CN109833295A - A kind of polymer micelle and preparation method thereof of cancer target and pH response - Google Patents
A kind of polymer micelle and preparation method thereof of cancer target and pH response Download PDFInfo
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Abstract
The invention discloses the polymer micelles and preparation method thereof that a kind of cancer target and pH are responded, including having the ligand modified first polymer carrier of targeting ability to kinds of tumor cells film surface biomarker phosphatidyl serine, the ligand is Annexin V, PSBP-6, hexapeptide, one of PGN635, the first polymer carrier is hydrophilic section, the amphipathic nature block polymer that hydrophobic section is formed, preparation method includes the preparation process for the first polymer carrier modified the targeting ligand that cell membrane surface biomarker phosphatidyl serine has affinity；Polymer micelle is actively accumulated in tumor tissues by enhancing infiltration retention effect, longer second polymer carrier depolymerizes in mixed micelle structure, expose targeting ligand, selectively targeted tumour cell, as antineoplastic drug carrier, there is high stability, high Drug loading capacity, long-acting slow-releasing and controlled-releasing ability, high targeting ability.
Description
Technical field
The invention belongs to tumour medicine targeting delivery vehicles and preparation method thereof technical fields, and in particular to be a kind of swollen
The polymer micelle and preparation method thereof of tumor targeting and pH response.
Background technique
Compared with normal cell, tumour cell is overexpressed some receptors, including folacin receptor, transforming growth factor receptor and
EGF-R ELISA realizes carrier active targeting energy through carrier surface ligands specific in conjunction with tumor surface receptor
Power.In carrier surface modified specificity ligand, drug tumour cell accumulating capability is improved, is increased by receptor mediated endocytosis
Add intake of the tumour cell to carrier, improving drug effect reduces toxic side effect.
Amphipathilic block polymer micelle refers to by the self assembly in aqueous solution of the block polymer with amphipathic structure
One kind of formation has core-shell structure, and using hydrophobic group as kernel, hydrophilic group is the molecule aggregate of shell.Its hydrophobic cores can
Insoluble drug is loaded as drug release reservoir, hydrophily shell can be reduced between micella and mononuclear phagocyte system and be interacted,
It avoids being absorbed by mononuclear macrophage, extends circulation time in vivo.Use Amphipathilic block polymer micelle anti-swollen as slightly solubility
Tumor medicine carrier has high stability, high Drug loading capacity, long-acting slow-releasing and controlled-releasing ability, high targeting ability.Become polymer micelle
One of the main approaches of insoluble anti-tumor medicament drug delivery system.
Phosphatidylserine (pHospHatidylserine, PS) belongs to the glycerophosphatide class in cytoplasma membrane phosphatide, is
Important one of the phospholipid composition of cytoplasma membrane, under normal physiological conditions, PS, which is generally concentrated, is distributed in cytoplasma membrane endite, but
It is scientific research personnel's discovery in recent years, PS, which turns up, to be present on the outside of kinds of tumor cells, such as lung cancer, breast cancer, bladder cancer, pancreas
Cancer, cutaneum carcinoma, metastatic encephaloma, carcinoma of the rectum etc., and the amount of its surface expression PS and the grade malignancy of tumour cell are positively correlated
Property.It has been reported that PS is efficiently used for the diagnosis and tumour cell targeting of cancer as tumour cell biomarker,
For targeted pancreatic cancer lung carcinoma cell etc..Research finds that the polypeptide FNFRLKAGQKIRFG (PSBP-0) containing 14 amino acid can be with
PS that cell membrane surface turns up is combined, but affinity is extremely low, after Gln in PSBP-0 amino acid sequence substituted with Ala obtain
The affinity of tumor cell surface PS is remarkably reinforced in PSBP-6.
Summary of the invention
Technical problem to be solved by the present invention lies in: the polymer micelle of a kind of cancer target and pH response, energy are provided
Enough selectively targeted tumour cells, improving anti-tumor drug drug effect reduces toxic side effect.
The present invention is to solve above-mentioned technical problem by the following technical programs:
A kind of polymer micelle of cancer target and pH response, including to kinds of tumor cells film surface biomarker phosphorus
Ester acyl serine have targeting ability ligand modified first polymer carrier, the ligand be Annexin V, PSBP-6,
One of hexapeptide, PGN635, the first polymer carrier are hydrophilic section, the amphipathic block that hydrophobic section is formed
Copolymer.
As an improvement of the above technical solution, the hydrophilic section is polyethylene glycol, in polyoxyethylene, polyvinylpyrrolidone
Any substance, the hydrophobic section be poly-dl-lactide, polystyrene, poly-epsilon-caprolactone, poly- benzyl asparatate,
Any substance in poly- benzyl glutamate, poly lactide-glycolide acid.
As an improvement of the above technical solution, the polymer micelle further includes being collectively formed to mix with the first vector
The second polymer carrier of micella；
Wherein, histidine protonates under the slightly sour environment of tumour in the second polymer carrier, structural instability, occurs
It depolymerizes.
As an improvement of the above technical solution, the second polymer carrier includes using polyhistidyl and polyethylene glycol group
At amphipathic nature block polymer.
As an improvement of the above technical solution, the second polymer carrier further includes the glue of pH sensitive chemicals key connection
Beam, the pH sensitive chemicals key are one of hydrazone bond, second dicarboxyl aldehyde key, β-carboxylic acyloxy amine key, original acid ester key.
Meanwhile the preparation method of the above-mentioned cancer target of this hair disclosure of the invention and the polymer micelle of pH response, preparation side
Method includes the first polymer modified the targeting ligand that cell membrane surface biomarker phosphatidyl serine has affinity
The preparation method of carrier；
Wherein, when in the ligand molecular structure that the phosphatidyl serine to turn up to tumour cell film surface has targeting ability
When containing active amino, preparation method includes the following steps:
1.1, it is activated using 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride and n-hydroxysuccinimide
Terminal carboxyl group in the first polymer carrier；
1.2, there is the carboxyl activated in step 1.1 with the phosphatidyl serine to turn up to tumour cell film surface affine
Amino is crosslinked in the targeting ligand of power；
1.3, it dialyses to the reaction product in step 1.2, acquired solution is freeze-dried up to outside to cell membrane surface
The phosphatidyl serine turned over has the carrier of targeting affinity；
Wherein, when in the ligand molecular structure that the phosphatidyl serine to turn up to tumour cell film surface has targeting ability
When without containing active amino, preparation method includes the following steps:
2.1, it is activated using 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride and n-hydroxysuccinimide
There is the terminal carboxyl group of the ligand of targeting ability to the phosphatidyl serine that tumour cell film surface is turned up；
2.2, it is condensed the amino in the carboxyl activated in step 2.1 and the first polymer carrier；
2.3, it dialyses to the reaction product of step 2.2, acquired solution is freeze-dried to turn up to cell membrane surface
Phosphatidyl serine have targeting affinity carrier.
As an improvement of the above technical solution, the preparation process including PSBP-6 modification first polymer carrier, and it is described
First polymer carrier is the amphipathic nature block polymer that poly-dl-lactide and polyethylene glycol are formed, preparation process such as I institute of formula
Show:
First polymer carrier is the preparation side for the amphipathic nature block polymer that poly-dl-lactide and polyethylene glycol are formed
Method, comprising the following steps:
PDLLA-PEG-COOH is dissolved in dimethyl sulfoxide by step 1.1, is added 1- ethyl-(3- dimethylaminopropyl)
Carbodiimide hydrochloride reacts 5 hours, adds n-hydroxysuccinimide and is stirred overnight at room temperature, obtains PDLLA-PEG-CONHS；
PSBP-6 is dissolved in dimethyl sulphoxide solution and is added dropwise to step 1.1 solution by step 1.2, adds triethylamine tune
PH to 8 or so is saved, is stirred at room temperature 3-5 days；
Step 1.3 dialyses step 1.2 acquired solution, and the racemic of PSBP-6 modification is obtained after acquired solution freeze-drying
The amphipathic nature block polymer that polylactic acid and polyethylene glycol are formed.
It as an improvement of the above technical solution, further include being protonated under the slightly sour environment of tumour, structural instability solves
Polymerization, and the preparation process of the second polymer carrier of mixed micelle can be formed with the first polymer carrier, described the
Dimerization object carrier is the amphipathic nature block polymer that polyhistidyl and polyethylene glycol are formed, and preparation process is as shown in formula II:
The second polymer carrier is the preparation packet for the amphipathic nature block polymer that polyhistidyl and polyethylene glycol are formed
Include following steps:
Step 2.1, N- (tertbutyloxycarbonyl) -1- (2,4- bis- that Boc-His (Dnp)-OHIPAl is dissolved in isopropanol
Nitrobenzophenone)-L-Histidine is dissolved in Isosorbide-5-Nitrae-dioxane, add solution after thionyl chloride Isosorbide-5-Nitrae-dioxane dilution
After being reacted 1.5 hours under nitrogen protection, enough anhydrous ethers are added and sufficiently precipitate, is dried in vacuo sediment 3-4 hours after suction filtration,
Product is dissolved in ether and is recrystallized with 100 DEG C of acetone, after acquired solution is filtered to remove insoluble matter, adds enough anhydrous ethers sufficiently heavy
It forms sediment, is dried in vacuo sediment 3-4 hours after suction filtration, obtains Dnp-NCAHcl；
Sodium carbonate is added after step 2.1 gained Dnp-NCAHcl to be dissolved in N,N-dimethylformamide dissolution in step 2.2
Hydrochloric acid is neutralized, after being stirred at room temperature 1 hour, methoxy poly (ethylene glycol) amine (mPEG-NH is added2) N,N-dimethylformamide solution
It is stirred to react under reduced pressure and is centrifuged and is filtered with filter paper after a week, solution is added enough ether and sufficiently precipitates, after standing overnight
Solution is poured out, bottom precipitation is dried in vacuo 1-2 days to obtain brown solid PEG-b-poly (Nim-Dnp-His)；
Step 2.3, by PEG-b-poly made from step 2.2 (Nim-Dnp-His) be added N,N-dimethylformamide it is molten
Xie Hou is added beta -mercaptoethanol and reacts 2-3 days, sloughs 2,4- dinitrophenyl and acquired solution is fitted into bag filter in dimethyl
It dialyses 6 hours in sulfoxide, transferring to dialysis in pure water, to dialyzate substantially without yellow, acquired solution is to obtain the final product poly- after being freeze-dried
Histidine and polyethylene glycol amphipathic nature block polymer.
It as an improvement of the above technical solution, further include that the phosphatidyl serine to turn up to tumour cell film surface has target
The preparation process of mixed micelle is formed to the ligand modified first polymer carrier and second polymer carrier of ability comprising
Following steps:
The phosphatidyl serine to turn up to tumour cell film surface is had to the first ligand modified polymerization of targeting ability
Object carrier and second polymer carrier are dissolved in dimethyl sulfoxide, and the sodium borate buffer solution that pH is 9 is added dropwise, is vigorously stirred 1
Hour, bag filter is transferred to after ice-bath ultrasonic and is dialysed with dobell's solution, and mixed micelle solution is obtained.
As an improvement of the above technical solution, further include method that core-shell structure mixed micelle contains drug:
The phosphatidyl serine to turn up to tumour cell film surface is had to the first ligand modified polymerization of targeting ability
Object carrier and second polymer carrier and drug are dissolved in dimethyl sulfoxide, and the sodium borate buffer solution that pH is 9 is added dropwise, acute
Strong stirring 1 hour, is transferred to bag filter and is dialysed with dobell's solution, must carry medicine mixed micelle solution after ice-bath ultrasonic.
The present invention has the advantage that compared with prior art
The present invention discloses a kind of cancer target and pH responsive polymer micella, is based on kinds of tumor cells film surface phosphatidyl
In the expression of serine height and the slightly sour environmental basis of tumor tissues, using the target with phosphatidyl serine with specific affinity
To ligand modified first polymer carrier, mixed micelle is collectively formed with the second polymer carrier of the response slightly sour environment of tumour.
Tumor vessel special construction make injection drug containing carrier solution by enhancing infiltration retention effect actively accumulation in
Tumor tissues, longer second polymer carrier depolymerizes in mixed micelle structure, exposes second polymer carrier end and repairs
The targeting ligand of decorations, selectively targeted tumour cell.
Meanwhile the invention discloses the preparation methods of above-mentioned tumour target polymer micella, including raw to cell membrane surface
The preparation process for the first polymer carrier that there is object marker phosphatidyl serine the targeting ligand of affinity to modify.
Tumour target polymer micella disclosed by the invention has high stability, high load medicine as antineoplastic drug carrier
Ability, long-acting slow-releasing and controlled-releasing ability, high targeting ability.
Detailed description of the invention
Fig. 1 is the hydrogen spectrogram of the PDLLA-PEG-COOH in the embodiment of the present invention；
Fig. 2 is the hydrogen spectrogram of the PDLLA-PEG-PSBP-6 in the embodiment of the present invention；
Fig. 3 is the hydrogen spectrogram of the PEG-pHis in the embodiment of the present invention；
Fig. 4 is the blank mixed micelle partial size of the PDLLA-PEG-PSBP-6 and PEG-pHis formation in the embodiment of the present invention
Distribution schematic diagram；
Fig. 5 is the load medicine mixed micelle partial size of the PDLLA-PEG-PSBP-6 and PEG-pHis formation in the embodiment of the present invention
Distribution schematic diagram；
Fig. 6 is the mixed micelle flow cytometric analysis results figure in the embodiment of the present invention；
Wherein, Fig. 6 includes the mixed micelle under the conditions of five kinds of different proportions of a1-a5, specifically:
A1:PSBP:PDLLA-PEG-COOH=1:1；
A2:PSBP:PDLLA-PEG-COOH=1:3；
A3:PSBP:PDLLA-PEG-COOH=1:5；
A4:PSBP:PDLLA-PEG-COOH=1:7；
A5: PSBP is not contained；
Fig. 7 is that the mixed micelle of the different graft ratios in the embodiment of the present invention absorbs the percentage result table of comparisons.
Specific embodiment
It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention
Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation
Example.
Embodiment 1: the preparation of cancer target and the polymer micelle of pH response:
Poly-dl-lactide that PDLLA-PEG-PSBP-6 described below, which is PSBP-6, to be modified as targeting ligand and poly-
Glycol copolymer, the pHis are polyhistidyl and ethylene glycol copolymer.
The preparation method of a kind of cancer target and the polymer micelle of pH response, the system including PDLLA-PEG-PSBP-6
Standby, PEG-pHis preparation, and PDLLA-PEG-PSBP-6 and PEG-pHis are prepared into mixed micelle.
The preparation process of above-mentioned PDLLA-PEG-PSBP-6 is as shown in formula I:
The preparation of above-mentioned PDLLA-PEG-PSBP-6 the following steps are included:
PDLLA-PEG-COOH is dissolved in dimethyl sulfoxide by step 1.1, is added 1- ethyl-(3- dimethylaminopropyl)
Carbodiimide hydrochloride reacts 5 hours, adds n-hydroxysuccinimide and is stirred overnight at room temperature, obtains PDLLA-PEG-CONHS；
PSBP-6 is dissolved in dimethyl sulphoxide solution and is added dropwise to step 1.1 solution by step 1.2, adds triethylamine tune
PH to 8 or so is saved, is stirred at room temperature 3-5 days；
Step 1.3 dialyses step 1.2 acquired solution, and the racemic of PSBP-6 modification is obtained after acquired solution freeze-drying
The amphipathic nature block polymer that polylactic acid and polyethylene glycol are formed.
The preparation process of PEG-pHis is as shown in formula II:
The preparation method of PEG-pHis the following steps are included:
Step 2.1, N- (tertbutyloxycarbonyl) -1- (2,4- bis- that Boc-His (Dnp)-OHIPAl is dissolved in isopropanol
Nitrobenzophenone)-L-Histidine is dissolved in Isosorbide-5-Nitrae-dioxane, add solution after thionyl chloride Isosorbide-5-Nitrae-dioxane dilution
After being reacted 1.5 hours under nitrogen protection, enough anhydrous ethers are added and sufficiently precipitate, is dried in vacuo sediment 3-4 hours after suction filtration,
Product is dissolved in ether and is recrystallized with 100 DEG C of acetone, after acquired solution is filtered to remove insoluble matter, adds enough anhydrous ethers sufficiently heavy
It forms sediment, is dried in vacuo sediment 3-4 hours after suction filtration, obtains Dnp-NCAHcl；
Sodium carbonate is added after step 2.1 gained Dnp-NCAHcl to be dissolved in N,N-dimethylformamide dissolution in step 2.2
Hydrochloric acid is neutralized, after being stirred at room temperature 1 hour, methoxy poly (ethylene glycol) amine (mPEG-NH is added2) N,N-dimethylformamide solution
It is stirred to react under reduced pressure and is centrifuged and is filtered with filter paper after a week, solution is added enough ether and sufficiently precipitates, after standing overnight
Solution is poured out, bottom precipitation is dried in vacuo 1-2 days to obtain brown solid PEG-b-poly (Nim-Dnp-His)；
Step 2.3, by PEG-b-poly made from step 2.2 (Nim-Dnp-His) be added N,N-dimethylformamide it is molten
Xie Hou is added beta -mercaptoethanol and reacts 2-3 days, sloughs 2,4- dinitrophenyl and acquired solution is fitted into bag filter in dimethyl
It dialyses 6 hours in sulfoxide, transferring to dialysis in pure water, to dialyzate substantially without yellow, acquired solution is to obtain the final product poly- after being freeze-dried
Histidine and polyethylene glycol amphipathic nature block polymer.
The hydrogen spectrum of PDLLA-PEG-COOH is as shown in Figure 1；
The hydrogen spectrum of PDLLA-PEG-PSBP-6 is as shown in Figure 2；
The hydrogen spectrum of PEG-pHis is as shown in Figure 3.
PDLLA-PEG-PSBP-6 and PEG-pHis are prepared into mixed micelle, the preparation method of mixed micelle includes following
Step:
The phosphatidyl serine to turn up to tumour cell film surface is had to the first ligand modified polymerization of targeting ability
Object carrier and second polymer carrier are dissolved in dimethyl sulfoxide, and the sodium borate buffer solution that pH is 9 is added dropwise, is vigorously stirred 1
Hour, bag filter is transferred to after ice-bath ultrasonic and is dialysed with dobell's solution, and mixed micelle solution is obtained.
The blank mixed micelle particle diameter distribution that PDLLA-PEG-PSBP-6 and PEG-pHis is formed is shown as shown in Figure 4.
Embodiment 2: the polymer micelle that cancer target and pH are responded contains the preparation of drug
The phosphatidyl serine to turn up to tumour cell film surface is had to the first ligand modified polymerization of targeting ability
Object carrier and second polymer carrier and drug are dissolved in dimethyl sulfoxide, and the sodium borate buffer solution that pH is 9 is added dropwise, acute
Strong stirring 1 hour, is transferred to bag filter and is dialysed with dobell's solution, must carry medicine mixed micelle solution after ice-bath ultrasonic.
Specifically, containing Japanese yew as carrier medicament, cancer target and the pH polymer micelle responded using taxol
The method that the preparation of alcohol contains drug is as follows:
PDLLA-PEG-PSBP-6, PEG-pHis and taxol are dissolved in dimethyl sulfoxide, the boric acid that pH is 9 is added dropwise
Sodium buffer solution is vigorously stirred 1 hour, and ice-bath ultrasonic is transferred to bag filter dobell's solution and dialyses 24 hours after 30 minutes leads to
The filtering of 0.45 micron membrane filter is crossed to get medicine mixed micelle solution is carried.
The load medicine mixed micelle particle diameter distribution that PDLLA-PEG-PSBP-6 and PEG-pHis is formed is as shown in Figure 5.
Embodiment 3:
The targeting ability of above-mentioned PDLLA-PEG-PSBP-6, PEG-pHis mixed micelle being prepared, uses with lower section
Method verifying:
PDLLA-PEG-PSBP-6, PEG-pHis mixed micelle target ability and investigate:
HELA cell, which uses, contains 10% fetal calf serum DMEM culture medium, and 37 DEG C of 5%CO2 are cultivated in incubator, take in pair
The cell in number growth period is tested, and is separately added into PDLLA-PEG-FITC, PDLLA-PEG-PSBP-6 and PEG-pHis and is formed
Mixed micelle and PDLLA-PEG-FITC, PDLLA-PEG-COOH and PEG-pHis formed mixed micelle, act on 4 hours
Afterwards, phosphate buffer washes away the fluorescent material not absorbed, analyzes two groups with stream type cell analyzer after 4% paraformaldehyde is fixed
Fluorescence intensity is absorbed, every group sets three multiple holes, as a result as shown in Figure 6.
As a result the PDLLA-PEG-PSBP-6 and PEG- being grafted with molar ratio PSBP-6:COOH-PEG-PDLLA=1:1
It is 83.7 ± 2 that pHis mixed micelle group, which absorbs percentage, and intake average fluorescent strength is 5729 ± 351.7；The intake of 1:3 grafting
Percentage is 89.3 ± 1.4, and intake average fluorescent strength is 6969 ± 170；The intake percentage of 1:5 grafting is 60.0 ± 3.2,
Absorbing average fluorescent strength is 4915 ± 252.6；The intake percentage of 1:7 grafting is 51.5 ± 0.8, absorbs average fluorescent strength
It is 4499.6 ± 50.6；It is 25.5 ± 1.76 that PDLLA-PEG-COOH and PEG-pHis mixed micelle group, which absorbs percentage, intake
Average fluorescent strength is 3263.7 ± 129.7, and the mixed micelle intake percentage result table of comparisons of above-mentioned difference graft ratio is such as
Shown in Fig. 7.
The result shows that:
Firstly, from Fig. 4-5: PEG-pHis has the targeting conveying capacity for tumor cell tissue；
Secondly, as shown in Figure 6: after PSBP-6 modification PEG-PDLLA, than unmodified PEG- under the same terms
PDLLA intake dramatically increases,
Finally, as shown in Figure 7: having with PSBP-6:COOH-PEG-PDLLA 1:3 grafting PDLLA-PEG-PSBP6 optimal
Targeting Effect, this is consistent with the characteristics of PSBP-6 set forth in the present invention.
In the present invention, have the ligand of targeting ability can also be in folic acid, PSBP-6, RGD cyclic peptide tumour cell
It is a kind of.
In the present invention, first polymer carrier PDLLA-PEG is also possible to the carrier material with hydrophilic section and hydrophobic section,
Hydrophilic section can for polyoxyethylene, polyvinylpyrrolidone etc., hydrophobic section can for l-lactic acid, polystyrene, poly- ε-oneself
Lactone, poly- benzyl asparatate, poly- benzyl glutamate, poly lactide-glycolide acid etc..
It can be poly group ammonia with the PDLLA-PEG-PSBP-6 pH sensitive material that mixed micelle is collectively formed in the present invention
Amphipathic nature block polymer, poly- N, N- diethyl amido ethylmethyl acrylate and the polyethylene glycol of acid and polyethylene glycol composition
The amphipathic nature block polymer of composition, poly- (4- or 2- vinylpyridine) and polyethylene glycol composition amphipathic nature block polymer,
Or the micella of pH sensitive chemicals key connection, including hydrazone bond, second dicarboxyl aldehyde key, β-carboxylic acyloxy amine key, original acid ester key.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. the polymer micelle of a kind of cancer target and pH response, which is characterized in that including raw to kinds of tumor cells film surface
Object marker phosphatidyl serine has the ligand modified first polymer carrier of targeting ability, and the ligand is Annexin
V, one of PSBP-6, hexapeptide, PGN635, the first polymer carrier are hydrophilic section, what hydrophobic section was formed
Amphipathic nature block polymer.
2. the polymer micelle of cancer target according to claim 1 and pH response, which is characterized in that the hydrophilic section is
Polyethylene glycol, polyoxyethylene, any substance in polyvinylpyrrolidone, the hydrophobic section is poly-dl-lactide, poly-
Styrene, poly-epsilon-caprolactone, poly- benzyl asparatate, poly- benzyl glutamate, any in poly lactide-glycolide acid
A kind of substance.
3. the polymer micelle of cancer target according to claim 1 to 2 and pH response, which is characterized in that described poly-
Closing object micella further includes the second polymer carrier that mixed micelle is collectively formed with the first vector；
Wherein, histidine protonates under the slightly sour environment of tumour in the second polymer carrier, structural instability, and depolymerization occurs
It closes.
4. the polymer micelle of cancer target according to claim 3 and pH response, which is characterized in that second polymerization
Object carrier includes the amphipathic nature block polymer of polyhistidyl and polyethylene glycol composition.
5. the polymer micelle of cancer target according to claim 4 and pH response, which is characterized in that second polymerization
Object carrier further includes the micella of pH sensitive chemicals key connection, and the pH sensitive chemicals key is hydrazone bond, second dicarboxyl aldehyde key, β-carboxylic
One of sour amido bond, original acid ester key.
6. the preparation method of a kind of cancer target a method as claimed in any one of claims 1 to 5 and the polymer micelle of pH response, special
Sign is that first including having the targeting ligand of affinity to modify to cell membrane surface biomarker phosphatidyl serine gathers
Close the preparation method of object carrier；
Wherein, contain when in the ligand molecular structure that the phosphatidyl serine to turn up to tumour cell film surface has targeting ability
When active amino, preparation method includes the following steps:
1.1, using described in 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride and n-hydroxysuccinimide activation
Terminal carboxyl group in first polymer carrier；
1.2, make the carboxyl activated in step 1.1 that there is affinity with the phosphatidyl serine to turn up to tumour cell film surface
Amino is crosslinked in targeting ligand；
1.3, it dialyses to the reaction product in step 1.2, acquired solution is freeze-dried to turn up to cell membrane surface
Phosphatidyl serine has the carrier of targeting affinity；
Wherein, it is free of when in the ligand molecular structure that the phosphatidyl serine to turn up to tumour cell film surface has targeting ability
When active amino, preparation method includes the following steps:
2.1, using 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride and n-hydroxysuccinimide activation to swollen
The phosphatidyl serine that oncocyte film surface is turned up has the terminal carboxyl group of the ligand of targeting ability；
2.2, it is condensed the amino in the carboxyl activated in step 2.1 and the first polymer carrier；
2.3, it dialyses to the reaction product of step 2.2, acquired solution is freeze-dried up to the phosphorus to turn up to cell membrane surface
Ester acyl serine has the carrier of targeting affinity.
7. the preparation method of cancer target according to claim 6 and the polymer micelle of pH response, which is characterized in that packet
The preparation process of PSBP-6 modification first polymer carrier is included, and the first polymer carrier is poly-dl-lactide and poly- second
The amphipathic nature block polymer that glycol is formed, preparation process is as shown in formula I:
First polymer carrier is the preparation method packet for the amphipathic nature block polymer that poly-dl-lactide and polyethylene glycol are formed
Include following steps:
PDLLA-PEG-COOH is dissolved in dimethyl sulfoxide by step 1.1, and 1- ethyl-(3- dimethylaminopropyl) carbon two is added
It imide hydrochloride reactant salt 5 hours, adds n-hydroxysuccinimide and is stirred overnight at room temperature, obtain PDLLA-PEG-CONHS；
PSBP-6 is dissolved in dimethyl sulphoxide solution and is added dropwise to step 1.1 solution by step 1.2, is added triethylamine and is adjusted pH
To 8 or so, it is stirred at room temperature 3-5 days；
Step 1.3 dialyses step 1.2 acquired solution, and the poly- cream of racemic of PSBP-6 modification is obtained after acquired solution freeze-drying
The amphipathic nature block polymer that acid is formed with polyethylene glycol.
8. the preparation method of cancer target according to claim 6 or 7 and the polymer micelle of pH response, feature exist
In further including being protonated under the slightly sour environment of tumour, structural instability depolymerizes, and can carry with the first polymer
Body forms the preparation process of the second polymer carrier of mixed micelle, and the second polymer carrier is polyhistidyl and poly- second two
The amphipathic nature block polymer that alcohol is formed, preparation process is as shown in formula II:
The second polymer carrier is the preparation method for the amphipathic nature block polymer that polyhistidyl and polyethylene glycol are formed,
The following steps are included:
Step 2.1, N- (tertbutyloxycarbonyl) -1- (2,4- dinitro that Boc-His (Dnp)-OHIPAl is dissolved in isopropanol
Phenyl)-L-Histidine is dissolved in Isosorbide-5-Nitrae-dioxane, add solution nitrogen after thionyl chloride Isosorbide-5-Nitrae-dioxane dilution
After lower reaction 1.5 hours of protection, enough anhydrous ethers, which are added, sufficiently to be precipitated, and is dried in vacuo sediment 3-4 hours after suction filtration, product
It is dissolved in ether and is recrystallized with 100 DEG C of acetone, after acquired solution is filtered to remove insoluble matter, enough anhydrous ethers is added sufficiently to precipitate,
It is dried in vacuo sediment 3-4 hours after suction filtration, obtains Dnp-NCAHcl；
Sodium carbonate neutralization is added after step 2.1 gained Dnp-NCAHcl to be dissolved in N,N-dimethylformamide dissolution in step 2.2
After being stirred at room temperature 1 hour, methoxy poly (ethylene glycol) amine (mPEG-NH is added in hydrochloric acid2) N,N-dimethylformamide solution decompression
Under the conditions of be stirred to react and be centrifuged and filtered with filter paper after a week, solution is added enough ether and sufficiently precipitates, will be molten after standing overnight
Liquid is poured out, and bottom precipitation is dried in vacuo 1-2 days to obtain brown solid PEG-b-poly (Nim-Dnp-His)；
Step 2.3, by PEG-b-poly made from step 2.2 (Nim-Dnp-His) be added n,N-Dimethylformamide dissolution after,
Beta -mercaptoethanol is added to react 2-3 days, sloughs 2,4- dinitrophenyl and acquired solution is fitted into bag filter in dimethyl sulfoxide
Middle dialysis 6 hours is transferred in pure water and is dialysed to dialyzate substantially without yellow, up to poly group ammonia after acquired solution freeze-drying
Acid and polyethylene glycol amphipathic nature block polymer.
9. the preparation method of cancer target according to claim 8 and the polymer micelle of pH response, which is characterized in that also
Phosphatidyl serine including turning up to tumour cell film surface has the ligand modified first polymer carrier of targeting ability
The preparation process of mixed micelle is formed with second polymer carrier comprising following steps:
There is the ligand modified first polymer of targeting ability to carry the phosphatidyl serine to turn up to tumour cell film surface
Body and second polymer carrier are dissolved in dimethyl sulfoxide, and the sodium borate buffer solution that pH is 9 is added dropwise, it is small to be vigorously stirred 1-2
When, bag filter is transferred to after ice-bath ultrasonic and is dialysed with dobell's solution, and mixed micelle solution is obtained.
10. the preparation method of cancer target according to claim 9 and the polymer micelle of pH response, which is characterized in that
Further include the method that core-shell structure mixed micelle contains drug:
There is the ligand modified first polymer of targeting ability to carry the phosphatidyl serine to turn up to tumour cell film surface
Body and second polymer carrier and drug are dissolved in dimethyl sulfoxide, and the sodium borate buffer solution that pH is 9 is added dropwise, acutely stirs
It mixes 1-2 hours, bag filter is transferred to after ice-bath ultrasonic and is dialysed with dobell's solution, medicine mixed micelle solution must be carried.
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| CN112107690A (en) * | 2020-10-16 | 2020-12-22 | 浙江工业大学 | Preparation method of camptothecin drug nanoparticles |
| CN112876673A (en) * | 2021-01-25 | 2021-06-01 | 山东大学 | PH-responsive nano copolymer carrier and preparation method and application thereof |
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