CN105273207B - Polycation inclusion compound, preparation method and the usage - Google Patents
Polycation inclusion compound, preparation method and the usage Download PDFInfo
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Abstract
The present invention relates to a kind of aminated poly (glycidyl methacrylate) modification two block Subjective and Objective polycation inclusion compound of adamantane of polymethylacrylic acid diisopropylaminoethyl ethyl ester modification beta cyclodextrin, and its synthetic method, and the cationic micelle and preparation method with dual acid-sensitivity prepared by above-mentioned polycation inclusion compound.Cationic micelle according to the present invention has dual acid-sensitivity, and the hydrophilic outer layer of micella can show electropositive in the case where pH is 7.4 neutral physiological condition with reference to proton, and the hydrophobic inner layer of micella can combine proton dissociation in weak acid environments of the pH less than 6.3.The average hydrodynamic particle diameter of cationic micelle is preferably 20 200 nanometers.The invention further relates to the application that the cationic micelle of the dual acid-sensitivity is used for nucleic acid drug conveying, is mainly used for inverse cancer cell multidrug resistance or suppresses cancer metastasis.
Description
Technical field
The present invention relates to a kind of polycation inclusion compound, and in particular to a kind of polymethylacrylic acid diisopropylaminoethyl ethyl ester is repaiied
Beta-cyclodextrin-aminated poly (glycidyl methacrylate) modification two block Subjective and Objective polycation inclusion compound of adamantane is adornd,
And its synthetic method, and the cationic micelle with dual acid-sensitivity prepared by above-mentioned polycation inclusion compound and preparation side
Method.The invention further relates to the application that the cationic micelle of the dual acid-sensitivity is used for nucleic acid drug conveying, it is mainly used for inverse
Turn cancer cell multidrug resistance or suppress cancer metastasis.
Background technology
Malignant tumour (cancer) has the double high features of low cure rate, recurrence rate and the death rate, it has also become threatens the mankind to be good for
One of major disease of health.In recent years, the ribonucleotide based on Cancer Molecular biology (RNA) interference therapy is cancer
Treatment is filled with new vitality.RNA interference (RNAi) therapy can suppress and cancer drug resistance or the relevant base of transfer in molecular level
Because of expression, so as to reverse cancer drug resistance or suppress cancer metastasis.But at present, the main problem of RNAi therapies is, how will
SiRNA is transferred efficiently into target cell.At present lead viral vector to be applied can make the siRNA of carrying efficiently transfect into
Enter cell, but have the disadvantage that, immune response can be produced to host cell, or even produce potential carcinogenicity.And apply nanometer
The non-virus carrier of technology can improve medicine stability, and increase medicine improves targeted drug delivery energy to the adhesion of biomembrane
Power, controls the release of medicine and changes medicine approach, is had received widespread attention simultaneously in terms for the treatment of transfer and drug resistant cancer
Remarkable effect is achieved, but its problem is its transfection efficiency well below viral vector.Such as Chinese patent
CN102030898A discloses a kind of ABC types amphiphilic biologically degradable polyester triblock copolymer, which can
Ball shaped nano grain is self-assembly of in water, for the nucleic acid drug such as dewatering medicament and deoxyribonucleotide or ribonucleotide
Common conveying.Chinese patent CN1281355A discloses a kind of Biodegradable mixed polymeric micelles based on gene delivery,
Its carrier can control the size and charge density for containing gene nano particle, improve gene delivery efficiency.However, above-mentioned carrier
Lack the selectivity to intraor extracellular difference sour environment, when being conveyed for nucleic acid drug, it is difficult to meet that extracellular stabilization is deposited
, it is intracellular quickly dissociate and discharge contained the requirement of nucleic acid drug, its application range and using effect all receive seriously
Limitation.Therefore, currently still need to constantly look for the new specific nano-carrier that gene delivery can be entered to cell.
The content of the invention
Based on background above, it is an aspect of the invention to provide a kind of polycation inclusion compound, it is poly- methyl-prop
Olefin(e) acid diisopropylaminoethyl ethyl ester modification beta-cyclodextrin-aminated poly (glycidyl methacrylate) modification adamantane, it is described poly-
Cation inclusion compound is as the amine shown in the beta-cyclodextrin and formula B of the polymethylacrylic acid diisopropylaminoethyl ethyl ester modification shown in formula A
The adamantane of base poly (glycidyl methacrylate) modification is formed through Host guest complexation,
Wherein, R1~R7In any 2~5 beRemaining is hydrogen, and n is the integer of 1-100, excellent
Select the integer of 5-30；M is the integer of 1-100, the preferably integer of 1-20；A is the integer of 0-10, the preferably integer of 0-4.
The concrete structure of the polycation inclusion compound is not completely clear and definite, but it was deduced that it can have substantially such as
It is aminated shown in the beta-cyclodextrin and formula B modified as the polymethylacrylic acid diisopropylaminoethyl ethyl ester shown in formula A shown in Fig. 6
The structure as shown in A-B that the adamantane of poly (glycidyl methacrylate) modification is formed through complexing.
Another aspect of the present invention is related to a kind of intermediate for preparing above-mentioned polycation inclusion compound, it is shown in formula A
The beta-cyclodextrin of polymethylacrylic acid diisopropylaminoethyl ethyl ester modification,
Wherein, R1~R7In any 2~5 beRemaining is hydrogen, and m is the integer of 1-100, excellent
Select the integer of 1-20.
The present invention formula A shown in polymethylacrylic acid diisopropylaminoethyl ethyl ester modification beta-cyclodextrin can by β-
Cyclodextrin is modified to obtain.
Another aspect of the present invention is related to another intermediate for preparing above-mentioned polycation inclusion compound, it is shown in formula B
Aminated poly (glycidyl methacrylate) modification adamantane,
Wherein, n is the integer of 1-100, the preferably integer of 5-30；A is the integer of 0-10, the preferably integer of 0-4.
The adamantane of the aminated poly (glycidyl methacrylate) modification shown in formula B can be by right
Adamantane methanol is modified to obtain.
Another aspect of the present invention is to provide a kind of method for preparing above-mentioned polycation inclusion compound, and this method includes mixing
Aminated polymethyl shown in the beta-cyclodextrin and formula B of polymethylacrylic acid diisopropylaminoethyl ethyl ester modification shown in formula A
The step of adamantane of acid glycidyl ester modification is to occur complexing.More specifically, the method can be carried out as follows：
Aminated poly (glycidyl methacrylate) modification adamantane is dissolved in deionized water, it is different to be then added to polymethylacrylic acid two
Complexing occurs in the hydrochloric acid solution of third amino ethyl ester modification beta-cyclodextrin and obtains target product.The method can also be into one
The step of step includes isolating and purifying polycation inclusion compound according to the present invention.Described isolate and purify can use, for example, super
The methods of filter, dialysis, carries out, but not limited to this.For example, can with deionized water dialyse 24 it is small when after freeze-drying obtain basis
The polycation inclusion compound of the present invention.
Another aspect of the present invention is to provide the polymethylacrylic acid diisopropylaminoethyl ethyl ester modification shown in a kind of formula A
The method of beta-cyclodextrin, described method includes following steps：
(1) beta-cyclodextrin is modified to obtain the beta-cyclodextrin initiator for being appropriate for living polymerization；
(2) above-mentioned beta-cyclodextrin initiator is carried out living polymerization with methacrylic acid diisopropylaminoethyl acetate monomer to obtain
The beta-cyclodextrin of poly- methyl diisopropylaminoethyl ethyl ester modification.
In the method for the beta-cyclodextrin of the polymethylacrylic acid diisopropylaminoethyl ethyl ester modification shown in above-mentioned formula A,
The method of the living polymerization is not particularly limited, as long as the target product of the present invention can be obtained, for example, it can be
Active free radical polymerization, such as atom transfer radical polymerization.
In a preferred embodiment, using atom transfer radical polymerization come synthesis type A shown in polymethyl
Sour diisopropylaminoethyl ethyl ester modifies beta-cyclodextrin, comprises the following steps that：
Step a：The synthesis of beta-cyclodextrin initiator
Beta-cyclodextrin and bromo alkyl acyl bromide reagent are obtained into beta-cyclodextrin initiator through esterification；
Wherein, R8~R14In any 2~5 beRemaining is hydrogen；
The reaction can carry out in the presence of organic solvent and organic base, and the organic solvent can be N, N- dimethyl
Acetamide, the organic base can be triethylamine.Preferably, the reactions steps are：Beta-cyclodextrin is dissolved in N, N- dimethyl
So that final concentration of 5-20w/v, anhydrous triethylamine is added into solution in acetamide, 2- bromine isobutyl group acylbromides and its is molten are weighed
2- bromine isobutyl group acylbromides are added drop-wise in beta-cyclodextrin solution under anhydrous methylene chloride, ice bath, it is small that 24 are reacted after completion of dropwise addition
When, with ether precipitation reaction product and with being rotated after acetone rinsing, obtain the beta-cyclodextrin initiator of bromine end-blocking.
In above-mentioned reaction, molar ratio=1 of triethylamine and beta-cyclodextrin:1 to 5:1, preferably 2:1.2- bromine isobutyl group acyls
Molar ratio=2 of bromine and beta-cyclodextrin:1 to 5:1, preferably 4:1.
Step b：Polymethylacrylic acid diisopropylaminoethyl ethyl ester modifies the synthesis of beta-cyclodextrin
The beta-cyclodextrin initiator obtained in step a is mixed with methacrylic acid diisopropylaminoethyl acetate monomer, in original
Polymerization occurs in the presence of sub- transferring free-radical polymerization catalyst ligand and catalyst and obtains polymethylacrylic acid diisopropylaminoethyl second
The beta-cyclodextrin of ester modification, wherein, R8~R14In beGroup be reacted to through above-mentioned
Group, remaining R8~R14Still it is hydrogen.
In above-mentioned reaction, the solvent, atom transfer radical polymerization catalyst and catalyst ligand can be ability
Common solvent, catalyst and catalyst ligand in domain；Wherein, beta-cyclodextrin initiator and methacrylic acid diisopropylaminoethyl
The ratio of acetate monomer is 1:30 to 1:200；And preferably, the solvent is volume ratio=10:1 to 1:10 N, N- dimethyl
Acetamide-isopropyl alcohol mixed solvent；The catalyst ligand is pentamethyl-diethylenetriamine, its dosage is beta-cyclodextrin macromolecular
4 times of molar ratios of initiator；The catalyst is cuprous bromide, its dosage and catalyst ligand equimolar ratio；Required reaction temperature
Spend for the arbitrary temp between 30-90 DEG C, preferably 40-60 DEG C.When reaction time is 1-24 small.
Another aspect of the present invention is to provide a kind of cationic micelle, and the cationic micelle includes poly- according to the present invention
Cation inclusion compound, it is preferably formed by polycation inclusion compound according to the present invention, wherein, the cationic micelle includes amine
Base poly (glycidyl methacrylate) cationization hydrophilic outer layer and polymethylacrylic acid diisopropylaminoethyl ethyl ester are hydrophobic interior
Layer.
The cationic micelle has dual acid-sensitivity, its hydrophilic outer layer can be in the case where pH be 7.4 neutral physiological condition
Electropositive is shown with reference to proton, the hydrophobic inner layer of micella can combine proton dissociation in weak acid environments of the pH less than 6.3.The sun
The average hydrodynamic particle diameter of ionic micelle is preferably 20-200 nanometers.
Another aspect of the present invention is to provide a kind of preparation method of the cationic micelle, it is characterized in that：Will be according to this
The polycation inclusion compound of invention is dissolved in 1-5 times of volume acidic buffer solution, which is instilled 10-50 bodies under ultrasound
In product alkaline buffer, the cationic micelle solution is obtained after purification through ultrafiltration or dialysis, wherein, acidic buffer
PH value is 1.0-5.0, and the pH value of alkaline buffer is 8.0-12.0.
The cationic micelle that the present invention is prepared by polycation inclusion compound by solution self-assembling method has core shell structure,
Including aminated poly (glycidyl methacrylate) cationization hydrophilic outer layer and polymethylacrylic acid diisopropylaminoethyl ethyl ester
Hydrophobic inner layer, the average hydrodynamic particle diameter of cationic micelle is preferably 20-200 nanometers, more preferably 20-60 nanometers.
The hydrophobic stratum nucleare and aquation outer layer of the cationic micelle have difference in functionality, possess corresponding cationic micelle
It is multi-functional.Poly (glycidyl methacrylate) carries level-one amine and secondary amine after ethylenediamine or its oligomer are aminated,
The block has strong proton buffer capacity, positively charged under neutral physiological condition, can adsorb elecrtonegativity core by electrostatic interaction
Sour medicine.Polymethylacrylic acid diisopropylaminoethyl ethyl ester block has sensitivity to acid (pKa 6.3), has under neutral physiological condition
There is hydrophobicity, form the hydrophobic inner core of cationic micelle.Polymethylacrylic acid diisopropylaminoethyl ethyl ester kernel is in cell faintly acid
Dissociated in environment, quick release is contained medicine.The cationic micelle has in pH=4.5-6.0 and 6.0-9.0 Liang Ge areas
Between sensitivity to acid.
It is a further aspect of the present invention to provide a kind of cationic micelle compound for being loaded with nucleic acid, it includes：According to this
The cationic micelle of invention；With the nucleic acid drug being supported on above-mentioned cationic micelle.
In the cationic micelle compound of nucleic acid is loaded with according to the present invention, the weight of the nucleic acid loaded preferably accounts for
The 1.0-20% of the cationic micelle compound weight.
In the cationic micelle compound of nucleic acid is loaded with according to the present invention, the nucleic acid is selected from small interference ribose core
Sour (siRNA) and short heparin ribonucleic acid (shRNA).
The cationic micelle compound for being loaded with nucleic acid according to the present invention has dual acid-sensitivity.
It is another aspect of the present invention to provide a kind of method for the cationic micelle compound for preparing and being loaded with nucleic acid, it is wrapped
Include the step of mixing cationic micelle with nucleic acid.
It is loaded with above-mentioned preparation in the method for cationic micelle compound of nucleic acid, preferably by cationic micelle and nucleic acid
Ratio using weight ratio as 1-30 mixes.A period of time can be stood after mixing, such as about 30 points can be stood at normal temperatures
Clock, but not limited to this.Thus the multilayer cationic micelle with dual acid-sensitivity for obtaining being loaded with nucleic acid drug is compound
Thing；Wherein, the weight of the nucleic acid drug loaded preferably accounts for the 1.0-20% of the cationic micelle compound weight.
Another aspect of the present invention is to provide a kind of use of the polycation inclusion compound as pharmaceutical carrier according to the present invention
On the way.Preferably, the pharmaceutical carrier is used in delivery of nucleic acids.
Another aspect of the present invention is to provide a kind of polycation inclusion compound according to the present invention and is preparing for delivering core
Purposes in the medicine of acid.The medicine can be used for inverse cancer cell multidrug resistance or suppress cancer metastasis.
The nucleic acid is small interference ribonucleic acid (siRNA) or short heparin ribonucleic acid (shRNA).
In application documents, unless otherwise indicated, all number ranges be included therein all numerical value for including and on
Lower limit, and it is included in all subranges in the number range.Such as the integer of 0-10 include 0,1,2,3,4,5,6,7,8,
9th, 10 and all subranges for being made of these numerical value.Other number ranges and so on.
Brief description of the drawings
Fig. 1 is that the methacrylic acid diisopropylaminoethyl ethyl ester synthesized in the embodiment of the present invention 6 modifies beta-cyclodextrin-second two
Amination poly (glycidyl methacrylate) modifies the nuclear magnetic resonance spectroscopy of adamantane polycation inclusion compound, wherein, A is second two
Amine aminated poly (glycidyl methacrylate) modification adamantane, B for polymethylacrylic acid diisopropylaminoethyl ethyl ester modified beta-
Cyclodextrin, and C modify beta-cyclodextrin-ethylenediamine polymethylacrylic acid shrink for polymethylacrylic acid diisopropylaminoethyl ethyl ester
Glyceride modifies adamantane inclusion compound polycation.
Fig. 2 is that the polymethylacrylic acid diisopropylaminoethyl ethyl ester synthesized in the embodiment of the present invention 6 modifies beta-cyclodextrin-second
Two amination poly (glycidyl methacrylate)s modify the acid-base titration curve of adamantane polycation inclusion compound.A is deionization
Water (H2O), B modifies adamantane for ethylenediamine poly (glycidyl methacrylate), and C is polymethylacrylic acid diisopropylaminoethyl
Ethyl ester modifies beta-cyclodextrin, and D and modifies the poly- methyl of beta-cyclodextrin-ethylenediamineization for polymethylacrylic acid diisopropylaminoethyl ethyl ester
Glycidyl acrylate modifies adamantane polycation inclusion compound.
Fig. 3 A are the Hydrodynamic diameter of each material prepared by the present invention, and Fig. 3 B are each material prepared by the present invention
Surface potential；Wherein, a modifies beta-cyclodextrin-ethylenediamine polymethyl for polymethylacrylic acid diisopropylaminoethyl ethyl ester
Acid glycidyl ester modifies the cationic micelle of adamantane polycation inclusion compound, and b is polymethylacrylic acid diisopropylaminoethyl second
The sun of ester modification beta-cyclodextrin-diethyl triamine poly (glycidyl methacrylate) modification adamantane polycation inclusion compound
Ionic micelle, c modify beta-cyclodextrin-triethylene tetramine polymethylacrylic acid contracting for polymethylacrylic acid diisopropylaminoethyl ethyl ester
Water glyceride modifies the cationic micelle of adamantane polycation inclusion compound, and d is polymethylacrylic acid diisopropylaminoethyl ethyl ester
Modify the sun of beta-cyclodextrin-tetraethylenepentamine poly (glycidyl methacrylate) modification adamantane polycation inclusion compound from
Sub- micella.
Fig. 4 is that the polymethylacrylic acid diisopropylaminoethyl ethyl ester that the embodiment of the present invention 6 synthesizes modifies beta-cyclodextrin-second two
The cationic micelle of amination poly (glycidyl methacrylate) modification adamantane polycation inclusion compound different poly- sun from
Son and the gel electrophoresis images evaluated under the mass ratio of pDNA-GFP nucleic acid binding ability.
Fig. 5 is contains the cationic micelle compound of pDNA-GFP transfection A549 lung carcinoma cells in the embodiment of the present invention 8 after,
Fluorescence microscope (OLYMPUS1X81 types inverted fluorescence microscope) testing result of egfp expression situation.
It is poly- that Fig. 6 for A-B types polymethylacrylic acid diisopropylaminoethyl ethyl ester of the present invention modifies beta-cyclodextrin-aminated
Glycidyl methacrylate modifies the structure diagram of adamantane polycation inclusion compound.
Embodiment
By specific examples below, the present invention will be described, but the present invention is from the restriction of these specific embodiments.
In the present invention, nmr spectrum is surveyed by Varian-MERCURY Plus-400 type hydrogen nuclear magnetic resonances spectrometer
Measure.PH value is measured by Sartorius PB-10 types pH meter, and secondary deionized water is by Advantage A10 type pure water meters
Prepare.The hydrodynamics particle diameter and surface potential of cationic micelle are measured by MALVERN NANO SIZER types particle instrument.Gel
Electrophoresis is completed using the DY-501 gel-electrophoretic apparatuses of Shanghai Wanda's science and technology equipment Co., Ltd, and gel photograph is by BIO RAD
Chemi DOCTM MP Imaging System types gel imager is shot.
2- bromine isobutyl groups acylbromide used, stannous chloride, cuprous bromide, methacrylic acid two are different in the embodiment of the present invention
Third amino ethyl ester and pentamethyl-diethylenetriamine are purchased from Sigma-Aldrich (China) company.Glycidyl methacrylate
Purchased from uncommon love (Shanghai) the chemical conversion industry Development Co., Ltd of ladder.Unless otherwise specified, remaining agents useful for same and solvent are purchased from state
Medicine group (Shanghai) chemical reagent Co., Ltd.
SiRNA and pDNA sequences are synthesized by Shanghai Ji Ma medicine biological techniques company.A549 lung carcinoma cells are purchased from the U.S.
ATCC cell banks, cell culture are purchased from Gibco companies with DMEM culture mediums and hyclone.
In this application, unless otherwise specified, device therefor and test method are the apparatus and method of this area routine.
Embodiment 1：The synthesis of beta-cyclodextrin initiator
Beta-cyclodextrin 4.5mmol is weighed, is dissolved under agitation in the anhydrous n,N-dimethylacetamide of 30ml, and adds three
Ethamine 22.5mmol.Weigh 2- bromine isobutyl group acylbromides 22.3mmol and be dissolved in the anhydrous DMAC N,N' dimethyl acetamides of 10ml.2- bromines is different
Butyl acylbromide solution is slowly dropped in beta-cyclodextrin solution, is slowly increased to room temperature after being stirred under ice bath, is continued stirring and reacted
Night.Question response terminates to be precipitated with ether, with acetone, obtains 4 Chagerdβcyclodextrins initiator 3.3g (yield 42.4%).1H NMR
(CDCl3)：δ=1.88 (24H ,-OCO-C (CH3)2Br), 3.00-6.00 (66H ,-OH and-CH- β-CD).
Embodiment 2：The synthesis of adamantane initiator
Weigh adamantane methanol 3.0g (18.0mmol) to be dissolved in 50ml dichloromethane, then triethylamine is added into solution
3ml(21.6mmol).Measure 2- bromine isobutyl group acylbromide 2.7ml (21.8mmol) and be dissolved in dichloromethane, be added dropwise under ice bath
In adamantane methanol solution.After completion of dropwise addition, continue to be stirred overnight, after solution concentrated by rotary evaporation, with silica gel chromatographic column to product
Purified, obtain adamantane initiator 4.6g (yields：81%).1H NMR(CDCl3)：δ=2.00 (3H, CH), 1.95 (6H,
CH2), 1.69 (6H ,-C (CH3)2Br), 1.58 (6H, CH2)。
Embodiment 3：Polymethylacrylic acid diisopropylaminoethyl ethyl ester modifies the synthesis of beta-cyclodextrin
The beta-cyclodextrin initiator 0.3g synthesized in Example 1 be dissolved in 1.5ml DMAC N,N' dimethyl acetamides stir to
Dissolving, adds 1ml methacrylic acid diisopropylaminoethyl ethyl esters (DPMA) and catalyst ligand pentamethyl-diethylenetriamine
(PMDETA) 132 μ l, add catalyst cuprous bromide 90mg under anaerobic, when 40 DEG C of stirring reactions 24 are small, peroxidating aluminium column,
Obtain polymethylacrylic acid diisopropylaminoethyl ethyl ester modification beta-cyclodextrin 1.14g (yields：83%).1H NMR(DMF)：δ=
1.88-2.25 (44H ,-OCO-C (CH3)2-CH2-CCH3Br), 2.74 (8H, CH), 3.08 (8H, CH2), 3.99 (8H, CH2),
3.00-6.00 (66H ,-OH and-CH- β-CD).Its nuclear magnetic data is as shown in the B in Fig. 1.
Embodiment 4：Poly (glycidyl methacrylate) modifies the synthesis of adamantane
The adamantane initiator 133mg synthesized in Example 2 is dissolved in 3ml DMAC N,N' dimethyl acetamides-isopropanol (9:
1) stirring adds glycidyl methacrylate (GC) 3ml, 88 μ l of pentamethyl-diethylenetriamine to dissolving in solution.Add
Enter catalyst cuprous bromide 60mg, when 40 DEG C of reactions 15 are small, peroxidating aluminium column, polymethylacrylic acid diisopropylaminoethyl is obtained after freezing
Ethyl ester modifies adamantane 810mg (yields：79%).1H NMR(CDCl3)：δ=4.29；3.78 (30H, CH2), 3.22 (15H,
CH), 2.83；2.62 (30H, CH2), 1.07 (45H, CH3)。
Embodiment 5：Ethylenediamine poly (glycidyl methacrylate) modifies the synthesis of adamantane
The poly (glycidyl methacrylate) modification adamantane (AD-PGC synthesized in Example 415) 100mg is dissolved in
In 2ml DMAC N,N' dimethyl acetamides.Ethylenediamine (DEA) 1ml is taken to be dissolved in 2ml DMAC N,N' dimethyl acetamides.By the poly- methyl of 2ml
Glycidyl acrylate modification adamantane solution is added drop-wise in ethylenediamine solution.When 40 DEG C of stirring reactions 12 are small, freeze-drying
Obtain ethylenediamine poly (glycidyl methacrylate) modification adamantane 130mg (yields：95%).In its nuclear magnetic data such as Fig. 1
A shown in.1H NMR(CDCl3)：δ=1.47-2.12 (28H ,-OCO-C (CH3)2-CH2-CCH3Br and-CH-AD),
3.27；3.42 (2H, CH2), 3.58 (4H, CH2), 4.16；4.41 (2H, CH2)。
Embodiment 6：Polymethylacrylic acid diisopropylaminoethyl ethyl ester modification beta-cyclodextrin-ethylenediamine polymethylacrylic acid contracting
Water glyceride modifies the synthesis of adamantane polycation inclusion compound
Weigh the ethylenediamine poly (glycidyl methacrylate) modification adamantane 330mg synthesized in embodiment 5
(0.1mmol), is dissolved in 5ml deionized waters, and the polymethylacrylic acid synthesized in the embodiment 3 of 3ml is slowly dropped under vortex
In the 1M HCl solutions of diisopropylaminoethyl ethyl ester modification beta-cyclodextrin 200mg (0.05mmol), freezed after super filter tube centrifugal concentrating
It is dry, obtain target product 260mg (yields：72%).Its nuclear magnetic data is as shown in the C in Fig. 1.
Embodiment 7：The preparation of dual acid-sensitive polycation micella
The polymethylacrylic acid diisopropylaminoethyl ethyl ester modification beta-cyclodextrin-ethylenediamine synthesized in embodiment 6 is weighed to gather
Glycidyl methacrylate modification adamantane polycation inclusion compound 0.50g (0.1mmol), is dissolved in the 1M salt of 5ml volumes
In acid solution, which is instilled in the 0.5M sodium hydroxide solutions of 50ml under ultrasound, ultrafiltration constant volume after purification, obtains institute
State polycation micellar solution.
Embodiment 8：Cationic micelle is conveyed for nucleic acid drug
The cationic micelle aqueous solution prepared in Example 7 is respectively 2.0 with weight ratio with pDNA-GFP:Isosorbide-5-Nitrae .0:1,
8:1 and 16:1 different proportion mixing, room temperature stand 30 minutes micelle complex for obtaining containing Plasmid DNA.A549 lung cancer
Cell spreads 24 orifice plates, and ten thousand/holes of 4-5, use after being incubated 24h, is 0.5ml DMEM complete mediums per hole.Phase is separately added into per hole
With the micelle complex of concentration.Inverted fluorescence microscope detects transfection after cell transfecting 60h.
EXPERIMENTAL EXAMPLE 1：The acid base titration experiment of A-B polycation inclusion compounds
The polymethylacrylic acid diisopropyl synthesized in the embodiment of the present invention 6 is measured using Sartorius PB-10 types pH meter
Amino ethyl ester modification beta-cyclodextrin-ethylenediamine poly (glycidyl methacrylate) modification adamantane polycation inclusion compound
Acid-base titration curve.The results are shown in Figure 2 for it.According to Fig. 2 as can be seen that polymethylacrylic acid diisopropyl ammonia in embodiment 6
Base ethyl ester modification beta-cyclodextrin-ethylenediamine poly (glycidyl methacrylate) modification adamantane polycation inclusion compound has
Proton buffer capacity in the range of two pH of 4.5-6.0 and 6.0-9.0, it was demonstrated that the polycation possesses dual acid-sensitivity,
It can meet that carrier is contained nucleic acid medicine for being extracellularly stabilized during nucleic acid drug conveying, quickly dissociating and discharge into the cell
The requirement of thing.
EXPERIMENTAL EXAMPLE 2：The hydrodynamics particle diameter of the dual acid-sensitive polycation micellas of A-B and the measure of surface potential
Handbook according to manufacturer determines the dual acid-sensitives of A-B using MALVERN NANO SIZER type particle instruments and gathers sun
The hydrodynamics particle diameter and surface potential of ionic micelle.The results are shown in Figure 3 for it.According to Fig. 3 as can be seen that prepared by the present invention
Dual acid-sensitive polycation micella hydrodynamics particle diameter and surface potential meet be used for nucleic acid drug convey, to target cell
The requirement of a large amount of conveying medicines.
EXPERIMENTAL EXAMPLE 3：The gel electrophoresis assay combined to nucleic acid of A-B polycation inclusion compounds
Using the plasmid deoxyribonucleic acid (pDNA-GFP) of enhanced green fluorescent protein, with different polycations with
The mass ratio of pDNA-GFP (is respectively 4:1、3.5:1、3.0:1、2.5:1、2.0:1、1.0:1 and 0.5:1) with Shanghai Wanda section
The DY-501 gel-electrophoretic apparatuses of skill equipment Co., Ltd make gel electrophoresis assay, and with BIO RAD Chemi DOCTM MP
Imaging System types gel imager obtains gel photograph.The results are shown in Figure 4 for it.According to Fig. 4 as can be seen that the present invention
Prepared polymethylacrylic acid diisopropylaminoethyl ethyl ester modification beta-cyclodextrin-ethylenediamine poly (glycidyl methacrylate)
Modification adamantane polycation micella can be with compressing pRNA completely during pDNA mass ratioes=1.0, it was demonstrated that it is with good core
Sour binding ability.
EXPERIMENTAL EXAMPLE 4：Contain the expression of the cationic micelle transfection A549 lung carcinoma cells of pDNA-GFP
A549 lung carcinoma cells are transfected using the cationic micelle that pDNA-GFP is contained in embodiment 8, are then shown using fluorescence
Micro mirror (OLYMPUS1X81 types inverted fluorescence microscope) detects egfp expression situation.Wherein, cationic micelle and matter
Grain DNA mass ratioes are 8.0, pDNA concentration 200ng/ holes, transfection time 60h.The results are shown in Figure 5 for it.It can be seen according to Fig. 5
Go out, the pDNA-GFP of package-contained has obtained good expression, i.e. carrier using the present invention achieves good transfection efficiency.
The A-B polycation inclusion compounds of the present invention are can be seen that with the result of EXPERIMENTAL EXAMPLE according to the above embodiments
And its cationic micelle formed possesses dual acid-sensitivity, conveying nucleic acid drug can be efficiently used for, can meet carrier
Extracellularly it is stabilized when being conveyed for nucleic acid drug, quickly dissociates and discharge the requirement for being contained nucleic acid drug into the cell, and
It is a kind of new and the more general Lipofectamin-2000 cationic liposomal transfection reagents of its transfection efficiency greatly improve
Stablize relatively, is special, safety gene vector system, can be efficiently used for the extensive use of RNAi technology treating cancer
In.
Claims (24)
1. a kind of polycation inclusion compound, it modifies beta-cyclodextrin-aminated poly- for polymethylacrylic acid diisopropylaminoethyl ethyl ester
Glycidyl methacrylate modifies adamantane, and the polycation inclusion compound is different as the polymethylacrylic acid two shown in formula A
The adamantane warp of aminated poly (glycidyl methacrylate) modification shown in the beta-cyclodextrin and formula B of the modification of third amino ethyl ester
Host guest complexation forms,
Wherein, R1~R7In any 2~5 beRemaining is hydrogen, and m is the integer of 1-100；N is 1-
100 integer；A is the integer of 0-10.
2. polycation inclusion compound according to claim 1, wherein, the n is the integer of 5-30；M is the integer of 1-20；
A is the integer of 0-4.
3. a kind of intermediate for being used to prepare polycation inclusion compound according to claim 1, it is the poly- first shown in formula A
The beta-cyclodextrin of base acrylic acid diisopropylaminoethyl ethyl ester modification,
Wherein, R1~R7In any 2~5 beRemaining is hydrogen, and m is the integer of 1-100.
4. a kind of method for preparing polycation inclusion compound according to claim 1, this method is included shown in hybrid A
Aminated polymethyl acid glycidyl shown in the beta-cyclodextrin and formula B of the modification of polymethylacrylic acid diisopropylaminoethyl ethyl ester
The step of adamantane of ester modification is to occur complexing,
5. a kind of method for the intermediate for preparing polycation inclusion compound according to claim 3, the described method includes such as
Lower step：
Step a：The synthesis of beta-cyclodextrin initiator
Beta-cyclodextrin and 2- bromine isobutyl group acylbromides are obtained into beta-cyclodextrin initiator through esterification；
Wherein, R8~R14In any 2~5 beRemaining is hydrogen；
Step b：Polymethylacrylic acid diisopropylaminoethyl ethyl ester modifies the synthesis of beta-cyclodextrin
The beta-cyclodextrin initiator obtained in step a is mixed with methacrylic acid diisopropylaminoethyl acetate monomer, is turned in atom
Move in the presence of catalysts for radical polymerization ligand and catalyst and polymerization occurs obtain polymethylacrylic acid diisopropylaminoethyl ethyl ester to repair
The beta-cyclodextrin of decorations, wherein, R8~R14In beGroup be reacted to through above-mentionedBase
Group, remaining R8~R14For hydrogen.
6. according to the method described in claim 5, wherein,
In step a, the molar ratio of 2- bromine isobutyl group acylbromides and beta-cyclodextrin is 2:1 to 5:1；
In stepb, the molar ratio of beta-cyclodextrin initiator and methacrylic acid diisopropylaminoethyl acetate monomer is 1:30 to 1:
200。
7. a kind of cationic micelle, the cationic micelle includes polycation inclusion compound according to claim 1 or 2,
Wherein, the cationic micelle includes aminated poly (glycidyl methacrylate) cationization hydrophilic outer layer and poly- methyl-prop
Olefin(e) acid diisopropylaminoethyl ethyl ester hydrophobic inner layer.
8. cationic micelle according to claim 7, wherein, the cationic micelle has dual acid-sensitivity, micella
Hydrophilic outer layer can show electropositive with reference to proton in the case where pH is 7.4 neutral physiological condition, the hydrophobic inner layer of micella can be
PH combines proton dissociation in the weak acid environment less than 6.3.
9. cationic micelle according to claim 7, wherein, the average hydrodynamic particle diameter of the cationic micelle is
20-200 nanometers.
10. cationic micelle according to claim 7, wherein, the average hydrodynamic particle diameter of the cationic micelle is
20-60 nanometers.
11. cationic micelle according to claim 7, wherein, the cationic micelle have in pH=4.5-6.0 and
6.0-9.0 the sensitivity to acid in two sections.
12. a kind of method for preparing the cationic micelle described in claim 7, it is characterized in that：By the poly- sun described in claim 1
Ion inclusion compound is dissolved in 1-5 times of volume acidic buffer solution, and resulting solution is instilled 10-50 volumes alkalescence under ultrasound delays
In fliud flushing, cationic micelle is obtained after purification through ultrafiltration or dialysis, wherein, the pH value of acidic buffer is 1.0-5.0, alkali
The pH value of property buffer solution is 8.0-12.0.
13. a kind of cationic micelle compound for being loaded with nucleic acid, it includes：Cationic micelle according to claim 7；
With the nucleic acid being supported on the cationic micelle.
14. the cationic micelle compound according to claim 13 for being loaded with nucleic acid, wherein, the nucleic acid is selected from small dry
Disturb ribonucleic acid (siRNA) and short heparin ribonucleic acid (shRNA).
15. the cationic micelle compound according to claim 13 for being loaded with nucleic acid, wherein, the weight of the nucleic acid loaded
Amount accounts for the 1.0-20% of the cationic micelle compound weight.
16. a kind of method for preparing the cationic micelle compound for being loaded with nucleic acid, it includes will be according to claim 7
The step of cationic micelle is mixed with nucleic acid.
17. according to the method for claim 16, wherein, the nucleic acid is selected from small interference ribonucleic acid (siRNA) and short liver
Plain ribonucleic acid (shRNA).
18. according to the method for claim 16, wherein, the weight of the nucleic acid loaded accounts for the cationic micelle compound
The 1.0-20% of weight.
19. purposes of the polycation inclusion compound according to claim 1 in the medicine for delivery of nucleic acids is prepared.
20. purposes according to claim 19, wherein, the nucleic acid is selected from small interference ribonucleic acid (siRNA) and short liver
Plain ribonucleic acid (shRNA).
21. purposes according to claim 19, wherein, the medicine is used for inverse cancer cell multidrug resistance or suppression cancer is thin
Dysuria with lower abdominal colic is moved.
22. purposes of the polycation inclusion compound according to claim 1 or 2 as pharmaceutical carrier in pharmacy.
23. purposes according to claim 22, wherein, the pharmaceutical carrier is used in delivery of nucleic acids.
24. purposes according to claim 23, wherein, the nucleic acid is small interference ribonucleic acid (siRNA) or short heparin
Ribonucleic acid (shRNA).
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