CN102796236A - Cationic triblock copolymer based on biodegradable polyphosphate ester and application thereof - Google Patents

Cationic triblock copolymer based on biodegradable polyphosphate ester and application thereof Download PDF

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CN102796236A
CN102796236A CN2012103051792A CN201210305179A CN102796236A CN 102796236 A CN102796236 A CN 102796236A CN 2012103051792 A CN2012103051792 A CN 2012103051792A CN 201210305179 A CN201210305179 A CN 201210305179A CN 102796236 A CN102796236 A CN 102796236A
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triblock copolymer
monomer
chlorine
phosphate
reaction
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倪沛红
卞姣
张明祖
何金林
梁珂
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Suzhou University
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Suzhou University
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Abstract

The invention discloses a preparation method of a cationic triblock copolymer based on biodegradable polyphosphate ester and application thereof in medicine carriers and gene vectors. Caprolactone is subjected to ring-opening polymerization by a micro-molecule initiator containing halogen on one terminal and hydroxyl group on the other terminal under the catalytic action of stannous octoate, so as to obtain a macro-molecule ATRP initiator containing hydroxyl group on one terminal and bromine or chlorine substituent on the other terminal, and then an annular phosphate ester monomer is subjected to ring-open polymerization, so as to obtain a diblock copolymer of the poly-caprolactone and poly-phosphate ester; and finally the diblock copolymer is subjected to solution ATRP reaction with pH sensitive nitrogen-contained monomers under the action of an ATRP catalyst and a ligand, so as to obtain a pH sensitive triblock copolymer based on poly-phosphate ester, which can be used as hydrophobic medicine carrier and gene vector.

Description

Cationic triblock copolymer and application thereof based on the biodegradable poly phosphate
Technical field
The invention belongs to field of biomedical polymer materials, be specifically related to a kind of cationic triblock copolymer, its preparation method based on the biodegradable poly phosphate with and as the application of pharmaceutical carrier and genophore.
Background technology
The whole world has more than 700 ten thousand people to lose one's life because of cancer stricken every year, and this numeral rises year by year.Treatment for cancer is the maximum test and the difficult problem of 21st century face of mankind.
Common chemotherapy is that cancer therapy drug is passed through intravenous delivery to whole body, though cancer cells is had restraining effect, also normal cell is had very big damage.The cancer therapy drug that develops into of nanotechnology provides opportunity to effective conveying of tumour.Utilize nano material to load cancer therapy drug as carrier; And nano-carrier carried out special processing (as connect targeted molecular, polymkeric substance has methods such as stimulating responsive); Can reach the purpose of targeted medicine; Aim at pathological tissues or organ more exactly, reduce the untoward reaction of medicine human body.
Treatment for cancer only relies on simple a kind of method to be difficult to reach ideal effect; Use the method for two or more different effects mechanism to combine; For cancer therapy is a kind of very promising method, and chemotherapy is combined with gene therapy has obtained increasing concern in recent years.
The treatment that gene therapy is acknowledged as range gene disease (comprise transmissible disease, with the disorder and the cancer of gene-correlation) has great potential, thereby receives extensive concern.Be prone to by nuclease digestion because great majority are used for the nucleic acid drug of gene therapy, therefore, need effective genophore nucleic acid drug to be transported to the cell position of expection.Wherein, Non-virus type positively charged ion genophore is owing to have a higher load efficiency, to DNA size and the number that is shifted not have restriction, prepare relatively simply, can reuse, the immunogenicity problem is less, the advantage of the more high aspect of security and receiving much concern.The kind of cationic polymer gene vector is a lot, and the polypeptide class is arranged: polylysine, polyglutamic acid and verivate thereof; Poly amine: polymine (PEI), Vestolen PP 7052 imines tree; Polymethacrylic acid; Polyamide-amide type tree; And some natural polymers such as chitosan, gelatin etc.Biodegradable cationic type polymkeric substance in recent years; For example: gather beta-amino acid esters, polylysine, amino derivative and contain amino block polymer etc.; Be widely used in field of gene, wherein particularly extensive as the research of non-viral gene vector with nitrogenous segmented copolymer.
In the prior art, the research as medicine and genophore mainly contains about amphipathic cationic segmented copolymer:
(1) self-assembly of PDMAEMA-b-PCL-b-PDMAEMA triblock copolymer forms micella, as the delivery vehicles of siRNA and anti-cancer medicine paclitaxel (PTX) (referring to Zhu C.H.; Jung S.Y.; Luo S.B.; Meng F.H.; Zhu X.L.; Park T.G.; Zhong Z.Y..Co-delivery of siRNA and paclitaxel into cancer cells by biodegradable cationic micelles based on PDMAEMA-PCL-PDMAEMA triblock copolymers.Biomaterials; 2010,31:2408-2416.);
(2) Polyethylenimine-graft-Poly ((PEC) amphiphilic graft polymers of ε-caprolactone) as the delivery vehicles of DNA and Zorubicin (DOX) (referring to Qiu L.Y.; Bae Y.H..Self-assembled polyethylenimine-graft-poly (the micelles as potential dual carriers of genes and anticancer drugs.Biomaterials of ε-caprolactone); 2007,28:4132-4142.)
(3) Methoxy polyethylene glycol-b-poly{N-[3-(dimethylamino) propyl] methacrylamide-co-[2-hydroxylethylmethacrylate-poly (ε-caprolactone)] } { MPEG-b-P [NDAPM-co-(HEMA-PCL)] } amphipathic cation type polymer be used for DNA and Zorubicin (DOX) delivery vehicles (referring to Zhu J.L.; Cheng H.; Jin Y.; Cheng S.X.; Zhang X.Z.; Zhuo R.X..Novel polycationic micelles for drug delivery and gene transfer.J.Mater.Chem., 2008,18:4433-4441.)
(4) Poly (ethylene oxide)-block-polyesters with grafted polyamines; For example: grafting has the spermine (PEO-b-P (CL-g-SP)) or the grafting tetraethylenepentamine (PEO-b-P (CL-g-TP)) of nitrogen-containing group on the side chain of PEO-b-PCL, can be used as medicine and DNA delivery vehicles.(referring to: Xiong X.B.;
Figure BDA00002055782100031
H.; Lavasanifar A..Biodegradable amphiphilic poly (ethylene oxide)-block-polyesters with grafted polyamines as supramolecular nanocarriers for efficient siRNA delivery.Biomaterials; 2009,30:242-253.)
Discovering in recent years; Poly phosphate (polyphosphoesters) is one type of polymkeric substance that main chain is a phosphate ester structure; Therefore the structural similitude of this base polymer and biomacromolecule nucleic acid, has fabulous biocompatibility; And through hydrolysis or under physiological condition, carry out enzymic digestion, can biological degradation.Poly phosphate has good application prospects aspect biomedical material.It should be noted that; Biodegradable polyesters class commonly used; Like polycaprolactone (PCL), polylactide (PLA), gather NSC 403079 (PGA), glycolide-lactide copolymer (PLGA) etc.; In biodegradable process, have carbonic acid gas and generate, can make that the local pH value reduces in the organism after water-soluble, thereby organism is produced certain spinoff.Moreover characteristics are unfavorable for its degraded because the polyester hydrophobicity is big, fusing point is high, crystallinity is strong etc.But the main chain of poly phosphate is a phosphate ester structure, and the product after the degraded does not produce carbonic acid gas, can not have side effects to organism.
So far also not having bibliographical information to be similar among the present invention will and can be used as the structure of the polycaprolactone segment and the triblock copolymer that pH sensitive polymer segment connects as one of oil-soluble medicine carrier based on biodegradable wetting ability poly phosphate segment, and be used for pharmaceutical carrier and genophore simultaneously.
Summary of the invention
The objective of the invention is to disclose a kind of preparation and application aspect pharmaceutical carrier and genophore thereof with cationic triblock copolymer of pH responsiveness of good biocompatibility and biodegradability.
General plotting of the present invention is: ring-opening polymerization (ROP) and two kinds of methods of ATRP (ATRP) are combined the preparation amphipathic three block copolymer.The small organic molecule that at first utilizes hydroxyl and halogen is as initiator; Under the katalysis of stannous octoate; To 6-caprolactone (ε-CL) carry out ring-opening polymerization, it is hydroxyl to obtain molecular chain one end, another end has the functional macromolecule initiator of bromine or chlorine substituent; And then utilize its terminal hydroxyl, and the annular phosphate monomer is carried out ring-opening polymerization, obtain the di-block copolymer that end contains bromine or chlorine substituent; At last, utilize it, carry out the ATRP solution reaction, obtain the cationic triblock copolymer of pH responsiveness with the pH sensitive monomer as macromole ATRP initiator.
For achieving the above object, technical scheme provided by the invention is: a kind of cationic triblock copolymer based on the biodegradable poly phosphate, and the chemical structural formula of this triblock copolymer is:
Figure BDA00002055782100041
In the formula, j is 20～100, and m is 20～80, and n is 20～100; X is bromine or chlorine;
R 1Group is selected from: a kind of in the end capped polyethylene oxide base of ethyl, sec.-propyl, butyl or monomethyl, wherein, the chemical structural formula of the end capped polyethylene oxide base of monomethyl is :-(CH 2CH 2O) kCH 3, k is 2～10 in the formula;
R 2Group is selected from:
Figure BDA00002055782100042
A kind of in
Figure BDA00002055782100043
;
R 3Group is selected from:
Figure BDA00002055782100051
A kind of in
Figure BDA00002055782100052
.
Above-mentioned cationic triblock copolymer based on the biodegradable poly phosphate had structurally both contained hydrophobic PCL block; Can wrap up hydrophobic anticancer drug; As pharmaceutical carrier, contain the responsive cation type polymer block of pH again, protonated back is positively charged under certain pH conditions; With electronegative DNA the static compound action taking place, can be used as genophore.
In the technique scheme, the number-average molecular weight scope of described triblock copolymer is: 8000～40000gmol -1
A kind of functional macromolecule initiator, molecular chain one end of functional macromolecule initiator contains hydroxyl, and the other end contains bromine or chlorine substituent, and the chemical structural formula of said functional macromolecule initiator is:
Figure BDA00002055782100053
In the formula, R 3Group is selected from:
Figure BDA00002055782100054
M is 20～80; X is bromine or chlorine.
The preparation method of above-mentioned cationic triblock copolymer based on the biodegradable poly phosphate may further comprise the steps:
(1) preparation annular phosphate monomer: with R 1-OH with
Figure BDA00002055782100055
React the synthetic R that has 1The annular phosphate monomer of side-chain radical
Figure BDA00002055782100061
(2) with having the brominated of terminal hydroxy group functional group or chlorine organic cpds HO-R 3-X carries out ring-opening polymerization to the cyclic monomer 6-caprolactone in the presence of octoate catalyst is stannous, obtain the functional macromolecule initiator of the brominated or chlorine substituent of molecule chain end;
(3) the functional macromolecule initiator that adopts step (2) to obtain; At first cause annular phosphate monomer
Figure BDA00002055782100062
through terminal hydroxyl and carry out ring-opening polymerization, preparation contains the di-block copolymer of poly phosphate and polycaprolactone block
Figure BDA00002055782100063
In the formula, j is 20～100, and m is 20～80, and X is bromine or chlorine;
(4) in the presence of ATRP catalyzer and catalyst ligand, carry out solution A TRP reaction with the responsive monomer of pH, obtain triblock copolymer;
Said ATRP catalyzer is selected from a kind of in cuprous chloride or the cuprous bromide;
Said catalyst ligand is selected from: a kind of in dipyridyl, five methyl diethylentriamine, Tetramethyl Ethylene Diamine or the hexamethyl Triethylenetetramine (TETA);
The responsive monomer of said pH is selected from: a kind of in methylacrylic acid-2-(dimethylamino) ethyl ester, methylacrylic acid-2-(diethylin) ethyl ester, methylacrylic acid-2-(diisopropylaminoethyl) ethyl ester, methylacrylic acid-2-(the N-tertiary butyl the is amino) ethyl ester;
The mol ratio of monomer and functional macromolecule initiator is (20～200): 1; The mol ratio of functional macromolecule initiator and ATRP catalyzer is (0.5～1): (1～2); The mol ratio of ATRP catalyzer and catalyst ligand is 1: (1～2).
Further technical scheme is carried out purification processes respectively after step (3) and step (4) completion, and said purification processes may further comprise the steps:
(1) purification processes of di-block copolymer: after step (3) reaction finishes; Rotary evaporation removes and desolvates; Using volume ratio is (10～15): 1 ice anhydrous diethyl ether/ice methanol mixed solvent deposition, remove unreacted monomer and homopolymer, and adopt the B suction filtration; Obtain solid product and place vacuum drying oven to be dried to constant weight, obtain white product;
(2) purification processes of triblock copolymer: (a) after step (4) reaction is accomplished, add THF reaction product is diluted, and under stirring condition, contact, make reaction terminating with air; (b) treat the solution becomes green after, make it through alkaline Al is housed 2O 3Pillar to remove the mantoquita in the solution; (c) rotary evaporation makes solution concentration, and again liquid concentrator being splashed into volume ratio is 1: in the mixed solvent of the ice normal hexane of (5～10) and ice ether, remove unreacted monomer and homopolymer; (d) the gained white depositions is dried to constant weight for 40～50 ℃ at vacuum drying oven, obtains milky viscous substance.
The present invention asks for protection the application of above-mentioned cationic triblock copolymer based on the biodegradable poly phosphate as pharmaceutical carrier and genophore simultaneously.
Wherein, contain hydrophobic biodegradability polycaprolactone (PCL) segment, can be used for wrapping up hydrophobic anticancer drug; Contain the responsive polycation segment of pH, under certain pH conditions, take place protonated positively chargedly, can electrostatic interaction take place with electronegative DNA, compression DNA is as genophore; And the poly phosphate segment has excellent biological compatibility, biodegradability and wetting ability, can make the self-assembly of formation particle-stabilised.
Because the pKa of PDMA is 8.0, under the pH7.4 condition, just can take place protonated, compound with DNA.
Because the enforcement of such scheme, the present invention compared with prior art has the following advantages:
1, the present invention has adopted good poly phosphate of biological degradability and biocompatibility and polycaprolactone respectively as wetting ability and hydrophobic segment; The pH sensitive polymer segment good with biocompatibility combines; Through the method that ring-opening polymerization and ATRP combine, obtained to have the pH response triblock copolymer of good biocompatibility and biodegradability.
But the triblock copolymer that 2, obtains among the present invention self-assembly in water forms micella, and the micellar hydrophobic inner core can be wrapped the loaded with anti-cancer medicine Zorubicin, and the micellar surface has positive charge simultaneously, can be compound with electronegative DNA, play the effect that gene is carried.Therefore, triblock copolymer disclosed by the invention can wrap loaded with anti-cancer medicine and DNA simultaneously, aspect treatment for cancer, has the potential application prospect.
Description of drawings
Fig. 1 is functional macromolecule initiator polycaprolactone (HEBI-PCL among the embodiment one 50) proton nmr spectra ( 1H NMR) spectrogram, solvent are CDCl 3
Fig. 2 is for containing the di-block copolymer (PEEP of poly phosphate among the embodiment two 50-b-PCL 50) proton nmr spectra ( 1H NMR) spectrogram, solvent are CDCl 3
Fig. 3 is triblock copolymer (PEEP among the embodiment three 50-b-PCL 50-b-PDMAEMA 25) proton nmr spectra ( 1H NMR) spectrogram, solvent are CDCl 3
Fig. 4 is HEBI-PCL among the embodiment one 50, PEEP among the embodiment two 50-b-PCL 50With PEEP among the embodiment three 50-b-PCL 50-b-PDMAEMA 25The elution curve of gel permeation chromatography (GPC) of polymkeric substance.
Fig. 5 is PEEP among the embodiment four 50-b-PCL 50-b-PDMAEMA 95(concentration is 1mg mL to the triblock copolymer micella -1) transmission electron microscope photo of self-assembly form in pH7.4 buffered soln.Wherein, (a) scale is 50nm, and (b) scale is 200nm.
Fig. 6 is PEEP among the embodiment five 50-b-PCL 50-b-PDMAEMA 95The triblock copolymer carrier micelle is at the buffered soln drug release curve of different pH values.
Fig. 7 be among the embodiment six at different N/P than under the condition, polymer P EEP 50-b-PCL 50-b-PDMAEMA 95Gel blocking electrophoretic image with the calf thymus DNA mixture.The N/P mol ratio of polymkeric substance and DNA is respectively 0,0.08,0.25,0.45,0.6,0.8,1.0,1.2,1.5,1.8,2.2,2.7,3.0,3.5 in 1～12 swimming lane.
Fig. 8 is among the embodiment seven, triblock copolymer PEEP 50-b-PCL 50-b-PDMAEMA 95In the presence of phosphodiesterase, the hydrogen nuclear magnetic resonance spectrogram of the degraded product that obtains under the different degradation times ( 1HNMR), solvent is CDCl 3(a) original copolymer, (b) degraded is 3 days, and (c) degraded is 6 days, and (d) degraded is 9 days.
The practical implementation method
Below in conjunction with embodiment and accompanying drawing the present invention is further described:
Embodiment one: macromole evocating agent HO-PCL 50The concrete compound method of-X
With the arm round-bottomed flask of putting into stirrer behind 120 ℃ of dry at least 24h of baking oven, glass stopper beyond the Great Wall, through emulsion tube link to each other with oil pump be evacuated to room temperature after, feed high-purity argon gas again, vacuumize three times so repeatedly again.
With the exsiccant syringe from arm, inject successively bromo acid-2-hydroxy methacrylate (HEBI) (0.12g, 0.57mmol), caprolactone (ε-CL) (3.89g, 34.12mmol) and stannous octoate (Sn (Oct) 2) (7.8mg) (CL calculates consumption according to the theoretical polymerization degree; ε-CL:Sn (Oct) 2=1:0.002, mass ratio), the 10mL dry toluene is a solvent.After in reaction flask, being full of argon gas, stirring reaction 12h in 90 ℃ oil bath.
After reaction finishes; Most of toluene solvant is revolved steaming remove, deposition is removed unreacted monomer twice in 150mL ice anhydrous methanol, places vacuum drying oven to dry to constant weight with the product behind the B suction filtration; Obtain white product HO-PCL-X, record productive rate and be approximately 90%.
Embodiment two: the di-block copolymer PEEP that contains poly phosphate 50-b-PCL 50Compound method
After the arm round-bottomed flask handled according to the method described above, be full of argon gas, take by weighing the back rapidly and add certain amount of H EBI-PCL 50(3g; 0.53mmol) and micro-stannous octoate (6mg); Inject the 10mL THF with the exsiccant syringe HEBI-PCL is dissolved fully, squeeze into a certain amount of monomer 2-oxyethyl group-2-oxygen-1,3 then; 2-dioxaphospholane (EEP) (5.52g, 34.45mmol) (EEP:Sn (Oct) 2=1:0.002, mass ratio), round-bottomed flask is full of argon gas and moves to stirring reaction 3h in 40 ℃ of oil baths.
Reaction finishes the back rotary evaporation and removes a large amount of THFs, with anhydrous diethyl ether/ice methyl alcohol (volume ratio is 10:1) deposition of 150mL ice, removes unreacted monomer and homopolymer.Place vacuum drying oven to be dried to constant weight the product that obtains behind the B suction filtration, obtain white product.
Embodiment three: cationic triblock copolymer PEEP 50-b-PCL 50-b-PDMAEMA 25Compound method
After the arm round-bottomed flask handled according to the method described above, be full of argon gas, take by weighing rapidly and add above-mentioned bi-block copolymer PEEP 50-b-PCL 50(4g, 0.30mmol), cuprous bromide (0.042g, 0.30mmol) and dipyridyl (0.187g; 0.6mol) (three's mol ratio is 1:1:2); Extraction gas is the condition of three guarantee system anaerobics repeatedly, adds 10mL solvent N, dinethylformamide (DMF) with the exsiccant syringe; Be stirred to mixture and dissolve fully, solution is dark-brown.
Then round-bottomed flask is moved in 50 ℃ the oil bath; Add methylacrylic acid-2-(dimethylamino) ethyl ester DMAEMA (1.884g with the exsiccant syringe again; 12mmol) (calculate consumption) according to the theoretical polymerization degree; The halogen that utilizes PCL segment end in two blocks causes the ATRP reaction, stirring reaction 24h as initiator.
After reaction finished, ingress of air made reaction terminating, and mixed with polymers solution makes solution pass through the Al of alkalescence after diluting with the 50mL THF 2O 3Pillar is removed the mantoquita in the solution; The mixed solution rotary evaporation is removed most of solvent and is made solution concentration; In the mixed solvent of 150mL ice normal hexane and ice ether (volume ratio is 1:5), precipitate to remove and desolvate and unreacted monomer; Product behind the suction filtration is dried to constant weight at vacuum drying oven, obtains white product.
In conjunction with embodiment one and embodiment two, to each step products therefrom utilize proton nmr spectra ( 1H NMR) and gel permeation chromatography (GPC) verify its polymer architecture, molecular weight and MWD.Fig. 1, Fig. 2 and Fig. 3 are respectively polymkeric substance HEBI-PCL 50, PEEP 50-b-PCL 50And PEEP 50-b-PCL 50-b-PDMAEMA 25The hydrogen nuclear magnetic resonance spectrogram, Fig. 4 is the GPC elution curve (HEBI-PCL of above-mentioned three kinds of polymkeric substance 50:
Figure BDA00002055782100111
=7560g mol -1,
Figure BDA00002055782100112
=1.09; PEEP 50-b-PCL 50:
Figure BDA00002055782100113
=17550gmol -1,
Figure BDA00002055782100114
=1.26; PEEP 50-b-PCL 50-b-PDMAEMA 25: =14280gmol -1,
Figure BDA00002055782100116
=1.38; ).
Embodiment four: adopt dialysis method to prepare the triblock copolymer micella
With 25mg polymer P EEP 50-b-PCL 50-b-PDMAEMA 25Join in the round-bottomed flask, add 3mL organic solvent N, dinethylformamide (DMF) stirred 4～6 hours, polymer chain is unfolded fully come, and under condition of stirring, utilized the micro-sampling pump with 2mL h -1Speed 9mL pH7.4 PBS buffered soln is added, treat complete sample introduction after, mixing solutions is changed in the dialysis tubing (MWCO 5000), organic solvent is gone fully thoroughly.Changing the solution after the dialysis in 25mL volumetric flask constant volume, is 1mg mL thereby obtain concentration -1Micellar solution.Utilize transmission electron microscope observing gained micellar pattern and size, as shown in Figure 5, triblock copolymer self-assembly in the aqueous solution forms micellar structure.
Embodiment five: polymer micelle loads the performance study of anticancer drugs, doxorubicin
Take by weighing the 25mg polymkeric substance respectively and 5mg anticancer drugs, doxorubicin (DOX) joins in the round-bottomed flask; Add 5mL organic solvent N (DMF), stirred 4～6 hours, the state that polymer chain is in unfold fully; Still be under the state of stirring then, utilize sampling pump with 2mL h -1Speed 10mL pH 7.4 PBS buffered soln are added, treat complete sample introduction after, mixing solutions is changed in the dialysis tubing, organic solvent is gone fully thoroughly.Changing the solution after the dialysis in 25mL volumetric flask constant volume, is 1mg mL thereby obtain concentration -1Polypeptide drug-loaded micelle solution.Get the 5mL polypeptide drug-loaded micelle solution in dialysis tubing, the outside buffered soln that adds the different pH values of 20mL is placed in the constant temperature oscillator, under the water bath with thermostatic control of 37 ° of C, discharges.Get 5mL dialysis tubing external solution at set intervals, replenish the buffered soln of the fresh identical pH value of 5mL simultaneously.Detect the content of the DOX that discharges with spectrophotofluorometer.The release profiles of polymer micelle under condition of different pH that is loaded with Zorubicin is as shown in Figure 6, and drug release rate is obviously faster than pH7.4 when pH5.0, and explanation can reach the purpose of controlled release.
Embodiment six: the situation of utilizing sepharose retardance electrophoresis observation pH response triblock copolymer compression DNA
Polymkeric substance and DNA sample are dissolved in respectively among 0.5 * TBE (Tris-borate buffer), and ultrasonic and stirring is dissolved it fully, and the ultimate density of said polymkeric substance and dna solution is 1mg mL -1
According to the N/P of different ratios, in a certain amount of dna solution, add required polymers soln, the whirlpool concussion makes fully and mixes.Polymers soln has positive electricity, behind the dna solution thorough mixing, through electrostatic interaction compression DNA, forms the complex solution of polymkeric substance and DNA.
Above-mentioned complex solution (8 μ L) is mixed with the appearance buffered soln (85% USP Kosher and 15% tetrabromophenol sulfonphthalein) of going up of 2 μ L, and point sample is to containing 0.5 μ g mL -1In 0.8% the sepharose of ethidium bromide.Electrophoretic voltage is 70V, and the time is 40min, and buffered soln is 0.5 * TBE.
Above-mentioned N/P is than the mol ratio for phosphorus atom among nitrogen-atoms and the DNA among the polymer P EEP-b-PCL-b-PDMAEMA.
Sepharose retardance electrophoresis result is as shown in Figure 7, when N/P than greater than 1.8 the time, phosphor strip disappears, and shows that polymkeric substance can be completely fixed DNA, the migration of retardance DNA.
Embodiment seven: the external degradation experiment of triblock copolymer in the presence of phosphodiesterase
Take by weighing 25mg triblock copolymer PEEP 50-b-PCL 50-b-PDMAEMA 95Be dissolved among the 2.5mLDMF, after the dissolving, the TrisHCl damping fluid (pH=8.8) of 10mL dropwise joined in the solution that is stirring fully.Then this solution is transferred in the dialysis tubing (MWCO 7000); Dialyse with the TrisHCl damping fluid; Remove organic solvent N, dinethylformamide (DMF), during every at a distance from damping fluid of replacing in 4 hours; Stop dialysis after 24 hours, triblock copolymer self-assembly in the TrisHCl damping fluid forms micella.With the polymer micelle solution dilution to 1.0mg mL -1, transfer in the centrifuge tube, in centrifuge tube, add phosphodiesterase (0.2mg mL simultaneously -1).Centrifuge tube is placed the water bath with thermostatic control vibrator, and keeping temperature is 37 ± 1 ℃, takes out at set intervals, drains solution is freezing, obtains a large amount of white solids (mixture of degraded product and Tris).White solid is dissolved in the 50mL dichloromethane solution, and throw out is Tris, removes by filter Tris, and filtrating is removed methylene dichloride through rotary evaporation, and products therefrom places vacuum drying oven to be dried to constant weight, and a small amount of white solid that obtains at last is PEEP 50-b-PCL 50-b-PDMAEMA 95Product after the triblock copolymer degraded.Utilize proton nmr spectra that degraded product is carried out structural characterization.As shown in Figure 8, along with the prolongation of degradation time, the characterization displacement study signal δ 4.10ppm and the 4.30ppm of poly phosphate PEEP segment proton reduce until disappearance gradually in the degraded product.Can prove that from the proton nmr spectra result phosphodiesterase has Degradation to the PEEP segment.

Claims (7)

1. cationic triblock copolymer based on the biodegradable poly phosphate, it is characterized in that: the chemical structural formula of this triblock copolymer is:
Figure FDA00002055782000011
In the formula, j is 20～100, and m is 20～80, and n is 20～100; X is bromine or chlorine;
R 1Group is selected from: a kind of in the end capped polyethylene oxide base of ethyl, sec.-propyl, butyl or monomethyl, wherein, the chemical structural formula of the end capped polyethylene oxide base of monomethyl is :-(CH 2CH 2O) kCH 3, k is 2～10 in the formula;
R 2Group is selected from:
Figure FDA00002055782000012
Figure FDA00002055782000013
in one;
R 3Group is selected from:
Figure FDA00002055782000014
Figure FDA00002055782000015
in one.
2. the cationic triblock copolymer based on the biodegradable poly phosphate according to claim 1 is characterized in that the number-average molecular weight scope of described triblock copolymer is: 8000～40000gmol -1
3. functional macromolecule initiator, it is characterized in that: molecular chain one end of functional macromolecule initiator contains hydroxyl, and the other end contains bromine or chlorine substituent, and the chemical structural formula of said functional macromolecule initiator is:
In the formula, R 3Group is selected from:
Figure FDA00002055782000022
Figure FDA00002055782000023
in one;
M is 20～80; X is bromine or chlorine.
4. the preparation method of the said cationic triblock copolymer based on the biodegradable poly phosphate of claim 1 is characterized in that, may further comprise the steps:
(1) preparation annular phosphate monomer: with R 1-OH with
Figure FDA00002055782000024
React the synthetic R that has 1The annular phosphate monomer of side-chain radical
Figure FDA00002055782000025
(2) with having the brominated of terminal hydroxy group functional group or chlorine organic cpds HO-R 3-X carries out ring-opening polymerization to the cyclic monomer 6-caprolactone in the presence of octoate catalyst is stannous, obtain the functional macromolecule initiator of the brominated or chlorine substituent of the described molecule chain end of claim 3;
(3) the functional macromolecule initiator that adopts step (2) to obtain; At first cause annular phosphate monomer
Figure FDA00002055782000031
through terminal hydroxyl and carry out ring-opening polymerization, preparation contains the di-block copolymer of poly phosphate and polycaprolactone
Figure FDA00002055782000032
In the formula, j is 20～100, and m is 20～80, and X is bromine or chlorine;
(4) in the presence of ATRP catalyzer and catalyst ligand, carry out solution A TRP reaction with the responsive monomer of pH, obtain triblock copolymer;
Said ATRP catalyzer is selected from a kind of in cuprous chloride or the cuprous bromide;
Said catalyst ligand is selected from: a kind of in dipyridyl, five methyl diethylentriamine, Tetramethyl Ethylene Diamine or the hexamethyl Triethylenetetramine (TETA);
The responsive monomer of said pH is selected from: a kind of in methylacrylic acid-2-(dimethylamino) ethyl ester, methylacrylic acid-2-(diethylin) ethyl ester, methylacrylic acid-2-(diisopropylaminoethyl) ethyl ester, methylacrylic acid-2-(the N-tertiary butyl the is amino) ethyl ester;
The mol ratio of monomer and functional macromolecule initiator is (20～200): 1; The mol ratio of functional macromolecule initiator and ATRP catalyzer is (0.5～1): (1～2); The mol ratio of ATRP catalyzer and catalyst ligand is 1: (1～2).
5. preparation method according to claim 4 is characterized in that: after step (3) and step (4) completion, carry out purification processes respectively, said purification processes may further comprise the steps:
(1) purification processes of di-block copolymer: after step (3) reaction finishes; Rotary evaporation removes and desolvates; Using volume ratio is (10～15): 1 ice anhydrous diethyl ether/ice methanol mixed solvent deposition, remove unreacted monomer and homopolymer, and adopt the B suction filtration; The solid product that obtains places vacuum drying oven to be dried to constant weight, obtains white product;
(2) purification processes of triblock copolymer: (a) after step (4) reaction is accomplished, add THF reaction product is diluted, and under stirring condition, contact, make reaction terminating with air; (b) treat the solution becomes blueness after, make it through alkaline Al is housed 2O 3Pillar to remove the mantoquita in the solution; (c) rotary evaporation makes solution concentration, and again liquid concentrator being splashed into volume ratio is 1: in the mixed solvent of the ice normal hexane of (5～10) and ice ether, remove unreacted monomer and homopolymer; (d) the gained white depositions is dried to constant weight for 40～50 ℃ at vacuum drying oven, obtains milky viscous substance.
6. the said cationic triblock copolymer based on the biodegradable poly phosphate of claim 1 is as the application of pharmaceutical carrier and genophore.
7. application according to claim 6 is characterized in that: through regulating the pH value of medium, the triblock copolymer self-assembly forms micella, and hydrophobicity PCL segment forms micellar nuclear, is used to wrap up hydrophobic drug; Wetting ability poly phosphate segment forms the micellar shell; Ester class segment with polymethyl acrylic acid nitrogen atom of pH responsiveness, protonated, positively charged through pH response generation, compound with electronegative DNA, compression DNA is as genophore.
CN2012103051792A 2012-08-24 2012-08-24 Cationic triblock copolymer based on biodegradable polyphosphate ester and application thereof Pending CN102796236A (en)

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CN104098777A (en) * 2014-07-10 2014-10-15 苏州大学 Tri-block polymer and preparation method thereof
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CN109749064A (en) * 2018-12-28 2019-05-14 广东工业大学 A kind of cationic antimicrobial polymer and preparation method thereof with self-degradation
CN111363328A (en) * 2020-05-12 2020-07-03 张中明 High-strength flame-retardant chitosan-polylactic acid composite material and preparation method thereof
CN115124669A (en) * 2022-05-27 2022-09-30 北京化工大学 Preparation method and application of linear block copolymer nano-carrier with double-carrier genes and medicines

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Title
《中国优秀硕士学位论文全文数据库 工程科技I辑》 20100909 张文玲 "生物相容的pH响应性三嵌段共聚物的合成及应用" 21、43-44、51-53、60-61 1-7 , 第01期 *
张文玲: ""生物相容的pH响应性三嵌段共聚物的合成及应用"", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 *

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Publication number Priority date Publication date Assignee Title
CN104098777A (en) * 2014-07-10 2014-10-15 苏州大学 Tri-block polymer and preparation method thereof
CN104095814A (en) * 2014-07-10 2014-10-15 苏州大学 Tri-block polymer micelle, preparation method and application
CN104098777B (en) * 2014-07-10 2017-01-04 苏州大学 A kind of triblock polymer and preparation method thereof
CN106589270A (en) * 2016-11-23 2017-04-26 同济大学 Preparation method of star polymer-based drug carrier material with fluorescence labeling and temperature responsiveness
CN106750328A (en) * 2016-11-23 2017-05-31 厦门大学 A kind of fluorine silicon polyphosphate and its preparation method and application
CN106750328B (en) * 2016-11-23 2019-07-30 厦门大学 A kind of fluorine silicon polyphosphate and its preparation method and application
CN109749064A (en) * 2018-12-28 2019-05-14 广东工业大学 A kind of cationic antimicrobial polymer and preparation method thereof with self-degradation
CN111363328A (en) * 2020-05-12 2020-07-03 张中明 High-strength flame-retardant chitosan-polylactic acid composite material and preparation method thereof
CN111363328B (en) * 2020-05-12 2022-08-23 山西浩博瑞新材料有限公司 High-strength flame-retardant chitosan-polylactic acid composite material and preparation method thereof
CN115124669A (en) * 2022-05-27 2022-09-30 北京化工大学 Preparation method and application of linear block copolymer nano-carrier with double-carrier genes and medicines
CN115124669B (en) * 2022-05-27 2024-03-26 北京化工大学 Preparation method and application of linear block copolymer nano-carrier for double-gene and medicine

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