CN104258418A - NGF gene-polycationic nano-particle composite as well as preparation method and application thereof - Google Patents
NGF gene-polycationic nano-particle composite as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an NGF gene-polycationic nano-particle composite as well as a preparation method and an application thereof. The nano-particle composite comprises an NGF gene substance and a polycation, wherein the polycation refers to polyspermineimidazole-4,5-diimine or polyspermineimidazole-4,5-diamide; and a mass ratio of the polycation to the NGF gene substance is 5-100. The nano-particle composite disclosed by the invention is low in toxicity and high in transfection efficiency, can give consideration to the stability before reaching target cells and the biological responsiveness after entering the target cells in the body circulation process, and the polycation can finish non-toxic metabolism in vivo. Therefore, the composite can be used for treating Parkinson's disease and complications thereof through local intracranial injection.
Description
Technical field
The present invention relates to non-viral gene vector technical field, specifically, relate to ngf gene-polycation nano particle composites and its preparation method and application.
Background technology
In the evolution of gene therapy, the research of gene delivery system and genophore is the focus that people pay close attention to all the time, and can genophore passenger gene be the key point whether gene therapy can apply clinically to appointed place safely and effectively.Genophore can be divided into virus based vector and the large class of non-viral type carrier two.Although what use was maximum at present remains virus based vector, its transfection efficiency can reach more than 90%, but there is the limitation self being difficult to overcome in viral vector, if induction host immune response and potential carcinogenecity, preparation is complicated, and the foreign DNA size that can load is limited, makes it apply and is very limited.So people start the research being conceived to non-viral type carrier.The method of current existing non-viral type vector-mediated gene transfection has naked DNA injection, calcium phosphate mediation, electrotransfer method, cationic-liposome and cationic polymer mediation etc., although former three does not have the shortcoming of viral vector, transfection efficiency is not fully up to expectations.Cationic liposomal transfection efficiency is higher, reproducible, but need remove serum during transfection, and in body, instability is a difficult problem for puzzlement medical circle always.From current development trend, cationic polymer carrier presents significant advantage in gene transfer.
Cationic polymer is also known as polycation, and kind is a lot, and what research was more at present has polypeptide class: polylysine, polyglutamic acid and derivant thereof; Poly amine: polymine, polyporpyleneimine dendrimer; Polymethacrylic acid: polyamidoamine tree, polyethyl methacrylate 2-(dimethylamine); And some natural polymers are as chitosan, gelatin etc.A common feature on these polymer architectures is containing much amino in molecule, can occur protonated at physiological ph, these protonated amino can in and the negative charge of DNA plasmid surface, make DNA molecular by the relatively little DNA particle of stretched out structure boil down to volume, or genes of interest is wrapped in wherein, make DNA from the degraded of nuclease, they can as genophore.
DNA transfer is mainly entered cell by endocytosis by cationic polymer-DNA complex, and form Inclusion (endosome), DNA discharges from Inclusion, enters in Cytoplasm, then enters in core further and transcribe, express.
Poly-spermine imidazoles-4,5-diimine (Polyspermine imidazole-4,5-imine, PSI) structural formula is as follows:
，
Poly-spermine imidazoles-4,5-diamides (Polyspermine imidazole-4,5-amide, PSIA) structural formula is as follows:
，
Above two kinds of compounds are polycation.
NGF albumen (being encoded by ngf gene) has neurotrophic activity to what grow with the motor neuron of maturation, it is the strongest in the middle of the gene that finds so far to the effect of motor neuron, the survival of motor neuron, differentiation and metabolic activity can be promoted, and have very strong repair to the motor neuron of damage after period of embryo or birth.NGF albumen to the neuron particularly obvious reparation damaging action that has of DOPA ammonia serotonergic neuron and motor neuron, for Parkinsonian treatment provides possibility.
Treat parkinson disease about the nano-particle complex using ngf gene and PSI or PSIA polycation to be formed at present have not been reported.
Summary of the invention
The object of the invention is for deficiency of the prior art, a kind of ngf gene-polycation nano particle composites is provided.
Of the present invention again one object be that the preparation method of described ngf gene-polycation nano particle composites is provided.
Another object of the present invention provides the purposes of described ngf gene-polycation nano particle composites.
For realizing above-mentioned first object, the technical scheme that the present invention takes is:
A kind of ngf gene-polycation nano particle composites, it comprises ngf gene material and polycation, and described polycation is poly-spermine imidazoles-4,5-diimine or poly-spermine imidazoles-4,5-diamides, the mass ratio of described polycation and ngf gene material is 5 ~ 100.
The mass ratio of described polycation and ngf gene material is 40 ~ 50.
It is prepared by following methods:
(1) said polycation solution is joined fast in the ngf gene solution of same volume, mix homogeneously;
(2) by mixed solution in incubated at room temperature 20 ~ 30 minutes, obtain ngf gene-polycation nano particle composites.
Described poly-spermine imidazoles-4,5-diimine structural formula is:
，
Wherein, n is 100 ~ 500.
Described poly-spermine imidazoles-4,5-diamides structural formula is:
，
Wherein, n is 100 ~ 500.
The particle diameter of described ngf gene-polycation nano particle composites is 100 ~ 300 nanometers.
For realizing above-mentioned second object, the technical scheme that the present invention takes is:
The preparation method of ngf gene as above-polycation nano particle composites, comprises the following steps:
(1) said polycation solution is joined fast in the ngf gene solution of same volume, mix homogeneously;
(2) by mixed solution in incubated at room temperature 20 ~ 30 minutes, obtain ngf gene-polycation nano particle composites.
Described poly-spermine imidazoles-4,5-diimine structural formula is:
，
Described poly-spermine imidazoles-4,5-diamides structural formula is:
，
Wherein, n is 100 ~ 500.
The particle diameter of described ngf gene-polycation nano particle composites is 100 ~ 300 nanometers.
In step (1), the speed of mixing is preferably 200 ~ 2000rpm, and incubation time is preferably 20 ~ 30 minutes.
For realizing above-mentioned 3rd object, the technical scheme that the present invention takes is:
The application of ngf gene as above-polycation nano particle composites in preparation treatment parkinson disease or unusual fluctuation disease drug.
Ngf gene of the present invention-polycation nano particle composites can directly intracranial injection in Parkinsonian's striatum, or intravenous injection, nasal-cavity administration, inhalation, be used for the treatment of parkinson disease or its unusual fluctuation disease, injected dose is that 0.5 mg is to 100 mg.
In the present invention, the gene order being used for the treatment of parkinson disease or its unusual fluctuation disease is not limited only to embodiment, as long as comprise the functional sequence of ngf gene.
The invention has the advantages that:
Compared with prior art, polycation of the present invention gathers spermine imidazoles-4,5-diimine and poly-spermine imidazoles-4,5-diamides can condense, carry ngf gene, biological responding after can taking into account the stability that arrives before target cell in vivo in cyclic process and entering target cell, and polycation self can complete the metabolism of Non-toxic in vivo, be therefore used for the treatment of parkinson disease and complication thereof by intracranial local injection.On the whole, strong in conjunction with DNA ability, that toxicity is little, transfection efficiency is high advantage is possessed.Compared to Virus delivery vehicles, devoid of risk, to have no side effect.Preparation method of the present invention can prepare the nano-particle complex that even particle size distribution and genetic stew are evenly distributed.
Detailed description of the invention
Below detailed description of the invention provided by the invention is elaborated.
The physicochemical property characterizing method of the present invention to ngf gene-polycation nano particle composites comprises: agarose gel electrophoresis, transmission electron microscope, dynamic light scattering and zeta potential test.It is PC-12 cell that ngf gene-polycation nano particle composites gene transfection and cytotoxicity test the cell selected.The PD mouse models that the therapeutic test of ngf gene-polycation nano particle composites to parkinson disease and unusual fluctuation disease thereof is selected.
In following examples, described PSIA and PSI structural formula is respectively:
，
，
Wherein, n is 100 ~ 500.
In following examples, the concrete sequence of ngf gene used is as shown in SEQ ID NO.1.
the preparation of embodiment 1 ngf gene-polycation nano particle composites
Take a certain amount of PSIA or PSI to be dissolved in the water, make concentration be 2.0 mg/ml, for subsequent use after the water film filtering of 0.45 μm; Draw a certain amount of ngf gene solution and be diluted with water to the ngf gene storing solution of 20 μ g/ml.When preparation PSIA or PSI and ngf gene nano-particle complex, according to a series of polycation of setting and the mass ratio of ngf gene, said polycation solution is diluted to respective concentration, join same volume fast again and in the fixing ngf gene solution of concentration, the final concentration of ngf gene is made to be 20 μ g/ml, last 1100rpm homogeneous mixture solotion, incubated at room temperature 25 minutes, obtain the nanoparticles solution of a series of polycation and ngf gene different quality ratio, the mass ratio of polycation and ngf gene is 5,7,10,20,40,50,100.
the agarose gel electrophoresis of embodiment 2 ngf genes-polycation nano particle composites
Take 1.0 g agaroses, add 100 ml 1 × TAE buffer, heating for dissolving in microwave oven, treat that temperature is down to 65 DEG C, add Ethidum Eremide (EB), be mixed with 1.0 % agarose solutions (containing 0.5 μ g/ml Ethidum Eremide), pour in glue groove, insert sample comb, room temperature is placed and within 0.5-1 hour, is waited gelling solid.Then, extract sample comb, in electrophoresis tank, add TAE buffer do not have gel, wait for loading.Then, with reference to the preparation method of the nanoparticle complex solution of embodiment 1, the nanoparticle complex solution of preparation different quality ratio, in nanoparticle complex solution, the mass ratio of PSIA or PSI and ngf gene is followed successively by 0, and 1,2,3,5,7,10,15,20,30,50,70,100.Marker selects DS
tM5000(100 – 5000 bp), loading 2 μ l; 6 × sample-loading buffer (bromjophenol blue-glycerol indicator, containing 0.25 % bromjophenol blue, 40 % glycerol) 1 μ l and nanoparticle complex solution 5 μ l Homogeneous phase mixing, loading 6 μ l.Electrophoresis 45 minutes under 110 mV voltages, is finally placed in ultraviolet gel imaging system and observes and record electrophoretogram.Electrophoresis pattern shows: when the mass ratio of PSIA or PSI and ngf gene is 1, the swimming in the electric field of ngf gene molecule is subject to partial block, along with the increase gradually of PSIA or PSI and ngf gene mass ratio, when the mass ratio of PSIA or PSI and ngf gene is between 5-7, polycation PSIA or PSI has almost blocked the migration of ngf gene molecule completely, even in larger quality than under condition, poly-spermine cation can wrap up ngf gene molecule more fully, does not observe the movement of ngf gene molecule in electrophoretogram completely.Experiment demonstrates PSIA or PSI and has the ability that more satisfactory cohesion ngf gene forms composite particles, thus plays the effect of protection ngf gene, is degraded before cytophagy to avoid it.
the transmission electron microscope observing of embodiment 3 ngf genes-polycation nano particle composites
The mass ratio preparing PSIA or PSI and ngf gene according to the preparation method of the nanoparticle complex solution of embodiment 1 is the nanoparticle complex solution of 40, and sample size is 100 μ l.First draw 10 μ l nanoparticle complex solution slowly to drip on 400 object copper mesh, naturally dry under room temperature, finally use transmission electron microscope to observe the form of sample, record transmission electron microscope picture.Transmission electron microscope picture shows: poly-spermine cation PSIA or PSI joins in ngf gene molecule, complex is formed by electrostatic force complexation, present the nano-particle that class is spherical, its form is regular, even, it is evident that ngf gene molecule is wrapped in inside polymer, the grain size of nano-particle is at 100-300 nm.
the particle size distribution of embodiment 4 ngf genes-polycation nano particle composites and zeta potential test
Prepare the nanoparticle complex solution of different quality ratio according to the preparation method of the nanoparticle complex solution of embodiment 1, PSIA or PSI of the nanoparticle complex solution of required mensuration and the mass ratio of ngf gene are followed successively by 5,10,15,20,30, sample size is 2 ml.At ambient temperature, first preheating Particle Size Analyzer 30 minutes, then draw 1 ml nanoparticles solution and add micro-example pond, then micro-example pond is put into the test trough of Particle Size Analyzer, arranging probe temperature is 25 DEG C, and medium is water, viscosity is 0.890 cP, and refractive index is 1.330.Particle size distribution for nano-particle is tested, and light scattering angle is 90 DEG C, and determined wavelength is 659.0 nm, each sample test 3 times, and each run time is 2 minutes, records meansigma methods and the polydispersity thereof of each sample particle diameter.For the zeta potential test of nano-particle, dielectric constant is 78.54, pH value is 7.0, Zeta potential analytical model is Smoluchowski equation (polar solvent: Henrys equation F (ka) is similar to 1.5), each sample test 3 times, each operation automatically 10 times, records meansigma methods and the mobility thereof of each sample zeta current potential.Result shows: the ngf gene-PSIA nano-particle of different quality ratio and the particle diameter of ngf gene-PSI Nanoparticles Nanoparticles are stabilized in 100-300 nm; The zeta Potential distribution of nano-particle is at 8-25mV.
the cell transfecting of embodiment 5 ngf genes-polycation nano particle composites
Select PC-12 neurocyte as the receptor studying PSIA or PSI and ngf gene nano-particle complex cell transfecting.According to (5-10) × 10
4the cell density of number/ml Cell sap turns 48 porocyte plates.For cell transfecting, PSIA or PSI and ngf gene nano-particle complex are added cell culture.First, the volume that every hole adds solution is 50 μ l, parallel 3 multiple holes, and the quality that every hole adds ngf gene is 500 ng, diluent media is phosphate buffered solution, PSIA or PSI of the nanoparticle complex solution of required investigation and the mass ratio of ngf gene are followed successively by 0,10,20,30,40,50.Then, 48 porocyte plates are taken out from incubator, suck culture fluid, every hole 200 μ l phosphate buffered solution are rinsed once, suck phosphate buffered solution again, every hole adds 250 μ l DMEM high glucose mediums (containing phenol red), is joined successively in cell plates by the nanoparticle complex solution of a series of different quality ratio subsequently, is placed in 37 DEG C, cultivates 4 hours in 5% cell culture incubator.Then, 48 porocyte plates are taken out from incubator, suck culture fluid, every hole 200 μ l phosphate buffered solution are rinsed once, suck phosphate buffered solution again, every hole adds 500 μ l containing the DMEM high glucose medium (containing phenol red) of 10 % hyclones, is finally placed in 37 DEG C again, cultivates in 5 % cell culture incubators.After 44 hours, measure the efficiency of cell transfecting.Meanwhile, PC-12 neurocyte growth rate method is adopted to measure the rate of increase of cell.Each sample parallel tests 3 times, mapping of averaging.Then, 48 porocyte plates are taken out from incubator, suck culture fluid, every hole 200 μ l phosphate buffered solution are rinsed once, suck phosphate buffered solution again, every hole adds 250 μ l DMEM high glucose mediums (without phenol red), the nanoparticles solution of a series of different quality ratio is joined successively in cell plates subsequently, is placed in 37 DEG C, cultivates 4 hours in 5 % cell culture incubators.Then, 48 porocyte plates are taken out from incubator, suck culture fluid, every hole 200 μ l phosphate buffered solution are rinsed once, suck phosphate buffered solution again, every hole adds 500 μ l containing the DMEM high glucose medium (containing phenol red) of 10% hyclone, is finally placed in 37 DEG C again, cultivates in 5% cell culture incubator.After 44 hours, measure the efficiency of cell transfecting.Each sample parallel tests 3 times, mapping of averaging.Statistical analysis adopts independent sample T inspection.The cell transfecting result of ngf gene-PSIA nano-particle complex (mass ratio of PSIA and ngf gene is 40) and ngf gene-PSI nano-particle complex (mass ratio of PSI and ngf gene is 40) is as shown in table 1.Can draw to draw a conclusion: PSIA or PSI possesses desirable conveying NGF(gene expression) ability.
The cell transfecting result of table 1 ngf gene-PSIA or PSI nano-particle complex
Note: *, ngf gene-PSIA carrier 1, ngf gene-PSIA carrier 2 respectively compared with PSIA vehicle group, P<0.05; #, ngf gene-PSI carrier 1, ngf gene-PSI carrier 2 respectively compared with PSI vehicle group, P<0.05.
the cytotoxicity test experiments of embodiment 6 PSIA or PSI
Adopt Thiazolyl blue method (MTT) quantitative expedition PSIA or PSI to PC-12 cytotoxicity.First, according to (5-10) × 10
4the cell density of number/ml Cell sap turns 96 porocyte plates, is placed in 37 DEG C, overnight incubation in 5 % cell culture incubators.Secondly, a series of variable concentrations (50,100,150 is prepared, 200,400,600,800 μ g/ml) PSIA or PSI gather spermine cationic solution, the volume that every hole adds poly-spermine cationic solution is 100 μ l, parallel 6 multiple holes, diluent media is DMEM high glucose medium (without phenol red), and required investigation PSIA or PSI gathers the cationic concentration of spermine and be followed successively by 50,100,150,200,400,600,800, unit is μ g/ml.Then, 96 porocyte plates are taken out from incubator, suck culture fluid, every hole 100 μ l phosphate buffered solution are rinsed once, suck phosphate buffered solution again, the poly-spermine cationic solution of above-mentioned a series of variable concentrations joined in cell plates successively, is placed in 37 DEG C, cultivate 4 hours in 5 % cell culture incubators.Then, 96 porocyte plates are taken out from incubator, suck culture fluid, every hole 100 μ l phosphate buffered solution are rinsed once, suck phosphate buffered solution again, every hole adds 112.5 μ l DMEM high glucose mediums (without phenol red) and 12.5 μ l MTT solution (5 mg/ml), continues to be placed in 37 DEG C, cultivates in 5 % cell culture incubators.After 6 hours, 96 porocyte plates are taken out from incubator, suck culture fluid, every hole adds 150 μ l dimethyl sulfoxide (molecular biosciences level), slightly shake up, treat that bluish violet crystal first is praised (formazan) and dissolved completely, finally, use multi-functional microplate reader working sample at the absorbance at 570 nm and 630 nm places.Each sample parallel tests 6 times, mapping of averaging, and statistical analysis adopts independent sample T inspection (PEI 25 KDa as a control group).Cell survival rate (percentage ratio) treats that the absorbance of test sample is compared Normocellular absorbance (namely the concentration of polymer solution is 0) and represented.The cytotoxicity test result of PSIA and PSI is as shown in table 2.Can draw to draw a conclusion: PSIA or PSI is very low to PC-12 cytotoxicity in a series of activities concentration range, cell survival rate is 87% ~ 99%.
The cytotoxicity test result of table 2 PSIA and PSI
embodiment 7 ngf genes-polycation nano particle composites treatment Parkinson disease model
1. unusual fluctuation disease (levodopa-induced dyskinesia, LID) model preparation
After the anaesthetized with pentobarbital of rats by intraperitoneal injection 3%, strict flat cranium position is fixed on rat brain stereotactic apparatus, iodophor disinfection skin of head after cropping, scalp is cut along median line, 15% hydrogen peroxide burns periosteum, expose skull suture and bregma, determine bregma coordinate, according to bag new people wait show " rat brain stereotaxic atlas ", determine right side medial forebrain bundle (medial forebrain bundle, MFB) injection site: 1. 3.7mm after bregma, 1.7mm on the right side of sagittal suture, 8.0mm under skull, front tooth line 2.4mm; 2. 4.4mm after bregma, 1.2mm on the right side of sagittal suture, 8.0mm under skull, front tooth line 2.4mm.By the above-mentioned injection site boring determined, extract 6-OHDA 6 μ l(with the microsyringe of 10 μ l specifications and be dissolved in 0.2% vitamin C normal saline, concentration 4 μ g/ μ l), often some injection 3 μ l, injection speed about 1 μ l/min, the slowly withdraw of the needle after let the acupuncture needle remain at a certain point 5min, move back 4mm let the acupuncture needle remain at a certain point again 5min, until exit completely, skin suture otch, puts cage and feeds.After 3 weeks, rats by intraperitoneal injection apomorphine (0.5mg/kg) induction rotates, and average speed >7 time/min is successful PD rat model.After utilizing 6-OHDA to prepare PD rat model, lumbar injection LDME/benserazide (50 mg/kg LDME and 25 mg/kg benserazides are dissolved in containing in 0.2% ascorbic sterilization normal saline) 4 weeks, preparation LID rat model.
2. model grouping and behavioristics measure
At random LID rat model is divided into: (I) ngf gene-PSIA vehicle group, (II) ngf gene-PSI vehicle group, (III) PSIA vehicle group, (IV) PSI vehicle group, (V) ngf gene group.LID rat intracranial injectable drug, dosage is 50mg/kg, in the medicine that I, II, III, IV group uses, PSIA or PSI concentration is 800 μ g/ml, and in I, II group, the mass ratio of PSIA or PSI and ngf gene is ngf gene concentration in 40, IV and V groups and is 20 μ g/ml.Each group of rat rotates through the induction of lumbar injection apomorphine, records rotating cycle in its 30 minutes, calculates the percent (%) shared by number of times that each group of rat average rotating cycle per minute and forelimb touch barrel wall.As shown in Table 3 and Table 4, the cylinder therapeutic effect of ngf gene-PSI nano-particle complex as shown in table 5 and table 6 for the cylinder therapeutic effect of ngf gene-PSIA nano-particle complex.Result shows that ngf gene-PSIA or PSI polycation nano particle composites significantly can reduce LID rat rotating cycle, and forelimb touches barrel wall to be increased.
Table 3 ngf gene-PSIA nano-particle complex is on the impact of number of revolutions
Note: *, compared with the ngf gene group of same time after injection, P<0.05; #, compared with the PSIA group of same time after injection, P<0.05.
Table 4 ngf gene-PSIA nano-particle complex touches a barrel impact for wall number of times to forelimb
Note: *, compared with the ngf gene group of same time after injection, P<0.05; #, compared with the PSIA group of same time after injection, P<0.05.
Table 5 ngf gene-PSI nano-particle complex is on the impact of number of revolutions
Note: *, compared with the ngf gene group of same time after injection, P<0.05; #, compared with the PSI group of same time after injection, P<0.05.
Table 6 ngf gene-PSI nano-particle complex touches a barrel impact for wall number of times to forelimb
Note: *, compared with the ngf gene group of same time after injection, P<0.05; #, compared with the PSI group of same time after injection, P<0.05.
In summary, PSIA or PSI of the present invention gathers spermine cation and can condense, carry nucleic acid drug, in vivo in cyclic process, biological responding after can taking into account the stability before arrival target cell and entering target cell, further, PSIA or PSI gathers the metabolism that spermine cation self can complete Non-toxic in vivo.
embodiment 8 investigates other class ngf gene-Poly-cation nano-particle complexes to the ethological impact of rat model of Parkinson disease
LID rat model is divided into: 1. of the present invention group: the method with reference to embodiment 1 prepares ngf gene-PSIA nano-particle complex, and wherein the mass ratio of PSIA and ngf gene is 40; 2. matched group one: the method with reference to embodiment 1 prepares ngf gene-PSIA nano-particle complex, and wherein the mass ratio of PSIA and ngf gene is 40, and the n of PSIA is 600-800; 3. matched group two: the method with reference to embodiment 1 prepares ngf gene-PSIA nano-particle complex, and wherein the mass ratio of PSIA and ngf gene is 35.Below respectively organize LID rat intracranial injected dose and be 50mg/kg, in medicine, PSIA concentration is 800 μ g/ml.Each group of rat rotates through the induction of lumbar injection apomorphine, records rotating cycle in its 30 minutes, calculates the percent (%) shared by number of times that each group of rat average rotating cycle per minute and forelimb touch barrel wall.Result is as shown in table 7 and table 8.
Table 7 respectively organizes nano-particle complex to the impact of number of revolutions
Note: *, compared with the matched group one of same time after injection, P<0.05; #, compared with the matched group two of same time after injection, P<0.05.
Table 8 respectively group nano-particle complex touches a barrel impact for wall number of times to forelimb
Note: *, compared with the matched group one of same time after injection, P<0.05; #, compared with the matched group two of same time after injection, P<0.05.
In addition, be also provided with experimental group: the method 4. with reference to embodiment 1 prepares ngf gene-PSI nano-particle complex, and wherein the mass ratio of PSI and ngf gene is 40; 5. the method with reference to embodiment 1 prepares ngf gene-PSI nano-particle complex, and wherein the mass ratio of PSI and ngf gene is 40, and the n of PSIA is 600-800; 6. the method with reference to embodiment 1 prepares ngf gene-PSI nano-particle complex, and wherein the mass ratio of PSI and ngf gene is 35.Result display experimental group, 4. when medication 4,8,12,16,20 days, 5. and 6. significantly can reduce the percent (P<0.05) shared by number of times that rat average rotating cycle (P<0.05) per minute and increase forelimb touch barrel wall compared to experimental group.Above result shows that PSIA, PSI are the good carrier of ngf gene.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
SEQUENCE LISTING
<110> Xinhua Hospital Attached to Medical School, Shanghai Jiaotong Univ.
<120> ngf gene-polycation nano particle composites and its preparation method and application
<130> /
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 726
<212> DNA
<213> artificial sequence
<400> 1
atgtccatgt tgttctacac tctgatcaca gcttttctga tcggcataca ggcggaacca 60
cactcagaga gcaatgtccc tgcaggacac accatccccc aagcccactg gactaaactt 120
cagcattccc ttgacactgc ccttcgcaga gcccgcagcg ccccggcagc ggcgatagct 180
gcacgcgtgg cggggcagac ccgcaacatt actgtggacc ccaggctgtt taaaaagcgg 240
cgactccgtt caccccgtgt gctgtttagc acccagcctc cccgtgaagc tgcagacact 300
caggatctgg acttcgaggt cggtggtgct gcccccttca acaggactca caggagcaag 360
cggtcatcat cccatcccat cttccacagg ggcgaattct cggtgtgtga cagtgtcagc 420
gtgtgggttg gggataagac caccgccaca gacatcaagg gcaaggaggt gatggtgttg 480
ggagaggtga acattaacaa cagtgtattc aaacagtact tttttgagac caagtgccgg 540
gacccaaatc ccgttgacag cgggtgccgg ggcattgact caaagcactg gaactcatat 600
tgtaccacga ctcacacctt tgtcaaggcg ctgaccatgg atggcaagca ggctgcctgg 660
cggtttatcc ggatagatac ggcctgtgtg tgtgtgctca gcaggaaggc tgtgagaaga 720
gcctga 726
Claims (10)
1. ngf gene-polycation nano particle composites, it is characterized in that, it comprises ngf gene material and polycation, described polycation is poly-spermine imidazoles-4,5-diimine or poly-spermine imidazoles-4,5-diamides, the mass ratio of described polycation and ngf gene material is 5 ~ 100.
2. ngf gene according to claim 1-polycation nano particle composites, is characterized in that, the mass ratio of described polycation and ngf gene material is 40 ~ 50.
3. ngf gene according to claim 1-polycation nano particle composites, it is characterized in that, it is prepared by following methods:
(1) said polycation solution is joined fast in the ngf gene solution of same volume, mix homogeneously;
(2) by mixed solution in incubated at room temperature 20 ~ 30 minutes, obtain ngf gene-polycation nano particle composites.
4. ngf gene according to claim 1-polycation nano particle composites, is characterized in that, described poly-spermine imidazoles-4,5-diimine structural formula is:
，
Wherein, n is 100 ~ 500.
5. ngf gene according to claim 1-polycation nano particle composites, is characterized in that, described poly-spermine imidazoles-4,5-diamides structural formula is:
，
Wherein, n is 100 ~ 500.
6. ngf gene according to claim 1-polycation nano particle composites, is characterized in that, the particle diameter of described ngf gene-polycation nano particle composites is 100 ~ 300 nanometers.
7. the preparation method of ngf gene according to claim 1-polycation nano particle composites, is characterized in that, comprise the following steps:
(1) said polycation solution is joined fast in the ngf gene solution of same volume, mix homogeneously;
(2) by mixed solution in incubated at room temperature 20 ~ 30 minutes, obtain ngf gene-polycation nano particle composites.
8. preparation method according to claim 7, is characterized in that,
Described poly-spermine imidazoles-4,5-diimine structural formula is:
，
Described poly-spermine imidazoles-4,5-diamides structural formula is:
，
Wherein, n is 100 ~ 500.
9. preparation method according to claim 7, is characterized in that, the particle diameter of described ngf gene-polycation nano particle composites is 100 ~ 300 nanometers.
10. the application of ngf gene according to claim 1-polycation nano particle composites in preparation treatment parkinson disease or unusual fluctuation disease drug.
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