CN102911368A - Amphiphilic triblock copolymer with acid sensitivity and preparation method and application thereof - Google Patents

Amphiphilic triblock copolymer with acid sensitivity and preparation method and application thereof Download PDF

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CN102911368A
CN102911368A CN2012104631600A CN201210463160A CN102911368A CN 102911368 A CN102911368 A CN 102911368A CN 2012104631600 A CN2012104631600 A CN 2012104631600A CN 201210463160 A CN201210463160 A CN 201210463160A CN 102911368 A CN102911368 A CN 102911368A
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polyoxyethylene glycol
reaction
sensitivity
acid
amphipathic
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CN102911368B (en
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倪沛红
王海蓉
何金林
张明祖
李飞
陶云锋
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Suzhou University
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Abstract

The invention discloses an amphiphilic triblock copolymer with acid sensitivity and a preparation method and an application thereof. The preparation method comprises the following steps of: modifying polyethylene glycol with a hydroxyl group at the end to enable the structure of polyethylene glycol to contain a diazido ethyl diacetal end group, namely polyethylene glycol contains the diazido ethyl diacetal group at the end; carrying out ring-opening polymerization on cyclic ester monomers by utilizing propargyl alcohol as an initiator and stannous caprylate as a catalyst to obtain biodegradable polyester with alkynyl at the single end; and finally, under the action of a 'click' chemical catalyst and ligand, carrying out 'click' chemical reaction on polyethylene glycol containing the diazido ethyl diacetal group at the end and the biodegradable polyester with alkynyl at the end to obtain the biodegradable amphiphilic triblock copolymer with acid sensitivity. The biodegradable amphiphilic triblock copolymer with acid sensitivity has good biocompatibility, biodegradability and acid sensitivity, and can be used as a drug carrier.

Description

Amphipathic three block copolymer, its preparation method and application with sensitivity to acid
Technical field
The invention belongs to field of biomedical polymer materials, be specifically related to a kind of biodegradable amphipathic three block copolymer with sensitivity to acid, its preparation method with and as the application of pharmaceutical carrier.
Background technology
The whole world has more than 700 ten thousand people to lose one's life because of cancer stricken every year, and this numeral rises year by year.The treatment of cancer is maximum test and the difficult problem of 21st century facing mankind.
Development and progress along with science and technology, human continuous exploration to related sciences such as oncology, molecular biology, pharmacy and chemistry, so that the research of antitumor drug has been had significant progress, many effective cancer therapy drugs are designed to develop.But there is toxic side effect in present most cancer therapy drug greatly and the shortcoming of poorly water-soluble, thereby has greatly limited the application of cancer therapy drug in clinical.
Amphipathic nature polyalcohol generally is comprised of hydrophilic and hydrophobic two portions, and they can form the micella with nucleocapsid structure in the aqueous solution.Wherein, kernel, the hydrophilic segment of hydrophobic polymer chain formation micella then form shell.Because most Effective Anti cancer drug all has very strong hydrophobicity, therefore, the hydrophobic cores of polymer micelle can be used for loading hydrophobic anticancer drug, improves the water-soluble of medicine and reduces toxic side effect; And hydrophilic shell can play the effect of stablizing micella, and has greatly improved the blood circulation time of carrier micelle.
In general, the pH value of the pathological tissues inside such as tumour and the inflammation all pH value than normal body fluid is low, presents slightly acidic.Therefore, can utilize these characteristics to carry out the design of pharmaceutical carrier, some labile components under acidic conditions are introduced in the amphipathic nature polyalcohol.The micella that they are assembled in the aqueous solution can drug loading and stable existence under neutrallty condition, but under acidic conditions, sensitivity to acid group or segment can make micella destroyed by fracture, thereby the medicine of its internal package can be discharged rapidly, these sensitivity to acid groups comprise acetal radical, hydrazone key, ortho-formiate etc.
In the prior art, have some are used as pharmaceutical carrier about the amphipathic multipolymer of sensitivity to acid report.But, as pharmaceutical carrier, should have good biocompatibility and biodegradability, and, carrier as antitumor drug, also should have following feature: can form stable polymer micelle in the aqueous solution, its hydrophobic cores can be loaded hydrophobic anticancer drug, and the wetting ability shell plays the effect of stable micella, raising micella blood circulation time; When carrier micelle arrives tumour or pathological tissues, can utilize the lower characteristics of pH value of local organization, destroy micella, discharge rapidly cancer therapy drug.
Therefore, need to seek the amphipathic multipolymer of more acid-sensitive sense.
Summary of the invention
The objective of the invention is, a kind of amphipathic three block copolymer with sensitivity to acid is provided, this multipolymer should have good biodegradability, the carrier of useful as anti-cancer agents thing; Another object of the present invention provides preparation method and the application of this amphipathic three block copolymer.
For achieving the above object, general plotting of the present invention is: ring-opening polymerization (ROP) and two kinds of methods of " click " chemical reaction are combined, and preparation has the amphipathic three block copolymer of sensitivity to acid.At first utilize the polyoxyethylene glycol (HO-PEG-OH) and vinyl chloride ethyl ether (CEVE) reaction of two terminal hydroxyls, and further prepare terminal two azidoethyls, the two acetal radical polyoxyethylene glycol (N that contain sensitivity to acid acetal radical and azido group with reaction of sodium azide 3- α-PEG- α-N 3); Then utilize propargyl alcohol to be catalyzer as initiator, stannous octoate, cyclic ester monomer is carried out ring-opening polymerization, obtain single terminal hydrophobicity biodegradable polyesters (Polyester-C ≡ C) with alkynyl; At last, utilize prepared N 3- α-PEG- α-N 3With Polyester-C ≡ C " click " chemical reaction occuring, obtains to have the biodegradable amphipathic three block copolymer of sensitivity to acid.
The concrete technical scheme of the present invention is: a kind of amphipathic three block copolymer with sensitivity to acid, expressed by the following chemical structure formula:
Figure 458192DEST_PATH_IMAGE001
；
In the formula, R 1Be selected from
Figure 793358DEST_PATH_IMAGE002
,
Figure 53438DEST_PATH_IMAGE003
Or
Figure 663411DEST_PATH_IMAGE004
In a kind of; N is that 30～90, m is 32～192.
In the technique scheme, the group with sensitivity to acid is acetal radical, and its chemical structural formula is:
Figure 161389DEST_PATH_IMAGE005
This biodegradable amphipathic three block copolymer with sensitivity to acid had structurally both contained hydrophobic polyester block, can be used for loading hydrophobic anticancer drug; Contain again the acetal radical that can be hydrolyzed under acidic conditions, acetal radical is hydrolyzed under certain pH conditions, and micella is destroyed, thereby discharges rapidly the hydrophobic anticancer drug that is wrapped in micella inside.
In the technique scheme, the number-average molecular weight scope of triblock copolymer is: 12000～24000 g mol -1
Above-mentioned preparation method with amphipathic three block copolymer of sensitivity to acid may further comprise the steps:
(1) take two ends as hydroxy-end capped polyoxyethylene glycol (HO-PEG-OH) with vinyl chloride ethyl ether (CEVE) as raw material, adopt anhydrous methylene chloride as solvent, under the catalysis of para-methylbenzenepyridinsulfonate sulfonate (PPTS), react the terminal polyoxyethylene glycol (Cl-that contains Dichloroethyl two acetal radicals of preparation α-PEG- α-Cl); Its chemical structural formula is:
；
The structural formula of described vinyl chloride ethyl ether is
Figure 447456DEST_PATH_IMAGE007
Wherein, two ends are that the number-average molecular weight scope of hydroxy-end capped polyoxyethylene glycol is 2000 ~ 12000 g mol -1, two ends are that the mol ratio of hydroxy-end capped polyoxyethylene glycol (HO-PEG-OH), para-methylbenzenepyridinsulfonate sulfonate (PPTS) and vinyl chloride ethyl ether (CEVE) is 5: 1: (25～50);
(2) end that obtains with step (1) contains polyoxyethylene glycol and the sodiumazide (NaN of Dichloroethyl two acetal radicals 3) reaction, the terminal polyoxyethylene glycol (N that contains two azidoethyls, two acetal radicals of preparation 3- α-PEG- α-N 3); Its structural formula is:
Figure 329961DEST_PATH_IMAGE008
；
Described end contains the polyoxyethylene glycol of Dichloroethyl two acetal radicals and the mol ratio of sodiumazide is 1: (2～10);
(3) use propargyl alcohol as initiator, stannous octoate [Sn (Oct) 2] be catalyzer, cyclic ester monomer is carried out ring-opening polymerization, obtain single terminal hydrophobicity biodegradable polyesters (Polyester-C ≡ C) with alkynyl, described cyclic ester monomer is selected from: a kind of in 6-caprolactone, rac-Lactide or the trimethylammonium cyclic carbonate ester;
The mol ratio of described propargyl alcohol and cyclic ester monomer is 1: (30～90);
(4) in the presence of " click " chemical reaction catalyst and part, the end that adopts step (2) to obtain contains the polyoxyethylene glycol (N of two azidoethyls, two acetal radicals 3- α-PEG- α-N 3) with the single terminal hydrophobicity biodegradable polyesters (Polyester-C ≡ C) with alkynyl that step (3) obtains, by the preparation of " clicks " chemical method, acquisition has the amphipathic three block copolymer (Polyester-of sensitivity to acid α-PEG- α-Polyester), its structural formula is:
Figure 213604DEST_PATH_IMAGE001
，
Wherein, end contains two azidoethyls, two acetal radical polyoxyethylene glycol, single end is (2.1～2.4) with the hydrophobicity biodegradable polyesters of alkynyl and the mol ratio of catalyzer: 1: 1; The mol ratio of catalyzer and part is 1: (1～2).
" click " chemical reaction be a kind of novel, simple, efficiently, synthetic method fast, this method has following distinguishing feature: (1) raw materials used and reagent easily obtains; (2) reaction conditions is simple; (3) productive rate is high; (4) stereoselectivity is good; (5) the product purification techniques is simple; (6) product stability is good.Numerous advantages is so that " click " chemical reaction is modified at polymer chemistry, material, be used widely in the field such as biomedical.So far also do not have bibliographical information to be similar among the present invention and by " click " chemical reaction hydrophilic polyglycol (PEG) segment is linked to each other by the acetal radical with sensitivity to acid with the hydrophobicity polyester segment, form the structure of ABA type triblock copolymer.
In the technique scheme, in the step (4), " click " chemical reaction catalyst is selected from cuprous chloride or cuprous bromide; Part is selected from: a kind of in dipyridyl, five methyl diethylentriamine, Tetramethyl Ethylene Diamine or the hexamethyl Triethylenetetramine (TETA).
Further technical scheme after step (1), step (2), step (3) and step (4) are finished, is carried out purification processes to product respectively, and described purge process may further comprise the steps:
(i) end contains the purifying of the polyoxyethylene glycol of Dichloroethyl two acetal radicals: after reaction finished, the adding massfraction was 5% Na 2CO 3Aqueous solution termination reaction, after adding the methylene dichloride dilution, water extraction 2～3 times, collect lower floor's organic phase, use anhydrous magnesium sulfate drying, the filtrate that filtration is obtained is concentrated, dropwise joins in the ice normal hexane, filter and place vacuum drying oven to be dried to constant weight the solid product of collecting, obtain white product;
Described Na 2CO 3With the mol ratio of para-methylbenzenepyridinsulfonate sulfonate be (25～50): 1; Methylene dichloride and Na 2CO 3The volume ratio of the aqueous solution is (4～8): 1;
(ii) end contains the purifying of the polyoxyethylene glycol of two azidoethyls, two acetal radicals: after reaction finishes, with reaction mixture by alkaline Al is housed 2O 3Pillar to remove the mantoquita in the solution, removal of solvent under reduced pressure, after adding the methylene dichloride dilution, lower floor's organic phase is collected in water extraction 2～3 times, uses anhydrous magnesium sulfate drying, the filtrate that filtration is obtained is concentrated, dropwise add volume ratio and be in 1: 1 the mixed solvent of ice normal hexane and ice ether, filter and place vacuum drying oven to be dried to constant weight the solid product of collecting, obtain white product;
(iii) purifying of single terminal hydrophobicity biodegradable polyesters with alkynyl: after reaction finishes, decompression remove portion solvent, concentrated solution is dropwise added in the ice ether, filter and place vacuum drying oven to be dried to constant weight the solid product of collecting, obtain white product;
(iv) has the purifying of the amphipathic three block copolymer of sensitivity to acid: after reaction finishes, add tetrahydrofuran (THF) diluting reaction mixed solution, and under the condition that stirs, contact with air, make reaction terminating, after solution becomes green, make it by alkaline Al is housed 2O 3Pillar, removal of solvent under reduced pressure, again concentrated solution is dropwise added in the ice ether, filter and the solid product of collecting is dissolved in tetrahydrofuran (THF) and is contained in the dialysis tubing that molecular weight cut-off is 12000～14000 Da with deionized water dialysis 48～96 h, at last, with resulting colourless transparent solution lyophilize in the dialysis tubing, obtain the solid product of white.
The mass ratio of described tetrahydrofuran (THF) and solid product is (10～50): 1.
The invention discloses a kind of intermediate, it is the polyoxyethylene glycol that end contains two azidoethyls, two acetal radicals, is expressed by the following chemical structure formula:
Figure 687310DEST_PATH_IMAGE008
；
In the formula, m is 32～192.
The present invention asks for protection simultaneously above-mentionedly has the amphipathic three block copolymer of sensitivity to acid as the application of stimulating responsive pharmaceutical carrier.
Amphipathic three block copolymer Polyester- α-PEG- α-Polyester can self-assembly form micella in the aqueous solution, the hydrophobicity polyester segment forms the nuclear of micella, is used for the parcel hydrophobic drug; Wetting ability PEG segment forms the shell of micella, plays the effect of stablizing micella; And has the acetal radical of sensitivity to acid between hydrophobicity polyester segment and the wetting ability PEG segment, hydrolytic cleavage can occur under solutions of weak acidity, thereby make micella destroyed, discharge rapidly the medicine that is loaded in the micella nuclear, thereby can be as stimulating efficiently the sensitive drug carrier.
Because the enforcement of such scheme, the present invention compared with prior art has the following advantages:
1, the present invention has adopted the good polyoxyethylene glycol of biocompatibility and biodegradable polyester respectively as wetting ability and hydrophobic segment, as the sensitivity to acid linkage section, adopt first " click " chemical method to prepare the sensitivity to acid amphipathic three block copolymer with good biocompatibility and biodegradability with the acetal radical that under acidic conditions, can be hydrolyzed.
But the biodegradable amphipathic three block copolymer self-assembly in water with sensitivity to acid that 2, obtains among the present invention forms stable polymer micelle, its hydrophobic cores can be loaded hydrophobic anticancer drug, the wetting ability shell plays the effect of stable micella, raising micella blood circulation time, when carrier micelle arrives tumour or pathological tissues, the reduction of local pH value can make the acetal radical fracture, micella is destroyed, thereby discharges cancer therapy drug rapidly.Therefore, the biodegradable amphipathic three block copolymer with sensitivity to acid disclosed by the invention can load and release anti-cancer medicine effectively, aspect the treatment of cancer, has good using value.
Description of drawings
Fig. 1 is that the polyoxyethylene glycol of two terminal hydroxyls among the embodiment one, the end with sensitivity to acid contain Dichloroethyl two acetal radical polyoxyethylene glycol (Cl- α-PEG 135- α-Cl) and embodiment two in end be two azidoethyls, two acetal radical polyoxyethylene glycol (N 3- α-PEG 135- α-N 3) the hydrogen nuclear magnetic resonance spectrogram ( 1H NMR), solvent is CDCl 3
Fig. 2 is single terminal polycaprolactone (PCL of alkynyl that is among the embodiment three 30Hydrogen nuclear magnetic resonance spectrogram-C ≡ C) ( 1H NMR), solvent is CDCl 3
Fig. 3 is the biodegradable amphipathic three block copolymer (PCL that has sensitivity to acid among the embodiment four 30- α-PEG 135- α-PCL 30) the hydrogen nuclear magnetic resonance spectrogram ( 1H NMR), solvent is CDCl 3
Fig. 4 is PCL among PCL-C ≡ C among polyoxyethylene glycol, the embodiment three and the embodiment four 30- α-PEG 135- α-PCL 30Gel permeation chromatography (GPC) elution curve.
Fig. 5 is Cl-among the embodiment one α-PEG 135- αN in-Cl and the enforcement two 3- α-PEG 135- α-N 3Infrared spectrogram (FT-IR).
Fig. 6 is N among the embodiment two 3- α-PEG 135- α-N 3, PCL among PCL-C ≡ C and the embodiment four among the embodiment three 30- α-PEG 135- α-PCL 30Infrared spectrogram (FT-IR).
Fig. 7 is PCL among the embodiment four 30- α-PEG 135- α-PCL 30(concentration is 0.06 mg mL to the micella that self-assembly forms in pH 7.4 buffered soln -1) transmission electron microscope photo.
Fig. 8 is PCL among the embodiment four 30- α-PEG 135- α-PCL 30(concentration is 0.06 mg mL to the micella that self-assembly forms in pH 7.4 buffered soln -1) the dynamic light scattering curve.
Fig. 9 is PCL among the embodiment seven 30- α-PEG 135- α-PCL 30The drug release curve of carrier micelle in the buffered soln of different pH values that forms.
Figure 10 is PCL among the embodiment seven 30- α-PEG 135- α-PCL 30The cytotoxicity test pattern of triblock copolymer.
Figure 11 is PCL among the embodiment six 30- α-PEG 135- α-PCL 30The cytotoxicity test curve of triblock copolymer carrier micelle and anticancer drugs, doxorubicin (DOX).
Specific implementation method
The invention will be further described below in conjunction with embodiment and accompanying drawing:
Embodiment one: the end with sensitivity to acid contains the polyoxyethylene glycol (Cl-of Dichloroethyl two acetal radicals α-PEG 135- α-Cl) synthetic.
At first pass through the polyoxyethylene glycol (HO-PEG of two terminal hydroxyls 135-OH) with para-methylbenzenepyridinsulfonate sulfonate (PPTS) azeotropic water removing in toluene, then adopt anhydrous methylene chloride as solvent, with vinyl chloride ethyl ether (CEVE) reaction, the terminal polyoxyethylene glycol (Cl-that contains Dichloroethyl two acetal radicals of preparation α-PEG 135- α-Cl).Concrete synthetic method is as follows:
Magnetic stir bar is being housed, in the 100 mL round-bottomed flasks of drying tube and atmospheric distillation plant, add polyoxyethylene glycol (6.01 g, 1.0 mmol), para-methylbenzenepyridinsulfonate sulfonate (PPTS, 0.5 g, 0.2mmol) and toluene 30 mL, heat azeotropic, under 110 ℃ of conditions, distill out the azeotrope of toluene and water.
HO-PEG will be housed 135The round-bottomed flask of-OH and PPTS adds anhydrous methylene chloride (CH after being cooled to room temperature 2Cl 250 mL), in constant pressure funnel, add vinyl chloride ethyl ether (CEVE, 1 mL, 10 mmol) and 10 mL anhydrous methylene chlorides, under 0 ℃ of condition, stir, and slowly drip vinyl chloride ethyl ether (CEVE), continue to stir at ambient temperature 30 min after dropwising, add 10 mL massfractions and be 10% aqueous sodium carbonate and make system be alkalescence, termination reaction.
In above-mentioned reaction system, add 60 mL CH 2Cl 2With twice of the saturated NaCl solution washing of 10 mL pH 10.0, leave standstill separatory, collect organic phase, add anhydrous magnesium sulfate drying, filtration, after filtrate is concentrated in the ice normal hexane precipitation, filter, three times repeatedly, drain with water pump, place again vacuum drying oven inner drying 24 h, obtain the polyoxyethylene glycol (Cl-that end contains Dichloroethyl two acetal radicals α-PEG 135- α-Cl, 5.37 g), productive rate 89%.
Embodiment two: end contains the polyoxyethylene glycol (N of two azidoethyls, two acetal radicals 3- α-PEG 135- α-N 3) synthetic.
In the round-bottomed flask of 50 mL, add successively Cl- α-PEG 135- α-Cl(5.03 g, 0.8 mmol), sodiumazide (0.57 g, 8mmol) and 10 mL dimethyl formamides (DMF).Reaction 40 h under 60 ℃ condition.Reaction system is passed through alkaline aluminium sesquioxide post (Al 2O 3) remove unreacted sodiumazide, then under 50 ℃, take out DMF with the vacuum oil pump, add 60 mL CH 2Cl 2Use PBS damping fluid (10 mL) washed twice of pH 10.0 after the dilution, separatory leaves standstill, collect organic phase, through anhydrous magnesium sulfate drying, filtration, icing normal hexane and icing (volume ratio 1:1) precipitation in the ether, filter after filtrate is concentrated, three times repeatedly, draining with water pump, place again vacuum drying oven inner drying 24 h, obtain the polyoxyethylene glycol (N that end contains two azidoethyls, two acetal radicals 3- α-PEG 135- α-N 3, 2.48 g), productive rate 49%.
Embodiment three: single end is the polycaprolactone (PCL of alkynyl 30-C ≡ C) synthetic method.
With the arm round-bottomed flask of putting into stirrer behind 120 ℃ of dry at least 24 h of baking oven, glass stopper beyond the Great Wall, by emulsion tube link to each other with oil pump be evacuated to room temperature after, pass into again high-purity argon gas, vacuumize three times so repeatedly.
Syringe with drying injects propargyl alcohol (0.12 g, 2.14 mmol) successively from arm, caprolactone (ε-CL) (10.1 g, 86.1 mmol) and stannous octoate [Sn (Oct) 2] (430 mg) (ε-CL calculates consumption according to the theoretical polymerization degree; [ε-CL]: [Sn (Oct) 2]=1:0.04, mass ratio), 25 mL dry toluenes are solvent.After in reaction flask, being full of argon gas, stirring reaction 4 h in 90 ℃ oil bath.
After reaction finishes, toluene solvant revolved to steam remove, in precipitation in the ice anhydrous diethyl ether, filter, repeatedly twice, remove unreacted monomer, place vacuum drying oven to dry to constant weight with the product behind the Büchner funnel suction filtration, obtain white product (PCL 30-C ≡ C, 8.79 g), recording productive rate is 87.4%.
Embodiment four: the biodegradable amphipathic three block copolymer (PCL with sensitivity to acid 30- α-PEG 135- α-PCL 30) synthetic.
After the method processing of arm round-bottomed flask according to embodiment three, be full of argon gas, add rapidly the polyoxyethylene glycol N that above-mentioned end contains two azidoethyls, two acetal radicals 3- α-PEG 135- α-N 3(366 mg, 0.06 mmol), single end are the polycaprolactone (PCL) of alkynyl 30-C ≡ C (300 mg, 0.1 mmol), cuprous bromide (0.014 g, 0.10 mmol) and five methyl diethylentriamine (21 μ L, 0.10 mmol), with oil pump take out, applying argon gas, three times to remove a bottle Air repeatedly, add 20 mL solvents tetrahydrofurane (THF) with dry syringe again, be stirred to mixture and dissolve fully, it is blue that solution is.Then round-bottomed flask is moved in 25 ℃ the oil bath stirring reaction 24 h.
After reaction finished, ingress of air made reaction terminating, and mixed with polymers solution makes solution lead to peralkaline Al after diluting with 100 mL methylene dichloride 2O 3Pillar is removed the mantoquita in the solution, and the desolventizing of mixed solution rotary evaporation makes solution concentrated, precipitates in 150 mL ice ether, product behind the suction filtration is dissolved in the 5 mL tetrahydrofuran (THF)s, deionized water is dialysed behind 48 h, and lyophilize obtains white product to constant weight.
In conjunction with the embodiments one, embodiment two, embodiment three and embodiment four, utilize respectively proton nmr spectra ( 1H NMR), structure, the molecular weight and molecualr weight distribution of infrared spectra (FT-IR) and gel permeation chromatography (GPC) checking resulting polymers.Fig. 1 is respectively polymkeric substance Cl- α-PEG 135- α-Cl, N 3- α-PEG 135- α-N 3The proton nmr spectra comparison diagram.Fig. 2 and Fig. 3 are respectively polymer PC L 30-C ≡ C, PCL 30- α-PEG 135- α-PCL 30The hydrogen nuclear magnetic resonance spectrogram.Fig. 4 is the GPC elution curve (PEG of three kinds of polymkeric substance 135:
Figure 125245DEST_PATH_IMAGE009
=6860 g mol -1,
Figure 77020DEST_PATH_IMAGE010
=1.08; PCL 30-C ≡ C: =3010 g mol -1,
Figure 194198DEST_PATH_IMAGE010
=1.19; PCL 30- α-PEG 135- α-PCL 30:
Figure 650587DEST_PATH_IMAGE009
=11900 g mol -1, =1.10; ).Fig. 5 is polyoxyethylene glycol, Cl- α-PEG 135- α-Cl and N 3- α-PEG 135- α-N 3Infrared spectrogram, be 2109cm in wave number -1Azido group (N appears in the place 3) absorption peak.Fig. 6 is N 3- α-PEG 135- α-N 3, PCL 30-C ≡ C and triblock copolymer PCL 30- α-PEG 135- α-PCL 30Infrared spectrogram, at wave number 3280 cm -1The place is the absorption peak of alkynyl (C ≡ C), wave number 1730 cm -1The place is carbonyl (absorption peak C=O).Therefrom can analyze reaction product and meet experimental design.
Embodiment five: adopt dialysis method to prepare the triblock copolymer micella
With 1.5 mg polymer PC L 30- α-PEG 135- α-PCL 30Join in the round-bottomed flask, add 2 mL organic solvent tetrahydrofurans (THF), stir 4～6 h, polymer chain is unfolded fully come, in the situation that stir, utilize the micro-sampling pump with 2 mL h -1Speed 10 mL pH, 7.4 PB buffered soln are added, behind complete sample introduction, mixing solutions is changed in the dialysis tubing (molecular weight cut-off is 7000 Da), organic solvent is dialysed fully removes.Changing the solution after the dialysis in 25 mL volumetric flasks constant volume, is 0.06 mg mL thereby obtain concentration -1Micellar solution.Fig. 7 is PCL 30- α-PEG 135- α-PCL 30(concentration is 0.06 mg mL to the micella that self-assembly forms in pH 7.4 buffered soln -1) transmission electron microscope photo, Fig. 8 is the dynamic light scattering curve, as shown in Figure 7, triblock copolymer self-assembly in the aqueous solution forms micellar structure.Fig. 8 is the dynamic light scattering test curve figure of corresponding micella particle diameter.
Embodiment six: polymer micelle loads the performance study of anticancer drugs, doxorubicin
Take by weighing respectively 25 mg polymkeric substance and 5 mg anticancer drugs, doxorubicin hydrochlorides (DOXHCl) join in the round-bottomed flask, add 5 mL organic solvent tetrahydrofurans (THF) and 5 μ L triethylamines are removed hydrochloric acid, stir 4～6 h, the state that polymer chain is in unfold fully, then still be under the state of stirring, utilize sampling pump with 2 mL h -1Speed 10 mL ultrapure waters are added, behind complete sample introduction, mixing solutions is changed in the dialysis tubing (molecular weight cut-off is 7000 Da), organic solvent is dialysed fully removes.Changing the solution after the dialysis in 50 mL volumetric flasks constant volume, is 0.5 mg mL thereby obtain concentration -1Polypeptide drug-loaded micelle solution.Get 5 mL polypeptide drug-loaded micelle solution in dialysis tubing, the outside buffered soln that adds the different pH values of 20 mL is placed in the constant temperature oscillator, discharges under 37 ℃ water bath with thermostatic control.Get at set intervals 5 mL dialysis tubing external solution, replenish simultaneously the buffered soln of the fresh identical pH value of 5 mL.Detect the content of the DOX that discharges with spectrophotofluorometer.Be loaded with the release profiles of polymer micelle under condition of different pH of Zorubicin as shown in Figure 9, drug release rate is obviously faster than pH 7.4 when pH 5.0, illustrate that this triblock polymer carrier micelle has obvious pH susceptibility, can reach the purpose of controllable release.
Embodiment seven: 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 that selection is in the active growth phase is inoculated in every hole and contains in 96 orifice plates of 100 μ L RPMI-1640 substratum, cultivates 24 h.Configure mother liquid concentration (5 mg mL with dialysis method -1) triblock copolymer PCL 30- a-PEG 135- a-PCL 30Micella, the micellar solution of a series of different concns is joined in 96 orifice plates, continue to cultivate 48 h.The MTT reagent that then adds 25 μ L, further cultivate 30 min after, with microplate reader (Bio-Rad model 680) under 490 nm, measures the correspondence absorbancy.The method of calculation of cell survival rate are: cell survival rate (Cell viability) (%)=[A] Test/ [A] Control* 100%, wherein [A] TestBe PCL 30- α-PEG 135- α-PCL 30There is the lower absorbancy that records in copolymer micelle, and [A] ControlBe the absorbancy that records in the situation that does not add multipolymer.Its mean value is got in each sample test three times.As shown in figure 10, the double-hydrophilic block copolymer that is synthesized does not have toxic side effect substantially to the HeLa cell, proves that double-hydrophilic block copolymer has good biocompatibility.
In addition, above-mentioned testing method also is applied to the prepared micella that is loaded with anticancer drugs, doxorubicin (DOX) of embodiment six, and compares with the cytotoxicity of free Zorubicin, and the result as shown in figure 11.In the situation that identical doxorubicin concentration, the toxicity of polymer medicament carrying micelle is less, proves that polymkeric substance has good biocompatibility.

Claims (7)

1. amphipathic three block copolymer with sensitivity to acid is characterized in that: expressed by the following chemical structure formula:
；
In the formula, R 1Be selected from
Figure 933865DEST_PATH_IMAGE002
,
Figure 2012104631600100001DEST_PATH_IMAGE003
Or
Figure 880699DEST_PATH_IMAGE004
In a kind of; N is that 30～90, m is 32～192.
2. the amphipathic three block copolymer with sensitivity to acid according to claim 1, it is characterized in that: the number-average molecular weight scope of triblock copolymer is: 12000～24000 g mol -1
3. the described preparation method with amphipathic three block copolymer of sensitivity to acid of claim 1 is characterized in that, may further comprise the steps:
(1) take two ends as hydroxy-end capped polyoxyethylene glycol and the vinyl chloride ethyl ether as raw material, adopt anhydrous methylene chloride as solvent, under the catalysis of para-methylbenzenepyridinsulfonate sulfonate, react the terminal polyoxyethylene glycol that contains Dichloroethyl two acetal radicals of preparation;
Wherein, two ends are that the number-average molecular weight scope of hydroxy-end capped polyoxyethylene glycol is 2000 ~ 12000 g mol -1, two ends are that the mol ratio of hydroxy-end capped polyoxyethylene glycol, para-methylbenzenepyridinsulfonate sulfonate and vinyl chloride ethyl ether is 5: 1: (25～50);
(2) end that obtains with step (1) contains polyoxyethylene glycol and the reaction of sodium azide of Dichloroethyl two acetal radicals, the terminal polyoxyethylene glycol that contains two azidoethyls, two acetal radicals of preparation;
Described end contains the polyoxyethylene glycol of Dichloroethyl two acetal radicals and the mol ratio of sodiumazide is 1: (2～10);
(3) be catalyzer with propargyl alcohol as initiator, stannous octoate, cyclic ester monomer is carried out ring-opening polymerization, obtain single terminal hydrophobicity biodegradable polyesters with alkynyl, described cyclic ester monomer is selected from: a kind of in 6-caprolactone, rac-Lactide or the trimethylammonium cyclic carbonate ester;
The mol ratio of described propargyl alcohol and cyclic ester monomer is 1: (30～90);
(4) in the presence of " click " chemical reaction catalyst and part, the end that adopts step (2) to obtain contains the polyoxyethylene glycol of two azidoethyls, two acetal radicals and the single terminal hydrophobicity biodegradable polyesters with alkynyl that step (3) obtains, by " click " chemical reaction, obtain to have the amphipathic three block copolymer of sensitivity to acid;
Wherein, end contains two azidoethyls, two acetal radical polyoxyethylene glycol, single end is (2.1～2.4) with the hydrophobicity biodegradable polyesters of alkynyl and the mol ratio of catalyzer: 1: 1; The mol ratio of catalyzer and part is 1: (1～2).
4. preparation method according to claim 3, it is characterized in that: in the step (4), " click " chemical reaction catalyst is selected from cuprous chloride or cuprous bromide; Part is selected from: a kind of in dipyridyl, five methyl diethylentriamine, Tetramethyl Ethylene Diamine or the hexamethyl Triethylenetetramine (TETA).
5. preparation method according to claim 3, it is characterized in that: after step (1), step (2), step (3) and step (4) are finished, respectively product is carried out purification processes, described purge process may further comprise the steps:
(i) end contains the purifying of the polyoxyethylene glycol of Dichloroethyl two acetal radicals: after reaction finished, the adding massfraction was 5% Na 2CO 3Aqueous solution termination reaction, after adding the methylene dichloride dilution, water extraction 2～3 times, collect lower floor's organic phase, use anhydrous magnesium sulfate drying, the filtrate that filtration is obtained is concentrated, dropwise joins in the ice normal hexane, filter and place vacuum drying oven to be dried to constant weight the solid product of collecting, obtain white product;
Described Na 2CO 3With the mol ratio of para-methylbenzenepyridinsulfonate sulfonate be (25～50): 1; Methylene dichloride and Na 2CO 3The volume ratio of the aqueous solution is (4～8): 1;
(ii) end contains the purifying of the polyoxyethylene glycol of two azidoethyls, two acetal radicals: after reaction finishes, with reaction mixture by alkaline Al is housed 2O 3Pillar to remove the mantoquita in the solution, removal of solvent under reduced pressure, after adding the methylene dichloride dilution, lower floor's organic phase is collected in water extraction 2～3 times, uses anhydrous magnesium sulfate drying, the filtrate that filtration is obtained is concentrated, dropwise add volume ratio and be in 1: 1 the mixed solvent of ice normal hexane and ice ether, filter and place vacuum drying oven to be dried to constant weight the solid product of collecting, obtain white product;
(iii) purifying of single terminal hydrophobicity biodegradable polyesters with alkynyl: after reaction finishes, removal of solvent under reduced pressure, concentrated solution is dropwise added in the ice ether, filter and place vacuum drying oven to be dried to constant weight the solid product of collecting, obtain white product;
(iv) has the purifying of the amphipathic three block copolymer of sensitivity to acid: after reaction finishes, add tetrahydrofuran (THF) diluting reaction mixed solution, and under the condition that stirs, contact with air, make reaction terminating, after solution becomes green, make it by alkaline Al is housed 2O 3Pillar, removal of solvent under reduced pressure, again concentrated solution is dropwise added in the ice ether, filter and the solid product of collecting is dissolved in tetrahydrofuran (THF) and is contained in the dialysis tubing that molecular weight cut-off is 12000～14000 Da with deionized water dialysis 48～96 h, at last, with resulting colourless transparent solution lyophilize in the dialysis tubing, obtain the solid product of white;
The mass ratio of described tetrahydrofuran (THF) and solid product is (10～50): 1.
6. claim 1 or 2 describedly has the amphipathic three block copolymer of sensitivity to acid as the application of stimulating responsive pharmaceutical carrier.
7. intermediate, it is characterized in that: it is the polyoxyethylene glycol that end contains two azidoethyls, two acetal radicals, is expressed by the following chemical structure formula:
Figure 2012104631600100001DEST_PATH_IMAGE005
；
In the formula, m is 32～192.
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