CN104368009A - Bifunctional-peptide-modified Alzheimer-disease targeting gene gradient-release compound and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of medicines, and discloses a bifunctional-peptide-modified Alzheimer-disease targeting gene gradient-release compound and a preparation method thereof. According to the preparation method, poly(ethylene glycol)-poly(aspartic acid) PEG-P[Asp(TEP)-chole] modified by cholesteryl and tetraethylenepentamine is synthesized, electrostatic adsorption effect and gene combination are employed, and a carrier material which is modified by RVG29 and Tet1 functional peptides and possesses great clinic application prospect is employed, so that the described brain-targeting drug-delivery system is prepared. The disclosed bifunctional-peptide-modified Alzheimer-disease targeting gene gradient-release compound is capable of enabling the gene-carrying drug-delivery system to penetrate blood brain barrier and specifically target nerve cells, helps to improve the trasfection efficiency of gene and provides a wound-free drug delivery manner for drug delivery of brain.

Description

The Alzheimer target gene that a kind of bifunctional peptide is modified is passed and is released complex and preparation method thereof

Technical field:

The present invention relates to medical art, be specifically related to the Alzheimer target gene that a kind of bifunctional peptide modifies and pass and release complex.

Background technology:

The harm of central nervous system disease increases year by year, one of the most serious disease having become harm humans health.Genomic medicine, due to the feature such as high specific of its molecular target, has become research macromolecular drug comparatively widely at present.But due to the restriction of blood brain barrier, genomic medicine independently cannot pass through blood brain barrier, needs by the administering mode such as the ventricles of the brain or intrathecal injection, and this invasive administering mode causes restriction to clinical administration.Therefore, exploitation has the novel gene drug delivery system of intravascular administration, has very important researching value.

Connect ligands specific polymer support can targeting to the tissue of corresponding receptor high expressed and cell.Brain active targeting research more widely receptor has TfR, Insulin receptor INSR, low density lipoprotein receptor, folic acid etc.But there is research to think that transferrins is not desirable brain guide molecule, there is Competitive assays in medicine and the receptors bind of transferrins guiding, receptor is substantially saturated by endogenous transferrins, cause guiding efficiency (Qian ZM on the low side, Li H, Sun H, et a1.Targeted drugdelivery via the transferring receptor-mediated endocytosispathway [J] .Pharmaco Rev, 2002,54 (4): 561-587.).

RVG29 is a kind of 29 amino acids formed polypeptide deriving from rabies virus glycoprotein, (Kumar P, Wu H, McBride J L, et al.Transvascular delivery of smallinterfering RNA to the central nervous system [J] .Nature, 2007,448 (7149): 39-43.) related mechanism research shows, RVG after acetylcholinergic receptor on BBB and gamma-aminobutyric acid receptor be combined, may can realize efficient vertigo fortune.Tet1 is affinity (the Kwon E J by the little peptide of 12 Amino acid profiles and neuronal cell GT1B receptor with height, Lasiene J, Jacobson BE, et a1.Targeted nonviral delivery vehicles to neural progenitor ceils in themouse subventricular zone [J] .Biomaterials, 2010,31 (8): 2417-2424.).

The macromolecular material that Polyethylene Glycol poly-aspartate (PEG-PBLA) is a kind of biodegradable, biocompatibility is better, toxicity is lower, the particularity of structure solves the problem of chemical modification.PEG-PBLA is a kind of electroneutral polymer, cannot with electronegative gene compound, therefore through modifying containing amino tetren, make macromolecular material positively charged, can effectively and gene compound.Meanwhile, tetren has certain buffer capacity, strengthens endosome escape effect, improves transfection efficiency.End group modifies the interaction between cholesteryl energy enhancing gene and macromolecule carrier, improves the stability of complex.

Summary of the invention:

The Alzheimer target gene that the object of the present invention is to provide a kind of bifunctional peptide to modify is passed and is released complex, and another object of the present invention is to provides this target gene to pass the preparation method releasing complex.

Main technical schemes of the present invention is, the Alzheimer target gene providing a kind of bifunctional peptide to modify is passed and is released complex, also claim to pass release system, first the present invention has synthesized the Polyethylene Glycol poly-aspartate PEG-P [Asp (TEP)-chole] of cholesteryl and tetren modification; Then by electrostatic adsorption and gene compound; Adopt the Brain targeting Functional Polypeptides such as RVG29 and Tet1 to modify carrier material afterwards, preparation forms aforesaid brain targeting drug delivery system.

Two kinds of brain function peptides connect by the present invention, form bifunctional peptide, with the common targeting of polymer composite in brain.The present invention can make to carry gene delivery system through blood brain barrier and specific targeting in neurocyte, improve the transfection efficiency of gene, for brain administration provides a kind of hurtless measure administering mode.

A first aspect of the present invention, is to provide the Alzheimer target gene that a kind of bifunctional peptide modifies and passs and release complex (passing release system), and this is passed and releases complex and release carrier and electronegative genomic constitution by passing:

Carrier is released in described passing, and comprises cationic polymer that PEG (Polyethylene Glycol) changes, the end group of cationic polymer connects RVG29 or Tet1;

Described electronegative gene and described passing release cationic polymer in carrier by positive and negative charge electrostatic attraction.

Described cationic polymer, preferably through the poly-aspartate of tetren modification.

The Alzheimer target gene that a kind of bifunctional peptide of the present invention is modified pass release complex structure as shown in figure 19.

Described RVG29 derives from the rabies virus glycoprotein with brain transport features, and by 29 Amino acid profiles, end adds cysteine, and its sequence is: YTIWMPENPRPGTPCDIFTNSRGKR ASNGC (SEQ ID NO:1).

Described Tet1 and the GT1B of neuronal cell surface has the affinity of height, can be specific in conjunction with GT1B receptor.Tet1 is by 12 Amino acid profiles, and end adds cysteine, and its sequence is: HLNILSTLWKYRC (SEQ ID NO:2).

Described electronegative gene is selected from medicative interference sequence, is implemented in the PEG end of described cationic polymer.Preferred BACE1 interference sequence, sequence is: GCTTTGTGGAGATGGTGGA (SEQ ID NO:3).

A second aspect of the present invention, the Alzheimer target gene being to provide the modification of a kind of bifunctional peptide passs the preparation method releasing complex, and the method comprises the steps:

The preparation of A, cationic polymer carrier

Be dissolved in oxolane by aspartic acid benzyl ester and triphosgene according to molecular proportion 1:1 ~ 5, be heated to 50 ~ 80 DEG C, magnetic agitation is to dissolving, and normal hexane precipitates, and obtains aspartic acid benzyl ester carboxylic acid anhydrides; End contains amino PEG derivant and aspartic acid benzyl ester carboxylic acid anhydrides, is dissolved in CH ₂cl ₂, 25 ~ 50 DEG C of reaction 24 ~ 72h, ether sedimentation obtains Polyethylene Glycol poly-aspartate (PEG-PBLA);

Utilize the acetal of PEG end in cationic polymer, in acetate buffer solution, dehydration forms aldehyde-base, with the sulfydryl in cysteine by covalently bound.

B, pass the preparation of release system

The cationic polymer carrier that steps A prepares and electronegative gene are dissolved in the PBS buffer of pH7.4, equal-volume vortex mixed 30s, what form 80 ~ 300 nanosizeds passs release system, and wherein cationic polymer carrier is 2 ~ 16 with the N/P ratio of electronegative gene.

Described electronegative gene, preferred plasmid DNA.

A third aspect of the present invention, is to provide Alzheimer target gene that above-mentioned bifunctional peptide modifies and passs and release the application of complex in preparation treatment Alzheimer disease drugs.

Cationic polymer PEG-PAsp (the TEP)-chole that the present invention builds, biocompatibility is better, and biological cells and tissues toxicity is lower.

The polyplex that PEG-PAsp (the TEP)-chole that the present invention adopts and gene are compounded to form, can promote that endosome is escaped, significantly improve neurocyte transfection efficiency.

Two peptide species RVG29 and Tet1 that the present invention adopts, can make the carrier system carrying gene by blood brain barrier, simultaneously selectively targeted in neurocyte, improve brain targeting in body.

The present invention selects BACE1 interference sequence to be implemented in plasmid, forms complex with two ligand modified cationic polymer, effectively can lower the BACE1 protein level in neurocyte Neuro-2a.

The present invention selects BACE1 interference sequence to be implemented in plasmid, forms complex with two ligand modified cationic polymer, after tail vein injection, significantly can improve the spatial memory capacity of Alzheimer disease model Mus.

Accompanying drawing illustrates:

Fig. 1 is the nucleus magnetic hydrogen spectrum figure of Ace-PEG-P [Asp (TEP)]-chloe;

Fig. 2 is the nucleus magnetic hydrogen spectrum figure of RVG29-PEG-P [Asp (TEP)]-chloe;

Fig. 3 is particle diameter and the zeta potential measurement result of the complex solution of different N/P;

Fig. 4 is particle diameter and the zeta potential measurement result of the complex solution of N/P16, and wherein, A is grain-size graph, and B is zeta potential diagram;

Fig. 5 is the gene encapsulating situation that agarose gel electrophoresis investigates different N/P, and wherein, pDNA is gymnoplasm grain.

Fig. 6 is the form of complex when observing N/P16 under transmission electron microscope;

Fig. 7 is the toxicity that mtt assay investigates different N/P complexes upon cell, and wherein, A figure is the survival rate of Neuro-2a cell, and B figure is the survival rate of b.End3 cell;

Fig. 8 is that CD68 SABC investigates the toxicity of polymer to tissue, and wherein, 1 ~ 7 is respectively brain, cerebellum, heart, liver, spleen, kidney, and A ~ C is respectively normal saline group, the 24h of administration after 7 days, the 72h of administration after 7 days;

Fig. 9 is that laser co-focusing observes b.End3 cell to the picked-up situation of cy3-pDNA;

Figure 10 is that flow cytometer detection by quantitative b.End3 cell is to the intake of different FITC-polyplex;

Figure 11 be different FITC-polyplex at different time, through the permeability of b.End3 cell monolayer film;

Figure 12 is the expression of green fluorescent protein in Neuro-2a cell;

Figure 13 is the expression of luciferase in Neuro-2a cell;

Figure 14 is that small animal living body imaging investigates the polymer of year DiR fluorescent dye at rat kidney tissue, is followed successively by PEG-PAsp (TEP)-chole/DiR group from left to right,

Tet1-PEG-PAsp (TEP)-chole/DiR group,

RVG29-PEG-PAsp (TEP)-chole/DiR group,

RVG29/Tet1-PEG-PAsp (TEP)-chole/DiR group;

Figure 15 is the expression of different polyplex in Mice Body that balb/c mouse tail vein injection carries luciferin prunus mume (sieb.) sieb.et zucc. reporter plasmid;

Figure 16 is after western blot detects the different polyplex groups applying to carry pGPU6/GFP/Neo/BACE1 plasmid, the protein content in Neuro-2a cell;

Figure 17 is the incubation period of employing Morris water maze evaluation double transgenic APP/PS1 Mus in training period;

Figure 18 is that double transgenic APP/PS1 Mus is at platform place quadrant time of staying proportion;

Figure 19 of the present inventionly passs the structural representation releasing complex, and wherein, A is the structural formula of carrier PEG-PAsp (TEP)-chole, and B is the structural formula after polypeptide is connected to carrier.

Detailed description of the invention:

Below in conjunction with the drawings and specific embodiments, the invention will be further described.Should be understood that following examples only for illustration of the present invention but not for limiting scope of the present invention.

Embodiment 1:

Then the aspartic acid benzyl ester (purchased from Tianjin Heowns Biochemical Technology Co., Ltd.) taking 4.46g under agitation adds 6g triphosgene, and normal hexane precipitates, and obtains aspartic acid benzyl ester carboxylic acid anhydrides (BLA-NCA).

Take Ace-PEG ₁₀₃-NH ₂(purchased from Xiamen Sainuo Bangge Biotechnology Co., Ltd.) 0.5g adds 250ml there-necked flask, adds about 50ml CH ₂cl ₂, after magnetic agitation is dissolved, then add BLA-NCA2.25g, nitrogen protection reaction 72h at 30 DEG C.Concentration of reaction solution, with 10 times of ether sedimentations, filter, vacuum drying, obtains Polyethylene Glycol-b-poly-aspartate (Ace-PEG-PBLA).

Take Ace-PEG-PBLA0.5g, measure tetren (purchased from Aladdin reagent) 10mL, add in DMF, oil bath constant temperature 40 DEG C, stir 24h.Concentrated hydrochloric acid adjusts pH to neutral, and dialysis 48h, lyophilization, obtains Ace-PEG-P [Asp (TEP)]-chloe.

Take Ace-PEG-P [Asp (TEP)]-chloe of 0.1g, add in DMSO and dissolve, cholesteryl chloroformate (purchased from lark prestige bio tech ltd) (dissolving with DMSO in advance) is added by degree of modification 15%, add triethylamine, 35 DEG C of oil baths, heating 12 ~ 24h, adjust pH to neutral, dialysis 24 ~ 48h, lyophilization, obtains end-product Ace-PEG-P [Asp (TEP)]-chloe.

Embodiment 2:

Ace-PEG-P [Asp (TEP)]-chloe is dissolved in acetate buffer solution (0.2M with polypeptide RVG29, Tet1 (purchased from Zhongtai Bio-Chem. Co., Ltd., Hangzhou) with mol ratio 1:5 respectively, pH=4.0) in, room temperature magnetic agitation 3 ~ 5d.PBS dialyses 24 ~ 48h, then changes to heat up in a steamer water and continue dialysis 48h, except free polypeptide, lyophilization respectively product RVG29-PEG-P [Asp (TEP)]-chloe and Tet1-PEG-P [Asp (TEP)]-chloe.

Embodiment 3:

Ace-PEG-P [Asp (TEP)]-chloe10mg of synthesis in embodiment 1 is dissolved in the deuterated DMSO of 0.5mL, characterize through nucleus magnetic hydrogen spectrum, result shows, δ 5.1 place is the proton peak of cholesteryl, obvious peak, δ 3.5 place is the proton peak of PEG, the success of explanation Macroscopic single crystal, is shown in Fig. 1.

RVG29-PEG-P [Asp (TEP)]-chloe10mg of synthesis in embodiment 2 is dissolved in 0.5mL heavy water, characterize through nucleus magnetic hydrogen spectrum, result shows, proton peak near δ 7.0 is the amino acid whose proton peak containing phenyl ring on polypeptide, aminoacid containing phenyl ring in RVG is phenylalanine (Phenylalanine, F), tyrosine (Tyrosine, Y), tryptophan (Tryptophane, W), proton peak near δ 1.0 disappears, more than polypeptide successful connection is described, sees Fig. 2.

Embodiment 4:

Take Ace-PEG-P [Asp (TEP)]-chole1mg of preparation in embodiment 1, be dissolved in 0.5mLPBS buffer (pH=7.4), for subsequent use.PGFP/GFP/Neo egfp expression plasmid DNA (purchased from Shanghai JiMa pharmacy Technology Co., Ltd) is dissolved in appropriate PBS buffer, is made into 100 μ g/mL solution.Precision measures above-mentioned cationic polymer storing solution, and is diluted to 0.5mL, is added drop-wise in equal-volume pDNA solution respectively, vortex 30s, obtains Ace-PEG-P [Asp (TEP)]-chole/pDNA (polyplex) and passs release system.

Embodiment 5:

RVG29-PEG-P [Asp (TEP)]-chole and Tet1-PEG-P [Asp (TEP)]-chloe of synthesis in embodiment 2 is dissolved in a certain amount of PBS buffer respectively, is made into the solution of 2mg/mL.PGFP/GFP/Neo egfp expression plasmid DNA is dissolved in above-mentioned buffer respectively, is made into 100 μ g/mL solution.By both according to different N/P, equal-volume mixing vortex 30s, makes cationic compound RVG29-polyplex and Tet1-polyplex.

Embodiment 6:

Take Ace-PEG-P [Asp (TEP)]-chole1mg of preparation in embodiment 1, be dissolved in 0.5mLPBS buffer (pH=7.4), for subsequent use.PGL3 luciferase reporter plasmid DNA (purchased from Shanghai JiMa pharmacy Technology Co., Ltd) is dissolved in appropriate PBS buffer, is made into 100 μ g/mL solution.Precision measures the above-mentioned cationic polymer storing solution of different volumes, and is diluted to 0.5mL, is added drop-wise in equal-volume pDNA solution respectively, vortex 30s, and Ace-PEG-P [Asp (TEP)]-chole/pDNA obtaining different N/P passs release system.

Embodiment 7:

RVG29-PEG-P [Asp (TEP)]-chole and Tet1-PEG-P [Asp (TEP)]-chloe of synthesis in embodiment 2 is dissolved in a certain amount of PBS buffer respectively, is made into the solution of 2mg/mL.PGL3 luciferase reporter plasmid DNA is dissolved in above-mentioned buffer respectively, is made into 100 μ g/mL solution.By both according to different N/P, equal-volume mixing vortex 30s, makes cationic compound RVG29-polyplex and Tet1-polyplex.

Embodiment 8:

Take Ace-PEG-P [Asp (TEP)]-chole1mg of preparation in embodiment 1, be dissolved in 0.5mLPBS buffer (pH=7.4), for subsequent use.Select recombiant plasmid pGPU6/GFP/Neo/BACE1 (purchased from Shanghai JiMa pharmacy Technology Co., Ltd), wherein BACE1 sequence is GCTTTGTGGAGATGGTGGA.

PGPU6/GFP/Neo/BACE1 is dissolved in appropriate PBS buffer, is made into 100 μ g/mL solution.Precision measures the above-mentioned cationic polymer storing solution of different volumes, and is diluted to 0.5mL, is added drop-wise in equal-volume pDNA solution respectively, vortex 30s, and Ace-PEG-P [Asp (TEP)]-chole/pDNA obtaining different N/P passs release system.

Embodiment 9:

RVG29-PEG-P [Asp (TEP)]-chole and Tet1-PEG-P [Asp (TEP)]-chloe of synthesis in embodiment 2 is dissolved in a certain amount of PBS buffer respectively, is made into the solution of 2mg/mL.Recombiant plasmid pGPU6/GFP/Neo/BACE1 is dissolved in above-mentioned buffer respectively, is made into 100 μ g/mL solution.By both according to different N/P, equal-volume mixing vortex 30s, makes cationic compound RVG29-polyplex and Tet1-polyplex.

Embodiment 10:

Take 10mg PEG-PAsp (TEP)-chole, Tet1-PEG-PAsp (TEP)-chole respectively, RVG29-PEG-PAsp (TEP)-chole, RVG29/Tet1-PEG-PAsp (TEP)-chole are dissolved in 2mL DMSO solvent, then add containing 100 μ g DiR solution, agitation and dropping is in 6mL deionized water water, and dialysis is except organic solvent.The centrifugal 40min of 4000r, the DiR dyestuff that removing is free.

Embodiment 11:

Prepare Ace-PEG-P [Asp (TEP)]-chole/DNA carrying gene of different N/P according to embodiment 4 method respectively, be diluted to 1mL respectively, Dynamic Light Scattering Determination particle diameter and zeta current potential.Result shows, and in research range, with the increase of N/P ratio, particle diameter reduces gradually, and zeta current potential increases gradually, sees Fig. 3, and wherein the particle diameter of N/P16 and current potential the results are shown in Figure 4.

Embodiment 12:

Ace-PEG-P [Asp (TEP)]-chole/DNA carrying gene of different N/P is prepared respectively according to embodiment 4 method; Take 0.2g agarose in conical flask, add 20mL TAE electrophoresis liquid, in microwave, high fire heating makes agarose dissolve completely, and room temperature is placed to about 60 DEG C, adds 5 μ L ethidium bromides, shakes even, slowly pour in electrophoresis tank, leaves standstill and makes gel.Add in loading hole by above-mentioned different polyplex, under 150V, swimming is about 20min, observes and take pictures under uviol lamp.

Result shows, and pDNA can be trapped in loading hole completely, and namely pDNA can be carried by bag completely, the results are shown in Figure 5.

Embodiment 13:

Prepare the polyplex carrying gene of N/P16 respectively according to embodiment 4 method, phosphotungstic acid dyes, and project the form of electric Microscopic observation particle, the results are shown in Figure 6, particle is spherical in shape, is uniformly dispersed, and particle diameter is about 70nm.

Embodiment 14:

The polyplex carrying gene of different N/P is prepared, by brain capillary endothelial cell (b.End3 cell) (pharmacology teaching and research room professor Rui Yaocheng gives) and mouse brain neuroma cell (Neuro-2a cell) (purchased from ATCC cell bank) respectively with 5 × 10 by the method for embodiment 4 ³individual/hole is inoculated in 96 orifice plates, and cell is placed in 37 DEG C, 5%CO2 incubator hatches 24h to adherent, with PBS rinsing 3 times, above-mentioned different polyplex is put in different hole, and blank group and Lipofectamine-2000 positive controls is set.In cell culture incubator, hatch 24h, change fresh culture medium, continue to cultivate 48h.

Adopt mtt assay to measure the different groups of toxicity to cell, result shows, and apply two kinds of cells survival rates of the polyplex of different N/P all more than 85%, illustrate, cytotoxicity is less, sees Fig. 7.

Embodiment 15:

Balb/c mouse tail vein injection Ace-PEG-PAsp (TEP)-chole30mg/kg, successive administration 7d, administration terminates rear 24h, 72h, de-neck is put to death, get brain, cerebellum, the heart, liver, spleen, lung, kidney, after CD68 immunohistochemical staining, microscopic examination is also taken pictures.

Shown in result, in heart, liver and kidney, a small amount of macrophage is had to produce.When there being inflammation, a large amount of glomerule mesangial cells can be produced, engulfing of glomerule is strengthened with scavenging action.But this reaction occurs over just the 24h after administration.Polymeric material may produce transient toxicity, through 72h, slowly reduces until with matched group without significant difference, see Fig. 8.

Embodiment 16:

The polyplex carrying Cy3-pDNA of N/P16 is prepared by the method for embodiment 4.BEnd.3 cell is with 5.0 × 10 ⁵cells/well is inoculated in confocal special culture dish, hatch 24h to cell attachment, suck culture medium, complex containing 2 μ g pDNA is incorporated in culture dish, add 1ml not containing the culture medium of serum, put into after cell culture incubator lucifuge cultivates 2h and take out, picked-up is stopped with ice-cold PBS, rinsing 3 times, then the paraformaldehyde of 4% fixes 20mim, PBS washs 3 times, add DiO cell membrane dyestuff again, incubated at room 10min, PBS rinsing 3 times, DAPI solution carries out nuclear targeting, after anti-fluorescence quenching mounting, observe under being placed in laser confocal microscope.

Result shows, and polyplex-RVG29 group and polyplex-Tet1/RVG29 group show the strongest fluorescence intensity, see Fig. 9.

Embodiment 17:

Take Ace-PEG-P [Asp (TEP)]-chole, Tet1-PEG-P [Asp (TEP)]-chole, RVG29-PEG-P [Asp (TEP)]-chole, Tet1/RVG29-PEG-P [Asp (TEP)] each 20mg of-chole, be dissolved in 5ml carbonate buffer solution (pH=9.0) respectively, the water solublity Fluorescein isothiocyanate (purchased from Sigma-Aldrich company) of 500 μ g is added in above-mentioned carbonate buffer solution, lucifuge stirs 12h, deionized water is dialysed, removing free water dissolubility Fluorescein isothiocyanate.

The polyplex of N/P16 is prepared according to the method for embodiment 4 and embodiment 5.The bEnd.3 cell that phase of taking the logarithm grows, cell counting count board counts, with 1.0 × 10 ⁶individual/hole is inoculated in confocal special culture dish, and overnight incubation is to cell attachment.Complex containing 2 μ g pDNA is added in culture dish, add 1ml not containing the culture medium of serum, put into after cell culture incubator lucifuge cultivates 2h and take out, stop picked-up with ice-cold PBS, trypsin digestion cell, the culture medium containing serum stops digestion, the centrifugal 3min of 1000r, PBS rinsing, then adds 500 μ L PBS resuspended, flow cytomery.

Statistical analysis, the uptake ratio of Tet1/RVG29-polyplex with RVG29-polyplex in cell is compared with the polyplex of polyplex, Tet1, there is significant difference, the RVG29-polyplex intake that wherein cell is right is 2.8 times of polyplex, after bEnd.3 cell Tet1/RVG29-polyplex is hatched, compared with polyplex, fluorescence intensity adds 1.4 times.The polyplex that aggregate amount in cell is modified than RVG29 is few, sees Figure 10.

Embodiment 18:

By b.End3 cell with 5 × 10 ⁵individual/hole is inoculated in Tranwell (12 orifice plates, 3 μm, inner membrance 1.12cm ²) upper strata, and adding 0.5mL culture fluid, lower floor adds 1.5mL culture fluid simultaneously, changes fresh medium after 2d, continues to cultivate, and approximately cultivates 5 ~ 7d.Measure every day across endothelial cell membrane resistance (transendothelial electrical resistance, TEER) after it is stable, prompting monofilm is formed.To the polyplex of FITC be marked respectively, be placed in the upper strata of Tranwell, and add culture medium to 500 μ L.

After 1,2,4,6,8h after administration, sample 500 μ L, be placed in 4 DEG C of refrigerators, (selecting fluorescent spectrophotometer assay) to be measured, and add 500 μ L fresh cultures.Sample tests result shows, after administration after 2h, four kinds of complex basically identical through efficiency, when after administration 4h, permeability without polypex and the Tet1-polyplex group of any modification substantially no longer raises, and the permeability of RVG29-polyplex and Tet1/RVG29-polyplex has and significantly raises, see Figure 11.

Embodiment 19:

Polyplex, Tet1-polyplex, RVG29-polyplex, Tet1/RVG29-polyplex of N/P16 is prepared according to the method for embodiment 6 and embodiment 7.Neuro-2a cell is with 5.0 × 10 ⁴individual/hole is inoculated in 24 orifice plates, and overnight incubation, to cell attachment, sucks culture medium, is incorporated in culture dish by the above-mentioned complex containing 1 μ g pDNA, adds 1mL not containing the culture medium of FBS, puts into 37 DEG C, 5%CO ₂cell culture incubator in continue cultivate 48h, period replaced medium.After cultivation terminates, discard culture medium, PBS rinsing three times, the paraformaldehyde then adding 4% fixes 20mim, is placed in fluorescence microscopy Microscopic observation.

Result shows, the fluorescence intensity of blank polyplex is more weak, and the fluorescence intensity of Tet1-polyplex group and RVG29-polyplex group is all stronger, perusal does not have obvious difference, two ligand modified group shows the strongest fluorescence intensity in cell, illustrate two ligand modified after be conducive to swallowing born of the same parents in particle, have higher transfection efficiency, see Figure 12.

Embodiment 20:

Polyplex, Tet1-polyplex, RVG29-polyplex, Tet1/RVG29-polyplex of N/P16 is prepared by the method for embodiment 6 and embodiment 7.Neuro-2a cell is with 1.0 × 10 ⁴individual/hole is inoculated in 96 orifice plates, and overnight incubation is to cell attachment.The above-mentioned complex containing 0.5 μ g pDNA is incorporated in culture dish, adds 1mL culture medium, put into 37 DEG C, 5%CO ₂cell culture incubator in continue cultivate 48h, period replaced medium.After cultivation terminates, CCLR cell lysis, 10000r centrifuging and taking supernatant, adopt luciferase detection system to measure, BCA method measures protein content.

Result shows, and the fluorescence intensity of blank polyplex is more weak, and Tet1-polyplex group is 3.3 times of unmodified group fluorescence intensity, two ligand modified group shows the strongest fluorescence intensity in cell, be 4.6 times for modification group, this is also consistent with the result of qualitative detection, sees Figure 13.

Embodiment 21:

Adopt luciferase reporter plasmid pGL3 as fluorescent probe.Polyplex, Tet1-polyplex, RVG29-polyplex, Tet1/RVG29-polyplex of N/P16 is prepared respectively by embodiment 5.Balb/c male mice is through tail intravenously administrable, and dosage is pDNA50 μ g.After administration 48h, de-neck is put to death, and gets cerebral tissue respectively on ice, rinses out residual blood with ice PBS, get portion of tissue and add lysate CCLR, mortar grinder, 10000r centrifuging and taking supernatant, and adopt luciferase detection system to measure, BCA method measures protein content.

Shown in result, in the brain in Tet1/RVG29-polyplex administration group, uciferase activity is significantly higher than polyplex group, sees Figure 14.

Embodiment 22:

The different particle carrying DiR fluorescent dye is prepared according to embodiment 8, naked caudal vein injects the different composite thing particle containing DiR (0.5mg/kg) respectively, chloral hydrate anesthesia after certain hour, small animal living body imaging system is adopted to observe and take pictures, result shows, the strongest at the fluorescence of nude mice brain through two ligand modified complex, and the fluorescence intensity of liver region relative to other organize weak, illustrate through two ligand modified after be conducive to particle and be enriched in brain, and reduce the gathering of other non-target site, see Figure 15.

Embodiment 23:

Take the logarithm phase Neuro-2a cell, cell counting count board counts, with 5 × 10 ⁵individual/porocyte is inoculated in 6 orifice plates, cell culture incubator overnight incubation.Discard culture medium, apply polyplex, polyplex-Tet1, polyplex-RVG29, polyplex-Tet1/RVG29 respectively, with blanc cell in contrast, continue to cultivate 48h.Then, add the cell pyrolysis liquid containing PMSF, on ice cracking 30min, collect albumen, under 4 DEG C of conditions, the centrifugal 10min of 10000r, gets supernatant.Get part supernatant for determining the protein quantity, another part adds protein loading buffer boiling water bath degeneration 5min.Get 30 μ g albumen, detect according to the method for western blot.

Testing result shows, and polyplex group and blank histone are on close level, and the protein level of polyplex-Tet1 group and polyplex-RVG29 is close, but decline all to some extent compared with blank group, and the protein level of polyplex-Tet1/RVG29 group is minimum, sees Figure 16.

Embodiment 24:

Administration is started when choosing double transgenic APP/PS1 Mus (purchased from Beijing Medical Li Hao bio tech ltd) Beijing 6 months.Tail intravenously administrable different composite thing (containing 100 μ g recombiant plasmid), with normal saline group and non-transgenic Mus for contrast, administration every other week, successive administration 4 times, one week after administration, carries out water maze Behavioral assessment.

The display of navigation study experimental result, mice is with the increase of training natural law, and incubation period shortens gradually.When training the 4th day, administration polyplex-Tet1/RVG29 group is compared with normal saline group, incubation period obviously shortens, there is significant difference, and also decrease to some degree incubation period of polyplex-Tet1/RVG29. group and polyplex group mice, but compared with polyplex-Tet1/RVG29 group, incubation period is longer and have significant difference, sees Figure 17.

Train after four days, the explorative experiment carried out for 5th day, result shows, administration group mice time scale shared by third quadrant all has improvement to a certain degree compared with normal saline group, but do not have administration polyplex-RVG29, polyplex-Tet1/RVG29 group mice high in the time scale of platform place quadrant, wherein polyplex-Tet1/RVG29 group proportion is the highest.Illustrate, after administration polyplex-Tet1/RVG29, mouse memory power has improvement to a certain degree, sees Figure 18.

Below the preferred embodiment of the invention is illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make all equivalent modification or replacement under the prerequisite without prejudice to the invention spirit, and these equivalent modification or replacement are all included in the application's claim limited range.

Claims (6)

1. the Alzheimer target gene that bifunctional peptide is modified is passed and is released a complex, it is characterized in that, this is passed and releases complex and release carrier and electronegative genomic constitution by passing:

Carrier is released in described passing, and comprises end group connection RVG29 and Tet1 of the cationic polymer of Pegylation, cationic polymer;

Described RVG29, its sequence is as shown in SEQ ID NO:1;

Described Tet1, its sequence is as shown in SEQ ID NO:2;

Described electronegative gene and described passing release cationic polymer in carrier by positive and negative charge electrostatic attraction.

2. the Alzheimer target gene that a kind of bifunctional peptide according to claim 1 is modified is passed and is released complex, and it is characterized in that, described cationic polymer, is the poly-aspartate modified through tetren.

3. the Alzheimer target gene that a kind of bifunctional peptide according to claim 1 is modified is passed and is released complex, it is characterized in that, described electronegative gene is selected from medicative interference sequence, is implemented in the PEG end of described cationic polymer.

4. the Alzheimer target gene that a kind of bifunctional peptide according to claim 3 is modified is passed and is released complex, and it is characterized in that, described electronegative gene is BACE1 interference sequence, and its sequence is as shown in SEQ ID NO:3.

5. the Alzheimer target gene that bifunctional peptide is modified passs the preparation method releasing complex, and it is characterized in that, the method comprises the steps:

The preparation of A, cationic polymer carrier

Be dissolved in oxolane by aspartic acid benzyl ester and triphosgene according to molecular proportion 1:1 ~ 5, be heated to 50 ~ 80 DEG C, magnetic agitation is to dissolving, and normal hexane precipitates, and obtains aspartic acid benzyl ester carboxylic acid anhydrides; End contains amino PEG derivant and aspartic acid benzyl ester carboxylic acid anhydrides, is dissolved in CH ₂cl ₂, 25 ~ 50 DEG C of reaction 24 ~ 72h, ether sedimentation obtains Polyethylene Glycol poly-aspartate PEG-PBLA;

Utilize the acetal of PEG end in cationic polymer, in acetate buffer solution, dehydration forms aldehyde-base, with the sulfydryl in cysteine by covalently bound;

B, pass the preparation releasing complex

The cationic polymer carrier that steps A prepares and electronegative gene are dissolved in the PBS buffer of pH7.4, equal-volume vortex mixed 30s, what form 80 ~ 300 nanosizeds passs release system, and wherein cationic polymer carrier is 2 ~ 16 with the N/P ratio of electronegative gene.

6. one kind as arbitrary in Claims 1-4 as described in bifunctional peptide modify Alzheimer target gene pass release complex preparation treatment Alzheimer disease drugs in application.