CN104368009B - A kind of Alzheimer disease target gene of bifunctional peptide modification, which is passed, releases compound and preparation method thereof - Google Patents
A kind of Alzheimer disease target gene of bifunctional peptide modification, which is passed, releases compound and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to pharmaceutical technology field, it is that the Alzheimer disease target gene of bifunctional peptide modification a kind of is passed and releases compound and preparation method thereof, the present invention has synthesized cholesteryl and the polyethylene glycol poly-aspartate PEG P [Asp (TEP) chole] of tetren modification；It is compound with gene by electrostatic adsorption；Carrier material is modified using RVG29 and the Tet1 Functional Polypeptides of great potential applicability in clinical practice, preparation forms foregoing brain targeting drug delivery system.The present invention can make load gene delivery system pass through blood-brain barrier and the specific transfection efficiency for targeting nerve cell, improving gene, and a kind of hurtless measure administering mode is provided for brain administration.
Description
Technical field：
The present invention relates to pharmaceutical technology field, and in particular to a kind of Alzheimer disease target gene of bifunctional peptide modification
Pass and release compound.
Background technology：
The harm of central nervous system disease increases year by year, have become the most serious for endangering human health disease it
One.Genomic medicine, due to its molecular target high specific the features such as, it has also become study relatively broad macromolecular medicine at present
Thing.But because the limitation of blood-brain barrier, genomic medicine can not be independently by blood-brain barrier, it is necessary to pass through the ventricles of the brain or intrathecal note
Administering mode, this invasive administering mode such as penetrate to cause to limit to clinical administration.Therefore, exploitation is with intravascular administration
Novel gene drug delivery system, there is critically important researching value.
The polymer support for connecting ligands specific can be targeted to the tissue and cell of the high expression of corresponding acceptor.Brain active target
There are TfR, insulin receptor, LDL receptor, folic acid etc. to the acceptor for studying wide.But there is research
It is not preferable brain guide molecule to think transferrins, and the medicine that transferrins is oriented to is combined with acceptor in the presence of competition suppression
System, acceptor by endogenous transferrins saturation, cause to be oriented to efficiency relatively low (Qian ZM, Li H, Sun H, et substantially
a1.Targeted drug delivery via the transferring receptor-mediated endocytosis
pathway[J].Pharmaco Rev,2002,54(4):561-587.)。
RVG29 is a kind of 29 amino acids formed polypeptides from rabies virus glycoprotein, (Kumar P, Wu H,
McBride J L,et al.Transvascular delivery of small interfering RNA to the
central nervous system[J].Nature,2007,448(7149):39-43.) related mechanism research shows that RVG can
After being combined with the acetylcholinergic receptor on BBB and GABA receptor, efficient brain functioning can be achieved.Tet1 is by 12
The small peptide of individual Amino acid profile have with neuronal cell GT1B acceptors height compatibility (Kwon E J, Lasiene J,
Jacobson B E,et a1.Targeted nonviral delivery vehicles to neural progenitor
ceils in the mouse subventricular zone[J].Biomaterials,2010,31(8):2417-
2424.)。
Polyethylene glycol poly-aspartate (PEG-PBLA) is a kind of biodegradable, biocompatibility is preferable, toxicity is relatively low
High polymer material, the particularity of structure solves the problems, such as chemical modification.It is a kind of polymer of electroneutral, can not be born with electricity
Property gene it is compound, therefore through the tetren modification containing amino, make high polymer material positively charged, can effectively and base
Because compound.Meanwhile tetren has certain buffer capacity, enhancing endosome escape effect, improves transfection efficiency.End
Base modification cholesteryl can strengthen the interaction between gene and macromolecule carrier, improve the stability of compound.
The content of the invention：
Passed it is an object of the invention to provide a kind of Alzheimer disease target gene of bifunctional peptide modification and release compound,
Pass another object of the present invention is to providing the target gene and release the preparation method of compound.
The Alzheimer disease target gene that the main technical schemes of the present invention are to provide a kind of modification of bifunctional peptide is passed and released
Compound, also referred to as passs release system, and the present invention has synthesized cholesteryl and the poly- day of polyethylene glycol of tetren modification first
Winter propylhomoserin PEG-P [Asp (TEP)-chole]；Then it is compound with gene by electrostatic adsorption；Afterwards using RVG29 with
The Brain targeting such as Tet1 Functional Polypeptides modify carrier material, and preparation forms foregoing brain targeting drug delivery system.
The present invention is to connect two kinds of brain function peptides, forms bifunctional peptide, brain is targeted jointly with polymer composite.This
Invention can make load gene delivery system through blood-brain barrier and the specific transfection effect for targeting nerve cell, improving gene
Rate, a kind of hurtless measure administering mode is provided for brain administration.
The first aspect of the present invention, be to provide a kind of modification of bifunctional peptide Alzheimer disease target gene pass release it is compound
Thing (passs release system), and this, which is passed, releases compound and release carrier and electronegative gene forms by passing：
Carrier is released in described passing, including cationic polymer, the end group of cationic polymer that PEG (polyethylene glycol) changes connect
Meet RVG29 or Tet1；
The cationic polymer that described electronegative gene is released with described passing in carrier is inhaled by positive and negative charge electrostatic
Draw.
Described cationic polymer, the poly-aspartate preferably modified through tetren.
A kind of Alzheimer disease target gene of bifunctional peptide modification of the present invention passs the structure such as Figure 19 for releasing compound
It is shown.
Described RVG29 derives from the rabies virus glycoprotein with brain functioning characteristic, by 29 Amino acid profiles, end
Cysteine is added, its sequence is：YTIWMPENPRPGTPCDIFTNSRGKR ASNGC(SEQ ID NO：1).
The described Tet1 and GT1B of neuronal cell surface has the compatibility of height, can specific combination GT1B by
Body.Tet1 adds cysteine by 12 Amino acid profiles, end, and its sequence is：HLNILSTLWKYRC(SEQ ID NO：
2)。
Described electronegative gene is selected from medicative interference sequence, is implemented in described cationic polymerization
The PEG ends of thing.It is preferred that BACE1 interference sequences, sequence are：GCTTTGTGGAGATGGTGGA(SEQ ID NO：3).
The second aspect of the present invention, be to provide a kind of modification of bifunctional peptide Alzheimer disease target gene pass release it is compound
The preparation method of thing, this method comprise the following steps：
A, the preparation of cationic polymer carrier
By aspartic acid benzyl ester and triphosgene according to molecular proportion 1:1~5 is dissolved in tetrahydrofuran, is heated to 50~80 DEG C,
Magnetic agitation n-hexane precipitation, obtains aspartic acid benzyl ester carboxylic acid anhydrides to dissolving；Contain the PEG derivatives and asparagus fern of amino in end
Propylhomoserin benzyl ester carboxylic acid anhydrides, is dissolved in CH2Cl2, 25~50 DEG C are reacted 24~72h, and ether precipitates to obtain polyethylene glycol poly-aspartate
(PEG-PBLA)；
Using the acetal of PEG ends in cationic polymer, it is dehydrated in acetate buffer solution and forms aldehyde-base, with half Guang
Sulfydryl in propylhomoserin passes through covalent attachment.
B, the preparation of release system is passed
The cationic polymer carrier that step A is prepared is dissolved in pH7.4 PBS with electronegative gene, etc.
Volume vortex mixed 30s, form 80~300 nanosizeds passs release system, wherein cationic polymer carrier with it is electronegative
The N/P ratios of gene are 2~16.
Described electronegative gene, preferred plasmid DNA.
The third aspect of the present invention, be to provide above-mentioned bifunctional peptide modification Alzheimer disease target gene pass release it is multiple
Compound is preparing the application in treating Alzheimer disease drugs.
Cationic polymer PEG-PAsp (TEP)-chole that the present invention is built, biocompatibility is preferable, and cell and group
It is relatively low to knit toxicity.
The polyplex that PEG-PAsp (the TEP)-chole that the present invention uses are compounded to form with gene, can promote endosome
Escape, significantly improves nerve cell transfection efficiency.
Two kinds of polypeptides RVG29 and Tet1 that the present invention uses, the carrier system of load gene can be made by blood-brain barrier, simultaneously
It is selectively targeted in nerve cell, improve internal brain targeting.
The present invention is implemented in plasmid from BACE1 interference sequences, is formed with double ligand modified cationic polymers multiple
Compound, it can effectively lower the BACE1 protein levels in nerve cell Neuro-2a.
The present invention is implemented in plasmid from BACE1 interference sequences, is formed with double ligand modified cationic polymers multiple
Compound, after tail vein injection, the spatial memory capacity of Alzheimer disease model mouse can be significantly improved.
Brief description of the drawings：
Fig. 1 is Ace-PEG-P [Asp (TEP)]-chloe nucleus magnetic hydrogen spectrum figure；
Fig. 2 is RVG29-PEG-P [Asp (TEP)]-chloe nucleus magnetic hydrogen spectrum figure；
Fig. 3 is the particle diameter and zeta potential measurement results of different N/P complex solution；
Fig. 4 is the particle diameter and zeta potential measurement results of N/P16 complex solution, wherein, A is grain-size graph, B zeta
Potential diagram；
Fig. 5 is the gene encapsulating situation that agarose gel electrophoresis investigates different N/P, wherein, pDNA is gymnoplasm grain.
Fig. 6 is the form of compound when N/P16 is observed under transmission electron microscope；
Fig. 7 is the toxicity that mtt assay investigates different N/P complexes upon cell, wherein, A figures are the existence of Neuro-2a cells
Rate, B figures are the survival rate of b.End3 cells；
Fig. 8 is that CD68 SABCs investigate toxicity of the polymer to tissue, wherein, 1~7 is respectively brain, cerebellum, the heart
Dirty, liver, spleen, kidney, A~C be respectively physiological saline group, administration 7 days after 24h, administration 7 days after 72h；
Fig. 9 is that laser co-focusing observes intake situation of the b.End3 cells to cy3-pDNA；
Figure 10 is that flow cytometer quantitatively detects intake of the b.End3 cells to different FITC-polyplex；
Figure 11 be different FITC-polyplex in different time, through the permeability of b.End3 cell monolayer films；
Figure 12 is expression quantity of the green fluorescent protein in Neuro-2a cells；
Figure 13 is expression quantity of the luciferase in Neuro-2a cells；
Figure 14 is that small animal living body imaging investigates the polymer for carrying DiR fluorescent dyes in rat kidney tissue, from left to right
PEG-PAsp (TEP)-chole/DiR groups are followed successively by,
Tet1-PEG-PAsp (TEP)-chole/DiR groups,
RVG29-PEG-PAsp (TEP)-chole/DiR groups,
RVG29/Tet1-PEG-PAsp (TEP)-chole/DiR groups；
Figure 15 is that balb/c mouse tail vein injections carry the different polyplex of luciferin plum reporter plasmid in Mice Body
Interior expression quantity；
Figure 16 is that western blot detections apply the different polyplex groups for carrying pGPU6/GFP/Neo/BACE1 plasmids
Afterwards, the protein content in Neuro-2a cells；
Figure 17 is to evaluate incubation period of the double transgenic APP/PS1 mouse in training period using Morris water mazes；
Figure 18 is double transgenic APP/PS1 mouse quadrant residence time proportion where platform；
Figure 19 passs the structural representation for releasing compound for the present invention's, wherein, A is carrier PEG-PAsp (TEP)-chole
Structural formula, B be polypeptide be connected to the structural formula after carrier.
Embodiment：
Below in conjunction with the drawings and specific embodiments, the invention will be further described.It should be understood that following examples are only used for
Illustrate rather than for limiting the scope of the present invention.
Embodiment 1：
Weigh 4.46g aspartic acid benzyl ester (being purchased from Tianjin Heowns Biochemical Technology Co., Ltd.) and then add under agitation
Enter 6g triphosgenes, n-hexane precipitation, obtain aspartic acid benzyl ester carboxylic acid anhydrides (BLA-NCA).
Weigh Ace-PEG103-NH2(being purchased from Xiamen Sainuo Bangge Biotechnology Co., Ltd.) 0.5g adds tri- mouthfuls of 250ml
Bottle, add 50ml or so CH2Cl2, after magnetic agitation dissolving, BLA-NCA2.25g is added, the nitrogen protection reaction at 30 DEG C
72h.Concentration of reaction solution, precipitated, filtered with 10 times of ether, vacuum drying, produce polyethylene glycol-b- poly-aspartates (Ace-
PEG-PBLA)。
Ace-PEG-PBLA 0.5g are weighed, measure tetren (being purchased from Aladdin reagent) 10mL, are added in DMF,
40 DEG C of oil bath constant temperature, stir 24h.Concentrated hydrochloric acid adjusts pH, and to neutrality, dialyse 48h, freeze-drying, produces Ace-PEG-P [Asp
(TEP)]。
0.1g Ace-PEG-P [Asp (TEP)] is weighed, adds in DMSO and dissolves, chloro-carbonic acid courage is added by degree of modification 15%
Sterol ester (being purchased from lark prestige bio tech ltd) (being dissolved in advance with DMSO), add triethylamine, 35 DEG C of oil baths, heating 12
~24h, adjusting pH, dialyse 24~48h, freeze-drying, produces end-product Ace-PEG-P [Asp (TEP)]-chloe to neutrality.
Embodiment 2：
Ace-PEG-P [Asp (TEP)]-chloe (is purchased from the biochemical limited public affairs of peptide in Hangzhou with polypeptide RVG29, Tet1 respectively
Department) with mol ratio 1:5 are dissolved in acetate buffer solution (0.2M, pH=4.0), 3~5d of room temperature magnetic agitation.PBS 24~
48h, then change distilled water and continue the 48h that dialyses, except free polypeptide, freeze-drying obtains product RVG29-PEG-P [Asp respectively
(TEP)]-chloe and Tet1-PEG-P [Asp (TEP)]-chloe.
Embodiment 3：
The Ace-PEG-P synthesized in embodiment 1 [Asp (TEP)]-chloe10mg is dissolved in the deuterated DMSO of 0.5mL, through core
Magnetic hydrogen stave is levied, and is as a result shown, is the proton peak of cholesteryl at δ 5.1, the proton peak that obvious peak is PEG at δ 3.5, explanation
Macroscopic single crystal success, is shown in Fig. 1.
The RVG29-PEG-P synthesized in embodiment 2 [Asp (TEP)]-chloe10mg is dissolved in 0.5mL heavy water, through nuclear-magnetism
Hydrogen stave is levied, and is as a result shown, the proton peak near δ 7.0 is the proton peak of the amino acid containing phenyl ring on polypeptide, contains phenyl ring in RVG
Amino acid for phenylalanine (Phenylalanine, F), tyrosine (Tyrosine, Y), tryptophan (Tryptophane, W),
Proton peak near δ 1.0 disappears, and polypeptide successful connection described above, sees Fig. 2.
Embodiment 4：
Ace-PEG-P [Asp (TEP)]-chole 1mg prepared in embodiment 1 are weighed, are dissolved in 0.5mL PBSs
(pH=7.4) it is, standby.PGPU6/GFP/Neo egfp expressions DNA (is purchased from Shanghai Ji agate pharmaceutical technology
Co., Ltd) it is dissolved in appropriate PBS, it is made into 100 μ g/mL solution.Precision measures above-mentioned cationic polymer deposit
Liquid, and be diluted to 0.5mL, is added drop-wise in isometric pDNA solution, vortex 30s respectively, produce Ace-PEG-P [Asp (TEP)]-
Chole/pDNA (polyplex) passs release system.
Embodiment 5：
By the RVG29-PEG-P synthesized in embodiment 2 [Asp (TEP)]-chole and Tet1-PEG-P [Asp (TEP)]-
Chloe is dissolved in a certain amount of PBS respectively, is made into 2mg/mL solution.By pGPU6/GFP/Neo green fluorescent proteins
Expression plasmid DNA is dissolved in above-mentioned buffer solution respectively, is made into 100 μ g/mL solution.By both according to different N/P, isometric mixing
Vortex 30s, cationic compound RVG29-polyplex and Tet1-polyplex is made.
Embodiment 6：
Ace-PEG-P [Asp (TEP)]-chole1mg prepared in embodiment 1 is weighed, is dissolved in 0.5mLPBS buffer solutions (pH
=7.4) it is, standby.PGL3 luciferase reporter plasmids DNA (being purchased from Shanghai JiMa pharmacy Technology Co., Ltd) is dissolved in suitable
In the PBS of amount, 100 μ g/mL solution are made into.Precision measures the above-mentioned cationic polymer storing solution of different volumes, and
0.5mL is diluted to, is added drop-wise to respectively in isometric pDNA solution, vortex 30s, produces different N/P Ace-PEG-P [Asp
(TEP)]-chole/pDNA passs release system.
Embodiment 7：
By the RVG29-PEG-P synthesized in embodiment 2 [Asp (TEP)]-chole and Tet1-PEG-P [Asp (TEP)]-
Chloe is dissolved in a certain amount of PBS respectively, is made into 2mg/mL solution.By pGL3 luciferase reporter plasmids
DNA is dissolved in above-mentioned buffer solution respectively, is made into 100 μ g/mL solution.Will both according to different N/P, it is isometric to mix vortex 30s,
Cationic compound RVG29-polyplex and Tet1-polyplex is made.
Embodiment 8：
Ace-PEG-P [Asp (TEP)]-chole1mg prepared in embodiment 1 is weighed, is dissolved in 0.5mLPBS buffer solutions (pH
=7.4) it is, standby.From recombinant plasmid pGPU6/GFP/Neo/BACE1 (being purchased from Shanghai JiMa pharmacy Technology Co., Ltd),
Wherein BACE1 sequences are GCTTTGTGGAGATGGTGGA.
PGPU6/GFP/Neo/BACE1 is dissolved in appropriate PBS, is made into 100 μ g/mL solution.Precision measures
The above-mentioned cationic polymer storing solution of different volumes, and 0.5mL is diluted to, it is added drop-wise to respectively in isometric pDNA solution, whirlpool
30s is revolved, Ace-PEG-P [Asp (TEP)]-chole/pDNA for producing different N/P passs release system.
Embodiment 9：
By the RVG29-PEG-P synthesized in embodiment 2 [Asp (TEP)]-chole and Tet1-PEG-P [Asp (TEP)]-
Chloe is dissolved in a certain amount of PBS respectively, is made into 2mg/mL solution.By recombinant plasmid pGPU6/GFP/Neo/
BACE1 is dissolved in above-mentioned buffer solution respectively, is made into 100 μ g/mL solution.Both are vortexed according to different N/P, isometric mixing
30s, cationic compound RVG29-polyplex and Tet1-polyplex is made.
Embodiment 10：
10mg PEG-PAsp (TEP)-chole, Tet1-PEG-PAsp (TEP)-chole, RVG29-PEG- are weighed respectively
PAsp (TEP)-chole, RVG29/Tet1-PEG-PAsp (TEP)-chole are dissolved in 2mL DMSO solvents, are then added and are contained
100 μ g DiR solution, for agitation and dropping in 6mL deionized water water, dialysis removes organic solvent.4000r centrifuges 40min, removes trip
From DiR dyestuffs.
Embodiment 11：
Prepare Ace-PEG-P [Asp (TEP)]-chole/DNA of different N/P load gene respectively according to the method for embodiment 4,
1mL, dynamic light scattering measure particle diameter and zeta current potentials are diluted to respectively.As a result show, in research range, with N/P than increasing
Add, particle diameter is gradually reduced, and zeta current potentials gradually increase, and sees Fig. 3, and wherein N/P16 particle diameter and current potential result is shown in Fig. 4.
Embodiment 12：
Prepare Ace-PEG-P [Asp (TEP)]-chole/DNA of different N/P load gene respectively according to the method for embodiment 4；
0.2g agaroses are weighed in conical flask, add 20mL TAE electrophoresis liquids, high fiery heat is completely dissolved agarose in microwave,
Room temperature is placed to about 60 DEG C, adds 5 μ L ethidium bromides, and rolling is even, is poured slowly into electrophoresis tank, and standing makes gel.By it is above-mentioned not
Added with polyplex in loading hole, swimming about 20min under 150V, observe and take pictures under uviol lamp.
As a result show, pDNA can be trapped in loading hole completely, i.e. pDNA can be contained completely, as a result see Fig. 5.
Embodiment 13：
Prepare the polyplex of N/P16 load gene respectively according to the method for embodiment 4, phosphotungstic acid dyeing, project and seen under Electronic Speculum
The form of particle is examined, as a result sees Fig. 6, particle is spherical in shape, is uniformly dispersed, particle diameter about 70nm.
Embodiment 14：
The polyplex of different N/P load gene is prepared as described in Example 4, by brain capillary endothelial cell
(b.End3 cells) (pharmacology teaching and research room professor Rui Yaocheng gives) and mouse brain nerve oncocyte (Neuro-2a cells) (are purchased from ATCC
Cell bank) respectively with 5 × 103Individual/hole is inoculated in 96 orifice plates, and cell is placed in into 37 DEG C, is incubated 24h extremely in 5%CO2 incubators
It is adherent, with PBS rinse 3 times, above-mentioned different polyplex are put in different holes, and set blank control group and
Lipofectamine-2000 positive controls.24h is incubated in cell culture incubator, changes fresh culture medium, continues to cultivate
48h。
The different groups of toxicity to cell is determined using mtt assay, as a result shows, applies two kinds of different N/P polyplex
Cells survival rate illustrates that cytotoxicity is smaller all more than 85%, sees Fig. 7.
Embodiment 15：
Balb/c mouse tail vein injections Ace-PEG-PAsp (TEP)-chole30mg/kg, successive administration 7d, administration knot
24h, 72h after beam, take off neck and put to death, take brain, cerebellum, the heart, liver, spleen, lung, kidney, after CD68 immunohistochemical stainings, microscope is seen
Examine and take pictures.
As a result it is shown, in heart, liver and kidney, there is a small amount of macrophage to produce.In the case where there is inflammation, meeting
Substantial amounts of glomerulus mesangial cell is produced, makes the phagocytosis and scavenging action enhancing of glomerulus.But this reaction occurs over just
24h after administration.Polymeric material, by 72h, is slowly reduced until with control group without notable there may be transient toxicity
Difference, see Fig. 8.
Embodiment 16：
N/P16 load Cy3-pDNA polyplex is prepared as described in Example 4.BEnd.3 cells are with 5.0 × 105Carefully
Born of the same parents/hole is inoculated in confocal special culture dishs, is incubated 24h to cell attachment, culture medium is sucked, by answering containing 2 μ g pDNA
Compound is incorporated in culture dish, and addition 1ml is free of the culture medium of serum, is put into cell culture incubator and is taken out after lucifuge culture 2h,
Intake is terminated with ice-cold PBS, is rinsed 3 times, then 4% paraformaldehyde fixation 20mim, PBS washing 3 times, it is thin to add DiO
After birth dyestuff, 10min being incubated at room temperature, PBS is rinsed 3 times, DAPI solution progress nuclear targeting, after anti-fluorescence quenching mounting,
It is placed under laser confocal microscope and observes.
As a result showing, polyplex-RVG29 groups and polyplex-Tet1/RVG29 groups show most strong fluorescence intensity,
See Fig. 9.
Embodiment 17：
Weigh 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, are dissolved in 5ml carbonate buffers respectively
Liquid (pH=9.0), 500 μ g water-soluble fluorescein isothiocynate (being purchased from Sigma-Aldrich companies) is added into above-mentioned carbonic acid
In salt buffer, lucifuge stirring 12h, deionized water dialysis, the water-soluble fluorescein isothiocynate that dissociates is removed.
Method according to embodiment 4 and embodiment 5 prepares N/P16 polyplex.Take the logarithm the phase growth bEnd.3 it is thin
Born of the same parents, cell counting count board counts, with 1.0 × 106Individual/hole is inoculated in confocal special culture dishs, is incubated overnight to cell and is pasted
Wall.Compound containing 2 μ g pDNA is added in culture dish, addition 1ml is free of the culture medium of serum, is put into cell culture incubator
Being taken out after lucifuge culture 2h, intake, trypsin digestion cell are terminated with ice-cold PBS, the culture medium containing serum terminates digestion,
1000r centrifuges 3min, and PBS rinsings, then plus 500 μ L PBS are resuspended, flow cytomery.
The uptake ratio of statistical analysis, Tet1/RVG29-polyplex and RVG29-polyplex in cell with
Polyplex, Tet1 polyplex are compared, and have significant difference, and the RVG29-polyplex intakes of wherein cell pair are
2.8 times of polyplex, after bEnd.3 cells Tet1/RVG29-polyplex is incubated, compared with polyplex, fluorescence intensity increases
1.4 times are added.Aggregate amount in cell is fewer than the polyplex that RVG29 is modified, and sees Figure 10.
Embodiment 18：
By b.End3 cells with 5 × 105Individual/hole be inoculated in Tranwell (12 orifice plates, 3 μm, inner membrance 1.12cm2) upper strata, and
0.5mL nutrient solutions are added, while lower floor adds 1.5mL nutrient solutions, changes fresh medium after 2d, continues to cultivate, about cultivate 5
~7d.Across the endothelial cell membrane resistance (transendothelial electrical resistance, TEER) of measure treats it daily
After stable, monofilm is prompted to be formed.FITC polyplex will be marked respectively, is placed in Tranwell upper strata, and is added
Add culture medium to 500 μ L.
After 1 after administration, 2,4,6,8h, 500 μ L are sampled, are placed in 4 DEG C of refrigerators, it is to be measured (to select fluorescence spectrophotometer light
Degree meter measure), and add 500 μ L fresh cultures.The measurement result of sample is shown, after administration after 2h, four kinds of compounds it is saturating
It is basically identical to cross efficiency, after 4h is administered, the permeability of the polypex and Tet1-polyplex groups without any modification is basic
No longer raise, and RVG29-polyplex and Tet1/RVG29-polyplex permeability has and significantly raised, and sees Figure 11.
Embodiment 19：
Method according to embodiment 6 and embodiment 7 prepares N/P16 polyplex, Tet1-polyplex, RVG29-
polyplex、Tet1/RVG29-polyplex.Neuro-2a cells are with 5.0 × 104Individual/hole is inoculated in 24 orifice plates, is incubated
Night to cell attachment, sucks culture medium, the above-mentioned compound for containing 1 μ g pDNA is incorporated in culture dish, addition 1mL is free of FBS
Culture medium, be put into 37 DEG C, 5%CO2Cell culture incubator in continue cultivate 48h, during which change culture medium.After culture terminates,
Culture medium is discarded, PBS is rinsed three times, and the paraformaldehyde for then adding 4% fixes 20mim, is placed in fluorescence microscopy Microscopic observation.
As a result show, blank polyplex fluorescence intensity is weaker, and Tet1-polyplex groups and RVG29-polyplex
The fluorescence intensity of group is all stronger, is virtually free from obvious difference, double ligand modified groups shown in cell it is most strong
Fluorescence intensity, illustrate it is double it is ligand modified after be advantageous to swallow born of the same parents in particle, have higher transfection efficiency, see Figure 12.
Embodiment 20：
N/P16 polyplex, Tet1-polyplex, RVG29- is prepared by the method for embodiment 6 and embodiment 7
polyplex、Tet1/RVG29-polyplex.Neuro-2a cells are with 1.0 × 104Individual/hole is inoculated in 96 orifice plates, is incubated
Night is to cell attachment.The above-mentioned compound for containing 0.5 μ g pDNA is incorporated in culture dish, 1mL culture mediums is added, is put into 37 DEG C,
5%CO2Cell culture incubator in continue cultivate 48h, during which change culture medium.After culture terminates, CCLR cell lysis,
10000r centrifuging and taking supernatants, determined using luciferase detecting system, BCA methods measure protein content.
As a result show, blank polyplex fluorescence intensity is weaker, and Tet1-polyplex groups are that unmodified group of fluorescence is strong
3.3 times of degree, double ligand modified groups show most strong fluorescence intensity in cell, are for 4.6 times of modification group, this is also with determining
Property detection result it is consistent, see Figure 13.
Embodiment 21：
Using luciferase reporter plasmid pGL3 as fluorescence probe.Prepare N/P16's by embodiment 5 respectively
polyplex、Tet1-polyplex、RVG29-polyplex、Tet1/RVG29-polyplex.Balb/c male mices are through tail
Intravenously administrable, dosage are pDNA50 μ g.After 48h is administered, takes off neck and put to death, take brain tissue respectively on ice, residual is rinsed out with ice PBS
Blood, take portion of tissue to add lysate CCLR, mortar grinder, 10000r centrifuging and taking supernatants are detected using luciferase and are
Unified test is determined, BCA methods measure protein content.
As a result shown, the intracerebral uciferase activity in Tet1/RVG29-polyplex administration groups is significantly higher than
Polyplex groups, are shown in Figure 14.
Embodiment 22：
The particle of different load DiR fluorescent dyes is prepared according to embodiment 8, nude mice tail vein is injected containing DiR (0.5mg/ respectively
Kg different composite thing particle), chloral hydrate anesthesia after certain time, is observed and is taken pictures using small animal living body imaging system,
As a result show, it is most strong in the fluorescence of nude mice brain through double ligand modified compounds, and the fluorescence intensity of liver region relative to
Other groups weak, illustrate through it is double it is ligand modified after be advantageous to particle and be enriched in brain, and reduce the aggregation of other non-target sites, see
Figure 15.
Embodiment 23：
Take the logarithm phase Neuro-2a cell, cell counting count board counts, with 5 × 105Individual/hole cell is inoculated in 6 orifice plates, cell
Incubator is incubated overnight.Discard culture medium, respectively apply polyplex, polyplex-Tet1, polyplex-RVG29,
Polyplex-Tet1/RVG29, using blanc cell as control, continue to cultivate 48h.Then, the cell cracking containing PMSF is added
Liquid, 30min being cracked on ice, collecting albumen, 10000r centrifuges 10min under the conditions of 4 DEG C, takes supernatant.Part supernatant is taken to be used for
Determining the protein quantity, another part add protein loading buffer boiling water baths denaturation 5min.30 μ g albumen are taken, according to
Western blot method is detected.
Testing result shows that polyplex groups approach with blank group protein level, polyplex-Tet1 groups and
Polyplex-RVG29 protein level is close, but has all declined compared with blank group, polyplex-Tet1/RVG29 groups
Protein level is minimum, sees Figure 16.
Embodiment 24：
Opened when choosing double transgenic APP/PS1 mouse (being purchased from Beijing Medical Li Hao bio tech ltd) Beijing 6 months
Begin to be administered.Tail vein administration different composite thing (containing 100 μ g recombinant plasmids), using physiological saline group and non-transgenic mouse as control,
It is administered every other week, successive administration 4 times, one week after administration, carries out water maze Behavioral assessment.
Navigation study experimental result shows that mouse is gradually shortened with the increase for training number of days, incubation period.Train the 4th day
When, compared with physiological saline group, incubation period substantially shortens administration polyplex-Tet1/RVG29 groups, has significant difference, and
Polyplex-Tet1/RVG29. group and the incubation period also decrease to some degree of polyplex group mouse, but with
Polyplex-Tet1/RVG29 groups are compared, and incubation period is longer and has significant difference, sees Figure 17.
After training four days, the 5th day explorative experiment carried out, as a result show, administration group mouse is in the time shared by third quadrant
Ratio all has a certain degree of improvement compared with physiological saline group, but without administration polyplex-RVG29, polyplex-Tet1/
The time scale of RVG29 group mouse quadrant where platform is high, wherein polyplex-Tet1/RVG29 groups proportion highest.
Illustrate, after polyplex-Tet1/RVG29 is administered, mouse memory power has a certain degree of improvement, sees Figure 18.
The preferred embodiment to the invention is illustrated above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit
Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (4)
1. a kind of Alzheimer disease target gene of bifunctional peptide modification, which is passed, releases compound, it is characterised in that this pass release it is compound
Thing releases carrier and electronegative gene forms by passing：
Carrier is released in described passing, including the cationic polymer of Pegylation, cationic polymer end group connection RVG29 and
Tet1；
Described RVG29, its sequence such as SEQ ID NO：Shown in 1；
Described Tet1, its sequence such as SEQ ID NO：Shown in 2；
Described cationic polymer, for the poly-aspartate modified through tetren；
Described electronegative gene is selected from medicative interference sequence, is implemented in described cationic polymer
PEG ends.
2. a kind of Alzheimer disease target gene of bifunctional peptide modification according to claim 1, which is passed, releases compound, its
It is characterised by, described electronegative gene is BACE1 interference sequences, its sequence such as SEQ ID NO：Shown in 3.
3. a kind of Alzheimer disease target gene of bifunctional peptide modification as claimed in claim 1 or 2, which is passed, releases compound
Preparation method, it is characterised in that this method comprises the following steps：
A, the preparation of cationic polymer carrier
By aspartic acid benzyl ester and triphosgene according to molecular proportion 1:1~5 is dissolved in tetrahydrofuran, is heated to 50~80 DEG C, magnetic force
Stirring n-hexane precipitation, obtains aspartic acid benzyl ester carboxylic acid anhydrides to dissolving；Contain the PEG derivatives and aspartic acid of amino in end
Benzyl ester carboxylic acid anhydrides, is dissolved in CH2Cl2, 25~50 DEG C are reacted 24~72h, and ether precipitates to obtain polyethylene glycol poly-aspartate PEG-
PBLA；
Using the acetal of PEG ends in cationic polymer, it is dehydrated in acetate buffer solution and forms aldehyde-base, with cysteine
In sulfydryl pass through covalent attachment；
B, the preparation for releasing compound is passed
The cationic polymer carrier that step A is prepared is dissolved in pH 7.4 PBS with electronegative gene, in equal volume
Vortex mixed 30s, form the release system of passing of 80~300 nanosizeds, wherein cationic polymer carrier and electronegative gene
N/P ratios be 2~16.
4. a kind of Alzheimer disease target gene of bifunctional peptide modification as described in claim 1 or 2, which is passed, releases compound
Preparing the application in treating Alzheimer disease drugs.
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| Multifunctional polyion complex micelle featuring enhanced stability, targetability, and endosome escapability for systemic siRNA delivery to subcutaneous model of lung cancer;Hyun Jin Kim et al;《Drug Deliv. and Transl. Res.》;20130918;第4卷(第1期);摘要、第53页左栏第2-3段、第54页左栏第3段至右栏第3段、第57页左栏第3段至右栏第1段、图1、图3 * |
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