CN102154349A - Cationized mulberry polysaccharide nanoparticle gene vectors and manufacturing method thereof - Google Patents

Cationized mulberry polysaccharide nanoparticle gene vectors and manufacturing method thereof Download PDF

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CN102154349A
CN102154349A CN 201010613484 CN201010613484A CN102154349A CN 102154349 A CN102154349 A CN 102154349A CN 201010613484 CN201010613484 CN 201010613484 CN 201010613484 A CN201010613484 A CN 201010613484A CN 102154349 A CN102154349 A CN 102154349A
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polysaccharides
folium mori
cationization
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CN102154349B (en
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徐希明
邓纹纹
余江南
王淼
曹霞
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Jiangsu University
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Abstract

The invention discloses cationized mulberry polysaccharide nanoparticle gene vectors, which are gene vectors combining mulberry polysaccharides modified by an amine compound and DNA plasmids, wherein the molecular weight distribution of the mulberry polysaccharides is 3 to 3.5*10<4>Da; and the mass ratio of the cationized mulberry polysaccharidesto the DNA plasmids is (0.5-100):1; the particle size of the plasmids is 50 to 150 nanometers; and the amine compounds is spermine, ethylenediamine or polyethyleneimine with a number-average molecular weight of 600Da to 2,000Da. The three kinds of cationized mulberry polysaccharides used in the invention all have good DNA plasmid bonding actions and can form biodegradable, high-efficiency and low-toxicity novel non-viral gene vector materials. And the preparation processes of the cationized mulberry polysaccharides are simple and the prices of the cationized mulberry polysaccharides are low. The invention discloses a manufacturing method of the cationized mulberry polysaccharide nanoparticle gene vectors.

Description

The polysaccharides of Folium Mori nanoparticle genophore and the method for making thereof of cationization
Technical field
The present invention relates to herbal polysaccharide, relate to genophore, be specifically related to a kind of polysaccharides of Folium Mori nanoparticle genophore of cationization.
Background technology
In recent years, along with the continuous development of biotechnology, gene therapy has become the effective means of the many major diseases of clinical treatment such as cancer, cardiovascular and cerebrovascular diseases, diabetes etc.Along with going deep into of gene therapy research, it is found that the key of gene therapy is that the suitable efficient gene carrier of selection is appropriate introduction method, makes goal gene obtain target importing, stabilizing effective expression, and body is not produced toxic side effect.At present, be used for Vectors in Gene Therapy and have two kinds: virus vector and non-virus carrier.Though virus vector is widely adopted in present gene therapy clinical and experimental study, the many deficiencies of himself, little and targeting specific difference etc. has limited its application as the capacity of potential carinogenicity, autoimmunization originality, goal gene.And non-virus carrier has that toxicity is low, immune response is little, be easy to modify to obtain advantage such as better target, more and more is subject to people's attention.Cationic polymers is the extremely genophore of people's concern of a class.The cationoid polymerisation species is a lot, studies the more polypeptide class that has at present: polylysine, polyglutamic acid and derivative thereof; Poly amine: polymine, polypropylene imines tree; Polymethacrylic acid: polymeric amide ~ amine dendritic thing and some natural polymers such as (reference: Wang Qin, Gong Yuefa etc. such as chitosan, gelatin.Gene Study on Cationic Polymers progress.ACAD J GCP, 2004,22(1): 180 ~ 185.).A common feature on these polymer architectures is that intramolecularly contains many amino, under physiological pH, can take place protonated, these protonated amino can in and the negative charge on DNA plasmid surface, make dna molecular by the less relatively dna particle of unfolded structure boil down to volume, or goal gene is wrapped in wherein, make DNA avoid the degraded of nuclease, they can be used as genophore.But there is the limitation of self in these cationic polymerss, as poly amine shortage biodegradability, polypeptide class potential immunogenicity etc. are arranged.A kind of synthetic materials polymine (PEI) is arranged at present, and high molecular (〉 20000 Da are found in test) the PEI transfection efficiency very high, but cytotoxicity is very big, and has the problem of degradation property; Small molecular weight PEI(600 Da, 1300 Da, 2000 Da) toxicity is very little, is easy to metabolism in vivo, almost do not have (the reference: Hongliang Huang, Hai Yu, Guping Tang, Qingqing Wang and Jun Li of transfection toxicity ,Low molecular weight polyethylenimine cross ~ linked by 2 ~ hydroxypropyl ~ γ ~ cyclodextrin coupled to peptide targeting HER2 as a gene delivery vector. Biomaterials, 2010,31 (7): 1830 ~ 1838).Also have the scholar to adopt quadrol, spermine, putrescine, aminated compoundss such as spermidine are modified gelatin (reference: TOSHIHIRO KUSHIBIKI, RYUJI TOMOSHIGE, KAZUNORI IWANAGA etal. In vitro transfection of plasmid DNA by cationized gelatin prepared from different amine compounds. Journal of Biomaterial Science, Polymer Edn, 2006,17 (6): 645 ~ 658.), dextran (reference: Hagit Eliyahu, Aviva Joseph, Tony Azzam et al. Dextran-spermine based polyplexes-Evaluation of transgene expression and of local and systemic toxicity in mice. Biomaterials 27 (2006) 1636 ~ 1645.) etc. macromolecular compound is studied its possibility as non-viral gene vector.
Polysaccharide, the natural high moleculer eompound as a kind of nature extensively exists extensively is present in the biologies such as plant, marine organisms and mushroom, has biodegradability and biocompatibility.Most of vegetable polysaccharidess all have biological activity, are one of important integral parts of organism.Because polysaccharide chain contains many activity hydroxies, and very high reactive behavior is arranged, and is easy to carry out chemically modified, thereby increases new biological function.Polysaccharides of Folium Mori is another effective active composition in the mulberry leaf of discovered in recent years, has hypoglycemic effect.
Summary of the invention
The present invention is based on polysaccharides of Folium Mori (Latin formal name used at school: Folium Mori), use diverse ways that it is carried out amination and modify, increase its positive polarity, make it be easy to become the efficient gene transport vehicle in conjunction with electronegative plasmid DNA.Be specially: the mulberry leaf crude drug is through water extract-alcohol precipitation, trichloroacetic acid method removes after the albumen, carry out separation and purification with AB-8 macroporous adsorbent resin and SephadexG-100 dextrane gel resin respectively, dialysis, freeze-drying, obtain after the purified polysaccharides of Folium Mori, use aminated compounds spermine, quadrol and small molecular weight PEI modified polysaccharide chain respectively, obtain the polysaccharides of Folium Mori of cationization.Then, the polysaccharides of Folium Mori of cationization combines with plasmid DNA and forms nano-complex.The present invention is with the combination to plasmid DNA of the polysaccharides of Folium Mori of three kinds of cationizations, stem cell transfection effect, cell adhesion contrasts, the liposome positive control is set simultaneously, experimental result shows that the keying action of the polysaccharides of Folium Mori of ethylene diamine-modified cationization and plasmid DNA is good, the polysaccharides of Folium Mori of the cationization that the stem cell transfection efficiency is modified apparently higher than spermine and the polysaccharides of Folium Mori of the cationization that small molecular weight PEI modifies, the stem cell transfection efficiency that also is higher than the positive control liposome, and the cell adhesion test shows that it has the good cell adhesion, and cell toxicity test shows its safety non-toxic.
The present invention adopts the method for chemically modified, provide a kind of based on cationization the genophore of high effect nontoxic.
Technical scheme of the present invention is as follows:
A kind of polysaccharides of Folium Mori nanoparticle genophore of cationization, it is the genophore of a kind of polysaccharides of Folium Mori of the cationization of modifying with aminated compounds in conjunction with the DNA plasmid, the molecular weight distribution of polysaccharides of Folium Mori is 3 ~ 3.5 * 10 4Da, wherein by mass ratio, the polysaccharides of Folium Mori of cationization: DNA plasmid=0.5 ~ 100:1, the particle diameter of plasmid are 50-150nm, described aminated compounds is that spermine, quadrol or number-average molecular weight are the polymine of 600Da-2000Da.
A kind of method for preparing the polysaccharides of Folium Mori nanoparticle genophore of above-mentioned cationization, it may further comprise the steps:
The preparation of step 1. oxidation polysaccharides of Folium Mori:
Take by weighing 0.5 ~ 1g purified polysaccharides of Folium Mori, be dissolved in 20ml ~ 50ml distilled water, add KIO 4, KIO 4With the mol ratio of monosaccharide units in the polysaccharide be: 0.5 ~ 5:1 is put into the darkroom rapidly, magnetic agitation, room temperature reaction 72h; Reaction solution adds 10 ~ 20ml ethylene glycol termination reaction, continues reaction 30min by aforementioned condition; With the reaction solution dialysis tubing (intercepting molecular weight〉3500Da) of packing into, 48h dialyses in distilled water; The dialyzate freeze-drying obtains oxidation polysaccharides of Folium Mori 0.4 ~ 0.9g.
Step 2. cationization preparation:
A. the preparation of the polysaccharides of Folium Mori of the cationization of spermine modification:
Take by weighing the oxidation polysaccharides of Folium Mori that 0.1 ~ 0.5g step 1 makes, be dissolved in 10 ~ 50ml distilled water; Take by weighing 0.2 ~ 1.5g spermine and be dissolved in the borate buffer solution (pH=9) of 5ml; The mol ratio of the aldehyde radical of spermine and oxidation of polysaccharides is 0.5 ~ 5:1, and the borate solution of spermine is slowly joined in the polysaccharides of Folium Mori solution with disposable syringe, carries out magnetic agitation simultaneously; After adding, magnetic agitation, room temperature reaction 24h; Afterwards, add 0.1 ~ 0.5g sodium borohydride in reaction solution, the same terms continues reaction 48h down; Add 0.1 ~ 0.5g sodium borohydride again in reaction solution, the total mass of adding sodium borohydride with the ratio of the quality of oxidation of polysaccharides is: 0.5 ~ 3:1, the same terms continue reaction 24h down; With the reaction solution dialysis tubing of packing into, dialysis 48h(intercepting molecular weight in distilled water〉3500Da); The dialyzate freeze-drying obtains the polysaccharides of Folium Mori 0.2 ~ 0.4g of the cationization that spermine modifies.
The preparation of the polysaccharides of Folium Mori of B. ethylene diamine-modified cationization
Get the oxidation polysaccharides of Folium Mori that 0.1 ~ 0.5g step 1 makes, be dissolved in 10 ~ 50ml distilled water; Get the borate buffer solution (pH=9) that quadrol is dissolved in 5ml, the mol ratio of the aldehyde radical of quadrol and oxidation of polysaccharides is 0.5 ~ 5:1; The borate solution of quadrol is slowly joined in the polysaccharides of Folium Mori solution with disposable syringe, carry out magnetic agitation simultaneously; After adding, magnetic agitation, room temperature reaction 24h; Afterwards, add 0.1 ~ 0.5g sodium borohydride in reaction solution, the same terms continues reaction 48h down; Add 0.1 ~ 0.5g sodium borohydride again in reaction solution, the total mass of adding sodium borohydride with the ratio of the quality of oxidation of polysaccharides is: 0.5 ~ 3:1, the same terms continue reaction 24h down; With the reaction solution dialysis tubing (intercepting molecular weight〉3500Da) of packing into, 48h dialyses in distilled water; The dialyzate freeze-drying obtains the polysaccharides of Folium Mori 0.2 ~ 0.4g of ethylene diamine-modified cationization.
C. the preparation of the polysaccharides of Folium Mori of the cationization of polyethylene imine beautify
Take by weighing 0.2 ~ 0.5g purified polysaccharides of Folium Mori, be dissolved in 10 ~ 15ml phosphate buffered saline buffer (pH=7); Linking agent with the activation hydroxyl, as: N, N '-carbonyl dimidazoles, benzotriazole carbonic ether, carbonylic imidazole, N, in the N '-two succinimido sulfuric ester any, be dissolved in the 5ml methylene dichloride, the mass ratio of polysaccharide and linking agent is: 0.5 ~ 3:1, under protection of nitrogen gas, at first add catalyst of triethylamine in polysaccharide liquid, the dichloromethane solution with the hydroxyl linking agent slowly adds in the polysaccharide soln again, and the limit edged stirs, in 30 ~ 100min, add, after adding, room temperature reaction 90 ~ 150min obtains the activatory polysaccharide soln; With number-average molecular weight is that the polymine of 600Da-2000Da is dissolved in the 10ml phosphate buffered saline buffer; the mass ratio of polysaccharide and polymine is 0.5 ~ 4:1; add catalyst of triethylamine; under lucifuge, nitrogen protection, room temperature condition, slowly join in the activatory polysaccharide liquid; the limit edged stirs; add at 120 ~ 150min; react 10h under lucifuge, the room temperature after adding; the solution of reaction after finishing obtains the polysaccharides of Folium Mori of the cationization of polyethylene imine beautify after dialysis (intercepting molecular weight〉3500Da), freeze-drying.
The preparation of the polysaccharides of Folium Mori nanoparticle genophore of step 3. cationization:
The polysaccharides of Folium Mori that takes by weighing the above-mentioned three kinds of cationizations of 10 ~ 40mg respectively is dissolved in 0.5 ~ 2ml distilled water, obtains the cationic polysaccharide storing solution.Respectively get cationic polysaccharide storing solution 50 μ l, dilute 100 times after, obtain cationic polysaccharide and use liquid; Get 10 μ l cationic polysaccharides application liquid and 10 μ l respectively and contain 0.1 ~ 3 μ g DNA plasmid solution, respectively at 55 ℃ of heating 30 ~ 60min; Again with the two mixing, vortex 30s promptly obtains the genophore of the polysaccharides of Folium Mori-DNA plasmid nano-complex of the cationization that aminated compounds modifies.
Beneficial effect
The polysaccharides of Folium Mori of three kinds of cationizations all has good DNA plasmid keying action.Sugar chain is in conjunction with after the aminated compounds, and positive charge increases, thereby can be good at combining by electrostatic interaction with electronegative DNA plasmid, and protection DNA plasmid is avoided the degraded of the inside and outside various enzymes of cell.Wherein, the polysaccharides of Folium Mori of ethylene diamine-modified cationization has best stem cell transfection effect, illustrates that through electrophoresis experiment and stem cell transfection experiment it has better combination and releasing effect to the DNA plasmid.And polysaccharides of Folium Mori has good biological activity, compares with the polysaccharide that was used for preparing cationic polymers in the past, has better biodegradability and biocompatibility, and preparation technology is simple, and is cheap.Lotus positive electricity polysaccharides of Folium Mori on its natural biological activity basis, increased new biological function, become the novel non-viral gene vector material of a kind of biodegradable, high-efficiency low-toxicity.
Description of drawings
Fig. 1 is preparation technology's schema of the polysaccharides of Folium Mori of ethylene diamine-modified cationization.
Fig. 2 is the electrophorogram of the polysaccharides of Folium Mori-DNA plasmid nano-complex of ethylene diamine-modified cationization, wherein:
Duct 1: naked pTGF β-1;
Duct 2 ~ 8: the polysaccharides of Folium Mori and the pTGF β-1(quality that are respectively cationization) than being: 1:1; 1:2; 1:5; 1:10; 1:30; 1:50; 1:70.
Fig. 3 is the transmission electron microscope picture of ethylene diamine-modified polysaccharides of Folium Mori-DNA plasmid nano-complex.
Fig. 4 is the size distribution figure of ethylene diamine-modified polysaccharides of Folium Mori-DNA plasmid nano-complex.
Fig. 5 is polysaccharides of Folium Mori-DNA plasmid nano-complex gene transfection design sketch that quadrol, spermine and PEI modify, wherein: the PEI post is represented with the positive control group of PEI-DNA plasmid nano-complex gene transfection effect (PEI molecular weight 25KD), polysaccharides of Folium Mori-DNA plasmid nano-complex gene transfection the effect of the cationization that the representative of mulberry leaf-quadrol post is ethylene diamine-modified, mulberry leaf-SP post represents the gene transfection effect of polysaccharides of Folium Mori-DNA plasmid nano-complex of the cationization of spermine modification; Mulberry leaf-PEI post is represented the polysaccharides of Folium Mori transfection effect of the cationization of PEI modification.
Embodiment
Material that following examples adopted and instrument:
Experiment material: mulberry leaf (Chinese medicinal materials, the big pharmacy of sesame woods, Zhengjiang City); 95% ethanol (Shandong Guang Yuan medicine company limited); Dehydrated alcohol, acetone, ether, ethylene glycol, trichoroacetic acid(TCA) (Chemical Reagent Co., Ltd., Sinopharm Group); AB-8 macroporous adsorbent resin (Anhui Samsung resin company limited); SephadexG-100 gel resin (Shanghai RiChu Bioscience company limited); KIO 4(Chemical Reagent Co., Ltd., Sinopharm Group); Spermine (Biosharp company, the U.S.), and quadrol (Sigma ~ Aldrich, USA), PEI(Sigma ~ Aldrich, USA); Sodium borohydride (Chemical Reagent Co., Ltd., Sinopharm Group); The big extraction reagent kit of no intracellular toxin plasmid (health is century); Rat TGF ~ β 1 ELISA Kit(Yantai Sai Ersi Bioisystech Co., Ltd).
Experiment equipment: magnetic stirring apparatus (the big-and-middle instrument plant in Jintan); Dialysis tubing (Biosharp company, the U.S.); Very low temperature supercentrifuge (Heareus, Germany); The dried machine of CHRIST lyophilize (BMH company, Germany), DY602S constant current constant voltage electrophoresis apparatus (Nanjing New Campus Biological Technology Institute); JEM ~ 2100 transmission electron microscopes (NEC); Rotary Evaporators (Heidolph company, Germany).
Embodiment 1.The preparation of purified polysaccharides of Folium Mori
Take by weighing dry mulberry leaf 120g, be ground into powder, cross 60 mesh sieves; Extract by following technology: solid-liquid ratio: 1:12, extract temperature: 85 ℃, extraction time 2h/ time, extraction time: 2 times; Merge vat liquor twice, the centrifugal 10min of 5000rpm gets supernatant liquor; Again the supernatant liquor rotary evaporation is concentrated into 1/6 of original volume, gets concentrated solution; Concentrated solution precipitates with 95% medical alcohol again, is 75%, 4 ℃ to determining alcohol and leaves standstill 12h; Collecting precipitation is used dehydrated alcohol, acetone, each washed twice of ether successively; Vacuum-drying obtains the mulberry leaf Crude polysaccharides.
Taking by weighing 3g mulberry leaf Crude polysaccharides, be dissolved in fully in the 40ml distilled water, is 3% to wherein adding 20% trichoroacetic acid(TCA) solution until the trichoroacetic acid(TCA) final concentration, the limit edged stirs, and after adding, 4 ℃ leave standstill 5h, centrifugal 3000r/min, 10min removes precipitation, and supernatant liquor neutralizes with 1M NaOH solution, rotary evaporation concentrates, dialysis, freeze-drying obtains removing proteic polysaccharides of Folium Mori.
Take by weighing 0.5g and remove albumen polysaccharides of Folium Mori afterwards, be dissolved in fully in the 10ml distilled water, 3000r/min, centrifugal 10min gets supernatant liquor, last AB-8 macroporous adsorptive resins, with distilled water as elutriant, flow velocity 6ml/min collects elutriant, adopts sulfuric acid-phynol method to follow the tracks of simultaneously and detects the polysaccharide content of collecting; The collection liquid that will contain polysaccharide merges, and rotary evaporation concentrates, and gets concentrated solution, dialysis, freeze-drying.
Take by weighing the polysaccharides of Folium Mori 0.2g that step 3 obtains, be dissolved in SephadexG-100 gel resin post, as elutriant, flow velocity 6ml/min collects elutriant with 0.1 mol/LNaCl solution, adopt sulfuric acid-phynol method to follow the tracks of simultaneously and detect the polysaccharide content of collecting, the collection liquid that will contain polysaccharide merges, and rotary evaporation concentrates, dialysis, freeze-drying obtains the polysaccharides of Folium Mori of purifying.
Embodiment 2.The preparation of oxidation polysaccharides of Folium Mori
Claim 0.5g purified polysaccharides of Folium Mori, be dissolved in the 30ml distilled water, add 0.8g KIO 4, be put into the darkroom rapidly, magnetic agitation, room temperature reaction 72h; Reaction solution adds 12ml ethylene glycol termination reaction, continues reaction 30min by aforementioned condition; With the reaction solution dialysis tubing of packing into, 48h dialyses in distilled water; The dialyzate freeze-drying obtains the oxidation polysaccharides of Folium Mori.
Embodiment 3.The preparation of the polysaccharides of Folium Mori of ethylene diamine-modified cationization
Get 0.3g oxidation polysaccharides of Folium Mori, be dissolved in the 20ml distilled water; Get the borate buffer solution (pH=9) that the 0.1ml quadrol is dissolved in 5ml; The borate solution of quadrol is slowly joined in the polysaccharides of Folium Mori solution with disposable syringe, carry out magnetic agitation simultaneously; After adding, magnetic agitation, room temperature reaction 24h; Afterwards, add the 0.4g sodium borohydride in reaction solution, the same terms continues reaction 48h down; Add the 0.4g sodium borohydride again in reaction solution, the same terms continues reaction 24h down; With the reaction solution dialysis tubing of packing into, 48h dialyses in distilled water; The dialyzate freeze-drying obtains the polysaccharides of Folium Mori of ethylene diamine-modified cationization.
Embodiment 4.The preparation of the genophore of ethylene diamine-modified polysaccharides of Folium Mori-DNA plasmid nano-complex
The polysaccharides of Folium Mori that takes by weighing the ethylene diamine-modified cationization of 30mg is dissolved in the 0.5ml distilled water, obtains the cationic polysaccharide storing solution.Get cationic polysaccharide storing solution 50 μ l, dilute 100 times after, obtain cationic polysaccharide and use liquid; Get 10 μ l cationic polysaccharides application liquid and 10 μ l and contain 0.2 μ g DNA plasmid solution, respectively at 55 ℃ of heating 30min; Again with the two mixing, vortex 30s promptly obtains the genophore of ethylene diamine-modified polysaccharides of Folium Mori-DNA plasmid nano-complex, and its transmission electron microscope is seen Fig. 3, and size distribution is seen Fig. 4.
Embodiment 5.
Get 0.5g oxidation polysaccharides of Folium Mori, be dissolved in the 30ml distilled water; Get the borate buffer solution (pH=9) that the 0.62ml quadrol is dissolved in 5ml; The borate solution of quadrol is slowly joined in the polysaccharides of Folium Mori solution with disposable syringe, carry out magnetic agitation simultaneously; After adding, magnetic agitation, room temperature reaction 24h; Afterwards, add the 0.5g sodium borohydride in reaction solution, the same terms continues reaction 48h down; Add the 0.5g sodium borohydride again in reaction solution, the same terms continues reaction 24h down; With the reaction solution dialysis tubing of packing into, 48h dialyses in distilled water; The dialyzate freeze-drying obtains the polysaccharides of Folium Mori of ethylene diamine-modified cationization.Electrophoresis detection proves: the polysaccharides of Folium Mori of originally executing the routine cationization for preparing is more effective to the parcel of plasmid DNA than the polysaccharides of Folium Mori of the cationization of embodiment 3 preparations.
Embodiment 6.
The polysaccharides of Folium Mori that takes by weighing the ethylene diamine-modified cationization of 20mg is dissolved in the 1ml distilled water, obtains the cationic polysaccharide storing solution.Get cationic polysaccharide storing solution 50 μ l, dilute 100 times after, obtain cationic polysaccharide and use liquid; Get 10 μ l cationic polysaccharides application liquid and 10 μ l and contain 0.4 μ g DNA plasmid solution, respectively at 55 ℃ of heating 30min; Again with the two mixing, vortex 30s promptly obtains the genophore of ethylene diamine-modified polysaccharides of Folium Mori-DNA plasmid nano-complex.Electrophoresis detection proves: the polysaccharides of Folium Mori of the cationization that embodiment 4 is prepared has better package action than the polysaccharides of Folium Mori of the cationization of present embodiment preparation to plasmid DNA.
Embodiment 7.The preparation of the polysaccharides of Folium Mori of the cationization of polyethylene imine beautify
Claim 0.2g purified polysaccharides of Folium Mori, be dissolved in the 20ml phosphate buffered saline buffer (Ph=7); Linking agent N with the activation hydroxyl, N '-carbonyl dimidazoles (Aldrich, the U.S.) 0.5g is dissolved in the 5ml methylene dichloride, under protection of nitrogen gas, at first adds catalyst of triethylamine 0.1ml in polysaccharide liquid, dichloromethane solution with the hydroxyl linking agent slowly adds in the polysaccharide soln again, the limit edged stirs, and adds in 30 ~ 100min, after adding, room temperature reaction 90 ~ 150min obtains the activatory polysaccharide soln; The PEI that with the 5.0g number-average molecular weight is 600Da or 2000Da respectively is dissolved in the 10ml phosphate buffered saline buffer; add catalyst of triethylamine; under lucifuge, nitrogen protection, room temperature condition, slowly join in the activatory polysaccharide liquid; the limit edged stirs; add at 120 ~ 150min; react 10h under the lucifuge, room temperature after adding, the solution after reaction is finished obtains the polysaccharides of Folium Mori of the cationization that the PEI of number-average molecular weight 600Da or 2000Da modifies after the dialysis freeze-drying.
With benzotriazole carbonic ether (Aldrich, the U.S.), carbonylic imidazole (Aldrich, the U.S.) or N, N '-two succinimido sulfuric ester (Aldrich, the U.S.) substitutes N, and N '-carbonyl dimidazoles carries out above-mentioned preparation, other step is constant, obtains identical result.
Embodiment 8.The preparation of the genophore of polysaccharides of Folium Mori-DNA plasmid nano-complex that PEI modifies
The polysaccharides of Folium Mori that takes by weighing the cationization of 40mg PEI modification is dissolved in the 1ml distilled water, obtains the cationic polysaccharide storing solution.Get cationic polysaccharide storing solution 50 μ l, dilute 100 times after, obtain cationic polysaccharide and use liquid; Get 10 μ l cationic polysaccharides application liquid and 10 μ l and contain 0.2 μ g DNA plasmid solution, respectively at 55 ℃ of heating 35min; Again with the two mixing, vortex 30s promptly obtains the genophore of polysaccharides of Folium Mori-DNA plasmid nano-complex that PEI modifies.
Embodiment 9.The preparation of the polysaccharides of Folium Mori of the cationization that spermine is modified
Take by weighing 0.2g oxidation polysaccharides of Folium Mori, be dissolved in the 10ml distilled water; Take by weighing the 0.5g spermine and be dissolved in the borate buffer solution (pH=9) of 5ml; The borate solution of spermine is slowly joined in the polysaccharides of Folium Mori solution with disposable syringe, carry out magnetic agitation simultaneously; After adding, magnetic agitation, room temperature reaction 24h; Afterwards, add the 0.2g sodium borohydride in reaction solution, the same terms continues reaction 48h down; Add the 0.2g sodium borohydride again in reaction solution, the same terms continues reaction 24h down; With the reaction solution dialysis tubing of packing into, in distilled water dialysis 48h(intercepting molecular weight greater than 3500Da); The dialyzate freeze-drying obtains the polysaccharides of Folium Mori of the cationization that spermine modifies.
Embodiment 10.The genophore of polysaccharides of Folium Mori-DNA plasmid nano-complex that spermine is modified
The polysaccharides of Folium Mori that takes by weighing the cationization of 20mg spermine modification is dissolved in the 1ml distilled water, obtains the cationic polysaccharide storing solution.Get cationic polysaccharide storing solution 50 μ l, dilute 100 times after, obtain cationic polysaccharide and use liquid; Get 10 μ l cationic polysaccharides application liquid and 10 μ l and contain 0.4 μ g DNA plasmid solution, respectively at 55 ℃ of heating 45min; Again with the two mixing, vortex 30s promptly obtains the genophore of polysaccharides of Folium Mori-DNA plasmid nano-complex that spermine modifies.
Embodiment 11.Agarose gel electrophoresis
Prepare 1% sepharose, add 0.5 μ g/ml bromination second pyridine, bed board, application of sample adopts the gel imaging system observations behind 80V electrophoresis 1.5h.Shown in figure two, the polysaccharides of Folium Mori of cationization can wrap up plasmid DNA preferably.
The electrophoretic step of agarose DNA is as follows:
Step 1. preparation 1% sepharose: take by weighing the 0.2g agarose and place Erlenmeyer flask, add 20ml 0.5 * TBE, bottleneck back-off small beaker.Microwave oven heated and boiled 3 times to agarose all melts, and shakes up, and promptly obtains 1.0% sepharose liquid.
The preparation of step 2. offset plate: synthetic glass inside groove in the electrophoresis chamber and glue trough washery is clean, dry.The synthetic glass inside groove is put into the glue groove, and put comb well in the fixed position.Treat that sepharose solution is cooled to about 65 ℃, add 0.5 μ g/ml ethidium bromide in sepharose liquid, mixing is poured the synthetic glass inside groove carefully into, is that coagulant liquid slowly launches, and forms even glue-line up to whole glass pane surface.Under the room temperature, level leaves standstill until gel solidifies fully, vertically extracts comb gently, and gel and inside groove are put into electrophoresis chamber together.
Step 3. is joined sample: adopt the polysaccharides of Folium Mori of the prepared cationization of embodiment 5, with some prepared row samples of the mass ratio of different cationic polysaccharides and plasmid DNA, being respectively lotus positive electricity polysaccharides of Folium Mori and plasmid DNA mass ratio is 2:1; 5:1; 10:1; 30:1; 50:1; 80:1; 100:1.
Step 4. application of sample: (polysaccharides of Folium Mori of cationization and the mass ratio of plasmid DNA are followed successively by from 8 ducts, the 2nd duct to the polysaccharides of Folium Mori that mixes a series of cationizations on point template: 2:1 with the polysaccharide-plasmid dna complex laminate samples of plasmid DNA different mass ratio; 5:1; 10:1; 30:1; 50:1; 80:1; 100:1) and sample-loading buffer, the 1st duct is free plasmid DNA.Respectively sample is added in the sample sulculus of offset plate with 10 μ l micropipets.
Step 5. electrophoresis: the gel slab after the oiling is switched on immediately and is carried out electrophoresis, voltage 60 ~ 100V, and sample is moved to negative pole (black) direction by anodal (redness).When tetrabromophenol sulfonphthalein moves to apart from the about 1cm in offset plate forward position place, stop electrophoresis.
After step 6. electrophoresis finishes, take out gel.
Step 7. observe to be taken a picture: observe under ultraviolet lamp, DNA exists and then demonstrates the fluorescent red-orange band, adopts the gel imaging system preservation of taking pictures.As seen the polysaccharides of Folium Mori of cationization has tangible package action (see figure 2) to the DNA plasmid.Among Fig. 2, be exposed plasmid in the duct 1, can be dyeed by the bromination second pyridine in the gel, under ultraviolet lamp, manifest orange fluorescence, in electrophoresis,, and 28 be that (mass ratio of cationization polysaccharide and plasmid DNA is followed successively by from 8 ducts, the 2nd duct to the: 2:1 with cationization polysaccharides of Folium Mori bonded plasmid to the duct from the duct towards positive pole migration; 5:1; 10:1; 30:1; 50:1; 80:1; 100:1), the 2nd ~ 4 duct is owing to the polysaccharide quantity not sufficient discharges the part plasmid to wrap up plasmid fully, tangible migration is arranged in gel, though the 5th duct does not have tangible DNA migration band, but show orange fluorescence at foramen primum, illustrate that plasmid partly exposes the surface at nano-complex; Increase (the 6th ~ 8 duct) along with polysaccharide quality proportion, owing to being wrapped up by the cationization polysaccharide fully, plasmid avoids being dyeed by the pyridine of bromination second, thereby can not manifest fluorescence, the positive charge of the cationization polysaccharide negative charge of plasmid that neutralized simultaneously, in electrophoresis to the positive pole migration and be not trapped in the former duct.
Embodiment 12.The mensuration of transfection effect
With TGF β-1 plasmid is reporter gene, according to the polysaccharides of Folium Mori ~ DNA plasmid nanoparticle genophore of three kinds of cationizations of embodiment one, two, three described preparations.Cultivate the SD rat bone marrow mesenchymal stem cells in 96 orifice plates, cell concn reaches 2 * 105/ml perfect medium/hole, after hatching 24 ~ 48h, replace former substratum with serum free medium, polysaccharides of Folium Mori-DNA plasmid the nano-complex that adds three kinds of cationizations respectively, liposome Lipofectamine-DNA plasmid composite, free plasmid, make every hole plasmid DNA amount be 0.2 μ g, and with the negative contrast of blank cell, after hatching 4h, serum free medium is replaced as the fresh blood serum medium that contains, continue to hatch 72h, Rat TGF-β 1 ELISA Kit detects the transfection effect.Shown in Fig. 5, the polysaccharides of Folium Mori that spermine and small molecular weight PEI modify all has certain transfection effect, but transfection efficiency less than liposome-DNA plasmid composite, the transfection efficiency of the polysaccharides of Folium Mori of ethylene diamine-modified cationization-DNA plasmid composite is the highest, and is higher than the transfection efficiency of liposome-DNA plasmid composite.The polysaccharides of Folium Mori that ethylene diamine-modified cationization is described is as the optimal selection of gene transfection carrier in the polysaccharide of these three kinds of lotus positive electricity.
The cell transfecting experimental procedure:
Step 1, stem cell separate and cultivate
Draw neck to put to death the SD rat, volume fraction is alcohol immersion 3 ~ 5min of 75%, and aseptic condition takes out shin bone and femur down; With its two ends metaphysis excision, expose medullary space, draw an amount of PBS cleaning down medullary space with asepsis injector; Marrow is gone out in piping and druming repeatedly; Medullary cell is fully disperseed; The marrow single cell suspension that is obtained slowly drips in the centrifuge tube of the Percoll parting liquid that presets (relative volume mass 1.073) along tube wall, and the volume ratio of marrow single cell suspension and parting liquid is 1:1; 2000rpm, centrifugal 20min, cloud cellular layer in the middle of drawing is with PBS washing 3 times; Again hanged cell, added perfect medium (containing the DMEM that volume fraction is 10% foetal calf serum), placed culturing bottle, 37 ℃ of volume fractions are 5% CO 2Cultivate in the incubator.
Step 2. cell transfecting
The transfection of the polysaccharides of Folium Mori of three kinds of cationizations-DNA plasmid nano-complex: get the polysaccharides of Folium Mori-DNA plasmid nano-complex (20 μ g/ml) of the cationization that quadrol, spermine and small molecular weight PEI modify respectively, add in 96 orifice plates (2.5 * 10 respectively 5/ hole) and jiggle and make its uniform mixing; Place 37 ℃ of CO 2Incubator is hatched 24 ~ 48h, with the transfection efficiency of liposome-DNA plasmid composite in contrast.

Claims (2)

1. the polysaccharides of Folium Mori nanoparticle genophore of a cationization is characterized in that: it is the genophore of a kind of polysaccharides of Folium Mori of modifying with aminated compounds in conjunction with the DNA plasmid, and the molecular weight distribution of polysaccharides of Folium Mori is 3 ~ 3.5 * 10 4Da, wherein by mass ratio, the polysaccharides of Folium Mori of cationization: DNA plasmid=0.5 ~ 100:1, the particle diameter of plasmid are 50-150nm, described aminated compounds is that spermine, quadrol or number-average molecular weight are the polymine of 600Da-2000Da.
2. method for preparing the polysaccharides of Folium Mori nanoparticle genophore of the described cationization of claim 1 is characterized in that it may further comprise the steps:
The preparation of step 1. oxidation polysaccharides of Folium Mori:
Take by weighing 0.5 ~ 1g purified polysaccharides of Folium Mori, be dissolved in 20ml ~ 50ml distilled water, add KIO 4, KIO 4With the mol ratio of monosaccharide units in the polysaccharide be: 0.5 ~ 5:1 is put into the darkroom rapidly, magnetic agitation, room temperature reaction 72h; Reaction solution adds 10 ~ 20ml ethylene glycol termination reaction, continues reaction 30min by aforementioned condition; With the reaction solution dialysis tubing of packing into, 48h dialyses in distilled water; The dialyzate freeze-drying obtains oxidation polysaccharides of Folium Mori 0.4 ~ 0.9g;
The preparation of the polysaccharides of Folium Mori of step 2. cationization:
A. the preparation of the polysaccharides of Folium Mori of the cationization of spermine modification:
Take by weighing the oxidation polysaccharides of Folium Mori that 0.1 ~ 0.5g step 1 makes, be dissolved in 10 ~ 50ml distilled water; Take by weighing 0.2 ~ 1.5g spermine and be dissolved in the borate buffer solution (pH=9) of 5ml; The mol ratio of the aldehyde radical of spermine and oxidation of polysaccharides is 0.5 ~ 5:1, and the borate solution of spermine is slowly joined in the polysaccharides of Folium Mori solution with disposable syringe, carries out magnetic agitation simultaneously; After adding, magnetic agitation, room temperature reaction 24h; Afterwards, add 0.1 ~ 0.5g sodium borohydride in reaction solution, the same terms continues reaction 48h down; Add 0.1 ~ 0.5g sodium borohydride again in reaction solution, the total mass of adding sodium borohydride with the ratio of the quality of oxidation of polysaccharides is: 0.5 ~ 3:1, the same terms continue reaction 24h down; With the reaction solution dialysis tubing of packing into, 48h dialyses in distilled water; The dialyzate freeze-drying obtains the polysaccharides of Folium Mori 0.2 ~ 0.4g of the cationization that spermine modifies;
The preparation of the polysaccharides of Folium Mori of B. ethylene diamine-modified cationization
Get the oxidation polysaccharides of Folium Mori that 0.1 ~ 0.5g step 1 makes, be dissolved in 10 ~ 50ml distilled water; Get the borate buffer solution (pH=9) that quadrol is dissolved in 5ml, the mol ratio of the aldehyde radical of quadrol and oxidation of polysaccharides is 0.5 ~ 5:1; The borate solution of quadrol is slowly joined in the polysaccharides of Folium Mori solution with disposable syringe, carry out magnetic agitation simultaneously; After adding, magnetic agitation, room temperature reaction 24h; Afterwards, add 0.1 ~ 0.5g sodium borohydride in reaction solution, the same terms continues reaction 48h down; Add 0.1 ~ 0.5g sodium borohydride again in reaction solution, the total mass of adding sodium borohydride with the ratio of the quality of oxidation of polysaccharides is: 0.5 ~ 3:1, the same terms continue reaction 24h down; With the reaction solution dialysis tubing of packing into, 48h dialyses in distilled water; The dialyzate freeze-drying obtains the polysaccharides of Folium Mori 0.2 ~ 0.4g of ethylene diamine-modified cationization;
C. the preparation of the polysaccharides of Folium Mori of the cationization of polyethylene imine beautify
Take by weighing 0.2 ~ 0.5g purified polysaccharides of Folium Mori, be dissolved in 10 ~ 15ml phosphate buffered saline buffer (pH=7); Linking agent with the activation hydroxyl, as: N, N '-carbonyl dimidazoles, benzotriazole carbonic ether, carbonylic imidazole, N, in the N '-two succinimido sulfuric ester any, be dissolved in the 5ml methylene dichloride, the mass ratio of polysaccharide and linking agent is: 0.5 ~ 3:1, under protection of nitrogen gas, at first add catalyst of triethylamine in polysaccharide liquid, the dichloromethane solution with the hydroxyl linking agent slowly adds in the polysaccharide soln again, and the limit edged stirs, in 30 ~ 100min, add, after adding, room temperature reaction 90 ~ 150min obtains the activatory polysaccharide soln; With number-average molecular weight is that the polymine of 600Da-2000Da is dissolved in the 10ml phosphate buffered saline buffer, the mass ratio of polysaccharide and polymine is 0.5 ~ 4:1, add catalyst of triethylamine, under lucifuge, nitrogen protection, room temperature condition, slowly join in the activatory polysaccharide liquid, the limit edged stirs, and adds at 120 ~ 150min, reacts 10h under lucifuge, the room temperature after adding, solution after reaction is finished obtains the polysaccharides of Folium Mori of the cationization of polyethylene imine beautify after dialysis, freeze-drying;
The preparation of the polysaccharides of Folium Mori nanoparticle genophore of step 3. cationization:
The polysaccharides of Folium Mori that takes by weighing the above-mentioned three kinds of cationizations of 10 ~ 40mg respectively is dissolved in 0.5 ~ 2ml distilled water, obtains the cationic polysaccharide storing solution;
Respectively get cationic polysaccharide storing solution 50 μ l, dilute 100 times after, obtain cationic polysaccharide and use liquid; Get 10 μ l cationic polysaccharides application liquid and 10 μ l respectively and contain 0.1 ~ 3 μ g DNA plasmid solution, respectively at 55 ℃ of heating 30 ~ 60min; Again with the two mixing, vortex 30s promptly obtains the genophore of the polysaccharides of Folium Mori-DNA plasmid nano-complex of the cationization that aminated compounds modifies.
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