CN114904012B - Active oxygen self-supplementing amphiphilic block copolymer-drug conjugate, preparation method and application thereof - Google Patents

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

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CN114904012B
CN114904012B CN202210461713.2A CN202210461713A CN114904012B CN 114904012 B CN114904012 B CN 114904012B CN 202210461713 A CN202210461713 A CN 202210461713A CN 114904012 B CN114904012 B CN 114904012B
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CN114904012A (en
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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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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
    • C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/11—Aldehydes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2438/00—Living radical polymerisation
    • C08F2438/03—Use of a di- or tri-thiocarbonylthio compound, e.g. di- or tri-thioester, di- or tri-thiocarbamate, or a xanthate as chain transfer agent, e.g . Reversible Addition Fragmentation chain Transfer [RAFT] or Macromolecular Design via Interchange of Xanthates [MADIX]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an active oxygen self-supplementing amphiphilic block copolymer-drug conjugate, a preparation method and application thereof. The structural formula of the active oxygen self-supplementing amphiphilic block copolymer-drug conjugate 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:wherein R is D Is R D OH removes the substituent after the hydroxyl group, R D OH is an antitumor drug molecule; the repeating unit B is:the active oxygen self-supplementing amphiphilic block copolymer-drug conjugate provided by the invention can solve the problem of insufficient drug release in ROS responsive DDS, and has very high application potential in development and application of antitumor drugs.

Description

Active oxygen self-supplementing 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 active oxygen self-supplementing amphiphilic block copolymer-drug conjugate, a preparation method and application thereof.
Background
Drug delivery systems (drug delivery system, DDS) refer to the form of administration of various drugs that people use in the course of controlling diseases. The design concept is to deliver the medicine to the necessary parts in the necessary time and the necessary quantity so as to achieve the maximum curative effect and the minimum toxic and side effect.
Currently, reactive Oxygen Species (ROS) -responsive DDS have been widely reported. As endogenous stimuli, ROS include superoxide (O 2 - ) Hypochlorite ion (OCl) - ) Peroxynitrite (ONOO) - ) Hydroxyl radical (. OH) and hydrogen peroxide (H) 2 O 2 ) And high levels of 0.1mM can be achieved in tumor cells, which are thousands of times higher than normal cells (-0.02. Mu.M). Such high levels of ROS in tumor cells have been used as a stimulus for application in drug delivery systems. However, in many cases, these ROS-responsive DDSs do not achieve adequate release of the drug. This low release efficiency may be due in part to the heterogeneity of ROS levels in different tumor tissues, or the lack of more intelligent, synergistic DDS.
Cinnamaldehyde (CA) is the major component of cinnamon and its safety has been approved by the united states Food and Drug Administration (FDA). CA is reported to be capable of producing ROS in mitochondria and inducing apoptosis of cancer cells by amplifying oxidative stress. However, the aldehyde groups in CA undergo rapid oxidation and have low antitumor efficacy, which hinders the application of CA in antitumor drugs. Chinese patent No. CN113072704A discloses a polythioacetal based on active oxygen self-amplification degradation and a preparation method and application thereof. The polysulfide acetal provided by the invention is derived from cinnamaldehyde, and has the following structural formula:
The structure of the polythioacetal can effectively protect aldehyde groups in CA from oxidation. Meanwhile, the thioacetal bond in the polythioacetal can be responded and broken in the presence of ROS, and the CA released after the thioacetal bond is responded and broken can generate ROS in cells, and the ROS generated by the CA further break the thioacetal chain in the polythioacetal, so that the effect of self-amplifying degradation is generated. The polythioacetal is used as a tumor drug carrier, is easy to be efficiently degraded by the induction of ROS in tumors, is favorable for rapidly releasing the drug into tumor cells, and opens up a new way for the effective release of the drug.
However, the action of CA to produce ROS needs to be performed in mitochondria, and the above polythioacetals do not have the effect of targeted enrichment into mitochondria of tumor cells, which limits the exertion of their self-amplifying degradation drug release action. 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 new drug delivery systems that load CA and antineoplastic agents and are capable of achieving targeted enrichment to the mitochondria.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an active oxygen self-supplementing amphiphilic block copolymer-drug conjugate, a preparation method and application thereof, and aims to provide an amphiphilic block copolymer-drug conjugate which can effectively target and enrich mitochondria of tumor cells, generate cascade amplification degradation and effectively release antitumor drugs in the tumor cells.
An active oxygen self-supplementing amphiphilic block copolymer-drug conjugate is characterized in that the structural formula of the active oxygen self-supplementing amphiphilic block copolymer-drug conjugate is shown as formula I:
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 2-10, preferably 5-10, the value of y is selected from 20-40,
the repeating unit A is:wherein R is D Is R D OH removes the substituent after the hydroxyl group, R D OH is an antitumor drug molecule;
the repeating unit B is:wherein R is B Selected from hydrogen or methyl, X is selected from O,
Preferably, said R D OH is taxol, docetaxel, doxorubicin, epirubicin, capecitabine, cabazitaxel, 2-methoxyestradiol, camptothecin, hydroxycamptothecin, 9-aminocamptothecin, topotecan or irinotecan.
Preferably, the repeating unit B is selected from
Preferably, said R D OH is taxol and the repeating unit B is selected from
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:
(2) The following compound B was prepared:
(3) Compound a, compound B and 2- (diisopropylamino) ethyl methacrylate prepare a compound of formula i by the following reaction:
Wherein n, R and R D The method of claim 1 or 2.
Preferably, the step (1) specifically includes the following steps:
(1.1) preparation of Compound TA-CA-NH according to the following reaction scheme 2 ：
(1.2) preparation of Compound R1-TA-CA-NH according to the following reaction scheme 2 ：
(1.3) the compound CTA-NPC was prepared according to the following reaction scheme:
(1.4) Compound A is prepared according to the following reaction scheme:
preferably, the step (2) specifically includes the following steps:
(2.1) the compound TA-CA was prepared according to the following reaction scheme:
(2.2) preparation of Compound MA-TA-CA according to the following reaction scheme:
(2.3) Compound B is prepared according to the following reaction scheme:
preferably, in the step (3), the feeding ratio of the compound A to the compound B to the 2- (diisopropylamino) ethyl methacrylate is 1 (20-40): 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 solution, 1, 4-dioxane, tetrahydrofuran or trifluoroethanol with the O volume ratio of 9:1;
and/or, the reaction conditions are: reacting for 16-48 hours at 45-85 ℃ in inert atmosphere.
The invention also provides a micelle which is formed by the conjugate.
Preferably, the particle size of the micelle is 100-200nm;
and/or the zeta potential of the micelle is-15 to-5 mV in the environment with the pH=7.4, the zeta potential of the micelle is +10 to +25mV in the environment with the pH=6.0, and the zeta potential of the micelle is +25 to +30mV in the environment with the pH=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-supplementing active oxygen based on positive feedback strategy. The conjugate has a pH-sensitive N, N-Diisopropylamine (DPA) moiety therein, which can combine with protons to form positively charged ammonium in the acidic environment of tumor cells. The DPA moiety in the copolymer facilitates transfer of the conjugate to the mitochondria, as positively charged nanoparticles can interact strongly with negatively charged mitochondrial membranes. Meanwhile, the conjugate has the characteristic of ROS response, can release anti-tumor drugs and CA in the environment of tumor cells, and the CA can further generate ROS in mitochondria, so that the conjugate is subjected to self-degradation, and the release of the anti-tumor drugs and the CA is further promoted. Based on the principle, the cooperation of the functional parts in the conjugate can obviously enhance the capability of releasing the antitumor drug, thereby enhancing the antitumor effect of the conjugate.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of MA-TA-CA-PTX in example 1;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of mPEG5k-NPC in example 1;
FIG. 3 is a schematic diagram of example 1mPEG5k-TA-CA-NH 2 Hydrogen nuclear magnetic resonance spectrum of (2);
FIG. 4 is a nuclear magnetic resonance hydrogen spectrum of mPEG5k-TA-CA-CTA in example 1;
FIG. 5 is a nuclear magnetic resonance hydrogen 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 nuclear magnetic resonance hydrogen 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 is a molecular weight test result of a polymer;
FIG. 12 shows Zeta potentials of micelles at different pH values;
FIG. 13 is a graph of 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 experiments;
FIG. 16 is a fluorescence image of 4T1 cells treated with PBS, CA, TA-CA-prodrug, TK-prodrug or prodrug for 2h, where the CA concentration is 12 μg/mL and the PTX concentration in each prodrug group is 48 μg/mL. Cellular ROS levels were detected by fluorescent probe DCFH-DA (green: DCFH). The scale bar is 50 mu m;
FIG. 17 is an IC50 of each prodrug to 4T1 cells;
FIG. 18 shows the intracellular localization of prodrug-treated 4T1 cells (Cy 5 concentration 0.5. Mu.g/mL) 2h later; wherein, lysacker Green and Mitotracker Green respectively mark mitochondria and lysosomes in 4T1 cells, and the proportion is 25 μm;
FIG. 19 is a CLSM image of mitochondrial membrane potential changes after 4T1 cells are exposed to PTX prodrug or CA 4h, scale bar 25 μm;
fig. 20 is an in vitro fluorescence imaging using Cy5 via the IVIS Spectrum system, monitoring drug distribution in major organs/tumors and survival time following dosing of tumor bearing mice, data expressed as mean±sd, n=3;
Fig. 21 is a graph of tumor proliferation following dosing of tumor-bearing mice (n=6), with tumor length and width recorded every other day;
fig. 22 is a graph of tumor growth inhibition calculated by isolated tumor mass (n=6);
FIG. 23 shows the change in body weight of tumor-bearing mice after treatment with different drugs;
fig. 24 h & e, immunohistochemistry and immunofluorescence staining for detection of tumor tissue necrosis, proliferation (Ki 67), angiogenesis (CD 31) 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, the reagents and materials used were commercially available unless otherwise specified.
EXAMPLE 1 Synthesis of conjugate mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA)
This example is synthesized by the following synthetic route to obtain a preferred structure of the conjugate of the invention mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA) (abbreviated as TA-CA-Prodrug):
in the above reaction formula, x=3 and y=25.
The method comprises the following specific steps:
step one:
cinnamaldehyde (CA, 10.00g,75.67 mmol) and beta-mercaptoethanol (11.82 g,151.33 mmol) were dissolved in 70mL THF, then ZrCl was added at 0deg.C 4 (3.53 g,15.13 mmol). The reaction mixture is reactedThe 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 (18.19 g) 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:
TA-CA-OH (1.00 g,3.70 mmol) was dissolved in 30mL dry THF containing triethylamine (TEA, 0.77mL,5.55 mmol) and a solution of methacryloyl chloride (0.39 g,3.70 mmol) in THF was added slowly to the reaction system under ice-bath. 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 resulting residue was purified by silica gel chromatography to give MA-TA-CA-OH (0.98 g) as a colorless oil in 78% yield. 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:
MA-TA-CA-OH (0.20 g,0.59 mmol) was dissolved in 20mL dry THF and containing TEA (0.16 mL,1.18 mmol) and a catalytic amount of DMAP was added. A solution of 4-nitrophenoxycarbonyl chloride (0.18 g,0.89 mmol) in THF was slowly added to the above mixture under ice-bath. 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 (530.80 mg,0.59 mmol) and DMAP (216.24 mg,1.77 mmol) 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 distilled off, and the obtained residue was purified by silica gel chromatography to give MA-TA-CA-PTX as a white solid (0.59 g) in 82% yield.
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:
CA (2.91 g,22.00 mmol) and 2, 2-trifluoro-N- (2-mercaptoethyl) acetamide (8.00 g,46.20 mmol) were dissolved in 100mL THF, aluminum chloride (1.47 g,11.00 mmol) was added under ice-bath and stirring continued under ice-bath for 10 min. Evaporating the solvent, purifying the crude product by silica gel chromatography to obtain TA-CA-NHCOCF 3 As a white solid (9.06 g), 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:
TA-CA-NHCOCF 3 (5.00 g,10.86 mmol) was dissolved in 50mL of methanol and 50mL of aqueous NaOH (2.17 g,54.29 mmol) was added. 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). Combining the organic phases and drying with anhydrous sodium sulfate, filtering, evaporating the solvent to obtain TA-CA-NH 2 Is a pale yellow viscous liquid (2.49 g) with a yield of 85%.
Step six:
CTA (0.30 g,1.07 mmol) was added to 30mL of dichloromethane containing 2- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 0.71g,1.88 mmol) and N, N-diisopropylethylamine (DIEA, 0.31mL,1.88 mmol) under ice-bath followed by 2-aminoethanol (65.60 mg,1.07 mmol). The reaction was continued at room temperature for 10 minutes. Saturated sodium bicarbonate and dilute H for solution Cl and saturated brine, and dried over anhydrous sodium sulfate. The solvent was distilled off, and the crude product was purified by means of a silica gel column chromatography to give CTA-OH as a violet viscous material (0.31 g) 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:
CTA-OH (0.30 g,0.93 mmol) was dissolved in 20mL dry THF containing triethylamine (0.26 mL,1.86 mmol) in the presence of a catalytic amount of DMAP. A solution of 4-nitrophenyl chloroformate (0.37 g,1.86 mmol) in THF was added dropwise to the above reaction system under ice-bath, and the reaction was continued overnight. The triethylamine hydrochloride solid formed was removed by filtration, and the organic solvent was distilled off. The crude product was purified by silica gel column chromatography to give CTA-NPC as a violet dope (0.27 g) 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:
in the presence of a catalytic amount of DMAP, mPEG5k-OH (10.00 g,2.00 mmol) was dissolved in 100mL of anhydrous DCM containing DIEA (3.96 mL,24.00 mmol). A solution of 4-nitrophenyl chloroformate (4.03 g,20.00 mmol) in DCM was added dropwise to the above reaction system under ice-bath, 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 (500 mL) to precipitate a solid. The dissolution-precipitation procedure was performed by cycling to give the desired product. After drying under vacuum, mPEG5k-NPC (9.53 g) was obtained as a white solid in 92% yield. mPEG5k-NPC 1 The H NMR spectrum is shown in FIG. 2.
Step nine:
a solution of mPEG5k-NPC (5 g,1mmol,20 mL) in DCM was added dropwise to TA-CA-NH 2 (0.81 g,3 mmol) in solution. At this time, the color of the solution turned yellow, and reacted at room temperature for 48 hours. The solvent was removed, the residue was dissolved in water for dialysis purification, and lyophilized. Finally, obtaining the product mPEG5k-TA-CA-NH 2 (4.58 g) as a white solid in 92% yield. mPEG5k-TA-CA-NH 2 A kind of electronic device 1 The H NMR spectrum is shown in FIG. 3.
Step ten:
mPEG5k-TA-CA-NH 2 (0.5 mmol) was dissolved in 25mL DCM. A solution of CTA-NPC in DCM (0.49 g,1mmol,25 mL) was added dropwise to the reaction with stirring, and the reaction was continued for 48 hours. After the completion of the reaction, the reaction solution was concentrated and added dropwise to diethyl ether (300 mL), whereby a pink solid was precipitated. After two cycles of dissolution-precipitation procedure, the product was obtained. After drying in vacuo, the mPEG5k-TA-CA-CTA was obtained as a pink solid in 70% yield. mPEG5k-TA-CA-CTA 1 The H NMR spectrum is shown in FIG. 4.
Step eleven:
mPEG5k-TA-CA-CTA (0.30 g,0.055 mmol), monomer MA-TA-CA-PTX (0.33 g,0.27 mmol) and 2- (diisopropylamino) ethyl methacrylate (0.35 g,1.64 mmol) were charged to a round bottom flask under an argon atmosphere. The round bottom flask was sealed and 4mL DMSO/H2O (9/1, v/v) containing initiator VA044 (7.05 mg,0.022 mmol) was injected into the reaction flask. After bubbling with argon for 30 minutes, the reaction mixture was stirred at 47 ℃ for 24 hours. Thereafter, the reaction was quenched with liquid nitrogen and the mixture was added dropwise to MeOH/H 2 O (400 mL,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 (300 mL) and the precipitate was collected. After vacuum drying, the final prodrug, mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA), was a pale pink solid in 65% yield. 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 shows that the conjugate shown in the structural formula of the product of the reaction formula is truly synthesized in the embodiment.
Example 2 preparation of mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA) micelle
mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA) (10 mg) 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, removing the organic solvent through dialysis overnight on deionized water to obtain the nano-scale 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)
This comparative example was synthesized by the following synthetic route to give mPEG5k-TK-block-poly (TK-PTX-co-DPA) (TK-Prodrug for short):
In the above reaction formula, x=3 and y=29.
This comparisonIn the examples, the synthesis of MA-TK-PTX was the same as that of MA-TA-CA-PTX in example 1, except that CA in the raw material was replaced with acetone. MA-TK-PTX 1 The H NMR spectrum is shown in FIG. 6.
TK-NH 2 Synthesis of (A) and TA-CA-NH in example 1 2 The synthesis method and the raw material amount (molar ratio) are the same, except that CA in the raw material is replaced by acetone. In this comparative example, the y value of the structural formula was different from that of example 1 because of the difference in polymerization degree.
mPEG5k-TK-NH 2 Synthesis of mPEG5k-TA-CA-NH from example 1 2 The synthesis method and the raw material dosage (molar ratio) are the same, and the difference is that TA-CA-NH in the raw material 2 Replaced by TK-NH 2 。
The synthesis of mPEG5k-TK-CTA was identical to that of example 1 in the method of mPEG5k-TA-CA-CTA synthesis and the amount of raw materials (molar ratio) except that mPEG5k-TA-CA-NH was used in the raw materials 2 Replaced by mPEG5k-TK-NH 2 . mPEG5k-TK-CTA 1 The H NMR spectrum is shown in FIG. 7.
The TK-Prodrug synthesis was the same as the TA-CA-Prodrug synthesis method and raw material usage (molar ratio) in example 1, except that mPEG5k-TA-CA-CTA in the raw material was replaced with mPEG5k-TK-CTA and MA-TA-CA-PTX was replaced with MA-TK-PTX. 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)
This comparative example was synthesized by the following synthetic route to give mPEG5k-block-poly (TK-PTX-co-DPA) (abbreviated as Prodrug):
in the above reaction formula, x=3 and y=26.
In this comparative example, the MA-TK-PTX was synthesized in the same manner as in comparative example 1.
The synthesis of mPEG5k-CTA was identical to that of example 1, except that the original one was usedmPEG5k-TA-CA-NH in the material 2 Replaced by mPEG5k-NH 2 . In this comparative example, the y value of the structural formula was different from that of example 1 because of the difference in polymerization degree. mPEG5k-CTA 1 The H NMR spectrum is shown in FIG. 9.
The synthesis of Prodrug was identical to the synthesis of TA-CA-Prodrug in example 1, and the amounts of raw materials (molar ratios) were the same, except that mPEG5k-TA-CA-CTA in the raw materials was replaced with mPEG5k-CTA, and MA-TA-CA-PTX was replaced with MA-TK-PTX. Prodrug 1 The H NMR spectrum is shown in FIG. 10.
To further illustrate the technical effects of the present invention, the physicochemical properties and activities of the conjugates prepared in the above examples and comparative examples are tested by experiments. In the discussion of 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
In order to calculate the polymer molecular weight, gel Permeation Chromatography (GPC) analysis was performed. 2mg of the amphiphilic polymer was dissolved in 800. Mu. L N, N-Dimethylformamide (DMF), and 200. Mu.LLiCl (0.2M in water) was added. After filtration, the mixed solution was used for GPC analysis. A mixed solution of DMF/LiCl (4/1, volume ratio) was used as the mobile phase at a flow rate of 0.5mL/min. The molecular weight of each polymer was determined by the standard curve of the Prussian blue standard. The results are shown in FIG. 11.
From the results, the molecular weight of the obtained polymer was significantly increased and the monodispersity was better compared to the macromolecular chain transfer agents (mPEG 5k-TA-CA-CTA, mPEG5k-TK-CTA, mPEG5 k-CTA), indicating that the polymerization reaction was successful.
Experimental example 2 micelle size and potential
The particle size and the potential of the micelle prepared in example 2 were studied in this experimental example.
The experimental method comprises the following steps: dynamic Light Scattering (DLS) experiments were used to measure the aqueous phase particle size of self-assembled nanoparticles and PALS method was used to detect their zeta potential. The prepared amphiphilic polymer nanoparticles were diluted to 1mg/mL with deionized water. The pH of 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 (bruc halv instruments, usa). The measurement parameters are as follows: detection angle=90°, equilibration time=60 s, measurement time=30 s, measurement temperature=25℃. Each sample was recorded three times.
The potential results for micelles at different pH are shown in FIG. 12, with the presence of the pH sensitive moiety DPA in the TA-CA-Prodrug, 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.
The particle size of the micelles at different pH is shown in FIG. 13, with the particle size of TA-CA-Prodrug being 150.72nm (PDI 0.24) at pH 7.4 and 173.24nm (PDI 0.22) at pH 6.2.
The micelles of example 2 showed a spherical morphology under Transmission Electron Microscopy (TEM), as shown in fig. 14, demonstrating that the TA-CA-drug self-assembly process formed a micelle structure.
Experimental example 3 in vitro drug Release experiment
The experimental method comprises the following steps: to study the relationship between the hydrogen peroxide concentration of the TA-CA-Prodrug release efficiency, the following test was performed: at 37 ℃ (n=3, n is the number of experimental replicates), 1.0mg/mL TA-CA-Prodrug micelles were suspended in 2.0mL containing different concentrations of H 2 O 2 (0, 0.01, 0.1 and 1 mM) in phosphate buffer (PBS, pH 7.4). In a given time interval, 100. Mu.L of the solution was taken out and diluted to 1mL with acetonitrile, and the measurement was performed by reverse phase high performance liquid chromatography (RP-HPLC, SHIMADZU, japan).
To assess the effect of the polymer structure on drug release efficiency, TK-Prodrug and Prodrug were suspended in a solution containing 0.1mM H 2 O 2 Is added to the PBS solution. Then, experiments were 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 the case of PTX and CA, the release amounts are negligible, which indicates that the drug release in normal tissues at low ROS levels is difficult for TA-CA-Prodrug, so that the DDS can reduce the PTX side effects. Under stimulation of ROS, peaks of PTX and CA were observed in HPLC chromatogramsThis demonstrates that TA-CA-Prodrug is able to release PTX and CA in response to ROS. At low ROS concentrations (0.01 mM), about 20% of PTX is released from TA-CA-Prodrug, which is very slow. As the concentration of ROS increases, both the amount of PTX released and the rate of release increase significantly, indicating that the drug release of TA-CA-Prodrug is positively correlated with ROS concentration. Therefore, the ROS consumption in cells is compensated in time, and the effective release of the medicine is facilitated.
Drug release curves for 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 experimental example, the mouse mammary tumor cell line (4T 1) was derived from China academy of sciences (Shanghai, china). ROS produced in 4T1 cells treated with TA-CA-pro, TK-Prodrug, prodrug or CA, respectively, were detected with fluorescent probes as dichlorofluorescein-diacetate (DCFH-DA). After 4T1 cells were treated and acted on for various times on different samples, they were stained with DCF-DA (μM) for 30min and then the cell images were observed under a fluorescence microscope.
The results are shown in FIG. 16. The green fluorescence intensity in cells treated by TA-CA-Prodrug is obviously stronger than that of cells treated by TK-Prodrug. ROS levels in TA-CA-Prodrug treated cells were 1.21 times higher than those in TK-Prodrug group cells. Among the three prodrugs, TA-CA-Prodrug induced ROS were highest in level, due in part to the release of two ROS-inducing compounds CA and PTX. Furthermore, due to the positive feedback of ROS, the rapid and sufficient release of PTX from TA-CA-Prodrug may also be responsible for intracellular ROS differences. Intracellular ROS levels in the Prodrug treated group were lower than those in the TK-Prodrug treated group, probably due to the slower rate of PTX release from the Prodrug, because there was no ROS-responsive linker in the co-backbone of the Prodrug.
Experimental example 5 cell viability assay
The experimental method comprises the following steps: 4T1 cells were seeded into 96-well plates at a density of 5000 cells per well. After 24h incubation, 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 48h. RPMI 1640 with 10% CCK-8 (Dojindo) was added to each well. The 96-well plate was placed in a cell incubator for 2.5 hours of continuous incubation, and absorbance was measured at 450 nm. IC50 values were calculated from GraphPad Prism 8.0.2. The IC50 value in this experimental example was 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. IC50 values of CA, TA-CA-Prodrug, TK-Prodrug and Prodrug calculated in Graphpad Prism were 3.93. Mu.g/mL, 1.31. Mu.g/mL, 4.32. Mu.g/mL and 7.13. Mu.g/mL, respectively, which were significantly higher than PTX (0.04. Mu.g/mL). TA-CA-Prodrug, TK-Prodrug and Prodrug are less cytotoxic than PTX because of their slow process of internalization by the cell and the time required for drug release.
Experimental example 6 prodrug subcellular localization
The experimental method comprises the following steps: 4T1 cells were plated on 8-well chamber slides (Cellvis), 5000 cells per well, and after 48h incubation the cells were treated with an equivalent amount of Cy5 (0.5. Mu.g/mL) in each case of TA-CA-prodrug, TK-prodrug or prodrug for 2h. Nuclei, lysosomes and mitochondria were stained with Hoechst 33342, lyster Green and Mitotracker Green, respectively, and images were collected under CLSM (NIKON). The Pearson correlation coefficient (Pcc), the Manders overlap coefficient (Moc), and the Manders co-localization coefficient were analyzed using image J software.
The results of the transport of PTX prodrugs within 4T1 cells are shown in fig. 18, and after 2h treatment of cells with Cy5 (red) loaded prodrugs, the nuclei, lysosomes and mitochondria of cells were fluorescently labeled with Hoechst 33342 (blue), lysotracker (green) and Mito-tracker (green) staining. The red fluorescent signal in CLSM images was generated by the pro-drug being internalized by the cell. Since lysosomes and mitochondria are labeled green, the co-localized regions of the prodrug and lysosomes or mitochondria should appear as yellow fluorescent. The green fluorescent signal (lysosomal tracer) and red fluorescent signal (prodrug) within the cell are interwoven together, but a very weak yellow fluorescent signal can be seen in fig. 18A. This result indicates that only a small amount of prodrug is transferred to lysosomes after internalization. However, in fig. 18B, a high overlap of red (prodrug) and green (mitochondrial) signals was found. The strong yellow fluorescent signal appears in the overlap region, indicating that the prodrug is transported mostly to the mitochondria rather than lysosomes. Since these prodrugs have a positive surface potential in late endosomes in an acidic environment, they may be able to escape from the endosome and interact with the negatively charged mitochondrial membrane. Furthermore, localization of the Prodrug in mitochondria facilitates drug and CA release from TA-CA-Prodrug. Mitochondria are one of the major organelles that produce ROS, and ROS levels around mitochondria tend to be high. After the TA-CA-Prodrug reaches the mitochondria, high ROS levels favor the release of CA and PTX by the TA-CA-Prodrug. Released CA may interact with mitochondria to generate more ROS, further promoting ROS-responsive drug release of the prodrug.
Experimental example 7 mitochondrial Membrane potential
The experimental method comprises the following steps: to detect changes in mitochondrial membrane potential, 4T1 cell mitochondria after treatment with different prodrugs were stained with JC-10 fluorescent probes. The treated cells were washed with phenol red-free RPMI 1640 medium. JC-10 was then dissolved in 37℃complete medium and stained in incubator darkness for 20min. The original medium was replaced with fresh phenol red-free RPMI 1640 medium containing 2% fetal bovine serum and observed under CLSM. Flow cytometer detection: the treated cells were trypsinized, collected by centrifugation, suspended using warm JC-10 solution, placed in an incubator for 20min and then examined by flow cytometry (FACS Aria II, becton Dickinson, USA).
The results are shown in FIG. 19. As these prodrugs accumulate in the mitochondria, ROS can be produced in the cell after release of CA and PTX. Thus, it is speculated that prodrugs may cause damage to mitochondria. JC-10 can assess mitochondrial function in 4T1 cancer cells by changes in mitochondrial membrane potential (Δψm). After 4h of drug and cell treatment, the cells were stained with JC-10 dye for 20min. The "J-monomers" that interact with the damaged mitochondrial membrane are shown as green fluorescent, while the "J-aggregates" that interact with the normal mitochondrial membrane are shown as red fluorescent. As shown in fig. 19, red fluorescence intensity was significantly reduced in the cells after the three prodrug treatments compared to the control group. A decrease in red fluorescence or an increase in green fluorescence indicates that the mitochondrial membrane is damaged by a high level of depolarization. CA and PTX released by TA-CA-Prodrug can rapidly generate ROS, which indicates that TA-CA-Prodrug has the strongest effect of disrupting mitochondrial function.
EXAMPLE 8 in vivo imaging Studies
The experimental method comprises the following steps: female BALB/c mice were derived from the Chengdu BioIndustry (Chengdu China). Animal study procedures were performed according to the guidelines for laboratory animal care and use of the Huaxi hospital and were approved by the animal ethics committee of the university of Sichuan. Cy5@TA-CA-Prodrug, cy5@TK-Prodrug or Cy5@Prodrug (Cy 5 dose 1 mg/kg) was administered to the 4T1 tumor-bearing mice via the tail vein. The biodistribution of these prodrugs was examined in BALB/c mice 4, 12, 24 or 24 hours after injection. The surgically excised organs/tissues (tumor, heart, spleen, liver, lung and kidney) were thoroughly rinsed in physiological saline and placed in an in vivo imaging system (PerkinElmer) for bright field and fluorescence imaging (0.5 s exposure time; excitation filter=640 nm, emission filter=680 nm).
The results are shown in FIG. 20. Cy5 signaling for 3 prodrugs occurred predominantly in the liver and spleen following intravenous prodrug injection in 4T1 tumor-bearing mice (fig. 20A and 20B). At 4h, the accumulation of TK-Prodrug in liver and spleen was higher than that of TA-CA-Prodrug and Prodrug, which may be related to larger TK-Prodrug particle size. Thus, TK-Prodrug may be taken up by the reticuloendothelial phagocytic system of the liver and spleen. TA-CA-Prodrug is enriched in the kidney due to smaller particle size and can be discharged through the kidney. Due to its small particle size, it is less taken up by the reticuloendothelial system. Thus, more TA-CA-Prodrug can circulate in the blood for longer periods of time, thereby increasing its enrichment at the tumor site. The Cy5 fluorescence signal of TA-CA-Prodrug at the tumor site is gradually enhanced along with the extension of time to 48h, which shows that the TA-CA-Prodrug has good aggregation and retention 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 anti-tumor Effect
The experimental method comprises the following steps: in vivo anti-tumor experiments are carried out by adopting female BALB/c mice, and the weight is 20+/-2 g. Each mouse was subcutaneously injected 1×10 6 4T1 cells for construction of 4T1 mammary tumor engrafting mouse model. When the tumor size reaches 50mm 3 About, an antitumor experiment was started. Mice were randomly divided into 5 groups of 6 mice each. The administration was once every other day, 5 times. Intravenous physiological saline, PTX, TA-CA-prodrug, TK-prodrug or prodrug (the same PTX dose is 10mg kg -1 ). At a pre-set time point, tumor volumes were assessed using vernier calipers. Tumor volume (mm) 3 ) The calculation formula is as follows, tumor volume=l×w 2 And/2, wherein L and W are the length and width of the tumor, respectively. In addition, body weight changes were recorded every other day.
The results are shown in FIGS. 21 and 22. By monitoring tumor volume, TA-CA-Prodrug has the strongest inhibitory effect on tumor growth, while TK-Prodrug and Prodrug have moderate inhibitory effect on tumor growth. Tumor Growth Inhibition (TGI) was calculated from the mass of the tumor. TGI was 21.9% in the free PTX treatment group and 30.5%, 34.3% and 58.9% in the Prodrug, TK-Prodrug and TA-CA-Prodrug treatment groups, respectively (FIG. 22). These results indicate that TA-CA-Prodrug has good anti-tumor effect, the IC50 is the lowest, and the biological distribution in tumor is higher. Although the accumulation of TK-Prodrug in the tumor was lower than that of Prodrug, the TGI of the TK-Prodrug treated group was slightly higher than that of the Prodrug treated group (FIG. 22). This is due to the presence of reactive TK groups in the copolymer backbone of TK-Prodrug that can be destroyed by ROS.
In another aspect, the change in body weight of the 4T1 tumor-bearing mice after different drug treatments is shown in FIG. 23. The body weight of mice treated with the different prodrugs did not change significantly, meaning that the prodrugs exhibited 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 various PTX prodrugs, the mice were sacrificed, tumors and internal organs were isolated, and fixed in 10% pbs buffer neutral formaldehyde for 24h, paraffin sections were prepared. The sections were then 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 acquired under an optical microscope. Tumor tissue sections were stained using the (Promega Corp, USA) read End fluorescence TUNEL system. The deoxyribonuclease treated sections served as positive controls. The sections were imaged with an optical microscope (Imager Z2, zeiss, germany).
The experimental results are shown in FIG. 24. After H & E staining, the sections in the prodrug group showed large area necrosis of tissue, decreased cell matrix, loose structure and fragmentation of nuclei. The TA-CA-Prodrug treatment group had the highest degree of histopathological damage. TUNEL staining detects apoptosis in situ of tumors, CD31 and Ki67 immunohistochemistry detects anti-angiogenic and proliferative capabilities of tumors. Among tumors treated with TA-CA-Prodrug, ki67 and CD31 positive cells were the lowest in number, indicating that TA-CA-Prodrug significantly inhibited proliferation and angiogenesis in tumor tissue. In addition, after TUNEL staining, the number of apoptotic cells in TA-CA-Prodrug treated tumors increased significantly. 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 has time dependence. Since the DPA moiety in the conjugate molecule can be protonated in an acidic microenvironment, it can escape from late endosomes and reach the mitochondria. Upon exposure to high levels of ROS produced by mitochondria, release of CA and PTX in the conjugate molecule is triggered. Released CA can interact with mitochondria to produce more ROS, resulting in a decrease in mitochondrial membrane potential and promoting more PTX release from prodrugs. The released PTX causes cell cycle arrest and inhibits its proliferation. The cascade of ROS feedback effects in this novel prodrug enhances the cell cycle arrest effect, ultimately leading to apoptosis of tumor cells. Compared with two control groups (TK-Prodrug and Prodrug-formed micelle), the micelle formed by the conjugate TA-CA-Prodrug prepared by the embodiment of the invention has an optimized copolymer structure and an ROS amplified chemotherapeutic effect, and has outstanding performance 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 the ROS responsive DDS, and has very high application potential in development and application of antitumor drugs.

Claims (6)

1. The active oxygen self-supplementing amphiphilic block copolymer-drug conjugate is characterized by being prepared by the following steps:
step one:
75.67mmol of cinnamaldehyde and 151.33mmol of beta-mercaptoethanol are dissolved in 70mL of THF, and 15.13mmol of ZrCl are added at 0deg.C 4 The method comprises the steps of carrying out a first treatment on the surface of the Stirring for 30 min at 0deg.C, and evaporating to remove solvent; purifying the crude product by silica gel chromatography to obtain TA-CA;
step two:
3.70 mmole of TA-CA was dissolved in 30mL of anhydrous THF containing 5.55 mmole of triethylamine, a solution of 3.70 mmole of methacryloyl chloride in THF was slowly added to the reaction system under ice bath, the reaction was continued in ice bath for 4 hours, the white solid of triethylamine hydrochloride was removed by filtration, the solvent in the filtrate was distilled off, and the resulting residue was purified by silica gel chromatography to give MA-TA-CA as a colorless oil;
step three:
0.59mmol of MA-TA-CA was dissolved in 20mL of dry THF containing 1.18 mmole of TEA and a catalytic amount of DMAP was added; slowly adding a solution of 0.89 mmole of 4-nitrophenoxycarbonyl chloride in THF under ice bath; the reaction system was stirred at room temperature overnight, the triethylamine hydrochloride solid formed was removed by filtration, and the filtrate was added dropwise to a solution of 0.59 mmole of PTX and 1.77 mmole of DMAP in methylene chloride, reaction 2 After 4 hours, the mixture was quenched with dilute HCl, saturated Na 2 CO 3 And brine, and drying over anhydrous sodium sulfate, evaporating the solvent, and purifying the obtained residue by silica gel chromatography to obtain MA-TA-CA-PTX;
step four:
22.00 mmole of CA and 46.20 mmole of 2, 2-trifluoro-N- (2-mercaptoethyl) acetamide were dissolved in 100ml of THF, 11.00 mmole of aluminum chloride was added under ice bath, and stirring under ice bath was continued for 10 minutes; evaporating the solvent, purifying the crude product by silica gel chromatography to obtain TA-CA-NHCOCF 3 ；
Step five:
10.86mmolTA-CA-NHCOCF 3 Dissolved in 50mL of methanol and 50mL of an aqueous solution containing 54.29 mmole of naoh was added, and the mixture was stirred for 30 minutes to completely deprotect the trifluoroacetyl group; removing methanol under reduced pressure, extracting the obtained solution with 100 mM DCM for 3 times, mixing the organic phases, drying with anhydrous sodium sulfate, filtering, and evaporating solvent to obtain TA-CA-NH 2 ；
Step six:
1.07mmol of CTA was added to 30mL of methylene chloride containing 1.88 mmole of 2- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate and 1.88 mmole of N, N-diisopropylethylamine in an ice bath, and then 1.07 mmole of 2-aminoethanol was added thereto, the reaction was continued at room temperature for 10 minutes, the solution was washed with saturated sodium hydrogencarbonate, diluted HCl and saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off, and the obtained crude product was purified by silica gel chromatography to give CTA-OH;
Step seven:
in the presence of a catalytic amount of DMAP, 0.93 mmole of CTA-OH was dissolved in 20mL of anhydrous THF containing 1.86 mmole of triethylamine; dropwise adding 1.86mmol of 4-nitrophenyl chloroformate in THF under ice bath into the reaction system, and continuing to react overnight; filtering to remove generated triethylamine hydrochloride solid, evaporating to remove organic solvent, and purifying the crude product by silica gel column chromatography to obtain CTA-NPC;
step eight:
2.00 mmole of mPEG5k-OH was dissolved in 100mL of anhydrous DCM containing 24.00 mmole of DIEA in the presence of a catalytic amount of DMAP; dropwise adding 20.00mmol of DCM solution of 4-nitrophenyl chloroformate into the reaction system under ice bath, continuously reacting for 24 hours at room temperature, evaporating the solvent under reduced pressure, dropwise adding the concentrated mixture into 500mL of diethyl ether, separating out solid, and circularly performing a dissolving-precipitating procedure to obtain a required product; drying under vacuum to obtain mPEG5k-NPC;
step nine:
a20 mM DCM solution containing 1mmol of mPEG5k-NPC was added dropwise to a solution containing 3mmolTA-CA-NH 2 In (2) for 48 hours at room temperature, removing the solvent, dissolving the residue in water for dialysis and purification, and freeze-drying to obtain the product mPEG5k-TA-CA-NH 2 ；
Step ten:
0.5mmol of PEG5k-TA-CA-NH 2 Dissolve in 25mL DCM; dropwise adding 25 mM DCM solution containing 1 mmole of CTA-NPC into the reaction system under stirring, and continuing to react for 48 hours; after the reaction is finished, the reaction solution is concentrated and is added into 300mL of diethyl ether in a dropwise manner, and pink solid is separated out; the dissolution-precipitation procedure is carried out twice by circulation to obtain a product; vacuum drying to obtain mPEG5k-TA-CA-CTA;
step eleven:
0.055mmol of monomer MA-TA-CA-PTX, 0.27mmol of monomer MA-TA-CA-PTX and 1.64mmol of 2- (diisopropylamino) ethyl methacrylate are added to a round bottom flask under argon atmosphere; the round bottom flask was sealed and 4mL of DMSO/H2O at a volume ratio of 9/1 containing 0.022mmol of initiator VA044 was injected into the reaction flask; after bubbling with argon for 30 minutes, the reaction mixture was stirred at 47 ℃ for 24 hours; the reaction was quenched with liquid nitrogen and the mixture was added dropwise to 400mL of MeOH/H1/1 by volume 2 In O, a precipitate was obtained, collected, dried in vacuo and redissolved in DCM; the resulting solution was added dropwise to 300mL of diethyl ether and the precipitate was collected; after drying in vacuo, the final conjugate mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA) was obtained.
2. The method of preparing the conjugate of claim 1, comprising the steps of:
Step one:
75.67mmol of cinnamaldehyde and 151.33mmol of beta-mercaptoethanol are dissolved in 70mL of THF, and 15.13mmol of ZrCl are added at 0deg.C 4 The method comprises the steps of carrying out a first treatment on the surface of the Stirring for 30 min at 0deg.C, and evaporating to remove solvent; the crude product is purified by silica gel chromatography to obtain TA-CA；
Step two:
3.70 mmole of TA-CA was dissolved in 30mL of anhydrous THF containing 5.55 mmole of triethylamine, a solution of 3.70 mmole of methacryloyl chloride in THF was slowly added to the reaction system under ice bath, the reaction was continued in ice bath for 4 hours, the white solid of triethylamine hydrochloride was removed by filtration, the solvent in the filtrate was distilled off, and the resulting residue was purified by silica gel chromatography to give MA-TA-CA as a colorless oil;
step three:
0.59mmol of MA-TA-CA was dissolved in 20mL of dry THF containing 1.18 mmole of TEA and a catalytic amount of DMAP was added; under ice bath, a solution of 0.89mmol of 4-nitrophenoxycarbonyl chloride in THF was slowly added; the reaction system was stirred at room temperature overnight, the solid triethylamine hydrochloride formed was removed by filtration, the filtrate was added dropwise to a solution of 0.59mmol of PTX and 1.77mmol of DMAP in methylene chloride, and after 24 hours the mixture was reacted with dilute HCl, saturated Na 2 CO 3 And brine, and drying over anhydrous sodium sulfate, evaporating the solvent, and purifying the obtained residue by silica gel chromatography to obtain MA-TA-CA-PTX;
Step four:
22.00mmol of CA and 46.20mmol of 2, 2-trifluoro-N- (2-mercaptoethyl) acetamide were dissolved in 100ml of THF, 11.00mmol of aluminum chloride was added under ice bath, and stirring under ice bath was continued for 10 minutes; evaporating the solvent, purifying the crude product by silica gel chromatography to obtain TA-CA-NHCOCF 3 ；
Step five:
10.86mmolTA-CA-NHCOCF 3 Dissolved in 50mL of methanol and 50mL of an aqueous solution containing 54.29 mmole of naoh was added, and the mixture was stirred for 30 minutes to completely deprotect the trifluoroacetyl group; removing methanol under reduced pressure, extracting the obtained solution with 100 mM DCM for 3 times, mixing the organic phases, drying with anhydrous sodium sulfate, filtering, and evaporating solvent to obtain TA-CA-NH 2 ；
Step six:
1.07mmol of CTA was added to 30mL of methylene chloride containing 1.88mmol of 2- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate and 1.88mmol of N, N-diisopropylethylamine under ice bath, and 1.07mmol of 2-aminoethanol was further added, the reaction system was allowed to react at room temperature for 10 minutes, the solution was washed with saturated sodium hydrogencarbonate, diluted HCl and saturated saline, dried over anhydrous sodium sulfate, and the solvent was distilled off, and the obtained crude product was purified by silica gel chromatography to give CTA-OH;
step seven:
in the presence of a catalytic amount of DMAP, 0.93mmol of CTA-OH was dissolved in 20mL of anhydrous THF containing 1.86mmol of triethylamine; dropwise adding 1.86mmol of 4-nitrophenyl chloroformate in THF under ice bath into the reaction system, and continuing to react overnight; filtering to remove generated triethylamine hydrochloride solid, evaporating to remove organic solvent, and purifying the crude product by silica gel column chromatography to obtain CTA-NPC;
Step eight:
2.00mmol of mPEG5k-OH was dissolved in 100mL of anhydrous DCM containing 24.00mmol of DIEA in the presence of a catalytic amount of DMAP; dropwise adding 20.00mmol of DCM solution of 4-nitrophenyl chloroformate into the reaction system under ice bath, continuously reacting for 24 hours at room temperature, evaporating the solvent under reduced pressure, dropwise adding the concentrated mixture into 500mL of diethyl ether, separating out solid, and circularly performing a dissolving-precipitating procedure to obtain a required product; drying under vacuum to obtain mPEG5k-NPC;
step nine:
20mL of DCM solution containing 1mmol of mPEG5k-NPC was added dropwise containing 3mmol of TA-CA-NH 2 In (2) for 48 hours at room temperature, removing the solvent, dissolving the residue in water for dialysis and purification, and freeze-drying to obtain the product mPEG5k-TA-CA-NH 2 ；
Step ten:
0.5mmol of mPEG5k-TA-CA-NH 2 Dissolve in 25mL DCM; dropwise adding 25mLDCM solution containing 1mmol CTA-NPC into the reaction system under stirring, and continuing to react for 48 hours; after the reaction is finished, the reaction solution is concentrated and is added into 300mL of diethyl ether in a dropwise manner, and pink solid is separated out; the dissolution-precipitation procedure is carried out twice by circulation to obtain a product; vacuum drying to obtain mPEG5k-TA-CA-CTA;
step eleven:
0.055mmol of mPEG5k-TA-CA-CTA, 0.27mmol of monomer MA-TA-CA-PTX and1.64mmol of 2- (diisopropylamino) ethyl methacrylate was added to a round bottom flask under argon atmosphere; the round bottom flask was sealed and 4mL of DMSO/H2O at a volume ratio of 9/1 containing 0.022mmol of initiator VA044 was injected into the reaction flask; after bubbling with argon for 30 minutes, the reaction mixture was stirred at 47 ℃ for 24 hours; the reaction was quenched with liquid nitrogen and the mixture was added dropwise to 400mL of MeOH/H1/1 by volume 2 In O, a precipitate was obtained, collected, dried in vacuo and redissolved in DCM; the resulting solution was added dropwise to 300mL of diethyl ether and the precipitate was collected; after drying in vacuo, the final conjugate mPEG5k-TA-CA-block-poly (TA-CA-PTX-co-DPA) was obtained.
3. A micelle, characterized in that: formed from the conjugate of claim 1.
4. A micelle as in claim 3 wherein: the particle size of the micelle is 100-200nm;
and/or the zeta potential of the micelle is-15 to-5 mV in the environment with the pH=7.4, the zeta potential of the micelle is +10 to +25mV in the environment with the pH=6.0, and the zeta potential of the micelle is +25 to +30mV in the environment with the pH=5.2.
5. Use of the conjugate of claim 1, or the micelle of claim 3 or 4 in the preparation of an antitumor drug.
6. A medicament, characterized in that: the conjugate is prepared by taking the conjugate as defined in claim 1 or the micelle as defined in claim 3 or 4 as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
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