CN106265510A - Multistage target polymer micelle of pH trigger-type release and preparation method thereof in a kind of tumor cell - Google Patents
Multistage target polymer micelle of pH trigger-type release and preparation method thereof in a kind of tumor cell Download PDFInfo
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Abstract
The present invention relates to multistage target polymer micelle of pH trigger-type release and preparation method thereof in a kind of tumor cell, this micelle is self-assembly of in aqueous medium by the hydrophobization modified polysaccharide polymer with endosome pH sensitivity characteristic.The present invention uses hyaluronic acid deoxycholic acid histidine polymer to be carrier, is prepared the endosome pH sensitive polymer micelle of tumor-targeting by ultrasonic method or dialysis, and bag carries insoluble anti-tumor medicament.The present invention utilizes passive target, CD44 receptor active targeting and the pH sensitivity targeting strategy coordination mechanism that EPR mediates, carry out " omnidistance targeting " from these four medicine conveying critical stages of blood long circulating, tumor tissue accumulation, cellular uptake and intracellular release to guide, realize multistage targeting drug delivery, it is effectively improved intracellular drug level, provides a kind of novel carrier and formulation strategies for improving the antitumor curative effect of insoluble anti-tumor medicament.
Description
Technical field
The invention belongs to macromolecular material and technical field of medicine, relate to pH trigger-type release in a kind of tumor cell
Multistage target polymer micelle and preparation method thereof.
Technical background
Chemotherapy is the effective ways of current clinical treatment tumor, but the multidrug resistance that anticarcinogen is produced by tumor cell
It it is the major reason causing chemotherapy failed.Classical tumor drug resistance mechanism is the drug-resistant protein that height is expressed, mainly P-sugar egg
In vain, by intracellular anticarcinogen " pump " to extracellular, cause intracellular levels of drugs to decline, produce drug resistance.Therefore, medicine carrying is utilized
System targeted delivery medicine enters in tumor cell, it is to avoid arranges outside P-glycoprotein, is reverse multidrug drug resistance, improves medicine tumor and controls
Treat the key issue that effect letter is to be solved.
Hydrophobization modifies the targeting hyaluronan with the receptor target effect of cytokine CD44
(hyaluronic acid, HA), the polymer micelle prepared has good biocompatibility, and Drug loading capacity is strong, good body
The features such as interior external stability and long circulating.HA micellar system can be passive with what delay (EPR) effect mediated by high-permeability
Targeting realizes the accumulation of tumor tissues, carries medicine and enters cell by the receptor-mediated endocytosis of CD44, it is achieved that it is in tumor
Tissue, effectively accumulation and the picked-up of tumor cell.
For tumor cell endosome low ph value feature, construct and have that to trigger the pH sensitivity micelle conveying of release Mechanisms anticancer
Medicine, achieves breakthrough in terms of reverse multidrug drug resistance.This intelligent delivery system can respond endosome acid pH and trigger
Release, and discharge medicine in Cytoplasm, the drug level being effectively improved in kytoplasm, the resistance mechanism of saturated tumor cell, 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
Going out good collaborative targeting drug delivery effect, improve the therapeutic efficiency of anticarcinogen and reduce its toxic and side effects, the targeting for cancer is controlled
Treat the approach providing new.
Summary of the invention
In view of hydrophobization modified polysaccharide polymer micelle shows excellent passive target, Drug loading capacity and long circulating spy
Property；The cancer target ability of active targeting strategy, and the positioning release medicine function of physical chemistry targeting strategy, the present invention combines quilt
Moving-target is to, active targeting and physical chemistry targeting mechanism, it is provided that the multistage target of a kind of tumor cell endosome pH trigger-type release
To polymer micelle, to realize the tumor tissues targeting accumulation of EPR effect mediation, the receptor-mediated active targeting of CD44 absorb into
Born of the same parents and endosome pH trigger release, combine reversing tumor drug resistance, improve the antitumor efficacy of medicine.
It is a further object to provide the multistage targeting polymerization of above-mentioned tumor cell endosome pH trigger-type release
The preparation method of thing micelle.
For achieving the above object, the present invention is achieved by the following technical solutions:
The multistage target polymer micelle of pH trigger-type release in a kind of tumor cell, it is characterised in that in this micelle includes having
Containing the hydrophobization modified polysaccharide polymer of body pH sensitivity characteristic, this polymer is self-assembly of in aqueous medium.
Described polymer has hydrophilic segment and hydrophobic segment, and wherein hydrophilic segment is to have 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 substitution value of described hydrophobic segment is 5%～30%.
Described pH sensing unit is histidine (histidine, His) or its analog.
Containing imidazole ring in the molecular structure of described histidine or its analog, its pH response range between 5.0～
Between 7.4.
The molecular weight of described hyaluronic acid is 1 × 103～1 × 107 Da。
Described polymer is prepared according to following processing step:
1) deoxycholic acid-histidine or the synthesis of its analog (Trt)
Deoxycholic acid (DOCA) is dissolved in reaction dissolvent, with 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and
N-Hydroxysuccinimide (NHS) is activator, activates 2～24 h under room temperature；
Histidine or its analog (Trt) are dissolved in reaction dissolvent, add triethylamine, be then slowly dropped to above-mentioned de-
In oxycholic acid mixed liquor, under 30～50 DEG C of temperature conditionss, stirring reaction 12～48 h, collect product, drying under reduced pressure, are taken off
Oxycholic acid-histidine or its analog (Trt)；
2) hyaluronic acid-deoxycholic acid-histidine or the synthesis of its analog (Trt)
Hyaluronic acid is dissolved in reaction dissolvent, stirring and dissolving under 40～60 DEG C of temperature conditionss, is cooled to room temperature；It is subsequently adding
1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-Hydroxysuccinimide, activate 2～5 h under ice bath；
Deoxycholic acid-the histidine or its analog (Trt) that step 1) are obtained are dissolved in reaction dissolvent, the most slowly drip
Being added in above-mentioned hyaluronic acid mixed liquor, under 40～70 DEG C of temperature conditionss, stirring reaction 6～12 h, continue stirring at room temperature
Reaction 24～48 h, collect product, product through dialysis purification, lyophilization, obtain hyaluronic acid-deoxycholic acid-histidine or
Its analog (Trt)；
3) hyaluronic acid-deoxycholic acid-histidine or the synthesis of its analog
By step 2) gained hyaluronic acid-deoxycholic acid-histidine or its analog (Trt) be dissolved in trifluoroacetic acid, add
Thioanisole, stirs 2～12 h, collects product under room temperature, product, through dialysis purification, lyophilization, obtains hyaluronic acid-deoxidation
Cholic acid-histidine or its analog.
Described reaction dissolvent is N, N-dimethylformamide, Methanamide, ethanol water and N, N-dimethylformamide
One or more combinations in aqueous solution.
In step 1), described deoxycholic acid is 1:1～2 with the mol ratio of histidine or its analog；
The mol ratio of deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-Hydroxysuccinimide be 1:1～
2:1～2；
The mol ratio of histidine or its analog (Trt) and triethylamine is 1:1～2；
Step 2) in, described hyaluronic acid with the mol ratio of deoxycholic acid-histidine or its analog (Trt) intermediate is
1:2～5,
Hyaluronic acid and 1-ethyl-(3-dimethylaminopropyl) carbodiimide, the mol ratio of N-Hydroxysuccinimide be 1:1～
5:1～5,
In step 3), hyaluronic acid-deoxycholic acid-histidine or its analog (Trt) and trifluoroacetic acid, thioanisole
Mol ratio is 1:1～5:1～5.
The preparation method of the multistage target polymer micelle of pH trigger-type release in above-mentioned tumor cell, it is characterised in that:
Ultrasonic method, dialysis or solvent evaporation method is used to prepare in the hydrophobization modified polysaccharide polymer with endosome pH sensitivity characteristic
Polymer micelle, bag carries insoluble anti-tumor medicament.
Concretely comprising the following steps of described ultrasonic method:
1) the described hydrophobization modified polysaccharide polymer with endosome pH sensitivity characteristic is dissolved in by the concentration of 1～5 mg/mL
In the phosphate buffer (phosphate buffer solution, PBS) of pH 7.4；
2), after being dissolved with organic solvent by insoluble anti-tumor medicament, said process 1 is instilled) in the polymer P BS of gained, through super
Sonication, prepared particle diameter is the carrier micelle of 10～1000 nm.
The detailed process of described dialysis is: by the described hydrophobization modified polysaccharide polymerization with endosome pH sensitivity characteristic
Thing 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 in bag, liquid is the medicine carrying that prepared particle diameter is 10～1000 nm
Micelle.
The detailed process of described solvent evaporation method is: modify transparent by the described hydrophobization with endosome pH sensitivity characteristic
Matter acid polymer and insoluble anti-tumor medicament are codissolved in organic solvent, add the PBS of pH 7.4, and stirring to organic solvent is waved
Going, prepared particle diameter is the carrier micelle of 10～1000 nm.
Described insoluble anti-tumor medicament is paclitaxel, amycin, camptothecine, hydroxy camptothecin, topotecan, reach sand replaces
More than the one or two kinds of in Buddhist nun, 5-fluorouracil, vincristine and cisplatin.
Described organic solvent is ethanol, oxolane, dimethyl sulfoxide, N, the one or many in N-dimethylformamide
Plant combination.
By above technical scheme, the invention has the beneficial effects as follows: this micelle can be used for preparation treatment tumor disease medicine
Purposes, intravenous administration is administered, in physiological environment (pH 7.4) and tumor cell liquid (pH 6.5～7.2), micellar structure outward
Completely, do not discharge or low release medicine, can accumulate in tumor tissues by the tumor vascular targeting that EPR effect mediates, go forward side by side one
The CD44 receptor active targeting mechanism by HA that walks is absorbed and is entered tumor cell；Final glue in endosome (pH 5.0～6.0)
Shu Xieju, triggers release, simultaneously endosome film rupture, and release medicine arrives Cytoplasm, improves intracellular drug level, saturated drug resistance
Mechanism, overcomes tumor multi-medicine drug-resistant, improves drugs against tumor effect.
In sum, the present invention uses hyaluronic acid-deoxycholic acid-histidine polymer to be carrier, by ultrasonic method, thoroughly
Analysis method or solvent evaporation method prepare the endosome pH sensitive polymer micelle of tumor-targeting, and bag carries slightly solubility antineoplastic agent
Thing.The present invention utilizes passive target, CD44 receptor active targeting and the pH sensitivity targeting strategy coordination mechanism that EPR mediates, from blood
These four medicine conveying critical stages of liquid long circulating, tumor tissue accumulation, cellular uptake and intracellular release carry out " omnidistance targeting "
Guide, it is achieved multistage targeting drug delivery, for improve insoluble anti-tumor medicament antitumor curative effect provide a kind of novel carrier and
Formulation strategies.
Accompanying drawing explanation
Fig. 1 is the synthetic route chart of HA-DOCA-His polymer；
Fig. 2 is HA-DOCA-His micelle grain size distribution at various ph values；
Fig. 3 is the HA-DOCA-His micelle carrying paclitaxel release profiles in different pH release medium；
Fig. 4 is HA-DOCA-His carrier micelle self assembly, be targeted to tumor tissue accumulation, tumor cell picked-up and intracellular in
Contain the acid-sensitive depolymerization of body and trigger release schematic diagram.
Detailed description of the invention
For better illustrating 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 DMFs, adds 0.29 g EDC and 0.18 g NHS, is stirred at room temperature 2
h.0.37 g trityl histidine methylester hydrochlorate (H-His (Trt)-OMe HCl) is dissolved in 20 mL DMF, adds 50
L triethylamine, is slowly dropped in DOCA mixed liquor, and mixed liquor is in 35 DEG C of stirring in water bath 24 h.Reaction mixture adds
NaHCO3Solution (pH 9～10), sucking filtration must precipitate, and drying under reduced pressure obtains DOCA-His (Trt).
0.1 g HA is dissolved in 5 mL dry formamide, 50 DEG C of heating for dissolving, is cooled to room temperature.Add 96 mg EDC and 58
Mg NHS, ice bath magnetic agitation 2 h.0.4 g DOCA-His (Trt) is dissolved in 5 mL dry DMF, is slowly dropped to HA mixing
In liquid, after 50 DEG C of stirring in water bath 6 h, continue stirring 24 h in room temperature.Reaction mixture dialyses 2～3 days (thoroughly in distilled water
Analysis bag molecular cut off: 3500), it is filtered to remove insoluble impurities, lyophilization, obtains HA-DOCA-His (Trt) white powder.
0.1 g HA-DOCA-His (Trt) is dissolved in 0.5 mL DMSO, adds 0.5 mL trifluoroacetic acid and 25 L benzene first
Thioether, is stirred at room temperature 2 h, and reaction mixture is dialysis 2 d(bag filter molecular cut offs in aqueous alkali (pH 9～10): 3500),
Distilled water is dialysed 2 days, is filtered to remove insoluble impurities, lyophilization, obtains HA-DOCA-His white powder.Reaction scheme 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 DMFs, adds 0.29 g EDC and 0.18 g NHS, is stirred at room temperature 24
h.0.37 g trityl histidine methylester hydrochlorate (H-His (Trt)-OMe HCl) is dissolved in 20 mL DMF, slowly drips
Being added in DOCA mixed liquor, mixed liquor is in 35 DEG C of stirring in water bath 24 h.Reaction mixture rotation is evaporated off organic solvent, and post separates,
Obtain DOCA-His (Trt).
0.1 g HA is dissolved in 5 mL dry formamide, 50 DEG C of heating for dissolving, is cooled to room temperature.Add 96 mg EDC and 58
Mg NHS, ice bath magnetic agitation 2 h.0.4 g DOCA-His (Trt) is dissolved in 5 mL dry DMF, is slowly dropped to HA mixing
In liquid, after 50 DEG C of stirring in water bath 6 h, continue stirring 24 h in room temperature.Reaction mixture dialyses 2～3 days (thoroughly in distilled water
Analysis bag molecular cut off: 3500), it is filtered to remove insoluble impurities, lyophilization, obtains HA-DOCA-His (Trt) white powder.
0.1 g HA-DOCA-His (Trt) is dissolved in 0.5 mL DMSO, adds 0.5 mL trifluoroacetic acid and 25 L benzene first
Thioether, is stirred at room temperature 2 h, and reaction mixture is dialysed 2 d(bag filter molecular cut offs in distilled water: 3500), in distilled water
Dialyse 2 days, be filtered to remove insoluble impurities, lyophilization, obtain HA-DOCA-His white powder.
The synthesis of example 3:HA-DOCA-His polymer
0.5 g DOCA is dissolved in 5 mL DMFs, adds 0.29 g EDC and 0.18 g NHS, is stirred at room temperature 24
h.0.37 g trityl histidine methylester (H-His (Trt)-OH) is dissolved in 20 mL DMF, is slowly dropped to DOCA mixing
In liquid, mixed liquor is in 35 DEG C of stirring in water bath 24 h.Reaction mixture rotation is evaporated off organic solvent, and post separates, and obtains DOCA-His
(Trt)。
0.1 g HA is dissolved in 5 mL dry formamide, 60 DEG C of heating for dissolving, is cooled to room temperature.Add 192 mg EDC and
116 mg NHS, ice bath magnetic agitation 5 h.0.8 g DOCA-His (Trt) is dissolved in 5 mL dry DMF, is slowly dropped to HA
In mixed liquor, 48 hs are stirred at room temperature.Reaction mixture adds ethanol removing impurity by means of precipitation, obtains HA-DOCA-His (Trt) white powder
End.
0.1 g HA-DOCA-His (Trt) is dissolved in 0.5 mL DMSO, adds 0.5 mL trifluoroacetic acid and 50 L benzene first
Thioether, is stirred at room temperature 12 h, and reaction mixture is dialysed 2 days (bag filter molecular cut off: 3500) in distilled water, in distilled water
Dialyse 2 days, be filtered to remove insoluble impurities, lyophilization, obtain HA-DOCA-His white powder.
HA-DOCA-His's1HNMR collection of illustrative plates is as in figure 2 it is shown, the NHCOCH of HA3Upper-CH3Chemical shift 1.95
Ppm, on 2,3,4 and 5 carbon, the chemical shift of hydrogen is at 3.0～4.0 ppm, and on 6 carbon the chemical shift of hydrogen 4.35～
4.45 ppm, belong to-CH in DOCA at 0.8～2.0 ppm3With-CH2-characteristic peak, return at 7.45 ppm and 8.82 ppm
Belong to the characteristic peak of-N=CH-and-N=CH=C-in His, it was demonstrated that DOCA-His has successfully been synthesized on HA.
Example 4: ultrasonic method preparation carries the HA-DOCA-His micelle of paclitaxel
HA-DOCA-His is dissolved in the PBS of pH 7.4, and concentration is 1～5 mg/mL, and magnetic agitation, to being completely dissolved, will be dissolved in
The paclitaxel (the 10% of HA-DOCA-His addition, 20% and 30%) of dehydrated alcohol (methanol, oxolane) is slowly added dropwise
State in polymer solution, after magnetic agitation 24～48 h, ice-bath ultrasonic (ultrasonic power 100～400 W, 2 s that work, intermittently 3
S) 2～20 min, micellar solution, through 0.45 m membrane filtration, prepares carrier micelle, and lyophilizing can obtain carrier micelle powder.
Example 5: dialysis preparation carries the HA-DOCA-His micelle of amycin
Weigh 5 mg doxorubicin hydrochlorides and be scattered in 1 mL DMSO(oxolane) in, adding 7 L triethylamines, stirring is to Ah mould
Element dissolves, and prepares amycin DMSO(oxolane) solution (5 mg/mL).HA-DOCA-His is dissolved in DMSO(10 mg/mL) in,
Add amycin (the 10% of HA-DOCA-His addition, 20% and 30%) DMSO(oxolane) solution, magnetic agitation under room temperature
Make it mix, be placed in bag filter (molecular cut off: 3500), immerse in the PBS of pH7.4 and dialyse 3 days, liquid warp in bag filter
0.45 m membrane filtration, prepares carrier micelle, and lyophilizing can obtain carrier micelle powder.
Example 6: solvent evaporation method preparation carries the HA-DOCA-His micelle of amycin
Weigh 5 mg doxorubicin hydrochlorides to be scattered in 1 mL methanol, add 7 L triethylamines, stir and dissolve to amycin, make
Obtain amycin methanol solution (5 mg/mL).HA-DOCA-His is dissolved in oxolane (10 mg/mL), adds amycin (HA-
The 10% of DOCA-His addition, 20% and 30%) methanol solution, magnetic agitation makes it mix, and instills the PBS of pH7.4, and magnetic force stirs
Mixing 24～48 h, micellar solution, through 0.45 m membrane filtration, prepares carrier micelle, and lyophilizing can obtain carrier micelle powder.
Example 7:HA-DOCA-His micelle pH sensitivity testing
HA-DOCA-His polymer is dissolved in the PBS of pH7.4, and concentration is 1 mg/mL.Particle size analyzer is used to measure the flat of micelle
All particle diameter and PI, with the HCl solution of 0.01 mol/L, regulation pH is 6.5,6.0,5.0 successively, often one pH value of regulation, sample
Solution magnetic agitation 30 min, stands 10 min, measures particle diameter, in triplicate, observes change of size situation.Result is shown in Fig. 3,
Micelle change of size when pH 7.4 and 6.5 is inconspicuous, and during pH 6.0 and pH 5.0, particle diameter and PI are increased dramatically, and show HA-
DOCA-His micelle has endosome pH(5.0～6.0) sensitivity.
Example 8: carry the release in vitro of the HA-DOCA-His micelle of paclitaxel
Dialysis is used to investigate the tablets in vitro of the carrier micelle prepared in example 4.Precision measures 3 mL and carries the HA-of paclitaxel
DOCA-His micelle, is placed in bag filter (molecular cut off: 3500), immerses 80 mL containing 2% Cremophor EL(w/v), pH
Being respectively in the PBS of 7.4,6.5,6.0,5.0, in 37 DEG C, 100 r/min shakings, respectively at 1,2,4,6,8,12,16,24,36
H and 48 h samples 3 mL, supplements the fresh medium of same pH, volume and temperature simultaneously.Sample through 0.45 m membrane filtration,
Discarding just filtrate, HPLC method measures content of taxol, and calculates cumulative release percent, and result is shown in Fig. 4.Carry the HA-of paclitaxel
DOCA-His micelle in the release medium that pH is 7.4,6.5,6.0,5.0, the cumulative release amount of 6 h is respectively 12.2%,
13.8%, 29.9% and 55.3%, show that HA-DOCA-His micelle is at outer the liquid (> pH of physiological environment (pH 7.4) and tumor cell
6.5) in the most stable, enter tumor cell endosome sour environment (pH 5.0～6.0) be capable of trigger release.
Claims (17)
1. the multistage target polymer micelle of pH trigger-type release in a tumor cell, it is characterised in that this micelle is by having
The hydrophobization modified polysaccharide polymer of endosome pH sensitivity characteristic is self-assembly of in aqueous medium.
2., according to the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 1, its feature exists
Having hydrophilic segment and hydrophobic segment in described polymer, wherein hydrophilic segment is the hyalomitome with CD44 receptor target characteristic
Acid；Hydrophobic segment is selected from deoxycholic acid, and connects endosome pH sensing unit at its end.
3., according to the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 2, its feature exists
Substitution value in described polymer hydrophobic segment is 5%～30%.
4., according to the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 2, its feature exists
PH sensing unit in described polymer is histidine or its analog.
5., according to the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 4, its feature exists
Containing imidazole ring in the molecular structure of described histidine or its analog, its pH response range between 5.0～7.4 it
Between.
6., according to the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 2, its feature exists
Molecular weight in described hyaluronic acid is 1 × 103～1 × 107 Da。
7., according to the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 1, its feature exists
Prepare according to following processing step in described polymer:
1) deoxycholic acid-histidine or the synthesis of its analog
Deoxycholic acid is dissolved in reaction dissolvent, with 1-ethyl-(3-dimethylaminopropyl) carbodiimide and hydroxysuccinimidyl acyl
Imines is activator, activates 2～24 h under room temperature；
Histidine or its analog are dissolved in reaction dissolvent, add triethylamine, be then slowly dropped to above-mentioned deoxidation gallbladder
In acid mixed liquor, under 30～50 DEG C of temperature conditionss, stirring reaction 12～48 h, collect product, drying under reduced pressure, obtain deoxidation gallbladder
Acid-histidine or its analog；
2) hyaluronic acid-deoxycholic acid-histidine or the synthesis of its analog
Hyaluronic acid is dissolved in reaction dissolvent, stirring and dissolving under 40～60 DEG C of temperature conditionss, is cooled to room temperature；It is subsequently adding
1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-Hydroxysuccinimide, activate 2～5 h under ice bath；
Deoxycholic acid-the histidine or its analog (Trt) that step 1) are obtained are dissolved in reaction dissolvent, the most slowly drip
Being added in above-mentioned hyaluronic acid mixed liquor, under 40～70 DEG C of temperature conditionss, stirring reaction 6～12 h, continue stirring at room temperature
Reaction 24～48 h, collect product, product through dialysis purification, lyophilization, obtain hyaluronic acid-deoxycholic acid-histidine or
Its analog (Trt)；
3) hyaluronic acid-deoxycholic acid-histidine or the synthesis of its analog
By step 2) gained hyaluronic acid-deoxycholic acid-histidine or its analog (Trt) be dissolved in trifluoroacetic acid, add
Thioanisole, stirs 2～12 h, collects product under room temperature, product, through dialysis purification, lyophilization, obtains hyaluronic acid-deoxidation
Cholic acid-histidine or its analog.
8. according to the multistage target polymer of pH trigger-type release in the tumor cell described in claim 7, it is characterised in that institute
Stating reaction dissolvent is in N, N-dimethylformamide, Methanamide, ethanol water and N, N-dimethylformamide in water
One or more combinations.
9. according to the multistage target polymer of pH trigger-type release in the tumor cell described in claim 7, it is characterised in that step
Rapid 1), in, described deoxycholic acid is 1:1～2 with the mol ratio of histidine or its analog；
The mol ratio of deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-Hydroxysuccinimide be 1:1～
2:1～2；
The mol ratio of histidine or its analog (Trt) and triethylamine is 1:1～2.
10. according to the multistage target polymer of pH trigger-type release in the tumor cell described in claim 7, it is characterised in that
Step 2) in, described hyaluronic acid is 1:2 with the mol ratio of deoxycholic acid-histidine or its analog (Trt) intermediate
～5,
Hyaluronic acid and 1-ethyl-(3-dimethylaminopropyl) carbodiimide, the mol ratio of N-Hydroxysuccinimide be 1:1～
5:1～5.
11. according to the multistage target polymer of pH trigger-type release in the tumor cell described in claim 7, it is characterised in that
In step 3), hyaluronic acid-deoxycholic acid-histidine or its analog (Trt) and trifluoroacetic acid, thioanisole mole
Ratio is 1:1～5:1～5.
The multistage target polymer glue of pH trigger-type release in 12. 1 kinds of tumor cells as described in any one of claim 1 ~ 11
The preparation method of bundle, it is characterised in that: the hydrophobization with endosome pH sensitivity characteristic is modified hyaluronic acid polymer and uses
Ultrasonic method, dialysis or solvent evaporation method prepare polymer micelle, and bag carries insoluble anti-tumor medicament.
13. according to the preparation side of the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 12
Method, it is characterised in that concretely comprising the following steps of described ultrasonic method:
1) the described hydrophobization with endosome pH sensitivity characteristic is modified hyaluronic acid polymer by the concentration of 1～5 mg/mL
It is dissolved in the phosphate buffer of pH 7.4；
2), after being dissolved with organic solvent by insoluble anti-tumor medicament, said process 1 is instilled) the polymer micelle solution of gained
In, sonicated, prepared particle diameter is the carrier micelle of 10～1000 nm.
14. according to the preparation side of the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 12
Method, it is characterised in that the detailed process of described dialysis is: the described hydrophobization with endosome pH sensitivity characteristic is modified thoroughly
Bright matter acid polymer and insoluble anti-tumor medicament are codissolved in organic solvent, are placed in the bag filter that molecular cut off is 3500,
Immersing in the phosphate buffer of pH 7.4, dialysis removes organic solvent and free drug, and in bag, liquid is prepared particle diameter
It it is the carrier micelle of 10～1000 nm.
15. according to the preparation side of the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 12
Method, it is characterised in that the detailed process of described solvent evaporation method is: the described hydrophobization with endosome pH sensitivity characteristic is repaiied
Decorations hyaluronic acid polymer and insoluble anti-tumor medicament are codissolved in organic solvent, add the phosphate buffer of pH 7.4, stir
Mixing and fling to organic solvent, prepared particle diameter is the carrier micelle of 10～1000 nm.
16. according to the preparation side of the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 12
Method, it is characterised in that described insoluble anti-tumor medicament be paclitaxel, amycin, camptothecine, hydroxy camptothecin, topotecan,
More than the one or two kinds of in Dasatinib, 5-fluorouracil, vincristine and cisplatin.
17. according to the system of the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 13-15
Preparation Method, it is characterised in that described organic solvent is ethanol, oxolane, dimethyl sulfoxide, N, in N-dimethylformamide
One or more combinations.
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