CN1456356A - Gene carrier for therapy of cerebral gliocyte diseases - Google Patents
Gene carrier for therapy of cerebral gliocyte diseases Download PDFInfo
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- CN1456356A CN1456356A CN 02114118 CN02114118A CN1456356A CN 1456356 A CN1456356 A CN 1456356A CN 02114118 CN02114118 CN 02114118 CN 02114118 A CN02114118 A CN 02114118A CN 1456356 A CN1456356 A CN 1456356A
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- ferric oxide
- nanometer particle
- oxide nanometer
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Abstract
A gene carrier for treating the cerebral gliacyte disease is prepared through preparing nanoparticles of iron oxide by hydrolysis, and modifying the surface of said nanoparticle with polylysine. Said nanoparticle can bind with and protect chain plasmid DNA, single-chain antisense oligonucleotide, and RNA for carrying exogenous gene to cerebral gliacyte for efficient expression.
Description
[technical field] the present invention relates to a kind of genophore, especially for the genophore of brain glial cell treatment of diseases.
[background technology] also lacking ideal Therapeutic Method aspect the brain glial cell treatment of diseases both at home and abroad at present.Gene therapy has limited its application clinically in default of ideal genophore.Genophore has viral vector and non-virus carrier at present.Viral vector exists immunogenicity, potential oncogenicity and load defectives such as segmental size is restricted.Non-viral transporter commonly used at present also exists the defective that is difficult to overcome, big as cytotoxicity, transfection efficiency is low, especially for brain glial cell disease, present non-virus carrier is because of passing through blood brain barrier, so almost can't be used for brain glial cell treatment of diseases.
[summary of the invention] is for improving at present owing to brain glial cell disease lacks the situation that ideal genophore hinders gene therapy, the invention provides a kind of nontoxic, safety is used for the preparation method and the using method thereof of the novel nano genophore of brain glial cell disease efficiently.
The present invention uses Hydrolyze method synthetic iron oxide magnetic nanoparticle, because the avirulent characteristic of its pair cell utilizes ferric oxide nanometer particle as genophore; through successfully combination of surface modification; concentrate, protection DNA, RNA, and transmit DNA efficiently in vivo and in vitro, RNA enters cell.Concrete technical parameter is as follows:
(1) with glucosan, ferrous chloride, iron chloride are pressed quality than glucosan: Fe
2+: Fe
3+Be 40～45: 60～70 ℃ of reactions of 1.0: 2.5～3 mixed, 1～2h, add an amount of ammonia, product high speed centrifugation 15～30min gets supernatant, makes ferric oxide nanometer particle with membrane filtration.
(2) ferric oxide nanometer particle is resuspended in the NaCl solution of 1.5mmol/L, the poly-D-lysine of quality such as adding, ultrasonic concussion 1hr, after centrifugal, abandon supernatant, 1.5mmol/L NaCl washing after be resuspended among the NaCl of 1.5mmol/L and make surface-active ferric oxide nanometer particle, become poly-D-lysine-ferric oxide nanometer particle.
With the genophore of surface-active ferric oxide nanometer particle as the disease treatment of brain glial cell, its using method is:
(1) be dissolved in plasmid DNA among the NaCl of 1.5mM by 10ug/ml～20ug/ml, the mass ratio of isopyknic poly-D-lysine-ferric oxide nanometer particle by 5: 1～10: 1 is added dropwise in the plasmid solution, concuss 10s, room temperature leaves standstill 1h, and complex is gone in the Balb/C mice body from tail vein injection.
(2) or with poly-D-lysine-ferric oxide nanometer particle and antisense oligonucleotide chain is to mix in 5: 1～10: 1 by mass ratio, the glutaraldehyde that adds phosphate buffer and 1.1% is at 25 ℃ of reaction 1hr, behind the high speed centrifugation, remove supernatant, precipitation is resuspended among the NaCl of 1.5mmol/L, complex is gone in the Balb/C mice body from tail vein injection.
Manufacture method of the present invention is simple, prepared ferric oxide nanometer particle can pass through blood brain barrier and distribute in a large number in the brain glial cell, can be used as the good genophore of brain glial cell disease gene treatment, this carrier also can be used for the transhipment of medicine simultaneously, and the iron oxide magnetic nano granule has superparamagnetism, and externally-applied magnetic field can further be realized targeted therapy.The nano-particle footpath grain of making is even, and this nano-particle can efficiently carry and protect DNA, and this nanometer delivery system pair cell avirulence is in vivo and in vitro with in the exogenous gene high-efficient transfered cell.
[description of drawings]
Fig. 1: poly-D-lysine-ferric oxide nanometer particle Electronic Speculum figure;
Fig. 2: with poly-D-lysine-ferric oxide nanometer particle intravenous injection BALB/C mice, put to death mice after 24 hours, get cerebral tissue and do the Electronic Speculum detection, the result shows: poly-D-lysine-ferric oxide nanometer particle distributes in a large number at the brain glial cell.
Fig. 3: poly-D-lysine-ferric oxide nanometer particle carries the proteic plasmid DNA of coding fluorescence or fluorescently-labeled antisense oligonucleotide in the BALB/C mice body is gone in intravenous injection, put to death mice after 24～48 hours, cerebral tissue is made section, and fluorescence microscope shows down: at cerebral tissue a large amount of fluorescence signals are arranged.
[specific embodiment]
1. glucosan, ferrous chloride, iron chloride is pressed glucosan: Fe
2+: Fe
3+Be 60 ℃ of reactions of mixed 1h of 40: 1.0: 2.7, add an amount of ammonia, product 12, the centrifugal 15min of 000r/min gets supernatant, makes ferric oxide nanometer particle with membrane filtration.(2) ferric oxide nanometer particle is resuspended in the NaCl solution of 1.5mM, the poly-D-lysine of quality such as adding, ultrasonic concussion 1hr, after centrifugal, abandon supernatant, 1.5mM NaCl washing after be resuspended in the ferric oxide nanometer particle of making this property of surface among the NaCl of 1.5mM, become poly-D-lysine-ferric oxide nanometer particle.
(a) reporter plasmid EGFP-C2 is dissolved in by 10ug/ml among the NaCl of 1.5mM, the mass ratio of isopyknic poly-D-lysine-ferric oxide nanometer particle by 10: 1 is added dropwise in the plasmid solution, concuss 10s, room temperature leaves standstill 1h.
(b) or with poly-D-lysine-ferric oxide nanometer particle and antisense oligonucleotide chain is to mix at 10: 1 by mass ratio, the glutaraldehyde that adds phosphate buffer and 1.1% is at 25 ℃ of reaction 1hr, behind the high speed centrifugation, remove supernatant, precipitation is resuspended among the NaCl of 1.5mM.
2. two kinds of complex of (a) and (b) gained are gone in the BALB/C mice body from tail vein injection respectively.Put to death mice, the cerebral tissue of mice is made section, fluorescence microscope is observed (Fig. 3) down.
Claims (3)
1. preparation method that is used for the genophore of brain glial cell disease treatment; it is characterized in that: the present invention uses Hydrolyze method synthetic iron oxide magnetic nanoparticle; utilize ferric oxide nanometer particle as genophore; through successfully combination of surface modification; concentrate; protection DNA, RNA, and transmit DNA efficiently in vivo and in vitro, RNA enters cell, concrete technical parameter is as follows:
(1) with glucosan, ferrous chloride, iron chloride are pressed quality than glucosan: Fe
2+: Fe
3+Be 40～45: 60～70 ℃ of reactions of 1.0: 2.5～3 mixed, 1～2h, add an amount of ammonia, product high speed centrifugation 15～30min gets supernatant, makes ferric oxide nanometer particle with membrane filtration;
(2) ferric oxide nanometer particle is resuspended in the NaCl solution of 1.5mM, the poly-D-lysine of quality such as adding, ultrasonic concussion 1hr, after centrifugal, abandon supernatant, 1.5mM NaCl washing after be resuspended among the NaCl of 1.5mM and make surface-active ferric oxide nanometer particle, become poly-D-lysine-ferric oxide nanometer particle.
2. described genophore of claim 1, it is characterized in that: its using method is: adopt poly-D-lysine that ferric oxide nanometer particle is carried out finishing, plasmid DNA 10ug/ml～20ug/ml is dissolved among the NaCl of 1.5mM, the mass ratio of isopyknic poly-D-lysine-ferric oxide nanometer particle by 10: 1 is added dropwise in the plasmid solution, concuss 10s, room temperature leaves standstill 1h.
3. described genophore of claim 1, it is characterized in that: its using method is: adopt the ferric oxide nanometer particle of modification to can be used as the double-stranded circular plasmid DNA, single stranded antisense oligonucleotides, the carrier of RNA and other medicine, is mixing in 10: 1 with poly-D-lysine-ferric oxide nanometer particle and antisense oligonucleotide chain by mass ratio, and the glutaraldehyde that adds phosphate buffer and 1.1% is at 25 ℃ of reaction 1hr, behind the high speed centrifugation, remove supernatant, precipitation is resuspended among the NaCl of 1.5mM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02114118 CN1456356A (en) | 2002-05-09 | 2002-05-09 | Gene carrier for therapy of cerebral gliocyte diseases |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02114118 CN1456356A (en) | 2002-05-09 | 2002-05-09 | Gene carrier for therapy of cerebral gliocyte diseases |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1306965C (en) * | 2004-06-25 | 2007-03-28 | 中南大学 | Production of gene carrier and use thereof |
| WO2007028607A3 (en) * | 2005-09-09 | 2007-05-18 | Aesculap Ag & Co Kg | Antimicrobial medical product, method for the production thereof and use thereof |
| CN101354938B (en) * | 2007-07-25 | 2012-04-04 | 北京海达丰科技有限公司 | Magnetic particle for separation as well as preparation method and application thereof |
| WO2016078576A1 (en) * | 2014-11-19 | 2016-05-26 | 正大天晴药业集团股份有限公司 | Method for preparing modified superparamagnetic ferric oxide |
| CN109731140A (en) * | 2018-12-27 | 2019-05-10 | 上海北陆医药科技有限公司 | A kind of long-acting gene expression cytoskeleton and preparation method thereof |
-
2002
- 2002-05-09 CN CN 02114118 patent/CN1456356A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1306965C (en) * | 2004-06-25 | 2007-03-28 | 中南大学 | Production of gene carrier and use thereof |
| WO2007028607A3 (en) * | 2005-09-09 | 2007-05-18 | Aesculap Ag & Co Kg | Antimicrobial medical product, method for the production thereof and use thereof |
| US8858972B2 (en) | 2005-09-09 | 2014-10-14 | Aesculap Ag | Antimicrobial medicotechnical product, process for its preparation and use |
| CN101354938B (en) * | 2007-07-25 | 2012-04-04 | 北京海达丰科技有限公司 | Magnetic particle for separation as well as preparation method and application thereof |
| WO2016078576A1 (en) * | 2014-11-19 | 2016-05-26 | 正大天晴药业集团股份有限公司 | Method for preparing modified superparamagnetic ferric oxide |
| CN109731140A (en) * | 2018-12-27 | 2019-05-10 | 上海北陆医药科技有限公司 | A kind of long-acting gene expression cytoskeleton and preparation method thereof |
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