CN106265510B - The multistage target polymer micella and preparation method thereof of pH trigger-type drug release in a kind of tumour cell - Google Patents
The multistage target polymer micella and preparation method thereof of pH trigger-type drug release in a kind of tumour cell Download PDFInfo
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Abstract
The present invention relates to the multistage target polymer micellas and preparation method thereof that pH trigger-type in a kind of tumour cell releases the drug, which is self-assembly of in an aqueous medium by the hydrophobization modified polysaccharide polymer with endosome pH sensitivity characteristic.The present invention uses hyaluronic acid-deoxycholic acid-histidine polymer for carrier, and the endosome pH sensitive polymer micella of tumor-targeting is prepared by ultrasonic method or dialysis, contains insoluble anti-tumor medicament.Passive target, CD44 receptor active targeting and the pH sensitivity that the present invention is mediated using EPR target strategy coordination mechanism, from blood long circulating, tumor tissue accumulation, cellular uptake and drug release intracellular, this four drug conveying critical stages carry out " whole process targeting " guidance, realize multistage targeting drug delivery, drug concentration intracellular is effectively improved, provides a kind of novel carrier and formulation strategies to improve the antitumor curative effect of insoluble anti-tumor medicament.
Description
Technical field
The invention belongs to high molecular material and technical field of medicine, it is related to pH trigger-type drug release in a kind of tumour cell
Multistage target polymer micella and preparation method thereof.
Technical background
Chemotherapy is the effective ways of current clinical treatment tumour, but the multidrug resistance that tumour cell generates anticarcinogen
It is the major reason for leading to chemotherapy failure.Classical tumor drug resistance mechanism is the drug-resistant protein of height expression, mainly P- sugar egg
It is white, by intracellular anticarcinogen " pump " to extracellularly, levels of drugs intracellular is caused to decline, generates drug resistance.Therefore, load medicine is utilized
System targeted delivery drug enters in tumour cell, avoids P- glycoprotein outlet, is reverse multidrug drug resistance, improves drug tumour and controls
Treat effect letter critical issue to be solved.
Hydrophobization modification has targeting polysaccharide-hyaluronic acid of cell factor CD44 receptor target effect
(hyaluronic acid, HA), polymer micelle obtained have good biocompatibility, and Drug loading capacity is strong, good body
The features such as interior external stability and long circulating.It is passive that HA micellar system can be mediated by high-permeability with delay (EPR) effect
The accumulation of tumor tissues is realized in targeting, is carried drug by the receptor-mediated endocytosis of CD44 and is entered cell, realizes it in tumour
The effective accumulation and intake of tissue, tumour cell.
For tumour cell endosome low ph value feature, the pH sensitivity micella conveying anticancer with triggering release Mechanisms is constructed
Medicine achieves breakthrough in terms of reverse multidrug drug resistance.The intelligent delivery system can respond the triggering of endosome acid pH
Drug release, and drug is discharged into cytoplasm, the drug concentration in cytoplasm is effectively improved, the resistance mechanism of tumour cell is saturated, gram
Take multidrug resistance.Therefore, receptor-mediated targeting drug delivery and pH sensitivity targeting drug release strategy are united and applied in micellar system, performance
Good collaboration targeting drug delivery effect out, improves the therapeutic efficiency of anticarcinogen and reduces its toxic side effect, control for the targeting of cancer
It treats and new approach is provided.
Summary of the invention
Excellent passive target is shown in view of hydrophobization modified polysaccharide polymer micelle, Drug loading capacity and long circulating are special
Property；The positioning release medicine function of cancer target ability and physical chemistry the targeting strategy of active targeting strategy, the present invention combine quilt
Moving-target targets mechanism to, active targeting and physical chemistry, provides a kind of multistage target of tumour cell endosome pH trigger-type drug release
To polymer micelle, with realize the tumor tissues targeting accumulation that EPR effect mediates, the receptor-mediated active targeting of CD44 absorb into
Born of the same parents and endosome pH triggering drug release, combine reversing tumor drug resistance, improve the antitumor efficacy of drug.
It is a further object to provide the multistage targeting polymerizations of above-mentioned tumour cell endosome pH trigger-type drug release
The preparation method of object micella.
For achieving the above object, the present invention is achieved by the following scheme:
The multistage target polymer micella of pH trigger-type drug release in a kind of tumour cell, it is characterised in that the micella includes tool
There is the hydrophobization modified polysaccharide polymer of endosome pH sensitivity characteristic, which is self-assembly of in an aqueous medium.
The polymer has hydrophilic segment and hydrophobic segment, and wherein hydrophilic segment is with CD44 receptor target characteristic
Hyaluronic acid (HA)；Hydrophobic segment is selected from deoxycholic acid (deoxycholic acid, DOCA), and connects endosome at its end
PH sensing unit.
The degree of substitution of the hydrophobic segment is 5%~30%.
The pH sensing unit is histidine (histidine, His) or its analogue.
Contain imidazole ring in the molecular structure of the histidine or its analogue, pH response range between 5.0~
Between 7.4.
The molecular weight of the hyaluronic acid is 1 × 103~1 × 107 Da。
The polymer is prepared according to following processing step:
1) synthesis of deoxycholic acid-histidine or its analogue (Trt)
Deoxycholic acid (DOCA) is dissolved in reaction dissolvent, with 1- ethyl-(3- dimethylaminopropyl) carbodiimide
(EDC) and HOSu NHS (NHS) is activator, activates 2~24 h at room temperature；
Histidine or its analogue (Trt) are dissolved in reaction dissolvent, triethylamine is added, is then slowly dropped to
It states in deoxycholic acid mixed liquor, 12~48 h is stirred to react under the conditions of 30~50 DEG C of temperature, collect product, be dried under reduced pressure, obtain
To deoxycholic acid-histidine or its analogue (Trt)；
2) synthesis of hyaluronic acid-deoxycholic acid-histidine or its analogue (Trt)
Hyaluronic acid is dissolved in reaction dissolvent, stirring and dissolving under the conditions of 40~60 DEG C of temperature is cooled to room temperature；Then
1- ethyl-(3- dimethylaminopropyl) carbodiimide and HOSu NHS is added, activates 2~5 h under ice bath；
Deoxycholic acid-histidine that step 1) obtains or its analogue (Trt) are dissolved in reaction dissolvent, then delayed
Slowly it is added drop-wise in above-mentioned hyaluronic acid mixed liquor, 6~12 h is stirred to react under the conditions of 40~70 DEG C of temperature, are continued at room temperature
24~48 h are stirred to react, product is collected, product obtains hyaluronic acid-deoxycholic acid-group ammonia through dialysis purification, freeze-drying
Acid or its analogue (Trt)；
3) synthesis of hyaluronic acid-deoxycholic acid-histidine or its analogue
Hyaluronic acid-deoxycholic acid-histidine obtained by step 2 or its analogue (Trt) are dissolved in trifluoroacetic acid,
Thioanisole is added, stirs 2~12 h at room temperature, collects product, product obtains hyaluronic acid-through dialysis purification, freeze-drying
Deoxycholic acid-histidine or its analogue.
The reaction dissolvent is N, N- dimethylformamide, formamide, ethanol water and N, N- dimethylformamide
One of aqueous solution or multiple combinations.
In step 1), the deoxycholic acid is 1:1~2 with the molar ratio of histidine or its analogue；
The molar ratio of deoxycholic acid, 1- ethyl-(3- dimethylaminopropyl) carbodiimide and HOSu NHS is
1:1~2:1~2；
The molar ratio of histidine or its analogue (Trt) and triethylamine is 1:1~2；
In step 2, mole of the hyaluronic acid and deoxycholic acid-histidine or its analogue (Trt) intermediate
Than for 1:2~5,
The molar ratio of hyaluronic acid and 1- ethyl-(3- dimethylaminopropyl) carbodiimide, HOSu NHS is
1:1~5:1~5,
In step 3), hyaluronic acid-deoxycholic acid-histidine or its analogue (Trt) and trifluoroacetic acid, benzene first sulphur
The molar ratio of ether is 1:1~5:1~5.
The preparation method for the multistage target polymer micella that pH trigger-type releases the drug in above-mentioned tumour cell, it is characterised in that:
By the hydrophobization modified polysaccharide polymer with endosome pH sensitivity characteristic using ultrasonic method, dialysis or solvent evaporation method preparation
Polymer micelle contains insoluble anti-tumor medicament.
The specific steps of the ultrasonic method are as follows:
1) the hydrophobization modified polysaccharide polymer with endosome pH sensitivity characteristic is pressed to the concentration of 1~5 mg/mL
It is dissolved in the phosphate buffer (phosphate buffer solution, PBS) of pH 7.4；
2) after dissolving insoluble anti-tumor medicament with organic solvent, the above process 1 is instilled) in resulting polymer P BS,
It is sonicated, the carrier micelle that partial size is 10~1000 nm is made.
The detailed process of the dialysis are as follows: polymerize the hydrophobization modified polysaccharide with endosome pH sensitivity characteristic
Object and insoluble anti-tumor medicament are codissolved in organic solvent, are placed in the bag filter that molecular cut off is 3500, immerse pH 7.4
PBS in, dialysis removes organic solvent and free drug, and liquid is the load medicine that partial size obtained is 10~1000 nm in bag
Micella.
The detailed process of the solvent evaporation method are as follows: the hydrophobization modification with endosome pH sensitivity characteristic is transparent
Matter acid polymer and insoluble anti-tumor medicament are codissolved in organic solvent, and the PBS of pH 7.4 is added, and stirring to organic solvent is waved
It goes, the carrier micelle that partial size is 10~1000 nm is made.
The insoluble anti-tumor medicament is taxol, adriamycin, camptothecine, hydroxycamptothecin, topotecan, replaces up to sand
One of Buddhist nun, 5 FU 5 fluorouracil, vincristine and cis-platinum are two or more.
The organic solvent is ethyl alcohol, tetrahydrofuran, dimethyl sulfoxide, N, one of N- dimethylformamide or more
Kind combination.
By above technical scheme, the beneficial effects of the present invention are: the micella can be used for preparing treatment tumor disease drug
Purposes, intravenous administration administration, in physiological environment (pH 7.4) and tumour cell external solution (pH 6.5~7.2), micellar structure
Completely, it does not discharge or low release drug, one can be gone forward side by side by tumor vascular targeting accumulation that EPR effect mediates in tumor tissues
Step enters tumour cell by the CD44 receptor active targeting mechanism intake of HA；The finally glue in endosome (pH 5.0~6.0)
Shu Xieju, triggering drug release, while endosome film ruptures, release drug reaches cytoplasm, improves drug concentration intracellular, is saturated drug resistance
Mechanism overcomes tumor multi-medicine drug-resistant, improves drugs against tumor effect.
In conclusion the present invention uses hyaluronic acid-deoxycholic acid-histidine polymer for carrier, by ultrasonic method, thoroughly
Analysis method or solvent evaporation method prepare the endosome pH sensitive polymer micella of tumor-targeting, contain slightly solubility antineoplastic
Object.Passive target, CD44 receptor active targeting and the pH sensitivity that the present invention is mediated using EPR target strategy coordination mechanism, from blood
This four drug conveying critical stages of liquid long circulating, tumor tissue accumulation, cellular uptake and drug release intracellular carry out " whole process targeting "
Multistage targeting drug delivery is realized in guidance, for improve the antitumor curative effect of insoluble anti-tumor medicament provide a kind of novel carrier and
Formulation strategies.
Detailed description of the invention
Fig. 1 is the synthetic route chart of HA-DOCA-His polymer；
Fig. 2 is the grain size distribution of HA-DOCA-His micella at various ph values；
Fig. 3 is release profiles of the HA-DOCA-His micella of load taxol in different pH dissolution mediums；
Fig. 4 is the self assembly of HA-DOCA-His carrier micelle, targeted to tumor tissue accumulation, tumour cell intake and cell
The interior acid-sensitive depolymerization triggering drug release schematic diagram of endosome.
Specific embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further, but protection scope of the present invention is not limited to that.
The synthesis of example 1:HA-DOCA-His polymer
0.5 g DOCA is dissolved in 5 mL n,N-Dimethylformamide, and 0.29 g EDC is added and 0.18 g NHS, room temperature are stirred
Mix 2 h.0.37 g trityl histidine methylester hydrochloride (H-His (Trt)-OMeHCl) is dissolved in 20 mL DMF, is added
Enter 50 μ L triethylamines, be slowly dropped in DOCA mixed liquor, mixed liquor is in 35 DEG C of 24 h of stirring in water bath.Add in reaction mixture
Enter NaHCO3Solution (pH 9~10) filters to obtain precipitating, is dried under reduced pressure to obtain DOCA-His (Trt).
0.1 g HA is dissolved in 5 mL dry formamides, and 50 DEG C of heating for dissolving are cooled to room temperature.96 mg EDC and 58 are added
Mg NHS, 2 h of ice bath magnetic agitation.0.4 g DOCA-His (Trt) is dissolved in 5 mL anhydrous DMFs, is slowly dropped to HA mixing
In liquid, after 50 DEG C of 6 h of stirring in water bath, continue to stir 24 h in room temperature.Reaction mixture dialyses 2~3 days (thoroughly in distilled water
Analysis bag molecular cut off: 3500), being filtered to remove insoluble impurities, is freeze-dried, obtains HA-DOCA-His (Trt) white powder.
0.1 g HA-DOCA-His (Trt) is dissolved in 0.5 mL DMSO, and 0.5 mL trifluoroacetic acid and 25 μ L benzene first are added
2 h are stirred at room temperature, reaction mixture 2 d(dialysis bag retention molecular weights of dialysis in buck (pH 9~10) in thioether: 3500),
It dialyses 2 days in distilled water, is filtered to remove insoluble impurities, be freeze-dried, obtain HA-DOCA-His white powder.Reaction route is such as
Shown in Fig. 1.
The synthesis of example 2:HA-DOCA-His polymer
0.5 g DOCA is dissolved in 5 mL n,N-Dimethylformamide, and 0.29 g EDC is added and 0.18 g NHS, room temperature are stirred
Mix 24 h.0.37 g trityl histidine methylester hydrochloride (H-His (Trt)-OMeHCl) is dissolved in 20 mL DMF, is delayed
Slowly it is added drop-wise in DOCA mixed liquor, mixed liquor is in 35 DEG C of 24 h of stirring in water bath.Reaction mixture revolving removes organic solvent, column
Separation, obtains DOCA-His (Trt).
0.1 g HA is dissolved in 5 mL dry formamides, and 50 DEG C of heating for dissolving are cooled to room temperature.96 mg EDC and 58 are added
Mg NHS, 2 h of ice bath magnetic agitation.0.4 g DOCA-His (Trt) is dissolved in 5 mL anhydrous DMFs, is slowly dropped to HA mixing
In liquid, after 50 DEG C of 6 h of stirring in water bath, continue to stir 24 h in room temperature.Reaction mixture dialyses 2~3 days (thoroughly in distilled water
Analysis bag molecular cut off: 3500), being filtered to remove insoluble impurities, is freeze-dried, obtains HA-DOCA-His (Trt) white powder.
0.1 g HA-DOCA-His (Trt) is dissolved in 0.5 mL DMSO, and 0.5 mL trifluoroacetic acid and 25 μ L benzene first are added
Thioether, is stirred at room temperature 2 h, and reaction mixture is dialysed 2 d(dialysis bag retention molecular weights in distilled water: 3500), in distilled water
Dialysis 2 days, is filtered to remove insoluble impurities, is freeze-dried, obtains HA-DOCA-His white powder.
The synthesis of example 3:HA-DOCA-His polymer
0.5 g DOCA is dissolved in 5 mL n,N-Dimethylformamide, and 0.29 g EDC is added and 0.18 g NHS, room temperature are stirred
Mix 24 h.0.37 g trityl histidine methylester (H-His (Trt)-OH) is dissolved in 20 mL DMF, is slowly dropped to DOCA
In mixed liquor, mixed liquor is in 35 DEG C of 24 h of stirring in water bath.Reaction mixture revolving removes organic solvent, and post separation obtains DOCA-
His (Trt)。
0.1 g HA is dissolved in 5 mL dry formamides, and 60 DEG C of heating for dissolving are cooled to room temperature.Be added 192 mg EDC and
116 mg NHS, 5 h of ice bath magnetic agitation.0.8 g DOCA-His (Trt) is dissolved in 5 mL anhydrous DMFs, is slowly dropped to HA
In mixed liquor, 48 h are stirred at room temperature.Ethanol precipitation removal of impurities is added in reaction mixture, obtains the white powder of HA-DOCA-His (Trt)
End.
0.1 g HA-DOCA-His (Trt) is dissolved in 0.5 mL DMSO, and 0.5 mL trifluoroacetic acid and 50 μ L benzene first are added
Thioether, is stirred at room temperature 12 h, and reaction mixture is dialysed 2 days (dialysis bag retention molecular weights: 3500), in distilled water in distilled water
Dialysis 2 days, is filtered to remove insoluble impurities, is freeze-dried, obtains HA-DOCA-His white powder.
HA-DOCA-His's1HNMR map as shown in Fig. 2, HA NHCOCH3Upper-CH3Chemical shift 1.95
Ppm, on 2,3,4 and 5 carbon the chemical shift of hydrogen in 3.0~4.0 ppm, and on 6 carbon the chemical shift of hydrogen 4.35~
- CH in DOCA is belonged at 4.45 ppm, 0.8~2.0 ppm3With-CH2Characteristic peak, return at 7.45 ppm and 8.82 ppm
Belong to-N in His=CH- and-N=CH=C- characteristic peak, it was demonstrated that DOCA-His has successfully been synthesized on HA.
Example 4: ultrasonic method preparation carries the HA-DOCA-His micella of taxol
HA-DOCA-His is dissolved in the PBS of pH 7.4, and concentration is 1~5 mg/mL, and magnetic agitation, will be molten to being completely dissolved
Solution is slowly added dropwise in the taxol (10%, 20% and the 30% of HA-DOCA-His additional amount) of dehydrated alcohol (methanol, tetrahydrofuran)
Enter in above-mentioned polymer solution, after 24~48 h of magnetic agitation, ice-bath ultrasonic (100~400 W of ultrasonic power, work 2 s,
Have a rest 3 s) 2~20 min, and carrier micelle is made through 0.45 μm of membrane filtration in micellar solution, and freeze-drying can obtain carrier micelle powder.
Example 5: dialysis preparation carries the HA-DOCA-His micella of adriamycin
Weigh 5 mg doxorubicin hydrochlorides and be scattered in 1 mL DMSO(tetrahydrofuran) in, 7 μ L triethylamines are added, stirring is extremely
Adriamycin dissolution, is made adriamycin DMSO(tetrahydrofuran) solution (5 mg/mL).HA-DOCA-His is dissolved in DMSO(10 mg/
ML in), adriamycin (10%, 20% and the 30% of HA-DOCA-His additional amount) DMSO(tetrahydrofuran is added) solution, magnetic at room temperature
Power stirring makes its mixing, is placed in bag filter and (molecular cut off: in 3500), immerses in the PBS of pH7.4 and dialyse 3 days, in bag filter
Carrier micelle is made through 0.45 μm of membrane filtration in liquid, and freeze-drying can obtain carrier micelle powder.
Example 6: solvent evaporation method preparation carries the HA-DOCA-His micella of adriamycin
It weighs 5 mg doxorubicin hydrochlorides to be scattered in 1 mL methanol, 7 μ L triethylamines is added, stir molten to adriamycin
Solution is made adriamycin methanol solution (5 mg/mL).HA-DOCA-His is dissolved in tetrahydrofuran (10 mg/mL), and adriamycin is added
(10%, 20% and the 30% of HA-DOCA-His additional amount) methanol solution, magnetic agitation make its mixing, instill the PBS of pH7.4, magnetic
Power stirs 24~48 h, and carrier micelle is made through 0.45 μm of membrane filtration in micellar solution, and freeze-drying can obtain carrier micelle powder.
Example 7:HA-DOCA-His micella pH sensitivity testing
HA-DOCA-His polymer is dissolved in the PBS of pH7.4, and concentration is 1 mg/mL.Micella is measured using particle size analyzer
Average grain diameter and PI, with the HCl solution of 0.01 mol/L, successively adjusting pH is 6.5,6.0,5.0, one pH value of every adjusting,
30 min of sample solution magnetic agitation stands 10 min, measures partial size, in triplicate, observes change of size situation.As a result see figure
3, micella change of size in pH 7.4 and 6.5 is unobvious, pH 6.0 and partial size and PI increased dramatically when pH 5.0, shows HA-
DOCA-His micella has endosome pH(5.0~6.0) sensibility.
Example 8: the release in vitro of the HA-DOCA-His micella of taxol is carried
Using the tablets in vitro of carrier micelle obtained in dialysis investigation example 4.Precision measures 3 mL and carries taxol
HA-DOCA-His micella is placed in bag filter and (molecular cut off: in 3500), immerses 80 mL and contain 2% Cremophor EL(w/
V), pH be respectively 7.4,6.5,6.0,5.0 PBS in, in 37 DEG C, 100 r/min shaking, respectively at 1,2,4,6,8,12,16,
24,36 h and 48 h sample 3 mL, while supplementing the fresh medium of same pH, volume and temperature.Sample is through 0.45 μm of filter membrane
Filtering discards primary filtrate, and HPLC method measures content of taxol, and calculates cumulative release percentage, as a result sees Fig. 4.Carry taxol
HA-DOCA-His micella pH be 7.4,6.5,6.0,5.0 dissolution medium in, the cumulative release amount of 6 h is respectively
12.2%, 13.8%, 29.9% and 55.3%, show HA-DOCA-His micella in physiological environment (pH 7.4) and tumour cell external solution
(> pH 6.5) in it is stable, into tumour cell endosome acidic environment (pH 5.0~6.0) can be realized triggering drug release.
Claims (13)
1. the multistage target polymer micella that pH trigger-type releases the drug in a kind of tumour cell, it is characterised in that the micella is by having
Phosphate-buffered of the hydrophobization modified polysaccharide polymer and insoluble anti-tumor medicament of endosome pH sensitivity characteristic in pH 7.4
It is self-assembly of in liquid, the polymer is prepared into according to following processing step with hydrophilic segment and hydrophobic segment, wherein parent
Water segment is the hyaluronic acid with CD44 receptor target characteristic, and hydrophobic segment is selected from deoxycholic acid, and at its end in connection
Contain the structure type of body pH sensing unit histidine,
1) deoxycholic acid-trityl histidine synthesis
Deoxycholic acid is dissolved in reaction dissolvent, with 1- ethyl-(3- dimethylaminopropyl) carbodiimide and hydroxysuccinimidyl acyl
Imines is activator, activates 2~24 h at room temperature；
Trityl histidine methylester hydrochloride is dissolved in reaction dissolvent, triethylamine is added, is then slowly dropped to above-mentioned de-
In oxycholic acid mixed liquor, it is stirred to react 12~48 h under the conditions of 30~50 DEG C of temperature, collects product, is dried under reduced pressure, is taken off
Oxycholic acid-trityl histidine；
2) hyaluronic acid-deoxycholic acid-trityl histidine synthesis
Hyaluronic acid is dissolved in reaction dissolvent, stirring and dissolving under the conditions of 40~60 DEG C of temperature is cooled to room temperature；Then it is added
1- ethyl-(3- dimethylaminopropyl) carbodiimide and HOSu NHS activate 2~5 h under ice bath；
Deoxycholic acid-trityl histidine that step 1) obtains is dissolved in reaction dissolvent, is then slowly dropped to above-mentioned
In bright matter acid mixed liquor, it is stirred to react 6~12 h under the conditions of 40~70 DEG C of temperature, continues to be stirred to react 24~48 at room temperature
H, collects product, and product obtains hyaluronic acid-deoxycholic acid-trityl histidine through dialysis purification, freeze-drying；
3) hyaluronic acid-deoxycholic acid-histidine synthesis；
Hyaluronic acid-deoxycholic acid-trityl histidine obtained by step 2 is dissolved in trifluoroacetic acid, thioanisole, room is added
Temperature is lower to stir 2~12 h, collects product, and product obtains hyaluronic acid-deoxycholic acid-group ammonia through dialysis purification, freeze-drying
Acid.
2. the multistage target polymer micella that pH trigger-type releases the drug in tumour cell described in accordance with the claim 1, feature exist
In the polymer hydrophobic segment degree of substitution be 5%~30%.
3. the multistage target polymer micella that pH trigger-type releases the drug in tumour cell described in accordance with the claim 1, feature exist
Contain imidazole ring in the molecular structure of histidine in the polymer, pH response range is between 5.0~7.4.
4. the multistage target polymer micella that pH trigger-type releases the drug in tumour cell described in accordance with the claim 1, feature exist
The molecular weight of hyaluronic acid is 1 × 10 in the polymer3~1 × 107 Da。
5. the multistage target polymer micella that pH trigger-type releases the drug in tumour cell described in accordance with the claim 1, feature exist
In the reaction dissolvent be N, N- dimethylformamide, formamide, ethanol water and N, N- dimethylformamide in water
One of or multiple combinations.
6. the multistage target polymer micella that pH trigger-type releases the drug in tumour cell described in accordance with the claim 1, feature exist
In in step 1), the molar ratio of the deoxycholic acid and trityl histidine methylester hydrochloride is 1:1~2；
The molar ratio of deoxycholic acid, 1- ethyl-(3- dimethylaminopropyl) carbodiimide and HOSu NHS be 1:1~
2:1~2；
The molar ratio of trityl histidine methylester hydrochloride and triethylamine is 1:1~2.
7. the multistage target polymer micella that pH trigger-type releases the drug in tumour cell described in accordance with the claim 1, feature exist
In, in step 2, the hyaluronic acid and deoxycholic acid-trityl histidine intermediate molar ratio are 1:2~5,
Hyaluronic acid and 1- ethyl-(3- dimethylaminopropyl) carbodiimide, HOSu NHS molar ratio be 1:1~
5:1~5.
8. the multistage target polymer micella that pH trigger-type releases the drug in tumour cell described in accordance with the claim 1, feature exist
In in step 3), the molar ratio of hyaluronic acid-deoxycholic acid-trityl histidine and trifluoroacetic acid, thioanisole is 1:1
~5:1~5.
9. the multistage target polymer micella of pH trigger-type drug release in a kind of such as described in any item tumour cells of claim 1 ~ 8
Preparation method, it is characterised in that: by the hydrophobization modified polysaccharide polymer with endosome pH sensitivity characteristic using ultrasonic method,
Dialysis or solvent evaporation method preparation contain the polymer micelle of insoluble anti-tumor medicament.
10. the preparation side for the multistage target polymer micella that pH trigger-type releases the drug in tumour cell according to claim 9
Method, it is characterised in that the specific steps of the ultrasonic method are as follows:
1) the hydrophobization modified polysaccharide polymer with endosome pH sensitivity characteristic is dissolved in by the concentration of 1~5 mg/mL
In the phosphate buffer of pH 7.4；
2) after dissolving insoluble anti-tumor medicament with organic solvent, the above process 1 is instilled) resulting polymer micelle solution
In, it is sonicated, the carrier micelle that partial size is 10~1000 nm is made；
The organic solvent is ethyl alcohol, tetrahydrofuran, dimethyl sulfoxide, N, one of N- dimethylformamide or a variety of groups
It closes.
11. the preparation side for the multistage target polymer micella that pH trigger-type releases the drug in tumour cell according to claim 9
Method, it is characterised in that the detailed process of the dialysis are as follows: the hydrophobization modification with endosome pH sensitivity characteristic is more
Glycopolymers and insoluble anti-tumor medicament are codissolved in organic solvent, are placed in the bag filter that molecular cut off is 3500, immerse
In the phosphate buffer of pH 7.4, dialysis removes organic solvent and free drug, and it is 10 that liquid, which is partial size obtained, in bag
The carrier micelle of~1000 nm；
The organic solvent is ethyl alcohol, tetrahydrofuran, dimethyl sulfoxide, N, one of N- dimethylformamide or a variety of groups
It closes.
12. the preparation side for the multistage target polymer micella that pH trigger-type releases the drug in tumour cell according to claim 9
Method, it is characterised in that the detailed process of the solvent evaporation method are as follows: repair the hydrophobization with endosome pH sensitivity characteristic
Decorations polysaccharide polymer and insoluble anti-tumor medicament are codissolved in organic solvent, the phosphate buffer of pH 7.4 are added, stirring is extremely
Organic solvent is flung to, and the carrier micelle that partial size is 10~1000 nm is made；
The organic solvent is ethyl alcohol, tetrahydrofuran, dimethyl sulfoxide, N, one of N- dimethylformamide or a variety of groups
It closes.
13. the preparation side for the multistage target polymer micella that pH trigger-type releases the drug in tumour cell according to claim 9
Method, it is characterised in that the insoluble anti-tumor medicament be taxol, adriamycin, camptothecine, hydroxycamptothecin, topotecan,
One of Dasatinib, 5 FU 5 fluorouracil, vincristine and cis-platinum are two or more.
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