CN109337929A - A kind of preparation method of the non-viral gene vector with core target function - Google Patents
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Abstract
The invention discloses a kind of preparation methods of non-viral gene vector with core target function, comprise the steps of: the synthesis of 1 generation PAMAM primitive D1, the synthesis of 2 generation SS-PAMAM primitive SS-D2, the synthesis of 3 generation SS-PAMAM primitive SS-D3, prepare [N-2- (formoxyl-ethyl)-maleimide]-SS-D3, it is slowly dropped into the PBS buffer solution of core target polypeptide, it is protected from light and is stirred to react for 24 hours, after reaction, reaction solution is transferred in bag filter, dialysis 24 hours, it is freeze-dried to obtain core gene targeting vector, the core gene targeting vector and plasmid or siRNA of synthesis are dissolved in PBS, it places 30 minutes after mixing evenly, form it into stable compound.The main object of the present invention is the genophore for synthesizing a kind of cell nucleus targeting, to solve the problems, such as that non-viral gene vector transfection efficiency is not high.
Description
Technical field
Loaded gene can be targeted and be transported to carefully the present invention relates to genomic medicine controlled-release technology field more particularly to one kind
The non-viral gene vector of karyon targeting further relates to the preparation method of the polymer nano granules with carrier material preparation.
Background technique
Cancer is that worldwide have very high disease incidence and lethality, with to tumor development molecular mechanism
Understanding deepens continuously, it is found that tumour is an extremely complex process, the inhibition of many genes, overexpression or prominent
Become the generation that all can lead to tumour.Therefore the gene therapy of tumour, which has become, captures and cures that tumour most wishes and challenge grinds
Study carefully field.The gene therapy system of tumour leads the gene of gene or therapeutic effect with normal function by specific mode
Enter into tumour target cell, to achieve the purpose that treat tumour.It is effective in addition to finding in the implementation process of therapy of tumor
Therapeutic gene outside, gene can be effectively delivered into tumor tissues, equally be oncotherapy by the carrier of a highly effective and safe
In a very important link.The genophore applied at present includes viral vectors and non-virus carrier two major classes.Due to right
Virus genomic understanding is more clear, and transfection efficiency is high, and viral vectors is widely used in gene therapy research and clinical examination
It tests.However its there are the problem of, can such as induce body to generate immune response to a certain degree, insertion mutation tumorigenesis, intoxicating wind
Danger, bearer capabilities are limited, prepare the disadvantages of multiplicity is not high, so that people will study more and more steering non-virus carriers.It is non-
Viral vectors mediates gene transfer with physico-chemical method, except have no infectiousness, do not limit bearer capabilities, chemical structure it is controllable and
It is easy to outside the advantages that largely preparing, compared with viral vectors, also there is low toxicity, low immune response, entrained target gene is not
The advantages such as host cell gene group are integrated into, therefore, non-virus carrier is quickly grown in recent years.Non-virus carrier mainly includes sun
Ionomer and liposome, in contrast, the advantage based on the non-virus carrier of polymer compared with cationic-liposome are
It is safer, is easy to mass production, and more stable.Till now, the either natural polymer sun of a large amount of synthesis
Ionophore has been investigated as genophore, this includes polyethyleneimine (PEI), chitosan, polyamide-amide dendrimer
(PAMAM) cationic polymers such as.However, polymer cation carrier transfection efficiency is this far away from Viral Delivery Systems
The common fault of class carrier, to find out its cause, being mainly caused by its DNA nucleus transfer efficiency is not high.Nucleus is non-in cell body
A kind of often important organelle is the main place of gene coding and transcription, and the main object of the present invention is to synthesize a kind of cell
Core target to genophore, to solve the problems, such as that non-viral gene vector transfection efficiency is not high.
Summary of the invention
In view of the above-mentioned problems, the purpose of the present invention is develop a kind of system of non-viral gene vector with core target function
Preparation Method.
The purpose of the invention is achieved by the following technical solution:
The synthesis of (1) 1 generation PAMAM primitive (D1): under ice-water bath, ethylenediamine is dissolved in methanol by nitrogen protection, is slowly dripped
Enter in the methanol solution of methyl acrylate.It after being added dropwise to complete, is first stirred at 0 DEG C, then heats to and reaction is stirred at room temperature for 24 hours.
After reaction, rotary evaporation removes unreacted methyl acrylate and methanol, and vacuum drying obtained for 0.5 generation containing alkynyl
PAMAM primitive D 0.5.Under ice-water bath, D 0.5 is dissolved in methanol by nitrogen protection, is slowly dropped into the methanol solution of ethylenediamine.
It after being added dropwise to complete, is first stirred to react at 0 DEG C, reaction is then stirred at room temperature for 24 hours.After reaction, rotary evaporation removes
Unreacted ethylenediamine and methanol, vacuum drying obtain D1.
The synthesis of (2) 2 generation SS-D2: under ice-water bath, D1 is dissolved in methanol by nitrogen protection, is slowly dropped into methyl acrylate
Methanol solution in.After being added dropwise to complete, 1h is stirred, then heats to and reaction is stirred at room temperature for 24 hours.After reaction, rotary evaporation
Unreacted methyl acrylate and methanol are removed, vacuum drying obtains SS-D1.5 primitive.Under ice-water bath, nitrogen protection, by SS-
D1.5 is dissolved in methanol, is slowly dropped into the methanol solution of cystamine.After being added dropwise to complete, under be stirred to react 1h, then at room temperature
It is stirred to react 48h.After reaction, rotary evaporation removes unreacted cystamine and methanol, and vacuum drying is obtained containing disulfide bond
SS-D2。
The synthesis of (3) 3 generation SS-D3: under ice-water bath, SS-D2 is dissolved in methanol by nitrogen protection, is slowly dropped into acrylic acid
In the methanol solution of methyl esters.After being added dropwise to complete, 1h first is stirred at 0 DEG C, then heats to and reaction is stirred at room temperature for 24 hours.Reaction knot
Shu Hou, rotary evaporation remove unreacted methyl acrylate and methanol, and vacuum drying obtains 2.5 generation SS-D2.5 primitives.Ice-water bath
Under, SS-D2.5 primitive is dissolved in methanol, is slowly dropped into the methanol solution of ethylenediamine by nitrogen protection.After 2h is added dropwise to complete,
It is stirred to react 1h, reaction 48h is then stirred at room temperature.After reaction, rotary evaporation removes unreacted ethylenediamine and first
Alcohol, vacuum drying obtain SS-D3.
(4) SS-D3 and triethanolamine are dissolved in methanol, are slowly added dropwise containing [N-2- (formoxyl-ethyl)]-Malaysia acyl
The methanol solution of imines is first stirred to react 1h at 0 DEG C, and reaction is then stirred at room temperature for 24 hours, and after reaction, rotation is steamed
Hair removes methanol, then that obtained product is soluble in water, is dialysed in bag filter for 24 hours, and freeze-drying obtains [N-2-
(formoxyl-ethyl)-maleimide]-SS-D3.
(5) [N-2- (formoxyl-ethyl)-maleimide]-SS-D3 is dissolved in the buffer solution of 7.4 PBS of pH,
Then it is slowly dropped into core target polypeptide PBS buffer solution, is protected from light and is stirred to react for 24 hours.After reaction, reaction solution is transferred to dialysis
In bag, dialyses 24 hours, be freeze-dried to obtain core gene targeting vector.
(6) the core gene targeting vector synthesized in step (5) and plasmid or siRNA are dissolved in PBS, after mixing evenly
It places 30 minutes, forms it into stable compound.
The addition mole of present invention methyl acrylate described in step 1 is equivalent to 4~8 times of ethylenediamine；Preferably
5 times.
The dosage of present invention ethylenediamine described in step 1 is 4~8 times of 0.5 mole of D.
The temperature of present invention reaction described in step 1 is preferably 0~5 DEG C.
The addition mole of methyl acrylate described in step 2 of the present invention is equivalent to 8~12 times of D1；Preferably 10
Times.
The dosage of cystamine described in step 2 of the present invention is 8~12 times of 1.5 mole of D.
The addition mole of methyl acrylate described in step 3 of the present invention is equivalent to 16~32 times of SS-D2；It is preferred that
It is 18 times.
The dosage of ethylenediamine described in step 3 of the present invention is 16~30 times of SS-D2.5 mole.
[N-2- (formoxyl-ethyl)]-maleimide dosage described in step 4 of the present invention is SS-D3 mole
1~2 times.
Bag filter molecule interception described in step 4 of the present invention is 3000~5000.
Core target polypeptide described in step 5 of the present invention is the trans- activating transcription factor of HIV-1.
Plasmid described in step 6 of the present invention can be p53, p16, APC, RB, DCC, and the siRNA can be VEGF-
SiRNA, Bcl-2-siRNA, survivin-siRNA.
Carrier-gene composite nano particle diameter of the preparation method preparation of nano-carrier of the invention is 100~130
Nanometer.
Polymer matrix of the invention is because of nano-carrier: the modification of penetratin takes gene vector material surface
The identification molecule of nucleus, can make more genes as carrier enters nucleus.Core target function is targeted to carrier carefully
Karyon.In addition to this, in the D2 layer formed by cystamine, connected by disulfide bond and D3 layers, disulfide bond can intelligent response it is swollen
Higher glutathione concentrations are in oncocyte to be broken, and then further increase the efficiency that gene enters in nucleus,
Further increase the transfection efficiency of carrier.
Detailed description of the invention
Fig. 1 is the nucleus magnetic hydrogen spectrum figure of Mal-SS-D3 and TAT-SS-D3.
Fig. 2 is TAT-D3/p53 and TAT-SS-D3/p53 compound transmission electron microscope picture.
Fig. 3 is the measurement of carrier/p53 compound Zeta potential.
Fig. 4 is the Laser Scanning Confocal Microscope figure and flow cytometer detection figure of the carrier of FITC label, (1) PAMAM-D3 (2) SS-
PAMAM-D3 (3)TAT-PAMAM-D3 (4)TAT-SS-PAMAM-D3。
Fig. 5 is the Laser Scanning Confocal Microscope figure and transfection efficiency (1) D3 (2) SS-D3 (3) TAT-D3 that carrier transfects peGFP-53
(4)TAT-SS-D3。
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate this hair
It is bright, rather than limit the scope of the invention.Under the premise of without departing substantially from technical solution of the invention, the present invention is made
Those of ordinary skill in the art's any change easy to accomplish fall within scope of the presently claimed invention.
Embodiment 1
The synthesis of (1) 1 generation PAMAM primitive (D1): under ice-water bath, 1g ethylenediamine is dissolved in methanol by nitrogen protection, is delayed
It is slow to instill in the 100mL methanol solution containing 4.48g methyl acrylate.After being added dropwise to complete, first stirs at 0 DEG C, then heat up
It is reacted for 24 hours to being stirred at room temperature.After reaction, rotary evaporation removes unreacted methyl acrylate and methanol, is dried in vacuo
PAMAM primitive D 0.5 to 0.5 generation containing alkynyl.Under ice-water bath, D 0.5 is dissolved in methanol, is slowly dropped into and contains by nitrogen protection
There is the methanol solution of 5g ethylenediamine.It after being added dropwise to complete, is first stirred to react at 0 DEG C, reaction is then stirred at room temperature for 24 hours.Instead
After answering, rotary evaporation removes unreacted ethylenediamine and methanol, and vacuum drying obtains D1.
The synthesis of (2) 2 generation SS-PAMAM primitives (D2): under ice-water bath, 2g D1 is dissolved in methanol by nitrogen protection, is delayed
It is slow to instill in the methanol solution containing 3.2g methyl acrylate.After being added dropwise to complete, 1h is stirred, then heats to and reaction is stirred at room temperature
24h.After reaction, rotary evaporation removes unreacted methyl acrylate and methanol, and vacuum drying obtained for 1.5 generations containing D1.5
Primitive.Under ice-water bath, D1.5 primitive is dissolved in 20mL methanol by nitrogen protection, and it is molten to be slowly dropped into the methanol containing 8.46g cystamine
In liquid.After being added dropwise to complete, under be stirred to react 1 h, be then stirred at room temperature reaction 48h.After reaction, rotary evaporation removes
Unreacted cystamine and methanol, vacuum drying obtain the D2 (SS-D2) containing disulfide bond
The synthesis of (3) 3 generation SS-D3: under ice-water bath, 4g SS-D2 is dissolved in methanol, is slowly dropped into and contains by nitrogen protection
In the methanol solution for having 2.57g methyl acrylate.After being added dropwise to complete, 1h first is stirred at 0 DEG C, then heats to and is stirred at room temperature instead
It should for 24 hours.After reaction, rotary evaporation removes unreacted methyl acrylate and methanol, and vacuum drying obtains 2.5 generation SS-
D2.5 primitive.Under ice-water bath, SS-D2.5 primitive is dissolved in methanol by nitrogen protection, is slowly dropped into containing 2.87g ethylenediamine
In methanol solution.After 2h is added dropwise to complete, it is stirred to react 1h, reaction 48h is then stirred at room temperature.After reaction, rotation is steamed
Hair removes unreacted ethylenediamine and methanol, and vacuum drying obtains SS-D3.
(4) 4g SS-D3 and 0.1g triethanolamine is dissolved in methanol, is slowly added dropwise containing 0.2g [N-2- (formoxyl-
Ethyl)] methanol solution of-maleimide, it is first stirred to react 1h at 0 DEG C, reaction is then stirred at room temperature for 24 hours, reaction
After, rotary evaporation removes methanol, and it is then that obtained product is soluble in water, it is dialysed in bag filter for 24 hours, freezing is dry
It is dry to obtain [N-2- (formoxyl-ethyl)-maleimide]-SS-D3 (Mal-SS-D3).
(5) 4g [N-2- (formoxyl-ethyl)-maleimide]-SS-D3 is dissolved in the buffer solution of pH 7.4PBS,
Then it is slowly dropped into containing 0.05g core target to peptide T AT PBS buffer solution, is protected from light and is stirred to react for 24 hours.After reaction, will
Reaction solution is transferred in bag filter, is dialysed 24 hours, is freeze-dried to obtain core gene targeting vector (TAT-SS-D3).
Embodiment 2
The present embodiment is that the p53 plasmid in embodiment 1 is changed to p16 plasmid, and other methods are the same as embodiment 1.
Embodiment 3
The present embodiment is that the p53 plasmid in embodiment 1 is changed to APC plasmid, and other methods are the same as embodiment 1.
Embodiment 4
The present embodiment is that the p53 plasmid in embodiment 1 is changed to DCC plasmid, and other methods are the same as embodiment 1.
Embodiment 5
The present embodiment is that the p53 plasmid in embodiment 1 is changed to VEGF-siRNA, and other methods are the same as embodiment 1.
Embodiment 6
The present embodiment is that the p53 plasmid in embodiment 1 is changed to Bcl-2-siRNA, and other methods are the same as embodiment 1.
Characterizing method:
Each polymer takes about 10mg to be dissolved in appropriate deuterated solvent, with 1H-NMR 400MHz Bruker NMR spectrum
Instrument is tested, and the variation of 1H chemical shift in post-consumer polymer structure is detected, and test result is shown in Fig. 1.Mal- is set forth in Fig. 1
The nucleus magnetic hydrogen spectrum figure of SS-D3 and TAT-SS-D3, the two compare, Mal-SS-D3 have at 6.65ppm one it is apparent bimodal,
This is the characteristic peak of double bond on maleic acid.And in the nucleus magnetic hydrogen spectrum figure of TAT-SS-D3, due to the sulfydryl and maleic acid on TAT
On double bond have occurred click-reaction, therefore double bond disappears, this illustrates that TAT has had successfully connected on SS-D3.
The pattern of micella is observed with transmission electron microscope (TEM), brief operation is as follows: taking 4 μ L samples (0.25mg/mL) drop pure
It on carbon film copper mesh, dries at room temperature, dyes 3min with 3% acetic acid uranium；Then it is observed, is examined at 120kV with transmission electron microscope
It surveys result and sees Fig. 2.Whether TAT-PAMAM-D3/p53 or TAT-SS-PAMAM-D3/p53 compound is all seen under Electronic Speculum
The pattern of spherical rule is observed.The difference is that TAT-SS-PAMAM-D3/p53, under the action of GSH, partial size is by original
100nm becomes 45nm effect, illustrates that disulfide bond is able to respond GSH, is broken under the action of GSH, and it is smaller to form partial size
Compound, this is more advantageous to compound and enters nucleus across nucleopore and play a role.
With the potential of Zeta potential detection compound, potential value takes the average value of measured value three times, and testing result is shown in Fig. 3.
By testing result it is found that either whether there is or not the effect of GSH, the Zeta potential value of TAT-D3 rises with the rising of N/P value,
Potential value variation is little after N/P value is greater than 10.And under the action of having GSH, even N/P value reaches 40, TAT-SS-D3
Zeta potential value remain as negative value, this is because disulfide bonds cause remaining sulfydryl to keep carrier surface, and sulfydryl is
It is electronegative.TAT-D3 does not have this responsiveness, because TAT-D3 does not have disulfide bond.
The carrier and Laser Scanning Confocal Microscope figure and flow cytometer detection figure after HeLa cell incubation 24 hours that Fig. 4 is FITC label,
TAT-SS-D3 group enters the most of nucleus as shown in Figure 4, flow cytometer detection as the result is shown 66.3% TAT-SS-D3 enter
Arrived in nucleus, and PAMAM-D3, SS-PAMAM-D3 and TAT-PAMAM-D3 only have 10.1%, 23.5% and 40.3% into
Enter into nucleus, illustrates that the modification of TAT can more promote carrier to enter in nucleus plus the connection of disulfide bond.
Fig. 5 is each carrier loaded peGFP-53 plasmid and cell be incubated for jointly 24 hours after the copolymerization coke that shoots it is micro-
Mirror photo and transfection efficiency testing result, from Laser Scanning Confocal Microscope figure it is found that expressing green fluorescent protein is most in TAT-SS-D3 group
More, in contrast, TAT-D3 group ranked second, and only observed a small amount of green fluorescent protein table in the figure of D3 group and SS-D3 group
It reaches.Flow cytometer detection transfect green fluorescent protein the results show that the transfection efficiency of D3 group only has 10.1%, SS-D3's and TAT-D3
Transfection efficiency is 21.5% and 34.3% respectively, and TAT-SS-D3 has reached 48.5%, this result and Laser Scanning Confocal Microscope are seen
It is observing the result is that similar, the modification for showing again TAT can be improved greatly plus the connection design of disulfide bond to be turned
Contaminate efficiency.
After each carrier loaded p53 plasmid, the apoptosis rate testing result that is detected through apoptosis detection reagent.SS-D3,
Caused apoptosis rate is respectively 11.3%, 24.6% and 42.7% after TAT-D3 and TAT-SS-D3 transfection p53, explanation
P53 plasmid can be efficiently transferred in cell by TAT-SS-D3, so as to cause more apoptosis of tumor cells.
Claims (8)
1. the preparation method of the non-viral gene vector with core target function, it is characterised in that: comprised the steps of: for (1) 1 generation
The synthesis of PAMAM primitive D1: under ice-water bath, ethylenediamine is dissolved in methanol by nitrogen protection, is slowly dropped into the first of methyl acrylate
It in alcoholic solution, after being added dropwise to complete, is first stirred at 0 DEG C, then heats to and reaction is stirred at room temperature for 24 hours, after reaction, rotation is steamed
Hair removes unreacted methyl acrylate and methanol, and vacuum drying obtains PAMAM primitive D0.5 of 0.5 generation containing alkynyl, ice-water bath
Under, D0.5 is dissolved in methanol by nitrogen protection, is slowly dropped into the methanol solution of ethylenediamine, after being added dropwise to complete, is first stirred at 0 DEG C
Reaction is mixed, reaction is then stirred at room temperature for 24 hours, after reaction, rotary evaporation removes unreacted ethylenediamine and methanol, very
Sky is dried to obtain D1；
The synthesis of (2) 2 generation SS-PAMAM primitive SS-D2: under ice-water bath, D1 is dissolved in methanol by nitrogen protection, is slowly dropped into third
In the methanol solution of e pioic acid methyl ester, after being added dropwise to complete, 1h is stirred, then heats to and reaction is stirred at room temperature for 24 hours, after reaction,
Rotary evaporation removes unreacted methyl acrylate and methanol, is dried in vacuo and obtains 1.5 generation primitives containing PAMAM, under ice-water bath, nitrogen
D1.5 is dissolved in 20mL methanol, is slowly dropped into the methanol solution of cystamine by gas shielded, after being added dropwise to complete, under be stirred to react 1h,
Then reaction 48h is stirred at room temperature, after reaction, rotary evaporation removes unreacted cystamine and methanol, is dried in vacuo
To the SS-D2 containing disulfide bond；
The synthesis of (3) 3 generation SS-PAMAM primitive SS-D3: under ice-water bath, SS-D2 is dissolved in methanol by nitrogen protection, is slowly dripped
Enter in the methanol solution of methyl acrylate, after being added dropwise to complete, first stirs 1h at 0 DEG C, then heat to and reaction is stirred at room temperature
For 24 hours, after reaction, rotary evaporation removes unreacted methyl acrylate and methanol, and vacuum drying obtains SS-D2.5 primitive,
Under ice-water bath, SS-D2.5 primitive is dissolved in methanol, is slowly dropped into the methanol solution of ethylenediamine by nitrogen protection, and 2h is dripped
Cheng Hou, is stirred to react 1h, and reaction 48h is then stirred at room temperature, and after reaction, rotary evaporation removes unreacted ethylenediamine
With methanol, vacuum drying obtains SS-D3；
(4) SS-D3 and triethanolamine are dissolved in methanol, are slowly added dropwise containing [N-2- (formoxyl-ethyl)]-maleimide
Methanol solution, first 1h is stirred to react at 0 DEG C, be then stirred at room temperature reaction for 24 hours, after reaction, rotary evaporation removes
Methanol is removed, it is then that obtained product is soluble in water, it is dialysed in bag filter for 24 hours, freeze-drying obtains [N-2- (formyl
Base-ethyl)-maleimide]-SS-D3；
(5) [N-2- (formoxyl-ethyl)-maleimide]-SS-D3 is dissolved in the PBS buffer solution that pH is 7.4, then
It is slowly dropped into the PBS buffer solution of core target polypeptide, is protected from light and is stirred to react for 24 hours, after reaction, reaction solution is transferred to bag filter
In, it dialyses 24 hours, is freeze-dried to obtain core gene targeting vector；
(6) the core gene targeting vector synthesized in step (5) and plasmid or siRNA are dissolved in PBS, place 30 after mixing evenly
Minute, form it into stable compound.
2. the preparation method of the non-viral gene vector according to claim 1 with core target function, it is characterised in that:
It ethylenediamine is dissolved in methanol in the step 1, is slowly dropped into the methanol solution of methyl acrylate, wherein methyl acrylate
It is added 4~8 times that mole is equivalent to ethylenediamine；D0.5 is dissolved in methanol in the step 1, is slowly dropped into the first of ethylenediamine
Alcoholic solution ethylenediamine, wherein the dosage of ethylenediamine is 4~8 times of D0.5 mole.
3. the preparation method of the non-viral gene vector according to claim 1 with core target function, it is characterised in that:
The addition mole of methyl acrylate described in step 2 is equivalent to 8~12 times of D1；The dosage of the cystamine is D1.5
8~12 times of mole.
4. the preparation method of the non-viral gene vector according to claim 1 with core target function, it is characterised in that:
The addition mole of methyl acrylate described in step 3 is equivalent to 16~32 times of SS-D2；The dosage of the ethylenediamine
It is 16~30 times of SS-D2.5 mole.
5. the preparation method of the non-viral gene vector according to claim 1 with core target function, it is characterised in that:
[N-2- (formoxyl-ethyl)]-maleimide dosage described in step 4 is 1~2 times of SS-D3 mole.
6. the preparation method of the non-viral gene vector according to claim 1 with core target function, it is characterised in that:
Bag filter molecule interception is 3000~5000 in the step 4 and step 5.
7. the preparation method of the non-viral gene vector according to claim 1 with core target function, it is characterised in that:
Core target polypeptide described in the step 5 is the trans- activating transcription factor of HIV-1.
8. the preparation method of the non-viral gene vector according to claim 1 with core target function, it is characterised in that:
Plasmid described in the step 6 can be p53, p16, APC, RB, DCC, and the siRNA can be VEGF-siRNA, Bcl-2-
SiRNA, survivin-siRNA.
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