CN104830903B - A kind of transgene carrier and its application based on polypeptide K12 - Google Patents
A kind of transgene carrier and its application based on polypeptide K12 Download PDFInfo
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Abstract
The genophore modified the present invention relates to a kind of polypeptide K12 and by K12, the polypeptide K12 sequences are：Lys‑Lys‑Lys‑Arg‑Lys‑Cys‑Gly‑Asn‑Lys‑Arg‑Thr‑Arg.First PEI is connected from poloxamer188 (P407), obtain the HMW PEI derivatives of multi-branched shape or network structure, select the small peptides of tLyP 1, it is connected with nuclear localization signal peptide NLS, synthesis is with the polypeptide K12 for targeting NRP and raising nuclear delivery ability, K12 and PEI derivatives are coupled using crosslinking technological, new non-viral gene vector P407 PEI K12 are constructed.Cytotoxicity and transfection experiment result show that not only toxicity is low by the new non-viral gene vector system P407 PEI K12 that the present invention is provided, and with stronger targeting, cell and internal transfection are superior to control group.
Description
Technical field
It is that the PEI derivatives P407-PEI of polypeptide K12 modifications turns base specifically the present invention relates to pharmaceutical technology field
Because of carrier and its application.
Background technology
Gene therapy is the novel method for the treatment of set up in recent years on technique for gene engineering and molecular genetics principle, because
The Basic of Biology that tumour betides development is gene mutation, so gene therapy turns into the most desired neck of work(gram tumour
Domain.Gene therapy has three important steps, i.e. target gene, transgene carrier and target cell.It is that gene is controlled that gene, which pours into system,
The core technology for the treatment of, the biggest problem faced at this stage is not yet to find preferable genophore.The carrier bag applied at present
Include viral vector and the major class of non-virus carrier two.Viral vector transfection efficiency is higher but has that carrying capacity is low, have potential safety
The problems such as threat, and non-virus carrier is quickly grown in recent years, especially cationic polymer.
Polyethyleneimine (polyethylenimine PEI) is that the most commonly used cationic polymer of Recent study is non-
Viral genetic vector, rich in cation, with powerful buffer capacity, there is the ability of stronger combination DNA and adherent cell.So
And, polyethyleneimine is used in the presence of two outstanding problems as genophore：There is lance in the firstth, transfection efficiency and cytotoxicity
Shield.Though small molecule PEI cytotoxicities are low, easily dissociated with DNA under physiological ion concentration, cause transfection poor；
Though molecular weight more than 20kd PEI have comparatively ideal transfection efficiency, due to PEI surfaces rich in positive charge and in vivo not
Degradability, causes HMW PEI to show stronger cytotoxicity.Second, polyethyleneimine targeting is poor：It is to utilize
The positive charge of itself institute's band, is combined with the negatively charged acceptor of cell surface by electrostatic interaction, so the selection of cell is special
The opposite sex is poor, and solve targeting sex chromosome mosaicism turns into the problem of being paid close attention to the most in non-virus carrier.In addition, polymer/DNA compounds are
Need to enter in nucleus, and dissociate to play a role by RNA.Therefore, cationic compound target in vivo how is improved
Can the nucleus delivery capability of cell turns into it be applied to one of clinical key.Poloxamer poloxame is a class by gathering
The PEO-PPO-PEO type nonionic triblock copolymers of oxygen ethene (PEO), polyoxypropylene (PPO) composition.Poloxamer188 (letter
Claim P407) it is made up of about 70% ethylene oxide and 30% propylene oxide, mean molecule quantity 11500 is Families of poloxamers
It is middle research the most extensively, be also a kind of most commonly used polymer.
Nuclear localization signal NLS is the architecture basics that functional protein enters nucleus in core, is that some albumen of mediation enter core
One section of abundant and necessary information segment, macromolecular substances can be by NLS mediation through nuclear Pore Complex active transport to cell
In core, the NLS of wherein most study is the T antigens from SV40, and major function sequence is KKKRKV.Can be by PEI's by NLS
Compound improves the transfection efficiency of compound in nuclear translocation to core.NPR is glioma tumor cell membrane and new green blood
Transmembrane protein present on endothelial cell film, very crucial effect is played in terms of the biology of tumour, is had proven at present
There is high expression in NRP acceptors, can be as pernicious swollen on the cells such as cancer of pancreas, stomach cancer, colon cancer, melanoma, oophoroma
One of mark of knurl.TLyP-1 is newfound target polypeptide, and amino acid sequence is CGNKRTR, can it is specific targeting and with
NRPs acceptors are efficiently combined, while can produce across Vascular permeability and tumour membrane penetration effect, have specificity and height affine to NPR acceptors
Property.Research shows that tLyP-1 selective can go back to the nest and tumour target position, is efficiently seeped into outside blood vessel, soaks to uniformity from intravascular
Moisten into mesenchyma stroma of tumors, and can enter through the cell membrane of tumour cell in nucleus.
Therefore, in theory can by tLyP-1 (sequence is CGNKRTR) introduce NLS sequences (KKKRK), synthesis containing tLyP-1,
NLS polypeptide tLyP-1-1-NLS (sequence is KKKRKCGNKRTR, is named as K12), for modifying P407-PEI derivatives, increases
It is in vivo to tumor cells selectivity by force, and improves nuclear delivery ability, so as to increase DNA internal transfection efficiency.
Chinese patent literature CN201110372844.5, discloses genophore and its preparation of a kind of bifunctional peptide modification
Methods and applications, the genophore of the invention is the polyethyleneimine by bifunctional peptide (RGDCTAT) and pluronic P123 modifications
Amine derivative coupling.Chinese patent literature CN201410766122.1 discloses a kind of base modified by Functional Polypeptides R11
Because of carrier and its preparation method and application, the genophore of the invention is by bifunctional peptide (RGDCNLS) and F68 P123
The polyethylenimine derivates coupling of modification is formed.But poloxamer188-the polyethylene modified on polypeptide tLyP-1-NLS
Imines, yet there are no report.
The content of the invention
The purpose of the present invention is that there is provided a kind of polypeptide for deficiency of the prior art.
Another purpose of the present invention is to provide a kind of genophore.
Another purpose of the present invention is to provide the preparation method of said gene carrier.
Fourth object of the present invention is to provide a kind of gene composite.
The 5th purpose of the present invention is to provide aforementioned polypeptides and the purposes of genophore.
To realize above-mentioned first purpose, the present invention is adopted the technical scheme that：
A kind of polypeptide, described polypeptid acid sequence is as shown in SEQ ID NO.1.
The polypeptide that the present invention is provided targets NRP transmembrane protein acceptors by nuclear localization signal peptide NLS (KKKRK) with having
TLyP-1 is constituted, and peptide sequence is KKKRKCGNKRTR.
To realize above-mentioned second purpose, the present invention is adopted the technical scheme that：
A kind of genophore, it is formed by polypeptide as described above and polyethylenimine derivates coupling, and described is poly-
Ethylenimine derivatives are the polyethyleneimine i.e. P407- polyethyleneimines of poloxamer188 modification, described poloxamer
407 be 1 with the mol ratio of polyethyleneimine:1-20；The molecular weight ranges of described polyethyleneimine are 600-70000Da.
Described poloxamer188 is 1 with polyethyleneimine mol ratio:1-10.
Described poloxamer188 is 1 with polyethyleneimine mol ratio:10.
Described polypeptide and the mol ratio of polyethylenimine derivates are 1-20:1；Preferably 2:1.
To realize above-mentioned 3rd purpose, the present invention is adopted the technical scheme that：
As above the preparation method of any described genophore, comprises the following steps：
(a) polypeptide as described above is synthesized；
(b) after poloxamer188 water removal, the triphosgene of twice of mole is added, N- hydroxyls are added after magnetic agitation reaction
Anhydrous triethylamine is added dropwise under succinimide, stirring condition, question response removes solvent afterwards completely, residue obtained to be dissolved in acetic acid
Ethyl ester, centrifuging and taking supernatant, revolving removes the poloxamer188 that must be activated after ethyl acetate, cooling and solidifying；
(c) poloxamer188 of activation is dissolved in anhydrous methylene chloride respectively with polyethyleneimine PEI, by two liquid of gained
Add simultaneously in the liquid of anhydrous methylene chloride bottom, magnetic stirrer over night after inflated with nitrogen saturation is dialysed, and is freezed, is obtained P407-PEI；
(d) surfactant is dissolved in dimethyl sulphoxide solution, polypeptide and step (c) institute prepared by step (a)
The P407-PEI of preparation is dissolved separately in PBS solution, and surfactant solution is added in P407-PEI liquid and obtains Malaysia acyl
The P407-PEI liquid of imidization, the polypeptide solution prepared is added in the P407-PEI liquid of maleimation, 4 DEG C of shakes
Reaction is stayed overnight, and centrifugation is lyophilized, both.
Described surfactant is SMCC, is that a class contains n-hydroxysuccinimide (NHS) active ester and Malaysia acyl
The bifunctional coupling agent of imines.
The present invention connects PEI from poloxamer188 first, obtains the HMW PEI of multi-branched shape or network structure
Derivative, then using transmembrane protein acceptor NRP the characteristics of human tumor cells and tumor neogenetic blood vessels height expression, selection is special
The tLyP-1 small peptides of affine this receptor, are connected with nuclear localization signal peptide NLS, and synthesis, which has, targets NRP and raising nuclear delivery energy
K12 and PEI derivatives, are coupled, so as to build new non-viral gene vector system by the polypeptide K12 of power using crosslinking technological
P407-PEI-K12。
To realize above-mentioned 4th purpose, the present invention is adopted the technical scheme that：
As above the compound that any described genophore is formed with DNA.
Described DNA contains reporter gene, antioncogene and/or cytokine gene.
To realize above-mentioned 5th purpose, the present invention is adopted the technical scheme that：
The application of polypeptide and genophore as described above in the medicine for preparing gene therapy.
The invention has the advantages that：
The present invention connects PEI from poloxamer188 first, obtains the HMW PEI of multi-branched shape or network structure
Derivative, then using transmembrane protein acceptor NRP the characteristics of human tumor cells and tumor neogenetic blood vessels height expression, selection is special
The tLyP-1 small peptides of affine this receptor, are connected with nuclear localization signal peptide NLS, and synthesis, which has, targets NRP and raising nuclear delivery energy
K12 and PEI derivatives, are coupled, so as to build new non-viral gene vector system by the polypeptide K12 of power using crosslinking technological
P407-PEI-K12.Cytotoxicity and transfection experiment result show, the new non-viral gene vector system that the present invention is provided
Not only toxicity is low by P407-PEI-K12, and with stronger targeting, cell and internal transfection are superior to control group.
Brief description of the drawings
Accompanying drawing 1 is P407-PEI-K12 cytotoxicity experiment results.Wherein, 3 pillars of the first cluster are from left to right from left to right
P123-PEI-K12, PEI 2KDa, PEI 25KDa are represented respectively.
Accompanying drawing 2 is P407-PEI-K12 in-vitro transfection experimental results.Wherein, 3 pillars of the first cluster are from left to right from left to right
P123-PEI-K12, PEI 25KDa, PEI 2KDa are represented respectively.
Embodiment
The embodiment that the present invention is provided is elaborated below in conjunction with the accompanying drawings, but embodiments of the invention are not only
It is limited to this.
Herein, SMCC is that a class contains the difunctional of n-hydroxysuccinimide (NHS) active ester and maleimide
Coupling agent, can by the compound respectively containing sulfydryl with amino it is bonded together with, its molecular structure is as follows.
The P407-PEI of the polypeptide K12 of embodiment 1 modifications preparation and functional verification (one)
1st, P407-PEI preparation
The poloxamer188 (P407,0.6mmol) after appropriate water removal is weighed, dry toluene and anhydrous methylene chloride is dissolved in
Mixed solution in, add 1.2mmol triphosgene, at room temperature magnetic agitation reaction stay overnight.Vacuum revolving removes solvent, then uses
Appropriate dry toluene and anhydrous methylene chloride dissolving, is then added under 2.0mmol n-hydroxysuccinimides, magnetic agitation, will
2.0mmol anhydrous triethylamines are added dropwise in reaction solution, continue stirring reaction about 4h.After question response is complete, by reacting liquid filtering
It is residue obtained to be dissolved in ethyl acetate and vacuum revolving removes solvent again, take supernatant after high speed centrifugation, rotary evaporation,
Ethyl acetate is flung to, reactant, the P407 after must both activating is cooled and solidified.It is dissolved in after PEI 2K (0.20g, 0.10mmol) water removals
In 10mL anhydrous methylene chlorides, the P407 (0.01mmol) after activation is dissolved in anhydrous methylene chloride, while above-mentioned two liquid is delayed
In slow instillation 10mL anhydrous methylene chlorides bottom liquid, room temperature magnetic stirrer over night after nitrogen saturation is dialysed, and is freezed, is obtained P407-
PEI。
2nd, polypeptide K12 synthesis Lys Lys-Lys-Arg-Lys-Cys-Gly-Asn-Lys-Arg-Thr-Arg
According to tLyP-1 amino acid sequence：Cys-Gly-Asn-Lys-Arg-Thr-Arg, NLS amino acid sequence：
Lys-Lys-Lys-Arg-Lys, by Shanghai gill, biochemical Co., Ltd uses Solid phase synthesis polypeptide tLyP-1-NLS, its amino
Acid sequence is:Lys-Lys-Lys-Arg-Lys-Cys-Gly-Asn-Lys-Arg-Thr-Arg (SEQ ID NO.1) is simultaneously named as
K12。
3rd, modifications of the polypeptide K12 to P407-PEI
SMCC is dissolved in DMSO solution, and NLS-tLyP-1 (polypeptide K12) and P407-PEI is dissolved separately in PBS solution
In, by SMCC liquid in molar ratio 2:1 is added dropwise in PEI liquid, stirring while adding, and 30mim is reacted at room temperature, passes through gel color
Compose post and remove uncombined SMCC, produce the P407-PEI liquid (maleimided P407-PEI) of maleimation.By K12
Liquid in molar ratio 2:1 is added in maleimided P407-PEI liquid, and 4 DEG C are shaken reaction and stayed overnight.Then with ultra-filtration centrifuge tube from
The heart, freezes, produces P407-PEI-K12.
4th, P407-PEI-K12 cell toxicity test
B16 cells are inoculated on 96 orifice plates, 48h is cultivated, cell confluency degree is reached 70%-80%.Before experiment, suck
Culture medium, various concentrations cationic polymer (4,6,8,16,24,32 μ g/mL, serum-free 1640 is used as solvent) is added per hole,
Continue to cultivate 72h, mtt assay detects cytotoxicity, and as can be seen from Figure 1, unmodified PEI 25K cytotoxicities are stronger, and P407-
PEI-K12 is almost non-toxic, it is ensured that P407-PEI-K12 as genophore feasibility.
The cell toxicity test result of table 1
5th, P407-PEI-K12 in-vitro transfection experiment
B16 cells are inoculated on 24 orifice plates, 48h is cultivated, cell confluency degree is reached 70%-80%.By P407-PEI-
K12 and luciferase reporter gene in mass ratio 5:1、10:1、20:1 and 30:1 is made compound, adds on 24 orifice plates, training
4h is supported, serum-containing media is replaced and continues to cultivate 48h, detect luciferin expression of enzymes, as can be seen from Figure 2, P407-PEI-K12
Show very strong transfection efficiency, significantly larger than control group PEI 2K, also the optimum expression than PEI 25K is high 1000 times, explanation
P407-PEI-K12, which can be very good to target cell, to be transfected.
The in-vitro transfection experimental result of table 2
6th, P407-PEI-K12 internal transfection experiment
Selection ICR experimental mouses set up B16 murine melanoma transplantation models, and experimental animal is divided into 2 groups：P407-
PEI-K12, w/w=30；P123-PEI-R11 (is formed with F68 P123- polyethyleneimine amine couplings by polypeptide R11, synthesized
Method is referring to patent 201410766122.1), w/w=30.It is report base with plasmid PGL3-Control (being purchased from Promega)
Cause, evaluates the organ luciferase expression situation such as mouse core, liver, spleen, lung, kidney after intravenously administrable 24h.Wherein, w/w=30 is represented
The mass ratio of cationic polymer and luciferase reporter gene is 30, as a result as shown in table 3.From table 3 it can be seen that
P407-PEI-K12 shows very strong transfection efficiency, and the expression intensity of luciferase is significantly larger than control group P123-PEI-
R11, is 15 times of P123-PEI-R11 expression intensities respectively at the heart, liver, spleen, lung, kidney.
Transfection efficiency experimental result in the body of table 3
The P407-PEI of the polypeptide K12 of embodiment 2 modifications preparation and functional verification (two)
P407-PEI-K12 preparation and in-vitro transfection experimental procedure be the same as Example 1, difference is：In the present embodiment
PEI molecular weight is 70KDa, and it is 1 to prepare P407 used during P407-PEI with PEI mol ratios:1, during synthesis P407-PEI-K12
Polypeptide K12 and P407-PEI used mol ratio is 5:1.Internal transfection experiment result shows：P407-PEI-K12 fluorescence is mould
The expression intensity of element is significantly larger than P123-PEI-R11, is P123-PEI-R11 expression intensities respectively at the heart, liver, spleen, lung, kidney
12,13,12,11,11 times.
The P407-PEI of the polypeptide K12 of embodiment 3 modifications preparation and functional verification (three)
P407-PEI-K12 preparation and in-vitro transfection experimental procedure be the same as Example 1, difference is：In the present embodiment
PEI molecular weight is 4KDa, and it is 1 to prepare P407 used during P407-PEI with PEI mol ratios:5, synthesis P407-PEI-K12 when institute
Polypeptide K12 and P407-PEI mol ratio are 1:1.Internal transfection experiment result shows：P407-PEI-K12 fluorescence mycins
Expression intensity be significantly larger than P123-PEI-R11, be P123-PEI-R11 expression intensities respectively at the heart, liver, spleen, lung, kidney
13rd, 14,11,12,12 times.
The P407-PEI of the polypeptide K12 of embodiment 4 modifications preparation and functional verification (four)
P407-PEI-K12 preparation and in-vitro transfection experimental procedure be the same as Example 1, difference is：In the present embodiment
PEI molecular weight is 600Da, and it is 1 to prepare P407 used during P407-PEI with PEI mol ratios:20, during synthesis P407-PEI-K12
Polypeptide K12 and P407-PEI used mol ratio is 20:1.Internal transfection experiment result shows：P407-PEI-K12 fluorescence is mould
The expression intensity of element is significantly larger than P123-PEI-R11, is P123-PEI-R11 expression intensities respectively at the heart, liver, spleen, lung, kidney
10,11,13,11,12 times.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, on the premise of the inventive method is not departed from, can also make some improvement and supplement, and these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (5)
1. a kind of genophore, it is characterised in that it is formed by polypeptide and polyethylenimine derivates coupling, described poly- second
Alkene imine derivative is the polyethyleneimine i.e. P407- polyethyleneimines of poloxamer188 modification, described poloxamer188
Mol ratio with polyethyleneimine is 1:10；The molecular weight of described polyethyleneimine is 2KDa, the polypeptid acid sequence
As shown in SEQ ID NO.1, described polypeptide and the mol ratio of polyethylenimine derivates are 2:1.
2. the preparation method of the genophore described in claim 1, it is characterised in that comprise the following steps：
(a) polypeptide as claimed in claim 1 is synthesized；
(b) after poloxamer188 water removal, the triphosgene of twice of mole is added, N- hydroxysuccinimidyls are added after magnetic agitation reaction
Anhydrous triethylamine is added dropwise under acid imide, stirring condition, question response removes solvent afterwards completely, residue obtained to be dissolved in acetic acid second
Ester, centrifuging and taking supernatant, revolving removes the poloxamer188 that must be activated after ethyl acetate, cooling and solidifying；
(c) poloxamer188 of activation is dissolved in anhydrous methylene chloride respectively with polyethyleneimine PEI, by two liquid of gained simultaneously
Add in the liquid of anhydrous methylene chloride bottom, magnetic stirrer over night after inflated with nitrogen saturation is dialysed, and is freezed, is obtained P407-PEI；
(d) surfactant is dissolved in dimethyl sulphoxide solution, the polypeptide and step (c) prepared by step (a) are prepared
P407-PEI be dissolved separately in PBS solution, surfactant solution is added in P407-PEI liquid and obtains maleimide
The P407-PEI liquid of change, the polypeptide solution prepared is added in the P407-PEI liquid of maleimation, and 4 DEG C are shaken reaction
Overnight, centrifuge and freeze, produce.
3. genophore and the compound of DNA formation described in claim 1.
4. compound according to claim 3, it is characterised in that described DNA containing reporter gene, antioncogene and/or
Cytokine gene.
5. application of the genophore in the medicine for preparing gene therapy described in claim 1.
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| EP3225246A1 (en) * | 2016-03-31 | 2017-10-04 | Klinikum der Universität München | Synthetic compound for improving efficiency of transfection |
| CN106480092B (en) * | 2016-10-20 | 2019-08-27 | 上海海洋大学 | A kind of PEGylated Fe based on Functional Polypeptides R9 modification3+/ PEI genophore and its preparation method and application |
| CN106801068B (en) * | 2017-01-12 | 2020-03-17 | 西安交通大学 | Non-viral gene vector capable of degrading poly-citrate through autofluorescence and preparation method thereof |
| CN109486827A (en) * | 2018-12-04 | 2019-03-19 | 南京林业大学 | A kind of apoferritin nanocages and preparation method thereof of tumor-homing cell-penetrating peptide tLyP-1 modification |
| CN110004180B (en) * | 2019-04-16 | 2023-08-18 | 上海海洋大学 | Gene vector modified by functional peptide A25 and preparation method and application thereof |
| CN111154002B (en) * | 2020-01-10 | 2022-11-22 | 上海海洋大学 | Bifunctional peptide K14, gene vector and co-drug delivery system |
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| CN103665169A (en) * | 2013-11-21 | 2014-03-26 | 上海海洋大学 | Three-function peptide-modified gene carrier as well as preparation method and application thereof |
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