Prodrug molecule based on zwitterionic polyamide-amine dendrimer, nano drug-loaded micelle and preparation method and application thereof

Abstract

The invention provides a prodrug molecule based on a zwitterionic polyamide-amine dendrimer, a nano drug-loaded micelle as well as a preparation method and application of the prodrug molecule and the nano drug-loaded micelle. The preparation method of the nano drug-loaded micelle comprises the following steps: after PAMAM is modified by maleic anhydride, thiolated paclitaxel, c (RGDfC) and thiolated histidine are grafted on PAMAM through sulfydryl alkenyl reaction, and hydrophobic anti-cancer drugs are entrapped by utilizing the self-assembly characteristic of PAMAM to form the nano drug-loaded micelle. The nano drug-loaded micelle has the advantages of high drug loading capacity, small toxic and side effects, uniform particle size distribution, good stability, capability of enhancing the bioavailability of hydrophobic drugs and the like, has pH responsiveness, and can realize targeted release of the drugs at tumor parts, so that the tumor inhibition effect is enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical nano drug delivery systems, and specifically relates to a prodrug molecule based on zwitterionized polyamide-amine dendrimers, a nano drug-loaded micelle, and a preparation method and application thereof. Background Art

[0002] The mortality and morbidity of cancer are both very high. According to statistics, millions of people die of cancer every year. Cancer has become one of the diseases with the highest mortality rate in the world, seriously affecting human health and even life. Conventional treatments for cancer include surgery, chemotherapy, radiotherapy, immunotherapy, and molecular targeted drugs, among which chemotherapy plays an important role in the combined treatment of other therapies. However, most traditional chemotherapy drugs have low water solubility, poor selectivity, and no specificity for tumor cells, which can lead to dysfunction of the body.

[0003] Therefore, it is necessary to modify and improve chemotherapy drugs to reduce the secondary damage to patients during treatment. Researchers have proposed using nano drug delivery carriers to improve the above shortcomings. Nano drug delivery carriers can improve the solubility and bioavailability of poorly soluble drugs, improve drug targeting, achieve drug enrichment in specific organs or tissues, and achieve sustained or even controlled release of drugs, thereby extending the circulation time in the body, reducing side effects, and improving the safety of drugs, thereby improving the treatment effect, greatly promoting the application of chemotherapy drugs in tumor treatment.

[0004] Dendrimers are monodisperse polymers with highly branched structures. They have controllable particle sizes, regular shapes, and surface active functional groups that are easily modified. Therefore, they have good application prospects in drug delivery. The presence of a large number of amino groups at the end of polyamidoamine dendrimers (PAMAM), which are often used as nano drug delivery carriers, makes them water-soluble. However, as the number of branching generations increases, the more amino groups at the end, the stronger the cationicity. Excessive cationicity can easily cause cell membrane rupture and even cell apoptosis, causing toxicity to cells. Therefore, researchers modified the terminal groups on the surface of PAMAM using different molecules to reduce cytotoxicity while retaining the original advantages. For example, glycosylation, acetylation, pegylation or peptide modification of PAMAM can neutralize cations to reduce cytotoxicity.

[0005] Zwitterionic groups refer to groups that can carry both positive and negative charges under different pH conditions. Their charge characteristics are closely related to pH. Zwitterionic groups are widely present in a variety of natural and synthetic compounds, such as amino acids, polymers, surfactants, etc. Because their charge state changes with pH and is pH-sensitive, zwitterionic groups have important applications in a variety of fields, especially in biomedicine, materials science, environmental monitoring, etc. At present, the main way to encapsulate drugs in dendritic macromolecules is to use their internal cavities as guest encapsulation, but this method has a particularly limited drug loading capacity, a low drug loading rate, and no targeted response mechanism, which makes the drug release relatively limited. Therefore, after PAMAM is modified with zwitterionic groups, its charge state changes under different pH conditions can be used to optimize its drug delivery effect. The tumor microenvironment is usually acidic, and the normal physiological environment pH is close to 7.4. Zwitterionic groups are electrically neutral at near-neutral pH values, while under low pH conditions, the amino group will be protonated and the carboxyl group will remain undissociated, so that the group as a whole exhibits a positive charge. Therefore, zwitterion-modified PAMAM can be protonated in the acidic microenvironment of the tumor, achieving a potential reversal from negative to positive, showing a positive charge, thereby enhancing the interaction with the negatively charged cell membrane, and further promoting the responsive release and targeted delivery of the drug.

[0006] Therefore, it is very necessary to develop a nanomicelle that can improve the drug loading capacity and responsive release of the drug at the tumor site based on zwitterionic modification of dendrimers. Summary of the invention

[0007] The purpose of the present invention is to provide a prodrug molecule based on zwitterionized polyamidoamine dendrimers, a nano drug-carrying micelle, and a preparation method and application thereof. The nano micelle uses zwitterion-modified dendrimer PAMAM as a carrier and can encapsulate hydrophobic anticancer drugs to form nano drug-carrying micelles. The nano micelle uses the hydrophobic drug paclitaxel as the hydrophobic end during the zwitterion modification process, and uses the α-terminal peptide that can specifically recognize the overexpressed α-terminal peptide in tumor cells. V The targeting peptide of β3 integrin {tumor targeting peptide, c(RGDfC)} and pH-sensitive zwitterionic groups are used as hydrophilic ends to self-assemble. Then, another small molecule hydrophobic anticancer drug is physically embedded to obtain a dual-drug nano-carrier micelle, which has the advantages of high drug loading, low toxicity and side effects, and can enhance the bioavailability of hydrophobic drugs. It is also pH-responsive and can achieve targeted release of drugs at the tumor site, thereby enhancing the tumor inhibition effect.

[0008] In order to achieve the above-mentioned purpose of the present invention, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a prodrug molecule based on zwitterionic polyamidoamine dendrimers, wherein the prodrug molecule has a structure as shown in the general formula (I):

[0010]

[0011] In the general formula (I), a, b and c represent the amount of thiolated paclitaxel, tumor targeting peptide and thiolated histidine grafted on the polyamidoamine dendrimer, respectively, and the sum of a, b and c is the same as the total number of primary amino groups on the surface of the polyamidoamine dendrimer; wherein the polyamidoamine dendrimer is denoted as PAMAM, the thiolated paclitaxel is denoted as PTX-SH, the tumor targeting peptide is denoted as c(RGDfC), and the thiolated histidine is denoted as HIS-SH.

[0012] It should be noted that PAMAM includes first-generation polyamide-amine dendrimers (G1 PAMAM), second-generation polyamide-amine dendrimers (G2 PAMAM) and third-generation polyamide-amine dendrimers (G3 PAMAM); when PAMAM in the general formula (I) is specifically G1 PAMAM, the sum of a, b and c is 8; when PAMAM in the general formula (I) is specifically G2 PAMAM, the sum of a, b and c is 16; when PAMAM in the general formula (I) is specifically G3 PAMAM, the sum of a, b and c is 32.

[0013] The second aspect of the present invention provides a method for preparing a prodrug molecule based on the zwitterionic polyamide-amine dendrimer as described above, the preparation method comprising: S1. maleic anhydride treatment of PAMAM to obtain a first modified dendrimer, denoted as MM; S2. modifying MM with PTX-SH so that MM has a hydrophobic end, to obtain a second modified dendrimer, denoted as M-MP; S3. modifying M-MP with c(RGDfC) so that M-MP has a hydrophilic end that can target and recognize tumor cells, to obtain a third modified dendrimer, denoted as M-MPR; S4. modifying M-MPR with HIS-SH so that M-MPR has a pH-sensitive hydrophilic end, to obtain a prodrug molecule based on the zwitterionic polyamide-amine dendrimer, denoted as M-MPRH.

[0014] Furthermore, S1 specifically includes the following steps: S11. PAMAM and maleic anhydride are mixed in a molar ratio of 1:2 to 5, dissolved with dimethyl sulfoxide, and stirred at room temperature for 24 hours to obtain a first reaction solution; S12. The first reaction solution is dialyzed twice with the first dialysate replaced once every 6 to 8 hours, and then dialyzed twice with the second dialysate replaced once every 2 hours, and the dialysate is taken out and freeze-dried to obtain MM; wherein the first dialysate is a boric acid-borax buffer solution with a pH of 9, and the second dialysate is water; optionally, in S12, the dialysis bag used for dialysis has a molecular weight cutoff of 1500 to 7000.

[0015] Furthermore, S2 specifically includes the following steps: S21. 3,3'-dithiodipropionic acid and paclitaxel are dissolved in dichloromethane, stirred at room temperature for 1 hour, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine are added, and the mixture is refluxed at room temperature for 48 hours; after the reaction is completed, column chromatography is performed to purify and collect the product to obtain dithiodipropionic acid-esterified paclitaxel, which is recorded as PTX-SS-PTX; S22. PTX-SS-PTX and dithiothreitol (DTT) are co-dissolved in dichloromethane, stirred at room temperature for 1 hour, and then triethylamine is added, and the reaction is stirred under a N2 atmosphere. 12h; after the reaction is completed, the organic phase is collected, and then washed, dried, and concentrated, and the product is collected by centrifugation after precipitation with petroleum ether to obtain PTX-SH; S23. MM and PTX-SH are mixed in a molar ratio of 1:1 to 15, dissolved in methanol, and stirred at room temperature for 24 to 48h under N2 protection to obtain a second reaction solution; the second reaction solution is dialyzed with methanol as the dialysis medium at a frequency of changing once every 6h, and the dialyzate is collected after 3-6 dialysis times, and vacuum dried to obtain M-MP; optionally, in S23, the dialysis bag used for dialysis has a molecular weight cutoff of 2000 to 7000.

[0016] Furthermore, S3 specifically includes the following steps: mixing M-MP and c(RGDfC) in a molar ratio of 1:1 to 10, dissolving them in a mixed solvent of methanol and water in a volume ratio of 10:1, and stirring the mixture at room temperature for 24 hours under N2 protection to obtain a third reaction liquid; dialyzing the third reaction liquid with methanol as the dialysis medium at a frequency of changing it every 6 hours, collecting the dialysate after 4 dialysis, and vacuum drying to obtain M-MPR; optionally, in S3, the dialysis bag used for dialysis has a molecular weight cutoff of 2000 to 7000.

[0017] Further, S4 specifically includes the following steps: S41. Mixing mercaptoethylamine hydrochloride with triphenylmethane, dissolving with trifluoroacetic acid, and reacting with stirring at room temperature for 2 hours to obtain a fourth reaction solution; rotary evaporating and washing the fourth reaction solution, collecting the organic phase, and drying and concentrating to obtain 2-(triphenylmethylthio)ethylamine; S42. α -N τ -triphenylmethylhistidine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were mixed and dissolved in dichloromethane, and then triethylamine was added. After stirring at room temperature for 2 hours, 2-(tritylthio)ethylamine was added and stirred at room temperature for 12 hours. After the reaction, the white solid byproduct was filtered and the filtrate was collected. After rotary evaporation and concentration, it was purified by column chromatography. The product was collected and vacuum dried to obtain N'-(2-(tritylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ -triphenylmethylhistamine amide; S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ -Triphenylmethylhistamine amide is mixed with triisopropylsilane, trifluoroacetic acid is added and stirred in an ice bath for 2 hours. After the reaction is completed, most of the trifluoroacetic acid is removed by rotary evaporation, and then cold ether is used for precipitation. After centrifugation, the precipitate is dissolved in water and the insoluble matter is filtered out, and the filtrate is freeze-dried to obtain HIS-SH; S44. M-MPR and HIS-SH are mixed in a molar ratio of 1:1 to 30, dissolved in methanol, and stirred at room temperature for 24 to 48 hours under N2 protection to obtain a fifth reaction solution; the fifth reaction solution is dialyzed with methanol as the dialysis medium at a frequency of changing once every 6 hours, and the dialysate is collected after 3-6 dialysis times and vacuum dried to obtain M-MPRH; optionally, in S44, the dialysis bag used for dialysis has a molecular weight cutoff of 2000 to 7000.

[0018] The third aspect of the present invention provides the use of the above-mentioned prodrug molecules based on zwitterionic polyamidoamine dendrimers in the field of nano drug delivery systems.

[0019] The fourth aspect of the present invention provides a nano drug-loaded micelle, wherein the nano drug-loaded micelle comprises the above-mentioned prodrug molecule based on zwitterionic polyamidoamine dendrimers encapsulating a hydrophobic anticancer drug.

[0020] The fifth aspect of the present invention provides a method for preparing the above-mentioned nano drug-loaded micelles, which comprises using PAMAM as a carrier and a hydrophobic anti-tumor drug PTX-SH as a hydrophobic end, which can specifically recognize α overexpressed in tumor cells. VThe c(RGDfC) of β3 integrin and the pH-sensitive HIS-SH were used as the hydrophilic ends to self-assemble into a prodrug molecule based on zwitterionic polyamidoamine dendrimers, and then another hydrophobic anticancer drug was physically encapsulated to obtain nano-drug-loaded micelles.

[0021] Furthermore, the preparation method comprises: S5. preparing nano drug-loaded micelles by encapsulating hydrophobic anticancer drugs with M-MPRH; S5 specifically comprises the following steps: S51. dissolving the hydrophobic anticancer drug in methanol to prepare a hydrophobic anticancer drug methanol solution; S52. dissolving M-MPRH in methanol to prepare an M-MPRH methanol solution; S53. mixing the M-MPRH methanol solution and the hydrophobic anticancer drug methanol solution in a mass ratio of 5 to 20:1, stirring at room temperature in the dark; 2 to 6 hours to obtain a sixth reaction liquid; S54. The sixth reaction liquid is slowly dripped into water for dialysis, the dialysis medium is water, and the dialysis is performed 3 times with a frequency of changing once every 6 hours. After the dialysis, the dialysate is collected to obtain nano-drug-loaded micelles; Optionally, in S52, the hydrophobic anticancer drugs include at least one of doxorubicin, camptothecin, fluorouracil, and dihydrochlorin e6; Optionally, in S54, the mass ratio of the reaction liquid to water is 1:0.5 to 3, and the dripping speed into the water is 0.5 to 1 mL / min.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects:

[0023] 1. The present invention provides a PAMAM carrier modified with a pH-sensitive zwitterionic group, which uses the anticancer drug paclitaxel as a hydrophobic end and a α- V The c(RGDfC) of β3 integrin and the pH-sensitive zwitterionic group serve as the hydrophilic end, so that it can form a prodrug molecule based on zwitterionic polyamidoamine dendrimers; and can physically embed hydrophobic anticancer drugs during the self-assembly process, thereby preparing a new type of nano-drug-carrying micelle;

[0024] 2. The prodrug molecules and nano-drug-loaded micelles based on zwitterionic polyamidoamine dendrimers provided by the present invention can specifically recognize α- V β3 integrin, thereby achieving targeting to tumor sites;

[0025] 3. The hydrophilic end of the prodrug molecule based on zwitterionic polyamidoamine dendrimer and the nano-drug-carrying micelle provided by the present invention contains pH-sensitive histidine, which can achieve the reversal of the surface potential from negative to positive at the tumor site;

[0026] 4. The nano drug-loaded micelles provided by the present invention have uniform particle size and high drug loading capacity;

[0027] 5. The drug-loaded nanoparticles provided by the present invention have good tumor inhibition effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the preparation of prodrug molecules and nano-drug-loaded micelles based on zwitterionic polyamidoamine dendrimers according to embodiment 1 of the present invention;

[0029] Figure 2 The hydrogen nuclear magnetic resonance spectra of PAMAM, MM, M-MP, M-MPR and M-MPRH obtained in Example 1 of the present invention;

[0030] Figure 3 The critical micelle concentration value of the M-MPRH@DOX nano-drug-loaded micelles in aqueous solution obtained in Example 1 of the present invention;

[0031] Figure 4 This is the UV-visible absorption spectrum of the M-MPRH@DOX nano-drug loaded micelles obtained in Example 1 of the present invention;

[0032] Figure 5 This is a fluorescence spectrum of the M-MPRH@DOX nanodrug-loaded micelles obtained in Example 1 of the present invention;

[0033] Figure 6 This is a transmission electron microscopy image of the M-MPRH@DOX nanodrug-loaded micelles obtained in Example 1 of the present invention;

[0034] Figure 7 The transmission electron microscopy image of the particle size distribution histogram of the M-MPRH@DOX nano-drug-loaded micelles obtained in Example 1 of the present invention;

[0035] Figure 8 This is a particle size distribution diagram of the M-MPRH@DOX nano-drug-loaded micelles obtained in Example 1 of the present invention in a phosphate buffer solution with a pH of 5.5 to 7.4;

[0036] Fig. 9 The Zeta potential diagram of the M-MPRH@DOX nano-drug-loaded micelles obtained in Example 1 of the present invention in a phosphate buffer solution with a pH of 5.5 to 7.4;

[0037] Fig.10 The particle size distribution diagram of the interaction between M-MPRH micelles and proteins obtained in Example 1 of the present invention;

[0038] Fig.11 This is a particle size distribution diagram of the interaction between M-MPRH@DOX nano-drug loaded micelles and proteins obtained in Example 1 of the present invention;

[0039] Fig.12This is a drug release curve of the M-MPRH@DOX nano-drug-loaded micelles obtained in Example 1 of the present invention in a phosphate buffer solution with a pH of 5.5 to 7.4;

[0040] Fig.13 This is a graph showing the weight of mice in an in vivo tumor inhibition experiment of the M-MPRH@DOX nano-drug-loaded micelles obtained in Example 1 of the present invention;

[0041] Fig.14 This is a real picture of mouse tumors in the tumor inhibition experiment of mice with M-MPRH@DOX nano-drug-loaded micelles obtained in Example 1 of the present invention;

[0042] Fig.15 This is a diagram of mouse tumor volume in an in vivo tumor inhibition experiment of the M-MPRH@DOX nano-drug-loaded micelles obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0043] The following will describe the implementation of the present invention in conjunction with embodiments with reference to the accompanying drawings.

[0044] Specifically, the present invention provides a method for preparing nano drug-loaded micelles that use zwitterion-modified dendrimer PAMAM as a carrier and can encapsulate hydrophobic anticancer drugs, such as Figure 1 As shown, it specifically includes the following steps:

[0045] S1. Maleic anhydride treatment of PAMAM to obtain MM, the specific steps are as follows:

[0046] S11. PAMAM and maleic anhydride are mixed in a molar ratio of 1:2 to 5, dissolved in dimethyl sulfoxide, and stirred at room temperature for 24 hours to obtain a first reaction solution;

[0047] S12. The first reaction liquid is dialyzed twice with the first dialysate at a frequency of changing once every 6 to 8 hours, and then the dialysate is dialyzed twice with the second dialysate at a frequency of changing once every 2 hours, and the dialysate is taken out and freeze-dried to obtain MM; wherein the first dialysate is a boric acid-borax buffer solution with a pH of 9, and the second dialysate is water;

[0048] Optionally, in S12, the molecular weight cutoff of the dialysis bag used for dialysis is 1500-7000.

[0049] S2. Modify MM with PTX-SH to give MM a hydrophobic end to obtain M-MP. The specific steps are as follows:

[0050] S21. 3,3'-dithiodipropionic acid (58.79 mg, 0.280 mmol) and paclitaxel (573.00 mg, 0.671 mmol) were dissolved in dichloromethane, stirred at room temperature for 1 h, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (128.63 mg, 0.671 mmol) and 4-dimethylaminopyridine (13.66 mg, 0.112 mmol) were added, and refluxed at room temperature for 48 h; after the reaction was completed, column chromatography was performed to purify and collect the product to obtain dithiodipropionic acid-esterified paclitaxel, which was recorded as PTX-SS-PTX;

[0051] S22. PTX-SS-PTX (292 mg, 0.155 mmol) and DTT (47.86 mg, 0.310 mmol) were dissolved in dichloromethane, stirred at room temperature for 1 h, and then triethylamine (86 μL, 0.621 mmol) was added, and the mixture was stirred under N2 atmosphere for 12 h; after the reaction, the organic phase was collected, washed, dried, and concentrated, and then the product was collected by centrifugation after precipitation with petroleum ether to obtain PTX-SH;

[0052] S23. MM and PTX-SH are mixed in a molar ratio of 1:1 to 15, dissolved in methanol, and reacted at room temperature under N2 protection for 24 to 48 hours to obtain a second reaction solution; the second reaction solution is dialyzed with methanol as the dialysis medium at a frequency of changing once every 6 hours, and the dialyzate is collected after 3 to 6 times of dialysis, and vacuum dried to obtain M-MP;

[0053] Optionally, in S23, the molecular weight cutoff of the dialysis bag used for dialysis is 2000-7000.

[0054] S3. Use c(RGDfC) to modify M-MP so that M-MP has a hydrophilic end that can target and recognize tumor cells, thereby obtaining M-MPR. The specific steps are as follows:

[0055] M-MP and c(RGDfC) were mixed in a molar ratio of 1:1 to 10, dissolved in a mixed solvent of methanol and water in a volume ratio of 10:1, and reacted with stirring at room temperature for 24 hours under N2 protection to obtain a third reaction liquid; the third reaction liquid was dialyzed with methanol as the dialysis medium at a frequency of changing once every 6 hours, and the dialysate was collected after 4 dialysis, and vacuum dried to obtain M-MPR.

[0056] Optionally, in S3, the molecular weight cutoff of the dialysis bag used for dialysis is 2000-7000.

[0057] S4. M-MPR is modified with HIS-SH to provide M-MPR with a pH-sensitive hydrophilic end to obtain M-MPRH. The specific steps are as follows:

[0058] S41. Mercaptoethylamine hydrochloride (2.30 g, 20.22 mmol) and triphenylmethane (5.64 g, 20.22 mmol) were mixed, dissolved with trifluoroacetic acid, and stirred at room temperature for 2 h to obtain a fourth reaction solution; the fourth reaction solution was subjected to rotary evaporation and washing, and the organic phase was collected and dried and concentrated to obtain 2-(triphenylmethylthio)ethylamine;

[0059] S42. N α -N τ -Triphenylmethylhistidine (800.00 mg, 2.50 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (480.06 mg, 2.50 mmol) and N-hydroxysuccinimide (288.20 mg, 2.50 mmol) were mixed and dissolved in dichloromethane, and then triethylamine (1.04 mL, 7.51 mmol) was added. After stirring at room temperature for 2 h, 2-(tritylthio)ethylamine (996.85 mg, 2.00 mmol) was added and stirred at room temperature for 12 h. After the reaction was completed, the white solid byproduct was filtered and the filtrate was collected. After rotary evaporation and concentration, it was purified by column chromatography. The product was collected and dried in vacuo to obtain N'-(2-(tritylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ -Triphenylmethylhistamine amide;

[0060] S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ -Triphenylmethylhistamine amide (150.00 mg, 0.188 mmol) was mixed with triisopropylsilane (240 μL, 1.17 mmol), and 6 mL of trifluoroacetic acid was added, followed by stirring in an ice bath for 2 h. After the reaction was completed, most of the trifluoroacetic acid was removed by rotary evaporation, and then cold ether was used for precipitation. After centrifugation, the precipitate was dissolved in water, and the insoluble matter was filtered out. The filtrate was freeze-dried to obtain HIS-SH;

[0061] S44. M-MPR and HIS-SH were mixed at a molar ratio of 1:1 to 30, dissolved in methanol, and stirred at room temperature for 24 to 48 hours under N2 protection to obtain a fifth reaction solution; the fifth reaction solution was dialyzed with methanol as the dialysis medium at a frequency of changing once every 6 hours, and the dialyzate was collected after 3-6 times of dialysis, and vacuum dried to obtain M-MPRH;

[0062] Optionally, in S44, the molecular weight cutoff of the dialysis bag used for dialysis is 2000-7000.

[0063] S5. Preparation of nano-drug loaded micelles by encapsulating hydrophobic anticancer drugs with M-MPRH, the specific steps are as follows:

[0064] S51. dissolving the hydrophobic anticancer drug in methanol to prepare a hydrophobic anticancer drug methanol solution having a concentration of 0.1 to 0.5 mg / mL;

[0065] S52. dissolving M-MPRH in methanol to prepare a 0.5-5 mg / mL M-MPRH methanol solution;

[0066] S53. The M-MPRH methanol solution and the hydrophobic anticancer drug methanol solution are mixed in a ratio of 5 to 20:1 of M-MPRH: hydrophobic anticancer drug mass ratio, and stirred at room temperature in the dark for 2 to 6 hours to obtain a sixth reaction solution;

[0067] S54. The sixth reaction solution was slowly dripped into water for dialysis, the dialysis medium was water, and the dialysis was performed 3 times at a frequency of replacement every 6 hours. After the dialysis, the dialysate was collected to obtain nano-drug-loaded micelles;

[0068] Optionally, in S52, the hydrophobic anticancer drug includes at least one of doxorubicin (DOX), camptothecin, fluorouracil, and chlorin e6;

[0069] Optionally, in S54, the mass ratio of the reaction liquid to water is 1:0.5-3, and the dripping speed into the water is 0.5-1 mL / min.

[0070] Example 1

[0071] S1. Maleic anhydride treatment of G1 PAMAM, the specific steps are as follows:

[0072] S11. G1 PAMAM and maleic anhydride were mixed in a molar ratio of 1:2 according to the primary amino group of PAMAM and maleic anhydride, dissolved in dimethyl sulfoxide, and stirred at room temperature for 24 hours;

[0073] S12. After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 1500 for dialyzing, the dialysis medium is a boric acid-borax buffer solution with a pH of 9, and the dialysis solution is replaced every 6-8 hours. After dialysis with the above buffer solution for 2 times, the dialysis medium is replaced with water, and the dialysis solution is replaced every 2 hours. After dialysis for 2 times, the dialysis is terminated, and the dialysis solution is taken out and freeze-dried to obtain MM;

[0074] S2. Modify MM with PTX-SH to synthesize M-MP so that it has a hydrophobic end. The specific steps are as follows:

[0075] S21. 3,3'-dithiodipropionic acid (58.79 mg, 0.280 mmol) and paclitaxel (573.00 mg, 0.671 mmol) were dissolved in dichloromethane, stirred at room temperature for 1 h, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (128.63 mg, 0.671 mmol) and 4-dimethylaminopyridine (13.66 mg, 0.112 mmol) were added thereto, and refluxed at room temperature for 48 h. After the reaction was completed, it was purified by column chromatography with a mobile phase of ethyl acetate: n-hexane = 2:1, and R f =0.36, and vacuum dried to obtain the product PTX-SS-PTX;

[0076] S22. PTX-SS-PTX (292 mg, 0.155 mmol) and DTT (47.86 mg, 0.310 mmol) were dissolved in dichloromethane, stirred at room temperature for 1 h, and triethylamine (86 μL, 0.621 mmol) was added thereto, and stirred under N2 atmosphere for 12 h. After the reaction, the organic phase was collected, washed, dried, concentrated, and precipitated with petroleum ether, and the product PTX-SH was collected by centrifugation;

[0077] S23. MM and PTX-SH were mixed at a molar ratio of 1:4, dissolved in methanol, and stirred at room temperature for 24 hours under N2 protection. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 2000 for dialysis. The dialysis medium was methanol. The dialysis solution was replaced every 6 hours. After dialysis 4 times, the dialysis solution was collected and vacuum dried to obtain the product M-MP;

[0078] S3. Use c(RGDfC) to modify M-MP to synthesize M-MPR, so that it has a hydrophilic end that can target and recognize tumor cells. The specific steps are to mix M-MP and c(RGDfC) at a molar ratio of 1:2, dissolve in a mixed solvent of methanol and water with a volume ratio of 10:1, and stir at room temperature under N2 protection for 24 hours. After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 2000 for dialysis. The dialysis medium is methanol. The dialysis solution is replaced every 6 hours. After dialysis 4 times, the dialysis solution is collected and vacuum dried to obtain the product M-MPR;

[0079] S4. M-MPR is modified with HIS-SH to synthesize M-MPRH so that it has a pH-sensitive hydrophilic end. The specific steps are as follows:

[0080] S41. Mercaptoethylamine hydrochloride (2.30 g, 20.22 mmol) and triphenylmethane (5.64 g, 20.22 mmol) were mixed, dissolved with trifluoroacetic acid, and stirred at room temperature for 2 h. After the reaction, the reaction solution was rotary evaporated and washed, and the organic phase was collected and dried and concentrated to obtain the product 2-(triphenylmethylthio)ethylamine;

[0081] S42. N α -N τ -Triphenylmethylhistidine (800.00 mg, 2.50 mmol), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (480.06 mg, 2.50 mmol) and N-hydroxysuccinimide (288.20 mg, 2.50 mmol) were mixed and dissolved in dichloromethane, and triethylamine (1.04 mL, 7.51 mmol) was added thereto. After stirring at room temperature for 2 h, 2-(triphenylmethylthio)ethylamine (996.85 mg, 2.00 mmol) was added thereto. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the white solid byproduct was filtered and the filtrate was collected. After rotary evaporation and concentration, column chromatography was used for purification. The mobile phase was ethyl acetate: dichloromethane = 1:1. R f = 0.6, and vacuum dried to obtain the product N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ -Triphenylmethylhistamine amide;

[0082] S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ -Triphenylmethylhistamine amide (150.00 mg, 0.188 mmol) was mixed with triisopropylsilane (240 μL, 1.17 mmol), and 6 mL of trifluoroacetic acid was added and stirred in an ice bath for 2 h. After the reaction was completed, most of the trifluoroacetic acid was removed by rotary evaporation, and then cold ether was used for precipitation. After centrifugation, the precipitate was dissolved in water and the insoluble matter was filtered out. The filtrate was freeze-dried to obtain the product N'-(2-mercaptoethyl)histamine amide (HIS-SH);

[0083] S44. M-MPR and HIS-SH were mixed at a molar ratio of 1:10, dissolved in methanol, and stirred at room temperature for 24 hours under N2 protection. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 2000 for dialysis. The dialysis medium was methanol. The dialysis solution was replaced every 6 hours. After dialysis 4 times, the dialysis solution was collected and vacuum dried to obtain the product M-MPRH;

[0084] S5. Prepare nano-drug-loaded micelles by encapsulating the hydrophobic anticancer drug DOX with M-MPRH, and the specific steps are as follows:

[0085] S51. Dissolve 500 μg of DOX hydrochloride in 1 mL of methanol, add 20 μL of triethylamine, and sonicate for 1 h in the dark to obtain a desalted DOX methanol solution;

[0086] S52. Dissolve M-MPRH in methanol to prepare a 5 mg / mL M-MPRH methanol solution;

[0087] S53. Mix the M-MPRH methanol solution and the DOX methanol solution at a mass ratio of 10:1 between M-MPRH and DOX, and stir at room temperature in the dark for 3 h;

[0088] S54. After the reaction is completed, the reaction solution is slowly dripped into water at a rate of 0.5 mL / min, with a volume ratio of reaction solution to water of 1:1. The solution is placed in a dialysis bag with a molecular weight cutoff of 500 for dialyzing. The dialysis medium is water. The dialysis solution is replaced every 6 hours for a total of 3 times. After the dialysis is completed, the dialysis solution is collected to obtain nano-drug-loaded micelles, specifically M-MPRH@DOX nano-drug-loaded micelles.

[0089] The hydrogen spectrum of M-MPRH is as follows Figure 2 As shown, the chemical shift at 7.3-8.2ppm in M-MP corresponds to the characteristic peak of PTX-SH, and compared with MM, it can be seen that the peak area at 5.8-6.4ppm is reduced, which further proves that PTX-SH is successfully modified to MM. In addition, 7.1-7.4ppm corresponds to the characteristic hydrogen of c(RGDfC), and the two sharp peaks at 7.3-7.4ppm and 8.5-8.7ppm correspond to the characteristic hydrogen of HIS-SH, and the peak corresponding to olefin hydrogen at 5.8-6.4ppm in M-MPRH completely disappears, which also proves that the double bond on MM is completely added, further proving the successful synthesis of M-MPRH. The drug loading rate of paclitaxel is 27.25%.

[0090] The critical micelle concentration of M-MPRH was determined by fluorescence spectrophotometer. Figure 3 As shown, the inflection point of the curve is the critical micelle concentration of M-MPRH micelles in aqueous solution. The calculated critical micelle concentration of M-MPRH micelles is 4.98 μg / mL. The size of the critical micelle concentration is related to the ratio of the hydrophilic and hydrophobic ends of the micelles. In M-MPRH, the hydrophilic end is the first generation polyamide-amine dendrimer modified by c(RGDfC) and HIS-SH, and the hydrophobic end is PTX-SH. The molar ratio of the two is 3:1.

[0091] UV-visible spectrophotometry was used to scan the full wavelength of DOX and M-MPRH@DOX nanodrug-loaded micelles. Figure 4As shown, the maximum absorption peak of DOX is 480nm, and the maximum absorption peak of the drug-loaded micelles is at 494nm. The two peak shapes are similar, and the absorption peak of DOX is red-shifted, indicating that M-MPRH successfully encapsulated DOX to form M-MPRH@DOX nano-drug-loaded micelles.

[0092] The DOX and M-MPRH@DOX nano-drug loaded micelles were scanned by fluorescence spectrophotometer at an excitation wavelength of 480 nm and an emission wavelength of 498 nm. Figure 5 As shown, the maximum excitation wavelength of both was 596nm, and the peak shape was similar, which further proved that M-MPRH successfully encapsulated DOX. The drug loading rate of DOX was 8.58%, so the total drug loading rate of the drug-loaded micelles reached 35.83%.

[0093] Transmission electron microscopy was used to characterize the morphology of drug-loaded micelles. Figure 6 , Figure 7 As shown, the drug-loaded micelles have a relatively regular morphology, a relatively uniform particle size distribution, and an average particle size of 24.03 nm.

[0094] Laser particle size analyzer was used to measure the effect of different pH on the particle size of M-MPRH micelles and M-MPRH@DOX nano-drug loaded micelles. Figure 8 As shown in the figure, at pH = 7.4, 6.5, and 5.5, the hydrodynamic particle sizes of M-MPRH micelles are 81.71±1.98nm, 93.06±6.48nm, and 114.98±22.11nm, respectively, and the hydrodynamic particle sizes of M-MPRH@DOX nano-drug-loaded micelles are 92.83±5.78nm, 112.73±11.92nm, and 147.07±4.94nm, respectively. As the pH decreases, the hydrodynamic particle size of the micelles gradually increases. This is mainly because the imidazole ring of histidine is pH-sensitive, which makes it protonated and positively charged in an acidic environment, thereby destroying the charge balance on the surface of the micelles, causing the micelles to dissociate and increase the particle size. This result shows that M-MPRH@DOX nano-drug-loaded micelles have pH responsiveness in the acidic microenvironment of tumor cells, which is conducive to achieving the characteristics of rapid drug release.

[0095] The effect of different pH values ​​on the potential of M-MPRH micelles and M-MPRH@DOX nano-drug-loaded micelles was determined by laser particle size analyzer. Fig. 9As shown in the figure, at pH = 7.4, 6.5, and 5.5, the Zeta potential of M-MPRH micelles was -5.65±1.16mV, 1.52±0.72mV, and 9.43±1.96mV, respectively, and the Zeta potential of M-MPRH@DOX nano-drug-loaded micelles was -10.59±1.23mV, 0.66±0.24mV, and 5.41±0.37mV, respectively. The above results show that compared with the pH of normal tissues, M-MPRH micelles and M-MPRH@DOX nano-drug-loaded micelles can achieve a surface potential reversal from negative to positive in the weakly acidic environment of tumor tissues. The positively charged micelles can have a strong electrostatic attraction with the negatively charged tumor cell membranes. Therefore, this effect can enhance the cell uptake of micelles to a certain extent, thereby delivering more anti-tumor drugs to the tumor site, and according to Figure 8 As is known, it can dissociate micelles to achieve specific accumulation of the encapsulated drugs at the tumor site. The reason for the above results is that at pH = 7.4, the partial dissociation of the carboxyl groups on the surface of M-MPRH makes the micelles negatively charged. When the pH is lowered, the dissociation of the carboxyl groups in the weakly acidic environment is inhibited, the protonation of the amino groups in the molecules is enhanced, and the protonation of the imidazole ring in the histidine is enhanced, so that the potential of the micelles is reversed and the band is positively charged.

[0096] The stability of M-MPRH micelles and M-MPRH@DOX nano-drug-loaded micelles in protein solution was determined by laser particle size analyzer. Fig.10 As shown in the figure, the hydrodynamic particle size of 0.5 mg / mL fibrinogen is about 26.28 nm, the hydrodynamic particle size of 1 mg / mL M-MPRH micelle is about 80.71 nm, and the hydrodynamic particle size of the mixed solution of 0.5 mg / mL fibrinogen + 1 mg / mL M-MPRH is about 50.18 nm. The hydrodynamic particle size of the M-MPRH mixed solution is between the two, indicating that there is no aggregation between M-MPRH and protein. Fig.11 As shown in the figure, the hydrodynamic particle size of 1 mg / mL M-MPRH@DOX nano-drug-loaded micelles is about 98.24 nm, and the hydrodynamic particle size of the mixed solution of 0.5 mg / mL fibrinogen + 1 mg / mL M-MPRH@DOX nano-drug-loaded micelles is about 69.63 nm. The hydrodynamic particle size of the mixed solution of M-MPRH@DOX nano-drug-loaded micelles is also between the two, which also shows that there is no aggregation between it and the protein, indicating that it has good stability in the protein solution.

[0097] The fluorescence intensity of M-MPRH@DOX nano-drug-loaded micelles at pH = 7.4, 6.5, and 5.5 was measured every 1 h by using a fluorescence spectrophotometer to calculate the cumulative drug release. Fig.12As shown in the figure, at pH = 7.4, 6.5, and 5.5, the cumulative release of M-MPRH@DOX nano-drug-loaded micelles in 27 h were 28.68%, 43.46%, and 70.68%, respectively. As the pH decreased, the cumulative drug release gradually increased, indicating that M-MPRH@DOX nano-drug-loaded micelles are pH-sensitive and can achieve specific drug release in the acidic microenvironment of the tumor.

[0098] The M-MPRH@DOX nano-drug-loaded micelles were used for tumor inhibition experiments in mice. Fig.13 As shown, 14 days after administration, the average body weight of mice in the normal saline group was 30.12 g, the average body weight of mice in the free DOX group was 23.04 g, and the average body weight of mice in the M-MPRH@DOX nanodrug-loaded micelle group was 31.02 g. By comparison, the body weight of mice in the M-MPRH@DOX nanodrug-loaded micelle group was similar to that in the normal saline group, and much higher than that in the free DOX group, indicating that the drug-loaded micelles have lower biological toxicity than free DOX. Fig.14 This is a picture of the actual mouse tumor obtained after 14 days of drug treatment. Fig.14 It can be seen that the tumor volume of mice in the M-MPRH@DOX nano-drug-loaded micelle group was the smallest. It can be seen that the tumors of mice treated with drug-loaded micelles were significantly inhibited compared with those in the normal saline group, and the tumor inhibition effect was better than that of the free DOX group. Fig.15 The average tumor volume of all mice during the 14-day experimental period is shown in Figure 2. Fig.15 As shown in Figure 1, at day 14, the average tumor volume of mice in the saline group without drug treatment was approximately 1229.81 mm 3 The average tumor volume of mice in the free DOX group was approximately 377.68 mm 3 The average tumor volume of mice in the M-MPRH@DOX nano-drug-loaded micelle group was about 87.19 mm 3 The calculated tumor inhibition rate of the drug-loaded micelle group was 92.91%, which was higher than the 69.29% of the free DOX group. Fig.14 In response, it was proved that M-MPRH@DOX nanodrug-loaded micelles had good anti-tumor effect.

[0099] Example 2

[0100] S1. Maleic anhydride treatment of G2 PAMAM, the specific steps are as follows:

[0101] S11. G2 PAMAM and maleic anhydride were mixed in a molar ratio of 1:3 according to the primary amino group of PAMAM and maleic anhydride, dissolved in dimethyl sulfoxide, and stirred at room temperature for 24 hours;

[0102] S12. After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 3500 for dialyzing, the dialysis medium is a boric acid-borax buffer solution with a pH of 9, and the dialysis solution is replaced every 8 hours. After dialysis with the above buffer solution for 2 times, the dialysis medium is replaced with water, and the dialysis solution is replaced every 2 hours. After dialysis for 2 times, the dialysis is terminated, and the dialysis solution is taken out and freeze-dried to obtain MM;

[0103] S2. Modify MM with PTX-SH to synthesize M-MP so that it has a hydrophobic end. The specific steps are as follows:

[0104] S21. 3,3'-dithiodipropionic acid (58.79 mg, 0.280 mmol) and paclitaxel (573.00 mg, 0.671 mmol) were dissolved in dichloromethane, stirred at room temperature for 1 h, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (128.63 mg, 0.671 mmol) and 4-dimethylaminopyridine (13.66 mg, 0.112 mmol) were added thereto, and refluxed at room temperature for 48 h. After the reaction was completed, it was purified by column chromatography with a mobile phase of ethyl acetate: n-hexane = 2:1, and R f =0.36, and vacuum dried to obtain the product PTX-SS-PTX;

[0105] S22. PTX-SS-PTX (292 mg, 0.155 mmol) and DTT (47.86 mg, 0.310 mmol) were dissolved in dichloromethane, stirred at room temperature for 1 h, and triethylamine (86 μL, 0.621 mmol) was added thereto, and stirred under N2 atmosphere for 12 h. After the reaction, the organic phase was collected, washed, dried, concentrated, and precipitated with petroleum ether, and the product PTX-SH was collected by centrifugation;

[0106] S23. MM and PTX-SH were mixed at a molar ratio of 1:8, dissolved in methanol, and stirred at room temperature for 24 hours under N2 protection. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 for dialysis. The dialysis medium was methanol. The dialysis solution was replaced every 6 hours. After dialysis 4 times, the dialysis solution was collected and vacuum dried to obtain the product M-MP;

[0107] S3. Use c(RGDfC) to modify M-MP to synthesize M-MPR, so that it has a hydrophilic end that can target and recognize tumor cells. The specific steps are to mix M-MP and c(RGDfC) at a molar ratio of 1:4, dissolve in a mixed solvent of methanol and water with a volume ratio of 10:1, and stir at room temperature under N2 protection for 24 hours. After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 3500 for dialysis. The dialysis medium is methanol. The dialysis solution is replaced every 6 hours. After dialysis 4 times, the dialysis solution is collected and vacuum dried to obtain the product M-MPR;

[0108] S4. M-MPR is modified with HIS-SH to synthesize M-MPRH so that it has a pH-sensitive hydrophilic end. The specific steps are as follows:

[0109] S41. Mercaptoethylamine hydrochloride (2.30 g, 20.22 mmol) and triphenylmethane (5.64 g, 20.22 mmol) were mixed, dissolved with trifluoroacetic acid, and stirred at room temperature for 2 h. After the reaction, the reaction solution was rotary evaporated and washed, and the organic phase was collected and dried and concentrated to obtain the product 2-(triphenylmethylthio)ethylamine;

[0110] S42. N α -N τ -Triphenylmethylhistidine (800.00 mg, 2.50 mmol), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (480.06 mg, 2.50 mmol) and N-hydroxysuccinimide (288.20 mg, 2.50 mmol) were mixed and dissolved in dichloromethane, and triethylamine (1.04 mL, 7.51 mmol) was added thereto. After stirring at room temperature for 2 h, 2-(triphenylmethylthio)ethylamine (996.85 mg, 2.00 mmol) was added thereto. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the white solid byproduct was filtered and the filtrate was collected. After rotary evaporation and concentration, column chromatography was used for purification. The mobile phase was ethyl acetate: dichloromethane = 1:1. R f = 0.6, and vacuum dried to obtain the product N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ -Triphenylmethylhistamine amide;

[0111] S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ-Triphenylmethylhistamine amide (150.00 mg, 0.188 mmol) was mixed with triisopropylsilane (240 μL, 1.17 mmol), and 6 mL of trifluoroacetic acid was added, followed by stirring in an ice bath for 2 h. After the reaction was completed, most of the trifluoroacetic acid was removed by rotary evaporation, and then cold ether was used for precipitation. After centrifugation, the precipitate was dissolved in water, and the insoluble matter was filtered out. The filtrate was freeze-dried to obtain the product HIS-SH;

[0112] S44. M-MPR and HIS-SH were mixed at a molar ratio of 1:20, dissolved in methanol, and stirred at room temperature for 24 hours under N2 protection. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 for dialysis. The dialysis medium was methanol. The dialysis solution was replaced every 6 hours. After dialysis 4 times, the dialysis solution was collected and vacuum dried to obtain the product M-MPRH;

[0113] S5. Prepare nano-drug-loaded micelles by encapsulating the hydrophobic anticancer drug DOX with M-MPRH, and the specific steps are as follows:

[0114] S51. Dissolve 500 μg of DOX hydrochloride in 1 mL of methanol, add 20 μL of triethylamine, and sonicate for 1 h in the dark to obtain a desalted DOX methanol solution;

[0115] S52. Dissolve M-MPRH in methanol to prepare a 5 mg / mL M-MPRH methanol solution;

[0116] S53. Mix the M-MPRH methanol solution and the DOX methanol solution at a mass ratio of 10:1 between M-MPRH and DOX, and stir at room temperature in the dark for 3 h;

[0117] S54. After the reaction, the reaction solution was slowly dripped into water at a rate of 0.5 mL / min, with a volume ratio of reaction solution to water of 1:2. The solution was placed in a dialysis bag with a molecular weight cutoff of 500 for dialyzing. The dialysis medium was water. The dialysis solution was replaced every 6 hours for a total of 3 times. After the dialysis, the dialysate was collected to obtain M-MPRH@DOX nano-drug-loaded micelles.

[0118] Example 3

[0119] S1. Maleic anhydride treatment of G3 PAMAM, the specific steps are as follows:

[0120] S11. G3 PAMAM and maleic anhydride were mixed in a molar ratio of 1:4 according to the primary amino group of PAMAM and maleic anhydride, dissolved in dimethyl sulfoxide, and stirred at room temperature for 24 hours;

[0121] S12. After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 7000 for dialyzing, the dialysis medium is a boric acid-borax buffer solution with a pH of 9, and the dialysis solution is replaced every 6-8 hours. After dialysis with the above buffer solution for 2 times, the dialysis medium is replaced with water, and the dialysis solution is replaced every 2 hours. After dialysis for 2 times, the dialysis is terminated, and the dialysis solution is taken out and freeze-dried to obtain MM;

[0122] S2. Modify MM with PTX-SH to synthesize M-MP so that it has a hydrophobic end. The specific steps are as follows:

[0123] S21. 3,3'-dithiodipropionic acid (58.79 mg, 0.280 mmol) and paclitaxel (573.00 mg, 0.671 mmol) were dissolved in dichloromethane, stirred at room temperature for 1 h, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (128.63 mg, 0.671 mmol) and 4-dimethylaminopyridine (13.66 mg, 0.112 mmol) were added thereto, and refluxed at room temperature for 48 h. After the reaction was completed, it was purified by column chromatography with a mobile phase of ethyl acetate: n-hexane = 2:1, and R f =0.36, and vacuum dried to obtain the product PTX-SS-PTX;

[0124] S22. PTX-SS-PTX (292 mg, 0.155 mmol) and DTT (47.86 mg, 0.310 mmol) were dissolved in dichloromethane, stirred at room temperature for 1 h, and triethylamine (86 μL, 0.621 mmol) was added thereto, and stirred under N2 atmosphere for 12 h. After the reaction, the organic phase was collected, washed, dried, concentrated, and precipitated with petroleum ether, and the product PTX-SH was collected by centrifugation;

[0125] S23. MM and PTX-SH were mixed at a molar ratio of 1:12, dissolved in methanol, and stirred at room temperature for 24 hours under N2 protection. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 7000 for dialysis. The dialysis medium was methanol. The dialysis solution was replaced every 6 hours. After dialysis 4 times, the dialysis solution was collected and vacuum dried to obtain the product M-MP;

[0126] S3. Use c(RGDfC) to modify M-MP to synthesize M-MPR, so that it has a hydrophilic end that can target and recognize tumor cells. The specific steps are to mix M-MP and c(RGDfC) at a molar ratio of 1:8, dissolve in a mixed solvent of methanol and water with a volume ratio of 10:1, and stir at room temperature under N2 protection for 24 hours. After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 7000 for dialysis. The dialysis medium is methanol. The dialysis solution is replaced every 6 hours. After dialysis 4 times, the dialysis solution is collected and vacuum dried to obtain the product M-MPR;

[0127] S4. M-MPR is modified with HIS-SH to synthesize M-MPRH so that it has a pH-sensitive hydrophilic end. The specific steps are as follows:

[0128] S41. Mercaptoethylamine hydrochloride (2.30 g, 20.22 mmol) and triphenylmethane (5.64 g, 20.22 mmol) were mixed, dissolved with trifluoroacetic acid, and stirred at room temperature for 2 h. After the reaction, the reaction solution was rotary evaporated and washed, and the organic phase was collected and dried and concentrated to obtain the product 2-(triphenylmethylthio)ethylamine;

[0129] S42. N α -N τ -Triphenylmethylhistidine (800.00 mg, 2.50 mmol), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (480.06 mg, 2.50 mmol) and N-hydroxysuccinimide (288.20 mg, 2.50 mmol) were mixed and dissolved in dichloromethane, and triethylamine (1.04 mL, 7.51 mmol) was added thereto. After stirring at room temperature for 2 h, 2-(triphenylmethylthio)ethylamine (996.85 mg, 2.00 mmol) was added thereto. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the white solid byproduct was filtered and the filtrate was collected. After rotary evaporation and concentration, column chromatography was used for purification. The mobile phase was ethyl acetate: dichloromethane = 1:1. R f = 0.6, and vacuum dried to obtain the product N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ -Triphenylmethylhistamine amide;

[0130] S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ-Triphenylmethylhistamine amide (150.00 mg, 0.188 mmol) was mixed with triisopropylsilane (240 μL, 1.17 mmol), and 6 mL of trifluoroacetic acid was added, followed by stirring in an ice bath for 2 h. After the reaction was completed, most of the trifluoroacetic acid was removed by rotary evaporation, and then cold ether was used for precipitation. After centrifugation, the precipitate was dissolved in water, and the insoluble matter was filtered out. The filtrate was freeze-dried to obtain the product HIS-SH;

[0131] S44. M-MPR and HIS-SH were mixed at a molar ratio of 1:30, dissolved in methanol, and stirred at room temperature for 24 hours under N2 protection. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 7000 for dialysis. The dialysis medium was methanol. The dialysis solution was replaced every 6 hours. After dialysis 4 times, the dialysis solution was collected and vacuum dried to obtain the product M-MPRH;

[0132] S5. Prepare nano-drug-loaded micelles by encapsulating the hydrophobic anticancer drug DOX with M-MPRH, and the specific steps are as follows:

[0133] S51. Dissolve 500 μg of DOX hydrochloride in 1 mL of methanol, add 20 μL of triethylamine, and sonicate for 1 h in the dark to obtain a desalted DOX methanol solution;

[0134] S52. Dissolve M-MPRH in methanol to prepare a 5 mg / mL M-MPRH methanol solution;

[0135] S53. Mix the M-MPRH methanol solution and the DOX methanol solution at a mass ratio of 10:1 between M-MPRH and DOX, and stir at room temperature in the dark for 3 h;

[0136] S54. After the reaction, the reaction solution was slowly dripped into water at a rate of 0.5 mL / min, with a volume ratio of reaction solution to water of 1:3. The solution was placed in a dialysis bag with a molecular weight cutoff of 500 for dialyzing. The dialysis medium was water. The dialysate was replaced every 6 hours for a total of 3 times. After the dialysis, the dialysate was collected to obtain M-MPRH@DOX nano-drug-loaded micelles.

[0137] Example 4

[0138] S1. Maleic anhydride treatment of G1 PAMAM, the specific steps are as follows:

[0139] S11. G1 PAMAM and maleic anhydride were mixed in a molar ratio of 1:5 according to the primary amino group of PAMAM and maleic anhydride, dissolved in dimethyl sulfoxide, and stirred at room temperature for 24 hours;

[0140] S12. After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 1500 for dialyzing, the dialysis medium is a boric acid-borax buffer solution with a pH of 9, and the dialysis solution is replaced every 6-8 hours. After dialysis with the above buffer solution for 2 times, the dialysis medium is replaced with water, and the dialysis solution is replaced every 2 hours. After dialysis for 2 times, the dialysis is terminated, and the dialysis solution is taken out and freeze-dried to obtain MM;

[0141] S2. Modify MM with PTX-SH to synthesize M-MP so that it has a hydrophobic end. The specific steps are as follows:

[0142] S21. 3,3'-dithiodipropionic acid (58.79 mg, 0.280 mmol) and paclitaxel (573.00 mg, 0.671 mmol) were dissolved in dichloromethane, stirred at room temperature for 1 h, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (128.63 mg, 0.671 mmol) and 4-dimethylaminopyridine (13.66 mg, 0.112 mmol) were added thereto, and refluxed at room temperature for 48 h. After the reaction was completed, it was purified by column chromatography with a mobile phase of ethyl acetate: n-hexane = 2:1, and R f =0.36, and vacuum dried to obtain the product PTX-SS-PTX;

[0143] S22. PTX-SS-PTX (292 mg, 0.155 mmol) and DTT (47.86 mg, 0.310 mmol) were dissolved in dichloromethane, stirred at room temperature for 1 h, and triethylamine (86 μL, 0.621 mmol) was added thereto, and stirred under N2 atmosphere for 12 h. After the reaction, the organic phase was collected, washed, dried, concentrated, and precipitated with petroleum ether, and the product PTX-SH was collected by centrifugation;

[0144] S23. M-MP and PTX-SH were mixed at a molar ratio of 1:4, dissolved in methanol, and stirred at room temperature for 24 hours under N2 protection. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 2000 for dialysis. The dialysis medium was methanol. The dialysis solution was replaced every 6 hours. After dialysis 4 times, the dialysis solution was collected and vacuum dried to obtain the product M-MP;

[0145] S3. Use c(RGDfC) to modify M-MP to synthesize M-MPR, so that it has a hydrophilic end that can target and recognize tumor cells. The specific steps are to mix M-MP and c(RGDfC) at a molar ratio of 1:2, dissolve in a mixed solvent of methanol and water with a volume ratio of 10:1, and stir at room temperature under N2 protection for 24 hours. After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 2000 for dialysis. The dialysis medium is methanol. The dialysis solution is replaced every 6 hours. After dialysis 4 times, the dialysis solution is collected and vacuum dried to obtain the product M-MPR;

[0146] S4. M-MPR is modified with HIS-SH to synthesize M-MPRH so that it has a pH-sensitive hydrophilic end. The specific steps are as follows:

[0147] S41. Mercaptoethylamine hydrochloride (2.30 g, 20.22 mmol) and triphenylmethane (5.64 g, 20.22 mmol) were mixed, dissolved with trifluoroacetic acid, and stirred at room temperature for 2 h. After the reaction, the reaction solution was rotary evaporated and washed, and the organic phase was collected and dried and concentrated to obtain the product 2-(triphenylmethylthio)ethylamine;

[0148] S42. N α -N τ -Triphenylmethylhistidine (800.00 mg, 2.50 mmol), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (480.06 mg, 2.50 mmol) and N-hydroxysuccinimide (288.20 mg, 2.50 mmol) were mixed and dissolved in dichloromethane, and triethylamine (1.04 mL, 7.51 mmol) was added thereto. After stirring at room temperature for 2 h, 2-(triphenylmethylthio)ethylamine (996.85 mg, 2.00 mmol) was added thereto. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the white solid byproduct was filtered and the filtrate was collected. After rotary evaporation and concentration, column chromatography was used for purification. The mobile phase was ethyl acetate: dichloromethane = 1:1. R f = 0.6, and vacuum dried to obtain the product N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ -Triphenylmethylhistamine amide;

[0149] S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ-Triphenylmethylhistamine amide (150.00 mg, 0.188 mmol) was mixed with triisopropylsilane (240 μL, 1.17 mmol), and 6 mL of trifluoroacetic acid was added, followed by stirring in an ice bath for 2 h. After the reaction was completed, most of the trifluoroacetic acid was removed by rotary evaporation, and then cold ether was used for precipitation. After centrifugation, the precipitate was dissolved in water, and the insoluble matter was filtered out. The filtrate was freeze-dried to obtain the product HIS-SH;

[0150] S44. M-MPR and HIS-SH were mixed at a molar ratio of 1:20, dissolved in methanol, and stirred at room temperature for 24 hours under N2 protection. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 2000 for dialysis. The dialysis medium was methanol. The dialysis solution was replaced every 6 hours. After dialysis 4 times, the dialysis solution was collected and vacuum dried to obtain the product M-MPRH;

[0151] S5. The hydrophobic anticancer drug camptothecin (CPT) was encapsulated by M-MPRH to prepare nano drug-loaded micelles. The specific steps are as follows:

[0152] S51. Dissolve 500 μg of camptothecin in 1 mL of methanol to prepare a 0.5 mg / mL camptothecin methanol solution;

[0153] S52. Dissolve M-MPRH in methanol to prepare a 5 mg / mL M-MPRH methanol solution;

[0154] S53. The M-MPRH methanol solution and the camptothecin methanol solution were mixed at a mass ratio of 8:1 for M-MPRH: camptothecin, and stirred at room temperature in the dark for 3 h;

[0155] S54. After the reaction is completed, the reaction solution is slowly dripped into water at a rate of 0.5 mL / min, with a volume ratio of the reaction solution to water of 1:0.5. The solution is placed in a dialysis bag with a molecular weight cutoff of 500 for dialyzing. The dialysis medium is water. The dialysis solution is replaced every 6 hours for a total of 3 dialysis cycles. After the dialysis is completed, the dialysis solution is collected to obtain M-MPRH@CPT nano-drug-loaded micelles.

[0156] Example 5

[0157] S1. Maleic anhydride treatment of G1 PAMAM, the specific steps are as follows:

[0158] S11. G1 PAMAM and maleic anhydride were mixed in a molar ratio of 1:2 according to the primary amino group of PAMAM and maleic anhydride, dissolved in dimethyl sulfoxide, and stirred at room temperature for 24 hours;

[0159] S12. After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 1500 for dialyzing, the dialysis medium is a boric acid-borax buffer solution with a pH of 9, and the dialysis solution is replaced every 6-8 hours. After dialysis with the above buffer solution for 2 times, the dialysis medium is replaced with water, and the dialysis solution is replaced every 2 hours. After dialysis for 2 times, the dialysis is terminated, and the dialysis solution is taken out and freeze-dried to obtain MM;

[0160] S2. Modify MM with PTX-SH to synthesize M-MP so that it has a hydrophobic end. The specific steps are as follows:

[0161] S21. 3,3'-dithiodipropionic acid (58.79 mg, 0.280 mmol) and paclitaxel (573.00 mg, 0.671 mmol) were dissolved in dichloromethane, stirred at room temperature for 1 h, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (128.63 mg, 0.671 mmol) and 4-dimethylaminopyridine (13.66 mg, 0.112 mmol) were added thereto, and refluxed at room temperature for 48 h. After the reaction was completed, it was purified by column chromatography with a mobile phase of ethyl acetate: n-hexane = 2:1, and R f =0.36, and vacuum dried to obtain the product PTX-SS-PTX;

[0162] S22. PTX-SS-PTX (292 mg, 0.155 mmol) and DTT (47.86 mg, 0.310 mmol) were dissolved in dichloromethane, stirred at room temperature for 1 h, and triethylamine (86 μL, 0.621 mmol) was added thereto, and stirred under N2 atmosphere for 12 h. After the reaction, the organic phase was collected, washed, dried, concentrated, and precipitated with petroleum ether, and the product PTX-SH was collected by centrifugation;

[0163] S23. M-MP and PTX-SH were mixed at a molar ratio of 1:4, dissolved in methanol, and stirred at room temperature for 24 hours under N2 protection. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 2000 for dialysis. The dialysis medium was methanol. The dialysis solution was replaced every 6 hours. After dialysis 4 times, the dialysis solution was collected and vacuum dried to obtain the product M-MP;

[0164] S3. Use c(RGDfC) to modify M-MP to synthesize M-MPR, so that it has a hydrophilic end that can target and recognize tumor cells. The specific steps are to mix M-MP and c(RGDfC) at a molar ratio of 1:2, dissolve in a mixed solvent of methanol and water with a volume ratio of 10:1, and stir at room temperature under N2 protection for 24 hours. After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 2000 for dialysis. The dialysis medium is methanol. The dialysis solution is replaced every 6 hours. After dialysis 4 times, the dialysis solution is collected and vacuum dried to obtain the product M-MPR;

[0165] S4. M-MPR is modified with HIS-SH to synthesize M-MPRH so that it has a pH-sensitive hydrophilic end. The specific steps are as follows:

[0166] S41. Mercaptoethylamine hydrochloride (2.30 g, 20.22 mmol) and triphenylmethane (5.64 g, 20.22 mmol) were mixed, dissolved with trifluoroacetic acid, and stirred at room temperature for 2 h. After the reaction, the reaction solution was rotary evaporated and washed, and the organic phase was collected and dried and concentrated to obtain the product 2-(triphenylmethylthio)ethylamine;

[0167] S42. N α -N τ -Triphenylmethylhistidine (800.00 mg, 2.50 mmol), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (480.06 mg, 2.50 mmol) and N-hydroxysuccinimide (288.20 mg, 2.50 mmol) were mixed and dissolved in dichloromethane, and triethylamine (1.04 mL, 7.51 mmol) was added thereto. After stirring at room temperature for 2 h, 2-(triphenylmethylthio)ethylamine (996.85 mg, 2.00 mmol) was added thereto. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the white solid byproduct was filtered and the filtrate was collected. After rotary evaporation and concentration, column chromatography was used for purification. The mobile phase was ethyl acetate: dichloromethane = 1:1. R f = 0.6, and vacuum dried to obtain the product N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ -Triphenylmethylhistamine amide;

[0168] S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ-Triphenylmethylhistamine amide (150.00 mg, 0.188 mmol) was mixed with triisopropylsilane (240 μL, 1.17 mmol), and 6 mL of trifluoroacetic acid was added, followed by stirring in an ice bath for 2 h. After the reaction was completed, most of the trifluoroacetic acid was removed by rotary evaporation, and then cold ether was used for precipitation. After centrifugation, the precipitate was dissolved in water, and the insoluble matter was filtered out. The filtrate was freeze-dried to obtain the product HIS-SH;

[0169] S44. M-MPR and HIS-SH were mixed at a molar ratio of 1:10, dissolved in methanol, and stirred at room temperature for 24 hours under N2 protection. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 2000 for dialysis. The dialysis medium was methanol. The dialysis solution was replaced every 6 hours. After dialysis 4 times, the dialysis solution was collected and vacuum dried to obtain the product M-MPRH;

[0170] S5. The hydrophobic anticancer drug fluorouracil (5-FU) was encapsulated in M-MPRH to prepare nano drug-loaded micelles. The specific steps are as follows:

[0171] S51. Dissolve 500 μg of fluorouracil in 1 mL of methanol to prepare a 0.5 mg / mL fluorouracil methanol solution;

[0172] S52. Dissolve M-MPRH in methanol to prepare a 5 mg / mL M-MPRH methanol solution;

[0173] S53. Mix the M-MPRH methanol solution and the fluorouracil methanol solution at a mass ratio of 5:1 between M-MPRH and fluorouracil, and stir at room temperature in the dark for 3 h;

[0174] S54. After the reaction is completed, the reaction solution is slowly dripped into water at a rate of 1 mL / min, with a volume ratio of reaction solution to water of 1:0.5. The solution is placed in a dialysis bag with a molecular weight cutoff of 500 for dialyzing. The dialysis medium is water. The dialysis solution is replaced every 6 hours for a total of 3 times. After the dialysis is completed, the dialysate is collected to obtain M-MPRH@5-FU nano-drug-loaded micelles.

[0175] Example 6

[0176] S1. Maleic anhydride treatment of G3 PAMAM, the specific steps are as follows:

[0177] S11. G3 PAMAM and maleic anhydride were mixed in a molar ratio of 1:5 according to the primary amino group of PAMAM and maleic anhydride, dissolved in dimethyl sulfoxide, and stirred at room temperature for 24 hours;

[0178] S12. After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 7000 for dialyzing, the dialysis medium is a boric acid-borax buffer solution with a pH of 9, and the dialysis solution is replaced every 6-8 hours. After dialysis with the above buffer solution for 2 times, the dialysis medium is replaced with water, and the dialysis solution is replaced every 2 hours. After dialysis for 2 times, the dialysis is terminated, and the dialysis solution is taken out and freeze-dried to obtain MM;

[0179] S2. Modify MM with PTX-SH to synthesize M-MP so that it has a hydrophobic end. The specific steps are as follows:

[0180] S21. 3,3'-dithiodipropionic acid (58.79 mg, 0.280 mmol) and paclitaxel (573.00 mg, 0.671 mmol) were dissolved in dichloromethane, stirred at room temperature for 1 h, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (128.63 mg, 0.671 mmol) and 4-dimethylaminopyridine (13.66 mg, 0.112 mmol) were added thereto, and refluxed at room temperature for 48 h. After the reaction was completed, it was purified by column chromatography with a mobile phase of ethyl acetate: n-hexane = 2:1, and R f =0.36, and vacuum dried to obtain the product PTX-SS-PTX;

[0181] S22. PTX-SS-PTX (292 mg, 0.155 mmol) and DTT (47.86 mg, 0.310 mmol) were dissolved in dichloromethane, stirred at room temperature for 1 h, and triethylamine (86 μL, 0.621 mmol) was added thereto, and stirred under N2 atmosphere for 12 h. After the reaction, the organic phase was collected, washed, dried, concentrated, and precipitated with petroleum ether, and the product PTX-SH was collected by centrifugation;

[0182] S23. M-MP and PTX-SH were mixed at a molar ratio of 1:15, dissolved in methanol, and stirred at room temperature for 24 hours under N2 protection. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 7000 for dialysis. The dialysis medium was methanol. The dialysis solution was replaced every 6 hours. After dialysis 4 times, the dialysis solution was collected and vacuum dried to obtain the product M-MP;

[0183] S3. Use c(RGDfC) to modify M-MP to synthesize M-MPR, so that it has a hydrophilic end that can target and recognize tumor cells. The specific steps are to mix M-MP and c(RGDfC) at a molar ratio of 1:10, dissolve in a mixed solvent of methanol and water with a volume ratio of 10:1, and stir at room temperature under N2 protection for 24 hours. After the reaction is completed, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 7000 for dialysis. The dialysis medium is methanol. The dialysis fluid is replaced every 6 hours. After dialysis 4 times, the dialysis fluid is collected and vacuum dried to obtain the product M-MPR;

[0184] S4. M-MPR is modified with HIS-SH to synthesize M-MPRH so that it has a pH-sensitive hydrophilic end. The specific steps are as follows:

[0185] S41. Mercaptoethylamine hydrochloride (2.30 g, 20.22 mmol) and triphenylmethane (5.64 g, 20.22 mmol) were mixed, dissolved with trifluoroacetic acid, and stirred at room temperature for 2 h. After the reaction, the reaction solution was rotary evaporated and washed, and the organic phase was collected and dried and concentrated to obtain the product 2-(triphenylmethylthio)ethylamine;

[0186] S42. N α -N τ -Triphenylmethylhistidine (800.00 mg, 2.50 mmol), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (480.06 mg, 2.50 mmol) and N-hydroxysuccinimide (288.20 mg, 2.50 mmol) were mixed and dissolved in dichloromethane, and triethylamine (1.04 mL, 7.51 mmol) was added thereto. After stirring at room temperature for 2 h, 2-(triphenylmethylthio)ethylamine (996.85 mg, 2.00 mmol) was added thereto. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the white solid byproduct was filtered and the filtrate was collected. After rotary evaporation and concentration, column chromatography was used for purification. The mobile phase was ethyl acetate: dichloromethane = 1:1. R f = 0.6, and vacuum dried to obtain the product N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ -Triphenylmethylhistamine amide;

[0187] S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ-Triphenylmethylhistamine amide (150.00 mg, 0.188 mmol) was mixed with triisopropylsilane (240 μL, 1.17 mmol), and 6 mL of trifluoroacetic acid was added, followed by stirring in an ice bath for 2 h. After the reaction was completed, most of the trifluoroacetic acid was removed by rotary evaporation, and then cold ether was used for precipitation. After centrifugation, the precipitate was dissolved in water, and the insoluble matter was filtered out. The filtrate was freeze-dried to obtain the product HIS-SH;

[0188] S44. M-MPR and HIS-SH were mixed at a molar ratio of 1:30, dissolved in methanol, and stirred at room temperature for 24 hours under N2 protection. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 7000 for dialysis. The dialysis medium was methanol. The dialysis solution was replaced every 6 hours. After dialysis 4 times, the dialysis solution was collected and vacuum dried to obtain the product M-MPRH;

[0189] S5. The hydrophobic photosensitizer dihydrochlorin e6 (Ce6) was encapsulated with M-MPRH to prepare nano-drug-loaded micelles. The specific steps are as follows:

[0190] S51. Dissolve 500 μg of dihydrochlorin e6 in 1 mL of methanol to prepare a 0.5 mg / mL dihydrochlorin e6 methanol solution; dissolve 500 μg of DOX in 1 mL of methanol to prepare a 0.5 mg / mL DOX methanol solution;

[0191] S52. Dissolve M-MPRH in methanol to prepare a 5 mg / mL M-MPRH methanol solution;

[0192] S53. Mix the M-MPRH methanol solution with the dihydrochlorin e6 and DOX methanol solutions at a mass ratio of M-MPRH: dihydrochlorin e6: DOX of 40:1:1, and stir at room temperature in the dark for 3 h;

[0193] S54. After the reaction, the reaction solution was slowly dripped into water at a rate of 1 mL / min, with a volume ratio of reaction solution to water of 1:3. The solution was placed in a dialysis bag with a molecular weight cutoff of 500 for dialyzing. The dialysis medium was water. The dialysis solution was replaced every 6 hours for a total of 3 times. After the dialysis, the dialysate was collected to obtain M-MPRH@Ce6@DOX nanodrug-loaded micelles.

[0194] Those skilled in the art will appreciate that the above examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made by those of ordinary skill in the art to the technical solution of the present invention should all fall within the scope of protection determined by the claims of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

Claims

1. A prodrug molecule based on zwitterionic polyamidoamine dendrimers, characterized in that: The prodrug molecule has a structure as shown in the general formula (I): In the general formula (I), a, b and c represent the amount of thiolated paclitaxel, tumor targeting peptide and thiolated histidine grafted on the polyamidoamine dendrimer, respectively, and the sum of a, b and c is the same as the total number of primary amino groups on the surface of the polyamidoamine dendrimer; The polyamidoamine dendrimer is denoted as PAMAM, thiolated paclitaxel is denoted as PTX-SH, the tumor targeting peptide is denoted as c(RGDfC), and thiolated histidine is denoted as HIS-SH.

2. The method for preparing a prodrug molecule based on zwitterionic polyamidoamine dendrimers according to claim 1, characterized in that: The preparation method comprises: S1. PAMAM is maleated with maleic anhydride to obtain the first modified dendrimer, denoted as MM; S2. Modify MM with PTX-SH to give MM a hydrophobic end, thereby obtaining a second modified dendrimer, denoted as M-MP; S3. Using c(RGDfC) to modify M-MP, so that M-MP has a hydrophilic end that can target and recognize tumor cells, and obtain a third modified dendrimer, denoted as M-MPR; S4. M-MPR is modified by using HIS-SH to provide M-MPR with a pH-sensitive hydrophilic end, thereby obtaining the prodrug molecule based on the zwitterionic polyamide-amine dendrimer, which is denoted as M-MPRH.

3. The method for preparing a prodrug molecule based on zwitterionic polyamidoamine dendrimers according to claim 2, characterized in that: The S1 specifically includes the following steps: S11. PAMAM and maleic anhydride are mixed in a molar ratio of 1:2 to 5, dissolved in dimethyl sulfoxide, and stirred at room temperature for 24 hours to obtain a first reaction solution; S12. The first reaction liquid is dialyzed twice with the first dialysate at a frequency of changing once every 6 to 8 hours, and then the dialysate is dialyzed twice with the second dialysate at a frequency of changing once every 2 hours, and the dialysate is taken out and freeze-dried to obtain the MM; wherein the first dialysate is a boric acid-borax buffer solution with a pH of 9, and the second dialysate is water; Optionally, in S12, the molecular weight cutoff of the dialysis bag used for dialysis is 1500 to 7000.

4. The method for preparing a prodrug molecule based on zwitterionic polyamidoamine dendrimers according to claim 2, characterized in that: The S2 specifically includes the following steps: S21. 3,3'-dithiodipropionic acid and paclitaxel were dissolved in dichloromethane, stirred at room temperature for 1 hour, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were added, and the mixture was refluxed at room temperature for 48 hours. After the reaction was completed, column chromatography was performed to purify and collect the product to obtain dithiodipropionic acid-esterified paclitaxel, which was recorded as PTX-SS-PTX. S22. PTX-SS-PTX and dithiothreitol were dissolved in dichloromethane, stirred at room temperature for 1 hour, and then triethylamine was added, and the reaction was stirred for 12 hours under N2 atmosphere; after the reaction, the organic phase was collected, washed, dried, and concentrated, and the product was collected by centrifugation after precipitation with petroleum ether to obtain the PTX-SH; S23. The MM and PTX-SH are mixed in a molar ratio of 1:1 to 15, dissolved in methanol, and stirred at room temperature for 24 to 48 hours under N2 protection to obtain a second reaction solution; the second reaction solution is dialyzed with methanol as the dialysis medium at a frequency of changing once every 6 hours, and the dialyzate is collected after 3 to 6 times of dialysis, and vacuum dried to obtain the M-MP; Optionally, in S23, the molecular weight cutoff of the dialysis bag used for dialysis is 2000-7000.

5. The method for preparing a prodrug molecule based on zwitterionic polyamidoamine dendrimers according to claim 2, characterized in that: The S3 specifically includes the following steps: The M-MP and c(RGDfC) are mixed at a molar ratio of 1:1 to 10, dissolved in a mixed solvent of methanol and water in a volume ratio of 10:1, and reacted at room temperature for 24 hours under N2 protection to obtain a third reaction solution; the third reaction solution is dialyzed with methanol as the dialysis medium at a frequency of changing once every 6 hours, and the dialyzate is collected after 4 dialysis, and vacuum dried to obtain the M-MPR; Optionally, in S3, the molecular weight cutoff of the dialysis bag used for dialysis is 2000-7000.

6. The method for preparing a prodrug molecule based on zwitterionic polyamidoamine dendrimers according to claim 2, characterized in that: The S4 specifically includes the following steps: S41. The mercaptoethylamine hydrochloride and triphenylmethane were mixed, dissolved with trifluoroacetic acid, and stirred at room temperature for 2 hours to obtain a fourth reaction solution; the fourth reaction solution was subjected to rotary evaporation and washing, and the organic phase was collected and dried and concentrated to obtain 2-(triphenylmethylthio)ethylamine; S42. N α -N τ -triphenylmethylhistidine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were mixed and dissolved in dichloromethane, and then triethylamine was added. After stirring at room temperature for 2 hours, the 2-(tritylthio)ethylamine was added and stirred at room temperature for 12 hours. After the reaction, the white solid byproduct was filtered and the filtrate was collected. After rotary evaporation and concentration, the filtrate was purified by column chromatography. The product was collected and vacuum dried to obtain N'-(2-(tritylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ -Triphenylmethylhistamine amide; S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-butyloxycarbonyl-N τ -Triphenylmethylhistamine amide is mixed with triisopropylsilane, trifluoroacetic acid is added and stirred in an ice bath for 2 hours, most of the trifluoroacetic acid is removed by rotary evaporation after the reaction is completed, and then cold ether is used for precipitation. After centrifugation, the precipitate is dissolved in water and the insoluble matter is filtered out, and the filtrate is freeze-dried to obtain the HIS-SH; S44. The M-MPR and HIS-SH are mixed in a molar ratio of 1:1 to 30, dissolved in methanol, and stirred at room temperature for 24 to 48 hours under N2 protection to obtain a fifth reaction solution; the fifth reaction solution is dialyzed with methanol as the dialysis medium at a frequency of changing once every 6 hours, and the dialyzate is collected after 3 to 6 times of dialysis, and vacuum dried to obtain the M-MPRH; Optionally, in S44, the molecular weight cutoff of the dialysis bag used for dialysis is 2000-7000.

7. Use of the prodrug molecule based on zwitterionic polyamidoamine dendrimer as claimed in claim 1 in the field of nano drug delivery system.

8. A nano drug-loaded micelle, characterized in that: The nano drug-loaded micelle comprises the hydrophobic anticancer drug encapsulated by the prodrug molecule based on the zwitterionic polyamidoamine dendrimer as claimed in claim 1.

9. The method for preparing the drug-loaded nano micelles according to claim 8, characterized in that: The preparation method includes using PAMAM as a carrier and a hydrophobic anti-tumor drug PTX-SH as a hydrophobic end, which can specifically recognize α- V The c(RGDfC) of β3 integrin and the pH-sensitive HIS-SH are used as the hydrophilic ends to self-assemble into the prodrug molecule based on the zwitterionic polyamidoamine dendrimer, and then another hydrophobic anticancer drug is physically embedded to obtain the nano drug-loaded micelle.

10. The method for preparing drug-loaded nano micelles according to claim 9, characterized in that: The preparation method comprises: S5. preparing the nano drug-loaded micelle by encapsulating the hydrophobic anticancer drug with M-MPRH; The S5 specifically includes the following steps: S51. dissolving the hydrophobic anticancer drug in methanol to prepare a hydrophobic anticancer drug methanol solution; S52. Dissolving M-MPRH in methanol to prepare an M-MPRH methanol solution; S53. The M-MPRH methanol solution and the hydrophobic anticancer drug methanol solution are mixed in a mass ratio of 5 to 20:1, and stirred at room temperature in the dark for 2 to 6 hours to obtain a sixth reaction solution; S54. The sixth reaction solution was slowly dripped into water for dialysis, the dialysis medium was water, and the dialysis was performed 3 times at a frequency of replacement every 6 hours. After the dialysis was completed, the dialysate was collected to obtain the nano-drug-loaded micelles; Optionally, in S52, the hydrophobic anticancer drug includes at least one of doxorubicin, camptothecin, fluorouracil, and chlorin e6; Optionally, in S54, the mass ratio of the reaction liquid to water is 1:0.5-3, and the dripping speed into the water is 0.5-1 mL / min.

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