CN109550057A - Active targeting type gene delivery nanoparticle and its preparation method and application - Google Patents
Active targeting type gene delivery nanoparticle and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of active targeting type gene delivery nanoparticles and its preparation method and application；It is made of cationic polymer, hyaluronic acid and negatively charged gene, cationic polymer and negatively charged gene form compound kernel, hyaluronic acid is coated on compound core surface by charge effect and chemical bonds effect, cationic polymer is PAsp (EDA), one of PAsp (DET), PAsp (TET) or PAsp (TEP) or they through one of chemically modified derivative.The hyaluronic acid that the present invention uses has excellent biocompatibility, not only reduce the cytotoxicity as caused by cationic polymer, energy active targeting acts on the hyaluronic acid specific receptor of tumor cell surface height expression simultaneously, it is delivered to allogenic gene more efficiently in tumour cell, improves cellular uptake and transfection efficiency.
Description
Technical field
The invention belongs to nanometer biotechnology field of gene, are related to nano meter biomaterial and genes delivery system, tool
Body be related to it is a kind of using hyaluronic acid as a kind of novel active targeting type gene delivery nanoparticle of target molecule and preparation method thereof and
Using.
Background technique
Gene therapy (gene therapy), which refers to, imports external source target gene in patient target cell, to correct or compensate
Because gene defect and it is abnormal caused by disease, to reach therapeutic purposes.By the development of nearly ten or twenty many years, gene therapy
Research has been achieved for many progress, and development trend is encouraging, especially as cancer this mankind's major disease
New treatment method, it has also become research hotspot both domestic and external.Gene delivery vector is the most critical part in gene therapy, packet
Include viral vectors and non-virus carrier two major classes.Although viral vectors has the advantages that transfection efficiency is high, have a series of
Safety issue (potential mutagenicity and high immunogenicity etc.), make its develop and application be restricted.It is non-viral
Genophore has the advantages that viral vectors is incomparable, as cytotoxicity is low, exempts from as the gene delivery vector of a new generation
Epidemic focus is low, genetic fragment unbounded size system, gene capacity value is high, easily prepared and modification, is convenient for saving and examining etc., because
And it is widely studied and is paid close attention to.
Non-virus carrier mainly includes cationic polymer and cationic-liposome two major classes, cationic polymer
(polycation) it can be combined by electrostatic interaction and compress nucleic acid, " proton sea can be passed through after entering acid inclusion body
It is continuous " it acts on and successfully escaping from inclusion body, premise and guarantee are provided to improve the transfection efficiency of gene.Cationic polymerization
Object have be easily-synthesized and be modified, non-immunogenicity, nucleic acid protect not to be degraded, be convenient for targeting and biology applicability change
Many advantages, such as property.
In general, the surface of compound can be with superfluous positive charge, so that compound after cationic polymer and nucleic acid are compound
Between object and negatively charged cell membrane sticking without specificity, and positive charge amount it is excessive when can to cell generate toxicity make
With in addition excessive positive charge easily occurs interaction with the albumen in blood and is extremely easy to clear by reticuloendothelial system (RES)
It removes, circulation time in vivo is short.Therefore, its targeting is solved, safety and stability just becomes urgent problem to be solved.
Summary of the invention
The technical problems to be solved by the invention are as follows: how to provide a kind of while there is hydrophily, stability and targeting
And the preparation and application of the genes delivery system of the hypotoxicity of high transfection efficiency.
The technical solution of the present invention is as follows: a kind of active targeting type gene delivery nanoparticle, by cationic polymer, hyalomitome
Sour and negatively charged gene composition, cationic polymer and negatively charged gene form compound kernel, and hyaluronic acid is logical
Cross charge effect and chemical bonds effect be coated on compound core surface, cationic polymer can for PAsp (EDA),
PAsp (DET), PAsp (TET) or PAsp (TEP) or their derivative.(Hirokuni Uchida,et al.,
J.Am.Chem. Soc.2014,136,12396-12405).This few cationoid polymer is applied to the delivering of internal alia gene
With many advantages such as efficient, less toxic and biodegradable, in conjunction with the change of targeted molecular and/or tumor microenvironment responsiveness
Key is learned, the active targeting type gene delivery nanoparticle that targeting significantly improves can be prepared.
Further, the molar ratio of phosphate radical is 0.01- in nitrogen-atoms and gene protonated in cationic polymer
200；COO in protonated nitrogen-atoms and hyaluronic acid in cationic polymer-Molar ratio be 0.01-100.Preferably,
The molar ratio of phosphate radical is 1-60 in protonated nitrogen-atoms and gene in cationic polymer；It can in cationic polymer
COO in the nitrogen-atoms and hyaluronic acid of protonation-Molar ratio be 0.1-20.
Further the molecular weight of PAsp (EDA) is 1.0-100KDa, and the molecular weight of PAsp (DET) is 1.0-100KDa,
The molecular weight of PAsp (TET) is 1.0-100KDa, and the molecular weight of PAsp (TEP) is 1.0-100KDa.Preferably, PAsp (EDA)
Molecular weight be 1.0-40KDa, the molecular weight of PAsp (DET) is 1.0-40KDa, and the molecular weight of PAsp (TET) is 1.0-
The molecular weight of 40KDa, PAsp (TEP) are 1.0-40KDa.
Further, cationic polymer is poly- [N '-(N- citric acid -2- amino-ethyl) aspartic acid], poly- { N '-[N-
(2- amino-ethyl) -2- amino-ethyl] aspartic acid, poly- (N- { N '-[N "-(2- amino-ethyl) -2- amino-ethyl] -2- ammonia
Base ethyl } aspartic acid) or poly- [N- (N '-N "-[N ' "-(2- amino-ethyl) -2- amino-ethyl] -2- amino-ethyl } -2- ammonia
Base ethyl) aspartic acid].
Further, the hyaluronic acid is hyaluronic acid or hyaluronic acid through chemically modified derivative, molecule
Amount is 5.0-2000KDa.
Further, the negatively charged gene is DNA or RNA or oligonucleotides.The DNA or RNA is each
Kind of reporter gene, antioncogene, gene editing tool gene or cytokine gene can recombinantly express in eukaryocyte
DNA or RNA or oligonucleotides.
Pass through chemical bond between hyaluronic acid target molecule and cationic polymer and negatively charged gene composite kernel
Connection.The chemical bond is ester bond, amido bond, nitrine key, triazole key and disulfide bond etc..
The invention also discloses a kind of preparation methods of active targeting type gene delivery nanoparticle, comprising the following steps:
Step 1: cationic polymer, negatively charged gene, hyaluronic acid and crosslinking agent are used into solvent appropriate respectively
It is configured to the solution of suitable concentration；
Step 2: it by the solution of cationic polymer solution and electronegative gene, is mixed by proper proportion, makes cation
Polymer and negatively charged gene pass through electrostatic force formation compound kernel.It is positively charged to be formed by composite surface
Lotus, partial size is in 10-200nm；
Hyaluronic acid solution is added into the complex solution of cationic polymer and gene according to proper proportion in step 3
Or the mixed solution of hyaluronic acid and crosslinking agent, it is coated on hyaluronic acid by charge effect and chemical bonds effect multiple
Object core surface is closed, forms the negatively charged partial size in surface in the nanoparticle of 20-400nm；
Wherein, the concentration of cationic polymer and hyaluronic acid is 0.1-10 μ g/ μ l, and the concentration of gene is 0.01-10 μ g/
μl；The molar ratio of phosphate radical is 0.01-200 in protonated nitrogen-atoms and gene in cationic polymer；Cationic polymerization
The molar ratio of COO- is 0.01-100 in protonated nitrogen-atoms and hyaluronic acid in object.
Further, the crosslinking agent can be selected from one of carbodiimides/NHS ester this major class or a variety of, preferably
For for 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride/n-hydroxysuccinimide (EDC/NHS)；Described
Solvent for preparing solution is buffer salt solution or water, and buffer salt solution is selected from Tris-HCl, HEPES or phosphate solution
In the mixed solution of any one or more.
It is defeated that delivering nanoparticle of the invention is applied to genomic medicine in the gene transfection and human or animal's body of inside and outside cell
The purposes sent.
Compared with prior art, the invention has the following advantages:
(1) novel nonviral gene delivery nanoparticle provided by the invention has hydrophily and low cytotoxicity, in physiology
Under the conditions of have stability, substantially increase the safety and stability of genes delivery system.
(2) novel gene delivery system provided by the invention have extensive gene it is adaptive, can be used for from tens bp to
The delivering of the different size of genetic fragment of bp up to ten thousand, is with a wide range of applications.
(3) novel gene delivery system provided by the invention has active targeting and high transfection efficiency, can be by external source
Property target gene efficiently imports in the highly expressed tumour cell of CD44 receptor, substantially increases the effective of genes delivery system
Property, guarantee is provided for the validity of therapy of tumor.
(4) exogenous therapeutic gene can more efficiently be imported CD44 by novel gene delivery system provided by the invention
In the highly expressed tumor tissues of receptor (such as lung cancer, liver cancer and melanoma), it is able to suppress the growth of malignant tumour, this is actively
The novel gene delivery system of targeting has the validity of tumor-targeting and oncotherapy.The novel gene delivery system, also
It can be applied to prepare polygenic joint delivering to treat tumour, while also can be used as base of the platform technology for other diseases
Because for the treatment of, there is wide potential applicability in clinical practice.
Detailed description of the invention
Fig. 1: PAsp (DET)/DNA/HA nanoparticle partial size and Zeta potential result；
Fig. 2: PAsp (DET)/DNA/HA nanoparticle TEM figure；
Fig. 3: PAsp (DET)/DNA/HA nanoparticle cytotoxicity experiment result；
Fig. 4: PAsp (DET)/DNA/HA nanoparticle cell transfection assays result；
Fig. 5: PAsp (DET)/DNA/HA nanoparticle cellular uptake experimental result；
Fig. 6: PAsp (DET)/DNA/HA nanoparticle is in mouse subcutaneous solid tumors model transgenic inhibiting tumor assay result；
Fig. 7: PAsp (DET)/DNA/HA nanoparticle is in mouse pulmonary metastases model transgenic inhibiting tumor assay result.
Specific embodiment
In following example, PAsp (EDA) is poly- [N '-(N- citric acid -2- amino-ethyl) aspartic acid], PAsp
(DET) be poly- { N '-[N- (2- amino-ethyl) -2- amino-ethyl] aspartic acid }, PAsp (TET) is poly- (N- { N '-[N " -
(2- amino-ethyl) -2- amino-ethyl] -2- amino-ethyl } aspartic acid), PAsp (TEP) is poly- [N- (N '-{ N "-[N ' " -
(2- amino-ethyl) -2- amino-ethyl] -2- amino-ethyl } -2- amino-ethyl) aspartic acid].HEPES is 4- ethoxy piperazine
Piperazine ethanesulfonic acid, Tris are trishydroxymethylaminomethane.EGFP plasmid is a kind of plasmid for encoding strong type green fluorescent protein
(plasmid DNA, pDNA), sFlt-1 plasmid are a kind of plasmid of encoding soluble Angiogenesis factor receptors.
Embodiment 1: cationic polymer/gene composite preparation
PAsp (DET) and sFlt-1 plasmid are dissolved separately in HEPES (10mM, pH7.4), the concentration of sFlt-1 plasmid is
0.05 μ g/ μ l is adjusted the concentration of PAsp (DET) solution by different N/P than demand.By PAsp (DET) solution and 2 times of volumes
Be vortexed 30 seconds after the mixing of sFlt-1 plasmid solution, that is, be prepared into the PAsp (DET) of the final concentration of 33.3 μ g/mL of sFlt-1 plasmid/
PDNA complex solution.With same method and steps, can with PAsp (EDA), PAsp (TET) and PAsp (TEP) or they
Derivative construct cationic polymer/pDNA compound.
Equally, according to above-mentioned same step and method, can with PAsp (EDA), PAsp (DET), PAsp (TET) and
PAsp (TEP) or their derivative and mRNA, siRNA, oligonucleotides etc. form cationic polymer/mRNA compound, sun
Ionomer/siRNA compound, cationic polymer/oligonucleotide complex etc..
The preparation of embodiment 2:PAsp (DET)/DNA/HA nanoparticle
In the embodiment, PAsp (DET) is poly- { N '-[N- (2- amino-ethyl) -2- amino-ethyl] aspartic acid }, and HA is
Hyaluronic acid is made to form chemistry key connection between HA and compound kernel with crosslinking agent.EDC is 1- ethyl-(3- dimethylamino
Base propyl) carbodiimide hydrochloride, NHS is n-hydroxysuccinimide.
It is respectively 2.0 μ g/ μ l that PAsp (DET) and EGFP plasmid Tris-HCl (10mM, pH7.4), which are configured to concentration,
With the solution of 0.05 μ g/ μ l.The mixed solution of HA, EDC and NHS are prepared with water, the concentration of HA, EDC and NHS are respectively 1.0 μ
G/ μ l, 0.15 μ g/ μ l and 0.25 μ g/ μ l.PAsp (DET) and EGFP plasmid solution are adjusted to required fit with Tris-HCl
Suitable concentration is vortexed 30 seconds after mixing PAsp (DET) and EGFP plasmid solution in suitable ratio, PAsp (DET)/DNA is made
Complex solution.The complex solution is added by proper ratio in order successively by EDC solution after being placed at room temperature for 30 minutes
The mixed liquor of the hyaluronic acid obtained after being added with NHS solution is placed at room temperature for 8 hours after being vortexed 30 seconds, can obtain hyaluronic acid
With the nanoparticle solution of cationic polymer amino residue crosslinking.The solution is set in super filter tube, after Tris-HCl buffer is added
Centrifugal purification can be used for the nanoparticle solution of inside and outside experiment to obtain the final product.
With the above preparation method, by N/P/COO-(phosphorus in protonated nitrogen-atoms/EGFP plasmid in cationic polymer
COO in acid group/hyaluronic acid-Molar ratio) be 20:1:20 and N/P is that 20:1 prepares nanoparticle and compound.With same
Method and steps, cation can be constructed with PAsp (EDA), PAsp (TET) and PAsp (TEP) or their derivative
Polymer/DNA/HA nanoparticle.Equally, according to above-mentioned same step and method, PAsp (EDA), PAsp can be used
(DET), it is poly- to form cation for PAsp (TET) and PAsp (TEP) or their derivative and mRNA, siRNA, oligonucleotides etc.
Close object/mRNA/HA, cationic polymer/siRNA/HA, cationic polymer/oligonucleotides/HA nano particle etc..
50 μ l are taken to be measured with laser particle analyzer to PAsp (DET)/DNA/HA nanoparticle.As a result as shown in Fig. 1.Knot
Fruit shows that the hydration partial size of nanoparticle is 150nm or so, and Zeta potential is -24mV or so.
With the above preparation method, by N/P/COO-(phosphorus in protonated nitrogen-atoms/EGFP plasmid in cationic polymer
COO in acid group/hyaluronic acid-Molar ratio) be 20:1:20 and N/P is that 20:1 prepares nanoparticle and compound, after negative staining
Pass through the form and size of transmission electron microscope observation compound.It is observed by transmission electron microscope.As a result as schemed
Shown in 2, the rounded particle of nanoparticle.
Embodiment 3: cytotoxicity compares
With the above preparation method, PAsp (DET)/DNA/HA nanoparticle is prepared, investigates different N/P/ with CCK-8 experiment
COO-The cytotoxicity of the gene delivery nanoparticle under ratio.The HUVEC/B16F10 cell pancreas of logarithmic growth phase will be in
After enzymic digestion, with 5 × 103The density in a/hole is inoculated in 96 orifice plates, every 50 μ l cell suspension of hole, in 37 DEG C, 5%CO2In incubator
It after culture 24 hours, then is separately added into and volume is adjusted to the sample solution of 50 μ l with culture medium, every kind of sample solution is with N/P
The solution of four kinds of difference N/P ratios is prepared for 5:1,10:1,20:1 and 40:1, the sample solution that each hole is added contains 1.0 μ g
EGFP plasmid, as a control group with the PEG-PAsp (DET) of the hypotoxicity and 25K PEI being more toxic.After culture 24 hours,
10 μ l CCK-8 solution are added in every hole, continue culture 2 hours, with the absorbance value (A) at microplate reader measurement 450nm.According to A
Value clicks formula and calculates cell survival rate:
Survival rate %=(Asample-A0/ABlank-A0) * 100, A in formulasampleThe mean absorbance values of each sample group are represented,
ABlankRepresent the mean absorbance values for not being loaded the blanc cell group of product, A0Represent the mean absorbance values for only having culture medium group.
As a result see A and B in Fig. 3.The result shows that PAsp (DET)/DNA compound toxicity is obvious low under the conditions of identical N/P ratio
In PEI/DNA compound, the cytotoxicity of the cation is substantially less than the PEI that transfection efficiency is high but is more toxic；Use hyalomitome
After acid modification PAsp (DET)/DNA compound, the cytotoxicity of cationic polymer further reduced.The experimental verification
PAsp prepared by the present invention (DET)/DNA compound and PAsp (DET)/DNA/HA significantly reduce existing product
Cytotoxicity is a kind of technological progress.
Gene of embodiment 4:PAsp (the DET)/DNA/HA nanoparticle for cell transfects
With the above preparation method, with N/P/COO-PAsp (DET)/DNA/HA nanoparticle is prepared for 20:1:20, is investigated external
Cellular uptake situation.Using the PEG-PAsp (DET) of non-active targeting as control.PEG-PAsp (DET)/DNA nanoparticle and
PAsp (DET)/DNA compound N/P ratio is 20:1.After B16F10 cell in logarithmic growth phase is digested with pancreatin,
With 3 × 104The density in a/hole is inoculated in 24 orifice plates, every 400 μ l cell suspension of hole, in 37 DEG C of 5%CO2In incubator after culture 24,
Culture medium is replaced with to the fresh RPMI1640 culture medium containing 10% fetal calf serum, the sample solution prepared is added, 37
DEG C, 5%CO2In incubator after culture 24, cell is collected, is detected with flow cytometer.Experimental result is shown in attached drawings 4.Experiment knot
Fruit shows that PAsp (DET)/DNA compound and PAsp (DET)/DNA/HA nanoparticle all have very high cellular uptake rate,
The former is because of the electrostatic interaction between positively charged compound and cell membrane, and the latter is the master because of hyaluronan molecule
Dynamic targeting, the cellular uptake rate of the two be all remarkably higher than the PEG- PAsp (DET) of the non-active targeting type of existing report/
DNA nanoparticle.
With the above preparation method, with N/P/COO-PAsp (DET)/DNA/HA nanoparticle is prepared for 20:1:20, is investigated external
Cell transfecting efficiency.Using the PEG-PAsp (DET) of non-active targeting as control.PEG-PAsp (DET)/DNA nanoparticle and
PAsp (DET)/DNA compound N/P ratio is 20:1, PAsp (DET)/DNA/HA N/P/COO-Than for 20:1:20.It will
After B16F10 cell in logarithmic growth phase is digested with pancreatin, with 3 × 104The density in a/hole is inoculated in 24 orifice plates, every hole
400 μ l cell suspensions, in 37 DEG C of 5%CO2In incubator after culture 24, culture medium is replaced with fresh containing 10% fetal calf serum
RPMI1640 culture medium, the sample solution (each sample contain 1.0 μ g EGFP plasmids) prepared is added, in 37 DEG C, 5%
CO2In incubator after culture 48, is observed and taken pictures with fluorescence microscope.Then cell is collected, is examined with flow cytometer
It surveys.As a result see Fig. 5.
The experimental results showed that with PAsp (the DET)/DNA/ prepared after hyaluronic acid decorated PAsp (DET)/DNA compound
Transfection efficiency can be improved in HA nanoparticle, and transfection efficiency is significantly higher than PEG-PAsp (DET)/DNA nanoparticle of non-active targeting
Control group.
Embodiment 5:PAsp (DET)/DNA/HA nanoparticle inhibiting tumor assay in mouse subcutaneous solid tumors model
It is subcutaneous that B16F10 cell strain is inoculated in Balb/C mouse, establishes melanoma subcutaneous tumors model, tumour is long to about
30 mm3When be grouped, PAsp (DET)/DNA/HA nanoparticle that injection is loaded with sFlt-1 plasmid gene in mouse tail vein carries out base
Because for the treatment of, unit dosage form is 20 μ gsFlt-1 plasmids, and administration number of times is 3 times, and delivery time is 2 days, i.e. the 0th, 3
It was respectively administered once with 6 days.Shape of tumor variation is observed, every the gross tumor volume of measurement in 1 day, and monitors mouse weight variation.
In addition PBS (0.1M pH7.4 phosphate buffer) group, sFlt-1 plasmid group and PAsp (DET)/sFlt-1 plasmid are set compound
Object group and PEG-PAsp (DET)/DNA nanoparticle group compare tumor killing effect.Tumor growth curve is shown in A in Fig. 6, with other three
Group is compared, and PAsp (DET)/sFlt-1 plasmid/HA nanoparticle group tumour growth is obviously suppressed, PAsp (DET)/sFlt-1 matter
Grain/HA nanoparticle group inhibitory rate is significantly higher than PAsp (DET)/sFlt-1 plasmid composite of non-active targeting to 65%
Group and PEG-PAsp (DET)/DNA nanoparticle group.Mouse weight change curve is shown in Fig. 6, PAsp (DET)/sFlt-1 plasmid/HA
Nanoparticle group has no obvious changes of weight, illustrates there is peace when PAsp (DET)/sFlt-1 plasmid/HA nanoparticle group vivo applications
Quan Xing.
Embodiment 6:PAsp (DET)/DNA/HA nanoparticle inhibiting tumor assay in mouse pulmonary metastases model
B16F10-luc cell strain is inoculated in Balb/C mouse by tail vein injection, establishes melanoma pulmonary metastases
Model, whether the formation of Pulmonary artery and growing state after intraperitoneal injection fluorescein substrate with small animal living body by being imaged
System (IVIS) detection, in mouse tail vein injection be loaded with PAsp (DET)/DNA/HA nanoparticle of sFlt-1 plasmid gene into
Row gene therapy, unit dosage form are 20 μ gsFlt-1 plasmids, and administration number of times is 3 times, and delivery time is 1 day, i.e., the
0, it is respectively administered once within 2 and 4 days.With IVIS measurement bioluminescence value to supervise lung tumor growth situation, and monitor Mice Body
Change again.In addition PBS (0.1M pH7.4 phosphate buffer) group, sFlt-1 plasmid group and PAsp (DET)/sFlt-1 are set
Plasmid composite group and PEG-PAsp (DET)/DNA nanoparticle group, compare the tumor killing effect of each group.With the growing state of tumour
The bioluminescence intensity value being positively correlated is shown in A in Fig. 7.PAsp (DET)/sFlt-1 plasmid/HA nanometers compared with other several groups
Grain group bioluminescence intensity value is significantly lower than other groups, and tumour growth is obviously suppressed.Through PAsp (DET)/sFlt-1 plasmid/
The mice tumors grew situation of HA nanoparticle treatment swells compared with the PEG-PAsp (DET) of non-active targeting/DNA nanoparticle group
Tumor growth is significant to be suppressed.Mouse weight change curve is shown in B in Fig. 7, and PAsp (DET)/sFlt-1 plasmid/HA nanoparticle group is not
See obvious changes of weight, shows the safety that PAsp (DET)/sFlt-1 plasmid/HA nanoparticle is applied in vivo.
Claims (10)
1. a kind of active targeting type gene delivery nanoparticle, which is characterized in that it is by cationic polymer, hyaluronic acid and with negative
The gene of charge forms, and cationic polymer and negatively charged gene form compound kernel, and hyaluronic acid is made by charge
It is coated on compound core surface with chemical bonds effect, cationic polymer is PAsp (EDA), PAsp (DET), PAsp
(TET) or one of PAsp (TEP) or they through one of chemically modified derivative.
2. a kind of active targeting type gene delivery nanoparticle according to claim 1, which is characterized in that cationic polymer
In in protonated nitrogen-atoms and gene the molar ratio of phosphate radical be 0.01-200；Protonated nitrogen in cationic polymer
The molar ratio of COO- is 0.01-100 in atom and hyaluronic acid.
3. a kind of active targeting type gene delivery nanoparticle according to claim 2, which is characterized in that cationic polymer
In in protonated nitrogen-atoms and gene the molar ratio of phosphate radical be 1-60；Protonated nitrogen-atoms in cationic polymer
Molar ratio with COO- in hyaluronic acid is 0.1-20.
4. a kind of preparation method of active targeting type nonviral gene delivery nanoparticle according to claim 1, feature
It is, the molecular weight of PAsp (EDA) is 1.0-100KDa, and the molecular weight of PAsp (DET) is 1.0-100KDa, PAsp's (TET)
Molecular weight is 1.0-100KDa, and the molecular weight of PAsp (TEP) is 1.0-100KDa.
5. a kind of active targeting type nonviral gene delivery nanoparticle according to claim 1, which is characterized in that cation
Polymer is poly- [N '-(N- citric acid -2- amino-ethyl) aspartic acid], poly- { N '-[N- (2- amino-ethyl) -2- amino second
Base] aspartic acid, poly- (N- { N '-[N "-(2- amino-ethyl) -2- amino-ethyl] -2- amino-ethyl } aspartic acid) or poly-
[N- (N '-N "-[N ' "-(2- amino-ethyl) -2- amino-ethyl] -2- amino-ethyl } -2- amino-ethyl) aspartic acid].
6. a kind of active targeting type gene delivery nanoparticle according to claim 1, which is characterized in that the hyalomitome
Acid is hyaluronic acid or hyaluronic acid through chemically modified derivative, molecular weight 5.0-2000KDa.
7. a kind of active targeting type gene delivery nanoparticle according to claim 1, which is characterized in that described is negatively charged
The gene of lotus is DNA or RNA.
8. a kind of preparation method of active targeting type gene delivery nanoparticle according to claim 1-7, special
Sign is, comprising the following steps:
Step 1: cationic polymer, negatively charged gene, hyaluronic acid and crosslinking agent are prepared with solvent appropriate respectively
At the solution of suitable concentration；
Step 2: it by the solution of cationic polymer solution and electronegative gene, is mixed by proper proportion, makes cationic polymerization
Object and negatively charged gene are by electrostatic force formation compound kernel, and partial size is in 10-200nm；
Hyaluronic acid solution or saturating is added according to proper proportion into the complex solution of cationic polymer and gene for step 3
The mixed solution of bright matter acid and crosslinking agent is coated on hyaluronic acid in compound by charge effect and chemical bonds effect
Core forms partial size in the nanoparticle of 20-400nm, wherein and the concentration of cationic polymer and hyaluronic acid is 0.1-10 μ g/ μ l,
The concentration of gene is 0.01-10 μ g/ μ l；In cationic polymer in protonated nitrogen-atoms and gene phosphate radical molar ratio
For 0.01-200；COO in protonated nitrogen-atoms and hyaluronic acid in cationic polymer-Molar ratio be 0.01-100.
9. a kind of preparation method of active targeting type gene delivery nanoparticle according to claim 8, which is characterized in that institute
Stating crosslinking agent can be selected from one of carbodiimides/NHS ester this major class or a variety of, described for preparing the solvent of solution
For buffer salt solution or water, any one or more of buffer salt solution in Tris-HCl, HEPES or phosphate solution
Mixed solution.
10. it is according to claim 1-7 delivering nanoparticle be applied to inside and outside cell gene transfection and people or
The purposes that genomic medicine conveys in animal body.
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