CN1306965C - Production of gene carrier and use thereof - Google Patents
Production of gene carrier and use thereof Download PDFInfo
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- CN1306965C CN1306965C CNB2004100233474A CN200410023347A CN1306965C CN 1306965 C CN1306965 C CN 1306965C CN B2004100233474 A CNB2004100233474 A CN B2004100233474A CN 200410023347 A CN200410023347 A CN 200410023347A CN 1306965 C CN1306965 C CN 1306965C
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- hydroxyapatite nanoparticle
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Abstract
The present invention relates to material for the field of medicine, particularly to a preparation method of gene carrier material. The present invention is characterized in that Ca(NO3)2 solution and (NH4)H2PO4 solution are mixed by the proportion of the volume ratio of 5 to 3 at the rotary speed of 1500 circles/minute; obtained precipitate is put in an autoclave and treated for 4 hours at the rotary speed of 200 mv and 200 circles/minute of 200 DEG C for obtaining a hydroxyapatite nanometer particle. The structure of the hydroxyapatite nanometer particle is similar to the structures of bones and teeth. The hydroxyapatite nanometer particle is biological ceramic material with good biocompatibility and can be prepared into requisite geometric forms according to application requirements. The material has large surface energy, strengthens DNA carrying capability, solves the problems of low transduction rate of a gene carrier, easy medicine leakage from liposomes, difficult preparation, etc., ensures dispersion stability in the process of use, and satisfies the effectiveness and the safety of gene disease treatment.
Description
Technical field:
The present invention relates to the material that medical domain is used, especially a kind of preparation method of gene vector material and application.
Background technology:
Gene therapy provides wide treatment prospect for the many important diseases of the mankind such as genetic diseases, tumor, cardiovascular disease and neurodegenerative diseases.Gene therapy is to import body in by carrier or other approach the external source genes of interest and demonstrate a kind of means that corresponding biological effect reaches the treatment disease in target tissue and target cell, and the treatment carrier that possesses effectiveness and safety is still the problem of making great efforts to capture.
Effectiveness:
(1) genes of interest that is mediated can enter desired target cell efficiently;
(2) do not destroyed by various complements and various enzyme in blood plasma or the histiocyte;
(3) transducible gene can see through nuclear membrane, enters nucleus, and is incorporated in the chromosomal DNA, thereby obtain genetically modified efficient, stably express and bring into play therapeutical effect.
Safety:
(1) carrier that is used for gene transfer can not cause the immunoreation that the host is serious;
(2) can not cause the infection of sexual cell;
(3) can not cause inserting the gene inactivation in site and gene mutation etc.
In order to overcome the defective of viral vector, a series of non-virus type carrier just is familiar with by people and is utilized.As cationic-liposome (lipofectin), poly-D-lysine (ploy-L-lysine), polymine (PEI), dendroid high polymer (dendrimers).Show multiple potential superioritys such as low toxicity, low immunity, high capacity, preparation easy to operate, easy, easy modification, making design and developing new better gene therapy vector becomes possibility.Non-virus carrier also is faced with many challenges at present, though higher as liposome gene transfection efficient on some cellular level, even inject tumor in vivo, its efficient is still not high, and does not have targeting.Therefore setting up a kind of non-virus, non-immunogenicity, have targeting transfer vector in the genosome of highly effective and safe of targeting, has been the task of top priority of gene therapy.
Summary of the invention:
The purpose of this invention is to provide a kind of hydroxyapatite as genophore, its structure is similar with tooth to bone, is the bioceramic material with good biocompatibility, is the treatment carrier that possesses effectiveness and safety in gene therapy.
Technical scheme of the present invention comprise hydroxyapatite nanoparticle preparation, the preparation of hydroxyapatite nanoparticle suspension and acute toxicity test, hydroxyapatite nanoparticle combines with DNA and nano-particle-dna complex in-vitro transfection, concrete preparation process is as follows:
(1) hydroxyapatite nanoparticle preparation:
With analytical pure Ca (NO
3)
2(NH
4) H
2PO
4Be dissolved in respectively in the deionized water, be made into the solution of 1mol/L, with Ca (NO
3)
2(NH
4) H
2PO
4Solution mixes under the rotating speed of 1500 commentaries on classics/min with 5: 3 ratio of volume ratio, and with the pH value of NaOH regulator solution in 11～12 scopes, stir after 10 hours aging 24 hours; Again the precipitate that obtains is put into autoclave, under 200 ℃, 200mv, 200 rev/mins rotating speed, handled 4 hours, obtain hydroxyapatite nanoparticle through cleaning, filtration, drying again;
(2) preparation of hydroxyapatite nanoparticle suspension and acute toxicity test:
Get hydroxyapatite nanoparticle 0.5g, put into centrifuge tube, add deionized water 20ml, prepare the hydroxyapatite suspension, and add dispersant, suspension is fully disperseed and stablize with ultrasonic dispersing.Be respectively the hydroxyapatite nanoparticle suspension tail vein injection white mice of 4.5mg/ml, 1.5mg/ml, 0.5mg/ml with Concentraton gradient, continuous two weeks are observed death and the toxic reactions of animals;
(3) hydroxyapatite nanoparticle biocompatibility experiment:
With the SGC-7901 cell inoculation in 96 hole tissue culturing plates; The nano-particle suspension that adds variable concentrations continues to carry out MTT mensuration behind the cultivation 48h.Colorimetry is determined great-hearted cell number, and variance analysis method adopts the SPSS9.0 statistical software to carry out.
(4) hydroxyapatite nanoparticle combines experiment with DNA:
Getting agar Icing Sugar system gel hydroxyapatite nanoparticle suspension dilution adds PEGFP-N1 and is mixed that (VORTEX2GENIE USA), leaves standstill 30min to 10min, and is centrifugal; With PEGFP-N11 μ g is contrast, gets the supernatant agarose gel electrophoresis and detects.The hydroxyapatite nanoparticle suspension also transfers to different pH value with it, it is put into the eppendorf pipe respectively mix with PEGFP-N1, is mixed, and room temperature leaves standstill 30min, and is centrifugal; Getting the supernatant agarose gel electrophoresis detects;
(5) nano-particle-dna complex in-vitro transfection cell experiment:
With the SGC-7901 cell by be inoculated in culture dish add be furnished with the DMEM culture medium culturing 24h of calf serum after, add the nano-particle dna complex at culture dish; With simple PEGFP-N1 expression plasmid DNA and with the bonded PEGFP-N1 expression plasmid of liposome DNA in contrast.Mixed culture is respectively at 24,48, observed result under the fluorescence microscope behind the 72h;
In the hydroxyapatite nanoparticle preparation method, the also available CaCI of raw material
2And H
3PO
4Be raw material, available HNO
3And NH
4NO
3Come the regulator solution pH value.
Hydroxyapatite nanoparticle is gone back portability antitumor and antiviral gene and is carried out antitumor and antiviral therapy except combining as the genophore with the PEGFP-N1 gene; Can be made into laboratory gene transfection trial-production commonly used; Combine with gene such as Moisture factor and can be made into skin-protection product, combine with somatomedin and can be used for the treatment burn.
The structure of the present invention's preparation is similar with tooth to bone, has bioceramic material---the hydroxyapatite gene carrier of good biocompatibility, has overcome other carrier material and has caused defective because of reasons such as biocompatibility differences.Nano-grade hydroxy apatite can be prepared into needed geometric shape, three dimensional structure, density, bore open, mechanical strength and purity according to the requirement of using, and has characteristics such as small-size effect, skin effect; Granule has big surface energy, strengthens the ability that it carries DNA, has solved that genophore transduction rate is low, the easy problem such as seepage and difficult preparation from liposome of medicine.By the nano-grade hydroxy apatite granule is carried out surface modification, guaranteed its dispersion stabilization in use, satisfied effectiveness and safety that gene is cured the disease.
Technology of the present invention is simple; raw material sources are extensive; prepared hydroxyapatite nanoparticle has excellent biological compatibility; no acute toxicity effect; can with complete combination of PEGFP-N1 expression plasmid DNA; DNA is had protective effect and DNA can be transported to organ or tissue and express, maximum transfection efficiency be about liposome transfection efficient 80%.
Description of drawings:
Fig. 1 process chart of the present invention;
Fig. 2 HA nano-particle transmission photo;
Fig. 3 HA nano-particle and SGC-7901 mixing with cells cultivation-excellent biological compatibility;
Fig. 4 HA nano-particle combines the experiment electrophoretic analysis and shows that DNA is absorbed by nano-particle with DNA;
Fig. 5 HA nano-particle combines back transfection SGC-7901 cell (the 100x visual field) with PEGFP-N1;
Fig. 6 hydroxyapatite nanoparticle is transfected into the cell transient expression as the gene transfection carrier with PEGFP-N1 expression plasmid DNA;
Behind Fig. 7 hydroxyapatite nanoparticle-dna complex tail vein injection white mice, the brain tissue slice egfp expression.
The specific embodiment:
Example 1:
1. hydroxyapatite nano powder preparation
With analytical pure Ca (NO
3)
2(NH
4) H
2PO
4Be dissolved in respectively in the deionized water, be made into the solution of 1mol/L, with Ca (NO
3)
2(NH
4) H
2PO
4Solution mixes under the rotating speed of 1500 commentaries on classics/min with 5: 3 ratio of volume ratio, and with the pH value of NaOH regulator solution in 11～12 scopes, stir after 10 hours aging 24 hours.Again the precipitate that obtains is put into autoclave, under 200 ℃, 200mv, 200 rev/mins rotating speed, handled 4 hours, obtain hydroxyapatite nanoparticle through cleaning, filtration, drying again.
2. hydroxyapatite nanoparticle suspension preparation
Get hydroxyapatite nanoparticle 0.5g, put into centrifuge tube, add deionized water 20ml; concentration is 25mg/ml, and usefulness ultrasonic dispersing 60min (UltrasonicHemogenizer-4710, USA); leave standstill and observe 2h, the hydroxyapatite nanoparticle suspension does not have layering, is emulsus.The nano-particle suspension is inserted the 50ml vial, preserve behind the autoclaving.Get the capable electronic microscope photos of 1ml nano-particle suspension sample.
3. the hydroxyapatite nanoparticle and the compatibility of SGC-7901 cell biological experiment
In 96 hole tissue culturing plates, 104 cells, one hole adds 10% calf serum DMEM culture medium culturing 24h and makes cell attachment with the SGC-7901 cell inoculation, displacement fresh culture, every hole 100 μ l.The nano-particle suspension that adds 50 μ l variable concentrations, concentration are from 0,7.5,15.6,31.2,62.3,125,250, to 500 μ g/ml, and vibration is mixed, continue to cultivate 48h after row MTT measure.With the filtration sterilization of MTT solution, be matched group with the normal growth cell, every hole adds 8 μ lMTT solution, cultivate 4h, abandon supernatant, add the every hole of dimethyl sulfoxide (DirehylSulfoxide) 100 μ l, microscopically is observed has the MTT crystallization to form, vibration 10min, every hole is got 100 μ l and is placed the OD assay plate, surveys the OD value, wavelength 595nm, colorimetry is determined great-hearted cell number, and variance analysis method adopts the SPSS9.0 statistical software to carry out.
4. hydroxyapatite nanoparticle combines experiment with DNA
Get agar Icing Sugar 1g; the system gel; the dilution of disinfectant hydroxyapatite nanoparticle suspension is 10 times; concentration 2.5mg/ml, surveying pH value is 6.5, respectively gets suspension 20 μ l and inserts in the 5 pipe eppendorf pipes; add not commensurability PEGFP-N1 (1 respectively; 3,5 μ g), the 10min (VORTEX2GENIE that is mixed; USA); leave standstill 30min, centrifugal (eppendorfCentrifuge5415R centrifuge, USA; 13200rpm; 10min), be contrast with PEGFP-N11 μ g, get the supernatant agarose gel electrophoresis and detect.Respectively get the hydroxyapatite nanoparticle suspension of 50 μ l and it is transferred to different pH value (2,7,12); It is put into the eppendorf pipe respectively mix with 2 μ gPEGFP-N1, the 10min that is mixed, room temperature leaves standstill 30min, and it is centrifugal that (eppendorfCentrifuge5415R centrifuge, 13200r/min 10min), get the supernatant agarose gel electrophoresis and detect.
5. nano-particle-dna complex in-vitro transfection cell experiment
With the SGC-7901 cell by 1 * 105 be inoculated in culture dish (CellCultureDish, 35 * 10mn2), add the DMEM culture medium culturing 24h be furnished with calf serum, microscopically is observed and is seen cell attachment and well-grown.With twice of 1xDMEM eluting, add the nano-particle dna complex by every culture dish transfection green fluorescence protein gene PEGFP-N1 expression plasmid DNA2 μ g, with simple PEGFP-N1 expression plasmid DNA2 μ g and with the bonded PEGFP-N1 expression plasmid of liposome DNA2 μ g in contrast, change 10% calf serum cultivation behind the Mixed culture 8h, respectively at 24,48, observed result under the fluorescence microscope behind the 72h.
6. the acute toxicity test of hydroxyapatite nanoparticle suspension
Kunming kind white mice, healthy anosis, 60, male and female half and half, body weight 17g-21g, behind the fasting that afternoon 16h (can't help water), be divided into matched group and experimental group at random, 15 every group, once give maximum volume 0.4ml/20g nano-particle suspension, Concentraton gradient is respectively 4.5mg/ml, 1.5mg/ml, 0.5mg/ml, and the tail vein injection white mice is observed death and the toxic reaction of two all animals.
7. hydroxyapatite nanoparticle and nano-particle-dna complex
Kunming kind white mice, healthy anosis, 30, male and female half and half, body weight 17g-21g, behind the fasting that afternoon 16h (can't help water), be divided into matched group and experimental group at random, every group 10, one group is the nano-particle suspension tail vein injection white mice of 1.5mg/ml with maximum volume 0.4ml/20g Concentraton gradient, use nano-particle-dna complex tail vein injection white mice for one group, complex is that the nano-particle suspension of volume 0.4ml/20g, Concentraton gradient 1.5mg/ml combines with PEGFP-N1 expression plasmid 1 μ g.Another group is contrast, normal saline 0.4ml tail vein injection white mice.Respectively at 24h, put to death animal behind the 48h, get white mice liver, kidney and cerebral tissue, put into frozen pipe and 2% glutaraldehyde solution respectively, whether capable respectively frozen section and electron micrograph have egfp expression and nano-particle to distribute in the tissues observed.
Claims (2)
1, a kind of preparation method of genophore is characterized in that:
(1) hydroxyapatite nanoparticle preparation:
With analytical pure Ca (NO
3)
2(NH
4) H
2PO
4Be dissolved in respectively in the deionized water, be made into the solution of 1mol/L, with Ca (NO
3)
2(NH
4) H
2PO
4Solution mixes under the rotating speed of 1500 commentaries on classics/min with 5: 3 ratio of volume ratio, and with the pH value of NaOH regulator solution in 11～12 scopes, stir after 10 hours aging 24 hours; Again the precipitate that obtains is put into autoclave, under 200 ℃, 200mv, 200 rev/mins rotating speed, handled 4 hours, obtain hydroxyapatite nanoparticle through cleaning, filtration, drying again;
(2) hydroxyapatite nanoparticle biocompatibility experiment:
With the SGC-7901 cell inoculation in 96 hole tissue culturing plates; The nano-particle suspension that adds variable concentrations continues to carry out MTT mensuration behind the cultivation 48h, and colorimetry is determined great-hearted cell number, and variance analysis method adopts the SPSS9.0 statistical software to carry out;
(3) hydroxyapatite nanoparticle combines experiment with DNA:
Get agar Icing Sugar system gel,, add the PEGFP-N1 10min that is mixed, leave standstill 30min hydroxyapatite nanoparticle suspension dilution, centrifugal; With PEGFP-N11 μ g is contrast, gets the supernatant agarose gel electrophoresis and detects; Get the hydroxyapatite nanoparticle suspension, and it is transferred to different pH value, put into the eppendorf pipe respectively and mix with PEGFP-N1, be mixed, room temperature leaves standstill 30min, and is centrifugal; Getting the supernatant agarose gel electrophoresis detects;
(4) nano-particle one dna complex in-vitro transfection cell experiment:
SGC one 7901 cell inoculations after the culture dish adding is furnished with the DMEM culture medium culturing 24h of calf serum, are added the nano-particle dna complex at culture dish; With simple PEGFP-N1 expression plasmid DNA and with the bonded PEGFP-N1 expression plasmid of liposome DNA in contrast; Mixed culture is respectively at 24,48, observed result under the fluorescence microscope behind the 72h.
2, the preparation method of genophore according to claim 1 is characterized in that:
1. hydroxyapatite nanoparticle preparation
With analytical pure Ca (NO
3)
2(NH
4) H
2PO
4Be dissolved in respectively in the deionized water, be made into the solution of 1mol/L, with Ca (NO
3)
2(NH
4) H
2PO
4Solution mixes under the rotating speed of 1500 commentaries on classics/min with 5: 3 ratio of volume ratio, and with the pH value of NaOH regulator solution in 11～12 scopes, stir after 10 hours aging 24 hours; Again the precipitate that obtains is put into autoclave, under 200 ℃, 200mv, 200 rev/mins rotating speed, handled 4 hours, obtain hydroxyapatite nanoparticle through cleaning, filtration, drying again;
2. hydroxyapatite nanoparticle suspension preparation
Get hydroxyapatite nanoparticle 0.5g, put into centrifuge tube, add deionized water 20ml, concentration is 25mg/ml, uses ultrasonic dispersing 60min, leaves standstill to observe 2h, and the hydroxyapatite nanoparticle suspension does not have layering, is emulsus; The nano-particle suspension is inserted the 50ml vial, preserve behind the autoclaving; Get 1ml nano-particle suspension sample and carry out electronic microscope photos;
3. the hydroxyapatite nanoparticle and the compatibility of SGC-7901 cell biological experiment
With the SGC-7901 cell inoculation in 96 hole tissue culturing plates, 10
4Cell one hole adds 10% calf serum DMEM culture medium culturing 24h and makes cell attachment, displacement fresh culture, every hole 100 μ l; The nano-particle suspension that adds 50 μ l variable concentrations, concentration are from 0,7.5,15.6,31.2,62.3,125,250, to 500 μ g/ml, and vibration is mixed, continue to cultivate 48h after row MTT measure; With the filtration sterilization of MTT solution, be matched group with the normal growth cell, every hole adds 8 μ lMTT solution, cultivate 4h, abandon supernatant, add the every hole of dimethyl sulfoxide 100 μ l, microscopically is observed has the MTT crystallization to form, vibration 10min, every hole is got 100 μ l and is placed the OD assay plate, surveys the OD value, wavelength 595nm, colorimetry is determined great-hearted cell number, and variance analysis method adopts the SPSS9.0 statistical software to carry out;
4. hydroxyapatite nanoparticle combines experiment with DNA
Get agar Icing Sugar 1g, the system gel, the dilution of disinfectant hydroxyapatite nanoparticle suspension is 10 times, concentration 2.5mg/ml, surveying pH value is 6.5, respectively gets suspension 20 μ l and inserts in the 5 pipe eppendorf pipes, add not commensurability PEGFP-N1 respectively, be respectively 1,3 and 5 μ g, 10min is mixed, leave standstill 30min, centrifugal, be contrast with PEGFP-N11 μ g, get the supernatant agarose gel electrophoresis and detect; Respectively get the hydroxyapatite nanoparticle suspension of 50 μ l and it is transferred to different pH value 2,7,12; It is put into the eppendorf pipe respectively mix with 2 μ gPEGFP-N1, the 10min that is mixed, room temperature leaves standstill 30min, and is centrifugal, gets the supernatant agarose gel electrophoresis and detects;
5. nano-particle-dna complex in-vitro transfection cell experiment
The SGC-7901 cell is pressed 1 * 10
5Be inoculated in culture dish, add the DMEM culture medium culturing 24h that is furnished with calf serum, microscopically is observed and is seen cell attachment and well-grown; With twice of 1 * DMEM eluting, add the nano-particle dna complex by every culture dish transfection green fluorescence protein gene PEGFP-N1 expression plasmid DNA2 μ g, with simple PEGFP-N1 expression plasmid DNA2 μ g and with the bonded PEGFP-N1 expression plasmid of liposome DNA2 μ g in contrast, change 10% calf serum cultivation behind the Mixed culture 8h, respectively at 24,48, observed result under the fluorescence microscope behind the 72h.
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|---|---|---|---|
| CNB2004100233474A CN1306965C (en) | 2004-06-25 | 2004-06-25 | Production of gene carrier and use thereof |
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|---|---|---|---|
| CNB2004100233474A CN1306965C (en) | 2004-06-25 | 2004-06-25 | Production of gene carrier and use thereof |
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| CN1712070A CN1712070A (en) | 2005-12-28 |
| CN1306965C true CN1306965C (en) | 2007-03-28 |
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| CNB2004100233474A Expired - Fee Related CN1306965C (en) | 2004-06-25 | 2004-06-25 | Production of gene carrier and use thereof |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101486452B (en) * | 2009-02-16 | 2012-01-04 | 重庆大学 | Preparation of nano hydroxylapatite powder |
| CN101773801B (en) * | 2009-12-31 | 2012-05-30 | 中国科学院上海硅酸盐研究所 | Dispersant for dispersing carbon nano-tube and preparation method of carbon nano-tube film |
| CN108060176A (en) * | 2017-11-23 | 2018-05-22 | 华中农业大学 | A kind of functional form genophore and DNA/ carrier complexes |
| CN108264373A (en) * | 2018-02-06 | 2018-07-10 | 付主枝 | The preparation method of medical reinforced porous biological ceramic material |
| CN111573647A (en) * | 2020-06-16 | 2020-08-25 | 北京市创伤骨科研究所 | Application of nano-hydroxyapatite in preventing or inhibiting metastasis and recurrence of osteosarcoma |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003048298A2 (en) * | 2001-12-05 | 2003-06-12 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Nanoparticles containing polymeric nucleic acid homologs, pharmaceutical compositions and articles of manufacture containing same and methods of use thereof |
| CN1456356A (en) * | 2002-05-09 | 2003-11-19 | 中南大学 | Gene carrier for therapy of cerebral gliocyte diseases |
| CN1462763A (en) * | 2003-06-09 | 2003-12-24 | 湖南大学 | Nano granule of polylysine amylum and its preparation method as well as application gene carrier |
| US20040076585A1 (en) * | 2002-10-16 | 2004-04-22 | Xianchang Gong | Nanoparticles of cyclic tetrapyrrolic compounds as gene and drug delivery carriers |
-
2004
- 2004-06-25 CN CNB2004100233474A patent/CN1306965C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003048298A2 (en) * | 2001-12-05 | 2003-06-12 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Nanoparticles containing polymeric nucleic acid homologs, pharmaceutical compositions and articles of manufacture containing same and methods of use thereof |
| CN1456356A (en) * | 2002-05-09 | 2003-11-19 | 中南大学 | Gene carrier for therapy of cerebral gliocyte diseases |
| US20040076585A1 (en) * | 2002-10-16 | 2004-04-22 | Xianchang Gong | Nanoparticles of cyclic tetrapyrrolic compounds as gene and drug delivery carriers |
| CN1462763A (en) * | 2003-06-09 | 2003-12-24 | 湖南大学 | Nano granule of polylysine amylum and its preparation method as well as application gene carrier |
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| CN1712070A (en) | 2005-12-28 |
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