CN106581691B - Restore targeting polyethylene glycol carbonic ester maytansine prodrug micelle, preparation method and the application of response - Google Patents

Restore targeting polyethylene glycol carbonic ester maytansine prodrug micelle, preparation method and the application of response Download PDF

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CN106581691B
CN106581691B CN201611099081.0A CN201611099081A CN106581691B CN 106581691 B CN106581691 B CN 106581691B CN 201611099081 A CN201611099081 A CN 201611099081A CN 106581691 B CN106581691 B CN 106581691B
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maytansine
prodrug
polycarbonate
polyethylene glycol
amphipathic
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CN106581691A (en
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钟志远
程茹
钟平
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Suzhou University
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Abstract

The invention discloses targeting polyethylene glycol carbonic ester maytansine prodrug micelle, preparation method and the applications of a kind of reduction response.The targeting polyethylene glycol carbonic ester maytansine prodrug micelle of reduction response is obtained by amphipathic ethylene glycol-polycarbonate maytansine prodrug polymer self assembly in buffer of amphipathic ethylene glycol-polycarbonate maytansine prodrug polymer and end bonding targeted molecular；The partial size of micella is 30~150 nanometers, and the drugloading rate of maytansine is 2~60wt.%.The targeting polycarbonate maytansine prodrug micelle of reduction response provided by the invention has targeting, amphipathic and biodegradability, Nano medication can be prepared by it, stability, improvement drug pharmacokinetics behavior and the raising drug bioavailability of the water solubility, enhancing drug of drug in cyclic process can be significantly improved；It can be applied in preparation malignant tumour such as melanoma target therapeutic agent.

Description

Restore targeting polyethylene glycol carbonic ester maytansine prodrug micelle, its preparation of response Method and application
Technical field
The present invention relates to a kind of polymeric prodrugs and its applications, and in particular to a kind of poly- second two of the targeting of reduction response Alcohol-polycarbonate maytansine prodrug, preparation method and the application in neoplasm targeted therapy drug is being prepared, is belonging to medical material Field.
Background technique
Malignant tumour has become the primary killers for threatening human health, and morbidity and mortality are just in rise year by year Trend.Tumor therapeuticing method mainly includes surgical resection, radiation therapy and chemotherapy at present.These treatment means exist obvious Deficiency: in therapeutic process can cause irreversible damage to body normal tissue, generate serious toxic side effect, give patient Bring great pain.Maytansine is a potent Antitubulin, to many malignant tumours such as breast cancer, melanin Tumor, Huppert's disease and lung cancer have very strong killing ability.Herceptin-maytansine antibody coupling matter (T-DM1) The treatment that FDA approval is used for advanced stage HER2 positive breast cancer was obtained in 2013.Have at present close to ten kinds based on maytansine Antibody drug conjugates enter the clinical experimental study of different phase.Although antibody drug conjugates have very excellent tumour Targeting ability and antineoplaston effect, but the further marketization is wanted still to suffer from the challenge of some essence, such as it is difficult Large-scale production, cost is excessively high, and tumour cell ingestion efficiency is low, and potential immune response and anticancer drug content are excessively low. In addition, T-DM1 may also lead to some adverse reactions such as nausea, musculoskeletal pain, hepatotoxicity wind agitation, heart damage and chromic fibrous lung Disease.
In the past few decades, polymeric prodrugs a kind of are widely recognized as from being suggested to have developed into so far by scientists Can be effective for the Nano medication of oncotherapy.It is worth noting that, having there are some Macromolecule Prodrugs to enter at present The clinical test of different phase, such as the taxol (Xyotax, Opaxio) and polymethylacrylic acid of polyglutamic acid derivatization Doxorubicin (PK1, PK2) of hydroxypropyl acrylate derivatization etc..But above-mentioned polymeric prodrugs are due to lacking the specificity choosing to tumour It selects and anticancer drug discharged slowly, clinical testing treatment effect is simultaneously not so good as people's will, leads to not clinical application.Therefore, it opens Hair have tumor-targeting, can in tumor locus quick release anticancer drug, and with good biological degradability polymerization Object prodrug is great to oncotherapy research significance.
Summary of the invention
The object of the present invention is to provide the targeting polyethylene glycol carbonic ester maytansine prodrug micelles and its system of reduction response Preparation Method, has targeting, and amphipathic and biodegradability can prepare Nano medication by it, can significantly improve drug Stability, improvement drug pharmacokinetics behavior and raising drug biology benefit water-soluble, enhancing drug is in cyclic process Expenditure；It can be applied in preparation malignant tumour such as melanoma target therapeutic agent.
In order to achieve the above objectives, a kind of specific technical solution of the present invention are as follows: targeting polyethylene glycol carbon of reduction response Acid esters maytansine prodrug micelle is bonded the two of targeted molecular by amphipathic ethylene glycol-polycarbonate maytansine prodrug and end The self assembly in buffer of parent's property polyethylene glycol carbonic ester maytansine prodrug obtains；
The amphipathic ethylene glycol-polycarbonate maytansine prodrug chemical structural formula is as follows:
Wherein, R2 is selected from one of following group:
；
Amphipathic ethylene glycol-polycarbonate maytansine prodrug chemical structural formula of the end bonding targeted molecular is such as Under:
Wherein, R1 is selected from one of following group:
；
R2 is selected from one of following group:
；
It is 20~80, y is 3~15 that the n, which is 68~454, x,；
R is targeted molecular, is selected from antibody molecule (Rituximab, trastuzumab, cetuximab, bevacizumab Deng), peptide molecule (cRGD, iRGD, GE11, cNGQ etc.), glycan molecule (galactolipin, hyaluronic acid etc.) or biological micromolecule (folic acid, biotin etc.).
In the present invention, by the amphiphilic of amphipathic ethylene glycol-polycarbonate maytansine prodrug and end bonding targeted molecular Property the self assembly in phosphate buffer (10 mM, pH 7.4) of polyethylene glycol carbonic ester maytansine prodrug obtain reduction response Targeting polyethylene glycol carbonic ester maytansine prodrug micelle；The amphipathic biodegradable poly second of end bonding targeted molecular Glycol-proportional region of the polycarbonate maytansine prodrug in entire micella is 0~60 wt.% does not include 0；It is poly- using two kinds Closing the micella that object prodrug is mixed to get has good Targeting Performance to tumour.
In above-mentioned technical proposal, amphipathic biodegradable polyethylene glycol carbonic ester maytansine prodrug, end bonding In the amphipathic Biodegradable butane diacid of targeted molecular-polycarbonate maytansine prodrug polymer, R2 unit and two sulphur pyrroles Pyridine carbonate unit is in random arrangement.The molecular weight ranges of two kinds of polymer are 9500~28000 g/mol；In order to increase glue Beam stability improves drug assemble level and release efficiency, amphipathic ethylene glycol-polycarbonate maytansine prodrug and end Amphipathic ethylene glycol-polycarbonate maytansine prodrug of bonding targeted molecular segment group other than the targeted molecular that end is bonded At consistent, the two self assembly by a certain percentage obtains the targeting polycarbonate maytansine prodrug micelle of reduction response.
In above-mentioned technical proposal, the grain of the targeting polyethylene glycol carbonic ester maytansine prodrug micelle of the reduction response Diameter is 30~150 nanometers, and the drugloading rate of maytansine is about 2~60wt.%。
In above-mentioned technical proposal, the preparation side of the targeting polyethylene glycol carbonic ester maytansine prodrug micelle of response is restored Method, comprising the following steps:
(1) in the presence of polyethylene glycol initiator, two thiopyridines carbonic ester of ring-opening copolymerization and the carbonic acid containing R2 group Ester obtains amphipathic Biodegradable butane diacid-polycarbonate；Then amphipathic ethylene glycol-polycarbonate and sulfhydrylation Maytansine carries out sulfydryl-two sulphur exchange reactions and obtains amphipathic ethylene glycol-polycarbonate maytansine prodrug；R2 is selected from following base One of group:
；
(2) in the presence of functionalized poly (ethylene glycol) initiator, two thiopyridines carbonic ester of ring-opening copolymerization and contain R2 group Carbonate monomer obtain amphipathic ethylene glycol-polycarbonate of end-functionalization；Then by the poly- second two of end-functionalization The polyethylene glycol carbonic ester micella of end-functionalization is prepared using solvent displacement for alcohol-polycarbonate, then by end Functionalized polyethylene glycol carbonic ester micella carries out addition reaction with targeted molecular and obtains the amphiphilic that end is bonded targeted molecular Property polyethylene glycol carbonic ester；Amphipathic ethylene glycol-polycarbonate and sulfhydrylation beauty of last end bonding targeted molecular It steps on element progress sulfydryl-two sulphur exchange reactions and obtains amphipathic ethylene glycol-polycarbonate maytansine of end bonding targeted molecular Prodrug；R2 is selected from one of following group:
；
(3) by the amphipathic second of amphipathic ethylene glycol-polycarbonate maytansine prodrug and end bonding targeted molecular Glycol-polycarbonate maytansine prodrug self assembly in buffer obtains the polyethylene glycol-with targeted molecular of reduction response Polycarbonate maytansine prodrug micelle.
In above-mentioned technical proposal, step (1) is specifically, in nitrogen environment, two thiopyridines carbonic esters contain R2 group Carbonic ester and macromolecular polyethylene glycol initiator are dissolved in the first solvent, the first catalyst are then added, in closed reactor In, ring-opening copolymerization reaction is carried out, amphipathic ethylene glycol-polycarbonate is obtained；In nitrogen environment, by amphipathic second two Alcohol-polycarbonate and sulfhydrylation maytansine are dissolved in the second solvent, and the second catalyst is added, and in sealing reactor, are carried out Sulfydryl-two sulphur exchange reactions, then dialysis obtains amphipathic ethylene glycol-polycarbonate maytansine prodrug；
Step (2) is specifically, in nitrogen environment, two thiopyridines carbonic esters, carbonic ester and functionalization containing R2 group Macromole evocating agent is dissolved in third solvent, and third catalyst is added thereto, and in closed reactor, it is total to carry out open loop Polymerization reaction obtains amphipathic ethylene glycol-polycarbonate of end-functionalization, then obtains end official using solvent displacement Amphipathic ethylene glycol-polycarbonate micella of energyization；Then in nitrogen environment, end-functionalization is added in targeted molecular It is reacted in amphipathic ethylene glycol-polycarbonate micellar aqueous solution, then dialyses, is dried to obtain the two of end bonding targeted molecular Parent's property polyethylene glycol carbonic ester；Then in nitrogen environment, by the amphipathic polyethylene glycol of end bonding targeted molecular Carbonic ester and sulfhydrylation maytansine are dissolved in the 4th solvent, and the 4th catalyst is added, and in sealing reactor, carry out sulfydryl- Two sulphur exchange reactions, then dialysis obtains amphipathic ethylene glycol-polycarbonate maytansine prodrug of end bonding targeted molecular；
Step (3) is specially that amphipathic ethylene glycol-polycarbonate maytansine prodrug is bonded targeted molecular with end Amphipathic ethylene glycol-polycarbonate maytansine prodrug is dissolved separately in the 5th solvent, buffer is added dropwise after mixing, then thoroughly Analysis obtains the targeting polyethylene glycol carbonic ester maytansine prodrug micelle of reduction response.
In above-mentioned technical proposal, in step (1), macromole evocating agent is that one end is methoxyl group, and the other end is the poly- of hydroxyl Ethylene glycol, the first catalyst are bis- (double trimethyl silicon substrates) amine zinc；First solvent is methylene chloride；Ring-opening copolymerization reaction temperature It is 40 DEG C, the time is 24 h；Second catalyst is glacial acetic acid；Second solvent is N,N-dimethylformamide or dimethyl sulfoxide； Sulfydryl-two sulphur exchange reactions temperature is 40 DEG C, and the time is 48 h.
In above-mentioned technical proposal, in step (2), the functionalization macromole evocating agent is the Bifunctionalized poly- second two in end Alcohol, third catalyst are bis- (double trimethyl silicon substrates) amine zinc；Third solvent is methylene chloride；Ring-opening copolymerization reaction temperature is 40 DEG C, the time is 24 h；4th solvent is N,N-dimethylformamide or dimethyl sulfoxide；Graft reaction temperature is 35 DEG C, the time For for 24 hours；4th catalyst is glacial acetic acid；4th solvent is N,N-dimethylformamide or dimethyl sulfoxide；The exchange of-two sulphur of sulfydryl Reaction temperature is 40 DEG C, and the time is 48 h；The targeted molecular is one such: antibody molecule (Rituximab, Trastuzumab, cetuximab, bevacizumab etc.), peptide molecule (cRGD, iRGD, GE11, cNGQ etc.), sugar Class molecule (galactolipin, hyaluronic acid etc.) and biological micromolecule (folic acid, biotin etc.)；The change of the functionalized poly (ethylene glycol) Learn structural formula are as follows:
Wherein, R1 is selected from one of following group:
Such as using maleimide functionality and cRGD-SH peptide molecule in the preparation process of targeted polymeric prodrugs Reaction can be bonded upper cRGD targeted molecular in amphipathic ethylene glycol-polycarbonate one end, make the glue being finally prepared Beam has corresponding Targeting Performance.Its reaction condition is mild, can efficiently react completely in aqueous solution.
In above-mentioned technical proposal, in step (3), the 5th solvent is n,N-Dimethylformamide or dimethyl sulfoxide；End Amphipathic ethylene glycol-polycarbonate maytansine prodrug of bonding targeted molecular accounts for the 0~60% of micella gross mass, and is not 0； The buffer is phosphate buffer.Preferably, the 5th solvent is n,N-Dimethylformamide；Amphipathic polyethylene glycol carbon Acid esters maytansine prodrug is bonded amphipathic ethylene glycol-polycarbonate maytansine prodrug of targeted molecular with end in the 5th solvent In concentration it is the same, be added dropwise buffer time be 5~30min.
In above-mentioned technical proposal, in step (1), after the completion of copolymerization, reaction solution is precipitated through anhydrous ether, is vacuumized Filtering and Vacuum dry filter cake obtain amphipathic ethylene glycol-polycarbonate；After two sulphur-sulfydryl exchange reaction, reaction solution It successively dialyses (MWCO 7000) in n,N-Dimethylformamide and deionized water, is then freeze-dried, obtains amphipathic second Glycol-polycarbonate maytansine prodrug, can be reserved in -20 DEG C of refrigerators.
In above-mentioned technical proposal, in step (2), after the completion of copolymerization, reaction solution is precipitated through anhydrous ether, is vacuumized Filtering and Vacuum dry filter cake obtain amphipathic ethylene glycol-polycarbonate of end-functionalization；Using dialysis by end official Amphipathic ethylene glycol-polycarbonate of energyization is self-assembly of the polyethylene glycol that surface has functional group in aqueous solution Carbonic ester micella；The micella is bonded with targeted molecular in aqueous solution, and reaction solution is dialysed in deionized water, and then freeze-drying obtains Amphipathic ethylene glycol-polycarbonate of end bonding targeted molecular；Upper maytansine point is bonded by two sulphur-sulfydryl exchange reaction Son, reaction solution is successively dialysed (MWCO 7000) in n,N-Dimethylformamide and deionized water after reaction, is then freezed It is dry, amphipathic ethylene glycol-polycarbonate maytansine prodrug of end bonding targeted molecular is obtained, can be reserved in -20 DEG C of ice In case.
In above-mentioned technical proposal, in step (3), dialyse 12 h(MWCO 7000 in PB buffer), obtain reduction response Targeting polyethylene glycol carbonic ester maytansine prodrug micelle.
The end of the hydrophilic section PEG of the targeting polyethylene glycol carbonic ester maytansine prodrug micelle of reduction response of the invention End can with the selectively targeted molecule of chemical coupling such as cRGD polypeptide, to have tomour specific targeting, be provided simultaneously with it is amphiphilic, Biocompatibility；Such polymeric prodrugs micella have good stability simultaneously can specifically active targeting to swell Tumor, such as melanoma.
The targeting polyethylene glycol carbonic ester maytansine prodrug micelle of reduction response disclosed by the invention, dynamic light scattering It is 30~150 nm that particle size analyzer (DLS), which measures partial size, and micella is uniformly distributed transmission electron microscope observing as the result is shown, maytansine in micella Drugloading rate be about 2~60wt.%.The prodrug micelle is stable in vivo, and circulation time is long, can be increased by passive and active targeting It is added in the enrichment of tumor locus and the intake of tumour cell, and enters after cancer cell under strong reducing property environment in the cell, two sulphur Key fast fracture, quick release go out drug, efficiently kill tumour cell.Therefore the invention also discloses the targets of above-mentioned reduction response To application of the polyethylene glycol carbonic ester maytansine prodrug micelle in preparation tumor therapeutic agent.
The invention also discloses a kind of anti-tumor nano drug, the targeting polyethylene glycol carbon including above-mentioned reduction response Acid esters maytansine prodrug micelle.
The invention also discloses a kind of targeting polyethylene glycol carbonic ester maytansine prodrug of reduction response, chemistry knots Structure formula is one of following chemical structural formula:
Wherein, R1 is selected from one of following group:
；
R2 is selected from one of following group
；
It is 20~80, y is 3~15 that the n, which is 68~454, x,；
R is targeted molecular.
The present invention further discloses the targeting polyethylene glycol carbonic ester maytansine prodrug micelles of above-mentioned reduction response to exist Application in oncotherapy.
The invention also discloses a kind of polymeric prodrugs micellas, by reduction responsiveness amphipathic ethylene glycol-polycarbonate Maytansine prodrug self assembly is prepared；
Reduction responsiveness amphipathic ethylene glycol-polycarbonate maytansine prodrug chemical structural formula is as follows:
Wherein, R2 is selected from one of following group:
；
It is 20~80, y is 3~15 that the n, which is 68~454, x,.
Due to the application of the above technical scheme, compared with prior art, the present invention having the advantage that
1. the targeting polyethylene glycol carbonic ester maytansine prodrug micelle of reduction response disclosed by the invention, by amphipathic Amphipathic ethylene glycol-polycarbonate maytansine of polyethylene glycol carbonic ester maytansine prodrug and end bonding targeted molecular Prodrug self assembly in buffer obtains, and ring-opening polymerisation, Michael addition reaction and sulfydryl-two sulphur exchange reactions are controllable, entirely Synthesis process step is simple, and reaction condition is mild, maintains anticancer drug activity, there is good reproducibility.
2. amphipathic ethylene glycol-polycarbonate maytansine prodrug disclosed by the invention and end are bonded targeted molecular Amphipathic ethylene glycol-polycarbonate maytansine prodrug can reach production grade synthesis, solve existing antibody drug conjugates It is extremely difficult to large-scale production, the problems such as cost is excessively high, there is good biodegradability and biocompatibility, be conducive to drug Industrialization.
3. the targeting polyethylene glycol carbonic ester maytansine prodrug micelle of reduction response disclosed by the invention, dynamic optical dissipate Penetrating particle size analyzer (DLS) and measuring partial size is 30~150 nm, and micella is uniformly distributed transmission electron microscope observing as the result is shown, and micella Sino-U.S. steps on The drugloading rate of element is about 2~60wt.%, solve the problems, such as that anticancer drug content is too low in existing antibody drug conjugates.
4. the targeting polyethylene glycol carbonic ester maytansine prodrug micelle of reduction response disclosed by the invention passes through two sulphur Key is connect with amphipathic biodegradable polymer main chain, can be self-assembly of micella, and disulfide bond is well protected in glue In beam, drug molecule maytansine is sufficiently stable in tumour cell external environment, is not easy to reveal, and the high concentration in tumour cell Glutathione drug molecule maytansine can be promoted rapidly to release, while can completely retain original maytansine Molecular structure retains the anti-tumor activity of drug well, realizes drug cycles loss small, aura position release mostly and drug effect High effect.
5. the targeting polyethylene glycol carbonic ester maytansine prodrug micelle surface of reduction response disclosed by the invention passes through Modification targeted molecular such as cRGD polypeptide enhances micella in the enrichment of tumor locus and penetrates, and enhancing tumour cell takes the photograph drug Ability is taken, this not only adds the utilization rates of drug, can also reduce drug to the toxic side effect of its hetero-organization.
6. the targeting polyethylene glycol carbonic ester maytansine prodrug micelle of reduction response disclosed by the invention, with free beauty It steps on element to compare, the maximal tolerance dose of maytansine can be significantly improved；It is high in the accumulation rate of tumor locus, have very to tumour cell Strong lethality, referring to the embodiment of the present invention, the targeting polyethylene glycol carbonic ester maytansine of reduction response disclosed by the invention Prodrug micelle has good inhibition to tumour growth during to lotus tumour C57BL/6 black mouse interior therapeutic.
Detailed description of the invention
Fig. 1 is polymer P EG-P (TMC- in embodiment oneco-PDSC nucleus magnetic hydrogen spectrum figure)；
Fig. 2 is PEG-P (TMC- in embodiment twog- SSDM1) nucleus magnetic hydrogen spectrum figure；
Fig. 3 is polymer MAL-PEG-P (TMC- in embodiment threeco-PDSC nucleus magnetic hydrogen spectrum figure)；
Fig. 4 is polymer cRGD-PEG-P (TMC- in example IVco-PDSC nucleus magnetic hydrogen spectrum figure)；
Fig. 5 is cRGD-PEG-P (TMC- in embodiment fiveg- SSDM1) nucleus magnetic hydrogen spectrum figure；
Fig. 6 is partial size and the transmission for the polyethylene glycol carbonic ester maytansine prodrug micelle that response is restored in embodiment six Electron microscope；
Fig. 7 is the polyethylene glycol carbonic ester maytansine prodrug glue that the cRGD polypeptide targeting of response is restored in embodiment seven The partial size and transmission electron microscope picture of beam；
Fig. 8 is the polyethylene glycol carbonic ester maytansine prodrug glue that the cRGD polypeptide targeting of response is restored in embodiment seven Beam stability study figure in 10% fetal calf serum and DMEM culture medium；
Fig. 9 is the polyethylene glycol carbonic ester maytansine prodrug glue that the cRGD polypeptide targeting of response is restored in embodiment seven The reduction response test figure of beam；
Figure 10 is polyethylene glycol polycarbonate maytansine prodrug micelle (cRGD-MMP, MMP) in embodiment ten in gluathione Release in vitro result figure under peptide triggering；
Figure 11 is free maytansine (free DM1) in embodiment 11, restores the poly- second two of the cRGD polypeptide targeting of response Alcohol-polycarbonate maytansine prodrug micelle (cRGD-MMP) and the polyethylene glycol carbonic ester maytansine prodrug of reduction response Toxic test results figure of the micella (MMP) to B16F10 cell；
Figure 12 is that polyethylene glycol carbonic ester maytansine prodrug micelle (cRGD-MMP, MMP) is right in embodiment 12 The flow cytometer showed cellular uptake figure of B16F10 cell；
Figure 13 is that polyethylene glycol carbonic ester maytansine prodrug micelle (cRGD-MMP, MMP) is right in embodiment 13 The confocal microscopy cellular uptake figure of B16F10 cell；
Figure 14 is polyethylene glycol carbonic ester maytansine prodrug micelle (cRGD-MMP, MMP) in embodiment 14 in mouse Blood circulation inside body result figure；
Figure 15 is the polyethylene glycol carbonic ester maytansine prodrug that the cRGD polypeptide targeting of response is restored in embodiment 15 Micella (cRGD-MMP) is in the intracorporal maximal tolerance dose result figure of normal mouse；
Figure 16 is to restore the polyethylene glycol carbonic ester maytansine prodrug micelle (MMP) of response just in embodiment 16 The intracorporal maximal tolerance dose result figure of normal mouse；
Figure 17 is the black mouse tumor growth in vivo volume change figure of each group lotus B16F10 tumour C57BL/6 in embodiment 16；
Figure 18 is the tumor size image results figure of the black mouse of each group lotus B16F10 tumour C57BL/6 in embodiment 16；
Figure 19 is the tumor size weight result figure of the black mouse of each group lotus B16F10 tumour C57BL/6 in embodiment 16；
Figure 20 is the survival results figure of the black mouse of each group lotus B16F10 tumour C57BL/6 in embodiment 16；
Figure 21 is the relative body weight result of variations figure of the black mouse of each group lotus B16F10 tumour C57BL/6 in embodiment 16.
Specific embodiment
One polymer of embodiment (PEG-P (TMC-co-PDSC synthesis))
In a nitrogen environment, 86.7 mg(0.32 mmol) two thiopyridines carbonate monomers (PDSC) and 81.6 mg(0.8 Mmol trimethylene carbonate (TMC)) is dissolved in 2 mL methylene chloride, is added in sealing reactor, 0.1 g is then added (0.02 mmol) CH3The methylene chloride of O-PEG-OH (5 K) and bis- (double trimethyl silicon substrates) the amine zinc of the catalyst of 0.5 mL is molten Liquid (0.1 mol/L), is sealed reactor, is transferred out of glove box, and after reacting 24 hours in 40 DEG C of oil baths, glacial acetic acid terminates anti- It answers, is precipitated in ice ether, eventually pass through filtering, vacuum drying obtains polymer P EG-P (TMC-co-PDSC).Yield is 81.5%.Nuclear-magnetism figure is shown in attached drawing 1,1H NMR (400 MHz, CDCl3): TMC moieties: δ (ppm) 2.05,4.23, PEG moieties: δ (ppm) 3.37,3.65, PDSC moieties: δ (ppm) 1.12,3.01,4.11,7.09, 7.65, 8.46.It is respectively 39 and 10.5 that the degree of polymerization that molecular weight is 11.8 kg/mol, TMC and PDSC, which is calculated, in nuclear-magnetism.
A variety of PEG-P (TMC- can be prepared using above-mentioned similar methodco-PDSC), material rate and characterization are shown in Table 1.
1 polymer P EG-P (TMC- of tableco-PDSC preparation condition, product nuclear-magnetism and GPC characterization result)
Two amphipathic ethylene glycol of embodiment-polycarbonate maytansine prodrug polymer (PEG-P (TMC-g- SSDM1)) Synthesis
Under nitrogen protection, is sequentially added in the three-necked flask of 100mL and be dissolved in 10mL n,N-Dimethylformamide (DMF) the polymer P EG-P (TMC- inco-PDSC) (bis- thiopyridines functional group of 100 mg, 0.088 mmol), at the same to its Middle (100 μ L) glacial acetic acid that catalytic amount is added, is then added dropwise the mercapto-functionalized maytansine of 25 mL into there-necked flask (DM1) DMF solution of (97.8 mg, 0.135 mmol), reactor are placed in 35 DEG C of oil bath, are stirred to react 48 hours Afterwards, it successively dialyses (Spectra/Pore, MWCO 7000) in DMF and water, freeze-drying, yield 75%.Nuclear-magnetism is the result shows that it is tied Structure is amphipathic ethylene glycol-polycarbonate maytansine prodrug polymer, and the drugloading rate of maytansine is 40wt.%.Nuclear-magnetism figure is shown in Attached drawing 2,1H NMR (600 MHz, DMSO-d 6 ): PEG: δ 3.37, 3.64; TMC: δ 4.24, 2.04; DM1: δ 5.22-7.24, 0.71-1.52, and 3.25-3.55.It is 18.1 kg/mol that molecular weight, which is calculated, in nuclear-magnetism.
Embodiment trimerization object (Mal-PEG-P (TMC-co-PDSC synthesis))
In a nitrogen environment, 86.7 mg(0.32 mmol) two thiopyridines carbonate monomers (PDSC) and 81.6 mg(0.8 Mmol trimethylene carbonate (TMC)) is dissolved in 2 mL methylene chloride, is added in sealing reactor, 0.1 g is then added The methylene chloride of (0.02 mmol) MAL-PEG-OH (5 K) and bis- (double trimethyl silicon substrates) the amine zinc of the catalyst of 0.5 mL is molten Liquid (0.1 mol/L) seals reactor, is transferred out of glove box, after 40 DEG C of oil baths are reacted 24 hours, glacial acetic acid terminates reaction, ice It is precipitated in ether, eventually passes through filtering, vacuum drying obtains polymer MAL-PEG-P (TMC-co-PDSC).Yield is 82.3%. Nuclear-magnetism figure is shown in attached drawing 3,1H NMR (400 MHz, CDCl3): PEG moieties: δ 3.63; TMC moieties: δ 4.23, 2.05; PDSC moieties: δ 8.46, 7.65, 7.09, 4.10, 3.01, 1.12, Maleimide group: δ 6.71.It is respectively 39 and 11 that the degree of polymerization that molecular weight is 12 kg/mol, TMC and PDSC, which is calculated, in nuclear-magnetism.
A variety of Mal-PEG-P (TMC- can be prepared using above-mentioned similar preparation methodco-PDSC), it is shown in Table 2.
2 polymer Mal-PEG-P (TMC- of tableco-PDSC preparation condition, product nuclear-magnetism and GPC characterization result)
Polymer (cRGD-PEG-P (the TMC- of example IV cRGD polypeptide end group bondingco-PDSC synthesis))
By MAL-PEG-P (TMC-co-PDSC) the n,N-Dimethylformamide (DMF) (2.5 of (50 mg, 3.84 μm of ol) ML solution) is prepared into MAL-PEG-P (TMC- by exchange of solvent methodco-PDSC micella), resulting polymers micellar concentration About 3 mg/mL are then transferred to micella in the there-necked flask of 100 mL, and are suitably bubbled and guarantee nitrogen environment, then to it Middle addition cRGD-SH(4 mg, 5.76 μm of ol), it is placed in 35 DEG C of oil bath pans and stirs 24 h, then dialyse in deionized water (Spectra/Pore, MWCO 3500), freeze-drying, and be stored in -20 DEG C of refrigerators.Yield 78%.Nuclear-magnetism figure is shown in attached drawing 4,1H NMR (400 MHz, DMSO-d 6 ): PEG: δ 3.37, 3.64; TMC: δ 4.24, 2.04; PDSC: δ 8.46, 7.65, 7.09, 4.10, 3.01, 1.12; cRGD: δ 6.83-7.60.The nuclear-magnetism of nuclear-magnetism maleimide as the result is shown Characteristic peak completely disappears, and the appearance of cRGD characteristic peak shows cRGD fully reacting.Two thiopyridines characteristic peaks in polymer simultaneously Integrated value do not reduce, show cRGD-SH not with two thiopyridines occur sulphur sulphur exchange side reaction.
Targeted polymeric prodrugs of the present invention are in preparation first by MAL-PEG-P (TMC-co-PDSC) polymer prepares plastic Beam be by two thiopyridines protective groups in polymer chain in micelle inner core, avoid cRGD-SH and two thiopyridines from occurring secondary anti- It answers, by cRGD-SH and MAL-PEG-P (TMC-co-PDSC after the maleimide functionality reaction that) polymer micelle has, thoroughly Analysis removes unreacted polypeptide, and then freeze-drying collects and obtains cRGD-PEG-P (TMC-co-PDSC) polymer.
Amphipathic ethylene glycol-polycarbonate maytansine prodrug polymer of five cRGD polypeptide end group of embodiment bonding (cRGD-PEG-P (TMC-g- SSDM1)) synthesis
Under nitrogen protection, is sequentially added in the three-necked flask of 50mL and be dissolved in 3mL n,N-Dimethylformamide (DMF) In cRGD polypeptide end group bonding polymer MAL-PEG-P (TMC-co-PDSC) (bis- thiopyridines of 40 mg, 0.036 mmol Functional group), while (30 μ L) glacial acetic acid of catalytic amount being added thereto, 10 mL sulfydryls are then added dropwise into there-necked flask The DMF solution of the maytansine (DM1) (39.9 mg, 0.054 mmol) of functionalization, reactor are placed in 35 DEG C of oil bath, stir After mixing reaction 48 hours, successively dialyse (Spectra/Pore, MWCO 7000) in DMF and water, freeze-drying, yield 73%.Nuclear-magnetism The result shows that its structure is amphipathic ethylene glycol-polycarbonate maytansine prodrug polymer of cRGD polypeptide end group bonding, beauty The drugloading rate for stepping on element is 40.2wt.%.Nuclear-magnetism figure is shown in attached drawing 5,1H NMR (600 MHz, DMSO-d 6 ): PEG: δ 3.37, 3.64; TMC: δ 4.24, 2.04; DM1: δ 5.22-7.24, 0.71-1.52, and 3.25-3.55, cRGD: δ 6.83-7.60.It is 18.6 kg/mol that molecular weight, which is calculated, in nuclear-magnetism.
By taking the 3rd group of polymer of the 3rd group of polymer of table 1 and table 2 as an example, further studied.
The preparation of the polyethylene glycol carbonic ester maytansine prodrug micelle (MMP) of the reduction response of embodiment six
The polyethylene glycol carbonic ester maytansine prodrug micelle (MMP) of reduction response is prepared by solvent method of replacing It arrives, for further analyzing and researching.It is real that 800 μ L phosphate buffer solutions (PB, 10 mM, pH 7.4) are added drop-wise to 200 μ L dropwise Apply the PEG-P (TMC- synthesized in example twog- SSDM1) DMF solution (5 mg/mL) in, be then charged into bag filter (MWCO 7000) dialyse 12 h in, at least changes five water, and dialysis medium is PB(10 mM, pH 7.4).Obtained micella size is by dynamic Light scattering particle size analyzer (DLS) is measured as 39 nm, and the very narrow PDI of particle diameter distribution is 0.09, sees Fig. 6, and as seen from the figure, TEM is surveyed Nano medication particle distribution it is uniform, and size measured with dynamic light scattering method it is close.
Seven cRGD of embodiment targets the preparation of polyethylene glycol carbonic ester maytansine prodrug micelle (cRGD-MMP)
The polyethylene glycol carbonic ester maytansine prodrug micelle (cRGD-MMP) of the cRGD polypeptide targeting of reduction response is by reality Apply the amphipathic ethylene glycol-polycarbonate maytansine prodrug polymer (PEG-P (TMC- synthesized in example twog- SSDM1)) and it is real Apply amphipathic ethylene glycol-polycarbonate maytansine prodrug polymer (cRGD- of the cRGD polypeptide end group bonding synthesized in example five PEG-P(TMC-g-SSDM1 it)) is prepared by solvent method of replacing, for further analyzing and researching.800 μ L phosphate are slow It rushes solution (PB, 10 mM, pH 7.4) and is added drop-wise to the PEG-P (TMC- of 160 μ L dropwiseg- SSDM1) DMF solution (5 mg/ ) and the cRGD-PEG-P (TMC- of 40 μ L mLg- SSDM1) DMF solution (5 mg/mL) mixed liquor in, be then charged into bag filter Dialyse 12 h in (MWCO 7000), at least changes five water, and dialysis medium is PB(10 mM, pH 7.4).Obtained Nano medication Size is measured as 45 nm by dynamic light scattering particle size analyzer (DLS), and the very narrow PDI of particle diameter distribution is 0.09, Fig. 7 is seen, by scheming It is found that TEM is measured, Nano medication particle distribution is uniform, and size measured with dynamic light scattering method it is close.Above-mentioned reduction response CRGD polypeptide targeting polyethylene glycol carbonic ester maytansine prodrug micelle (cRGD-MMP) 10% fetal calf serum and The partial size and particle diameter distribution remained unchanged in the presence of DMEM culture medium, referring to Fig. 8, but in the case where simulating tumour cell reducing environment Disulfide bond fast fracture, referring to Fig. 9.
Eight fluorescence DOX of embodiment contains the preparation of polyethylene glycol carbonic ester maytansine prodrug micelle (DOX-MMP)
The polyethylene glycol carbonic ester maytansine prodrug micelle (DOX- for the reduction response that fluorescence adriamycin (DOX) contains MMP it) is prepared by solvent method of replacing.800 μ L phosphate buffer solutions (PB, 10 mM, pH 7.4) are added drop-wise to dropwise PEG-P (the TMC- synthesized in 200 μ L embodiments twog- SSDM1) DMF solution (5 mg/mL) and 20 μ L DOX DMF In the mixed liquor of solution (5 mg/mL), 12 h that dialyse are then charged into bag filter (MWCO 7000), five water are at least changed, dialysed Medium is PB(10 mM, pH 7.4).Obtained Nano medication size is measured as 44 by dynamic light scattering particle size analyzer (DLS) Nm, the very narrow PDI of particle diameter distribution are 0.12.
The polyethylene glycol carbonic ester maytansine of the cRGD polypeptide targeting for the reduction response that nine fluorescence DOX of embodiment is contained The preparation of prodrug micelle (DOX-cRGD-MMP)
The polyethylene glycol carbonic ester maytansine prodrug micelle of the cRGD polypeptide targeting for the reduction response that fluorescence DOX is contained (DOX-cRGD-MMP) amphipathic ethylene glycol-polycarbonate maytansine prodrug polymer (PEG-P by being synthesized in embodiment two (TMC-g- SSDM1)) and embodiment five in synthesize cRGD polypeptide end group bonding amphipathic ethylene glycol-polycarbonate U.S.A step on Plain prodrug polymer (cRGD-PEG-P (TMC-co-PDSC it)) is prepared by solvent method of replacing.800 μ L phosphate are slow It rushes solution (PB, 10 mM, pH 7.4) and is added drop-wise to the PEG-P (TMC- of 160 μ L dropwiseg- SSDM1) DMF solution (5 mg/ ML), the cRGD-PEG-P (TMC- of 40 μ Lg- SSDM1) DMF solution (5 mg/mL) and 20 μ L DOX DMF solution (5 Mg/mL) in mixed liquor, 12 h that dialyse are then charged into bag filter (MWCO 7000), at least change five water, dialysis medium is PB (10 mM, pH 7.4).Obtained Nano medication size is measured as 49 nm, partial size by dynamic light scattering particle size analyzer (DLS) Being distributed very narrow PDI is 0.19.
The polyethylene glycol carbonic ester maytansine prodrug micelle of the cRGD polypeptide targeting of the reduction response of embodiment ten (cRGD-MMP) and reduction response polyethylene glycol carbonic ester maytansine prodrug micelle (MMP) release in vitro behavioral study
CRGD-MMP and MMP Nano medication solution is prepared by solvent displacement, micellar concentration is 0.8 mg/ mL.Two micellas carry out the release experiment of drug at 37 DEG C in two different dissolution mediums, including PB (pH 7.4, 10 mM) and 10 mM GSH PB (7.4,10 mM of pH) solution.By cRGD-MMP the and MMP Nano medication solution of 0.5 mL It is fitted into bag filter (MWCO 12000), is placed in the corresponding dissolution medium of 30 mL.At the time point of each setting, take out The dissolution medium of 5 mL, and add the fresh medium of corresponding 5 mL.It is remained in the burst size and Nano medication of maytansine (DM1) Surplus is measured by high performance liquid chromatography, and each release experiment carries out three times in parallel, and final reality is that experiment is resulting Average value.Attached drawing 10 is the relationship of maytansine (DM1) cumulative release amount and time, it can be seen from the figure that it is swollen that simulation is added In oncocyte after GSH, release is signifi-cantly more rapidly than the sample for not adding GSH, illustrates the Nano medication for no matter targeting or not targeting In the presence of the GSH of 10 mM, it can be released effectively drug.
11 mtt assay of embodiment surveys the polyethylene glycol carbonic ester maytansine of free maytansine (DM1), reduction response The polyethylene glycol carbonic ester maytansine prodrug micelle (cRGD- of the cRGD polypeptide targeting of prodrug micelle (MMP) and reduction response MMP) to the toxicity of B16F10 melanoma cells
Test used by cell be mainly α v beta 3 receptor overexpression melanoma cells (B16F10), the cell from Chinese Academy of Sciences's Shanghai cell bank is bought.Culture solution used in B16F10 cell is DMEM culture solution, contains 10% tire ox blood simultaneously Clearly, 100 IU/mL penicillin and 100 μ g/mL streptomysins.Incubator environment is 5% carbon dioxide and 37 DEG C of constant temperature.
The concentration range of free maytansine (DM1), MMP and cRGD-MMP is 0.001 μ g DM1 in experimentation Equiv./mL to 10 μ g DM1 equiv./mL.Specific step is as follows: 80 μ L B16F10 cell liquid being first laid on 96 orifice plates In culture plate, and the final densities of cell are 3 × 103A/hole, and be placed in incubator overnight, the monolayer coverage of cell Reach 80% or so.Then to free maytansine (DM1), MMP and the cRGD- that the various concentration that 20 μ L have diluted is added in every hole MMP Nano medication solution, is placed in incubator after cultivating 4 h, removes original culture medium and that 100 μ L are added into every hole is new Fresh culture solution.After continuing to be placed in incubator and cultivating 68 h, 20 μ L 3- (4,5- dimethylthiazole -2) -2 are added into every hole, The PBS solution (5mg/mL) of 5- diphenyltetrazolium bromide bromide (MTT) continues 4 h of culture and moves back except supernatant, and uses 150 μ L DMSO dissolves the crystallization of purple first a ceremonial jade-ladle, used in libation, measures absorption of each hole at 490 nm using microplate reader (BioTek) after dissolving completely Value.Every group of experiment carries out four times in parallel, and final experimental results are four averaging of income values.Comparative survival rate of cells is experiment Group obtains compared with blanc cell control group is in the absorption value at 490 nm.
Cell survival rate (%)=(OD490nm sample/OD490nm control) × 100%
Figure 11 is free maytansine (DM1), MMP and cRGD-MMP are to the toxotests of B16F10 melanoma tumor cells Result figure, the results showed that, polycarbonate maytansine prodrug of the invention has preferably antitumor cell effect, especially cRGD- MMP has significantly superior different cellkilling capacity.
Embodiment 12 observes polyethylene glycol carbonic ester maytansine prodrug micelle using flow type analyzer (FACS) (cRGD-MMP, MMP) is to B16F10 melanoma cells cell endocytic behavioral study
The specific steps of which are as follows: first 1mL B16F10 cell liquid is laid in 6 orifice plates culture plate, and the final densities of cell It is 4 × 105A/hole, and be placed in incubator overnight, the monolayer coverage of cell reaches 80% or so.Fluorescence will then be contained The cRGD-MMP(DOX-cRGD-MMP of DOX) solution (DOX concentration: 10 μ g/mL) be added in every hole, cultivate in the incubator After 4 hours, culture solution is removed and with the trypsin digestion and cell for containing 0.03 % (w/v) EDTA, subsequent repetitive operation is centrifuged PBS is washed twice, and will be finally dispersed in cell liquid in 500 μ LPBS；DOX-MMP is equally operated.Figure 12 be MMP and Endocytosis behavior streaming result figure of the cRGD-MMP to B16F10 melanoma tumor cells, the results showed that polycarbonate of the invention Maytansine prodrug has good cell endocytic ability；Especially cRGD-MMP has significantly superior different cell endocytic ability.
The use of embodiment 13 Laser Scanning Confocal Microscope (CLSM) observation polycarbonate maytansine prodrug micelle (cRGD-MMP, MMP) to B16F10 melanoma cells cell endocytic behavioral study
Specific step is as follows for laser co-focusing experiment: 400 μ L B16F10 cell liquid being first laid on 24 orifice plate culture plates In, and the final densities of cell are 3 × 104A/hole, and be placed in incubator overnight, the monolayer coverage of cell reaches 80% Left and right.The cRGD-MMP(DOX-cRGD-MMP that will then fluorescence DOX be contained) solution (DOX concentration: 10 μ g/mL) be added to often Kong Zhong after cultivating 1 or 4 hour in the incubator, removes culture solution and with PBS repeated washing 3 times.Then with 4% paraformaldehyde Room temperature fix 15 min of cell, PBS repeated washing 3 times；Then with 4 ', 6- diamidino -2-phenylindone (DAPI) to nucleus Dye 10 min, PBS repeated washing 3 times；DOX-MMP is equally operated.Figure 13 is MMP and cRGD-MMP to B16F10 melanin The endocytosis behavior CLSM result figure of struma oncocyte, the results showed that polycarbonate maytansine prodrug of the invention has good thin The ability of gulping down intracellular；Especially cRGD-MMP has significantly superior different cell endocytic ability.
The polyethylene glycol carbonic ester maytansine prodrug micelle of the cRGD polypeptide targeting of the reduction response of embodiment 14 (cRGD-MMP) and the polyethylene glycol carbonic ester maytansine prodrug micelle (MMP) of reduction response is dynamic in the intracorporal drug of mouse Mechanics study
All zoopery operations meet University Of Suzhou's animal experimental center and University Of Suzhou's animal protection and use committee member The approval regulation of meeting, the experimental animal of selection is the black mouse of C57BL/6, female, 5 week old, and weight is 16 ~ 18 grams.Blood in vivo In liquid circulation experiment, cRGD-MMP and MMP Nano medication is by the way that in tail vein injection to Kunming mouse body, every group has 3 Experimental animal.Two groups of Nano medication animal blood taking times are set as 0.05,0.25,0.5,1,2,4,8,12 and 24 h, Mei Geshi Between the blood sampling volume put be 30 μ L.Blood sample is weighed after taking blood, blood weight is accurately calculated by difference assay, to each 200 μ L methanol solution, ultrasound crack it sufficiently in blood sample, after add 0.6 mL dimethyl sulfoxide, be placed in shaking table and place Overnight, after being then centrifuged for (min of 13000 r.p.m. × 20), supernatant liquor is taken, and be added into supernatant liquor excessive DTT is finally measured sample using high performance liquid chromatography (HPLC).
%ID/g=(FL sample × (V Qula leads to+V dimethyl sulfoxide))/(M blood × FL standard specimen × V standard specimen × standard sample dilution times Number) × 100%.
Figure 14 is MMP and cRGD-MMP Nano medication in the intracorporal pharmacokinetics results figure of mouse, as a result, it has been found that this hair Bright polyethylene glycol carbonic ester maytansine prodrug Nano medication all has longer circulation time.
The polyethylene glycol carbonic ester maytansine prodrug micelle of the cRGD polypeptide targeting of the reduction response of embodiment 15 (cRGD-MMP) and the polyethylene glycol carbonic ester maytansine prodrug micelle (MMP) of reduction response is resistance in the intracorporal maximum of mouse By dose study
By cRGD-MMP and MMP Nano medication by single tail vein injection to mouse body, two groups of Nano medications are given Pharmaceutical quantities are followed successively by 4,6,8 mg DM1 equiv./kg, in addition there are one group of mouse by injecting normal saline as control. Mouse weight and survival rate variation are then recorded in next 10 days, while whether also observation mouse significant pathology occurs Property feature, if action latens slow, back becomes rickets, and excrement and eye discharge are abnormal, and whether skin has anomalous variation etc..Most Big tolerance dose is defined as being administered in 10 day observation period that there is no dead or great physiological drug toxic side effect or Mice Bodies It reduces again and weighs 15% no more than substance.
Figure 15 and Figure 16 is respectively cRGD-MMP and MMP Nano medication in the intracorporal maximum tolerated dose result figure of mouse. As a result, it has been found that the maximum tolerated dose of two groups of Nano medications is 6 mg DM1 equiv./kg, higher than the existing freely beauty measured Step on the maximum tolerated dose (1 mg DM1 equiv./kg) of element.
16 polyethylene glycol carbonic ester maytansine prodrug micelle (cRGD-MMP, MMP) of embodiment is black to lotus B16F10 The antitumor research of melanoma mouse
CRGD-MMP Nano medication selects tumor size in the antitumor research experiment of lotus B16F10 melanoma mouse For 30 ~ 50 mm3Animal start to test, modeling method is as follows: in tumor-bearing mice modeling, we use yellow Jackets first (80 mg/kg) through intraperitoneal injection of anesthesia animal, then by B16F10 cell suspension, (50 μ L contain 8 × 106A cell) it is percutaneous Under be injected into back of mice upper position.
It is equipped with six groups altogether, is cRGD-MMP Nano medication (2.4 mg DM1 equiv./kg) respectively, cRGD-MMP nanometers Drug (1.6 mg DM1 equiv./kg), cRGD-MMP Nano medication (0.8 mg DM1 equiv./kg), MMP Nano medication (2.4 mg DM1 equiv./kg), free maytansine drug (0.8 mg/kg) and physiological saline group (PBS), every group has 8 Animal.Dosage rate are as follows: be administered once for every 2 days, be administered 3 times in total, wherein the timing definition of administration is the 0th day for the first time. In therapeutic process, the weight and gross tumor volume of every 1-2 days measurement mouse, the measurement method of gross tumor volume are as follows: with vernier caliper point Not Ce Liang tumour length (L), width (W) and thickness (H), then the volume (V) of tumour can calculate are as follows: V=(L × W × H)/2. Relative tumour volume is to pass through V/V0(V0For the gross tumor volume of measurement in the 0th day) be calculated, and relative body weight variation is to pass through M/M0(M0The nude mice weight weighed for the 0th day) be calculated.During experiment is investigated, there is natural death or tumour in mouse Volume is more than 1000 mm3It is determined as experimental endpoints.When to 10 days treatment ends, 3 mouse are taken out from every group, remaining 5 The time-to-live of ob mouse lotus knurl mouse is taken pictures and is weighed to the tumour for taking out all mouse, tumor control rate Calculation method are as follows:
Tumor control rate (%)=((physiological saline group tumour average weight-each experimental group tumour average weight)/physiology salt Water group tumour average weight) × 100
After treatment end, gross tumor volume in every group being in medium sized animal and puts to death one, dissection peels tumour, and It takes out main organs (heart, liver, spleen, lung, kidney), does histotomy analysis.Specific method is: each group is woven in Fu Er After fixing 48 hours in Malin's solution, specimens paraffin embedding slices are carried out, are dyed with h and E (H&E).Finally use optics Microscope (Olympus BX41 microscope) carries out observation and takes pictures.
Figure 17 to Figure 21 is the antitumor result of study figure of each group lotus B16F10 melanoma mouse.Physiology salt as the result is shown The tumour growth of water group is most fast, and free maytansine group has certain anti-tumor activity, cRGD-MMP Nano medication (2.4 mg DM1 Equiv./kg) there is strongest anti-tumor activity, tumor control rate reaches 97.5%, than the tumor suppression of MMP Nano medication Rate (87.5%) is substantially higher；The increase with dosage is found simultaneously, and the inhibitory effect of tumour is better；Mouse time-to-live result CRGD-MMP Nano medication is also shown with the longest time-to-live；Weight is not over the course for the treatment of for all groups of mouse simultaneously It changes a lot.
17 polymer of embodiment (PEG-P (CL-co-PDSC synthesis))
In a nitrogen environment, 86.7 mg(0.32 mmol) two thiopyridines carbonate monomers (PDSC) and 91.2 mg(0.8 Mmol caprolactone (CL)) is dissolved in 2 mL methylene chloride, is added in sealing reactor, 0.1 g(0.02 mmol is then added) CH3Dichloromethane solution (0.1 mol/ of O-PEG-OH (5 K) and bis- (double trimethyl silicon substrates) the amine zinc of the catalyst of 0.5 mL L), then reactor is sealed, is transferred out of glove box, after reacting 24 hours in 40 DEG C of oil baths, glacial acetic acid terminates reaction, ice It is precipitated in ether, eventually passes through filtering, vacuum drying obtains polymer P EG-P (TMC-co-PDSC).Yield is 85.5%.Nuclear-magnetism It is respectively 40 and 10.2 that the degree of polymerization that molecular weight is 11.8 kg/mol, CL and PDSC, which is calculated,.
18 amphipathic ethylene glycol of embodiment-polycarbonate maytansine prodrug polymer (PEG-P (CL-g- SSDM1)) Synthesis
Under nitrogen protection, is sequentially added in the three-necked flask of 100mL and be dissolved in 10mL n,N-Dimethylformamide (DMF) the polymer P EG-P (CL- inco-PDSC) (bis- thiopyridines functional group of 100 mg, 0.088 mmol), at the same to its Middle (100 μ L) glacial acetic acid that catalytic amount is added, is then added dropwise the mercapto-functionalized maytansine of 25 mL into there-necked flask (DM1) DMF solution of (97.8 mg, 0.135 mmol), reactor are placed in 35 DEG C of oil bath, are stirred to react 48 hours Afterwards, it successively dialyses (Spectra/Pore, MWCO 7000) in DMF and water, freeze-drying, yield 79%.Molecule is calculated in nuclear-magnetism Amount is 18.1 kg/mol.
19 polymer of embodiment (Mal-PEG-P (CL-co-PDSC synthesis))
In a nitrogen environment, 86.7 mg(0.32 mmol) two thiopyridines carbonate monomers (PDSC) and 91.2 mg(0.8 Mmol caprolactone (CL)) is dissolved in 2 mL methylene chloride, is added in sealing reactor, 0.1 g(0.02 mmol is then added) Dichloromethane solution (0.1 mol/ of MAL-PEG-OH (5 K) and bis- (double trimethyl silicon substrates) the amine zinc of the catalyst of 0.5 mL L), then reactor is sealed, is transferred out of glove box, after reacting 24 hours in 40 DEG C of oil baths, glacial acetic acid terminates reaction, ice It is precipitated in ether, eventually passes through filtering, vacuum drying obtains polymer MAL-PEG-P (TMC-co-PDSC).Yield is 85.5%. It is respectively 40 and 11.1 that the degree of polymerization that molecular weight is 12 kg/mol, CL and PDSC, which is calculated, in nuclear-magnetism.
Polymer (FA-PEG-P (the CL- of 20 folic acid end group of embodiment bondingco-PDSC synthesis))
By MAL-PEG-P (CL-co-PDSC) the n,N-Dimethylformamide (DMF) (2.5 of (50 mg, 3.84 μm of ol) ML solution) is prepared into MAL-PEG-P (CL- by exchange of solvent methodco-PDSC micella), resulting polymers micellar concentration is about For 3 mg/mL, then micella is transferred in the there-necked flask of 100 mL, and is suitably bubbled and guarantees nitrogen environment, then thereto FA-SH(2.9 mg, 5.76 μm of ol is added), it is placed in 35 DEG C of oil bath pans and stirs 24 h, then dialyse in deionized water (Spectra/Pore, MWCO 3500), freeze-drying, and be stored in -20 DEG C of refrigerators.Yield 78%.Nuclear-magnetism Malaysia acyl as the result is shown The nuclear-magnetism characteristic peak of imines completely disappears, and the appearance of FA characteristic peak shows folic acid fully reacting.Two sulphur pyrroles in polymer simultaneously The integrated value of pyridine characteristic peak is not reduced, shows that FA-SH does not occur sulphur sulphur with two thiopyridines and exchanges side reaction.
Amphipathic ethylene glycol-polycarbonate maytansine prodrug polymer of 21 folic acid end group of embodiment bonding (FA-PEG-P (TMC-g- SSDM1)) synthesis
Under nitrogen protection, is sequentially added in the three-necked flask of 50mL and be dissolved in 3mL n,N-Dimethylformamide (DMF) In folic acid end group bonding polymer FA-PEG-P (CL-co-PDSC) (bis- thiopyridines function of 40 mg, 0.036 mmol Group), while (30 μ L) glacial acetic acid of catalytic amount being added thereto, 10 mL sulfydryl functions are then added dropwise into there-necked flask The DMF solution of the maytansine (DM1) (39.9 mg, 0.054 mmol) of change, reactor are placed in 35 DEG C of oil bath, and stirring is anti- After answering 48 hours, successively dialyse (Spectra/Pore, MWCO 7000) in DMF and water, freeze-drying, yield 73%.Nuclear-magnetism result Show that its structure is amphipathic ethylene glycol-polycarbonate maytansine prodrug polymer of folic acid end group bonding, the load of maytansine Dose is 40.6wt.%。
Polymer (Gal-PEG-P (the CL- of 20 digalactosyl end group of embodiment bondingco-PDSC synthesis))
By MAL-PEG-P (CL-co-PDSC) the n,N-Dimethylformamide (DMF) (2.5 of (50 mg, 3.84 μm of ol) ML solution) is prepared into MAL-PEG-P (CL- by exchange of solvent methodco-PDSC micella), resulting polymers micellar concentration is about For 3 mg/mL, then micella is transferred in the there-necked flask of 100 mL, and is suitably bubbled and guarantees nitrogen environment, then thereto Gal-SH(2.5 mg, 5.76 μm of ol is added), it is placed in 35 DEG C of oil bath pans and stirs 24 h, then dialyse in deionized water (Spectra/Pore, MWCO 3500), freeze-drying, and be stored in -20 DEG C of refrigerators.Yield 78%.Nuclear-magnetism Malaysia acyl as the result is shown The nuclear-magnetism characteristic peak of imines completely disappears, and the appearance of Gal characteristic peak shows folic acid fully reacting.Two sulphur pyrroles in polymer simultaneously The integrated value of pyridine characteristic peak is not reduced, shows that Gal-SH does not occur sulphur sulphur with two thiopyridines and exchanges side reaction.
Amphipathic ethylene glycol-polycarbonate maytansine prodrug polymer of 21 galactolipin end group of embodiment bonding (FA-PEG-P (TMC-g- SSDM1)) synthesis
Under nitrogen protection, is sequentially added in the three-necked flask of 50mL and be dissolved in 3mL n,N-Dimethylformamide (DMF) In galactolipin end group bonding polymer Gal-PEG-P (CL-co-PDSC) (bis- thiopyridines official of 40 mg, 0.036 mmol Can roll into a ball), while (30 μ L) glacial acetic acid of catalytic amount being added thereto, 10 mL sulfydryl function are then added dropwise into there-necked flask The DMF solution of the maytansine (DM1) (39.9 mg, 0.054 mmol) of energyization, reactor are placed in 35 DEG C of oil bath, stirring After reaction 48 hours, successively dialyse (Spectra/Pore, MWCO 7000) in DMF and water, freeze-drying, yield 73%.Nuclear-magnetism knot Fruit shows that its structure is amphipathic ethylene glycol-polycarbonate maytansine prodrug polymer of folic acid end group bonding, maytansine Drugloading rate is 40.6wt.%。
Change different types of targeted molecular or the polymer containing targeted molecular, based on this, according to above-mentioned implementation The preparation method of example, can prepare the targeting polycarbonate maytansine prodrug micelle of a variety of reduction responses, and concrete application result is shown in Table 3-5, ratio are the mass ratio of 1 polymer of table and 2 polymer of table.
The cRGD targeting polyethylene glycol carbonic ester maytansine prodrug micelle of the reduction response of table 3 is to lotus melanoma mouse Antitumor activity
The folate-targeted polyethylene glycol carbonic ester maytansine prodrug micelle of the reduction response of table 4 is to lotus KB cytoma mouse Antitumor activity
The galactolipin targeting polyethylene glycol carbonic ester maytansine prodrug micelle of the reduction response of table 5 is to lotus HepG2 cytoma The antitumor activity of mouse
The targeting polyethylene glycol carbonic ester maytansine prodrug micelle of reduction response disclosed by the invention has following Advantage: the dissolubility of maytansine in water is greatly strengthened；The EPR effect and active targeting that micella has make it in lesion The concentration at position is greatly improved, and then improves the bioavilability of drug；It is enough steady during blood circulation It is fixed, significantly reduce nonspecific drug release behavior；Drugloading rate is very big horizontally to be improved and controllable；Especially in the present invention The advantages of targeting the advantages of multi-functional polymeric prodrugs micella had not only contained polymeric prodrugs but also enumerating micella.Compared to general Logical polymeric prodrugs, it improves the enrichment and penetration capacity in tumor locus significantly, improves taking the photograph by tumour cell Ability is taken, and drug molecule can fast and effeciently discharge in tumour cell.

Claims (8)

1. a kind of targeting polyethylene glycol carbonic ester maytansine prodrug micelle of reduction response, by amphipathic polyethylene glycol carbon Amphipathic ethylene glycol-polycarbonate maytansine prodrug of acid esters maytansine prodrug and end bonding targeted molecular is in buffer Self assembly obtains；It is characterized in that, the preparation of the targeting polyethylene glycol carbonic ester maytansine prodrug micelle of the reduction response Method the following steps are included:
(1) in the presence of polyethylene glycol initiator, it is total to two thiopyridines carbonic esters and the carbonate monomer open loop containing R2 group Polymerization obtains amphipathic Biodegradable butane diacid-polycarbonate；Then amphipathic ethylene glycol-polycarbonate and sulfhydrylation Maytansine carry out sulfydryl-two sulphur exchange reactions obtain amphipathic ethylene glycol-polycarbonate maytansine prodrug；The R2 group Selected from one of following group:
；
(2) in the presence of functionalized poly (ethylene glycol) initiator, make two thiopyridines carbonic esters and the carbonate monomer containing R2 group Ring-opening copolymerization obtains amphipathic ethylene glycol-polycarbonate of end-functionalization；Then by the polyethylene glycol-of end-functionalization The polyethylene glycol carbonic ester micella of end-functionalization is prepared using solvent displacement for polycarbonate, then by end official The polyethylene glycol carbonic ester micella of energyization obtains end with targeted molecular progress addition reaction and is bonded the amphipathic of targeted molecular Polyethylene glycol carbonic ester；Amphipathic ethylene glycol-polycarbonate of last end bonding targeted molecular and the U.S.A of sulfhydrylation are stepped on Before element progress sulfydryl-two sulphur exchange reactions obtain amphipathic ethylene glycol-polycarbonate maytansine of end bonding targeted molecular Medicine；The R2 group is selected from one of following group:
；
(3) by the amphipathic ethylene glycol-of amphipathic ethylene glycol-polycarbonate maytansine prodrug and end bonding targeted molecular The self assembly in buffer of polycarbonate maytansine prodrug obtains the polyethylene glycol carbonic acid with targeted molecular of reduction response Ester maytansine prodrug micelle.
2. restoring the targeting polyethylene glycol carbonic ester maytansine prodrug micelle of response according to claim 1, feature exists In: in the targeting polycarbonate maytansine prodrug micelle of the reduction response, end is bonded the amphipathic second two of targeted molecular Alcohol-polycarbonate maytansine prodrug mass percent is greater than 0 and is less than or equal to 60%；The buffer is phosphate buffer；It is described The partial size of the targeting polyethylene glycol carbonic ester maytansine prodrug micelle of reduction response is 30~150 nanometers；The reduction response Targeting polyethylene glycol carbonic ester maytansine prodrug micelle in, the drugloading rate of maytansine is 2~60wt.%。
3. restoring the targeting polyethylene glycol carbonic ester maytansine prodrug micelle of response according to claim 1, feature exists In:
For step (1) specifically, in nitrogen environment, two thiopyridines carbonic esters, the carbonic ester containing R2 group and polyethylene glycol are molten Then the first catalyst is added in the first solvent in solution, in closed reactor, carry out ring-opening copolymerization reaction, obtain amphiphilic Property polyethylene glycol carbonic ester；In nitrogen environment, amphipathic ethylene glycol-polycarbonate and sulfhydrylation maytansine are dissolved In the second solvent, the second catalyst is added, in sealing reactor, carries out sulfydryl-two sulphur exchange reactions, then dialysis obtains Amphipathic ethylene glycol-polycarbonate maytansine prodrug；
Step (2) is specifically, in nitrogen environment, two thiopyridines carbonic esters, the carbonic ester containing R2 group and functionalized poly second Glycol is dissolved in third solvent, and third catalyst is added thereto, and in closed reactor, it is anti-to carry out ring-opening copolymerization It answers, obtains amphipathic ethylene glycol-polycarbonate of end-functionalization；In nitrogen environment, end official is added in targeted molecular Addition reaction is carried out in amphipathic ethylene glycol-polycarbonate micellar aqueous solution of energyization, then dialyses, be dried to obtain end key Close amphipathic ethylene glycol-polycarbonate of targeted molecular；Then in nitrogen environment, by the amphiphilic of end bonding targeted molecular Property polyethylene glycol carbonic ester and sulfhydrylation maytansine be dissolved in the 4th solvent, be added the 4th catalyst, sealing reactor In, sulfydryl-two sulphur exchange reactions are carried out, then dialysis obtains the amphipathic polyethylene glycol carbonic acid of end bonding targeted molecular Ester maytansine prodrug；
Amphipathic ethylene glycol-polycarbonate maytansine prodrug specifically, is bonded the amphiphilic of targeted molecular by step (3) with end Property polyethylene glycol carbonic ester maytansine prodrug is dissolved separately in the 5th solvent, and buffer is added dropwise after mixing, then dialyses To the targeting polyethylene glycol carbonic ester maytansine prodrug micelle of reduction response.
4. restoring the targeting polyethylene glycol carbonic ester maytansine prodrug micelle of response according to claim 3, feature exists In:
In step (1), the first catalyst is bis- (double trimethyl silicon substrates) amine zinc；First solvent is methylene chloride；Ring-opening copolymerization The temperature of reaction is 40 DEG C, and the time is 24 h；Second catalyst is glacial acetic acid；Second solvent be N,N-dimethylformamide or Dimethyl sulfoxide；Sulfydryl-two sulphur exchange reactions temperature is 40 DEG C, and the time is 48 h；
In step (2), third catalyst is bis- (double trimethyl silicon substrates) amine zinc；Third solvent is methylene chloride；Ring-opening copolymerization Reaction temperature is 40 DEG C, and the time is 24 h；4th solvent is N,N-dimethylformamide or dimethyl sulfoxide；The temperature of addition reaction Degree is 35 DEG C, and the time is for 24 hours；4th catalyst is glacial acetic acid；4th solvent is N,N-dimethylformamide or dimethyl sulfoxide； Sulfydryl-two sulphur exchange reactions temperature is 40 DEG C, and the time is 48 h；The targeted molecular is antibody molecule, peptide molecule, sugar Son or biological micromolecule；The chemical structural formula of the functionalized poly (ethylene glycol) are as follows:
Wherein, R1 is following group:
In step (3), the 5th solvent is n,N-Dimethylformamide or dimethyl sulfoxide；End is bonded the amphipathic of targeted molecular Polyethylene glycol carbonic ester maytansine prodrug accounts for the 0~60% of micella quality, does not include 0；The buffer is phosphate buffer.
5. the targeting polyethylene glycol carbonic ester maytansine prodrug micelle for restoring response described in claim 1 is controlled in preparation tumour Treat the application in drug.
6. a kind of Nano medication, the targeting polycarbonate maytansine prodrug micelle including restoring response described in claim 1.
7. a kind of amphipathic ethylene glycol-polycarbonate maytansine prodrug, it is characterised in that: the amphipathic polyethylene glycol The preparation method of carbonic ester maytansine prodrug the following steps are included:
(1) in the presence of polyethylene glycol initiator, it is total to two thiopyridines carbonic esters and the carbonate monomer open loop containing R2 group Polymerization obtains amphipathic Biodegradable butane diacid-polycarbonate；Then amphipathic ethylene glycol-polycarbonate and sulfhydrylation Maytansine carry out sulfydryl-two sulphur exchange reactions obtain amphipathic ethylene glycol-polycarbonate maytansine prodrug；The R2 group Selected from one of following group:
；
(2) in the presence of functionalized poly (ethylene glycol) initiator, make two thiopyridines carbonic esters and the carbonate monomer containing R2 group Ring-opening copolymerization obtains amphipathic ethylene glycol-polycarbonate of end-functionalization；Then by the polyethylene glycol-of end-functionalization The polyethylene glycol carbonic ester micella of end-functionalization is prepared using solvent displacement for polycarbonate, then by end official The polyethylene glycol carbonic ester micella of energyization obtains end with targeted molecular progress addition reaction and is bonded the amphipathic of targeted molecular Polyethylene glycol carbonic ester；Amphipathic ethylene glycol-polycarbonate of last end bonding targeted molecular and the U.S.A of sulfhydrylation are stepped on Before element progress sulfydryl-two sulphur exchange reactions obtain amphipathic ethylene glycol-polycarbonate maytansine of end bonding targeted molecular Medicine；The R2 group is selected from one of following group:
。
8. the ethylene glycol of amphipathic described in claim 7-polycarbonate maytansine prodrug answering in preparation tumor therapeutic agent With.
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