Paclitaxel derivative modified based on zwitterionic polypeptide, prodrug nanocarrier micelles and preparation method thereof

Abstract

The application provides a zwitterionic polypeptide modified paclitaxel derivative, a prodrug nanocarrier micelle and a preparation method thereof, and belongs to the field of biological medicines. The preparation method comprises the following steps: mixing a zwitterionic polypeptide containing a sulfhydryl group with 2,2'-dithiodipyridine in a polar solvent, stirring and reacting, crystallizing, and obtaining a polypeptide derivative containing a disulfide bond; and then dissolving the polypeptide derivative and a sulfhydrylated paclitaxel (PTX-SH) in a polar solvent, adding glacial acetic acid to adjust the pH, stirring and reacting, and obtaining the paclitaxel derivative. The paclitaxel derivative is used for preparing a prodrug nanocarrier micelle by loading a hydrophobic anticancer drug, the prodrug nanocarrier micelle has a long blood circulation time in the body, a high drug loading capacity, small toxic side effects, excellent glutathione response and fast on-demand controlled release characteristics, and good tumor inhibition effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to a zwitterionic polypeptide modified paclitaxel derivative, a prodrug nanocarrier micelle and a preparation method thereof. BACKGROUND

[0002] Paclitaxel is a natural secondary metabolite from Taxus. It has a specific effect on the multiple ovarian cancer, uterine cancer and breast cancer in recent years. It stabilizes microtubules by promoting the polymerization of tubulin dimers and preventing them from depolymerizing, thereby inhibiting the normal dynamic reorganization of the microtubule network and affecting the cell function split during the interphase and mitotic phase. In addition, paclitaxel can also cause abnormal arrangement of microtubule 'bundles' and affect the division of tumor cells. However, paclitaxel has poor water solubility and is not easily absorbed by the human body, which limits its therapeutic effect. In 1992, the U.S. Food and Drug Administration (FDA) approved paclitaxel injection (Taxol) for clinical use, which added castor oil to improve its water solubility, but it produced serious nephrotoxicity and neurotoxicity after administration. In 2005, the albumin-bound paclitaxel preparation (Abraxance) began to be used in clinical practice, but it has the disadvantages of high cost, short half-life and fast elimination.

[0003] Therefore, it is very necessary to provide a paclitaxel preparation with low cost, long half-life and slow elimination. SUMMARY

[0004] The present application aims to provide a zwitterionic polypeptide modified paclitaxel derivative, a prodrug nanocarrier micelle and a preparation method thereof. The zwitterionic polypeptide modified on the surface of the prodrug nanocarrier micelle can prolong the blood circulation time of the hydrophobic anticancer drug in the body, has high drug loading capacity, small toxic and side effects, excellent glutathione (r-glutamyl cysteingl+glycine, GSH) response and fast on-demand controlled release characteristics, and good tumor inhibition effect.

[0005] In order to achieve the above-mentioned purpose of the present application, the following technical solutions are adopted:

[0006] In a first aspect, the present application provides a zwitterionic polypeptide modified paclitaxel derivative, which has a structure as shown in general formula (I):

[0007]

[0008] In the general formula (I), R1 and R2 are selected from the group consisting of amino acid residues with carboxyl-containing side chains and amino acid residues with amino-containing side chains, and the ratio of the amino and carboxyl contained in R1 and R2 is 1:1; n represents 1-40. The paclitaxel derivative is modified on the surface with a zwitterionic polypeptide, which can self-assemble into a nano-drug. Compared with the chemotherapeutic drugs used in the clinic, the self-assembled nano-drug has a long in vivo circulation time, low toxic side effects and high tumor inhibition effect.

[0009] Further, in the preferred embodiment of the present application, in the general formula (I), the amino acid residues with carboxyl-containing side chains include glutamic acid residues and aspartic acid residues; and the amino acid residues with amino-containing side chains include lysine residues, histidine residues, arginine residues, glutamine residues and asparagine residues.

[0010] In a second aspect, the present application provides a preparation method of the zwitterionic polypeptide-modified paclitaxel derivative as described above, which comprises:

[0011] In a polar solvent, the thiol-containing zwitterionic polypeptide is mixed and reacted with 2,2'-dithiodipyridine, and the product after the reaction is crystallized to obtain a polypeptide derivative containing a disulfide bond;

[0012] The polypeptide derivative containing a disulfide bond is then dissolved in a polar solvent at a molar ratio of 1:1.5-5 with thiolated paclitaxel, ice acetic acid is added to adjust the pH to 4.5-6.5, and after stirring and reaction, the zwitterionic polypeptide-modified paclitaxel derivative is obtained;

[0013] The thiol-containing zwitterionic polypeptide has a structure as shown in the general formula (II):

[0014]

[0015] The polypeptide derivative containing a disulfide bond has a structure as shown in the general formula (III):

[0016]

[0017] In the general formula (II) and the general formula (III), R1 and R2 are selected from the group consisting of amino acid residues with carboxyl-containing side chains and amino acid residues with amino-containing side chains, and the ratio of the amino and carboxyl contained in R1 and R2 is 1:1; n represents 1-40.

[0018] Further, in the preferred embodiment of the present application, in the reaction for preparing the polypeptide derivative containing a disulfide bond, the molar ratio of the thiol-containing zwitterionic polypeptide to 2,2'-dithiodipyridine is 1:1.5-5.

[0019] Further, in the preferred embodiment of the present application, in the step of mixing and reacting the thiol group-containing zwitterionic polypeptide with 2,2'-dithiodipyridine, specifically comprising: first mixing 2,2'-dithiodipyridine with trifluoroacetic acid to form a mixed solution; then adding the thiol group-containing zwitterionic polypeptide into the mixed solution for reaction. Preferably, the reaction temperature is 25°C, and the reaction time is 4-6 h. More preferably, in the preferred embodiment of the present application, the above-mentioned reaction for preparing the polypeptide derivative is: dissolving the thiol group-containing zwitterionic polypeptide and 2,2'-dithiodipyridine in a polar solvent such as methanol at a molar ratio of 1:1.5-5, and protecting with nitrogen. After stirring in a 25°C water bath for 4-6 h, rotary evaporation is performed, crystallization is performed in an ether solution, and vacuum drying is performed to obtain the polypeptide derivative containing a disulfide bond.

[0020] The reaction equation of the polypeptide derivative is shown in reaction formula (I):

[0021]

[0022] Further, in the preferred embodiment of the present application, the molar ratio of trifluoroacetic acid to 2,2'-dithiodipyridine is 10-10000:1.

[0023] Preferably, in the preferred embodiment of the present application, the reaction for preparing the paclitaxel derivative using the polypeptide derivative containing a disulfide bond and thiolated paclitaxel is: dissolving the polypeptide derivative containing a disulfide bond and thiolated paclitaxel in a polar solvent such as methanol at a molar ratio of 1:1.5-5. Then, glacial acetic acid is added to adjust the pH to 4.5-6.5, stirring is performed at 25°C for 12-24 h, dialysis bag purification is performed, and the solvent is removed to obtain the zwitterionic polypeptide-based paclitaxel derivative.

[0024] The reaction equation of the paclitaxel derivative is shown in reaction formula (II):

[0025]

[0026] wherein: n is any integer from 1 to 40. R1 and R2 have two combinations: one is that R1 is a side chain of an amino acid containing a carboxyl group such as glutamic acid or aspartic acid, and R2 is a side chain of an amino acid containing an amino group such as lysine, glutamine or asparagine; the other is that R1 is a side chain of an amino acid containing an amino group such as lysine, glutamine or asparagine, and R2 is a side chain of an amino acid containing a carboxyl group such as glutamic acid or aspartic acid, and the two combinations only need to ensure that the proportion of the side chain structures containing an amino group and a carboxyl group of R1 and R2 is 1:1.

[0027] In a third aspect, the present application provides a prodrug nanocarrier micelle based on paclitaxel derivative, which comprises the zwitterionic polypeptide-modified paclitaxel derivative as described above, and a hydrophobic anticancer drug and a polar solvent medium.

[0028] In a fourth aspect, the present application provides a preparation method of the prodrug nanocarrier micelle based on paclitaxel derivative as described above, which comprises: dissolving the zwitterionic polypeptide-modified paclitaxel derivative and the hydrophobic anticancer drug in a polar solvent medium respectively to obtain a polar solvent containing the zwitterionic polypeptide-modified paclitaxel derivative and a polar solvent containing the hydrophobic anticancer drug; and then performing self-assembly on the polar solvent containing the zwitterionic polypeptide-modified paclitaxel derivative and the polar solvent containing the hydrophobic anticancer drug, and then dialyzing to obtain the prodrug nanocarrier micelle based on paclitaxel derivative.

[0029] Further, the hydrophobic anticancer drug comprises one or more of doxorubicin, paclitaxel, docetaxel, camptothecin, 10-hydroxycamptothecin, plinabulin, and photosensitizer chlorin e6.

[0030] Further, the zwitterionic polypeptide-modified paclitaxel derivative is dissolved in methanol to form a paclitaxel derivative solution with a concentration of 4-10 mg / mL, and then the polar solution containing the hydrophobic anticancer drug and the paclitaxel derivative solution are fully mixed according to a molar ratio of 1:(2-20), and then dialyzed to obtain the prodrug nanocarrier micelle based on paclitaxel derivative.

[0031] The effects of the present application are as follows:

[0032] 1. The zwitterionic polypeptide used in the present application contains a thiol group at one end, which is connected to the chemotherapeutic drug paclitaxel through 2,2'-dithiodipyridine as a bridge in the form of a chemical bond. The chemical bond is sensitive to GSH and can be broken in the high-concentration GSH environment of tumor tissues, so that the hydrophobic anticancer drug is rapidly released. The prodrug nanocarrier micelle prepared in this way has the advantages of high drug loading and good GSH response and rapid on-demand controlled release characteristics in tumor cells.

[0033] 2. The zwitterionic polypeptide used in the present application contains amino groups and carboxyl groups in the side chain, and the ratio of amino groups to carboxyl groups is 1:1. The zwitterionic polypeptide composed of amino groups and carboxyl groups with a ratio of 1:1 has excellent anti-protein non-specific adsorption performance. After being modified to paclitaxel to form a prodrug and then being prepared into a prodrug nanocarrier micelle, the toxicity of the hydrophobic anticancer drug at normal tissues can be reduced, the blood circulation time of the hydrophobic anticancer drug in the body can be prolonged, and the prodrug nanocarrier micelle has good in-vivo biocompatibility.

[0034] 3、The surface of the prepared prodrug nano-drug-loaded micelles is modified with a layer of zwitterionic polypeptide, which forms a dense water layer, so that the affinity between the nano-micelles and normal cells is very low, and the nano-micelles have a slightly positive surface potential under the slightly acidic conditions of tumors due to the pH sensitivity of the zwitterionic polypeptide, so that the affinity of the nano-micelles to tumor cells is significantly higher than that to normal cells, so that the prodrug nano-drug-loaded micelles have better tumor targeting in the mouse body due to the pH responsiveness of the polypeptide.

[0035] 4、The particle size of the prepared prodrug nano-drug-loaded micelles can be controlled below 200nm, so that the high permeability and retention effect of tumors can be used to improve the enrichment amount of hydrophobic anticancer drugs at the tumor site, thereby obtaining a very good tumor inhibition effect. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Synthesis of paclitaxel derivative and preparation of prodrug nano-drug-loaded micelles in Example 1 of the present application;

[0037] Figure 2 Nuclear magnetic resonance hydrogen spectrum of (EK)3-C, (EK)3-C-SS-PY and E-SS-P obtained in Example 1 of the present application;

[0038] Figure 3 Critical micelle concentration value of E-SS-P micelles obtained in Example 1 of the present application in aqueous solution;

[0039] Figure 4 UV-visible absorption spectrum of E-SS-P-DOX drug-loaded micelles obtained in Example 1 of the present application;

[0040] Figure 5 Transmission electron micrograph of E-SS-P micelles obtained in Example 1 of the present application;

[0041] Figure 6 Transmission electron micrograph of E-SS-P micelles obtained in Example 1 of the present application;

[0042] Figure 7 Zeta potential graph of E-SS-P-DOX drug-loaded micelles obtained in Example 1 of the present application in pH 5.5-7.4 phosphate buffer solution;

[0043] Figure 8 Hydrodynamic particle size distribution histogram of E-SS-P-DOX drug-loaded micelles obtained in Example 1 of the present application in 0.1mM, 1mM and 5mM phosphate buffer solution;

[0044] Figure 9 Body weight change graph of experimental mice in Example 1 of the present application for 14 days;

[0045] Figure 10 Figure 2 is a graph showing the change in tumor volume of the experimental mice in Experimental Example 1 of the present application over 14 days;

[0046] Figure 11 Figure 3 is a photograph showing the tumor site of the experimental mice in Experimental Example 1 of the present application after 14 days of administration;

[0047] Figure 12 Figure 4 is a graph showing the blood circulation time curve of the E-SS-P-DOX drug-loaded micelles in mice in Experimental Example 1 of the present application;

[0048] Figure 13 Figure 5 is a graph showing the drug release curve of the E-SS-P-DOX drug-loaded micelles in 0.1 mM, 1 mM and 5 mM phosphate buffer solutions in Experimental Example 1 of the present application. DETAILED DESCRIPTION

[0049] The inventors found that researchers have attempted different nano delivery systems to improve the water solubility and anti-tumor activity of paclitaxel, including drug delivery systems such as micelles, liposomes and nanocrystals. The method of using nano drug carriers to encapsulate or chemically bond paclitaxel to deliver paclitaxel has improved the bioavailability of paclitaxel to some extent, but there are still problems of insufficient bioavailability and generally low drug loading. The prodrug strategy can effectively improve the poor physicochemical properties of the therapeutic drug, such as poor stability and low solubility. The nano-particle drug delivery system has obvious advantages in long circulation time, effective tumor targeting and controllable drug release. The prodrug-based nano drug-loaded micelles integrate the advantages of the prodrug strategy and the nano carrier, and exhibit particularly high drug loading and low toxic side effects, which can provide a new solution to the problems of poor water solubility and low drug loading commonly faced by paclitaxel. In addition, in order to induce the effectiveness of paclitaxel, i.e. the anti-tumor activity of paclitaxel, the prodrug-based nano micelles based on paclitaxel must be bioactivated to the active parent paclitaxel drug in the body, especially in tumor cells, which requires that the prodrug-based nano micelles based on paclitaxel can rapidly release the active parent paclitaxel drug on demand in tumor cells. In addition, the prodrug-based nano micelles based on paclitaxel can also be used to encapsulate hydrophobic anticancer drugs, which can significantly improve the poor solubility and large toxic side effects of hydrophobic anticancer drugs, further improving the bioavailability and tumor inhibition effect of hydrophobic anticancer drugs.

[0050] Embodiments of the present application will be described in detail below with reference to Examples, but those skilled in the art will understand that the following Examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If specific conditions are not specified in the Examples, they are carried out under conventional conditions or according to the manufacturer's recommendations. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0051] Example 1

[0052] The present embodiment provides a zwitterionic polypeptide-modified paclitaxel derivative, and a prodrug nanocarrier micelle formed by loading doxorubicin therein, and a preparation method thereof, as shown in the following scheme, specifically comprising: Figure 1

[0053] (1) 3,3'-dithiodipropionic acid (118 mg, 0.56 mmol) and paclitaxel (1144 mg, 1.34 mmol) were added to a round-bottom flask containing 12 mL of dichloromethane. After sealing, stirring was performed at room temperature for 1 h, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (276 mg, 1.34 mmol) and 4-dimethylaminopyridine (28 mg, 0.22 mmol) were added. After sealing, refluxing was performed for 48 h. After the reaction was completed, the mixture was concentrated, and column chromatography was performed to purify the mixture. After purification, the target product paclitaxel dimer (PTX-S-S-PTX) was obtained.

[0054] (2) In a round-bottom flask containing 1 mL of dichloromethane, PTX-S-S-PTX (37 mg, 0.02 mmol) and dithiothreitol (7 mg, 0.04 mmol) were added. After sealing, stirring was performed at room temperature for 1 h, and then triethylamine (8 mg, 0.08 mmol) was added to the reaction mixture. After sealing, refluxing was performed under N2 for 12 h. After the reaction was completed, the organic phase was removed and washed, dried, filtered, and concentrated. Finally, the concentrated solution was precipitated by petroleum ether to obtain the target product mercaptanized paclitaxel (PTX-SH).

[0055] (3) In a round-bottom flask containing trifluoroacetic acid, the molar ratio of the trifluoroacetic acid to 2,2'-dithiodipyridine was 3000:1. Zwitterionic polypeptide (EK)3-C composed of glutamic acid (E) and lysine (K) and 2,2'-dithiodipyridine were added, and the molar ratio of the zwitterionic polypeptide (EK)3-C to 2,2'-dithiodipyridine was 1:3.5. After stirring under N2 for 20 min, sealing was performed, and reaction was performed at room temperature for 4 h. After the reaction was completed, the solution was concentrated, and precipitated by cold ether to obtain a polypeptide derivative containing a disulfide bond ((EK)3-C-SS-PY). In a round-bottom flask containing 3 mL of methanol, (EK)3-C-SS-PY and PTX-SH were added, and the molar ratio of (EK)3-C-SS-PY to PTX-SH was 1:4. Glacial acetic acid was added to adjust the pH to 6.0. After stirring under N2 for 20 min, sealing was performed, and reaction was performed at room temperature for 12 h. After the reaction was completed, the solution was concentrated, and precipitated by cold petroleum ether to obtain the zwitterionic polypeptide-modified paclitaxel derivative (E-SS-P).

[0056] The nuclear magnetic resonance spectrum of E-SS-P is shown in the following figure, Figure 2 Figure 2 ​​On the E-SS-P nuclear magnetic spectrum, the characteristic peaks of PTX-SH appeared at chemical shifts of 1.1, 3.1, 3.6 and 7.46-8.13 ppm, which indicated that PTX-SH was successfully connected to (EK)3-C. In addition, the disappearance of peaks at 5.7 and 6.3 ppm further proved the successful synthesis of E-SS-P.

[0057] (4) 5 μL of triethylamine was added dropwise to 1 mL of methanol solution containing 186 μg of doxorubicin hydrochloride (DOX-HCl), and it was treated in the dark. Then it was ultrasonically treated for 1 h to obtain a DOX methanol solution. A 10 mg / mL E-SS-P methanol solution was prepared, and then the above-mentioned DOX methanol solution and E-SS-P methanol solution were mixed in a molar ratio of 1:5, and ultrasonically treated for 3 h at room temperature in the dark. After ultrasonic treatment, the above-mentioned mixture was added dropwise to 1.5 mL of water, and then dialyzed against water in a dialysis bag with a molecular weight cut-off of 500, using 0.5 L of water each time, and dialyzed for 8 h each time, for a total of 3 times. After dialysis, E-SS-P-DOX drug-loaded micelles were obtained.

[0058] The prepared nanomicelles were characterized, as shown in Figures 3 to 8

[0059] The critical micelle concentration value of E-SS-P was determined by fluorescence spectrophotometry, as shown in Figure 3 The inflection point of the curve is the critical micelle concentration value of the E-SS-P blank micelle aqueous solution, and the calculated critical micelle concentration of the blank micelles is 40.74 μg / mL. The size of the critical micelle concentration value is related to the ratio of the hydrophilic end and the hydrophobic end. At this time, in E-SS-P, one end is the zwitterionic peptide (EK)3-C molecule, and the other end is the modified paclitaxel, and the molar ratio of the two is 1:1.

[0060] The doxorubicin and E-SS-P-DOX drug-loaded micelles were scanned by ultraviolet-visible spectrophotometry, as shown in Figure 4 The highest absorption peak of doxorubicin is about 480 nm, and the highest absorption peak of the drug-loaded micelles is about 520 nm. The absorption peak of doxorubicin is red-shifted, indicating that the E-SS-P-DOX drug-loaded micelles successfully encapsulate doxorubicin.

[0061] The blank micelles were characterized by transmission electron microscopy, as shown in Figure 5 , Figure 6 The E-SS-P micelles have regular morphology and are approximately spherical, with an average particle size of 24.99 nm and a relatively uniform particle size distribution.

[0062] The effect of different pH on the zeta potential of E-SS-P-DOX drug-loaded micelles was determined by laser particle size analyzer, as shown in Figure 7 ​As shown, the Zeta potential of E-SS-P-DOX micelles at pH 7.4, 6.5 and 5.5 is -7.3, 5.0 and 16.5 mV, respectively. These results show that the surface potential of E-SS-P-DOX micelles is reversed from negative to positive in the weakly acidic environment of tumor tissue compared with the pH in normal tissue, which can enhance the uptake of E-SS-P-DOX micelles by cells to some extent, and further deliver more antitumor drugs to the tumor site. The reason for this result is that the dissociation of carboxyl groups causes E-SS-P-DOX micelles to carry a small negative charge at pH = 7.4, while under weakly acidic conditions, the dissociation of carboxyl groups of E-SS-P-DOX micelles is inhibited and the protonation of amino groups is enhanced, so that the Zeta potential of E-SS-P-DOX micelles is positive.

[0063] Figure 8 E-SS-P-DOX drug-loaded micelles were placed in phosphate buffer solutions with GSH concentrations of 0.1 mM, 1 mM and 5 mM, and the hydrodynamic particle size was tested by a particle size potential instrument. As can be seen from the figure, the hydrodynamic particle size of E-SS-P-DOX micelles was measured to be 190 nm, 396 nm and 955 nm at GSH levels of 0.1 mM, 1 mM and 5 mM, respectively. It was found by comparison that the hydrodynamic particle size of E-SS-P-DOX micelles gradually increased as the GSH concentration increased. This is mainly because the breakage of -SS- caused by the increase of GSH concentration, which in turn caused the destruction of E-SS-P-DOX micelles. This result shows that E-SS-P-DOX micelles have the characteristics of rapid on-demand controlled release in tumor cells.

[0064] Example 2

[0065] The present embodiment provides a zwitterionic polypeptide modified paclitaxel derivative, and a prodrug nanodrug-loaded micelle formed by loading doxorubicin therefrom, specifically comprising:

[0066] (1) 3,3'-dithiodipropionic acid (118 mg, 0.56 mmol) and paclitaxel (1144 mg, 1.34 mmol) were added to a round-bottom flask containing 12 mL of dichloromethane. After sealing, stirring was carried out at room temperature for 1 h, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (276 mg, 1.34 mmol) and 4-dimethylaminopyridine (28 mg, 0.22 mmol) were added, the reaction was sealed and refluxed for 48 h. After the reaction was completed, the mixture was concentrated, and column chromatography was performed to purify it. After purification, the target product PTX-S-S-PTX was obtained.

[0067] (2) In a round bottom flask containing 1 mL of dichloromethane, PTX-S-S-PTX (37 mg, 0.02 mmol) and dithiothreitol (7 mg, 0.04 mmol) were added. After sealing, it was stirred at room temperature for 1 h, then triethylamine (8 mg, 0.08 mmol) was added to the reaction mixture, sealed and refluxed under N2for 12 h. After the reaction was completed, the organic phase was taken out and washed, dried, filtered and concentrated, and finally the concentrated solution was precipitated by petroleum ether to obtain the target product PTX-SH.

[0068] (3) In a round bottom flask containing trifluoroacetic acid, the molar ratio of trifluoroacetic acid to 2,2'-dithiodipyridine was 10:1, and the zwitterionic polypeptide (EK) 1-C composed of glutamic acid (E) and lysine (K) and 2,2'-dithiodipyridine were added, the molar ratio of ion polypeptide (EK) 1-C to 2,2'-dithiodipyridine was 1:3, and N2was stirred for 20 min, then sealed and reacted at room temperature for 4 h. After the reaction was completed, the solution was concentrated, and precipitated with cold ether to obtain a disulfide-containing polypeptide derivative ((EK) 1-C-SS-PY). In a round bottom flask containing 3 mL of methanol, (EK) 1-C-SS-PY and PTX-SH were added, the molar ratio of (EK) 1-C-SS-PY to PTX-SH was 1:4, then glacial acetic acid was added to adjust the pH to 6.5, N2was stirred for 20 min, then sealed and reacted at room temperature for 12 h. After the reaction was completed, the solution was concentrated, and precipitated with cold petroleum ether to obtain a paclitaxel derivative based on zwitterionic polypeptide modification ((EK) 1-C-SS-PTX).

[0069] (4) 5 μL of triethylamine was added dropwise to 186 μg of DOX·HCl in 1 mL of methanol, and it was treated in the dark. Then it was ultrasonically treated for 1 h to obtain a DOX methanol solution. A 4 mg / mL (EK) 1-C-SS-PTX methanol solution was prepared, and then the above DOX methanol solution and the (EK) 1-C-SS-PTX methanol solution were mixed in a molar ratio of 1:20, ultrasonically treated in the dark at room temperature for 3 h. After ultrasonic treatment, the mixture was added to 1.5 mL of water in the form of slow dripping, and then dialyzed against water, 0.5 L of water was used each time, and dialyzed for 8 h each time, for a total of 3 times. After dialysis, (EK) 1-C-SS-PTX-DOX drug-loaded micelles were obtained.

[0070] Example 3

[0071] The present embodiment provides a zwitterionic polypeptide-modified paclitaxel derivative, and a prodrug nanodrug-loaded micelle formed by loading doxorubicin therefrom, and specifically comprises:

[0072] (1) 3,3'-dithiodipropionic acid (118 mg, 0.56 mmol) and paclitaxel (1144 mg, 1.34 mmol) were added to a round-bottom flask containing 12 mL of dichloromethane. The flask was sealed and stirred at room temperature for 1 h. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (276 mg, 1.34 mmol) and 4-dimethylaminopyridine (28 mg, 0.22 mmol) were added, the flask was sealed, and the reaction was refluxed for 48 h. After the reaction was completed, the mixture was concentrated and purified by column chromatography to obtain the target product PTX-SS-PTX.

[0073] (2) PTX-SS-PTX (37 mg, 0.02 mmol) and dithiothreitol (7 mg, 0.04 mmol) were added to a round-bottom flask containing 1 mL of dichloromethane. The flask was sealed and stirred at room temperature for 1 h. Triethylamine (8 mg, 0.08 mmol) was then added to the reaction mixture. The mixture was sealed and refluxed under N2 for 12 h. After the reaction, the organic phase was removed and washed, dried, filtered, and concentrated. The concentrate was precipitated with petroleum ether to obtain the target product, PTX-SH.

[0074] (3) In a round-bottom flask containing trifluoroacetic acid, the molar ratio of trifluoroacetic acid to 2,2'-dithiodipyridine is 4000:1, and zwitterionic peptides (EK) containing glutamic acid (E) and lysine (K) are added respectively. 14 -C and 2,2'-dithiodipyridine, zwitterionic peptide (EK) 14 The molar ratio of -C and 2,2'-disulfide dipyridine was 1:2.5, and N2 was stirred for 20 minutes, and then the mixture was sealed and reacted at room temperature for 4 hours. After the reaction was completed, the solution was concentrated and precipitated with cold ether to obtain a disulfide bond-containing polypeptide derivative ((EK) 14 -C-SS-PY). In a round-bottom flask containing 3 mL of methanol, (EK) 14 -C-SS-PY, PTX-SH, (EK) 14 The molar ratio of -C-SS-PY and PTX-SH was 1:5, and then glacial acetic acid was added to adjust the pH to 4.5, and N2 was stirred for 20 minutes, and then sealed and reacted at room temperature for 12 hours. After the reaction was completed, the solution was concentrated and precipitated with cold petroleum ether to obtain a paclitaxel derivative modified with a zwitterionic peptide ((EK) 14 -C-SS-PTX).

[0075] (4) Pipette 5 μL of triethylamine and add it dropwise to 1 mL of methanol solution containing 186 μg of DOX·HCl. Protect the solution from light. Then, ultrasonicate the solution for 1 hour to obtain a methanol solution containing DOX. Prepare 5 mg / mL (EK) 14methanol solution of C-SS-PTX, then the above DOX methanol solution and (EK) 14 The C-SS-PTX methanol solution was mixed in a molar ratio of 1:15, and then ultrasonicated for 3 h at room temperature in the dark. After ultrasonication, the mixture was added dropwise into 1.5 mL of water, and then dialyzed against water in a dialysis bag with a molecular weight cut-off of 500, using 0.5 L of water each time, for 8 h each time, for 3 times. After dialysis, (EK) 14 C-SS-PTX-DOX drug-loaded micelles.

[0076] Example 4

[0077] The present example provides a zwitterionic polypeptide-modified paclitaxel derivative, and a prodrug nanodrug-loaded micelle formed by loading doxorubicin therein, and specifically comprises:

[0078] (1) 3,3'-dithiodipropionic acid (118 mg, 0.56 mmol) and paclitaxel (1144 mg, 1.34 mmol) were added to a round-bottom flask containing 12 mL of dichloromethane. After sealing, stirring was performed at room temperature for 1 h, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (276 mg, 1.34 mmol) and 4-dimethylaminopyridine (28 mg, 0.22 mmol) were added. After sealing, refluxing was performed for 48 h. After the reaction was completed, the mixture was concentrated, and then column chromatography was performed to purify the mixture. After purification, the target product PTX-S-S-PTX was obtained.

[0079] (2) In a round-bottom flask containing 1 mL of dichloromethane, PTX-S-S-PTX (37 mg, 0.02 mmol) and dithiothreitol (7 mg, 0.04 mmol) were added. After sealing, stirring was performed at room temperature for 1 h, and then triethylamine (8 mg, 0.08 mmol) was added to the reaction mixture. After sealing, refluxing was performed under N2 for 12 h. After the reaction was completed, the organic phase was removed and washed, dried, filtered, and concentrated. Finally, the concentrated solution was precipitated by petroleum ether to obtain the target product PTX-SH.

[0080] (3) In a round-bottom flask containing trifluoroacetic acid, the molar ratio of the trifluoroacetic acid to 2,2'-dithiodipyridine was 8000:1, and a zwitterionic polypeptide (DQ) 30 C and 2,2'-dithiodipyridine, and a zwitterionic polypeptide (DQ) 30 The molar ratio of C to 2,2'-dithiodipyridine was 1:5, and stirring was performed under N2 for 20 min. After sealing, reaction was performed at room temperature for 4 h. After the reaction was completed, the solution was concentrated, and then precipitated by cold ether to obtain a polypeptide derivative containing a disulfide bond ((DQ) 30-C-SS-PY). In a round bottom flask containing 3 mL of methanol, (DQ) 30 -C-SS-PY, PTX-SH, (DQ) 30 The molar ratio of -C-SS-PY and PTX-SH was 1:5, then the pH was adjusted to 4.5 by adding glacial acetic acid, stirred under N2for 20 min, and then sealed and reacted at room temperature for 12 h. After the reaction was completed, the solution was concentrated, and precipitated with cold petroleum ether to obtain a paclitaxel derivative modified by a zwitterionic polypeptide ((DQ) 30 -C-SS-PTX).

[0081] (4) 5 μL of triethylamine was added dropwise to 1 mL of methanol solution containing 186 μg of DOX-HCl, and it was protected from light. Then it was ultrasonically treated for 1 h to obtain a DOX-containing methanol solution. A 4 mg / mL (DQ) 30 -C-SS-PTX) methanol solution was prepared, and the above DOX methanol solution and (DQ) 30 -C-SS-PTX methanol solution were mixed at a molar ratio of 1:8, and ultrasonically treated at room temperature for 3 h in the dark. After ultrasonic treatment, the mixture was added dropwise to 1.5 mL of water, and then dialyzed against water in a dialysis bag with a molecular weight cutoff of 500, using 0.5 L of water each time, and dialyzed for 8 h each time, for a total of 3 times. After dialysis, (DQ) 30 -C-SS-PTX-DOX drug-loaded micelles were obtained.

[0082] Example 5

[0083] The present embodiment provides a zwitterionic polypeptide-modified paclitaxel derivative, and a prodrug nanodrug-loaded micelle formed by loading doxorubicin therein, and specifically comprises:

[0084] (1) 3,3'-dithiodipropionic acid (118 mg, 0.56 mmol) and paclitaxel (1144 mg, 1.34 mmol) were added to a round bottom flask containing 12 mL of dichloromethane. After sealing, it was stirred at room temperature for 1 h, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (276 mg, 1.34 mmol) and 4-dimethylaminopyridine (28 mg, 0.22 mmol) were added, sealed, and refluxed for 48 h. After the reaction was completed, the mixture was concentrated, and then purified by column chromatography to obtain the target product PTX-S-S-PTX.

[0085] (2) In a round bottom flask containing 1 mL of dichloromethane, PTX-S-S-PTX (37 mg, 0.02 mmol) and dithiothreitol (7 mg, 0.04 mmol) were added. After sealing, it was stirred at room temperature for 1 h, then triethylamine (8 mg, 0.08 mmol) was added to the reaction mixture, sealed and refluxed under N2for 12 h. After the reaction was completed, the organic phase was taken out and washed, dried, filtered and concentrated, and finally the concentrated solution was precipitated by petroleum ether to obtain the target product PTX-SH.

[0086] (3) In a round bottom flask containing trifluoroacetic acid, the molar ratio of trifluoroacetic acid to 2,2'-dithiodipyridine was 2000:1, and the zwitterionic polypeptide (DH)3-C composed of aspartic acid (D) and histidine (H) and 2,2'-dithiodipyridine were added, the molar ratio of zwitterionic polypeptide (DH)3-C to 2,2'-dithiodipyridine was 1:1.5, and N2was stirred for 20 min, then sealed and reacted at room temperature for 4 h. After the reaction was completed, the solution was concentrated, and the dithio-containing polypeptide derivative ((DH)3-C-SS-PY) was precipitated with cold ether. In a round bottom flask containing 3 mL of methanol, (DH)3-C-SS-PY and PTX-SH were added, the molar ratio of (DH)3-C-SS-PY to PTX-SH was 1:1.5, then glacial acetic acid was added to adjust the pH to 6.5, N2was stirred for 20 min, then sealed and reacted at room temperature for 12 h. After the reaction was completed, the solution was concentrated, and the paclitaxel derivative based on zwitterionic polypeptide modification ((DH)3-C-SS-PTX) was precipitated with cold petroleum ether.

[0087] (4) 5 μL of triethylamine was added dropwise to 186 μg of DOX·HCl in 1 mL of methanol, and it was protected from light. Then it was ultrasonically treated for 1 h to obtain a DOX methanol solution. A 5 mg / mL (DH)3-C-SS-PTX methanol solution was prepared, and then the above DOX methanol solution and (DH)3-C-SS-PTX methanol solution were mixed in a molar ratio of 1:5, ultrasonically treated for 3 h at room temperature under light protection. After ultrasonic treatment, the mixture was added to 1.5 mL of water in the form of slow dripping, and then dialyzed against water, 0.5 L of water was used each time, and dialysis was performed for 8 h each time, for a total of 3 times. After dialysis was completed, (DH)3-C-SS-PTX-DOX drug-loaded micelles were obtained.

[0088] Example 6

[0089] The present embodiment provides a zwitterionic polypeptide-modified paclitaxel derivative, and a prodrug nanodrug-loaded micelle formed by loading camptothecin, plerixafor and chlorin e6 thereon, specifically comprising:

[0090] (1) 3,3'-dithiodipropionic acid (118 mg, 0.56 mmol) and paclitaxel (1144 mg, 1.34 mmol) were added to a round-bottom flask containing 12 mL of dichloromethane. The flask was sealed and stirred at room temperature for 1 h. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (276 mg, 1.34 mmol) and 4-dimethylaminopyridine (28 mg, 0.22 mmol) were added, the flask was sealed, and the reaction was refluxed for 48 h. After the reaction was completed, the mixture was concentrated and purified by column chromatography to obtain the target product PTX-SS-PTX.

[0091] (2) PTX-SS-PTX (37 mg, 0.02 mmol) and dithiothreitol (7 mg, 0.04 mmol) were added to a round-bottom flask containing 1 mL of dichloromethane. The flask was sealed and stirred at room temperature for 1 h. Triethylamine (8 mg, 0.08 mmol) was then added to the reaction mixture. The mixture was sealed and refluxed under N2 for 12 h. After the reaction, the organic phase was removed and washed, dried, filtered, and concentrated. The concentrate was precipitated with petroleum ether to obtain the target product, PTX-SH.

[0092] (3) In a round-bottom flask containing trifluoroacetic acid, the molar ratio of trifluoroacetic acid to 2,2'-disulfide dipyridine is 10000:1, and zwitterionic peptides (DR) containing aspartic acid (D) and arginine (R) are added respectively. 40 -C and 2,2'-dithiodipyridine, zwitterionic peptide (DR) 40 The molar ratio of -C and 2,2'-disulfide dipyridine was 1:5, stirred under N2 for 20 minutes, and then sealed and reacted at room temperature for 4 hours. After the reaction was completed, the solution was concentrated and precipitated with cold ether to obtain a polypeptide derivative containing a disulfide bond (DR) 40 -C-SS-PY). In a round-bottom flask containing 3 mL of methanol, (DR) 40 -C-SS-PY, PTX-SH, (DR) 40 The molar ratio of -C-SS-PY and PTX-SH was 1:5, and then glacial acetic acid was added to adjust the pH to 4.5, and N2 was stirred for 20 minutes, and then sealed and reacted at room temperature for 12 hours. After the reaction was completed, the solution was concentrated and precipitated with cold petroleum ether to obtain a paclitaxel derivative (DR) based on the zwitterionic peptide modification. 40 -C-SS-PTX).

[0093] (4) 0.5 mg of camptothecin (CPT) was added into 1 mL of methanol to prepare a 0.5 mg / mL camptothecin methanol solution, 0.5 mg of palbociclib (PB) was added into 1 mL of methanol to prepare a 0.5 mg / mL palbociclib methanol solution, and 0.5 mg of chlorin e6 (Ce6) was added into 1 mL of methanol to prepare a 0.5 mg / mL chlorin e6 methanol solution. 7 mg / mL (DR) 40 The methanol solution of C-SS-PTX, and then the above-mentioned camptothecin methanol solution, palbociclib methanol solution, chlorin e6 methanol solution and (DR) 40 The methanol solution of C-SS-PTX was mixed in a molar ratio of 1:1:1:9, and ultrasonically treated at room temperature for 3 h in the dark. After the ultrasonic treatment, the mixture was added dropwise into 1.5 mL of water, and then dialyzed against water in a dialysis bag with a molecular weight cut-off of 500, using 0.5 L of water each time, for 8 h each time, for 3 times. After the dialysis, (DR) 40 C-SS-PTX-CPT-PB-Ce6 drug-loaded micelles.

[0094] Example 7

[0095] The present embodiment provides a zwitterionic polypeptide-modified paclitaxel derivative and a prodrug nanodrug-loaded micelle formed by loading camptothecin therefrom, and specifically comprises:

[0096] (1) 3,3'-dithiodipropionic acid (118 mg, 0.56 mmol) and paclitaxel (1144 mg, 1.34 mmol) were added to a round-bottom flask containing 12 mL of dichloromethane. After sealing, stirring was performed at room temperature for 1 h, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (276 mg, 1.34 mmol) and 4-dimethylaminopyridine (28 mg, 0.22 mmol) were added, the flask was sealed, and refluxing was performed for 48 h. After the reaction was completed, the mixture was concentrated, and column chromatography was performed to purify the mixture. After the purification, the target product PTX-S-S-PTX was obtained.

[0097] (2) In a round-bottom flask containing 1 mL of dichloromethane, PTX-S-S-PTX (37 mg, 0.02 mmol) and dithiothreitol (7 mg, 0.04 mmol) were added. After sealing, stirring was performed at room temperature for 1 h, and then triethylamine (8 mg, 0.08 mmol) was added to the reaction mixture. After sealing, refluxing was performed under N2 for 12 h. After the reaction was completed, the organic phase was removed and washed, dried, filtered and concentrated. Finally, the concentrated solution was precipitated by petroleum ether to obtain the target product PTX-SH.

[0098] (3) In a round bottom flask containing trifluoroacetic acid, the molar ratio of trifluoroacetic acid to 2,2'-dithiodipyridine was 9000:1, and the zwitterionic polypeptide (EH) composed of glutamic acid (E) and histidine (H) was added respectively 40 -C and 2,2'-dithiodipyridine, zwitterionic polypeptide (EH) 40 -C and 2,2'-dithiodipyridine, zwitterionic polypeptide (EH) 40 -C-SS-PY). In a round bottom flask containing 3 mL of methanol, (EH) 40 -C-SS-PY, PTX-SH, (EH) 40 -C-SS-PY and PTX-SH at a molar ratio of 1:3, then add glacial acetic acid to adjust the pH to 5.5, stir for 20 min under N2, then seal and react at room temperature for 12 h. After the reaction is completed, the solution is concentrated, and precipitated with cold petroleum ether to obtain a camptothecin derivative modified based on zwitterionic polypeptide (EH) 40 -C-SS-PTX).

[0099] (4) 1 mg of camptothecin (CPT) was added to 1 mL of methanol to prepare a 1 mg / mL camptothecin methanol solution. 9 mg / mL (EH) 40 -C-SS-PTX was prepared, then the above camptothecin methanol solution and (EH) 40 -C-SS-PTX methanol solution were mixed at a molar ratio of 1:6, and ultrasonic treatment was carried out under dark conditions at room temperature for 3 h. After ultrasonic treatment, the mixture was added to 1.5 mL of water in a slow dripping manner, and then a dialysis bag with a molecular weight cutoff of 500 was used for water dialysis, with 0.5 L of water each time, and dialysis was carried out for 8 h each time, for a total of 3 times. After dialysis, (EH) 40 -C-SS-PTX-CPT drug-loaded micelles.

[0100] Example 8

[0101] The present embodiment provides a zwitterionic polypeptide modified paclitaxel derivative and a prodrug nano drug-loaded micelle formed by loading chlorin e6 thereon, and specifically comprises:

[0102] (1) 3,3'-dithiodipropionic acid (118 mg, 0.56 mmol) and paclitaxel (1144 mg, 1.34 mmol) were added to a round bottom flask containing 12 mL of dichloromethane. The flask was sealed and stirred at room temperature for 1 h, then l-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (276 mg, 1.34 mmol) and 4-dimethylaminopyridine (28 mg, 0.22 mmol) were added, the flask was sealed and refluxed for 48 h. After the reaction was completed, the mixture was concentrated and purified by column chromatography. The purified product was PTX-S-S-PTX.

[0103] (2) PTX-S-S-PTX (37 mg, 0.02 mmol) and dithiothreitol (7 mg, 0.04 mmol) were added to a round bottom flask containing 1 mL of dichloromethane. The flask was sealed and stirred at room temperature for 1 h, then triethylamine (8 mg, 0.08 mmol) was added to the reaction mixture, the flask was sealed and refluxed under N2for 12 h. After the reaction was completed, the organic phase was removed and washed, dried, filtered and concentrated. The concentrated solution was precipitated by petroleum ether to obtain the target product PTX-SH.

[0104] (3) In a round bottom flask containing trifluoroacetic acid, the molar ratio of trifluoroacetic acid to 2,2'-dithiodipyridine was 1500:1. Zwitterionic polypeptide (EQ)5-C composed of glutamic acid (E) and glutamine (Q) and 2,2'-dithiodipyridine were added, the molar ratio of zwitterionic polypeptide (EQ)5-C to 2,2'-dithiodipyridine was 1:1.5. The flask was stirred under N2for 20 min, then sealed and reacted at room temperature for 4 h. After the reaction was completed, the solution was concentrated and precipitated with cold ether to obtain a disulfide-containing polypeptide derivative ((EQ)5-C-SS-PY). In a round bottom flask containing 3 mL of methanol, (EQ)5-C-SS-PY and PTX-SH were added, the molar ratio of (EQ)5-C-SS-PY to PTX-SH was 1:1.5. Glacial acetic acid was added to adjust the pH to 4.8. The flask was stirred under N2for 20 min, then sealed and reacted at room temperature for 12 h. After the reaction was completed, the solution was concentrated and precipitated with cold petroleum ether to obtain a paclitaxel derivative based on zwitterionic polypeptide modification ((EQ)5-C-SS-PTX).

[0105] (4) 1 mg of chlorin e6 (Ce6) was added to 1 mL of methanol to prepare a 1 mg / mL chlorin e6 methanol solution. A 5 mg / mL (EQ)5-C-SS-PTX methanol solution was prepared, and then the chlorin e6 methanol solution and the (EQ)5-C-SS-PTX methanol solution were mixed in a molar ratio of 1:15, and ultrasonicated for 3 h at room temperature in the dark. After the ultrasonication, the mixture was added dropwise to 1.5 mL of water, and then dialyzed against water in a dialysis bag with a molecular weight cut-off of 500, using 0.5 L of water each time, for 8 h each time, for 3 times. After the dialysis, a (EQ)5-C-SS-PTX-Ce6 drug-loaded micelle was obtained.

[0106] Example 9

[0107] The present example provides a zwitterionic polypeptide-modified paclitaxel derivative, and a prodrug nanodrug-loaded micelle formed by loading paclitaxel and chlorin e6 therein, and specifically comprises:

[0108] (1) 3,3'-dithiodipropionic acid (118 mg, 0.56 mmol) and paclitaxel (1144 mg, 1.34 mmol) were added to a round-bottom flask containing 12 mL of dichloromethane. After sealing, stirring was performed at room temperature for 1 h, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (276 mg, 1.34 mmol) and 4-dimethylaminopyridine (28 mg, 0.22 mmol) were added, and the reaction was performed under reflux for 48 h. After the reaction was completed, the mixture was concentrated, and then column chromatography was performed to purify the mixture, and the target product PTX-S-S-PTX was obtained after the purification.

[0109] (2) PTX-S-S-PTX (37 mg, 0.02 mmol) and dithiothreitol (7 mg, 0.04 mmol) were added to a round-bottom flask containing 1 mL of dichloromethane. After sealing, stirring was performed at room temperature for 1 h, and then triethylamine (8 mg, 0.08 mmol) was added to the reaction mixture, and the reaction was performed under reflux for 12 h under N2. After the reaction was completed, the organic phase was removed and washed, dried, filtered, and concentrated, and finally the target product PTX-SH was obtained by precipitating the concentrated solution with petroleum ether.

[0110] (3) A zwitterionic polypeptide (EQ) composed of glutamic acid (E) and glutamine (Q) and 2,2'-dithiodipyridine were added to a round-bottom flask containing trifluoroacetic acid in a molar ratio of 7000:1. 30 -C and 2,2'-dithiodipyridine, and a zwitterionic polypeptide (EQ) 30- C and 2,2'-dithiodipyridine in a molar ratio of 1:5, stirring under N2for 20 min, and then sealing and reacting at room temperature for 4 h. After the reaction is completed, the solution is concentrated, and precipitated with cold diethyl ether to obtain a disulfide bond-containing polypeptide derivative ((EQ) 30 - C-SS-PY) in a round-bottom flask containing 3 mL of methanol, and then adding (EQ) 30 - C-SS-PY, PTX-SH, (EQ) 30 - C-SS-PY and PTX-SH in a molar ratio of 1:4.5, and then adding glacial acetic acid to adjust the pH to 4.7, stirring under N2for 20 min, and then sealing and reacting at room temperature for 12 h. After the reaction is completed, the solution is concentrated, and precipitated with cold petroleum ether to obtain a zwitterionic polypeptide-modified paclitaxel derivative ((EQ) 30 - C-SS-PTX).

[0111] (4) 1 mg of paclitaxel (PTX) was added to 1 mL of methanol to prepare a 1 mg / mL paclitaxel methanol solution, and 1 mg of chlorin e6 (Ce6) was added to 1 mL of methanol to prepare a 1 mg / mL chlorin e6 methanol solution. A 5 mg / mL (EQ) 30 - C-SS-PTX) was prepared, and then the above paclitaxel methanol solution, chlorin e6 methanol solution, and (EQ) 30 - C-SS-PTX methanol solution were mixed in a molar ratio of 1:1:5, and ultrasonicated at room temperature for 3 h in the dark. After the ultrasonication was completed, the mixture was added dropwise to 1.5 mL of water, and then dialyzed against water in a dialysis bag with a molecular weight cut-off of 500, using 0.5 L of water each time, for 8 h each time, for 3 times. After the dialysis was completed, (EQ) 30 - C-SS-PTX-PTX-Ce6 drug-loaded micelles were obtained.

[0112] Example 10

[0113] The present embodiment provides a zwitterionic polypeptide-modified paclitaxel derivative, and a prodrug nanodrug-loaded micelle formed by loading pipobroman and chlorin e6 therein, and specifically comprises:

[0114] (1) 3,3'-dithiodipropionic acid (118 mg, 0.56 mmol) and paclitaxel (1144 mg, 1.34 mmol) were added to a round-bottom flask containing 12 mL of dichloromethane. The flask was sealed and stirred at room temperature for 1 h. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (276 mg, 1.34 mmol) and 4-dimethylaminopyridine (28 mg, 0.22 mmol) were added, the flask was sealed, and the reaction was refluxed for 48 h. After the reaction was completed, the mixture was concentrated and purified by column chromatography to obtain the target product PTX-SS-PTX.

[0115] (2) PTX-SS-PTX (37 mg, 0.02 mmol) and dithiothreitol (7 mg, 0.04 mmol) were added to a round-bottom flask containing 1 mL of dichloromethane. The flask was sealed and stirred at room temperature for 1 h. Triethylamine (8 mg, 0.08 mmol) was then added to the reaction mixture. The mixture was sealed and refluxed under N2 for 12 h. After the reaction, the organic phase was removed and washed, dried, filtered, and concentrated. The concentrate was precipitated with petroleum ether to obtain the target product, PTX-SH.

[0116] (3) In a round-bottom flask containing trifluoroacetic acid, the molar ratio of trifluoroacetic acid to 2,2'-disulfide dipyridine is 4000:1, and a zwitterionic polypeptide (EN) containing glutamic acid (E) and asparagine (N) is added respectively. 10 -C and 2,2'-dithiodipyridine, zwitterionic peptide (EN) 10 The molar ratio of -C and 2,2'-disulfide dipyridine was 1:4.5, and N2 was stirred for 20 minutes, and then the mixture was sealed and reacted at room temperature for 4 hours. After the reaction was completed, the solution was concentrated and precipitated with cold ether to obtain a disulfide bond-containing polypeptide derivative ((EN) 10 -C-SS-PY). In a round-bottom flask containing 3 mL of methanol, (EN) 10 -C-SS-PY, PTX-SH, (EN) 10 The molar ratio of -C-SS-PY and PTX-SH was 1:4, and then glacial acetic acid was added to adjust the pH to 5.8, and N2 was stirred for 20 minutes, and then sealed and reacted at room temperature for 12 hours. After the reaction was completed, the solution was concentrated and precipitated with cold petroleum ether to obtain a paclitaxel derivative modified with a zwitterionic peptide ((EN) 10 -C-SS-PTX).

[0117] (4) 0.5 mg of palbociclib (PB) was added to 1 mL of methanol to prepare a 0.5 mg / mL palbociclib methanol solution, and 0.5 mg of chlorin e6 (Ce6) was added to 1 mL of methanol to prepare a 0.5 mg / mL chlorin e6 methanol solution. The 10 mg / mL (EN) 10 The methanol solution of C-SS-PTX, and then the above-mentioned palbociclib methanol solution, chlorin e6 methanol solution and (EN) 10 The C-SS-PTX methanol solution was mixed in a molar ratio of 1:2:6, and ultrasonicated for 3 h at room temperature in the dark. After ultrasonication, the mixture was added dropwise to 1.5 mL of water, and then dialyzed against water in a dialysis bag with a molecular weight cut-off of 500, using 0.5 L of water each time, for 8 h each time, for 3 times. After dialysis, (EN) 10 C-SS-PTX-PB-Ce6 drug-loaded micelles.

[0118] Example 11

[0119] The present example provides a zwitterionic polypeptide-modified paclitaxel derivative, and a prodrug nanodrug-loaded micelle formed by loading docetaxel therein, specifically comprising:

[0120] (1) 3,3'-dithiodipropionic acid (118 mg, 0.56 mmol) and paclitaxel (1144 mg, 1.34 mmol) were added to a round-bottom flask containing 12 mL of dichloromethane. After sealing, stirring was performed at room temperature for 1 h, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (276 mg, 1.34 mmol) and 4-dimethylaminopyridine (28 mg, 0.22 mmol) were added. After sealing, refluxing was performed for 48 h. After the reaction was completed, the mixture was concentrated, and column chromatography was performed to purify the same. After purification, the target product PTX-S-S-PTX was obtained.

[0121] (2) PTX-S-S-PTX (37 mg, 0.02 mmol) and dithiothreitol (7 mg, 0.04 mmol) were added to a round-bottom flask containing 1 mL of dichloromethane. After sealing, stirring was performed at room temperature for 1 h, and triethylamine (8 mg, 0.08 mmol) was added to the reaction mixture. After sealing, refluxing was performed under N2 conditions for 12 h. After the reaction was completed, the organic phase was removed and washed, dried, filtered and concentrated. Finally, the concentrated solution was precipitated by petroleum ether to obtain the target product PTX-SH.

[0122] (3) In a round bottom flask containing trifluoroacetic acid, the molar ratio of trifluoroacetic acid to 2,2'-dithiodipyridine was 3200:1, and the zwitterionic polypeptide (DK) composed of aspartic acid (D) and lysine (K) was added respectively 15 -C and 2,2'-dithiodipyridine, zwitterionic polypeptide (DK) 15 -C and 2,2'-dithiodipyridine, zwitterionic polypeptide (DK) 15 -C-SS-PY) In a round bottom flask containing 3 mL of methanol, (DK) 15 -C-SS-PY, PTX-SH, (DK) 15 -C-SS-PY and PTX-SH, and then 1 mL of glacial acetic acid was added to adjust the pH to 6.5, stirred for 20 min under N2, and then sealed and reacted at room temperature for 12 h. After the reaction was completed, the solution was concentrated, and precipitated with cold petroleum ether to obtain a zwitterionic polypeptide-modified paclitaxel derivative ((DK) 15 -C-SS-PTX).

[0123] (4) 1 mg of docetaxel (Taxotere, TX) was added to 1 mL of methanol to prepare a 1 mg / mL docetaxel methanol solution. 8 mg / mL of (DK) 15 -C-SS-PTX) was prepared, and then the above docetaxel methanol solution and (DK) 15 -C-SS-PTX) were mixed at a molar ratio of 1:20, and ultrasonicated for 3 h at room temperature in the dark. After ultrasonication, the mixture was added to 1.5 mL of water in a slow dripping manner, and then dialyzed against water in a dialysis bag with a molecular weight cutoff of 500, using 0.5 L of water each time, and dialyzed for 8 h each time, for a total of 3 times. After dialysis, (DK) 15 -C-SS-PTX-TX drug-loaded micelles.

[0124] Example 12

[0125] The present embodiment provides a zwitterionic polypeptide-modified paclitaxel derivative, and a prodrug nanodrug-loaded micelle formed by loading it with plinabulin, and specifically comprises:

[0126] (1) 3,3'-dithiodipropionic acid (118 mg, 0.56 mmol) and paclitaxel (1144 mg, 1.34 mmol) were added to a round-bottom flask containing 12 mL of dichloromethane. The flask was sealed and stirred at room temperature for 1 h. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (276 mg, 1.34 mmol) and 4-dimethylaminopyridine (28 mg, 0.22 mmol) were added, the flask was sealed, and the reaction was refluxed for 48 h. After the reaction was completed, the mixture was concentrated and purified by column chromatography to obtain the target product PTX-SS-PTX.

[0127] (2) PTX-SS-PTX (37 mg, 0.02 mmol) and dithiothreitol (7 mg, 0.04 mmol) were added to a round-bottom flask containing 1 mL of dichloromethane. The flask was sealed and stirred at room temperature for 1 h. Triethylamine (8 mg, 0.08 mmol) was then added to the reaction mixture. The mixture was sealed and refluxed under N2 for 12 h. After the reaction, the organic phase was removed and washed, dried, filtered, and concentrated. The concentrate was precipitated with petroleum ether to obtain the target product, PTX-SH.

[0128] (3) In a round-bottom flask containing trifluoroacetic acid, the molar ratio of trifluoroacetic acid to 2,2'-disulfide dipyridine is 6000:1, and zwitterionic peptides (DK) containing aspartic acid (D) and lysine (K) are added respectively. 20 -C and 2,2'-dithiodipyridine, zwitterionic peptide (DK) 20 The molar ratio of -C and 2,2'-disulfide dipyridine was 1:4.5, and N2 was stirred for 20 minutes, and then the mixture was sealed and reacted at room temperature for 4 hours. After the reaction was completed, the solution was concentrated and precipitated with cold ether to obtain a disulfide bond-containing polypeptide derivative ((DK) 20 -C-SS-PY). In a round-bottom flask containing 3 mL of methanol, (DK) 20 -C-SS-PY, PTX-SH, (DK) 20 The molar ratio of -C-SS-PY and PTX-SH was 1:4, and then glacial acetic acid was added to adjust the pH to 4.7, and N2 was stirred for 20 minutes, and then sealed and reacted at room temperature for 12 hours. After the reaction was completed, the solution was concentrated and precipitated with cold petroleum ether to obtain a paclitaxel derivative (DK) based on the modification of the zwitterionic peptide. 20 -C-SS-PTX).

[0129] (4) Prepare a 1 mg / mL palbociclib methanol solution by adding 1 mg of palbociclib (PB) to each mL of methanol. Prepare a 7 mg / mL (DK) solution. 20C-SS-PTX methanol solution, then the above pumiriclib methanol solution and (DK) 20 C-SS-PTX methanol solution was mixed at a molar ratio of 1:5, and ultrasonic treatment was performed under dark conditions at room temperature for 3 h. After the ultrasonic treatment was completed, the above mixture was added dropwise into 1.5 mL of water, and then dialysis was performed against water in a dialysis bag with a molecular weight cut-off of 500, using 0.5 L of water each time, for 8 h each time, for 3 times. After the dialysis was completed, (DK) 20 C-SS-PTX-PB drug-loaded micelles.

[0130] Example 13

[0131] The present example provides a zwitterionic polypeptide-modified paclitaxel derivative, and a prodrug nanodrug-loaded micelle formed by loading 10-hydroxycamptothecin therein, and specifically comprises:

[0132] (1) 3,3'-dithiodipropionic acid (118 mg, 0.56 mmol) and paclitaxel (1144 mg, 1.34 mmol) were added to a round-bottom flask containing 12 mL of dichloromethane. After sealing, stirring was performed at room temperature for 1 h, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (276 mg, 1.34 mmol) and 4-dimethylaminopyridine (28 mg, 0.22 mmol) were added, the flask was sealed, and reflux reaction was performed for 48 h. After the reaction was completed, the mixture was concentrated, and column chromatography was performed to purify the mixture, and the target product PTX-S-S-PTX was obtained after the purification.

[0133] (2) In a round-bottom flask containing 1 mL of dichloromethane, PTX-S-S-PTX (37 mg, 0.02 mmol) and dithiothreitol (7 mg, 0.04 mmol) were added. After sealing, stirring was performed at room temperature for 1 h, and then triethylamine (8 mg, 0.08 mmol) was added to the reaction mixture, the flask was sealed, and reflux reaction was performed under N2 conditions for 12 h. After the reaction was completed, the organic phase was removed and washed, dried, filtered, and concentrated, and finally the concentrated solution was precipitated by petroleum ether to obtain the target product PTX-SH.

[0134] (3) In a round-bottom flask containing trifluoroacetic acid, the molar ratio of the trifluoroacetic acid to 2,2'-dithiodipyridine was 9000:1, and a zwitterionic polypeptide (DN) 35 C and 2,2'-dithiodipyridine, zwitterionic polypeptide (DN) 35 C and 2,2'-dithiodipyridine, zwitterionic polypeptide (DN)35 -C-SS-PY) in a round bottom flask containing 3 mL of methanol. (DN) 35 -C-SS-PY, PTX-SH, (DN) 35 The molar ratio of -C-SS-PY and PTX-SH was 1:5, and then glacial acetic acid was added to adjust the pH to 4.5. After stirring for 20 min under N2, the reaction was sealed and reacted at room temperature for 12 h. After the reaction was completed, the solution was concentrated, and precipitated with cold petroleum ether to obtain a 10-hydroxycamptothecine derivative ((DN) 35 -C-SS-PTX).

[0135] (4) 1 mg of 10-hydroxycamptothecine (HPT) was added to 1 mL of methanol to prepare a 1 mg / mL 10-hydroxycamptothecine methanol solution. A 10 mg / mL (DN) 35 -C-SS-PTX solution in methanol, and then the above 10-hydroxycamptothecine methanol solution and (DN) 35 -C-SS-PTX methanol solution were mixed at a molar ratio of 1:2, and ultrasonicated for 3 h at room temperature in the dark. After ultrasonication, the mixture was added to 1.5 mL of water in a slow dripping manner, and then dialyzed against water in a dialysis bag with a molecular weight cutoff of 500. Each time, 0.5 L of water was used, and each dialysis was 8 h. The dialysis was repeated three times. After dialysis, (DN) 35 -C-SS-PTX-HPT drug-loaded micelles.

[0136] The following is an example of the preparation of the prodrug nanodrug-loaded micelles E-SS-P-DOX micelles prepared in Example 1, which are used to further illustrate the performance of the prodrug nanodrug-loaded micelles.

[0137] Experimental Example 1 In vivo tumor inhibition experiment in mice

[0138] I. Experimental method

[0139] Kunming female mice (body weight 18-22 g) were inoculated with cervical cancer cells (U14). U14 cells were inoculated into the abdominal cavity of healthy Kunming female mice, and after 5-7 days, the ascites was taken out under sterile conditions, and the cell number was diluted to 5 x 10 6U14 cells were subcutaneously injected into the right hind leg of Kunming female mice at a dose of 200 μL (2.5 mg / mL). Three days after tumor inoculation, the mice were randomly divided into five groups, each group of six mice, namely Saline group, free PTX group, free DOX group, E-SS-P group and E-SS-P-DOX group. The mice were administered by tail vein injection every other day at a dose of 200 μL (2.5 mg / mL, calculated by the concentration of DOX). The body weight and tumor size of the mice were recorded every day during the experiment. On the 15th day, the mice were sacrificed after anesthesia and photographed, and the tumors were dissected and weighed.

[0140] II. Experimental results

[0141] The in vivo tumor inhibition experiment results are shown in Figure 9 , Figure 10 and Figure 11 .

[0142] Figure 9 Figure 1 is the change in body weight of mice injected with the prepared prodrug nanocarrier micelles for 14 days in vivo. In the experiment, it can be observed that the body weight of mice injected with free DOX decreased significantly after the 8th day of administration, while the body weight of mice injected with E-SS-P-DOX micelles and normal saline did not show a significant downward trend. From the mouse weight experiment, it can be shown that the prepared E-SS-P-DOX micelles have lower toxicity compared to free doxorubicin.

[0143] Figure 10 Figure 2 is the change in tumor volume of mice injected with the prepared nanomicelles for 14 days in vivo. In the experiment, it can be observed that after 7 tail vein injections, as shown in the figure, the tumor volumes of the Saline, PTX, DOX, E-SS-P micelles and E-SS-P-DOX micelles groups were 1289.3 mm 3 , 789.3 mm 3 , 370.8 mm 3 , 569.3 mm 3 and 20.9 mm 3 , respectively. The tumor inhibition rate of the E-SS-P-DOX micelles group was 98.4%, which indicates that the E-SS-P-DOX micelles have good tumor inhibition performance.

[0144] Figure 11 Figure 3 is a photograph of the tumor site of mice injected with the prepared nanomicelles for 14 days in vivo. From Figure 10 and Figure 11 , it can be shown that the prodrug nanocarrier micelles have good tumor inhibition effect.

[0145] Experimental Example 2 Plasma clearance experiment

[0146] I. Experimental process:

[0147] The E-SS-P-DOX prodrug nanocarrier micelles prepared in Example 1 were used for plasma clearance experiment, which was mainly used to observe the content change of drug in the blood of mice over time. 150 μL of E-SS-P-DOX micelles and DOX (2.5 mg / kg (mass of DOX / mass of mouse)) containing normal saline were injected into the mice through the tail vein respectively. From the initial time of drug injection, 100 μL of blood samples were collected by the method of orbital blood collection at 2 min, 30 min, 1 h, 2 h, 4 h, 6 h, 9 h, 12 h and 24 h respectively. Then, the blood samples were centrifuged to separate the serum part of the supernatant. After that, the fluorescence intensity values of the serum samples were accurately measured and recorded by fluorescence spectroscopy at a specific excitation wavelength of 480 nm and an emission wavelength of 595 nm. The relative contents of E-SS-P-DOX micelles and DOX in the serum at the corresponding time were calculated.

[0148] II. Experimental results:

[0149] The results are shown in Figure 12 The graph shows that the prodrug nanocarrier micelles modified by the zwitterionic polypeptide have a longer blood circulation time in mice than free doxorubicin hydrochloride.

[0150] Example 3 in vitro release experiment

[0151] The E-SS-P-DOX prodrug nanocarrier micelles prepared in Example 1 were used for in vitro release experiment.

[0152] The E-SS-P-DOX micelles containing 100 μg of DOX were added to the dialysis bag with a molecular weight cut-off of 1500, and then the dialysis bag was immersed in PBS buffer solution with GSH concentrations of 0.1 mM, 1 mM and 5 mM respectively. Every 30 min, 2 mL of sample was taken out in PBS buffer solution with different GSH concentrations, and then the corresponding volume and GSH concentration of PBS buffer solution was added.

[0153] The results are shown in Figure 13 As shown in the graph, when the GSH concentration was 0.1 mM, 1.0 mM and 5.0 mM respectively, the cumulative release amount of E-SS-P-DOX micelles in 48 h was 20.6%, 46.2% and 81.3% respectively. As the GSH concentration increased from 0.1 mM to 1.0 mM and then to 5.0 mM, the cumulative release amount of E-SS-P-DOX micelles in 48 h increased from 20.6% to 46.2% and then to 81.3%, which indicated that E-SS-P-DOX micelles could be effectively reduced by GSH in the tumor microenvironment, thereby achieving the specific release of PTX and DOX. The experimental results showed that E-SS-P-DOX micelles had the characteristics of fast on-demand controlled release in tumor cells.

[0154] The above-described embodiments are merely intended to describe the preferred embodiments of the present application, and are not intended to limit the scope of the present application. Various changes and modifications made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the scope of protection of the present application.

Claims

1. A paclitaxel derivative modified with a zwitterionic peptide, characterized in that: The paclitaxel derivative modified with zwitterionic polypeptide has a structure as shown in the general formula (I): In the general formula (I), R1 and R2 are selected from the group consisting of amino acid residue side chains containing carboxyl groups and amino acid residue side chains containing amino groups, and the ratio of amino groups to carboxyl groups in R1 and R2 is 1:1; n represents 1 to 40; In the general formula (I), the amino acid residue side chains containing carboxyl groups are glutamic acid residues and aspartic acid residues; the amino acid residue side chains containing amino groups are lysine residues, histidine residues, arginine residues, glutamine residues and asparagine residues.

2. The method for preparing a paclitaxel derivative modified with a zwitterionic polypeptide according to claim 1, wherein: The preparation method comprises: In a polar solvent, a zwitterionic peptide containing a thiol group is mixed with 2,2'-disulfide dipyridine for reaction, and the reaction product is crystallized to obtain a peptide derivative containing a disulfide bond; Then, the disulfide bond-containing polypeptide derivative and thiolated paclitaxel are dissolved in a polar solvent at a molar ratio of 1:1.5-5, glacial acetic acid is added to adjust the pH to 4.5-6.5, and the mixture is stirred for reaction to obtain the zwitterionic polypeptide-modified paclitaxel derivative; The zwitterionic polypeptide containing a thiol group has a structure as shown in the general formula (II): The disulfide bond-containing polypeptide derivative has a structure as shown in general formula (III): In general formula (II) and general formula (III), R1 and R2 are selected from the group consisting of amino acid residue side chains containing carboxyl groups and amino acid residue side chains, and the ratio of amino groups to carboxyl groups in R1 and R2 is 1:1; n represents 1 to 40.

3. The method for preparing a paclitaxel derivative modified with a zwitterionic polypeptide according to claim 2, wherein: In the reaction for preparing the disulfide bond-containing polypeptide derivative, the molar ratio of the thiol-containing zwitterionic polypeptide to 2,2'-disulfide dipyridine is 1:1.5-5.

4. The method for preparing a paclitaxel derivative modified with a zwitterionic polypeptide according to claim 2, wherein: The step of mixing the thiol-containing zwitterionic polypeptide with 2,2'-dithiodipyridine for reaction specifically includes: first mixing 2,2'-dithiodipyridine with trifluoroacetic acid to form a mixed solution; and then adding the thiol-containing zwitterionic polypeptide to the mixed solution for reaction.

5. The method for preparing a paclitaxel derivative modified with a zwitterionic polypeptide according to claim 4, characterized in that: The molar ratio of trifluoroacetic acid to 2,2'-disulfide dipyridine is 10 to 10000:

1.

6. A prodrug nano-carrying micelle based on paclitaxel derivatives, characterized in that: The prodrug nano-drug-loaded micelle comprises the zwitterionic polypeptide-modified paclitaxel derivative according to claim 1, a hydrophobic anticancer drug and a polar solvent medium.

7. The method for preparing the paclitaxel derivative-based prodrug nano-micelles according to claim 6, characterized in that: The preparation method comprises: dissolving the zwitterionic peptide-modified paclitaxel derivative and the hydrophobic anticancer drug according to claim 1 or 2 in a polar solvent medium respectively to obtain a polar solvent containing the zwitterionic peptide-modified paclitaxel derivative and a polar solvent containing the hydrophobic anticancer drug; then self-assembling the polar solvent containing the zwitterionic peptide-modified paclitaxel derivative and the polar solvent containing the hydrophobic anticancer drug, and dialyzing to obtain the prodrug nano-carrier micelles based on the paclitaxel derivative.

8. The method for preparing the paclitaxel derivative-based prodrug nano-loaded micelles according to claim 7, characterized in that: The hydrophobic anticancer drug includes one or more of doxorubicin, paclitaxel, docetaxel, camptothecin, 10-hydroxycamptothecin, palbociclib, and photosensitizer chlorin e6.

9. The method for preparing the prodrug nano-micelles based on paclitaxel derivatives according to claim 8, characterized in that: The zwitterionic peptide-modified paclitaxel derivative is dissolved in methanol to form a paclitaxel derivative solution with a concentration of 4 to 10 mg / mL. The polar solution containing the hydrophobic anticancer drug and the paclitaxel derivative solution are then fully mixed at a molar ratio of 1:(2 to 20) and dialyzed to obtain the paclitaxel derivative-based prodrug nano-loaded micelles.

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