNA of new reductive band sulfydryl.Under the 6000rpm condition centrifugal 10 minutes,, heavily disperse three times in the 100mM sodium acetate (pH=7.5) at 30mM HEPES.Obtain the complex of strand siRNA and gold nano spherical shell, thereby the common carrier that has made up medicine and gene is.
Embodiment 3:
A. the preparation of cationic-liposome: DPPC, MPPC, the DPPE-PEG2000 weight ratio is 90:0:10, all the other implementation steps are identical with the preparation process of cationic liposome among the embodiment 1.
B. the formation of gold nanoshell: add the chlorauric acid solution of 18uL 100nM and the ascorbic acid solution of 27uL 500nM, the gold nano spherical shell absworption peak of formation is 1064nm; Add 7uL chlorauric acid solution and 10.5uL ascorbic acid solution, forming gold nano spherical shell absworption peak is 655nm.The sample that the makes 12h that in PBS solution, dialyses again.
C. being connected of gene and gold nano spherical shell: with SH-miR-21 (20 μ mol/L) with TE buffer (10mmol/L; The Tris-HCL of PH=8.0; 1mmol/L EDTA) is diluted to certain concentration; The miR-21 concentration of band sulfydryl is 2 μ mol/L, according to pre-designed volume ratio (64:1,16:1,2:1), the SH-miR-21 of same amount is mixed in TE buffer solution with the gold nano spherical shell; Incubated at room obtains both complex after 20 minutes, thereby the common carrier that has made up medicine and gene is.
Embodiment 4:
A. the preparation of cationic-liposome: DPPC, MPPC, the DPPE-PEG2000 weight ratio is 45:45:10, all the other implementation steps are identical with the preparation process of cationic liposome among the embodiment 1.
B. the formation of gold nanoshell: chlorauric acid solution is different with the concentration that ascorbic acid solution adds, and the golden thickness of the shell of formation is all different with particle diameter, and the absworption peak that the plasma resonance effect produces is also different.The gold nano spherical shell absworption peak that forms among the embodiment 1 is 1064nm, in this example, adds 7uL chlorauric acid solution and 10.5uL ascorbic acid solution, and forming gold nano spherical shell absworption peak is 655nm.The sample that the makes 12h that in PBS solution, dialyses again.
C. being connected of gene and gold nano spherical shell: with SH-miR-21 (20 μ mol/L) with TE buffer (10mmol/L; The Tris-HCL of PH=8.0; 1mmol/L EDTA) is diluted to certain concentration; The miR-21 concentration of band sulfydryl is 2 μ mol/L, according to pre-designed volume ratio (64:1,16:1,2:1), the SH-miR-21 of same amount is mixed in TE buffer solution with the gold nano spherical shell; Incubated at room obtains both complex after 20 minutes, thereby the common carrier that has made up medicine and gene is.
Embodiment 5:
A. the preparation of cationic-liposome: DPPC, MPPC, the DPPE-PEG2000 weight ratio is 45:45:10, all the other implementation steps are identical with the preparation process of cationic liposome among the embodiment 1.
B. the formation of gold nanoshell: chlorauric acid solution is different with the concentration that ascorbic acid solution adds, and the golden thickness of the shell of formation is all different with particle diameter, and the absworption peak that the plasma resonance effect produces is also different.The gold nano spherical shell absworption peak that forms among the embodiment 1 is 1064nm, in this example, adds 7uL chlorauric acid solution and 10.5uL ascorbic acid solution, and forming gold nano spherical shell absworption peak is 655nm.The sample that the makes 12h that in PBS solution, dialyses again.
C. with SH-miR-21 (20 μ mol/L) with TE buffer (10mmol/L; The Tris-HCL of PH=8.0 1mmol/LEDTA) is diluted to certain concentration, and the miR-21 concentration of band sulfydryl is 2 μ mol/L; According to pre-designed volume ratio (64:1,16:1,2:1); The SH-miR-21 of same amount is mixed in TE buffer solution with the gold nano spherical shell, and incubated at room obtains both complex after 20 minutes, thereby the common carrier that has made up medicine and gene is.
Embodiment 6:
A. the preparation of cationic-liposome: DPPC; MPPC, the DPPE-PEG2000 weight ratio is 45:45:10, the PBS solution of adding; Forming concentration is the lipid soln of 60nM; Add doxorubicin hydrochloride, make that doxorubicin hydrochloride concentration is 10nM, all the other implementation steps are identical with the preparation process of cationic liposome among the embodiment 1.
B. the formation of gold nanoshell: chlorauric acid solution is different with the concentration that ascorbic acid solution adds, and the golden thickness of the shell of formation is all different with particle diameter, and the absworption peak that the plasma resonance effect produces is also different.In this example, add 7uL chlorauric acid solution and 10.5uL ascorbic acid solution, forming gold nano spherical shell absworption peak is 655nm.The sample that the makes 12h that in PBS solution, dialyses again.
C. (10mmol/L, the Tris-HCL of PH=8.0 1mmol/LEDTA) are diluted to certain concentration with the TE buffer with SH-miR-21 (20 μ mol/L); The miR-21 concentration of band sulfydryl is 2 μ mol/L; According to pre-designed volume ratio (64:1,32:1,, 16:1,8:1,2:1), the SH-miR-21 of same amount is mixed in TE buffer solution with the gold nano spherical shell; Incubated at room obtains both complex after 20 minutes, thereby the common carrier that has made up medicine and gene is.
Used siRNA gene order is:
The microRNA sequence of the used band sulfydryl of 5 '-HO-(CH2) 6-SS-(CH2) 6-spacer18-ACCCUGAAGUU-CAUCUGCACCACdCdG-(C-H2) 6-NH2 is:
UAGCUUAUCAGACUGAUGUUGU
Claims (4)
1. with the cationic liposome the common carrier of the medicine gene system of the gold nano hollow ball shell structure of template formation; It is characterized in that: utilize the cationic liposome to carry hydrophilic medicine; At cationic-liposome outerwrap gold; Form the gold nano spherical shell structure of inner medicine carrying, gene is connected on the surface of gold nano spherical shell structure through sulfydryl, thereby the gold nano spherical shell that makes up inner medicine carrying, outside year gene is total to carrier system.
2. system as claimed in claim 1 is characterized in that described medicine is the hydrophilic cancer therapy drug of doxorubicin hydrochloride.
3. system as claimed in claim 1 is characterized in that described gene is all genes of being with sulfydryls or can modifying out sulfydryl.
4. make up claim 1 gold nano hollow ball shell structure the medicine gene altogether carrier system method, it is characterized in that step is following:
A. dipalmitoyl phosphatidyl choline and single palm fibre are plucked phosphatidyl choline and between 1: 1 and 1: 0, feed intake, add and be modified with 1, the Macrogol 2000 of two (diphenylphosphine) ethane of 2-according to weight ratio; 1; The quality of the Macrogol 2000 of two (diphenylphosphine) ethane of 2-accounts for 5% of gross mass, and above-mentioned substance is dissolved in dibastic sodium phosphate-sodium dihydrogen phosphate buffer, makes that phospholipid concentration concentration is 40-60nM; Add doxorubicin hydrochloride, make that doxorubicin hydrochloride concentration is 5-10nM.Circulating frozen melts, and extrudes through the polycarbonate membrane of 100nm, forms the cationic-liposome of medicine carrying; Get the liposome solutions 1mL of above-mentioned preparation, adding 7-18uL concentration is the 100nM chlorauric acid solution, adds the ascorbic acid solution that 10.5uL-27uL concentration is 500nM again, forms the gold nano spherical shell structure;
B. the strand siRNA (siRNA) of 50nmol is reduced in the buffer solution of pH=3.8 and obtain being with sulfydryl strand siRNA; Join in the 1ml gold nano spherical shell solution; Under the 6000rpm condition centrifugal 10 minutes; In 30mM hydroxyethyl piperazine second sulfacid 100mM, pH=7.5 SAS, heavily disperse three times, form the gold nano spherical shell that the medicine gene carries altogether.
5. method as claimed in claim 4; It is characterized in that said step b. is: the gene SH-miR-21 of directly tailor-made band sulfydryl; The gene of band sulfydryl is dissolved in the alkaline buffer solution; Concentration is 2umol/L, according to gold nano spherical shell liquor capacity than 64: 1 with 2: 1 scope in mix the gold nano spherical shell that formation medicine gene carries altogether.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103599549A (en) * | 2013-11-22 | 2014-02-26 | 大连民族学院 | SiRNA (Small interfering Ribonucleic Acid)/anti-cancer drug combined transferring composite supporter and preparation method and application thereof |
| CN103599070A (en) * | 2013-11-26 | 2014-02-26 | 上海交通大学 | Preparation method of temperature and fluorescence probe of lipidosome loaded with gold nanocluster and anti-cancer drug |
| CN104922069A (en) * | 2015-05-28 | 2015-09-23 | 燕山大学 | Nanometer gold spherical shell photosensitive liposome and preparation method for same |
| CN104984339A (en) * | 2015-06-26 | 2015-10-21 | 燕山大学 | Oleanolic acid liposome coated with nanogold spherical shell and preparation method thereof |
| CN105561322A (en) * | 2015-12-30 | 2016-05-11 | 东北师范大学 | Multifunctional gold coated chitosan nano-material and preparation method thereof |
| CN106806901A (en) * | 2016-12-19 | 2017-06-09 | 同济大学 | A kind of isotope of redox-sensitive type VEGF siRNA/ chicken egg whites nano particles and its preparation |
| CN106891018A (en) * | 2017-03-05 | 2017-06-27 | 北京化工大学 | A kind of nanoporous gold grain and preparation method thereof |
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2012
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103599549A (en) * | 2013-11-22 | 2014-02-26 | 大连民族学院 | SiRNA (Small interfering Ribonucleic Acid)/anti-cancer drug combined transferring composite supporter and preparation method and application thereof |
| CN103599070A (en) * | 2013-11-26 | 2014-02-26 | 上海交通大学 | Preparation method of temperature and fluorescence probe of lipidosome loaded with gold nanocluster and anti-cancer drug |
| CN103599070B (en) * | 2013-11-26 | 2015-05-20 | 上海交通大学 | Preparation method of temperature and fluorescence probe of lipidosome loaded with gold nanocluster and anti-cancer drug |
| CN104922069A (en) * | 2015-05-28 | 2015-09-23 | 燕山大学 | Nanometer gold spherical shell photosensitive liposome and preparation method for same |
| CN104922069B (en) * | 2015-05-28 | 2019-04-09 | 燕山大学 | A kind of nano gold spherical shell photosensitive liposomes and preparation method thereof |
| CN104984339A (en) * | 2015-06-26 | 2015-10-21 | 燕山大学 | Oleanolic acid liposome coated with nanogold spherical shell and preparation method thereof |
| CN104984339B (en) * | 2015-06-26 | 2017-11-24 | 燕山大学 | A kind of oleanolic acid liposome of nano gold spherical shell cladding and preparation method thereof |
| CN105561322A (en) * | 2015-12-30 | 2016-05-11 | 东北师范大学 | Multifunctional gold coated chitosan nano-material and preparation method thereof |
| CN106806901A (en) * | 2016-12-19 | 2017-06-09 | 同济大学 | A kind of isotope of redox-sensitive type VEGF siRNA/ chicken egg whites nano particles and its preparation |
| CN106891018A (en) * | 2017-03-05 | 2017-06-27 | 北京化工大学 | A kind of nanoporous gold grain and preparation method thereof |
| CN106891018B (en) * | 2017-03-05 | 2019-10-18 | 北京化工大学 | A kind of nanoporous gold particle and preparation method thereof |
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Application publication date: 20121205 |