CN105832668B - Folate-targeted acid-sensitive core based on polyphosphate is crosslinked carrier micelle - Google Patents

Folate-targeted acid-sensitive core based on polyphosphate is crosslinked carrier micelle Download PDF

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CN105832668B
CN105832668B CN201610322771.1A CN201610322771A CN105832668B CN 105832668 B CN105832668 B CN 105832668B CN 201610322771 A CN201610322771 A CN 201610322771A CN 105832668 B CN105832668 B CN 105832668B
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folate
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倪沛红
胡健
何金林
张明祖
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Suzhou University
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Abstract

The invention discloses a kind of, and the folate-targeted acid-sensitive core based on polyphosphate is crosslinked carrier micelle.First, amphipathic phosphoric acid ester block copolymer is prepared using " one kettle way " open loop；Its terminal hydroxyl is modified again, obtains the block copolymer of modified with folic acid；Then, hydroxyl modified on tetraethylene glycol is contained to the molecule of acetal radical and azido for end；Finally, acid-sensitive core is prepared using " CuAAC " click-reaction and is crosslinked carrier micelle.Core crosslinking micella prepared by the present invention can contain hydrophobic anticancer drug by self assembly, and in acid condition, due to the fracture of acetal radical, micellar structure be caused to be destroyed, and release package-contained drug, may be used as controllable in drug release carrier.Core crosslinking micella disclosed by the invention may be used as efficient controllable in drug release carrier, have good application value in biomaterial and biomedicine field.

Description

Folate-targeted acid-sensitive core based on polyphosphate is crosslinked carrier micelle
The application is that application No. is the divisional application of 2014103660130 Chinese invention patent application, the Shens of original application Please day be：On July 29th, 2014, application No. is：2014103660130, it is entitled：Folate-targeted based on polyphosphate Acid-sensitive core is crosslinked carrier micelle and preparation method thereof.
Technical field
The invention belongs to field of biomedical polymer materials, and in particular to a kind of folate-targeted acid based on polyphosphate The degradable core of sensitive biological is crosslinked carrier micelle.
Background technology
Amphipathic copolymer is generally made of hydrophilic and hydrophobic segment, due to its can be self-assembled into aqueous solution it is a variety of Shape nano-particle, such as micella, vesica, nanometer rods and thin slice have in fields such as biological medicine, supermolecule and nanosecond science and technology Wide application prospect.It is well known that most anticancer drugs all has a very strong hydrophobicity, and amphipathic copolymer self assembly The micella of the nucleocapsid structure of formation has hydrophobic cores, can be used for containing hydrophobic anticancer drug, and hydrophilic shell It is water-soluble can to play the role of stable micella, increase drug, and greatly improves the circulation time of carrier micelle in vivo.
The amphipathic copolymer carrier micelle of tradition mainly contains hydrophobic anticancer drug by physically trapping mode.When its note There are certain thermodynamic phases after being mapped in vivo, in vivo in cyclic process, since body fluid dilutes（Less than critical micell Concentration value）And body fluid complex environment the effects that, micellar structure is more easily damaged, and anticancer drug is made easily to be diffused out out of micella Come, causes drug relatively low in cancerous issue position enrichment degree, seriously affect therapeutic effect.Meanwhile the toxicity of drug is to human body Bring many adverse reactions.In recent years, document report mostly uses crosslinking method to reduce anticancer drug in vivo in cyclic process Diffusion, increase drug cancerous issue position enrichment.
There are mainly two types of the crosslinked methods of micella：" physical crosslinking method（Physical cross-links）" and " chemistry is handed over Connection method（Chemical cross-links）”.Physical crosslinking micella is mainly formed by non-covalent bond effect, such as：Electrostatic is made With, hydrogen bond, coordinate bond, hydrophobic effect and supermolecule complexing etc.；And micella is chemically crosslinked mainly by covalent bond effect shape At i.e. functional copolymer molecule interchain reacts to each other or add functional small molecule crosslinking agent and copolymer chain Learn reaction.According to the difference of micella crosslinking sites, crosslinking micella can be divided into " core crosslinking micella（Core cross-linked micelle）" and " shell is crosslinked micella（Shell cross-linked micelle）", this crosslinking micella is as pharmaceutical carrier It can be stabilized in cyclic process in vivo.
Although the research of pharmaceutical carrier has had considerable progress, it is applicable to the product category of clinical test It is limited, it is difficult to carry out drug release in specific time and specific position with expected speed after pharmaceutical carrier reaches in vivo, This becomes the biggest obstacle of limitation pharmaceutical carrier development.Cross-linking nanoparticles with good stability are in vivo in cyclic process It is effective to extend circulation time, but after entering cancer cell, carrier micelle will be faced with expected speed when specific Between and specific position carry out drug delivery problems, this become limitation pharmaceutical carrier development biggest obstacle.In this regard, researcher sets Meter develops intelligent response nano-particle pharmaceutical carrier, and this kind of pharmaceutical carrier is stimulated by from external environment（Including temperature Degree, Redox Condition, light and pH value etc.）When, respective change can occur for property, and using this point, researcher designs synthesis A variety of stimulating responsive nano-particle pharmaceutical carriers.When by environmental stimuli, nano particle structure can be destroyed, thus will The drug release contained inside it comes out, and reaches the controlled release of drug.
In general, in human body the pH value of different tissues and organelle can difference, such as：Blood and normal tissue site PH value be generally 7.4, the pH value of tumour and diseased region is 6.5 or so, and the pH in endosome and lysosome is even up to 5.0~5.5.Using this feature, the polymer micelle with acid-sensitive can be designed, these micellas can be in preparation process Hydrophobic drug is contained, into after body-internal-circulation, carrier micelle can be stabilized under the conditions of normal structure and body fluid, and be arrived Up under the acidic environments such as tumor locus, acid sensitive group can change, and micellar structure is caused to be destroyed, the drug release that will be contained Out, reach controlled drug release purpose.Common acid sensitive group includes mainly two kinds：One kind being reversible acid sensitive group, Including-COOH ,-NH2With-SO3H etc. is mainly acted on by protonation under different pH environment and deprotonation；Separately One is irreversible sour breaking bonds, including acetal radical, ketal group, hydrazone bond, oxime key and orthoformate etc., in acid condition, Irreversible chemical bond rupture can occur for these chemical bonds.
Traditional cross-linked structure micella is worked as poly- mostly using polyethylene glycol and polyester with good biocompatibility When molecular weight glycol is larger, it is difficult to it is internal by being metabolized discharge, and polyphosphate is similar to the structure of large biological molecule nucleic acid, There is fabulous biocompatibility, moreover, in vivo in the presence of phosphodiesterase, phosphate can hydrolyze, and have good biology Degradability；In addition, traditional cross-linked structure micella mostly use ester bond or reduction sensitive structure carry out Drug controlled release, have no by Acid sensitive group introduces the report of core crosslinking micella.
Ideal pharmaceutical carrier should have good biocompatibility and biodegradability, also, as antitumor The carrier of drug should also have following features：Stable polymer micelle, hydrophobic cores can be formed in aqueous solution Hydrophobic anticancer drug can be contained, hydrophily shell plays the role of stable micella；It in vivo can be to avoid in cyclic process The diffusion of drug and the aggregation of carrier improve micella circulation time in vivo；When carrier micelle reaches tumour or pathological tissues, Active or passive target can be utilized, cancer cell is efficiently entered；Meanwhile micella enters after cancer cell and should have " intelligence " loud Ying Xing reaches the control release of drug under cancer cell particular surroundings.Although having been achieved for growing for the research of pharmaceutical carrier The progress of foot, but the development of pharmaceutical carrier still suffers from huge challenge.
Therefore, it is necessary to the core crosslinking micellar structures for researching and developing novel to be used for pharmaceutical carrier.
Invention content
The object of the present invention is to provide a kind of, and the folate-targeted acid-sensitive core based on polyphosphate is crosslinked carrier micelle, the core Being crosslinked carrier micelle has good biocompatibility, biodegradability and sensitivity to acid, may be used as stimulating responsive medicine Objects system.
The present general inventive concept is：By ring-opening polymerisation（ROP）With the cycloaddition reaction of end alkynyl radical and nitrine（CuAAC）Connection With the acid-sensitive biodegradable core for synthesizing folate-targeted is crosslinked carrier micelle.First, it is to cause with the small molecule of hydroxyl Agent carries out ring-opening polymerisation to cyclic phosphate esters monomer, obtains amphipathic phosphoric acid ester block copolymer；Then, with folic acid pair The block copolymer terminal hydroxyl is modified, and the polyphosphate block copolymer of modified with folic acid is obtained；Later, from bishydroxy It closes object and the small molecule that azido and acetal radical are contained in end is prepared；Finally, hydrophobic anticancer drug, side chain are contained into alkynyl Modified with folic acid polyphosphate block copolymer and end contain the small molecule self assembly in aqueous solution of azido, and carry out " CuAAC " click-reaction obtains the core crosslinking carrier micelle with acid-sensitive.
In order to achieve the above objectives, the technical solution adopted by the present invention is that, a kind of folate-targeted based on polyphosphate is acid-sensitive Feel core and be crosslinked carrier micelle, preparation method is as follows：
(1) polyphosphate block copolymer is prepared：In inert atmosphere, in 1,8- diazabicylos [5.4.0], 11 carbon- Under the catalytic action of 7- alkene, using dichloromethane as solvent, under the conditions of 10~50 DEG C, using isopropanol as initiator, with cyclic annular phosphorus Acid estersRing-opening polymerisation is carried out for monomer, is reacted 30 seconds~60 minutes；Add cyclic phosphate ester monomer, the reaction was continued 30 seconds~60 minutes；Obtain polyphosphate block copolymer；
In annular phosphate monomer structure formula, R1For CH2Or CH2CH2；R2For methyl, ethyl, isopropyl, butyl or single first One kind in the polyethylene oxide base of base sealing end；The chemical structural formula of the polyethylene oxide base of monomethyl sealing end is： (CH2CH2O)xCH3, x=2~10 in formula；M=20~90, n=20~90；
The initiator,、And 11 carbon -7- alkene of 1,8- diazabicylos [5.4.0] Molar ratio is 1: (20~90): (20~90): (0.1~2)；
Above-mentioned reaction equation is as follows：
(2) the polyphosphate block copolymer of modified with folic acid is prepared：Under the catalytic action of dicyclohexylcarbodiimide, With folic acid andNHOSu NHS is raw material, using dimethyl sulfoxide as solvent, in the presence of 4-dimethylaminopyridine, in 10 ~40 DEG C are reacted 8~16 hours, add the polyphosphate block copolymer of step (1) preparation, stirring, the reaction was continued 8~48 Hour, obtain the polyphosphate block copolymer of modified with folic acid；
The polyphosphate block copolymer, folic acid,NHOSu NHS, dicyclohexylcarbodiimide and 4- diformazans The molar ratio of aminopyridine is 1: (1~2): (1.2~3): (1.2~3): (0.1~1)；
Above-mentioned reaction equation is as follows：
R in formula1For CH2Or CH2CH2；R2For the polyethylene oxide base of methyl, ethyl, isopropyl, butyl or monomethyl sealing end In one kind；The chemical structural formula of the polyethylene oxide base of monomethyl sealing end is：(CH2CH2O)xCH3, x=2 in formula~ 10；M=20~90, n=20~90；
(3) the acid-sensitive small molecule of azido sealing end is prepared：Under the catalytic action of 4- toluenesulfonic acid pyridines, with HO-R3- OH and 2- chloroethyl vinyl ethers are raw material, under the conditions of ice-water bath, are reacted 0.5~1 hour, and chlorine functional group envelope is prepared The small molecule at end；Then the small molecule of above-mentioned chlorine functional group dead-end, sodium azide are addedN, NDimethyl formamide solution In, it is reacted 30~50 hours in 40~80 DEG C, obtains the acid-sensitive small molecule of azido sealing end；
The HO-R3In-OH, R3For (CH2CH2O)yCH2CH2, wherein y=0~7；
The structural formula of the small molecule of the chlorine functional group dead-end is；
The HO-R3The molar ratio of-OH, 2- chloroethyl vinyl ethers and 4- toluenesulfonic acid pyridines are 1: (4~20): (0.1~0.5)；
The small molecule of the chlorine functional group dead-end and the molar ratio of sodium azide are 1: (4~40)；
Above-mentioned reaction equation is as follows：
(4) it prepares the folate-targeted acid-sensitive core based on polyphosphate and is crosslinked carrier micelle：In inert atmosphere, with hydrophobic Property anticancer drug, step (2) prepare modified with folic acid polyphosphate block copolymer and step (3) prepare azido sealing end Acid-sensitive small molecule be raw material, in cycloaddition click-reaction catalyst, catalyst ligand and the reproducibility of end alkynyl radical and nitrine In the presence of substance, with water/N, NDimethylformamide mixed liquor is solvent, is reacted 18~30 hours in 25~40 DEG C, obtains base It is crosslinked carrier micelle in the folate-targeted acid-sensitive core of polyphosphate；
The alkynyl of the polyphosphate block copolymer of the modified with folic acid, the acid-sensitive small molecule of azido sealing end, Terminal Acetylenes The molar ratio of the cycloaddition click-reaction catalyst of base and nitrine, catalyst ligand and reducing substances is 1: 0.5: 1: (1~2) : (1~2).
In above-mentioned technical proposal, the inert atmosphere is nitrogen or argon gas atmosphere.
In above-mentioned technical proposal, with anhydrous methylene chloride when preparing the small molecule of chlorine functional group dead-end in the step (3) For solvent.
In above-mentioned technical proposal, the polyphosphate block of hydrophobic anticancer drug and modified with folic acid is total in the step (4) The mass ratio of polymers is 1: (2~10).
In above-mentioned technical proposal, in the step (4) hydrophobic anticancer drug be selected from adriamycin, Epi-ADM, taxol, Bortezomib, Aclarubicin, pirarubicin, daunorubicin hydrochloride, Semustine, plicamycin, mitomycin, idarubicin or One kind in levamisol；The cycloaddition click-reaction catalyst of end alkynyl radical and nitrine is selected from stannous chloride, cuprous bromide or iodine Change it is cuprous in one kind；Catalyst ligand is selected from bipyridyl, five methyl diethylentriamine, tetramethylethylenediamine or hexamethyl three One kind in ethylene tetra；The one kind of reducing substances in vitamin C, sodium ascorbate, Vitamin C calcium or citric acid.
In above-mentioned technical proposal, after the completion of step (1)~(4), purification processes, the purification are carried out to product respectively Process includes the following steps：
1) purification of polyphosphate block copolymer：After ring-opening polymerization, by reaction product concentrated by rotary evaporation, then will Concentrate is instilled in methanol/ether mixed liquor and is precipitated, and pours out supernatant, remaining thick liquid is dissolved in methanol solution, then turns It moves on in bag filter, is placed in deionized water and dialyses 24~72 hours, be then freeze-dried, obtain polyphosphate block copolymerization Object；
2) purification of the polyphosphate block copolymer of modified with folic acid：After reaction, reaction solution is filtered, concentration, it will Concentrate is transferred in bag filter, is placed in deionized water and is dialysed 24~72 hours, is taken out, freeze-drying, then uses dichloromethane Dissolving, filtering, concentration, obtain faint yellow thick liquid, by the faint yellow thick liquid of gained air drying 24 in vacuum drying oven ~36 hours, obtain the polyphosphate block copolymer of modified with folic acid；
3) purification of small molecule：
(1) purification of the small molecule of chlorine functional group dead-end：After reaction, it is water-soluble that the sodium carbonate that mass fraction is 5% is added Liquid terminates reaction, is diluted with dichloromethane, adds saturated sodium-chloride phosphate buffer solution, stood after oscillation, separating lower layer has Machine phase, water phase are extracted with dichloromethane again, merge organic phase, and organic phase solution will be steamed through drying, filtering, concentration, air-distillation Resultant product air drying 24~36 hours in vacuum drying oven are evaporated, weak yellow liquid is obtained, are small point of chlorine functional group dead-end Son；
(2) purification of the acid-sensitive small molecule of azido sealing end：After reaction, by reaction product filtering, concentration, then will Concentrate is dissolved in dichloromethane solution, and saturated sodium-chloride phosphate buffer solution is added, is stood after oscillation, it is organic to separate lower layer Phase, water phase are extracted with dichloromethane again, merge organic phase, organic phase solution is through drying, filtering, concentration, using thin layer chromatography Isolated weak yellow liquid, by products therefrom in vacuum drying oven air drying 24~36 hours, obtain azido sealing end Acid-sensitive small molecule；
4) purification of the folate-targeted acid-sensitive core crosslinking carrier micelle based on polyphosphate：It, will after click-reaction Reaction product is transferred in bag filter, is placed in deionized water and is dialysed 24~72 hours, constant volume obtains the leaf based on polyphosphate Acid targeting acid-sensitive core is crosslinked carrier micelle.
In above-mentioned technical proposal, the pH value of the saturated sodium-chloride phosphate buffer solution is 10.0.
The folate-targeted acid-sensitive core crosslinking carrier micelle based on polyphosphate of the present invention is with the folded of acid-sensitive small molecule The alkynyl of nitrogen base and phosphate block polymer is crosslinking points, forms cross-linked structure.
In core crosslinking carrier micelle disclosed by the invention, the amphipathic phosphoric acid ester block copolymer of modified with folic acid is with good Good biocompatibility and biodegradability can contain hydrophobic anticancer containing hydrophilic and hydrophobic segment by self assembly Drug；It is formed by carrier micelle to be chemically crosslinked hydrophobic part using the functional molecular containing acetal radical, prepares knot The core that structure is stablized is crosslinked carrier micelle.In vivo in cyclic process, chemical crosslinking structure effectively extends circulation time in vivo, And folate-targeted mediates after entering cancer cell, and in the cell under acid condition, acetal radical fracture, the chemical crosslinking point of carrier micelle It destroys, in lysosome in the presence of hydrolase, polyphosphate hydrolysis causes the nucleocapsid structure of micella to destroy, releases and wrapped Drug is carried, efficient controlled drug delivery systme is may be used as.
Since said program uses, compared with prior art, the present invention haing the following advantages：
1. the present invention uses " one kettle way " thinking for the first time, by ring-opening polymerisation（ROP）It is anti-with the cycloaddition of end alkynyl radical and nitrine It answers（CuAAC）Combination has synthesized the folate-targeted acid-sensitive core crosslinking carrier micelle based on polyphosphate；The micella is with good Good biocompatibility and the polyphosphate of biodegradability, in aqueous solution can be certainly respectively as its hydrophilic and hydrophobic segment Assembling forms micella, and hydrophobic cores can be used for containing hydrophobic anticancer drug.
2. targeted molecular folic acid is bonded to amphipathic copolymer end by the present invention, is mediated during gulping down in the cell and carry medicine Micella enters cancer cell, increases utilization ratio of drug.
3. the present invention carries out the amphipathic phosphoric acid ester block copolymer of the folate-targeted of acquisition using acid-sensitive sense molecule Chemical crosslinking extends micella circulation time in vivo, and the acid-sensitive acetal radical site that core crosslinking micella contains is in acyclic acidic It is broken under border, polyphosphate main chain can hydrolyze in the presence of enzyme, and micellar structure is caused to be destroyed, and release package-contained drug, reach To the purpose for the treatment of tumour；Therefore, core crosslinking carrier micelle disclosed by the invention may be used as efficient controllable in drug release system, There is good application value in biomaterial and biomedicine field.
Description of the drawings
Fig. 1 is polyphosphate block copolymer in embodiment one（PBYP43-b-PEOP41-OH）Hydrogen nuclear magnetic resonance spectrogram, Solvent is deuterochloroform（CDCl3）；
Fig. 2 is the polyphosphate block copolymer of modified with folic acid in embodiment two（PBYP43-b-PEOP41-FA）, embodiment Polyphosphate block copolymer in one（PBYP43-b-PEOP41-OH）And folic acid（FA）Ultraviolet spectrogram, solvent is ethyl alcohol；
Fig. 3 is the acid-sensitive tetraethylene glycol of chlorine functional group dead-end in embodiment three（Cl-a-TEG-a-Cl）It is blocked with azido Acid-sensitive tetraethylene glycol（N3-a-TEG-a-N3）Hydrogen nuclear magnetic resonance spectrogram, solvent is deuterochloroform（CDCl3）；
Fig. 4 is N in embodiment three3-a-TEG-a-N3Carbon-13 nmr spectra figure, solvent is deuterochloroform（CDCl3）；
Fig. 5 is that acid-sensitive core is crosslinked in embodiment five（ACCL）The transmission electron microscope photo and dynamic light scattering curve of micella；
Fig. 6 is that acid-sensitive core is crosslinked in embodiment five（ACCL）The hydrogen nuclear magnetic resonance spectrogram of micella, solvent are deuterochloroform （CDCl3）；
Fig. 7 is that folate-targeted acid-sensitive core is crosslinked in embodiment seven（ACCL-FA）The transmission electron microscope photo of carrier micelle and Dynamic light scattering curve；
Fig. 8 is that folate-targeted acid-sensitive core is crosslinked accumulative drug of the carrier micelle under different pH condition in embodiment ten Release profiles；
Fig. 9 is uncrosslinked micella in embodiment 11（A）With crosslinking micella（B）To the toxotest figure and leaf of L929 cells Toxotest figure of the acid targeting crosslinking micella to KB cells（C）；
Figure 10 is the adriamycin to dissociate in embodiment 11（DOX）, folate-targeted acid-sensitive core crosslinking（ACCL-FA）Carry medicine Micella and the crosslinking of acid-sensitive core（ACCL）Cytotoxicity curve of the carrier micelle to KB cells；
Figure 11 is that KB cells shine the endocytosis that carrier micelle is crosslinked whether there is or not the acid-sensitive core of folate-targeted in embodiment 12 Piece figure.
Specific implementation mode
The invention will be further described with reference to the accompanying drawings and embodiments：
Embodiment one：Polyphosphate block copolymer（PBYP-b-PEOP-OH）Preparation
Ampoule bottle equipped with stirrer is placed on drying at least 24 hours in 120 DEG C of baking ovens, takes out, ampere bottle is connected to On biexhaust pipe, it is cooled to room temperature with oil pump pumping, repeats to take out inflation three times, nitrogen is finally full of, under nitrogen protection, into ampoule bottle Sequentially add 11 carbon -7- alkene of 1,8- diazabicylos [5.4.0]（DBU, 0.2738 g, 0.18 mmol）, dichloromethane （CH2Cl2, 1.5 mL）, isopropanol（IPA, 0.1121 g, 0.12 mmol）With monomer 2- alkynes butyl -2- oxos -1,3,2- two Oxygen phospholane（BYP, 0.8453 g, 4.8 mmol）, reaction bulb is put into and is set as in 25 DEG C of oil baths, lower reaction 30 is stirred Minute.After reaction, monomer 2- ethyl-2-oxos -1,3,2- dioxaphospholane are added（EOP, 0.73 g, 4.8 mmol）, continue at reaction 30 minutes in 25 DEG C of oil baths.
After ring-opening reaction, product is concentrated, then concentrate is instilled into the methanol/ether mixed liquor that volume ratio is 1: 10 （100 mL）Middle precipitation twice, pours out supernatant, gained thick liquid is dissolved in methanol solution, molecular cut off is then transferred to For 3500 g mol-1It in bag filter, is placed in deionized water and dialyses 24 hours, be freeze-dried, obtain glutinous thick liquid, it is as poly- Phosphoric acid ester block copolymer（PBYP43-b-PEOP41-OH）, yield 81.3%.Using nuclear magnetic resonance spectroscopy（1H NMR）To its carry out Characterization, attached drawing 1 are above-mentioned PBYP43-b-PEOP41The nuclear magnetic resonance spectroscopy of-OH（1H NMR）It is total to demonstrate polyphosphate block for figure The chemical constitution of polymers.
Embodiment two：The polyphosphate block copolymer of modified with folic acid（PBYP-b-PEOP-FA）Preparation
By equipped with stirrer side tube flask and after ground glass stopper handles according to one method of embodiment, nitrogen is full of, logical Under condition of nitrogen gas, folic acid is sequentially added（FA, 0.0159 g, 0.036 mmol）, dimethyl sulfoxide（DMSO, 10 mL）, dicyclohexyl Carbodiimide（DCC, 0.0096 g, 0.0468 mmol）、NHOSu NHS（NHS, 0.0054 g, 0.0468 mmol） And 4-dimethylaminopyridine（DMAP, 0.0057 g, 0. 0468 mmol）.Reaction bulb is full of nitrogen, is stirred to react in 25 DEG C 12 hours.After reaction, then PBYP is weighed43-b-PEOP41-OH（0.4161 g, 0.0300 mmol）It adds in reaction solution, The reaction was continued 24 hours.
After reaction, it is 3500 g mol product to be transferred to molecular cut off-1In bag filter, in deionized water thoroughly Analysis 24 hours, freeze-drying, obtains faint yellow thick liquid, adds dichloromethane（100 mL）Dissolving, filtering, concentration, will Gained thick liquid air drying 36 hours in vacuum drying oven obtain the polyphosphate block copolymer of modified with folic acid （PBYP43-b-PEOP41-FA）, yield 77.8%.Using ultraviolet specrophotometer（UV-Vis）It is characterized.Attached drawing 2 is Folic acid, PBYP43-b-PEOP41- OH and PBYP43-b-PEOP41The ultraviolet spectrogram of-FA.Within the scope of 220~400 nm of wavelength, The maximum absorption band of folic acid is in 283 nm, PBYP43-b-PEOP41- OH is in the range without maximum absorption band, and PBYP43-b- PEOP41The maximum absorption band of-FA is in 278 nm, this is because auxochrome group after carboxyl is bonded with hydroxyl on polyphosphate on folic acid It changes, leads to the absorption peak blue shift of folic acid.
Embodiment three：The acid-sensitive tetraethylene glycol of azido sealing end（N3-a-TEG-a-N3）Preparation
Weigh tetraethylene glycol（TEG, 0.97 g, 5 mmol）With 4- toluenesulfonic acid pyridines（PPTS, 0.2513 g, 1 mmol）It adds in the 100 mL side tube flasks equipped with magnetic stir bar, toluene is added（20 mL）, azeotropic water removing twice, then by azeotropic Mixture is dissolved in anhydrous methylene chloride（30 mL）, under the conditions of ice-water bath, 2- chloroethyl vinyl ethers are slowly added dropwise（CEVE, 2.5 mL, 25 mmol）And anhydrous methylene chloride（10 mL）In mixed liquor to side tube flask, after completion of dropwise addition, ice-water bath stirring is anti- It answers 0.5 hour.
After reaction, the aqueous sodium carbonate that mass fraction is 5% is added and terminates reaction, uses dichloromethane（30 mL）It is dilute It releases, adds saturated sodium-chloride phosphate buffer solution（10.0,10 mL of pH）, stood after oscillation, separate lower layer's organic phase, water phase Dichloromethane is used again（20 mL）Extraction merges organic phase, with anhydrous sodium sulfate drying, filtering, concentration, adds toluene（20 mL）Unreacted 2- chloroethyl vinyl ethers are azeotroped off, air drying 36 is small in vacuum drying oven by gained weak yellow liquid When, obtain the acid-sensitive tetraethylene glycol of chlorine functional group dead-end（Cl-a-TEG-a-Cl）, yield 74.9%.
By Cl-a-TEG-a-Cl（1.025 g, 2.5 mmol）, sodium azide（NaN3, 1.625 g, 25 mmol）WithN,NDimethylformamide（DMF, 10 mL）It adds to successively in 50 mL round-bottomed flasks, reaction bulb is moved in 60 DEG C of oil baths, stir Reaction 40 hours.
After reaction, product is filtered, concentration, then concentrate is dissolved in dichloromethane（100 mL）In, saturation is added Sodium chloride phosphate buffer solution（10.0,10 mL of pH）, stood after oscillation, separate lower layer's organic phase, water phase uses dichloromethane again （20 mL）Extraction merges organic phase, isolated yellowish using thin layer chromatography with anhydrous sodium sulfate drying, filtering, concentration Color liquid, by products therefrom in vacuum drying oven air drying 36 hours, obtain azido sealing end acid-sensitive tetraethylene glycol（N3-a-TEG-a-N3）, yield 72.2%.Using nuclear magnetic resonance spectroscopy（1H NMR）And carbon-13 nmr spectra（13C NMR）To product into Row characterizes, in attached drawing 3（A）With（B）Respectively Cl-a-TEG-a- Cl and N3-a-TEG-a-N3Nuclear magnetic resonance spectroscopy（1H NMR） Figure, attached drawing 4 are Cl-a-TEG-aThe carbon-13 nmr spectra of-Cl（13C NMR）Figure, the results show that being successfully prepared in embodiment three Two kinds of functional moleculars.
Example IV：Non- core crosslinking（UCCL）The preparation of micella
By PBYP43-b-PEOP41-OH（0.010 g）WithN, NDimethylformamide（2 mL）It adds to equipped with stirrer In round-bottomed flask, under continuous stirring, with microsyringe with slow speed（3 mL•h-1）Deionized water is added（20 mL）, wait for After complete sample introduction, stir 12 hours.
After reaction, it is 3500 g mol reaction solution to be transferred to molecular cut off-1Bag filter in, be placed in deionization It dialyses 24 hours in water, the solution after dialysis is settled to 25 mL, obtain non-core crosslinking（UCCL）Micella.
Embodiment five：Acid-sensitive core is crosslinked（ACCL）The preparation of micella
By PBYP43-b-PEOP41-OH（0.010 g, 0. 72 mmol）、N3-a-TEG-a-N3（6.5 mg, 0.0155 mmol）WithN, NDimethylformamide（2 mL）It adds in the round-bottomed flask equipped with stirrer, under continuous stirring, use is micro Injector is with slow speed（3 mL•h-1）Deionized water is added（20 mL）, after complete sample introduction, stir 12 hours, then add successively Enter cuprous bromide（CuBr, 4.4 mg, 0.031 mmol）, five methyl diethylentriamine（PMDETA, 1.3 mL, 0.062 mmol）And vitamin C（5.5 mg, 0.031 mmol）, sealing, pumping inflation are three times.Reaction bulb full of nitrogen and is moved to 25 DEG C In oil bath, it is stirred to react 24 hours.
After reaction, it is 3500 g mol reaction solution to be transferred to molecular cut off-1Bag filter in, be placed in deionization It dialyses 24 hours in water, the solution after dialysis is settled to 25 mL with water, obtain the crosslinking of acid-sensitive core（ACCL）Micella.Respectively Using transmission electron microscope（TEM）And dynamic light scattering（DLS）Micella pattern and particle size are characterized, attached drawing 5 is acid-sensitive Feel the transmission electron microscope photo of the self assembly of core crosslinking micella in deionized water（A）With dynamic light scattering curve（B）, as a result table Bright, acid-sensitive core is crosslinked micella structure spherical in shape in aqueous solution, and grain size is in 160 rans.Using nuclear magnetic resonance spectroscopy（1H NMR）Core cross-linked structure is characterized, attached drawing 6 is the nuclear magnetic resonance spectroscopy that acid-sensitive core is crosslinked micella（1H NMR）Figure, crosslinking After reaction, the proton peak intensity very little on PBYP segments and TEG molecules, and PEOP segment proton peaks intensity does not change substantially, The results show that being successfully prepared acid-sensitive core cross-linked structure.
Embodiment six：Folate-targeted acid-sensitive core is crosslinked（ACCL-FA）The preparation of micella
By PBYP43-b-PEOP41-FA（0.010 g, 0.72 mmol）、N3-a-TEG-a-N3（6.5 mg, 0.0155 mmol）WithN, NDimethylformamide（2 mL）It adds in the round-bottomed flask equipped with stirrer, under continuous stirring, use is micro Injector is with slow speed（3 mL•h-1）Deionized water is added（20 mL）, after complete sample introduction, stir 12 hours, then add successively Enter cuprous bromide（CuBr, 4.4 mg, 0.031 mmol）, five methyl diethylentriamine（PMDETA, 1.3 mL, 0.062 mmol）And vitamin C（5.5 mg, 0.031 mmol）, sealing, pumping inflation are three times.Reaction bulb full of nitrogen and is moved to 25 DEG C In oil bath, it is stirred to react 24 hours.
After reaction, it is 3500 g mol reaction solution to be transferred to molecular cut off-1Bag filter in, be placed in deionization It dialyses 24 hours in water, the solution after dialysis is settled to 25 mL with water, obtain the crosslinking of folate-targeted acid-sensitive core（ACCL- FA）Micella.
Embodiment seven：The core crosslinking micella for containing anticancer drug is prepared using dialysis
By PBYP43-b-PEOP41- OH or PBYP43-b-PEOP41-FA（0.010 g, 0. 72 mmol）、N3-a-TEG-a- N3（6.5 mg, 0.0155 mmol）WithN, NDimethylformamide（2 mL）It adds in the round-bottomed flask equipped with stirrer, amount Take 0.4 mL anticancer drugs, doxorubicins（DOX）Dimethyl sulfoxide (DMSO)（DMSO）Solution（5 mg•mL-1）It adds in mixed solution, Under lasting stirring, with microsyringe with slow speed（3 mL•h-1）Deionized water is added（20 mL）, after complete sample introduction, stir It mixes 12 hours, sequentially adds cuprous bromide（4.4 mg, 0.031 mmol）, five methyl diethylentriamine（1.3 mL, 0.062 mmol）And vitamin C（5.5 mg, 0.031 mmol）, sealing, pumping inflation are three times.Reaction bulb full of nitrogen and is moved Into 25 DEG C of oil baths, it is stirred to react 24 hours.
After reaction, it is 3500 g mol reaction solution to be transferred to molecular cut off-1Bag filter in, in deionized water Middle dialysis 24 hours, 25 mL are settled to by the solution after dialysis, respectively obtain the crosslinking of acid-sensitive core（ACCL）Carrier micelle and leaf Acid targeting acid-sensitive core crosslinking（ACCL-FA）Carrier micelle.Transmission electron microscope and dynamic light scattering is respectively adopted to carrier micelle shape Looks and particle size are characterized, and attached drawing 7 is that folate-targeted acid-sensitive core is crosslinked the self assembly of carrier micelle in deionized water Transmission electron microscope photo（A）With dynamic light scattering curve（B）, the results showed that, carrier micelle structure spherical in shape in aqueous solution, grain Diameter is in 165 rans.
Embodiment eight：The core crosslinking micella for containing anticancer drug is prepared using dialysis
By PBYP43-b-PEOP41-FA（0.010 g, 0. 72 mmol）、N3-a-TEG-a-N3（6.5 mg, 0.0155 mmol）WithN, NDimethylformamide（2 mL）It adds in the round-bottomed flask equipped with stirrer, measures 1 mL anticancer drug Japanese yews Alcohol（PTX）Dimethyl sulfoxide (DMSO)（DMSO）Solution（5 mg•mL-1）It adds in mixed solution, under continuous stirring, uses micro-sampling Device is with slow speed（3 mL•h-1）Deionized water is added（20 mL）, after complete sample introduction, stir 12 hours, sequentially add bromine Change cuprous（4.4 mg, 0.031 mmol）, five methyl diethylentriamine（1.3 mL, 0.062 mmol）And vitamin C（5.5 Mg, 0.031 mmol）, sealing, pumping inflation are three times.Reaction bulb full of nitrogen and is moved in 25 DEG C of oil baths, it is small to be stirred to react 24 When.
After reaction, it is 3500 g mol reaction solution to be transferred to molecular cut off-1Bag filter in, in deionized water Middle dialysis 24 hours, 25 mL are settled to by the solution after dialysis, obtain the crosslinking of folate-targeted acid-sensitive core（ACCL-FA）Carry medicine Micella.
Embodiment nine：The core crosslinking micella for containing anticancer drug is prepared using dialysis
By PBYP43-b-PEOP41-FA（0.010 g, 0. 72 mmol）、N3-a-TEG-a-N3（6.5 mg, 0.0155 mmol）WithN, NDimethylformamide（2 mL）It adds in the round-bottomed flask equipped with stirrer, it is left to measure 0.2 mL anticancer drugs Revolve imidazoles（LMS）Dimethyl sulfoxide (DMSO)（DMSO）Solution（5 mg•mL-1）It adds in mixed solution, under continuous stirring, use is micro Injector is with slow speed（3 mL•h-1）Deionized water is added（20 mL）, after complete sample introduction, stir 12 hours, then add successively Enter cuprous bromide（4.4 mg, 0.031 mmol）, five methyl diethylentriamine（1.3 mL, 0.062 mmol）And vitamin C （5.5 mg, 0.031 mmol）, sealing, pumping inflation are three times.Reaction bulb full of nitrogen and is moved in 25 DEG C of oil baths, is stirred to react 24 hours.
After reaction, it is 3500 g mol reaction solution to be transferred to molecular cut off-1Bag filter in, in deionized water Middle dialysis 24 hours, 25 mL are settled to by the solution after dialysis, respectively obtain the crosslinking of acid-sensitive core（ACCL）Carrier micelle and leaf Acid targeting acid-sensitive core crosslinking（ACCL-FA）Carrier micelle.
Embodiment ten：Acid-sensitive core crosslinking micella contains the extracorporeal releasing experiment of anticancer drugs, doxorubicin
Take 5 mL embodiments five prepare folate-targeted core crosslinking polypeptide drug-loaded micelle solution in molecular cut off be 12000~ 14000 g•mol-1Bag filter in, which is put into the centrifuge tube that capacity is 30 mL, different pH value are added in outside Buffer solution（20 mL）, centrifuge tube is placed in 37 DEG C of constant temperature oscillators and carries out release experiment.Take 5 mL saturating at regular intervals Bag external solution is analysed, while supplementing the buffer solution of 5 mL same pH.Release adriamycin is detected using sepectrophotofluorometer Content.It is loaded with Cumulative release profile such as attached drawing of the folate-targeted acid-sensitive core crosslinking micella of adriamycin under condition of different pH Shown in 8, the results showed that, drug rate of release under the conditions of 5.0 pH is significantly faster than that pH 7.4, it is seen that carrier micelle has certain Sensitivity to acid, drug controlled release effect can be reached.
Embodiment 11：Cytotoxicity test
Cytotoxicity test selects human body fibroblast（L929 cells）And KB cell（KB cells）Into Row, L929 cell culture is added with 10% fetal calf serum（FBS）DMEM culture mediums in, be put in 37 DEG C, 5% CO2, relative humidity To be cultivated in 90% incubator.Cell inoculation of the selection in the active growth phase contains the 96 of 100 μ L DMEM culture mediums in every hole In orifice plate, cultivate 24 hours.KB cells are cultivated two weeks or more in RPMI-1640 (-) FA culture mediums without folic acid, then are carried out Experiment.
Configure certain density testing sample solution：Non- core crosslinking（UCCL）Micella, 1000 mg L-1；Acid-sensitive core is crosslinked （ACCL）Micella, 1000 mg L-1；Folate-targeted acid-sensitive core is crosslinked（ACCL-FA）Micella, 1000 mg L-1；Adriamycin, 30 mg•L-1；Folate-targeted acid-sensitive core is crosslinked（ACCL-FA）Carrier micelle, doxorubicin concentration are 30 mg L-1；Acid-sensitive core is handed over Connection（ACCL）Carrier micelle, doxorubicin concentration are 30 mg L-1.A series of various concentration sample solutions are added in 96 orifice plates, Continue culture 48 hours；The MTT reagents of 25 μ L are subsequently added into, after further cultivating 4 hours, use microplate reader（Bio-Rad 680） Corresponding absorbance is measured under 570 nanometers.Cell survival rate is calculated according to following formula：
Cell survival rate（Cell viability）（%）= [A]test/[A]control × 100
In formula, [A]testFor added with the absorbance measured under the conditions of sample to be tested, [A]controlTo be not added with sample blank control Under the conditions of the absorbance that measures.Each sample test three times, takes its average value.
In attached drawing 9（A）、（B）With（C）Respectively non-core crosslinking（UCCL）Micella and the crosslinking of folate-targeted acid-sensitive core （ACCL-FA）Micella is crosslinked the toxotest and folate-targeted acid-sensitive core of L929 cells（ACCL-FA）Micella is thin to KB The toxotest of born of the same parents, the results showed that, two kinds of polymer micella has lower toxicity and good biocompatibility；
Attached drawing 10 is the adriamycin to dissociate（DOX）, folate-targeted acid-sensitive core crosslinking（ACCL-FA）Carrier micelle and acid-sensitive Feel core crosslinking（ACCL）Carrier micelle shows to kill with the increase of doxorubicin concentration, sample to the Toxic test results of KB cells The ability of dead cancer cell constantly enhances, moreover, under identical doxorubicin concentration, carrier micelle specific ionization adriamycin has stronger kill The ability of dead cancer cell, and folate-targeted acid-sensitive core crosslinking carrier micelle has strongest anticancer effect.
Embodiment 12：Cell endocytic is tested
KB cell culture is added with 10% fetal calf serum（FBS）RPMI-1640 (-) FA culture mediums in, be put in 37 DEG C, 5% CO2, relative humidity be 90% incubator in cultivate.Select the cell inoculation in the active growth phase in base thickness for 0.17 mm's In culture vessel with glass bottom, culture keeps its adherent in 12 hours.Culture dish is transferred in the cell culture system of living cells work station, Using containing 10 mL polypeptide drug-loaded micelle solutions（Folate-targeted acid-sensitive core is crosslinked（ACCL-FA）Carrier micelle, the crosslinking of acid-sensitive core （ACCL）Carrier micelle, doxorubicin concentration：30 mg•L-1）Culture medium（1 mL）Displace the culture medium in culture dish（Finally Doxorubicin concentration：0.3 mg•L-1）, endocytic processes of the cell to carrier micelle are detected using living cells work station.Attached drawing 11 is KB Cell is to whether there is or not the endocytosis photos that the acid-sensitive core of folate-targeted is crosslinked carrier micelle, the results showed that, the acid-sensitive of folate-targeted Core crosslinking carrier micelle enters the speed of KB cells faster than the carrier micelle of not modified with folic acid.

Claims (7)

1. a kind of folate-targeted acid-sensitive core based on polyphosphate is crosslinked carrier micelle, which is characterized in that described to be based on poly- phosphorus Preparing for the folate-targeted acid-sensitive core crosslinking carrier micelle of acid esters is as follows：
(1) polyphosphate block copolymer is prepared：In inert atmosphere, in 1,8- diazabicylos [5.4.0], 11 carbon -7- alkene Catalytic action under, using dichloromethane as solvent, under the conditions of 10~50 DEG C, using isopropanol as initiator, with annular phosphateRing-opening polymerisation is carried out for monomer, is reacted 30 seconds~60 minutes；Add cyclic phosphate ester monomer, The reaction was continued 30 seconds~60 minutes；Obtain polyphosphate block copolymer；
In annular phosphate monomer structure formula, R1For CH2Or CH2CH2；R2It is sealed for methyl, ethyl, isopropyl, butyl or monomethyl The chemical structural formula of one kind in the polyethylene oxide base at end, the polyethylene oxide base of monomethyl sealing end is：(CH2CH2O)xCH3, x=2~10 in formula；
The initiator,、And mole of 11 carbon -7- alkene of 1,8- diazabicylos [5.4.0] Than being 1: (20~90): (20~90): (0.1~2)；
(2) the polyphosphate block copolymer of modified with folic acid is prepared：Under the catalytic action of dicyclohexylcarbodiimide, with leaf Acid andNHOSu NHS is raw material, using dimethyl sulfoxide as solvent, in the presence of 4-dimethylaminopyridine, in 10~40 DEG C reaction 8~16 hours, add step (1) preparation polyphosphate block copolymer, stirring, the reaction was continued 8~48 hours, Obtain the polyphosphate block copolymer of modified with folic acid；
The polyphosphate block copolymer, folic acid,NHOSu NHS, dicyclohexylcarbodiimide and 4- dimethylaminos The molar ratio of pyridine is 1: (1~2): (1.2~3): (1.2~3): (0.1~1)；
(3) the acid-sensitive small molecule of azido sealing end is prepared：Under the catalytic action of 4- toluenesulfonic acid pyridines, with HO-R3- OH and 2- chloroethyl vinyl ethers are raw material, under the conditions of ice-water bath, react 0.5~1 hour, prepare the small of chlorine functional group dead-end Molecule；Then the small molecule of above-mentioned chlorine functional group dead-end, sodium azide are addedN, NIn dimethyl formamide solution, in 40 ~80 DEG C are reacted 30~50 hours, and the acid-sensitive small molecule of azido sealing end is obtained；
The HO-R3In-OH, R3For (CH2CH2O)yCH2CH2, wherein y=0~7；
The structural formula of the small molecule of the chlorine functional group dead-end is；
The HO-R3The molar ratio of-OH, 2- chloroethyl vinyl ethers and 4- toluenesulfonic acid pyridines are 1: (4~20): (0.1 ~0.5)；
The small molecule of the chlorine functional group dead-end and the molar ratio of sodium azide are 1: (4~40)；
(4) it prepares the folate-targeted acid-sensitive core based on polyphosphate and is crosslinked carrier micelle：It is anti-with hydrophobicity in inert atmosphere The acid of azido sealing end prepared by the polyphosphate block copolymer and step (3) of modified with folic acid prepared by cancer drug, step (2) Sensitive small molecule is raw material, in cycloaddition click-reaction catalyst, catalyst ligand and the reducing substances of end alkynyl radical and nitrine In the presence of, with water/N, NDimethylformamide mixed liquor is solvent, is reacted 18~30 hours in 25~40 DEG C, is obtained based on poly- The folate-targeted acid-sensitive core of phosphate is crosslinked carrier micelle；
The alkynyl of the polyphosphate block copolymer of the modified with folic acid, the acid-sensitive small molecule of azido sealing end, end alkynyl radical with The molar ratio of the cycloaddition click-reaction catalyst of nitrine, catalyst ligand and reducing substances is 1: 0.5: 1: (1~2): (1 ~2)；
The mass ratio of the polyphosphate block copolymer of hydrophobic anticancer drug and modified with folic acid is 1 in the step (4): (2~ 10) ；
In the step (4) hydrophobic anticancer drug be selected from adriamycin, Epi-ADM, taxol, bortezomib, Aclarubicin, One kind in pirarubicin, daunorubicin hydrochloride, Semustine, plicamycin, mitomycin, idarubicin or levamisol.
2. the folate-targeted acid-sensitive core based on polyphosphate is crosslinked carrier micelle according to claim 1, it is characterised in that： Using anhydrous methylene chloride as solvent when preparing the small molecule of chlorine functional group dead-end in the step (3).
3. the folate-targeted acid-sensitive core based on polyphosphate is crosslinked carrier micelle according to claim 1, it is characterised in that： It is sub- to be selected from stannous chloride, cuprous bromide or iodate for the cycloaddition click-reaction catalyst of end alkynyl radical and nitrine in the step (4) One kind in copper；Catalyst ligand is selected from bipyridyl, five methyl diethylentriamine, tetramethylethylenediamine or hexamethyl Sanya second One kind in urotropine.
4. the folate-targeted acid-sensitive core based on polyphosphate is crosslinked carrier micelle according to claim 1, it is characterised in that： The one kind of reducing substances in vitamin C, sodium ascorbate, Calcium Ascorbate or citric acid in the step (4).
5. the folate-targeted acid-sensitive core based on polyphosphate is crosslinked carrier micelle according to claim 1, it is characterised in that： The inert atmosphere is nitrogen or argon gas atmosphere.
6. the folate-targeted acid-sensitive core based on polyphosphate is crosslinked carrier micelle according to claim 1, it is characterised in that： After the completion of step (1)~(4) reaction, purification processes are carried out to product respectively, the purification process includes the following steps：
1) purification of polyphosphate block copolymer：After ring-opening polymerization, by reaction product concentrated by rotary evaporation, then will concentration Drop enters to be precipitated in methanol/ether mixed liquor, pours out supernatant, remaining thick liquid is dissolved in methanol solution, is then transferred to In bag filter, it is placed in deionized water and dialyses 24~72 hours, be then freeze-dried, obtain polyphosphate block copolymer；
2) purification of the polyphosphate block copolymer of modified with folic acid：After reaction, reaction solution is filtered, concentration, will be concentrated Liquid is transferred in bag filter, is placed in deionized water and is dialysed 24~72 hours, is taken out, freeze-drying, then dissolved with dichloromethane, Filtering, concentration, obtain faint yellow thick liquid, air drying 24~36 is small in vacuum drying oven by the faint yellow thick liquid of gained When, obtain the polyphosphate block copolymer of modified with folic acid；
3) purification of small molecule：
(1) purification of the small molecule of end modified acetal radical：After reaction, the aqueous sodium carbonate that mass fraction is 5% is added Reaction is terminated, is diluted with dichloromethane, is added saturated sodium-chloride phosphate buffer solution, stood after oscillation, it is organic to separate lower layer Phase, water phase are extracted with dichloromethane again, merge organic phase, and organic phase solution will be distilled through drying, filtering, concentration, air-distillation Resultant product air drying 24~36 hours in vacuum drying oven, obtain weak yellow liquid, are small point of end modified acetal radical Son；
(2) purification of the acid-sensitive small molecule of azido sealing end：After reaction, by reaction product filtering, concentration, then will concentration Liquid is dissolved in dichloromethane solution, and saturated sodium-chloride phosphate buffer solution is added, is stood after oscillation, separates lower layer's organic phase, water It is mutually extracted again with dichloromethane, merges organic phase, organic phase solution is detached through drying, filtering, concentration using thin layer chromatography To weak yellow liquid, by products therefrom in vacuum drying oven air drying 24~36 hours, obtain azido sealing end acid-sensitive Small molecule；
4) purification of the folate-targeted acid-sensitive core crosslinking carrier micelle based on polyphosphate：After click-reaction, it will react Product is transferred in bag filter, is placed in deionized water and is dialysed 24~72 hours, and constant volume obtains the folic acid target based on polyphosphate It is crosslinked carrier micelle to acid-sensitive core.
7. the folate-targeted acid-sensitive core based on polyphosphate is crosslinked carrier micelle according to claim 6, it is characterised in that： The pH value of the saturated sodium-chloride phosphate buffer solution is 10.0.
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