CN114904012A - Active oxygen self-complementary amphiphilic block copolymer-drug conjugate, preparation method and application thereof - Google Patents

Active oxygen self-complementary amphiphilic block copolymer-drug conjugate, preparation method and application thereof Download PDF

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CN114904012A
CN114904012A CN202210461713.2A CN202210461713A CN114904012A CN 114904012 A CN114904012 A CN 114904012A CN 202210461713 A CN202210461713 A CN 202210461713A CN 114904012 A CN114904012 A CN 114904012A
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罗奎
王兵
陈凯
朱红艳
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West China Hospital of Sichuan University
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Abstract

The invention belongs to the technical field of medicines, and particularly relates to an amphiphilic block copolymer-medicine conjugate with self-supplemented active oxygen, a preparation method and application thereof. The structural formula of the amphiphilic block copolymer-drug conjugate with active oxygen self-supplementation is shown as a formula I. R is a molecular chain formed by polymerizing a repeating unit A and a repeating unit B, wherein the repeating unit A is as follows:
Figure DDA0003621958340000011
wherein R is D Is R D OH, said R being a substituent after removal of the hydroxyl group D OH is an anti-tumor drug molecule; the repeating unit B is:
Figure DDA0003621958340000012
the amphiphilic block copolymer-drug conjugate with self-supplemented active oxygen provided by the invention can solve the problem of insufficient drug release in ROS-responsive DDS, and has very high application potential in the development and application of antitumor drugs.
Figure DDA0003621958340000013

Description

Active oxygen self-complementary amphiphilic block copolymer-drug conjugate, preparation method and application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an amphiphilic block copolymer-medicine conjugate with self-supplemented active oxygen, a preparation method and application thereof.
Background
Drug Delivery Systems (DDSs) refer to the form of administration of various drugs that people use in the prevention and treatment of disease. The design concept is to deliver the drug to the necessary site in the necessary amount and time to achieve the maximum therapeutic effect and the minimum toxic and side effects.
Currently, Reactive Oxygen Species (ROS) -responsive DDS have been widely reported. As endogenous stimulators, ROS include superoxide (O) 2 - ) Hypochlorite ion (OCl) - ) Peroxynitrite (ONOO) - ) Hydroxyl radical (. OH) and hydrogen peroxide (H) 2 O 2 ) And can reach high levels of 0.1mM in tumor cells, which is higher than that of normal cells (0.02. mu.M)One thousand times higher. Such high levels of ROS in tumor cells have been used as a stimulus in drug delivery systems. However, in many cases, these ROS-sensitive DDSs do not achieve sufficient release of the drug. This low release efficiency can be partly attributed to heterogeneity of ROS levels in different tumor tissues, or lack of a more intelligent, more synergistic DDS.
Cinnamaldehyde (CA) is the major component in cinnamon and its safety has been approved by the U.S. Food and Drug Administration (FDA). CA is reported to be able to generate ROS in mitochondria and induce cancer cell apoptosis by amplifying oxidative stress. However, aldehyde groups in CA are rapidly oxidized and have low antitumor efficacy, which hinders the use of CA in antitumor drugs. Chinese patent CN113072704A A polysulfide acetal degraded by self-amplification based on active oxygen, its preparation method and application disclose a polysulfide acetal degraded by self-amplification based on active oxygen, its preparation method and application. The polythioacetal provided by the invention is derived from cinnamaldehyde, and has the following structural formula:
Figure BDA0003621958320000011
the structure of the polythioacetal can effectively protect aldehyde groups in CA from being oxidized. Meanwhile, the thioacetal bond in the polythioacetal can respond to the breakage in the presence of ROS, CA released after the thioacetal bond responds to the breakage can generate ROS in cells, and the ROS generated by CA further breaks the thioacetal chain in the polythioacetal to generate the self-amplification degradation effect. The polythioacetal is used as a tumor drug carrier, is easily induced by ROS in tumors to be efficiently degraded, is beneficial to quickly releasing the drugs in tumor cells, and opens up a new way for the effective release of the drugs.
However, the action of CA to generate ROS needs to be carried out in mitochondria, and the above-mentioned polythioacetal does not have the effect of targeting enrichment into the mitochondria of tumor cells, which limits the exertion of the effect of releasing drugs from amplified degradation. And the hydrophilic and hydrophobic structural units of the polymer are not clear, so that a stable nano cavity is difficult to form for encapsulating the antitumor drug. Therefore, there is a need to provide a new drug delivery system, which can carry CA and antitumor drugs and realize the function of targeted enrichment to mitochondria.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an amphiphilic block copolymer-drug conjugate with self-supplemented active oxygen, a preparation method and application thereof, and aims to provide a conjugate which can effectively target and enrich to tumor cell mitochondria, can generate cascade amplification degradation, and can effectively release antitumor drugs in tumor cells.
An active oxygen self-complementary amphiphilic block copolymer-drug conjugate, which is characterized in that the structural formula is shown as formula I:
Figure BDA0003621958320000021
wherein the value of n is selected from 43-227,
r is a molecular chain formed by polymerizing x repeating units A and y repeating units B, the value of x is selected from 2 to 10, preferably 5 to 10, the value of y is selected from 20 to 40,
the repeating unit A is:
Figure BDA0003621958320000031
wherein R is D Is R D OH as a substituent after removal of the hydroxyl group, the R D OH is an anti-tumor drug molecule;
the repeating unit B is:
Figure BDA0003621958320000032
wherein R is B Selected from hydrogen or methyl, X is selected from O,
Figure BDA0003621958320000033
Preferably, said R D OH is paclitaxel, docetaxel, adriamycin, epirubicin, capecitabine, cabazitaxel, 2-methoxyestradiol, camptothecin, hydroxycamptothecin, 9-aminocamptothecin, topotecan or irinotecanAnd (3) taking litikang.
Preferably, the repeating unit B is selected from
Figure BDA0003621958320000034
Figure BDA0003621958320000035
Preferably, said R is D OH is taxol and the repeating unit B is selected from
Figure BDA0003621958320000041
n＝113，x＝3，y＝25。
The invention also provides a preparation method of the conjugate, which comprises the following steps:
(1) the following compound a was prepared:
Figure BDA0003621958320000042
(2) the following compound B was prepared:
Figure BDA0003621958320000043
(3) compound a, compound B and 2- (diisopropylamino) ethyl methacrylate the compound of formula i is prepared by the following reaction:
Figure BDA0003621958320000044
wherein n, R and R D As claimed in claim 1 or 2.
Preferably, the step (1) specifically comprises the following steps:
(1.1) preparation of Compound TA-CA-NH according to the following reaction scheme 2 ：
Figure BDA0003621958320000051
(1.2) preparation of Compound R1-TA-CA-NH according to the following reaction scheme 2 ：
Figure BDA0003621958320000052
(1.3) preparation of the Compound CTA-NPC according to the following reaction scheme:
Figure BDA0003621958320000053
(1.4) preparation of Compound A according to the following reaction scheme:
Figure BDA0003621958320000054
preferably, the step (2) specifically comprises the following steps:
(2.1) preparation of compound TA-CA according to the following reaction:
Figure BDA0003621958320000055
(2.2) preparation of compound MA-TA-CA according to the following reaction scheme:
Figure BDA0003621958320000056
(2.3) preparation of Compound B according to the following reaction scheme:
Figure BDA0003621958320000061
preferably, in the step (3), the feeding ratio of the compound A, the compound B and the 2- (diisopropylamino) ethyl methacrylate is the molar ratio of 1 (20-40) to 2-7;
and/or the reaction is carried out under the action of an initiator, wherein the initiator is selected from at least one of VA044, AIBN, ACVA or V501;
and/or the reaction is carried out in a solvent selected from DMSO and H 2 At least one of mixed liquid with the volume ratio of O to O of 9:1, 4-dioxane, tetrahydrofuran or trifluoroethanol;
and/or the reaction conditions are as follows: reacting for 16-48 hours at 45-85 ℃ in an inert atmosphere.
The invention also provides a micelle formed by the conjugate.
Preferably, the particle size of the micelle is 100-200 nm;
and/or the zeta potential of the micelle is-15 to-5 mV in an environment with a pH of 7.4, the zeta potential of the micelle is +10 to +25mV in an environment with a pH of 6.0, and the zeta potential of the micelle is +25 to +30mV in an environment with a pH of 5.2.
The invention also provides application of the conjugate or the micelle in preparation of antitumor drugs.
The invention also provides a medicine which is prepared by taking the conjugate or the micelle as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
The invention designs and synthesizes the amphiphilic block copolymer-drug conjugate with self-supplemented active oxygen based on a positive feedback strategy. The conjugate has a pH sensitive N, N-Diisopropylamine (DPA) moiety, and the DPA can be combined with protons to form positively charged ammonium in the acidic environment of tumor cells. Since positively charged nanoparticles can strongly interact with negatively charged mitochondrial membranes, the DPA moiety in the copolymer facilitates the transfer of the conjugate to the mitochondria. Meanwhile, the conjugate has ROS response characteristics, and can release antitumor drugs and CA in a tumor cell environment, and CA can further generate ROS in mitochondria to generate self-degradation effect on the conjugate, so that the release of the antitumor drugs and CA is further promoted. Based on the principle, the synergistic cooperation of all functional parts in the conjugate can obviously enhance the capability of the conjugate to release the anti-tumor drugs, thereby enhancing the anti-tumor efficacy of the conjugate.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of MA-TA-CA-PTX in example 1;
FIG. 2 is a NMR spectrum of mPEG5k-NPC in example 1;
FIG. 3 shows mPEG5k-TA-CA-NH in example 1 2 Hydrogen spectrum of Nuclear Magnetic Resonance (NMR);
FIG. 4 is a NMR spectrum of mPEG5k-TA-CA-CTA of example 1;
FIG. 5 is a NMR spectrum of mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA) in example 1;
FIG. 6 is a nuclear magnetic resonance hydrogen spectrum of MA-TK-PTX in comparative example 1;
FIG. 7 is a nuclear magnetic resonance hydrogen spectrum of mPEG5k-TK-CTA in comparative example 1;
FIG. 8 is a nuclear magnetic resonance hydrogen spectrum of mPEG5k-TK-block-poly (TK-PTX-co-DPA) in comparative example 1;
FIG. 9 is a NMR spectrum of mPEG5k-CTA in comparative example 2;
FIG. 10 is a nuclear magnetic resonance hydrogen spectrum of mPEG5k-block-poly (TK-PTX-co-DPA) in comparative example 2;
FIG. 11 shows the results of molecular weight measurements on polymers;
FIG. 12 is Zeta potential of micelles at different pH;
FIG. 13 is the particle size of micelles at different pH;
FIG. 14 is a TEM image of micelles prepared in example 2;
FIG. 15 shows the results of in vitro prodrug release assays;
FIG. 16 is a fluorescent image of 4T1 cells treated with PBS, CA, TA-CA-prodrug, TK-prodrug, or prodrug for 2h, with a CA concentration of 12 μ g/mL and a PTX concentration of 48 μ g/mL in each prodrug group. Cellular ROS levels were detected by fluorescent probe DCFH-DA (Green: DCFH). Scale bar 50 μm;
figure 17 is the IC50 of each prodrug on 4T1 cells;
FIG. 18 shows the intracellular localization of 4T1 cells treated with the prodrug 2h later (Cy5 concentration 0.5. mu.g/mL); wherein, the Lystracker Green and the Mitotracker Green mark mitochondria and lysosomes in 4T1 cells respectively, and the scale bar is 25 μm;
FIG. 19 is a CLSM image of mitochondrial membrane potential changes following exposure of 4T1 cells to PTX prodrug or CA 4h, scale bar: 25 μm;
fig. 20 is an in vitro fluorescence imaging using Cy5 by IVIS Spectrum system, monitoring drug distribution in major organs/tumors and survival time after tumor-bearing mice dosing, data expressed as mean ± SD, n ═ 3;
figure 21 is a graph of tumor proliferation after dosing in tumor-bearing mice (n-6), with tumor length and width recorded every other day;
figure 22 is a graph of tumor growth inhibition calculated from the isolated tumor mass (n-6);
FIG. 23 shows the body weight changes of tumor-bearing mice treated with different drugs;
figure 24H & E, immunohistochemistry and immunofluorescence staining detected tumor tissue necrosis, proliferation (Ki67), angiogenesis (CD31) and apoptosis, scale bar: 50 μm, data expressed as mean ± SD,. P <0.001,. P <0.01,. P < 0.05.
Detailed Description
In the following examples and experimental examples, reagents and materials used are commercially available unless otherwise specified.
Example 1 Synthesis of conjugate mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA)
In this example, a preferable structure mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA) (TA-CA-Prodrug for short) of the conjugate of the invention is synthesized by the following synthetic route:
Figure BDA0003621958320000081
Figure BDA0003621958320000091
in the above reaction formula, x is 3 and y is 25.
The method comprises the following specific steps:
the method comprises the following steps:
Figure BDA0003621958320000092
cinnamaldehyde (CA,10.00g,75.67mmol) and beta-mercaptoethanol (11.82g,151.33mmol) were dissolved in 70mL THF, then ZrCl was added at 0 deg.C 4 (3.53g,15.13 mmol). The reaction mixture was stirred at 0 ℃ for a further 30 minutes and the solvent was removed by evaporation. The crude product was purified by silica gel chromatography to give TA-C A-OH as a white solid (18.19g) in 89% yield. 1 H NMR(400MHz,DMSO-d 6 ):δ7.49-7.44(m,2H),7.33(t,J＝7.4Hz,2H),7.29-7.23(m,1H),6.59(d,J＝15.6Hz,1H),6.18(dd,J＝15.6,9.0Hz,1H),4.82(m,3H),3.56(m,4H),2.74-2.60(m,4H). 13 C NMR(100MHz,DMSO-d 6 ):δ136.27,130.80,129.12,128.54,128.32,126.93,61.24,51.31,33.79.HR-MS(ESI):[M+Na] + calcd for C 13 H 18 O 2 S 2 293.0640,found 293.0644.
Step two:
Figure BDA0003621958320000101
TA-CA-OH (1.00g,3.70mmol) was dissolved in 30mL dry THF containing triethylamine (TEA,0.77mL,5.55mmol) and a solution of methacryloyl chloride (0.39g,3.70mmol) in THF was added slowly to the reaction under ice. The reaction was continued in an ice bath for 4 hours. The white solid of triethylamine hydrochloride was removed by filtration, and the solvent was distilled off from the filtrate. The residue was purified by silica gel chromatography to give MA-TA-CA-OH (colorless oil)0.98g), yield 78%. 1 H NMR(400MHz,DMSO-d 6 ):δ7.50-7.44(m,2H),7.36-7.32(m,2H),7.29-7.29(m,1H),6.61(d,J＝15.6Hz,1H),6.21(dd,J＝15.6,9.0Hz,1H),6.04(m,1H),5.70(m,1H),4.86(d,J＝9.0Hz,1H),4.28(t,J＝6.6Hz,2H),3.57(t,J＝6.8Hz,2H),2.97-2.82(m,2H),2.75-2.62(m,2H),1.90-1.85(m,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ166.73,136.16,136.09,131.17,129.11,128.39,128.14,126.97,126.50,63.90,61.20,51.30,33.82,29.82,18.43.HR-MS(ESI):[M+Na] + calcd for C 17 H 22 O 3 S 2 361.0903,found 361.0907.
Step three:
Figure BDA0003621958320000102
MA-TA-CA-OH (0.20g,0.59mmol) was dissolved in 20mL of anhydrous THF containing TEA (0.16mL,1.18mmol) and neutralized and a catalytic amount of DMAP was added. A solution of 4-nitrophenoxycarbonyl chloride (0.18g, 0.89mmol) in THF was slowly added to the mixture while cooling on ice. The reaction was stirred at room temperature overnight. The resulting triethylamine hydrochloride solid was removed by filtration and the filtrate was added dropwise to a solution of PTX (530.80mg, 0.59mmol) and DMAP (216.24mg, 1.77mmol) in Dichloromethane (DCM). After 24 hours of reaction, the mixture was washed with dilute HCl, saturated Na2CO3, and brine, and dried over anhydrous sodium sulfate. The solvent was evaporated and the resulting residue was purified by silica gel chromatography to give MA-TA-CA-PTX as a white solid (0.59g) in 82% yield.
Of MA-TA-CA-PTX 1 The H NMR spectrum is shown in FIG. 1. 1 H NMR(400MHz,DMSO-d 6 ):δ7.99(d,J＝7.1Hz,2H),7.86(d,J＝7.2Hz,2H),7.73(t,J＝7.2Hz,1H),7.66(t,J＝7.4Hz,2H),7.55(t,J＝7.2Hz,1H),7.51-7.42(m,7H),7.37-7.30(m,2H),7.27(t,J＝7.2Hz,1H),7.21-7.17(m,1H),6.62(dd,J＝15.6,7.6Hz,1H),6.31(s,1H),6.22(dd,J＝15.6,9.0Hz,1H),6.03(s,1H),5.85(s,1H),5.68(s,1H),5.55(t,J＝8.6Hz,1H),5.42(d,J＝7.1Hz,1H),5.36(d,J＝8.6Hz,1H),4.96-4.87(m,3H),4.67(s,1H),4.38-4.23(m,4H),4.15-4.09(m,1H),4.04-3.97(m,2H),3.59(d,J＝7.2Hz,1H),2.95-2.85(m,4H),2.39-2.21(m,4H),2.10(d,J＝2.4Hz,3H),1.86(s,3H),1.81(s,3H),1.69-1.60(m,1H),1.50(s,4H),1.03-1.01(m,6H). 13 C NMR(100MHz,DMSO-d 6 ):δ202.81,170.10,169.37,169.15,166.75,166.72,165.63,154.11,139.62,137.37,136.07,136.01,135.99,134.42,133.88,132.01,131.63,130.32,130.02,129.21,129.11,128.77,128.50,128.00,127.87,127.62,127.03,126.51,84.03,80.66,77.08,75.70,75.09,74.88,71.55,71.10,70.87,67.84,63.71,57.82,54.33,51.29,46.47,43.37,36.95,34.77,29.89,29.83,29.49,26.75,22.99,21.81,21.10,18.41,14.38,10.21.HR-MS(ESI):[M+Na] + calcd for C 65 H 71 NO 18 S 2 1240.4005,found 1240.4014.
Step four:
Figure BDA0003621958320000111
CA (2.91g, 22.00mmol) and 2,2, 2-trifluoro-N- (2-mercaptoethyl) acetamide (8.00g, 46.20mmol) were dissolved in 100mL THF, aluminum chloride (1.47g, 11.00mmol) was added under ice bath, and stirring was continued for 10 min under ice bath. Evaporating to remove solvent, and purifying the obtained crude product by silica gel chromatography to obtain TA-CA-NHCOCF 3 It was a white solid (9.06g) in 89% yield. 1 H NMR(400MHz,DMSO-d 6 ):δ7.48(d,J＝7.3Hz,2H),7.35(t,J＝7.0Hz,2H),7.30-7.24(m,1H),6.64(d,J＝15.6Hz,1H),6.20(dd,J＝15.6,9.0Hz,1H),4.85(d,J＝8.9Hz,1H),3.46-3.38(m,4H),2.76(m,4H). 13 C NMR(100MHz,DMSO-d 6 ):δ156.47,156.11,135.69,131.13,128.65,128.00,127.20,126.57,117.30,114.44,50.10,29.32.HR-MS(ESI):[M+Na] + calcd for C 17 H 17 F 2 N 2 O 2 S 2 483.0606,found 483.0608.
Step five:
Figure BDA0003621958320000112
mixing TA-CA-NHCOCF 3 (5.00g,10.86mmol) was dissolved in 50mL of methanolTo this was added 50mL of aqueous NaOH (2.17g,54.29 mmol). The mixture was stirred for 30 minutes to completely deprotect the trifluoroacetyl group. Methanol was removed under reduced pressure and the resulting solution was extracted 3 times with DCM (100 mL). Mixing organic phases, drying with anhydrous sodium sulfate, filtering, and evaporating to remove solvent to obtain TA-CA-NH 2 It was a pale yellow viscous liquid (2.49g) with a yield of 85%.
Step six:
Figure BDA0003621958320000121
CTA (0.30g,1.07mmol) was added to 30mL of dichloromethane containing 2- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU,0.71g,1.88mmol) and N, N-diisopropylethylamine (DIEA,0.31mL,1.88mmol) while cooling on ice, and 2-aminoethanol (65.60mg, 1.07mmol) was added. The reaction was continued at room temperature for 10 minutes. The solution was washed with saturated sodium hydrogencarbonate, dilute HCl and saturated brine, and dried over anhydrous sodium sulfate. The solvent was distilled off, and the resulting crude product was purified by means of a silica gel column chromatography to give CTA-OH as a purple viscous substance (0.31g) in a yield of 90%. 1 H NMR(400MHz,DMSO-d 6 ):δ7.91(d,J＝7.2Hz,2H),7.69(t,J＝7.4Hz,1H),7.51(t,J＝8.0Hz,2H),3.40(q,J＝6.0Hz,2H),3.12(q,J＝6.0Hz,2H),2.49-2.34(m,4H),1.91(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ229.02,175.28,149.37,138.79,134.20,131.61,123.93,64.99,51.58,46.83,43.47,38.59,35.77,28.35.HR-MS(ESI):[M+Na] + calcd for C 15 H 18 N 2 O 2 S 2 345.0702,found 345.0688.
Step seven:
Figure BDA0003621958320000122
CTA-OH (0.30g,0.93mmol) was dissolved in 20mL of anhydrous THF containing triethylamine (0.26mL,1.86mmol) in the presence of catalytic amounts of DMAP. A THF solution of 4-nitrophenyl chloroformate (0.37g, 1.86mmol) was added dropwise to the above reaction system while cooling on iceAnd the reaction was continued overnight. The resulting triethylamine hydrochloride solid was removed by filtration, and the organic solvent was evaporated. The crude product was purified by silica gel column chromatography to give CTA-NPC as a purple viscous substance (0.27g) in 60% yield. 1 H NMR(400MHz,CDCl 3 ):δ8.26(d,J＝9.2Hz,2H),7.89(d,J＝7.6Hz,2H),7.56(t,J＝7.4Hz,1H),7.40-7.37(m,4H),4.38(t,J＝5.2Hz,2H),3.68-3.65(m,2H),2.73-2.54(m,3H),2.48-2.38(m,1H),1.94(s,3H). 13 C NMR(100MHz,CDCl 3 )δ222.44,170.73,155.29,152.37,145.47,145.34,144.41,133.07,128.56,126.61,125.31,121.72,67.85,45.99,38.61,38.52,33.93,31.84,24.26.HR-MS(ESI):[M+Na] + calcd for C 22 H 21 N 3 O 6 S 2 510.0764,found 510.0765.
Step eight:
Figure BDA0003621958320000131
mPEG5k-OH (10.00g, 2.00mmol) was dissolved in 100mL of anhydrous DCM containing DIEA (3.96mL, 24.00mmol) in the presence of a catalytic amount of DMAP. A solution of 4-nitrophenyl chloroformate (4.03g, 20.00mmol) in DCM was added dropwise to the above reaction system while cooling on ice, and the reaction was continued at room temperature for 24 hours, after which the solvent was distilled off under reduced pressure. The concentrated mixture was added dropwise to diethyl ether (500mL) to precipitate a solid. The desired product is obtained by recycling the dissolution-precipitation procedure. After drying under vacuum, mPEG5k-NPC (9.53g) was obtained as a white solid in 92% yield. Of mPEG5k-NPC 1 The H NMR spectrum is shown in FIG. 2.
Step nine:
Figure BDA0003621958320000132
a solution of mPEG5k-NPC (5g,1mmol,20mL) in DCM was added dropwise to TA-CA-NH 2 (0.81g, 3mmol) in water. At this time, the solution became yellow in color and reacted at room temperature for 48 hours. The solvent was removed and the residue was purified by dialysis in water and freeze-dried. Finally, the process is carried out in a batch,obtaining a product mPEG5k-TA-CA-NH 2 (4.58g) as a white solid, 92% yield. mPEG5k-TA-CA-NH 2 Is/are as follows 1 The H NMR spectrum is shown in FIG. 3.
Step ten:
Figure BDA0003621958320000133
mPEG5k-TA-CA-NH 2 (0.5mmol) was dissolved in 25mL DCM. A solution of CTA-NPC in DCM (0.49g, 1mmol, 25mL) was added dropwise to the reaction system with stirring, and the reaction was continued for 48 hours. After completion of the reaction, the reaction mixture was concentrated and added dropwise to diethyl ether (300mL) to precipitate a pink solid. The product was obtained after two cycles of dissolution-precipitation procedure. After drying in vacuo, mPEG5k-TA-CA-CTA was obtained as a pink solid in 70% yield. Preparation of mPEG5k-TA-CA-CTA 1 The H NMR spectrum is shown in FIG. 4.
Step eleven:
Figure BDA0003621958320000141
mPEG5k-TA-CA-CTA (0.30g,0.055mmol), monomer MA-TA-CA-PTX (0.33g,0.27mmol) and 2- (diisopropylamino) ethyl methacrylate (0.35g,1.64mmol) were added to a round-bottom flask under an argon atmosphere. The round bottom flask was sealed and 4mL of DMSO/H2O (9/1, v/v) containing initiator VA044(7.05mg,0.022mmol) was poured into the reaction flask. After bubbling with argon for 30 minutes, the reaction mixture was stirred at 47 ℃ for 24 hours. Thereafter, quench the reaction with liquid nitrogen and add the mixture dropwise to MeOH/H 2 O (400mL, 1/1, v/v) to give a precipitate, which was collected, dried in vacuo, and redissolved in DCM. The solution was added dropwise to diethyl ether (300mL) and the precipitate was collected. After drying in vacuo, the final prodrug mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA) was a pale pink solid in 65% yield. Of mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA) 1 The H NMR spectrum is shown in FIG. 5, and the nuclear magnetic characteristic peaks of each structural unit can be correspondingly found in the spectrum, which indicates that the example indeed synthesizes the compound of the above formulaThe product is conjugate shown in structural formula.
Example 2 preparation of mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA) micelles
mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA) (10mg) was dissolved in 100. mu. L N, N-dimethylacetamide (DMAc), and the resulting solution was slowly added to 4mL of distilled water under sonication. Finally, the organic solvent is removed by dialysis overnight against deionized water to obtain the nanoscale mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA) micelle.
Comparative example 1 Synthesis of conjugate mPEG5k-TK-block-poly (TK-PTX-co-DPA)
The comparison example is synthesized by the following synthetic route to obtain mPEG5k-TK-block-poly (TK-PTX-co-DPA) (TK-Prodrug for short):
Figure BDA0003621958320000151
in the above reaction formula, x is 3 and y is 29.
In this comparative example, the synthesis of MA-TK-PTX was performed in the same manner and in the same amount (molar ratio) as those of MA-TA-CA-PTX in example 1, except that CA in the starting material was replaced with acetone. Of MA-TK-PTX 1 The H NMR spectrum is shown in FIG. 6.
TK-NH 2 Synthesis of (D) and TA-CA-NH in example 1 2 The synthesis method and the amounts (molar ratios) of the raw materials of (1) were the same, except that the CA in the raw materials was replaced with acetone. It should be noted that in this comparative example, the reason why the value of y in the structural formula is different from that in example 1 is the difference in the degree of polymerization.
mPEG5k-TK-NH 2 Was synthesized with mPEG5k-TA-CA-NH from example 1 2 The synthesis method and the amount (molar ratio) of the raw materials are the same, and the difference is that TA-CA-NH in the raw materials 2 Replacement by TK-NH 2 。
The synthesis of mPEG5k-TK-CTA was performed in the same manner as mPEG5k-TA-CA-CTA in example 1, except that mPEG5k-TA-CA-NH was used as a raw material 2 Replacement with mPEG5k-TK-NH 2 . Preparation of mPEG5k-TK-CTA 1 The H NMR spectrum is shown in FIG. 7Shown in the figure.
TK-Prodrug was synthesized according to the same method and amounts (molar ratio) of the raw materials as those used in TA-CA-Prodrug in example 1, except that mPEG5k-TA-CA-CTA in the raw materials was replaced by mPEG5k-TK-CTA, and MA-TA-CA-PTX was replaced by MA-TK-PTX. Of TK-Prodrug 1 The H NMR spectrum is shown in FIG. 8.
Comparative example 2 Synthesis of conjugate mPEG5k-block-poly (TK-PTX-co-DPA)
The comparison example is synthesized by the following synthetic route to obtain mPEG5k-block-poly (TK-PTX-co-DPA) (Prodrug for short):
Figure BDA0003621958320000161
in the above reaction formula, x is 3 and y is 26.
In this comparative example, the synthesis of MA-TK-PTX was performed in the same manner as in comparative example 1.
The synthesis of mPEG5k-CTA was identical to the synthesis method and the raw material amount (molar ratio) of mPEG5k-TA-CA-CTA in example 1, except that mPEG5k-TA-CA-NH was added to the raw material 2 Replacement with mPEG5k-NH 2 . It should be noted that in this comparative example, the reason why the value of y in the structural formula is different from that in example 1 is the difference in the degree of polymerization. Of mPEG5k-CTA 1 The H NMR spectrum is shown in FIG. 9.
Prodrug was synthesized according to the same method and using amount (molar ratio) of raw materials as TA-CA-Prodrug in example 1, except that mPEG5k-TA-CA-CTA in the raw materials was replaced by mPEG5k-CTA, and MA-TA-CA-PTX was replaced by MA-TK-PTX. Of Prodrug 1 The H NMR spectrum is shown in FIG. 10.
To further illustrate the technical effects of the present invention, physicochemical properties and activities of the conjugates prepared in the above examples and comparative examples were tested by experiments. In the discussion of the experimental results below, TA-CA-Prodrug, TK-Prodrug and Prodrug are also collectively referred to as "prodrugs" or "PTX prodrugs".
Experimental example 1 GPC experiment
To calculate the polymer molecular weight, Gel Permeation Chromatography (GPC) analysis was performed. 2mg of amphiphilic polymer was dissolved in 800. mu. L N, N-Dimethylformamide (DMF), followed by the addition of 200. mu.LLICl (0.2M aqueous solution). After filtration, the mixed solution was used for GPC analysis. A mixed solution of DMF/LiCl (4/1, volume ratio) was used as a mobile phase at a flow rate of 0.5 mL/min. The molecular weight of each polymer was determined by standard curve of prussian blue standards. The results are shown in FIG. 11.
From the results, compared with each macromolecular chain transfer agent (mPEG5k-TA-CA-CTA, mPEG5k-TK-CTA and mPEG5k-CTA), the molecular weight of the obtained polymer is obviously increased, and the monodispersity is better, which indicates that the polymerization reaction is successfully carried out.
Experimental example 2 micelle particle diameter and potential
This experimental example investigated the particle size and potential of the micelles prepared in example 2.
The experimental method comprises the following steps: dynamic Light Scattering (DLS) experiments were used to measure the aqueous phase particle size of the self-assembled nanoparticles, for which the PALS method was used to detect the zeta potential. The prepared amphiphilic polymer nanoparticles were diluted to 1mg/mL with deionized water. The pH of the deionized water was adjusted by 1mol/L dilute hydrochloric acid and 1mol/L sodium hydroxide, and measured with a pH meter. The solution was then transferred to a cuvette and measured by an all-round multi-angle particle size analyzer and a high sensitivity zeta potentiometer (brookfield instruments, usa). The measurement parameters are: the detection angle is 90 °, the equilibration time is 60s, the measurement time is 30s, and the measurement temperature is 25 ℃. Each sample was recorded in triplicate.
Potential results for micelles at different pH As shown in FIG. 12, the zeta potential increased from-10.32 mV at pH 7.4 to +23.23mV at pH 6.0 and +27.70mV at pH 5.2 due to the presence of the pH sensitive moiety DPA in the TA-CA-Prodrug.
The particle size of the micelles at different pH is shown in FIG. 13, and the particle size of TA-CA-Prodrug is 150.72nm (PDI 0.24) at pH 7.4 and 173.24nm (PDI 0.22) at pH 6.2.
The micelle of example 2 showed a spherical morphology under Transmission Electron Microscopy (TEM), as shown in fig. 14, illustrating that the TA-CA-produge self-assembly process forms a micelle structure.
Experimental example 3 in vitro drug Release test
The experimental method comprises the following steps: to investigate the relationship between the TA-CA-Prodrug release efficiency hydrogen peroxide concentrations, the following tests were performed: 1.0mg/mL TA-CA-Prodrug micelles were suspended in 2.0mL solutions containing different concentrations of H at 37 deg.C (n-3, n is the number of experimental repetitions) 2 O 2 (0, 0.01, 0.1 and 1mM) in phosphate buffered saline (PBS, pH 7.4). At given time intervals, 100. mu.L of the solution was taken out and diluted to 1mL with acetonitrile, and measured by reversed phase high performance liquid chromatography (RP-HPLC, SHIMADZU, Japan).
To evaluate the effect of polymer structure on drug release efficiency, TK-Prodrug and Prodrug were suspended in a suspension containing 0.1mM H 2 O 2 In PBS solution of (a). Then, the experiment was performed in a similar procedure as described above.
The results are shown in FIG. 15 in the absence of ROS (H) 2 O 2 ) In this case, the release of PTX and CA is negligible, indicating that release of drug from normal tissues at low ROS levels is more difficult with TA-CA-Prodrug, and thus this DDS can reduce the side effects of PTX. Under the stimulation of ROS, peaks of PTX and CA were observed in the HPLC chromatogram, demonstrating that TA-CA-Prodrug is able to release PTX and CA in response to ROS. At low ROS concentrations (0.01mM), about 20% of PTX was released from the TA-CA-Prodrug at a very slow rate. With the increase of ROS concentration, the released PTX amount and the release rate are both increased remarkably, indicating that the drug release of TA-CA-Prodrug is in positive correlation with the ROS concentration. Therefore, the ROS consumption in the cells is compensated in time, and the effective release of the medicine is facilitated.
Drug release profiles of TA-CA-Prodrug, TK-Prodrug and Prodrug were compared at the same ROS concentration. TA-CA-Prodrug and TK-Prodrug release more PTX than Prodrug.
Experimental example 4 in vitro ROS detection
The experimental method comprises the following steps: in this example, the mouse mammary gland tumor cell line (4T1) originated from Chinese academy of sciences (Shanghai, China). ROS produced in 4T1 cells treated with TA-CA-Prodrug, TK-Prodrug, Prodrug or CA, respectively, were detected with a fluorescent probe as dichlorofluorescein-diacetate (DCFH-DA). After treating 4T1 cells for various samples and applying them for various times, they were stained with DCF-DA (. mu.M) for 30min, and then the cell images were observed under a fluorescent microscope.
The results are shown in FIG. 16. The green fluorescence intensity in the cells treated by the TA-CA-Prodrug is obviously stronger than that of the cells treated by the TK-Prodrug. The ROS level in the cells treated with TA-CA-Prodrug was 1.21 times higher than that in the TK-Prodrug group. Of the three prodrugs, TA-CA-produgs induced the highest level of ROS, partly due to the release of TA-CA-produgs of two ROS-inducible compounds CA and PTX. Furthermore, the rapid and sufficient release of PTX from TA-CA-produgs due to the positive feedback of ROS may also be responsible for intracellular ROS differences. The intracellular ROS levels in the produg-treated group were lower than in the TK-produg-treated group, probably due to the slower release rate of PTX from produg, since there was no ROS-responsive linker in the copolymeric backbone of produg.
Experimental example 5 cell viability assay
The experimental method comprises the following steps: 4T1 cells were seeded in 96-well plates at a density of 5000 cells per well. After 24h incubation, the adherent cells were treated with PTX, TA-CA-Prodrug, TK-Prodrug or Prodrug (PTX concentration 0-90. mu.g/mL) dissolved in fresh medium for 48 h. RPMI 1640 containing 10% CCK-8(Dojindo) was added to each well. The 96-well plate was placed in a cell incubator for further incubation for 2.5h, and then absorbance was measured at 450 nm. IC50 values were calculated from GraphPad Prism 8.0.2. IC50 values in this experimental example were calculated as PTX mass.
The results are shown in FIG. 17. There was a correlation between cell viability after treatment with the three prodrugs and the concentration of PTX in the three prodrugs. The IC50 values calculated in Graphpad Prism for CA, TA-CA-Prodrug, TK-Prodrug and Prodrug were 3.93. mu.g/mL, 1.31. mu.g/mL, 4.32. mu.g/mL and 7.13. mu.g/mL, respectively, significantly higher than PTX (0.04. mu.g/mL). TA-CA-Prodrug, TK-Prodrug and Prodrug are less cytotoxic than PTX due to the slow process of internalization by cells and the time required for drug release.
EXAMPLE 6 prodrug subcellular localization
The experimental method comprises the following steps: 4T1 cells were plated on 8-well chamber slides (Cellvis) for culture at 5000 cells per well and after 48h incubation the cells were treated with an equal amount of Cy5 (0.5. mu.g/mL) of the TA-CA-prodrug, TK-prodrug or prodrug for 2 h. Nuclei, lysosomes and mitochondria were stained with Hoechst 33342, lysracer Green and Mitotracker Green, respectively, and images were taken under clsm (nikon). Pearson correlation coefficients (Pcc), Manders overlap coefficients (Moc), and Manders co-localization coefficients were analyzed using image J software.
The results of the transport of PTX prodrug in 4T1 cells are shown in fig. 18, where cells were treated with prodrug loaded with Cy5 (red) for 2h and then stained with Hoechst 33342 (blue), Lysotracker (green) and Mito-tracker (green) to fluorescently label the nucleus, lysosomes and mitochondria of the cells. The red fluorescent signal in CLSM images is generated by a prodrug that is internalized by the cell. Since lysosomes and mitochondria are labeled green, the co-localized region of the prodrug and lysosomes or mitochondria should appear yellow fluorescence. The intracellular green fluorescent signal (lysosomal tracer) and red fluorescent signal (prodrug) were interwoven, but a very weak yellow fluorescent signal can be seen in fig. 18A. This result suggests that only a small amount of the prodrug is translocated to lysosomes after internalization. However, a high overlap of the red signal (prodrug) and the green signal (mitochondria) was found in fig. 18B. The overlap region shows a strong yellow fluorescence signal indicating that the prodrug is mostly transported to mitochondria rather than lysosomes. Since these prodrugs have a positive surface potential in late endosomes under acidic conditions, these prodrugs may be able to escape from the endosomes and interact with the negatively charged mitochondrial membrane. Furthermore, localization of the Prodrug in the mitochondria favors the drug and CA release of TA-CA-produgs. Mitochondria are one of the major organelles that produce ROS, and the level of ROS around mitochondria tends to be high. After TA-CA-Prodrug reaches mitochondria, high ROS levels favor TA-CA-Prodrug release of CA and PTX. The released CA can interact with mitochondria to generate more ROS, further promoting ROS-responsive drug release from the prodrug.
Experimental example 7 mitochondrial membrane potential
The experimental method comprises the following steps: to detect changes in mitochondrial membrane potential, 4T1 cells treated with different prodrugs were stained with JC-10 fluorescent probe for mitochondria. The treated cells were washed with phenol red free RPMI 1640 medium. JC-10 was then dissolved in complete medium at 37 ℃ and stained in an incubator in the dark for 20 min. The original medium was replaced with fresh phenol red-free RPMI 1640 medium containing 2% fetal bovine serum and observed under CLSM. Flow cytometry detection: the treated cells were trypsinized, harvested by centrifugation, suspended in warmed JC-10 solution, placed in an incubator for 20min and then detected by flow cytometry (FACS Asia II, Becton Dickinson, USA).
The results are shown in FIG. 19. Since these pro-drugs accumulate in the mitochondria, and the pro-drugs release CA and PTX, ROS can be produced intracellularly. Therefore, it is presumed that the prodrug may cause damage to mitochondria. JC-10 can assess mitochondrial function of 4T1 cancer cells by changes in mitochondrial membrane potential (. DELTA.. psi.m). After 4h of drug and cell treatment, the cells were stained with JC-10 dye for 20 min. "J-monomers" that interact with damaged mitochondrial membranes appear green fluorescent, while "J-aggregates" that interact with normal mitochondrial membranes appear red fluorescent. As shown in fig. 19A, the red fluorescence intensity was significantly reduced in the three prodrug-treated cells compared to the control. A decrease in red fluorescence or an increase in green fluorescence indicates that the mitochondrial membrane is compromised by a high level of depolarization. The ROS can be rapidly generated by CA and PTX released by the TA-CA-Prodrug, which indicates that the TA-CA-Prodrug has the strongest effect of destroying the functions of mitochondria.
Example 8 in vivo imaging Studies
The experimental method comprises the following steps: female BALB/c mice were from the institute for Hedgehog Biology (Chinese Chengdu). Animal study procedures were performed according to the guidelines for laboratory animal care and use in the western hospital, and approved by the animal ethics committee of the university of sichuan. 4T1 tumor-bearing mice were dosed via tail vein with Cy5@ TA-CA-Prodrug, Cy5@ TK-Prodrug or Cy5@ Prodrug (Cy5 dose 1 mg/kg). The biodistribution of these prodrugs was examined in BALB/ c mice 4, 12, 24 or 24h after injection. Surgically excised organs/tissues (tumor, heart, spleen, liver, lung and kidney) were thoroughly washed in saline and placed in an in vivo imaging system (PerkinElmer) for bright field and fluorescence imaging (0.5s exposure time; excitation filter 640nm, emission filter 680 nm).
The results are shown in FIG. 20. Following intravenous injection of the prodrug in 4T1 tumor-bearing mice, Cy5 signals for 3 prodrugs appeared predominantly in the liver and spleen (fig. 20A and 20B). At 4h, TK-Prodrug accumulates in the liver and spleen more than TA-CA-Prodrug and Prodrug, which may be associated with a larger TK-Prodrug particle size. Thus, TK-Prodrug may be taken up by the reticuloendothelial phagocytosis system of the liver and spleen. The TA-CA-Prodrug is concentrated in the kidney due to small particle size and can be excreted through the kidney. Due to the small particle size, it is less taken up by the reticuloendothelial system. Thus, more TA-CA-produgs can circulate in the blood for a longer period of time, thereby increasing their enrichment at the tumor site. The Cy5 fluorescence signal of TA-CA-Prodrug at the tumor site was gradually increased with time to 48h, indicating that TA-CA-Prodrug was well aggregated and retained at the tumor site. However, the Cy5 fluorescence signals of Prodrug and TK-Prodrug in tumors peaked at 24h and then gradually declined (FIG. 20B).
Experimental example 9 in vivo antitumor Effect
The experimental method comprises the following steps: female BALB/c mice with a body weight of 20 + -2 g were used for in vivo antitumor experiments. Each mouse was injected subcutaneously with 1X 10 6 4T1 cells, used to construct a 4T1 mammary tumor transplantation mouse model. When the tumor size reaches 50mm 3 On the other hand, an antitumor experiment was started. The mice were randomly divided into 5 groups of 6 mice each. The medicine is administered once every other day for 5 times. Intravenous saline, PTX, TA-CA-prodrug, TK-prodrug or prodrug (same PTX dose 10mg kg) -1 ). Tumor volume was assessed using a vernier caliper at a pre-set time point. Tumor volume (mm) 3 ) The calculation formula is that the tumor volume is L multiplied by W 2 L and W are the length and width of the tumor, respectively. In addition, changes in body weight were recorded every other day.
The results are shown in FIGS. 21 and 22. By monitoring the tumor volume, TA-CA-Prodrug showed the strongest inhibition of tumor growth, while TK-Prodrug and Prodrug showed moderate inhibition of tumor growth. Tumor growth inhibition rate (TGI) was calculated from the mass of the tumor. TGI was 21.9% in the free PTX treated group and 30.5%, 34.3% and 58.9% in produgg, TK-produgs and TA-CA-produgs treated groups, respectively (figure 22). These results indicate that TA-CA-Prodrug has a good anti-tumor effect with the lowest IC50 and a higher biodistribution in tumors. Although the accumulation of TK-Prodrug in the tumor was lower than Prodrug, the TGI was slightly higher in the TK-Prodrug treated group than in the Prodrug treated group (FIG. 22). This is due to the presence of reactive TK groups in the TK-produgs copolymer backbone, which can be destroyed by ROS.
In another aspect, the body weight changes of 4T1 tumor-bearing mice after different drug treatments are shown in FIG. 23. The body weight of mice treated with the different prodrugs did not change significantly, which means that the prodrugs showed lower systemic toxicity.
Example 10 hematoxylin-eosin staining, immunohistochemistry and TUNEL staining
The experimental method comprises the following steps: after 16 days of treatment of mice with each PTX prodrug, mice were sacrificed, tumors and internal organs were isolated, fixed in 10% PBS buffer neutral formaldehyde for 24h, and paraffin sections were prepared. Then, the sections were stained with hematoxylin and eosin (H & E). Histological changes of the sections were observed under an optical microscope. Tumor sections were stained with anti-CD 31 and anti-ki 67 antibodies and immunohistochemical images were collected under light microscopy. Tumor tissue sections were stained using the Dead End fluorescent TUNEL system (Promega Corp, USA). The dnase treated sections served as positive controls. The sections were photographed using an optical microscope (Imager Z2, Zeiss, Germany).
The results of the experiment are shown in FIG. 24. After H & E staining, large-area necrosis of tissues, reduction of intercellular substance, loose structure and nucleus fragmentation can be seen in the sections in the former Chinese medicine group. The TA-CA-Prodrug treated group showed the highest degree of histopathological damage. TUNEL staining detects apoptosis in situ of tumors, CD31 and Ki67 immunohistochemistry detects anti-angiogenic and proliferative capacity of tumors. The numbers of Ki67 and CD31 positive cells were the lowest in tumors treated with TA-CA-produgs, indicating that TA-CA-produgs significantly inhibited tumor tissue proliferation and angiogenesis. In addition, the number of apoptotic cells in TA-CA-produgs treated tumors was significantly increased after TUNEL staining. These results indicate that the inhibition of tumor proliferation and metastasis by TA-CA-Prodrug includes effective induction of tumor tissue apoptosis, inhibition of tumor tissue cell proliferation and angiogenesis.
As can be seen from the above examples and experimental examples, the micelle formed by the conjugate of the present invention can be endocytosed by 4T1 cells, and is time-dependent. Since the DPA moiety of the conjugate molecule can be protonated in the acidic microenvironment, it can escape from late endosomes before reaching the mitochondria. The release of CA and PTX in the conjugate molecule is triggered upon exposure to high levels of mitochondrial generated ROS. The released CA can interact with mitochondria to produce more ROS, resulting in a decrease in mitochondrial membrane potential and facilitating the release of more PTX from the prodrug. The released PTX causes cell cycle arrest and inhibits its proliferation. The cascade ROS feedback effect in this novel prodrug enhances the cell cycle arrest effect, eventually leading to tumor cell apoptosis. Compared with two control groups (micelles formed by TK-Prodrug and Prodrug), the micelle formed by the conjugate TA-CA-Prodrug prepared by the embodiment of the invention has an optimized copolymer structure and ROS amplified chemotherapy effect, and has prominent expression in eliminating tumor of tumor-bearing mice.
Therefore, the micelle formed by the conjugate provided by the invention can solve the problem of insufficient drug release in ROS-responsive DDS, and has very high application potential in the development and application of antitumor drugs.

Claims (10)

1. An active oxygen self-complementary amphiphilic block copolymer-drug conjugate, which is characterized in that the structural formula is shown as formula I:
Figure FDA0003621958310000011
wherein the value of n is selected from 43-227,
r is a molecular chain formed by polymerizing x repeating units A and y repeating units B, the value of x is selected from 2 to 10, the value of y is selected from 20 to 40,
the repeating unit A is:
Figure FDA0003621958310000012
wherein R is D Is R D OH as a substituent after removal of the hydroxyl group, the R D OH is an anti-tumor drug molecule;
the repeating unit B is:
Figure FDA0003621958310000013
wherein R is B Selected from hydrogen or methyl, X is selected from O,
Figure FDA0003621958310000014
2. The conjugate of claim 1, wherein: the R is D OH is paclitaxel, docetaxel, adriamycin, epirubicin, capecitabine, cabazitaxel, 2-methoxyestradiol, camptothecin, hydroxycamptothecin, 9-aminocamptothecin, topotecan or irinotecan.
3. The conjugate according to claim 1, wherein: the repeating unit B is selected from
Figure FDA0003621958310000021
4. The conjugate of claim 1, wherein: the R is D OH is taxol and the repeating unit B is selected from
Figure FDA0003621958310000022
n＝113，x＝3，y＝25。
5. A method for preparing a conjugate according to any one of claims 1 to 4, comprising the steps of:
(1) the following compound a was prepared:
Figure FDA0003621958310000023
(2) the following compound B was prepared:
Figure FDA0003621958310000024
(3) compound a, compound B and 2- (diisopropylamino) ethyl methacrylate the compound of formula i is prepared by the following reaction:
Figure FDA0003621958310000031
wherein n, R and R D The method according to any one of claims 1 to 4.
6. The method according to claim 5, wherein the step (1) comprises the steps of:
(1.1) preparation of Compound TA-CA-NH according to the following reaction scheme 2 ：
Figure FDA0003621958310000032
(1.2) preparation of Compound R1-TA-CA-NH according to the following reaction scheme 2 ：
Figure FDA0003621958310000033
(1.3) preparation of Compound CTA-NPC according to the following reaction scheme:
Figure FDA0003621958310000034
(1.4) preparation of Compound A according to the following reaction scheme:
Figure FDA0003621958310000035
and/or, the step (2) specifically comprises the following steps:
(2.1) preparation of compound TA-CA according to the following reaction scheme:
Figure FDA0003621958310000041
(2.2) preparation of compound MA-TA-CA according to the following reaction scheme:
Figure FDA0003621958310000042
(2.3) preparation of Compound B according to the following reaction scheme:
Figure FDA0003621958310000043
7. a micelle, characterized by: formed from a conjugate according to any one of claims 1 to 4.
8. A plurality of micelles of claim 7, wherein: the particle size of the micelle is 100-200 nm;
and/or the zeta potential of the micelle is-15 to-5 mV in an environment with a pH of 7.4, the zeta potential of the micelle is +10 to +25mV in an environment with a pH of 6.0, and the zeta potential of the micelle is +25 to +30mV in an environment with a pH of 5.2.
9. Use of the conjugate according to any one of claims 1 to 4, or the micelle according to claim 7 or 8 for the preparation of an anti-tumor medicament.
10. A medicament, characterized by: the conjugate as claimed in any one of claims 1 to 4, or the micelle as claimed in claim 8 or 9, as an active ingredient, together with pharmaceutically acceptable excipients.
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