CN108531514A - Endogenous hyperbranched poly spermine cationic gene carriers and the preparation method and application thereof - Google Patents
Endogenous hyperbranched poly spermine cationic gene carriers and the preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of endogenous hyperbranched poly spermine cationic gene carriers and the preparation method and application thereof.The preparation method of the genophore includes the following steps：(1) endogenous monomer is dissolved in organic solvent, protective agent is added under the conditions of anhydrous and oxygen-free, be stirred to react under the conditions of 20~30 DEG C, obtain protected endogenous monomer I；(2) endogenous monomer I is soluble in water, biodegradable bridging agent is added, addition reaction is carried out under the conditions of 60~80 DEG C, adding ether makes product precipitate, and obtains polymer II；(3) deprotection agent is added into polymer II to be reacted, filters, freeze-drying obtains endogenous hyperbranched poly spermine cationic gene carriers.The genophore obtained in the present invention has both biodegradable and non-toxic metabolic, can agglomerate, convey nucleic acid drug, it is compound with nucleic acid drug, can get the nano-complex particle for treating DME, realizes safe and efficient gene therapy.
Description
Technical field
The invention belongs to bio-medical engineering material field, more particularly to a kind of endogenous hyperbranched poly spermine cation base
Because of carrier and the preparation method and application thereof.
Background technology
In all complication of diabetes, the diabetic retinopathy illness rate that is averaged is up to 50%.When diabetes shadow
Ring retina after i.e. complicated with diabetes retinopathy, wherein diabetic macular edema (diabetic macular edema,
DME) be then diabetic's visual impairment first cause, late cases can cause patient complete because of traction property total detachment of retina
Full blindness.Gene therapy becomes the relatively treatment strategies of the DME of tool potentiality at present, especially the RNA interference technology, not only for
It studies the functions such as the gene expression and regulation of organism and provides new method, while also being opened up for the research of DME, prevention and treatment
New way.In terms of gene therapy, RNAi (RNA interference) is the powerful tool of silence specific gene, is widely used in
Each research field.In terms of the Therapy study of DME, using VEGF (vascular endothelial growth factor) and its receptor VEGFR as target base
Cause imports the siRNA or dsRNA that synthesize in vitro, as a result shows that corresponding siRNA can obviously reduce VEGF or VEGFR
Expression, effectively mitigate the inducing actions that are formed to DME of VEGF, inhibition eye DME is formed, to treat DME.Compared to more traditional
Mode of drug, gene therapy have incomparable advantage：(1) spy of carrier can be utilized by gene delivery technique
Property orientation transfected target cells；(2) product of gene therapy expression is endogenous protein, the tolerance phase of safety and body
Exogenous drug will be got well；(3) part repeatedly can be avoided in the expression anti-angiogenesis purpose factor of local long-time stable
Complication caused by injection is possible[1-4]。
One of key technology of gene therapy is the safe and efficient genophore of selection.Genophore partitivirus carrier and non-
Viral vectors two major classes.Virus gene carrier is to substitute the Disease-causing gene in virus with therapeutic gene, due to control virus
Gene into cell, transmitter loss and coding into nuclear fractions is also on viral vectors, so viral vectors is excellent
Point is that expression of target gene is efficient, but also has immunogenicity simultaneously, proto-oncogene may be activated to cause tumour and be difficult to largely make
The shortcomings of standby, to limit the clinical application of viral vectors.The advantage of non-viral gene vector is that immunogenicity is weak, prepares
Convenient, the requirement limitation to genetic material is few.Nonviral gene delivery carrier uses liposome, polyethyleneimine more at present
(PEI), dendroid polyamide-amine (PAMAM) etc., generally existing carrier toxicity is big, difficult to degrade or catabolite toxicity is big etc. lacks
Point.The gene delivery vector for preparing a kind of safe and efficient and degradable metabolism, is field of gene important class urgently to be resolved hurrily
Topic[5-7]。
Dissaving polymer is a kind of highly branched macromolecular with three-dimensional globular or near-spherical structure, this kind of to divide greatly
Son has the advantages that many uniquenesses：Molecule interchain is without entanglement, good dissolubility energy, internal cavities are big, a large amount of active reaction bases
Group[8].Structural advantages based on dissaving polymer, being applied to genophore field has prominent superiority：Hyperbranched polymerization
Object cation contains a large amount of amine at different levels on surface and inside, in addition its three-dimensional stereochemical structure and good molecule toughness make
Hard-packed spherical shape can be formed as genophore with DNA or siRNA by obtaining it, and transfection efficiency greatly improves.As reported in the recent period
The dendroid polylysine in road is modified amphiphilic hyperbranched polyglycidyl ether derivative and transmits control altogether for drug and gene
Treat the research of tumour[9]Gene therapy research etc. with redox response over-branched polyamidoamine derivative for tumour,
Good therapeutic effect is achieved, but from the point of view of current report, synthon selected by hyper branched polycation carrier is mostly outside human body
Source molecule, safety and cytotoxicity all need further to improve, and the safety of its catabolite need long-term investigation.
Spermine is the polyamino molecule of full-time cohesion gene in human sperm；In recent years, using spermine as framework construction list
Linear poly- spermine cation carrier (polyaminoacid ester of the spermine as skeleton of member[10], poly- (thiamine disulfide)[11]、SPE-alt-PEG
Poly- spermine[12]) all show it is higher than the gene transfection activity of the several orders of magnitude of PEI and thin less than more than ten of order of magnitude of PEI
Cellular toxicity[13]。
How to obtain with good biocompatibility, biodegradable and non-toxic metabolic hyperbranched genophore has become
For current biological engineering in medicine Material Field important topic urgently to be resolved hurrily.So far, by dissaving structure and endogenous list
Body is combined, and the hyperbranched cationic gene carriers of structure endogenous and its application have not been reported.
Invention content
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of endogenous hyperbranched poly essence
The preparation method of amine cation genophore.
Another object of the present invention is to provide the endogenous hyperbranched poly spermine cation bases that the method is prepared
Because of carrier.
It is still another object of the present invention to provide the applications of the endogenous hyperbranched poly spermine cationic gene carriers.
The purpose of the invention is achieved by the following technical solution：A kind of endogenous hyperbranched poly spermine cationic gene carriers
Preparation method, include the following steps：
(1) endogenous monomer is dissolved in organic solvent, protective agent is added under the conditions of anhydrous and oxygen-free, then 20~30
It is stirred to react under the conditions of DEG C, obtains protected endogenous monomer I；
(2) the endogenous monomer I obtained in step (1) is soluble in water, biodegradable bridging agent is added, 60~
Addition reaction is carried out under the conditions of 80 DEG C, waits for that ether is added after reaction makes product precipitate, and obtains polymer II；
(3) deprotection agent is added in the polymer II obtained to step (2), is then stirred under the conditions of 30~50 DEG C anti-
It answers, waits for filtering after reaction, be freeze-dried, obtain endogenous hyperbranched poly spermine cationic gene carriers.
Endogenous monomer described in step (1) is at least one of spermine, spermidine and arginine.
Organic solvent described in step (1) is chloroform；Preferably anhydrous chloroform.
Protective agent described in step (1) is in trifluoroacetic acid and 2- acetyl -5,5- dimethyl -1,3- cyclohexylamine diketone
It is at least one.
Endogenous monomer and protectant molar ratio described in step (1) are 1:2~4.
The condition being stirred to react described in step (1) is：300~700rpm stirs 24~36h；Preferably：At 25 DEG C
Under the conditions of, 500~600rpm stirring for 24 hours.
Biodegradable bridging agent described in step (2) is trimethylolpropane trimethacrylate and three hydroxyls containing ester bond
At least one of ethylbutane triacrylate.
The molar ratio of endogenous monomer I and biodegradable bridging agent described in step (2) are 3~5:1.
The condition of addition reaction described in step (2) is：400~700rpm stirs 5~7d (day)；Preferably：500~
600rpm stirs 6~6.5d (day).
The temperature of addition reaction described in step (2) is preferably 65 DEG C.
Deprotection agent described in step (3) is triethylamine, ammonium hydroxide, imidazoles, N-Methyl pyrrolidone and DMF (N, N- bis-
At least one of methylformamide)；The deprotection liquid that preferably imidazoles, N-Methyl pyrrolidone and DMF are mixed to get.
The dosage of the imidazoles is preferably calculated by every gram (g) imidazoles proportioning 5ml deprotection liquid.
The volume ratio of the N-Methyl pyrrolidone and DMF is preferably 4:1.
The dosage of deprotection agent described in step (3) preferably matches 0.6mg polymerizations by every milliliter of (ml) deprotection agent
Object II is calculated.
The condition being stirred to react described in step (3) is：300~700rpm stirs 4~6h；Preferably：500~
600rpm stirs 4~5h.
The temperature being stirred to react described in step (3) is preferably 40 DEG C.
Being filtered into described in step (3) uses molecular cut off to be filtered for 30000~60000 super filter tube,
Centrifugation rate is 4000~6000rpm；Molecular cut off is preferably used to be filtered for 40000~60000 super filter tube,
Centrifugation rate is 4000~5000rpm；
A kind of endogenous hyperbranched poly spermine cationic gene carriers, are prepared by method described in any one of the above embodiments
It arrives.
The endogenous hyperbranched poly spermine cationic gene carriers are being prepared for treating DME (Diabetic Maculars
Oedema) nano-complex particle in application.
A kind of nano-complex particle for treating DME, to carry above-mentioned endogenous hyperbranched poly spermine cation gene
Body after mixing, is incubated 20~30 minutes and obtains with nucleic acid drug at room temperature.
The nucleic acid drug is at least one of DNA vaccination, DNA, siRNA and microRNA.
The mass ratio of the endogenous hyperbranched poly spermine cationic gene carriers and nucleic acid drug is 1:1~30.
The nano-complex particle (the hyperbranched cation of endogenous and nucleic acid drug compound) for treating DME
Grain size is 100~300 nanometers.
The present invention has the following advantages and effects with respect to the prior art：
(1) present invention using endogenous monomer as basic structural unit, first it is protected with protective agent, then with it is biodegradable
Bridging agent carries out Michael addition reaction, then is deprotected, and last ultrafiltration removal of impurities, freeze-drying is prepared and obtains a kind of product
High income, the hyperbranched cationic gene carriers of endogenous of easy control of structure.
(2) Michael's addition is synthesized the hyperbranched genophore of endogenous by making with the electrostatic of nucleic acid drug by the present invention
With, be prepared treatment DME nano-complex particle, belong to for the first time synthesis the hyperbranched cationic gene carriers of endogenous.
(3) genophore of the invention can agglomerate, convey nucleic acid drug, and better quality is than the grain size after compound 100
Ran can be discharged therapeutic gene by proton sponge effect, to reach therapeutic effect.
(4) endogenous monomer is combined by the present invention with dissaving structure, have both it is biodegradable with non-toxic metabolic (
Toxicity is not shown in cytotoxicity experiment), the hyperbranched genophore of endogenous is built, is expected to realize safe and efficient gene
Treatment.
(5) genophore itself of the invention can complete non-toxic metabolic in vivo, therefore can lead to intravenous injection for more
The gene therapy of kind disease.
(6) material composition of the present invention is simple, raw material is easy to get, good biocompatibility is that it is preparing biological medicine engineering material
Application provide support, be expected to be used widely in bio-medical engineering material field.
Description of the drawings
Fig. 1 is the synthetic route chart of hyperbranched poly spermine.
Fig. 2 is the nucleus magnetic hydrogen spectrum figure of hyperbranched poly spermine in embodiment 2.
Fig. 3 is the grain-size graph of hyperbranched poly spermine and pDNA compounds in embodiment 4.
Fig. 4 is the potential diagram of hyperbranched poly spermine and pDNA compounds in embodiment 4.
Fig. 5 is the agarose gel electrophoresis figure of hyperbranched poly spermine and pDNA compounds in embodiment 6.
Fig. 6 be in embodiment 8 hyperbranched poly spermine to retinal endothelial cell toxicity figure.
Specific implementation mode
With reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Embodiment 1：The preparation of hyperbranched poly spermine cation
(1) it weighs 2mg spermine to be dissolved in 5ml anhydrous chloroforms, 2- acetyl -5,5- diformazans is utilized under the conditions of anhydrous and oxygen-free
Base -1,3- cyclohexylamine diketone (Dde-OH) (5.4mg) protects two primary amine group.It is 600rpm, temperature in rotating speed
It is reacted 24 hours under conditions of being 25 DEG C.It waits for cleaning to obtain protected spermine monomer after reaction；Wherein, spermine and Dde-
The molar ratio of OH is 1:3.
(2) it weighs (5mg) protected spermine monomer to be dissolved in 10mL water, trimethylolpropane trimethacrylate is added
(0.96mg) is stirred to react 6 days under conditions of rotating speed is 600rpm, temperature is 65 DEG C.It is anhydrous by being added after reaction
Ether makes polymer precipitate；10ml is added to the polymer of precipitation again and is deprotected liquid (imidazoles of 2.01g, the N- methylpyrroles of 8mL
Alkanone, the DMF of 2mL) it is stirred to react 4 hours (40 DEG C, 600rpm)；Product ultrafiltration is washed four times after reaction, then in rotating speed
Unreacted spermine monomer and trimethylolpropane tris propylene are removed by the super filter tube of molecular cut off 40000 under 4000rpm
Acid esters.Freeze-drying, obtains hyperbranched poly spermine；Wherein, protected spermine monomer and trimethylolpropane trimethacrylate
Molar ratio be 3:1.
Embodiment 2：The hydrogen spectrum nuclear-magnetism characterization of hyperbranched poly spermine cation
The hyperbranched poly spermine cation that embodiment 1 obtains is dissolved in deuterated water and carries out hydrogen spectrum nuclear-magnetism characterization.Such as Fig. 2,
The chemical shift of hydrogen atom in hyperbranched poly spermine cation is marked out, and Fig. 2 results prove that the step is reacted successfully
Synthesized hyperbranched poly spermine cation.
Embodiment 3：The preparation of hyperbranched poly spermine cation and pDNA nano-complexes
(1) weigh in embodiment 1 that hyperbranched poly spermine cation obtained is soluble in water, obtain hyperbranched poly spermine sun from
Sub- solution；
(2) pDNA (DNA, Suzhou Ji Ma Pharmaceutical Technology Inc.) is configured to the reserve liquid of 1mg/ml；
(3) hyperbranched poly spermine cation and pDNA nano-complexes are prepared：According to setting a series of mass ratioes (1,5,
10,15,20,30) hyperbranched poly spermine cationic solution, is diluted into 1mg/ml, 1,5,10,15,20 are added in each test tube,
The poly- spermine cationic solution of 30ml, then the pDNA reserve liquids of 1ml are rapidly joined, it is incubated 30 minutes at 25 DEG C, it is class to obtain one
The different nano-complex of surface charge property.
Embodiment 4：The test of hyperbranched poly spermine cation and the grain size and Zeta potential of pDNA nano-complexes
Not homogeneity is prepared according to the preparation method of the hyperbranched poly spermine cation of embodiment 3 and pDNA nano-complexes
The mass ratio of the nanoparticle complex solution of amount ratio, required hyperbranched poly spermine cation and pDNA are respectively 5,7,10,15,
20,30.Sample size is 1ml.At ambient temperature, nanometer laser particle size analyzer is preheated first 20 minutes, then draw lml nano particles
Micro-example pond is added in solution, and then micro-example pond is put into the test trough of Particle Size Analyzer, and setting test temperature is 25
DEG C, medium is ultra-pure water.Particle diameter distribution test for nano particle, each sample test 3 times, each run time are 2 points
Clock records the average value and its polydispersity of each sample particle diameter.Zeta potential test for nano particle, each sample are surveyed
Examination 3 times, each automatic running 12 times records the average value and its mobility of each sample Zeta potential.
As a result：The particle diameter distribution of hyperbranched poly spermine and pDNA compounds is as shown in figure 3, Zeta potential test result such as figure
Shown in 4, as can be seen from the figure mass ratio is steady more than 15 hyperbranched poly spermine cation and the grain size of pDNA nano-complexes
100~300 nanometers are scheduled on, the Zeta potential of nano particle is distributed in 20~30mV.
Embodiment 5：The transmission electron microscope observing of hyperbranched poly spermine cation and pDNA nano-complexes
Mass ratio is prepared according to the preparation method of the hyperbranched poly spermine cation of embodiment 3 and pDNA nano-complexes
For 30 nano particle.First drawing 10 μ l nanoparticles solutions, slowly drop uses transmitted electron on the copper mesh of 400 mesh after drying
Microscope observes the pattern grain size of nano particle (easily and intuitively show) of sample.Experimental result and dynamic light scattering measurement
Particle size results be consistent.
Embodiment 6：Different quality than hyperbranched poly spermine cation and pDNA nano-complexes Ago-Gel electricity
Swimming
1g agaroses are weighed, are added in 100ml 1 × TAE buffer solutions, being heated in micro-wave oven keeps agarose particle complete
Fully dissolved when being cooled to 50~60 DEG C, is added ethidium bromide (EB) solution, jiggles, mixing.Wait for that coagulant liquid is cooled to 50 DEG C of left sides
When right, gel slot is poured into, gel thicknesses are generally 5~8mm.It is put into comb in gel slot, it is careful to extract after gel shaped
Comb carefully takes out gel, is put into the electrophoresis tank for filling electrophoretic buffer.Electrophoretic buffer liquid level is set to be higher by 2~3mm of gel,
Wait for loading.With reference to embodiment 3 method, prepare different quality than hyperbranched poly spermine cation and pDNA it is nano combined
Object, mass ratio are respectively 1,3,5,10,20,30,50.5 μ l samples to be tested are taken with liquid-transfering gun, are uniformly mixed with sample-loading buffer,
It is carefully added in gel loading hole.Open electrophoresis apparatus, voltage 100V, electrophoresis 30min.After the completion of electrophoresis, gel is taken out, is set
In observing electrophoresis result on ultraviolet transilluminator.
Interpretation of result：Fig. 5 electrophoretograms show that pDNA divides when the mass ratio of hyperbranched poly spermine cation and pDNA are 5
Son swimming in the electric field is by partial block, with gradually increasing for hyperbranched poly spermine cation and pDNA mass ratioes, when
When the mass ratio of hyperbranched poly spermine cation and pDNA are reached between 20~30, the retardance completely of hyperbranched poly spermine cation
The migration of pDNA, or even under the conditions of the mass ratio of bigger hyperbranched poly spermine cation can be wrapped up more fully
PDNA does not observe the movement of pDNA in electrophoretogram completely, the experiment proves that hyperbranched poly spermine cation have it is more satisfactory
Cohesion plasmid pDNA form the abilities of composite particles and swallowed in cell to avoid it to play the effect of protection pDNA
Before be degraded.
Embodiment 7：The degradation of hyperbranched poly spermine cation and metabolic process
The hyperbranched poly spermine cation for weighing 2mg is dissolved in the PBS buffer solutions (pH=7.4) of 10ml, and one was taken every 2 days
Secondary sample continuously takes 20 days.Samples taken is put into -20 DEG C of refrigerator and preserves, and sample collection uses gel permeation chromatography afterwards together
(GPC) molecular weight for measuring each sample, using weight average molecular weight as ordinate, draws the drop of polymer using the time as abscissa
Solution curve studies its degradability.Test the degradation capability of hyperbranched poly spermine cation.
Experimental result：As time increases, the molecular weight of hyperbranched poly spermine is obviously reduced.At the 10th day, point
Son amount only initial 40%.At the 20th day, its molecular weight only had initial 15%.The experimental results showed that hyperbranched poly spermine has
Good biological degradability.
Embodiment 8：The cytotoxicity assay of hyperbranched poly spermine cation
The cytotoxicity of hyperbranched poly spermine cation is measured using CCK-8 methods.After filtration sterilization prepared by embodiment 1
The PEI (polyetherimide) of obtained hyperbranched poly spermine cation and contrast sample molecular weight 25000, is pressed with DMEM culture mediums
After certain concentration gradient (0.1,1,5,10,50,100 μ g/ml) dilution, degrees of fusion is added to up in 80% human retina
Hyperbranched poly is added per hole for co-incubation in chrotoplast (human retina endothelial cell, Shanghai and sequence bio tech ltd)
The volume of spermine cationic solution is 100 μ l, 3 multiple holes of each concentration.After 24 hours, the thin of material is measured using CCK-8 methods
Cellular toxicity.
The results are shown in Figure 6 for the cytotoxicity test of hyperbranched poly spermine.When hyperbranched poly spermine reaches 100 μ g/ml,
Cell still maintains 80% or more survival rate；And compare PEI and then show apparent cytotoxicity, cell survival rate has obviously
Concentration-dependent relation.It can obtain to draw a conclusion：Hyperbranched poly spermine is thin to human retina endothelium within the scope of working concentration
Born of the same parents do not have toxicity, there is good biocompatibility.
Embodiment 9：The cell transfecting of hyperbranched poly spermine cation and pDNA nano-complexes
The human retina endothelial cell of logarithmic phase growth is chosen as recipient cell, adjustment cell concentration is with every hole 105It is a
It is inoculated in 24 porocyte culture plates, in 37 DEG C, 5%CO2Under the conditions of culture to cell fusion degree be 70%.Then Opti- is changed
MEM serum-free cell culture mediums are incubated 2 hours, while by the free serum culture of hyperbranched poly spermine cation and pDNA compounds
Liquid is added in culture dish, it is ensured that the content of DNA is 1 μ g in per hole；Hyperbranched poly spermine cation and pDNA nanometers of quality
Than being respectively 5,10,20,30,50.Using PEI-25k/DNA compounds as positive controls, naked pDNA is as negative control group.
Then 24 orifice plates are placed in incubator, in 37 DEG C, 5%CO2In the environment of cultivate 4 hours after, culture solution is changed, addition contains
The culture solution of serum continues culture 24 hours.The expression of qualitative research green fluorescent protein under fluorescence microscope.Then
Culture solution is sucked out, trypsin digestion cell is added and culture medium termination pancreatin effect is added, cell sample suspends again after centrifugation
In PBS, using flow cytometric analysis cell transfecting number, the i.e. efficiency gene transfection of sample.
Experimental result：With the increase of mass ratio, transfection efficiency is in rising trend.And when mass ratio is 30, turn
Contaminate efficiency it is more taller than positive control (40%) go out 5%.But when mass ratio is 50, transfection efficiency is slightly declined.We
It is set to be difficult to discharge since carrier too closely wraps pDNA when thinking, thus transfection efficiency reduces.
Embodiment 10：Hyperbranched poly spermine cationic gene carriers transport VEGF-siRNA in human retina endothelial cell
The influence of vegf protein
Select VEGF-siRNA (Suzhou Ji agate gene) as nucleic acid drug.It is with mass ratio by human retina endothelial cell
30 hyperbranched poly spermine cation co-cultures 24 hours with VEGF-siRNA nano-complexes.Later by cells rinsed with PBS
Three times, being cracked on ice 30 minutes with SDS lysis buffers keeps cell cracking abundant, then by 4 DEG C 13000rpm from
The heart 15 minutes collects supernatant.Using Bio-Rad protein determinations, protein concentration is measured.Pass through lauryl sodium sulfate
Polyacrylamide gel electrophoresis protein isolate.Then at 300mA by protein delivery to pvdf membrane, with 5% skim milk by film
Closing 1 hour.Film is incubated overnight together with VEGF antibody at 4 DEG C later.Then film is incubated with secondary antibody at 25 DEG C to 1 small
When, it is washed three times with TBST solution.Later, sample is detected using chemiluminescence visualizer, GAPDH albumen is as a contrast.Experiment
The result shows that the hyperbranched poly spermine cationic gene carriers for being loaded with VEGF-siRNA can lower the table of vegf protein well
It reaches.This proves that therapeutic gene safely and efficiently can be transported affected area by bionic nano red blood cell genophore.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
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Claims (10)
1. a kind of preparation method of endogenous hyperbranched poly spermine cationic gene carriers, which is characterized in that include the following steps：
(1) endogenous monomer is dissolved in organic solvent, protective agent is added under the conditions of anhydrous and oxygen-free, then in 20~30 DEG C of items
It is stirred to react under part, obtains protected endogenous monomer I；
(2) the endogenous monomer I obtained in step (1) is soluble in water, biodegradable bridging agent is added, at 60~80 DEG C
Under the conditions of carry out addition reaction, wait for that ether is added after reaction makes product precipitate, and obtains polymer II；
(3) deprotection agent is added in the polymer II obtained to step (2), is then stirred to react, waits under the conditions of 30~50 DEG C
It filters after reaction, is freeze-dried, obtains endogenous hyperbranched poly spermine cationic gene carriers.
2. the preparation method of endogenous hyperbranched poly spermine cationic gene carriers according to claim 1, feature exist
In：
Endogenous monomer described in step (1) is at least one of spermine, spermidine and arginine.
3. the preparation method of endogenous hyperbranched poly spermine cationic gene carriers according to claim 1, feature exist
In：
Protective agent described in step (1) be in trifluoroacetic acid and 2- acetyl -5,5- dimethyl -1,3- cyclohexylamine diketone at least
It is a kind of；
Biodegradable bridging agent described in step (2) is trimethylolpropane trimethacrylate and trihydroxyethyl butane 3 third
At least one of olefin(e) acid ester；
Deprotection agent described in step (3) is at least one in triethylamine, ammonium hydroxide, imidazoles, N-Methyl pyrrolidone and DMF
Kind.
4. the preparation method of endogenous hyperbranched poly spermine cationic gene carriers according to claim 1, feature exist
In：
Endogenous monomer and protectant molar ratio described in step (1) are 1:2~4；
The molar ratio of endogenous monomer I and biodegradable bridging agent described in step (2) are 3~5:1.
5. the preparation method of endogenous hyperbranched poly spermine cationic gene carriers according to claim 1, feature exist
In：
Organic solvent described in step (1) is chloroform；
The condition being stirred to react described in step (1) is：300~700rpm stirs 24~36h；
The condition of addition reaction described in step (2) is：400~700rpm stirs 5~7d；
The condition being stirred to react described in step (3) is：300~700rpm stirs 4~6h；
Being filtered into described in step (3) uses molecular cut off to be filtered for 30000~60000 super filter tube, centrifugation
Rate is 4000~6000rpm.
6. a kind of endogenous hyperbranched poly spermine cationic gene carriers, it is characterised in that：Pass through any one of Claims 1 to 5
The method is prepared.
7. the endogenous hyperbranched poly spermine cationic gene carriers described in claim 6 are preparing the nanometer for treating DME
Application in compound particle.
8. a kind of nano-complex particle for treating DME, it is characterised in that：For the endogenous described in claim 6 is surpassed
Branched poly- spermine cationic gene carriers are incubated 20~30 minutes with nucleic acid drug and obtain after mixing, at room temperature.
9. the nano-complex particle according to claim 8 for treating DME, it is characterised in that：
The nucleic acid drug is at least one of DNA vaccination, DNA, siRNA and microRNA.
10. the nano-complex particle according to claim 8 for treating DME, it is characterised in that：
The mass ratio of the endogenous hyperbranched poly spermine cationic gene carriers and nucleic acid drug is 1:1~30.
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