CN102807657A - Amphiphilic waterborne segmented copolymer medicament carrier with pH responsiveness and biodegradability and preparation thereof - Google Patents
Amphiphilic waterborne segmented copolymer medicament carrier with pH responsiveness and biodegradability and preparation thereof Download PDFInfo
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- CN102807657A CN102807657A CN2012103050906A CN201210305090A CN102807657A CN 102807657 A CN102807657 A CN 102807657A CN 2012103050906 A CN2012103050906 A CN 2012103050906A CN 201210305090 A CN201210305090 A CN 201210305090A CN 102807657 A CN102807657 A CN 102807657A
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Abstract
The invention discloses an amphiphilic waterborne segmented copolymer medicament carrier with pH responsiveness and biodegradability and a preparation method thereof. An amphiphilic waterborne segmented copolymer consists of a chain segment which is modified with polyphosphoester and polymethacrylate hydroxyethyl and contains carboxyl. A polyanion chain segment can be reversely converted into a hydrophilic or hydrophobic chain segment by adjusting the pH value of a medium. The amphiphilic polymer is self-assembled into a micelle under the condition of certain PH, and a shell of the micelle formed by a hydrophilic polyphosphoester chain segment can be used for better stabilizing the micelle. A hydrophobic polyanionic chain segment is used for forming an inner core of the micelle, is used for coating a hydrophobic medicament, and serves as a medicament carrier. The polyphosphoester chain segment has high biocompatibility and high biodegradability, and long in-vivo circulation of a medicament-carrying micelle can be realized. Under a weakly alkaline condition, the aims of dissociating the micelle, releasing a medicament and realizing controllable release are fulfilled.
Description
Technical field
The invention belongs to field of biomedical polymer materials, be specifically related to double-hydrophilic block copolymer pharmaceutical carrier of a kind of pH of having responsiveness and biodegradability and preparation method thereof.
Background technology
Double-hydrophilic block copolymer (Double Hydrophilic Block Copolymers) is one type of special segmented copolymer; Form by two kinds of dissimilar hydrophilic segments; Wherein a kind of segment keeps the dissolved state all the time in water; And another segment can change physics or chemical property when in water, receiving the stimulation of external environment, is converted into hydrophobic state by hydrophily.External stimulus mainly is divided into two types: physical stimulation and chemical stimulation.Physical stimulation has temperature, magnetic field, light etc.; Chemical stimulation has pH, ionic strength etc.Double-hydrophilic block copolymer can design synthetic various structure as required; Therefore obtained in a lot of fields using widely, for example: the nano-reactor of delivery system, genophore, mineral template, crystal growth modifier, synthetic metal-sol etc.
The double-hydrophilic block copolymer of pH response is one type of very important segmented copolymer; Contain the responsive segment of pH in this base polymer, under the condition that pH changes, change hydrophobic state into by hydrophily; Therefore under certain pH conditions; The responsive segment of pH can self-assembly form micellar nuclear, and another kind of segment keeps hydrophily in water, form the micellar shell.
In the prior art, mainly contain about the research of pH responsiveness double-hydrophilic multipolymer:
(1) Armes seminar has synthesized pH response " both sexes " double-hydrophilic block copolymer of PDMAEMA-b-PDEAEMA; Investigate pH and change influence (referring to Structure of pH-Dependent Block Copolymer Micelles:Charge and Ionic Strength Dependence, Lee A.S. to its self-assembly behavior; B ü t ü n V.; Vamvakaki M.; Armes S.P.; Pople J.A.; Gast A.P.Macromolecules 2002,35,8540-8551.)
(2) the brave seminar of Liu's generation has synthesized pH response " both sexes " double-hydrophilic block copolymer of PVB-b-PMEMA; Investigate pH and change influence (referring to Micelle Formation and Inversion Kinetics of a Schizophrenic Diblock Copolymer, Wang D. to its self-assembly behavior; Yin J.; Zhu Z.Y.; Ge Z.S.; Liu H.W.; Armes S.P.; Liu S.Y.Macromolecules 2006,39,7378-7385.)
(3) Discher seminar has synthesized the temperature sensitive double-hydrophilic block copolymer of PEG-b-PNIPAm; Investigate temperature variation to the influence of its self-assembly behavior (referring to Temperature-Controlled Assembly and Release from Polymer Vesicles of Poly (ethylene oxide)-block-poly (N-isopropylacrylamide), Qin S.H.; Geng Y.; Discher D.E.; Yang S.Adv.Mater.2006,18,2905-2909.)
(4) Theato seminar has synthesized based on the part of PNIPAm and has contained temperature, the light of nitrogen benzide and TEMPO, the multipolymer of the triple sensitivities of reduction (referring to Multi-responsive copolymers:using thermo-; Light-and redox stimuli as three independent inputs towards polymeric information processing, Schattling P.; Jochuma F.D.; Theato P.Chem.Commun.2011,47,8859-8861.)
(5) Huang space seminar dawn has synthesized PPEGMA-b-P2VP double-hydrophilic multipolymer through the method for SET-LRP and ATRP; The behavior of having studied its self-assembly is (referring to Synthesis of Well-Defined pH-Responsive PPEGMEA-g-P2VP Double Hydrophilic Graft Copolymer via Sequential SET-LRP and ATRP, Zhai S.J.; Song X.M.; Yang D.; Chen W.L.; Hu J.H.; Lu G.L.; Huang X.Y.Journal of Polymer Science:part A:polymer science 2011,49,4055-4064.)
Poly phosphate (polyphosphoesters) is one type of polymkeric substance that main chain is a phosphate ester structure.Discovering in recent years is that the polyester of carbon-chain structure is different with main chain, and therefore the structural similitude of this base polymer and biomacromolecule (like nucleic acid) has fabulous biocompatibility.Through hydrolysis or under physiological condition, carry out enzymic digestion, poly phosphate can biological degradation, and its side chain can be modified by various functional groups, makes on its structure to have variety, thereby has potential application in a lot of fields.It should be noted that; Biodegradable polyester commonly used; Like polycaprolactone (PCL), POLYACTIC ACID (PLA) etc., main chain contains carbonyl and carbon-oxygen bond, in biodegradable process, has carbonic acid gas and generates; Can make partial pH value reduction in the organism after water-soluble, thereby organism is produced certain spinoff.But the main chain of poly phosphate is a phosphate ester structure, and the material after the degraded can not have side effects to organism.
Research to the poly phosphate segmented copolymer in the prior art mainly contains:
(1) the responsive amphipathic nature block polymer PEEP-SS-PCL of reduction; The conveying of using it for cancer therapy drug is (referring to Redox-Responsive Nanoparticles from the Single Disulfide Bond-Bridged Block Copolymer as Drug Carriers for Overcoming Multi-Drug Resistance in Cancer Cells, Wang Y.C.; Feng Wang F.; Sun T.M.; Wang J.Bioconjugate Chem.2011,22,1939-1945.)
(2) based on the amphipathic three block copolymer mPEG-b-PCL-b-PPEEA of poly phosphate; Self-assembly forms micella, and the double carrier that is used for anticancer drugs, doxorubicin (DOX) and siRNA is (referring to Simultaneous Delivery of siRNA and Paclitaxel via a " Two-in-One " Micelleplex Promotes Synergistic Tumor Suppression.Sun T.M.; Du J.Z.; Yao Y.D.; Mao C.Q.; Dou S.; Huang S.Y.; Zhang, P.Z.; Leong K.W.; Song E.W.; Wang J.ACS NANO 2011,5,1483-1494.)
(3) side chain contains the poly phosphate that can click the chemical reaction group and the amphipathic nature block polymer of aliphatic polyester; Self-assembly formation micella in water (referring to: Syntheses and Characterization of Block Copolymers of Poly (aliphatic ester) with Clickable Polyphosphoester, Wang Y.C.; Yuan Y.Y.; Wang F.; Wang J..Journal of Polymer Science:Part A 2011,49,487-494.)
(4) based on the thermosensitive polymer of poly phosphate (referring to Novel Thermoresponsive Polymers Having Biodegradable Phosphoester Backbones, Iwasaki Y.; Wachiralarpphaithoon C.; Akiyoshi K.Macromolecules 2007,40,8136-8138.)
(5) Yan De high mountain seminar has synthesized and contains arm and be the hyperbranched thing HPHSEP-star-PEP of poly phosphate with nuclear; Link to each other with reductibility S-S key between arm and the nuclear; Self-assembly forms the responsive micella of reduction; The conveying that is used for anticancer drugs, doxorubicin (DOX) is (referring to Redox Responsive Polyphosphate Nanosized Assemblies:A Smart Drug Delivery Platform for Cancer Therapy, Liu J.Y.; Pang Y.; Huang W.; Zhu Z.Y.; Zhu X.Y.; Zhou Y.F.; Yan D.Y.Biomacromolecules 2011,12,2407-2415.)
(6) inventor has also applied for the Chinese invention patent of relevant poly phosphate structure before, obtains the authorization.The patent No. is that China's invention of ZL 200810022216.2 is applied for a patent, and discloses a kind of biodegradable pH responsiveness aquagel and preparation method thereof.At first utilize the method for ring-opening polymerization to prepare the poly phosphate that the two ends of molecular chain contain carbon-carbon double bond, it as the macromolecules cross-linking agent, is carried out copolymerization with pH responsiveness monomer again, form the hydrogel that has the pH responsiveness, contains poly phosphate.The patent No. is the Chinese invention patent of ZL200910034471.3; Disclose a kind of double-hydrophilic block copolymer PEEP-b-PDMAEMA that contains biodegradable poly phosphate and the response of pH/ temperature dual and preparation method thereof, studied the self-assembly behavior under its different pH and the temperature condition.
But, also do not have so far to have described in document or patent report such as the present invention pH responsiveness and biodegradability double-hydrophilic block copolymer structure and as the application of pharmaceutical carrier.Double-hydrophilic block copolymer in the process like self-assembly in water, is avoided the use of organic solvent because of its particular performances, makes it at biomedical aspect the potential using value arranged.
Summary of the invention
The objective of the invention is to double-hydrophilic block copolymer pharmaceutical carrier that discloses a kind of pH of having responsiveness and biodegradability and preparation method thereof.
General plotting of the present invention is: the technology that adopts ring-opening polymerization (ROP), ATRP (ATRP) and side chain terminal to modify successively, preparation double-hydrophilic block copolymer.Utilize at first that an end is halogen-containing, the small organic molecule HO-R of the other end hydroxyl
2-X under the katalysis of stannous octoate, carries out ring-opening polymerization to the annular phosphate monomer as initiator, obtains the functional polyalkylene SULPHOSUCCINIC ACID ESTER macromole evocating agent HO-PPE-X that molecule chain end has bromine or chlorine substituent; The halogen that utilizes the HO-PPE-X chain end then under the effect of ATRP catalyzer and catalyst ligand, carries out solution A TRP reaction with Rocryl 400 (HEMA) monomer as macromole ATRP initiator; Last PHEMA segment pendant hydroxyl group and acid anhydrides generation esterification, acquisition contains the double-hydrophilic di-block copolymer of poly phosphate and pH responsiveness.This double-hydrophilic di-block copolymer is under certain pH conditions, and self-assembly forms micellar structure, can wrap and carry hydrophobic drug, and micella dissociates under weak basic condition, discharges medicine, realizes the purpose of controlled release.
For achieving the above object, technical scheme provided by the invention is: have the double-hydrophilic block copolymer pharmaceutical carrier of pH responsiveness and biodegradability, its chemical structural formula is:
In the formula, m is 20～50, and n is 40～200; 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:
A kind of among
;
R
3Group is selected from :-CH
2CH
2-or
In a kind of.
Adopt ATRP (ATRP), ring-opening polymerization (ROP) and side chain esterification to modify the method preparation of coupling.
The number-average molecular weight scope of the wetting ability segmented copolymer described in the technique scheme is: 4000～50000g mol
-1
In actual mechanical process,, can make with extra care raw material and solvent earlier for guaranteeing the success of preparation; Its method is: mix with the long-pending deionized water of triploid earlier before the monomer Rocryl 400 uses, use twice of n-hexane extraction then after, with adding in the aqueous solution of lower floor after sodium hydrogencarbonate stirs 1 hour; Adding sodium-chlor saltouts; The Rocryl 400 that obtains is used anhydrous magnesium sulfate drying 24h, remove by filter the sal epsom post precipitation, under the protection of high pure nitrogen, carry out underpressure distillation; Solvents tetrahydrofurane after dry 3 days, adds UVNUL MS-40 as indicator with solid potassium hydroxide, carries out anaerobic with the sodium silk and is back to and is grape, steams before the use, steams existing usefulness at present.
Above-mentioned double-hydrophilic block copolymer with pH responsiveness and biodegradability; The poly phosphate segment that had both contained good biocompatibility and biodegradability; As hydrophilic segment; Contain the responsive polyanion electrolyte segment of pH again, through regulating the pH value of medium, the polyanion segment is reversible change to be hydrophilic or hydrophobic segment.Such double-hydrophilic polymkeric substance is under certain pH conditions, and self-assembly forms micella, and hydrophilic poly phosphate segment forms the micellar shell, and hydrophobic polyanion segment forms the micellar kernel, is used to wrap up hydrophobic medicine, as pharmaceutical carrier.Under weak basic condition, micella dissociates, and discharges medicine, realizes the purpose of controlled release.
For achieving the above object, technical scheme provided by the invention is: a kind of functional macromolecule initiator is characterized in that: molecular chain one end of functional polyalkylene SULPHOSUCCINIC ACID ESTER macromole evocating agent contains hydroxyl; The other end contains bromine or chlorine substituent, and the chemical structural formula of said macromole evocating agent is:
In the formula, m is 20～50; 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:
A kind of among
.
The preparation method of above-mentioned functions property poly phosphate macromole evocating agent HO-PPE-X comprises following concrete steps:
(1) preparation annular phosphate monomer: with R
1-OH with
(COP) react the synthetic R that has
1The annular phosphate monomer of side substitution group
R wherein
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.
(2) preparation functional macromolecule ATRP initiator: utilize to have the brominated of terminal hydroxy group functional group or chlorine organic cpds HO-R
2-X carries out ring-opening polymerization to the annular phosphate monomer in the presence of octoate catalyst is stannous, obtain the functional macromolecule initiator of the brominated or chlorine substituent of molecule chain end.
Wherein, organic cpds HO-R
2R among the-X
2Be selected from:
A kind of among
; X is selected from bromine or chlorine.
The method for preparing the double-hydrophilic block copolymer of the above-mentioned pH of having responsiveness and biodegradability comprises following concrete steps:
(1) utilizes the SULPHOSUCCINIC ACID ESTER of functional polyalkylene described in technique scheme macromole evocating agent HO-PPE-X terminal bromine or cl radical; Under the effect of ATRP catalyzer and catalyst ligand; With monomer Rocryl 400 (HEMA;
) carry out solution A TRP reaction; Obtain the di-block copolymer PPE-b-PHEMA of poly phosphate and poly hydroxy ethyl acrylate, its chemical structural formula is:
In the formula, m is 20～50, and n is 40～200; 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:
A kind of among
;
In the technique scheme, said ATRP catalyzer is selected from a kind of in cuprous bromide or the cuprous chloride;
In the technique scheme, said catalyst ligand is selected from a kind of in dipyridyl, pentamethyl-trimethylene triamine, Tetramethyl Ethylene Diamine or the hexamethyl Triethylenetetramine (TETA);
In the technique scheme, said monomer Rocryl 400 [M] with the mol ratio of poly phosphate macromole ATRP initiator [I] is: [M]: [I]=40: 1～200: 1; Macromole ATRP initiator [I] with the mol ratio of catalyzer [Cat] is: [I]: [Cat]=0.5: 1～1: 2; Catalyzer [Cat] with the mol ratio of catalyst ligand [Ligand] is: [Cat]: [Ligand]=1: 1～1: 2.
(2) hydroxyl that utilizes prepared di-block copolymer PPE-b-PHEMA side chain respectively with maleic anhydride (SA;
) or 1; 2-cyclohexyl dicarboxylic acid acid anhydride (CDA;
) the generation esterification; Obtain parents' water di-block copolymer (being abbreviated as PPE-b-PSEMA or PPE-b-PCEMA) that side chain has carboxyl, its chemical structural formula is:
In the formula, m is 20～50, and n is 40～200; 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:
A kind of among
;
R
3Group is selected from :-CH
2CH
2-or
In a kind of.
Further in the technical scheme, reaction is carried out purification processes after accomplishing, and said purification processes may further comprise the steps:
(1) purification processes of PPE-b-PHEMA di-block copolymer: reaction finishes the back rotary evaporation and removes a large amount of solvents, with the THF deposition, removes unreacted monomer and homopolymer.The product that post precipitation is obtained with deionized water dissolving after dialysis (select dialysis tubing interception 1000～5000g mol for use
-1) 2～3 days, with the dialysis solution lyophilize, obtain white product at last.
(2) the double-hydrophilic block copolymer PPE-b-PSEMA of pH response or the purification processes of PPE-b-PCEMA: after reaction finishes; Reaction soln is precipitated in the ice anhydrous diethyl ether except that desolvating and excessive acid anhydrides; After the repeated precipitation 3 times, the product that post precipitation is obtained with deionized water dissolving after dialysis (select dialysis tubing interception 3500～7000g mol for use
-1) 2～3 days, clean until solvent and raw material are removed fully, with the dialysis solution lyophilize, obtain white product at last, promptly said pH response double-hydrophilic block copolymer.
Because the enforcement of such scheme, the present invention compared with prior art has advantage:
1, the present invention adopts through ring-opening polymerization, ATRP (ATRP) and side chain esterification and modifies coupling technique; Obtain the double-hydrophilic di-block copolymer; Wherein, the poly phosphate with good biocompatibility and biological degradability is as hydrophilic segment, the polymethacrylate segment that side group is modified; Owing to contain carboxyl in the structural unit, thereby the good pH responsiveness of tool.
2, the double-hydrophilic block copolymer of the present invention's acquisition avoids the use of organic solvent in self assembling process, only regulates and control the behavior of its self-assembly through regulating medium pH, thereby can form polymer micelle, better it is applied to the biological medicine aspect.
3, the di-block copolymer useful as drug carrier that obtains among the present invention; This double-hydrophilic block copolymer had both contained the poly phosphate segment of good biocompatibility and biodegradability; As hydrophilic segment, contain the responsive polyanion electrolyte segment of pH again.Through regulating the pH value of medium, the polyanion segment is reversible change to be hydrophilic or hydrophobic segment.Such double-hydrophilic block copolymer is under certain pH conditions, and self-assembly forms micella, and hydrophilic poly phosphate segment forms the micellar shell, can better stablize micella, realizes carrier micelle long circulation in vivo.Hydrophobic polyanion segment forms the micellar kernel, is used to wrap up hydrophobic medicine, as pharmaceutical carrier.Under weak basic condition, micella dissociates, and discharges medicine, realizes the purpose of controlled release.
Description of drawings
Fig. 1 is macromole ATRP initiator (HEBI-PEEP among the embodiment one
32) proton nmr spectra (
1H NMR), solvent is CDCl
3
Fig. 2 is di-block copolymer (PEEP among the embodiment two
32-b-PHEMA
128) proton nmr spectra (
1H NMR), solvent is DMSO-d
6
Fig. 3 is double-hydrophilic block copolymer (PEEP among the embodiment three
32-b-PSEMA
128) proton nmr spectra (
1H NMR), solvent is DMSO-d
6
Fig. 4 is PEEP among the embodiment four
32-b-PSEMA
128The Electronic Speculum figure of double-hydrophilic block copolymer pH responsiveness self-assembled micelle, wherein, (a) pH 3.3, and (b) pH 5.0, and (c) pH 7.4;
Fig. 5 is PEEP among the embodiment five
32-b-PSEMA
128The double-hydrophilic block copolymer carrier micelle is the medicine release profiles under condition of different pH;
Fig. 6 be among the embodiment six different segmented copolymers (PEEP-b-PSEMA) in external toxotest result to the HeLa cell.
Embodiment
Below in conjunction with embodiment and accompanying drawing the present invention is further described:
Embodiment one: preparation macromole ATRP initiator PEEP
32The concrete compound method of-Br:
(1) the arm round-bottomed flask that will put into stirrer behind 120 ℃ of dry at least 24 h 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.
(2) with the exsiccant syringe from arm, inject successively a certain amount of initiator bromo acid-2-hydroxy methacrylate (HEBI) (0.14g, 0.66mmol), 2-oxyethyl group-2-oxygen-1,3,2-dioxaphospholane (EEP) monomer (4g, 26mmol) and Sn (Oct)
2(EEP monomer: Sn (Oct)
2=1: 0.002, mass ratio), with anhydrous tetrahydro furan (15mL) as solvent.After in reaction flask, being full of argon gas, stirring reaction 1h in 40 ℃ oil bath.
(3) after reaction finishes, most of solvents tetrahydrofurane is revolved steaming remove, deposition is removed unreacted monomer twice in the ice anhydrous diethyl ether, places vacuum drying oven to dry to constant weight product, obtains transparent products, records productive rate and is approximately 80%.
The structure of above-mentioned monomer EEP is following:
Embodiment two: di-block copolymer PEEP
32-b-PHEMA
128Concrete preparation method:
(a) the arm round-bottomed flask is handled according to the method described above after, be full of argon gas, take by weighing rapidly and add PEEP
32-Br (2.25g, 0.45mmol), cuprous bromide (0.06g, 0.45mmol) and dipyridyl (0.14g; 0.45mmol) (three's mol ratio is 1: 1: 1), extraction gas after three times, is injected into 5mL solvent anhydrous methanol repeatedly; Be stirred to mixture and dissolve fully, it is brown that solution is.
(b) round-bottomed flask is moved in the oil bath, (8.7g 67.5mmol), utilizes PEEP to add monomer Rocryl 400 (HEMA) with the dry injection device again
32Bromine terminal in the-Br homopolymer causes ATRP reaction, stirring reaction 6h in 30 ℃ oil bath as initiator.
(c) after reaction finishes; Ingress of air makes reaction terminating; Most of solvent is removed in mixed with polymers solution rotating evaporation, in THF, precipitate to remove to desolvate and unreacted monomer, the product that post precipitation is obtained with deionized water dissolving after dialysis (select dialysis tubing interception 3500g mol for use
-1) 2～3 days, with the dialysis solution lyophilize, obtain white product at last, i.e. di-block copolymer PEEP
32-b-PHEMA
128
Embodiment three: pH response double-hydrophilic block copolymer PEEP
32-b-PSEMA
128Concrete compound method:
(1) the arm round-bottomed flask is handled according to the method described above after, be full of argon gas, add rapidly and be dissolved in the above-mentioned bi-block copolymer PEEP in the 10mL anhydrous pyridine
32-b-PHEMA
128(3g, the mole number of hydroxyl (HO-) is 16.64mmol) adds succinyl oxide under magnetic agitation
(3.328g, 33.28mmol) (mol ratio of hydroxyl and succinyl oxide is 1: 2) reacted 48h at normal temperatures.
(2) after reaction finishes, reaction soln precipitated to remove in 150mL ice anhydrous diethyl ether desolvates and unreacted monomer, after the repeated precipitation 3 times, the product that post precipitation is obtained with deionized water dissolving after dialysis (select dialysis tubing interception 3500g mol for use
-1) 2～3 days, with the dialysis solution lyophilize, obtain white product at last, i.e. PEEP
32-b-PSEMA
128Di-block copolymer.
Embodiment one, example two and routine three products therefroms through proton nmr spectra checking structure, prove to successfully synthesize the purpose product that concrete characterization result is seen Fig. 1～3.
Embodiment four: preparation double-hydrophilic block copolymer micella
Method 1: the carboxylic di-block copolymer PEEP-b-PSEMA of 5mg is joined in the round-bottomed flask; Add 5mL basic soln (pH=7～8), stirred 4～6 hours, polymer chain is unfolded fully come; Under condition of stirring, utilize the micro-sampling pump with 0.5mL h
-1Speed with 0.5mol L
-1Hydrochloric acid soln add, treat complete sample introduction after, behind the ultrasonic 10min, stirred 2～3 days, be 1mg mL thereby obtain concentration
-1Micellar solution.
Method 2: the carboxylic di-block copolymer of 5mg is added in the round-bottomed flask, directly add the buffered soln of the different pH values of 5mL, behind the ultrasonic 10min, stirred 2～3 days, thus the micellar solution of obtaining.
Utilize transmission electron microscope observing gained micellar pattern and size, concrete outcome is seen Fig. 4.From figure, can see, change the pH value of medium, carboxylic double-hydrophilic di-block copolymer PEEP
32-b-PSEMA
128Self-assembly forms the big or small micella of different-grain diameter.Table 1 is the size distribution test data of self-assembled micelle, also can find out from table 1 data, and in pH 5.0 buffered soln, the PSEMA segment is not charged, has hydrophobicity, and the particle diameter of particle diameter ratio in pH 7.4 buffered soln is little.This is that PSEMA segment part is electronegative, becomes hydrophilic radical because in the medium of pH 7.4, and the micella particle diameter that therefore forms increases.
Table 1
Embodiment five: adopt dialysis method to prepare carrier micelle
Take by weighing 25mg PEEP respectively
32-b-PSEMA
128Di-block copolymer and 5mg Naproxen Base (a kind of anti-inflammatory, analgesic, analgesic medicine) join in the round-bottomed flask, add 5mL organic solvent DMF; Stirred 4～6 hours, the state that polymer chain is in unfold fully still is under the state of stirring then; Utilize sampling pump ultrapure water to be added with the speed of 2mL/h; After treating complete sample introduction, 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 37 ℃ water bath with thermostatic control, discharges.Get 5mL dialysis tubing external solution at set intervals, replenish the fresh buffer of the identical pH value of 5mL simultaneously.The content of the Naproxen Base that discharges with UV spectrophotometer measuring.It is as shown in Figure 5 to wrap the release profiles of polymer micelle under condition of different pH that is loaded with Naproxen Base.Drug release rate explains that faster than rate of release in the medium of pH 5.0 parents' water block copolymer micelle has the pH responsiveness, can reach the purpose of controlled release medicine when pH 7.4.
Embodiment six: the cytotoxicity test
Human cervical carcinoma cell (HeLa cells) is cultivated in the RPMI-1640 substratum that is supplemented with 10% foetal calf serum (FBS), placed 37 ℃, 5% CO
2Cultivate the periodic replacement nutrient solution in the incubator of (relative humidity is 90%).The cell inoculation that selection is in the active growth phase contains in 96 orifice plates of 100 μ LRPMI-1640 substratum in every hole, cultivates 24h.Dispose mother liquid concentration (5mg mL with dialysis method
-1) double-hydrophilic di-block copolymer PEEP
32-b-PSEMA
128And PEEP
32-b-PSEMA
180Micella, the micellar solution of a series of different concns is joined in 96 orifice plates, continue to cultivate 48h.The MTT reagent that then adds 25 μ L, further cultivate 30min after, under 490nm, measure corresponding absorbancy with ELIASA (Bio-Rad model 680).The method of calculation of cell survival rate are: cell survival rate (Cell viability) (%)=[A]
Test/ [A]
Control* 100%, wherein [A]
TestBe the absorbancy that in the presence of polymkeric substance, records, and [A]
ControlThe absorbancy that records under the situation for addition polymerization compound not.Its MV is got in each sample test three times.As shown in Figure 6, institute's synthetic double-hydrophilic block copolymer does not have toxic side effect basically to the HeLa cell, proves that double-hydrophilic block copolymer has good biocompatibility.
Claims (8)
1. double-hydrophilic block copolymer with pH responsiveness and biodegradability is characterized in that: said segmented copolymer, and its chemical structural formula is:
In the formula, m is 20～50, and n is 40～200; 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, the chemical structural formula of the end capped polyethylene oxide base of said monomethyl is :-(CH
2CH
2O)
kCH
3, k is 2～10 in the formula;
R
2Group is selected from:
In a kind of;
R
3Group is selected from :-CH
2CH
2-or
In a kind of.
2. the double-hydrophilic block copolymer with pH responsiveness and biodegradability according to claim 1 is characterized in that, the number-average molecular weight of described double-hydrophilic block copolymer
Scope is: 4000～50000g mol
-1
3. a poly phosphate macromole evocating agent is characterized in that, its chemical structural formula is:
In the formula, m is 20～50; 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, the chemical structural formula of the end capped polyethylene oxide base of said monomethyl is :-(CH
2CH
2O)
kCH
3, k is 2～10 in the formula;
R
2Group is selected from:
In a kind of.
4. a method for preparing the double-hydrophilic block copolymer of the said pH of having responsiveness of claim 1 and biodegradability is characterized in that, comprises the following steps:
(1) preparation annular phosphate monomer: with R
1-OH with
React the synthetic R that has
1The annular phosphate monomer of side substitution group
(2) preparation functional polyalkylene SULPHOSUCCINIC ACID ESTER macromole evocating agent: utilize HO-R
2-X, the annular phosphate monomer that in the presence of octoate catalyst is stannous, step (1) is obtained carries out ring-opening polymerization, obtains the poly phosphate macromole evocating agent;
(3) terminal bromine or the cl radical of poly phosphate macromole evocating agent that utilizes step (2) to obtain; Under the effect of ATRP catalyzer and catalyst ligand; Carry out solution A TRP reaction with the monomer Rocryl 400; Obtain the di-block copolymer PPE-b-PHEMA of poly phosphate and poly hydroxy ethyl acrylate, its chemical structural formula is:
Said ATRP catalyzer is selected from a kind of in cuprous bromide or the cuprous chloride;
Said catalyst ligand is selected from a kind of in dipyridyl, pentamethyl-trimethylene triamine, Tetramethyl Ethylene Diamine or the hexamethyl Triethylenetetramine (TETA);
The mol ratio of monomer and macromole evocating agent is (40～200): 1; The mol ratio of macromole evocating agent and catalyzer is (0.5～1): (1～2); The mol ratio of catalyzer and catalyst ligand is 1: (1～2);
(4) utilize the pendant hydroxyl group and maleic anhydride or 1 of the di-block copolymer PPE-b-PHEMA that step (3) obtains, 2-cyclohexyl dicarboxylic acid acid anhydride generation esterification, the acquisition side chain has the double-hydrophilic block copolymer of carboxyl, and its chemical structural formula is:
In the chemical formula of above-mentioned each step, m is 20～50, and n is 40～200; 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 said monomethyl is :-(CH
2CH
2O)
kCH
3, k is 2～10 in the formula;
R
2Group is selected from::
In a kind of;
R
3Group is selected from :-CH
2CH
2-or
In a kind of.
5. method according to claim 4 is characterized in that: after the reaction of step (3) and step (4) is accomplished, carry out purification processes respectively, said purification processing method is:
(a) after the reaction of step (3) is accomplished; Rotary evaporation removes and desolvates, and with the THF deposition, removes unreacted monomer and homopolymer; The product that post precipitation is obtained was dialysed 2～3 days after with deionized water dissolving; With the dialysis solution lyophilize, obtain white product at last, be the required di-block copolymer of step (3);
(b) after the reaction of step (4) is accomplished; Reaction soln is precipitated in the ice anhydrous diethyl ether except that desolvating and unreacted monomer; After the repeated precipitation 3 times, the product that post precipitation is obtained was dialysed 2～3 days after with deionized water dissolving, at last with the dialysis solution lyophilize; Obtain white product, promptly said double-hydrophilic block copolymer.
6. method according to claim 5 is characterized in that: it is 3500g mol that the dialysis tubing interception is selected in the dialysis in step (a) and the step (b) for use
-1
7. the said application of claim 1 with double-hydrophilic block copolymer of pH responsiveness and biodegradability as pharmaceutical carrier loading hydrophobic drug.
8. application according to claim 7; It is characterized in that: through regulating the pH value of medium, self-assembly forms micella, and hydrophilic poly phosphate segment forms the micellar shell; Hydrophobic polyanion segment forms the micellar kernel, is used to wrap up hydrophobic medicine; Under weak basic condition, micella dissociates, and discharges medicine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104173282A (en) * | 2014-07-29 | 2014-12-03 | 苏州大学 | Polyphosphoester-based folate-targeted acid-sensitive core-crosslinked drug-loaded micelle and preparation method thereof |
| CN108503844A (en) * | 2018-05-03 | 2018-09-07 | 苏州大学 | Reduction responsive type copolymer and the preparation method and application thereof based on poly- disulfide and polyphosphate |
| CN108832175A (en) * | 2017-11-07 | 2018-11-16 | 江汉大学 | A kind of method for preparing polymer electrolytes |
| CN113621114A (en) * | 2021-08-11 | 2021-11-09 | 四川冠山科技有限公司 | Amphiphilic block copolymer wormlike micelle and preparation method and application thereof |
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| WO2007109584A1 (en) * | 2006-03-16 | 2007-09-27 | University Of Washington | Temperature-and ph-responsive polymer compositions |
| CN101649034A (en) * | 2009-08-31 | 2010-02-17 | 苏州大学 | pH/temperature response double hydrophilic block copolymer |
| CN101328251B (en) * | 2008-06-27 | 2010-06-09 | 苏州大学 | Biodegradable temperature/pH dual responsiveness aquagel |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007109584A1 (en) * | 2006-03-16 | 2007-09-27 | University Of Washington | Temperature-and ph-responsive polymer compositions |
| CN101328251B (en) * | 2008-06-27 | 2010-06-09 | 苏州大学 | Biodegradable temperature/pH dual responsiveness aquagel |
| CN101649034A (en) * | 2009-08-31 | 2010-02-17 | 苏州大学 | pH/temperature response double hydrophilic block copolymer |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104173282A (en) * | 2014-07-29 | 2014-12-03 | 苏州大学 | Polyphosphoester-based folate-targeted acid-sensitive core-crosslinked drug-loaded micelle and preparation method thereof |
| CN105832668A (en) * | 2014-07-29 | 2016-08-10 | 苏州大学 | Folic acid-targeted acid sensitive core-crosslinked drug-loaded micelles based on polyphosphoester |
| CN104173282B (en) * | 2014-07-29 | 2017-03-29 | 苏州大学 | Folate-targeted acid-sensitive core crosslinking carrier micelle based on poly phosphate and preparation method thereof |
| CN105832668B (en) * | 2014-07-29 | 2018-09-25 | 苏州大学 | Folate-targeted acid-sensitive core based on polyphosphate is crosslinked carrier micelle |
| CN108832175A (en) * | 2017-11-07 | 2018-11-16 | 江汉大学 | A kind of method for preparing polymer electrolytes |
| CN108503844A (en) * | 2018-05-03 | 2018-09-07 | 苏州大学 | Reduction responsive type copolymer and the preparation method and application thereof based on poly- disulfide and polyphosphate |
| CN108503844B (en) * | 2018-05-03 | 2020-08-04 | 苏州大学 | Reduction-sensitive copolymer based on polydisulfide and polyphosphate ester and preparation method and application thereof |
| CN113621114A (en) * | 2021-08-11 | 2021-11-09 | 四川冠山科技有限公司 | Amphiphilic block copolymer wormlike micelle and preparation method and application thereof |
| CN113621114B (en) * | 2021-08-11 | 2023-06-20 | 四川冠山科技有限公司 | Amphiphilic block copolymer worm-like micelle and preparation method and application thereof |
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