Prodrug molecules based on zwitterionic polyamidoamine dendrimers, nano-drug-loaded micelles, and preparation methods and applications thereof

Abstract

This invention provides a prodrug molecule, drug-loaded nanomicelles, and their preparation method and applications based on zwitterionic polyamide-amine dendritic macromolecules. The preparation method of the drug-loaded nanomicelles includes: modifying PAMAM with maleic anhydride, then grafting thiolated paclitaxel, c(RGDfC), and thiolated histidine onto PAMAM via a thioolefin reaction, utilizing its self-assembly properties to encapsulate hydrophobic anticancer drugs to form drug-loaded nanomicelles. These drug-loaded nanomicelles possess advantages such as high drug loading capacity, low toxicity, uniform particle size distribution, good stability, and enhanced bioavailability of hydrophobic drugs. Furthermore, they exhibit pH responsiveness, enabling targeted drug release at tumor sites, thereby enhancing tumor-suppressing effects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomedicine delivery system technology, specifically relating to a prodrug molecule based on zwitterionic polyamide-amine dendritic macromolecules, nanodrug-loaded micelles, their preparation methods and applications. Background Technology

[0002] Cancer has a high mortality and incidence rate. Statistics show that millions of people die from cancer globally each year, making it one of the leading causes of death worldwide, severely impacting human health and even life. Conventional cancer treatments include surgery, chemotherapy, radiotherapy, immunotherapy, and molecularly targeted drugs, with chemotherapy playing a crucial role in combination with other therapies. However, most traditional chemotherapy drugs have low water solubility, poor selectivity, and lack specificity for tumor cells, leading to organ dysfunction.

[0003] Therefore, it is necessary to modify and improve chemotherapy drugs to reduce secondary harm to patients during treatment. Researchers have proposed using nanomedicine delivery carriers to overcome these shortcomings. Nanomedicine delivery carriers can improve the solubility and bioavailability of poorly soluble drugs, enhance drug targeting, and achieve drug accumulation in specific organs or tissues. This enables sustained or even controlled release of drugs, thereby prolonging circulation time in the body, reducing side effects, improving drug safety, and ultimately enhancing therapeutic efficacy, greatly promoting the application of chemotherapy drugs in cancer treatment.

[0004] Dendrimers, as monodisperse polymers with highly branched structures, offer controllable particle size and regular shape, and their surface functional groups are easily modified, thus showing promising applications in drug delivery. Polyamidoamine (PAMAM), commonly used as a nanoparticle drug delivery carrier, possesses excellent water solubility due to the presence of numerous terminal amino groups. However, with increasing branching generations, the more terminal amino groups there are, the stronger the cationicity becomes. Excessive cationicity can easily cause cell membrane rupture and even apoptosis, leading to cytotoxicity. Therefore, researchers have modified the terminal groups on the PAMAM surface using different molecules to reduce cytotoxicity while retaining its original advantages. For example, glycosylation, acetylation, polyethylene glycol modification, or peptide modification of PAMAM can neutralize cations and reduce cytotoxicity.

[0005] Amphoteric groups are groups that can simultaneously carry positive and negative charges under different pH conditions. Their charge characteristics are closely related to pH. Amphoteric groups are widely found in various natural and synthetic compounds, such as amino acids, polymers, and surfactants. Because their charge state changes with pH, ​​exhibiting pH sensitivity, amphoteric groups have important applications in various fields, especially in biomedicine, materials science, and environmental monitoring. Currently, the main method for drug loading in dendritic macromolecules is to use their internal cavities as guest carriers. However, this method has limited drug loading capacity, low drug loading rate, and lacks a targeted response mechanism, resulting in relatively limited drug release. Therefore, modifying PAMAM with zwitterionic groups can utilize the changes in its charge state under different pH conditions to optimize its drug delivery effect. The tumor microenvironment is usually acidic, with a normal physiological pH close to 7.4. Zwitterionic groups are electroneutrally neutral at near-neutral pH values, while under low pH conditions, amino groups are protonated, and carboxyl groups remain undissociated, thus making the group as a whole positively charged. Therefore, PAMAM modified with zwitterions can undergo protonation in the acidic tumor microenvironment, achieving a potential reversal from negative to positive and exhibiting a positive charge, thereby enhancing its interaction with the negatively charged cell membrane and promoting responsive drug release and targeted delivery.

[0006] Therefore, it is essential to develop nanomicelles that can simultaneously enhance the drug loading capacity and responsive release of dendritic macromolecules at tumor sites by modifying them with zwitterionic ionization. Summary of the Invention

[0007] The present invention aims to provide a prodrug molecule, drug-loaded nanomicelles, and their preparation method and application based on zwitterionic polyamide-amine dendritic macromolecules. These nanomicelles use zwitterionic-modified dendritic PAMAM as a carrier to encapsulate hydrophobic anticancer drugs. During the zwitterionic modification process, the hydrophobic drug paclitaxel is used as the hydrophobic end of the nanomicelles to specifically recognize α-amino acids overexpressed by tumor cells. V The β3 integrin targeting peptide {tumor-targeting peptide, c(RGDfC)} and pH-sensitive zwitterionic groups are used as hydrophilic ends, enabling self-assembly. Then, another small-molecule hydrophobic anticancer drug is physically encapsulated to obtain dual-drug-loading nanomicelles. These nanomicelles have advantages such as high drug loading capacity, low toxicity, and enhanced bioavailability of hydrophobic drugs. Furthermore, their pH responsiveness allows for targeted drug release at the tumor site, thereby enhancing the tumor-suppressing effect.

[0008] To achieve the above-mentioned objectives of the present invention, the present invention adopts the following technical solution:

[0009] A first aspect of the present invention provides a prodrug molecule based on a zwitterionic polyamide-amine dendritic macromolecule, the prodrug molecule having a structure as shown in general formula (I):

[0010]

[0011] In general formula (I), a, b, and c represent the amounts of thiolated paclitaxel, tumor-targeting peptide, and thiolated histidine grafted onto the polyamide-amine dendrimer, respectively. The sum of a, b, and c is the same as the total number of primary amino groups on the surface of the polyamide-amine dendrimer. The polyamide-amine dendrimer is denoted as PAMAM, thiolated paclitaxel as PTX-SH, tumor-targeting peptide as c(RGDfC), and thiolated histidine 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 general formula (I) is specifically G1 PAMAM, the sum of a, b, and c is 8; when PAMAM in general formula (I) is specifically G2 PAMAM, the sum of a, b, and c is 16; when PAMAM in 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 a zwitterionic polyamide-amine dendritic macromolecule as described above. The preparation method includes: S1. maleic anhydride-modifying PAMAM to obtain a first modified dendritic macromolecule, denoted as MM; S2. modifying MM with PTX-SH to give MM a hydrophobic end, to obtain a second modified dendritic macromolecule, denoted as M-MP; S3. modifying M-MP with c(RGDfC) to give M-MP a hydrophilic end that can target and recognize tumor cells, to obtain a third modified dendritic macromolecule, denoted as M-MPR; S4. modifying M-MPR with HIS-SH to give M-MPR a pH-sensitive hydrophilic end, to obtain a prodrug molecule based on a zwitterionic polyamide-amine dendritic macromolecule, denoted as M-MPRH.

[0014] Further, 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 solution is dialyzed twice with the first dialysate replaced every 6 to 8 hours, and then dialyzed twice with the second dialysate replaced every 2 hours, and the dialysate is then removed and freeze-dried to obtain MM; wherein, the first dialysate is a boric acid-borax buffer solution with pH=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.

[0015] Further, S2 specifically includes the following steps: S21. Dissolve 3,3'-dithiodipropionic acid and paclitaxel in dichloromethane, stir at room temperature for 1 h, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, and reflux at room temperature for 48 h; after the reaction is completed, perform column chromatography purification and collect the product to obtain dithiodipropionic acid paclitaxel, denoted as PTX-SS-PTX; S22. Dissolve PTX-SS-PTX and dithiothreitol (DTT) together in dichloromethane, stir at room temperature for 1 h, then add triethylamine, and stir the reaction under N2 atmosphere. 12h; After the reaction is complete, collect the organic phase, wash, dry, and concentrate it, precipitate it with petroleum ether, and centrifuge to collect the product to obtain PTX-SH; S23. Mix MM and PTX-SH in a molar ratio of 1:1 to 15, dissolve them in methanol, and stir at room temperature under N2 protection for 24 to 48h to obtain the second reaction solution; Dialyze the second reaction solution with methanol as the dialysis medium, changing it every 6h, and collect the dialysate after 3 to 6 dialysis cycles. Dry it under vacuum to obtain M-MP; Optionally, in S23, the molecular weight cutoff of the dialysis bag used for dialysis is 2000 to 7000.

[0016] Further, S3 specifically includes the following steps: M-MP and c(RGDfC) are 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 stirred at room temperature under N2 protection for 24 hours to obtain a third reaction solution; the third reaction solution is dialyzed with methanol as the dialysis medium and replaced every 6 hours, and after dialysis 4 times, the dialysate is collected, vacuum dried, and M-MPR is obtained; optionally, in S3, the molecular weight cutoff of the dialysis bag used for dialysis is 2000 to 7000.

[0017] Further, S4 specifically includes the following steps: S41. Mix mercaptoethylamine hydrochloride with triphenylchloromethane, dissolve in trifluoroacetic acid, stir at room temperature for 2 hours to obtain the fourth reaction solution; rotary evaporate and wash the fourth reaction solution, collect the organic phase, dry and concentrate to obtain 2-(triphenylmethylthio)ethylamine; S42. N α -N τ Triphenylmethylhistidine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide were dissolved in dichloromethane, then triethylamine was added, and the mixture was stirred at room temperature for 2 hours. 2-(triphenylmethylthio)ethylamine was then added, and the mixture was stirred at room temperature for 12 hours. After the reaction was complete, the white solid byproduct was filtered off, the filtrate was collected, concentrated by rotary evaporation, and purified by column chromatography. The product was collected and dried under vacuum to obtain N'-(2-(triphenylmethylthio)ethyl)-N α -tert-Butoxycarbonyl-N τ -Triphenylmethylhistamide; S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-Butoxycarbonyl-N τ - Triphenylmethylhistamine amide and triisopropylsilane are mixed, and trifluoroacetic acid is added. The mixture is stirred in an ice bath for 2 hours. After the reaction is complete, most of the trifluoroacetic acid is removed by rotary evaporation. The mixture is then precipitated with cold diethyl ether. After centrifugation, the precipitate is dissolved in water and the insoluble matter is filtered off. 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 and dissolved in methanol. The mixture is stirred at room temperature under N2 protection for 24 to 48 hours to obtain the fifth reaction solution. The fifth reaction solution is dialyzed with methanol as the dialysis medium and replaced every 6 hours. After dialysis 3 to 6 times, the dialysate is collected and dried under vacuum to obtain M-MPRH. Optionally, in S44, the molecular weight cutoff of the dialysis bag used for dialysis is 2000 to 7000.

[0018] A third aspect of the present invention provides the application of prodrug molecules based on zwitterionic polyamide-amine dendritic macromolecules as described above in the field of nanomedicine delivery systems.

[0019] A fourth aspect of the present invention provides a nano-drug-loaded micelle comprising a prodrug molecule based on a zwitterionic polyamide-amine dendritic macromolecule as described above, encapsulating a hydrophobic anticancer drug.

[0020] The fifth aspect of this invention provides a method for preparing the drug-loaded nanomicelles as described above. This method includes using PAMAM as a carrier and the hydrophobic antitumor drug PTX-SH as a hydrophobic end, which can specifically recognize α-cells overexpressed by tumor cells. VAfter the c(RGDfC) of β3 integrin and the pH-sensitive HIS-SH serve as hydrophilic ends, they self-assemble to form a prodrug molecule based on a zwitterionic polyamide-amine dendritic macromolecule, and then physically encapsulate another hydrophobic anticancer drug to obtain drug-loaded nanomicelles.

[0021] Furthermore, the preparation method includes: S5. Preparing drug-loaded nanomicelles by encapsulating hydrophobic anticancer drugs with M-MPRH; 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. Mixing the M-MPRH methanol solution and the hydrophobic anticancer drug methanol solution at a mass ratio of 5-20:1, and stirring at room temperature in the dark. After 2–6 hours, the sixth reaction solution is obtained; S54. The sixth reaction solution is slowly added dropwise to water for dialysis. The dialysis medium is water, and the solution is changed every 6 hours for 3 dialysis cycles. After dialysis, the dialysis solution is collected to obtain drug-loaded nanomicelles; Optionally, in S52, the hydrophobic anticancer drug includes at least one of doxorubicin, camptothecin, fluorouracil, and dihydroporphyrin e6; Optionally, in S54, the mass ratio of the reaction solution to water is 1:0.5–3, and the dropping rate into the water is 0.5–1 mL / min.

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

[0023] 1. This invention provides a PAMAM carrier modified with pH-sensitive zwitterionic groups, which uses the anticancer drug paclitaxel as a hydrophobic end to specifically recognize α-carbohydrate overexpressed by tumor cells. V The c(RGDfC) of β3 integrin and its pH-sensitive zwitterionic group serve as hydrophilic ends, enabling it to form prodrug molecules based on zwitterionic polyamide-amine dendritic macromolecules; and it can physically encapsulate hydrophobic anticancer drugs during self-assembly, thus producing novel drug-loaded nanomicelles.

[0024] 2. The prodrug molecule and drug-carrying nanomicelles based on zwitterionic polyamide-amine dendritic macromolecules provided by this invention can specifically recognize tumor-overexpressed α-amino acids. V β3 integrin, thereby achieving targeted therapy to the tumor site;

[0025] 3. The prodrug molecule and nano-drug-carrying micelles based on zwitterionic polyamide-amine dendritic macromolecules provided by this invention contain pH-sensitive histidine at their hydrophilic ends, which can achieve a reversal of the surface potential from negative to positive at the tumor site.

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

[0027] 5. The drug-loaded nanomicelles provided by this invention have a good tumor-suppressing effect. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the preparation of prodrug molecules and nano-drug-carrying micelles based on zwitterionic polyamide-amine dendritic macromolecules in 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 Embodiment 1 of the present invention;

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

[0031] Figure 4 The UV-Vis absorption spectrum of M-MPRH@DOX drug-loaded nanomicelles obtained in Example 1 of this invention;

[0032] Figure 5 The fluorescence spectrum of M-MPRH@DOX nanomedicine micelles obtained in Example 1 of this invention;

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

[0034] Figure 7 The image shown is a transmission electron microscope (TEM) image of the M-MPRH@DOX drug-loaded nanomicelles obtained in Example 1 of this invention, along with their particle size distribution histogram.

[0035] Figure 8 The particle size distribution of M-MPRH@DOX drug-loaded nanomicelles obtained in Example 1 of this invention in a phosphate buffer solution with pH = 5.5 to 7.4 is shown.

[0036] Figure 9 The image shows the zeta potential of the M-MPRH@DOX drug-loaded nanomicelles obtained in Example 1 of this invention in a phosphate buffer solution with pH = 5.5 to 7.4.

[0037] Figure 10 This is a particle size distribution diagram of the interaction between M-MPRH micelles and proteins obtained in Example 1 of the present invention;

[0038] Figure 11 The particle size distribution diagram of the interaction between M-MPRH@DOX drug-loaded nanomicelles and proteins obtained in Example 1 of this invention is shown.

[0039] Figure 12The image shows the drug release curve of the M-MPRH@DOX nano-drug-loaded micelles obtained in Example 1 of this invention in a phosphate buffer solution with pH = 5.5 to 7.4.

[0040] Figure 13 This is a graph showing the body weight of mice in an in vivo tumor suppression experiment using M-MPRH@DOX drug-loaded nanomicelles obtained in Example 1 of this invention.

[0041] Figure 14 This is a photograph of a mouse tumor in an in vivo tumor suppression experiment using M-MPRH@DOX drug-loaded nanomicelles obtained in Example 1 of this invention.

[0042] Figure 15 This is a graph showing the tumor volume in mice during an in vivo tumor suppression experiment using M-MPRH@DOX nanomedicine micelles obtained in Example 1 of this invention. Detailed Implementation

[0043] The embodiments of the present invention will be described below with reference to the accompanying drawings and examples.

[0044] Specifically, this invention provides a method for preparing drug-carrying nanomicelles that use zwitterionic-modified dendritic macromolecule PAMAM as a carrier to encapsulate hydrophobic anticancer drugs, such as... Figure 1 As shown, it specifically includes the following steps:

[0045] S1. PAMAM is maleic anhydride-treated 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 the first reaction solution.

[0047] S12. Dialyze the first reaction solution twice, replacing the first dialysate every 6-8 hours. Then, dialyze twice more using the second dialysate every 2 hours. After dialysis, remove the dialysate and freeze-dry it to obtain MM. The first dialysate is a boric acid-borax buffer solution with pH=9, and the second dialysate is water.

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

[0049] S2. Modify MM using PTX-SH to give it a hydrophobic end, resulting in 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 and stirred at room temperature for 1 h. 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 the mixture was refluxed at room temperature for 48 h. After the reaction was completed, the product was purified by column chromatography and collected to obtain dithiodipropionic acid paclitaxel, denoted as PTX-SS-PTX.

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

[0052] S23. Mix MM and PTX-SH in a molar ratio of 1:1 to 15, dissolve them in methanol, and stir the reaction at room temperature under N2 protection for 24 to 48 hours to obtain a second reaction solution. Dialyze the second reaction solution with methanol as the dialysis medium, changing it every 6 hours. After dialysis 3 to 6 times, collect the dialysis solution, dry it under vacuum, and obtain M-MP.

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

[0054] S3. Modify M-MP with c(RGDfC) to give M-MP a hydrophilic end that can target and recognize tumor cells, thus 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 and dissolved in a mixed solvent of methanol and water in a volume ratio of 10:1. The mixture was stirred at room temperature under N2 protection for 24 hours to obtain the third reaction solution. The third reaction solution was dialyzed with methanol as the dialysis medium and replaced every 6 hours. After dialysis 4 times, the dialysate was collected, dried under vacuum, and M-MPR was obtained.

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

[0057] S4. Modify M-MPR using HIS-SH to give it a pH-sensitive hydrophilic end, thus obtaining M-MPRH. The specific steps are as follows:

[0058] S41. Mercaptoethylamine hydrochloride (2.30 g, 20.22 mmol) was mixed with triphenylchloromethane (5.64 g, 20.22 mmol), dissolved in trifluoroacetic acid, and stirred at room temperature for 2 h to obtain a fourth reaction solution; the fourth reaction solution was rotary evaporated, washed, and the organic phase was collected, dried and concentrated to obtain 2-(triphenylthio)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. Then, triethylamine (1.04 mL, 7.51 mmol) was added, and the mixture was stirred at room temperature for 2 h. Next, 2-(triphenylmethylthio)ethylamine (996.85 mg, 2.00 mmol) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the white solid byproduct was filtered off, the filtrate was collected, concentrated by rotary evaporation, and purified by column chromatography. The product was collected and dried under vacuum to give N'-(2-(triphenylmethylthio)ethyl)-N α -tert-Butoxycarbonyl-N τ -Triphenylmethylhistamine;

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

[0061] S44. Mix M-MPR and HIS-SH in a molar ratio of 1:1 to 30, dissolve them in methanol, and stir the mixture at room temperature under N2 protection for 24 to 48 hours to obtain the fifth reaction solution. Dialyze the fifth reaction solution with methanol as the dialysis medium, changing it every 6 hours. After dialysis 3 to 6 times, collect the dialysis solution, dry it under vacuum, and 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 drug-loaded nanomicelles by encapsulating hydrophobic anticancer drugs with M-MPRH, the specific steps are as follows:

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

[0065] S52. Dissolve M-MPRH in methanol to prepare an M-MPRH methanol solution of 0.5–5 mg / mL;

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

[0067] S54. The sixth reaction solution was slowly dripped into water for dialysis. The dialysis medium was water. The solution was changed every 6 hours for 3 dialysis cycles. After dialysis, the dialysis solution 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 dihydroporphyrin e6;

[0069] Optionally, in S54, the mass ratio of the reaction solution to water is 1:0.5 to 3, and the dropping rate into the water is 0.5 to 1 mL / min.

[0070] Example 1

[0071] S1. Maleate G1 PAMAM, the specific steps are as follows:

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

[0073] S12. After the reaction is complete, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 1500 for dialysis. The dialysis medium is a boric acid-borax buffer solution with pH=9. The dialysis medium is changed every 6-8 hours. After dialysis twice with the above buffer solution, the dialysis medium is changed to water. The dialysis medium is changed every 2 hours. After dialysis twice, the dialysis is stopped. The dialysis solution is taken out and freeze-dried to obtain MM.

[0074] S2. Modify MM with PTX-SH to synthesize M-MP, giving it 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 and stirred at room temperature for 1 h. 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 the mixture was refluxed at room temperature for 48 h. After the reaction was completed, the mixture was purified by column chromatography using ethyl acetate:n-hexane = 2:1 as the mobile phase. R was collected. f The substance with a concentration of 0.36 was 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 together in dichloromethane and stirred at room temperature for 1 h. Triethylamine (86 μL, 0.621 mmol) was then added, and the mixture was stirred under a nitrogen atmosphere for 12 h. After the reaction was complete, the organic phase was collected, washed, dried, concentrated, precipitated with petroleum ether, and the product PTX-SH was collected by centrifugation.

[0077] S23. Mix MM and PTX-SH in a molar ratio of 1:4, dissolve in methanol, and react at room temperature under N2 protection with stirring for 24 h. After the reaction is complete, place the reaction solution in a dialysis bag with a molecular weight cutoff of 2000 for dialysis. The dialysis medium is methanol. The dialysis solution is changed every 6 h. After dialysis 4 times, the dialysis solution is collected and dried under vacuum to obtain the product M-MP.

[0078] S3. M-MP was modified with c(RGDfC) to synthesize M-MPR, giving it a hydrophilic end that can target and recognize tumor cells. Specifically, M-MP and c(RGDfC) were mixed in a molar ratio of 1:2 and dissolved in a mixed solvent of methanol and water in a volume ratio of 10:1. The mixture was then stirred at room temperature under N2 protection for 24 hours. 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, and the dialysis solution was changed every 6 hours. After dialysis four times, the dialysis solution was collected and vacuum dried to obtain the product M-MPR.

[0079] S4. M-MPR was modified using HIS-SH to synthesize M-MPRH, giving it a pH-sensitive hydrophilic end. The specific steps are as follows:

[0080] S41. Mercaptoethylamine hydrochloride (2.30 g, 20.22 mmol) was mixed with triphenylchloromethane (5.64 g, 20.22 mmol), dissolved in trifluoroacetic acid, and stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was rotary evaporated, washed, and the organic phase was collected, dried, and concentrated to obtain the product 2-(triphenylthio)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. Triethylamine (1.04 mL, 7.51 mmol) was added, and the mixture was stirred at room temperature for 2 h. Then, 2-(triphenylmethylthio)ethylamine (996.85 mg, 2.00 mmol) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, a white solid byproduct was filtered off, and the filtrate was collected. The filtrate was concentrated by rotary evaporation and purified by column chromatography using ethyl acetate:dichloromethane (1:1) as the mobile phase. R was collected. f The substance with a concentration of 0.6 was dried under vacuum to give the product N'-(2-(triphenylmethylthio)ethyl)-N α -tert-Butoxycarbonyl-N τ -Triphenylmethylhistamine;

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

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

[0084] S5. Preparation of drug-loaded nanomicelles by encapsulating the hydrophobic anticancer drug DOX with M-MPRH, 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 in the dark for 1 h 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 M-MPRH methanol solution and DOX methanol solution at a mass ratio of M-MPRH:DOX of 10:1 and stir at room temperature in the dark for 3 hours.

[0088] S54. After the reaction is complete, the reaction solution is slowly added to water at a rate of 0.5 mL / min, with a volume ratio of 1:1 between the reaction solution and water. The solution is then placed in a dialysis bag with a molecular weight cutoff of 500 for dialysis. The dialysis medium is water, and the dialysis solution is changed every 6 hours for a total of 3 dialysis cycles. After dialysis, the dialysis solution is collected to obtain drug-loaded nanomicelles, specifically M-MPRH@DOX drug-loaded nanomicelles.

[0089] The proton spectrum of M-MPRH is as follows Figure 2 As shown, the chemical shifts at 7.3–8.2 ppm in M-MP correspond to the characteristic peaks of PTX-SH, and compared to MM, the peak area at 5.8–6.4 ppm is reduced, further proving that PTX-SH successfully modified MM. Furthermore, 7.1–7.4 ppm corresponds to the characteristic hydrogens of c(RGDfC), and the two sharp peaks at 7.3–7.4 ppm and 8.5–8.7 ppm correspond to the characteristic hydrogens of HIS-SH. The complete disappearance of the peak corresponding to the olefinic hydrogen at 5.8–6.4 ppm in M-MPRH also proves that the double bond on MM was completely added, further confirming the successful synthesis of M-MPRH. The paclitaxel loading rate was 27.25%.

[0090] The critical micelle concentration of M-MPRH was determined using a fluorescence spectrophotometer, such as... 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 critical micelle concentration is related to the ratio of hydrophilic to hydrophobic ends of the micelles. In M-MPRH, the hydrophilic end is a first-generation polyamide-amine dendritic macromolecule modified with c(RGDfC) and HIS-SH, and the hydrophobic end is PTX-SH. The molar ratio of the two is 3:1.

[0091] Full-wavelength scanning of DOX and M-MPRH@DOX drug-loaded nanomicelles was performed using UV-Vis spectrophotometry, such as... Figure 4As shown, the maximum absorption peak of DOX is at 480 nm, and the maximum absorption peak of the drug-loaded micelles is at 494 nm. The peak shapes of the two are similar, and the absorption peak of DOX undergoes a red shift, indicating that M-MPRH successfully encapsulates DOX to form M-MPRH@DOX nano-drug-loaded micelles.

[0092] The fluorescence spectrophotometer was used to scan DOX and M-MPRH@DOX drug-loaded nanomicelles 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 596 nm, and their peak shapes were similar, further proving that M-MPRH successfully encapsulated DOX. The DOX loading rate was 8.58%, so the total drug loading rate of the drug-loaded micelles reached 35.83%.

[0093] The morphology of drug-loaded micelles was characterized using transmission electron microscopy, such as... Figure 6 , Figure 7 As shown, the drug-loaded micelles have a relatively regular morphology and a relatively uniform particle size distribution, with an average particle size of 24.03 nm.

[0094] The effect of different pH values ​​on the particle size of M-MPRH micelles and M-MPRH@DOX drug-loaded nanomicelles was determined using a laser particle size analyzer. Figure 8 As shown, the hydrodynamic particle sizes of M-MPRH micelles were 81.71±1.98 nm, 93.06±6.48 nm, and 114.98±22.11 nm at pH = 7.4, 6.5, and 5.5, respectively, while those of M-MPRH@DOX drug-loaded nanomicelles were 92.83±5.78 nm, 112.73±11.92 nm, and 147.07±4.94 nm, respectively. The hydrodynamic particle size of the micelles gradually increased with decreasing pH. This is mainly because the imidazole ring of histidine is pH-sensitive; in an acidic environment, it protonates and becomes positively charged, thereby disrupting the surface charge balance of the micelles and causing dissociation, resulting in larger particle sizes. These results indicate that M-MPRH@DOX drug-loaded nanomicelles have pH-responsive capabilities in the acidic microenvironment of tumor cells, which is beneficial for achieving rapid drug release.

[0095] The effect of different pH values ​​on the potential of M-MPRH micelles and M-MPRH@DOX drug-loaded nanomicelles was determined using a laser particle size analyzer. Figure 9As shown, at pH = 7.4, 6.5, and 5.5, the Zeta potentials of M-MPRH micelles were -5.65±1.16 mV, 1.52±0.72 mV, and 9.43±1.96 mV, respectively, and the Zeta potentials of M-MPRH@DOX drug-loaded nanomicelles were -10.59±1.23 mV, 0.66±0.24 mV, and 5.41±0.37 mV, respectively. These results indicate that compared to the pH of normal tissue, M-MPRH micelles and M-MPRH@DOX drug-loaded nanomicelles can achieve a surface potential reversal from negative to positive in the weakly acidic environment of tumor tissue. The positively charged micelles can generate a strong electrostatic attraction with the negatively charged tumor cell membrane. Therefore, this effect can enhance the uptake of micelles by cells to a certain extent, thereby delivering more anti-tumor drugs to the tumor site. Figure 8 It is known that this can cause micelle dissociation, thereby achieving specific accumulation of the encapsulated drug at the tumor site. The reason for this result is that at pH=7.4, the partial dissociation of the carboxyl groups on the M-MPRH surface makes the micelles negatively charged. When the pH decreases, the dissociation of the carboxyl groups is inhibited in a weakly acidic environment, and the protonation of the amino groups and the imidazole ring in the histidine is enhanced, thereby causing the micelles to undergo a potential reversal and become positively charged.

[0096] The stability of M-MPRH micelles and M-MPRH@DOX drug-loaded nanomicelles in protein solutions was determined using a laser particle size analyzer. Figure 10 As shown, the hydrodynamic particle size of 0.5 mg / mL fibrinogen is approximately 26.28 nm, the hydrodynamic particle size of 1 mg / mL M-MPRH micelles is approximately 80.71 nm, and the hydrodynamic particle size of the mixed solution of 0.5 mg / mL fibrinogen + 1 mg / mL M-MPRH is approximately 50.18 nm. The hydrodynamic particle size of the M-MPRH mixed solution falls between the two, indicating that M-MPRH does not aggregate with the protein. Figure 11 As shown, the hydrodynamic particle size of 1 mg / mL M-MPRH@DOX drug-loaded micelles is approximately 98.24 nm, and the hydrodynamic particle size of the mixed solution of 0.5 mg / mL fibrinogen + 1 mg / mL M-MPRH@DOX drug-loaded micelles is approximately 69.63 nm. The hydrodynamic particle size of the M-MPRH@DOX drug-loaded micelle mixture is also between the two, indicating that it does not aggregate with proteins and demonstrates its good stability in protein solutions.

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

[0098] In vivo tumor suppression experiments were conducted on M-MPRH@DOX drug-loaded nanomicelles in mice. Figure 13 As shown, after 14 days of administration, the average weight of mice in the saline group was 30.12 g, the average weight of mice in the free DOX group was 23.04 g, and the average weight of mice in the M-MPRH@DOX nano-drug-loaded micelle group was 31.02 g. The comparison shows that the weight of mice in the M-MPRH@DOX nano-drug-loaded micelle group was similar to that in the saline group and much higher than that in the free DOX group, indicating that the drug-loaded micelles have lower biotoxicity compared to free DOX. Figure 14 To obtain images of mouse tumors, one mouse was randomly selected from each group after 14 days of treatment, dissected, and the tumor was removed and photographed. Figure 14 It can be seen that the tumor volume of mice in the M-MPRH@DOX nano-drug-loaded micelle group was the smallest, indicating that the tumors of mice treated with drug-loaded micelles were significantly inhibited compared with those in the saline group, and the tumor inhibition effect was better than that of the free DOX group. Figure 15 A graph showing the average tumor volume of all mice over a 14-day experimental period, as shown below. Figure 15 As shown, at 14 days, the average tumor volume of mice in the untreated saline group was approximately 1229.81 mm. 3 The average tumor volume in the free DOX group mice was approximately 377.68 mm. 3 The average tumor volume in mice in the M-MPRH@DOX nanoparticle drug-loaded micelle group was approximately 87.19 mm. 3 Calculations showed that the tumor inhibition rate of the drug-loaded micelle group was 92.91%, which was higher than that of the free DOX group (69.29%). Figure 14 This demonstrates that M-MPRH@DOX drug-loaded nanomicelles have a good tumor-suppressing effect.

[0099] Example 2

[0100] S1. Maleate G2 PAMAM, the specific steps are as follows:

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

[0102] S12. After the reaction is complete, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 3500 for dialysis. The dialysis medium is a boric acid-borax buffer solution with pH=9. The dialysis medium is changed every 8 hours. After dialysis twice using the above buffer solution, the dialysis medium is changed to water. The dialysis medium is changed every 2 hours. After dialysis twice, the dialysis is stopped. The dialysis solution is taken out and freeze-dried to obtain MM.

[0103] S2. Modify MM with PTX-SH to synthesize M-MP, giving it 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 and stirred at room temperature for 1 h. 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 the mixture was refluxed at room temperature for 48 h. After the reaction was completed, the mixture was purified by column chromatography using ethyl acetate:n-hexane = 2:1 as the mobile phase. R was collected. f The substance with a concentration of 0.36 was 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 together in dichloromethane and stirred at room temperature for 1 h. Triethylamine (86 μL, 0.621 mmol) was then added, and the mixture was stirred under a nitrogen atmosphere for 12 h. After the reaction was complete, the organic phase was collected, washed, dried, concentrated, precipitated with petroleum ether, and the product PTX-SH was collected by centrifugation.

[0106] S23. Mix MM and PTX-SH in a molar ratio of 1:8, dissolve in methanol, and react at room temperature under N2 protection with stirring for 24 h. After the reaction is complete, place the reaction solution in a dialysis bag with a molecular weight cutoff of 3500 for dialysis. The dialysis medium is methanol. Change the dialysis solution every 6 h. After dialysis 4 times, collect the dialysis solution and dry it under vacuum to obtain the product M-MP.

[0107] S3. M-MP was modified with c(RGDfC) to synthesize M-MPR, giving it a hydrophilic end that can target and recognize tumor cells. Specifically, M-MP and c(RGDfC) were mixed at a molar ratio of 1:4 and dissolved in a mixed solvent of methanol and water at a volume ratio of 10:1. The mixture was then stirred at room temperature under N2 protection for 24 hours. 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, and the dialysis solution was changed every 6 hours. After dialysis four times, the dialysis solution was collected and vacuum dried to obtain the product M-MPR.

[0108] S4. M-MPR was modified using HIS-SH to synthesize M-MPRH, giving it a pH-sensitive hydrophilic end. The specific steps are as follows:

[0109] S41. Mercaptoethylamine hydrochloride (2.30 g, 20.22 mmol) was mixed with triphenylchloromethane (5.64 g, 20.22 mmol), dissolved in trifluoroacetic acid, and stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was rotary evaporated, washed, and the organic phase was collected, dried, and concentrated to obtain the product 2-(triphenylthio)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. Triethylamine (1.04 mL, 7.51 mmol) was added, and the mixture was stirred at room temperature for 2 h. Then, 2-(triphenylmethylthio)ethylamine (996.85 mg, 2.00 mmol) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, a white solid byproduct was filtered off, and the filtrate was collected. The filtrate was concentrated by rotary evaporation and purified by column chromatography using ethyl acetate:dichloromethane (1:1) as the mobile phase. R was collected. f The substance with a concentration of 0.6 was dried under vacuum to give the product N'-(2-(triphenylmethylthio)ethyl)-N α -tert-Butoxycarbonyl-N τ -Triphenylmethylhistamine;

[0111] S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-Butoxycarbonyl-N τ- Triphenylmethylhistamide (150.00 mg, 0.188 mmol) was mixed with triisopropylsilane (240 μL, 1.17 mmol), and 6 mL of trifluoroacetic acid was added. The mixture was stirred in an ice bath for 2 h. After the reaction was completed, most of the trifluoroacetic acid was removed by rotary evaporation. The mixture was then precipitated with cold diethyl ether. After centrifugation, the precipitate was dissolved in water and the insoluble matter was filtered off. 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 reacted at room temperature under N2 protection with stirring for 24 h. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 for dialysis. The dialysis medium was methanol, and the dialysis solution was changed every 6 h. After dialysis 4 times, the dialysis solution was collected and dried under vacuum to obtain the product M-MPRH.

[0113] S5. Preparation of drug-loaded nanomicelles by encapsulating the hydrophobic anticancer drug DOX with M-MPRH, 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 in the dark for 1 h 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 M-MPRH methanol solution and DOX methanol solution at a mass ratio of M-MPRH:DOX of 10:1 and stir at room temperature in the dark for 3 hours.

[0117] S54. After the reaction is complete, the reaction solution is slowly added to water at a rate of 0.5 mL / min, with a volume ratio of reaction solution to water of 1:2. The solution is placed in a dialysis bag with a molecular weight cutoff of 500 for dialysis. The dialysis medium is water. The dialysis solution is changed every 6 hours, and a total of 3 dialysis cycles are performed. After the dialysis is completed, the dialysis solution is collected to obtain M-MPRH@DOX nano-drug-loaded micelles.

[0118] Example 3

[0119] S1. Maleate G3 PAMAM, the specific steps are as follows:

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

[0121] S12. After the reaction is complete, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 7000 for dialysis. The dialysis medium is a boric acid-borax buffer solution with pH=9. The dialysis medium is changed every 6-8 hours. After dialysis twice with the above buffer solution, the dialysis medium is changed to water. The dialysis medium is changed every 2 hours. After dialysis twice, the dialysis is stopped. The dialysis solution is taken out and freeze-dried to obtain MM.

[0122] S2. Modify MM with PTX-SH to synthesize M-MP, giving it 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 and stirred at room temperature for 1 h. 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 the mixture was refluxed at room temperature for 48 h. After the reaction was completed, the mixture was purified by column chromatography using ethyl acetate:n-hexane = 2:1 as the mobile phase. R was collected. f The substance with a concentration of 0.36 was 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 together in dichloromethane and stirred at room temperature for 1 h. Triethylamine (86 μL, 0.621 mmol) was then added, and the mixture was stirred under a nitrogen atmosphere for 12 h. After the reaction was complete, the organic phase was collected, washed, dried, concentrated, precipitated with petroleum ether, and the product PTX-SH was collected by centrifugation.

[0125] S23. Mix MM and PTX-SH in a molar ratio of 1:12, dissolve in methanol, and react at room temperature under N2 protection with stirring for 24 h. After the reaction is complete, place the reaction solution in a dialysis bag with a molecular weight cutoff of 7000 for dialysis. The dialysis medium is methanol. The dialysis solution is changed every 6 h. After dialysis 4 times, the dialysis solution is collected and dried under vacuum to obtain the product M-MP.

[0126] S3. M-MP was modified with c(RGDfC) to synthesize M-MPR, giving it a hydrophilic end that can target and recognize tumor cells. Specifically, M-MP and c(RGDfC) were mixed at a molar ratio of 1:8 and dissolved in a mixed solvent of methanol and water at a volume ratio of 10:1. The mixture was then stirred at room temperature under N2 protection for 24 hours. 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, and the dialysis solution was changed every 6 hours. After dialysis four times, the dialysis solution was collected and vacuum dried to obtain the product M-MPR.

[0127] S4. M-MPR was modified using HIS-SH to synthesize M-MPRH, giving it a pH-sensitive hydrophilic end. The specific steps are as follows:

[0128] S41. Mercaptoethylamine hydrochloride (2.30 g, 20.22 mmol) was mixed with triphenylchloromethane (5.64 g, 20.22 mmol), dissolved in trifluoroacetic acid, and stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was rotary evaporated, washed, and the organic phase was collected, dried, and concentrated to obtain the product 2-(triphenylthio)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. Triethylamine (1.04 mL, 7.51 mmol) was added, and the mixture was stirred at room temperature for 2 h. Then, 2-(triphenylmethylthio)ethylamine (996.85 mg, 2.00 mmol) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, a white solid byproduct was filtered off, and the filtrate was collected. The filtrate was concentrated by rotary evaporation and purified by column chromatography using ethyl acetate:dichloromethane (1:1) as the mobile phase. R was collected. f The substance with a concentration of 0.6 was dried under vacuum to give the product N'-(2-(triphenylmethylthio)ethyl)-N α -tert-Butoxycarbonyl-N τ -Triphenylmethylhistamine;

[0130] S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-Butoxycarbonyl-N τ- Triphenylmethylhistamide (150.00 mg, 0.188 mmol) was mixed with triisopropylsilane (240 μL, 1.17 mmol), and 6 mL of trifluoroacetic acid was added. The mixture was stirred in an ice bath for 2 h. After the reaction was completed, most of the trifluoroacetic acid was removed by rotary evaporation. The mixture was then precipitated with cold diethyl ether. After centrifugation, the precipitate was dissolved in water and the insoluble matter was filtered off. 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 reacted at room temperature under N2 protection with stirring for 24 h. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 7000 for dialysis. The dialysis medium was methanol, and the dialysis solution was changed every 6 h. After dialysis 4 times, the dialysis solution was collected and dried under vacuum to obtain the product M-MPRH.

[0132] S5. Preparation of drug-loaded nanomicelles by encapsulating the hydrophobic anticancer drug DOX with M-MPRH, 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 in the dark for 1 h 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 M-MPRH methanol solution and DOX methanol solution at a mass ratio of M-MPRH:DOX of 10:1 and stir at room temperature in the dark for 3 hours.

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

[0137] Example 4

[0138] S1. Maleate G1 PAMAM, the specific steps are as follows:

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

[0140] S12. After the reaction is complete, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 1500 for dialysis. The dialysis medium is a boric acid-borax buffer solution with pH=9. The dialysis medium is changed every 6-8 hours. After dialysis twice with the above buffer solution, the dialysis medium is changed to water. The dialysis medium is changed every 2 hours. After dialysis twice, the dialysis is stopped. The dialysis solution is taken out and freeze-dried to obtain MM.

[0141] S2. Modify MM with PTX-SH to synthesize M-MP, giving it 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 and stirred at room temperature for 1 h. 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 the mixture was refluxed at room temperature for 48 h. After the reaction was completed, the mixture was purified by column chromatography using ethyl acetate:n-hexane = 2:1 as the mobile phase. R was collected. f The substance with a concentration of 0.36 was 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 together in dichloromethane and stirred at room temperature for 1 h. Triethylamine (86 μL, 0.621 mmol) was then added, and the mixture was stirred under a nitrogen atmosphere for 12 h. After the reaction was complete, the organic phase was collected, washed, dried, concentrated, precipitated with petroleum ether, and the product PTX-SH was collected by centrifugation.

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

[0145] S3. M-MP was modified with c(RGDfC) to synthesize M-MPR, giving it a hydrophilic end that can target and recognize tumor cells. Specifically, M-MP and c(RGDfC) were mixed in a molar ratio of 1:2 and dissolved in a mixed solvent of methanol and water in a volume ratio of 10:1. The mixture was then stirred at room temperature under N2 protection for 24 hours. 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, and the dialysis solution was changed every 6 hours. After dialysis four times, the dialysis solution was collected and vacuum dried to obtain the product M-MPR.

[0146] S4. M-MPR was modified using HIS-SH to synthesize M-MPRH, giving it a pH-sensitive hydrophilic end. The specific steps are as follows:

[0147] S41. Mercaptoethylamine hydrochloride (2.30 g, 20.22 mmol) was mixed with triphenylchloromethane (5.64 g, 20.22 mmol), dissolved in trifluoroacetic acid, and stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was rotary evaporated, washed, and the organic phase was collected, dried, and concentrated to obtain the product 2-(triphenylthio)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. Triethylamine (1.04 mL, 7.51 mmol) was added, and the mixture was stirred at room temperature for 2 h. Then, 2-(triphenylmethylthio)ethylamine (996.85 mg, 2.00 mmol) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, a white solid byproduct was filtered off, and the filtrate was collected. The filtrate was concentrated by rotary evaporation and purified by column chromatography using ethyl acetate:dichloromethane (1:1) as the mobile phase. R was collected. f The substance with a concentration of 0.6 was dried under vacuum to give the product N'-(2-(triphenylmethylthio)ethyl)-N α -tert-Butoxycarbonyl-N τ -Triphenylmethylhistamine;

[0149] S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-Butoxycarbonyl-N τ- Triphenylmethylhistamide (150.00 mg, 0.188 mmol) was mixed with triisopropylsilane (240 μL, 1.17 mmol), and 6 mL of trifluoroacetic acid was added. The mixture was stirred in an ice bath for 2 h. After the reaction was completed, most of the trifluoroacetic acid was removed by rotary evaporation. The mixture was then precipitated with cold diethyl ether. After centrifugation, the precipitate was dissolved in water and the insoluble matter was filtered off. 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 reacted at room temperature under N2 protection with stirring for 24 h. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 2000 for dialysis. The dialysis medium was methanol, and the dialysis solution was changed every 6 h. After dialysis 4 times, the dialysis solution was collected and dried under vacuum to obtain the product M-MPRH.

[0151] S5. Preparation of drug-loaded nanomicelles by encapsulating the hydrophobic anticancer drug camptothecin (CPT) with M-MPRH, 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. Mix the M-MPRH methanol solution and the camptothecin methanol solution at a mass ratio of M-MPRH:camptothecin of 8:1, and stir at room temperature in the dark for 3 hours.

[0155] S54. After the reaction is complete, the reaction solution is slowly added to water at a rate of 0.5 mL / min. The volume ratio of the reaction solution to water is 1:0.5. The solution is placed in a dialysis bag with a molecular weight cutoff of 500 for dialysis. The dialysis medium is water. The dialysis solution is changed every 6 hours and dialysis is performed 3 times in total. After the dialysis is completed, the dialysis solution is collected to obtain M-MPRH@CPT drug-loaded nanomicelles.

[0156] Example 5

[0157] S1. Maleate G1 PAMAM, the specific steps are as follows:

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

[0159] S12. After the reaction is complete, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 1500 for dialysis. The dialysis medium is a boric acid-borax buffer solution with pH=9. The dialysis medium is changed every 6-8 hours. After dialysis twice with the above buffer solution, the dialysis medium is changed to water. The dialysis medium is changed every 2 hours. After dialysis twice, the dialysis is stopped. The dialysis solution is taken out and freeze-dried to obtain MM.

[0160] S2. Modify MM with PTX-SH to synthesize M-MP, giving it 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 and stirred at room temperature for 1 h. 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 the mixture was refluxed at room temperature for 48 h. After the reaction was completed, the mixture was purified by column chromatography using ethyl acetate:n-hexane = 2:1 as the mobile phase. R was collected. f The substance with a concentration of 0.36 was 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 together in dichloromethane and stirred at room temperature for 1 h. Triethylamine (86 μL, 0.621 mmol) was then added, and the mixture was stirred under a nitrogen atmosphere for 12 h. After the reaction was complete, the organic phase was collected, washed, dried, concentrated, precipitated with petroleum ether, and the product PTX-SH was collected by centrifugation.

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

[0164] S3. M-MP was modified with c(RGDfC) to synthesize M-MPR, giving it a hydrophilic end that can target and recognize tumor cells. Specifically, M-MP and c(RGDfC) were mixed in a molar ratio of 1:2 and dissolved in a mixed solvent of methanol and water in a volume ratio of 10:1. The mixture was then stirred at room temperature under N2 protection for 24 hours. 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, and the dialysis solution was changed every 6 hours. After dialysis four times, the dialysis solution was collected and vacuum dried to obtain the product M-MPR.

[0165] S4. M-MPR was modified using HIS-SH to synthesize M-MPRH, giving it a pH-sensitive hydrophilic end. The specific steps are as follows:

[0166] S41. Mercaptoethylamine hydrochloride (2.30 g, 20.22 mmol) was mixed with triphenylchloromethane (5.64 g, 20.22 mmol), dissolved in trifluoroacetic acid, and stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was rotary evaporated, washed, and the organic phase was collected, dried, and concentrated to obtain the product 2-(triphenylthio)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. Triethylamine (1.04 mL, 7.51 mmol) was added, and the mixture was stirred at room temperature for 2 h. Then, 2-(triphenylmethylthio)ethylamine (996.85 mg, 2.00 mmol) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, a white solid byproduct was filtered off, and the filtrate was collected. The filtrate was concentrated by rotary evaporation and purified by column chromatography using ethyl acetate:dichloromethane (1:1) as the mobile phase. R was collected. f The substance with a concentration of 0.6 was dried under vacuum to give the product N'-(2-(triphenylmethylthio)ethyl)-N α -tert-Butoxycarbonyl-N τ -Triphenylmethylhistamine;

[0168] S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-Butoxycarbonyl-N τ- Triphenylmethylhistamide (150.00 mg, 0.188 mmol) was mixed with triisopropylsilane (240 μL, 1.17 mmol), and 6 mL of trifluoroacetic acid was added. The mixture was stirred in an ice bath for 2 h. After the reaction was completed, most of the trifluoroacetic acid was removed by rotary evaporation. The mixture was then precipitated with cold diethyl ether. After centrifugation, the precipitate was dissolved in water and the insoluble matter was filtered off. 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 reacted at room temperature under N2 protection with stirring for 24 h. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 2000 for dialysis. The dialysis medium was methanol, and the dialysis solution was changed every 6 h. After dialysis 4 times, the dialysis solution was collected and dried under vacuum to obtain the product M-MPRH.

[0170] S5. The preparation of drug-loaded nanomicelles by encapsulating the hydrophobic anticancer drug fluorouracil (5-FU) with M-MPRH is 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 M-MPRH:fluorouracil of 5:1, and stir at room temperature in the dark for 3 hours.

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

[0175] Example 6

[0176] S1. Maleate G3 PAMAM, the specific steps are as follows:

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

[0178] S12. After the reaction is complete, the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 7000 for dialysis. The dialysis medium is a boric acid-borax buffer solution with pH=9. The dialysis medium is changed every 6-8 hours. After dialysis twice with the above buffer solution, the dialysis medium is changed to water. The dialysis medium is changed every 2 hours. After dialysis twice, the dialysis is stopped. The dialysis solution is taken out and freeze-dried to obtain MM.

[0179] S2. Modify MM with PTX-SH to synthesize M-MP, giving it 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 and stirred at room temperature for 1 h. 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 the mixture was refluxed at room temperature for 48 h. After the reaction was completed, the mixture was purified by column chromatography using ethyl acetate:n-hexane = 2:1 as the mobile phase. R was collected. f The substance with a concentration of 0.36 was 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 together in dichloromethane and stirred at room temperature for 1 h. Triethylamine (86 μL, 0.621 mmol) was then added, and the mixture was stirred under a nitrogen atmosphere for 12 h. After the reaction was complete, the organic phase was collected, washed, dried, concentrated, 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 reacted at room temperature under N2 protection with stirring for 24 h. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 7000 for dialysis. The dialysis medium was methanol, and the dialysis solution was changed every 6 h. After dialysis 4 times, the dialysis solution was collected and dried under vacuum to obtain the product M-MP.

[0183] S3. M-MP was modified with c(RGDfC) to synthesize M-MPR, giving it a hydrophilic end that can target and recognize tumor cells. Specifically, M-MP and c(RGDfC) were mixed at a molar ratio of 1:10 and dissolved in a mixed solvent of methanol and water at a volume ratio of 10:1. The mixture was then stirred at room temperature under N2 protection for 24 hours. 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, and the dialysis solution was changed every 6 hours. After dialysis four times, the dialysis solution was collected and vacuum dried to obtain the product M-MPR.

[0184] S4. M-MPR was modified using HIS-SH to synthesize M-MPRH, giving it a pH-sensitive hydrophilic end. The specific steps are as follows:

[0185] S41. Mercaptoethylamine hydrochloride (2.30 g, 20.22 mmol) was mixed with triphenylchloromethane (5.64 g, 20.22 mmol), dissolved in trifluoroacetic acid, and stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was rotary evaporated, washed, and the organic phase was collected, dried, and concentrated to obtain the product 2-(triphenylthio)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. Triethylamine (1.04 mL, 7.51 mmol) was added, and the mixture was stirred at room temperature for 2 h. Then, 2-(triphenylmethylthio)ethylamine (996.85 mg, 2.00 mmol) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, a white solid byproduct was filtered off, and the filtrate was collected. The filtrate was concentrated by rotary evaporation and purified by column chromatography using ethyl acetate:dichloromethane (1:1) as the mobile phase. R was collected. f The substance with a concentration of 0.6 was dried under vacuum to give the product N'-(2-(triphenylmethylthio)ethyl)-N α -tert-Butoxycarbonyl-N τ -Triphenylmethylhistamine;

[0187] S43. N'-(2-(triphenylmethylthio)ethyl)-N α -tert-Butoxycarbonyl-N τ- Triphenylmethylhistamide (150.00 mg, 0.188 mmol) was mixed with triisopropylsilane (240 μL, 1.17 mmol), and 6 mL of trifluoroacetic acid was added. The mixture was stirred in an ice bath for 2 h. After the reaction was completed, most of the trifluoroacetic acid was removed by rotary evaporation. The mixture was then precipitated with cold diethyl ether. After centrifugation, the precipitate was dissolved in water and the insoluble matter was filtered off. 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 reacted at room temperature under N2 protection with stirring for 24 h. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 7000 for dialysis. The dialysis medium was methanol, and the dialysis solution was changed every 6 h. After dialysis 4 times, the dialysis solution was collected and dried under vacuum to obtain the product M-MPRH.

[0189] S5. Nanoparticles of drug-loaded micelles were prepared by encapsulating the hydrophobic photosensitizer dihydroporphyrin e6 (Ce6) with M-MPRH. The specific steps are as follows:

[0190] S51. Dissolve 500 μg of dihydroporphyrin E6 in 1 mL of methanol to prepare a 0.5 mg / mL dihydroporphyrin 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 dihydroporphyrin e6 and DOX methanol solution at a mass ratio of M-MPRH:dihydroporphyrin e6:DOX of 40:1:1, and stir at room temperature in the dark for 3 hours.

[0193] S54. After the reaction is complete, the reaction solution is slowly added to water at a rate of 1 mL / min. The volume ratio of the reaction solution to water is 1:3. The solution is placed in a dialysis bag with a molecular weight cutoff of 500 for dialysis. The dialysis medium is water. The dialysis solution is changed every 6 hours and dialysis is performed 3 times in total. After the dialysis is completed, the dialysis solution is collected to obtain M-MPRH@Ce6@DOX nano-drug-loaded micelles.

[0194] Those skilled in the art will understand that the above embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Various modifications and improvements made to the technical solutions of this invention by those skilled in the art without departing from the spirit of the invention should fall within the protection scope defined by the claims. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

Claims

1. A prodrug molecule based on a zwitterionic polyamidoamine dendrimer characterized in that, The prodrug molecule has a structure as shown in general formula (I): In general formula (I), a, b and c respectively represent the amount of thiolated paclitaxel, tumor targeting peptide and thiolated histidine grafted on the polyamidoamine dendrimer, 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.

2. The method of making a prodrug molecule based on a zwitterionic polyamido-amine dendrimer of claim 1, wherein, The preparation method comprises: S1. Maleic anhydride is used to modify PAMAM to obtain a first modified dendrimer, denoted as M-M; S2. PTX-SH is used to modify M-M to make M-M have a hydrophobic end, to obtain a second modified dendrimer, denoted as M-MP; S3. c(RGDfC) is used to modify M-MP to make M-MP have a hydrophilic end capable of targeting recognition of tumor cells, to obtain a third modified dendrimer, denoted as M-MPR; S4. HIS-SH is used to modify M-MPR to make M-MPR have a pH-sensitive hydrophilic end, to obtain the prodrug molecule based on the zwitterionic polyamidoamine dendrimer, denoted as M-MPRH.

3. The method of claim 2, wherein the prepolymer is a zwitterionic polyamidoamine dendrimer.

3. The method of claim 2, wherein the prepolymer is a zwitterionic polyamidoamine dendrimer. The S1 specifically comprises the following steps: S11. PAMAM is mixed with maleic anhydride at a molar ratio of 1:2-5, dimethyl sulfoxide is used for dissolution, and stirring reaction is carried out at room temperature for 24 h to obtain a first reaction solution; S12. The first reaction solution is dialyzed, dialyzed twice with a first dialysis solution at a frequency of replacement every 6-8 h, and then dialyzed twice with a second dialysis solution at a frequency of replacement every 2 h to end the dialysis, and the obtained dialysis solution is taken out and freeze-dried to obtain the M-M; wherein the first dialysis solution is a boric acid-borax buffer solution with pH=9, and the second dialysis solution is water; In the S12, the dialysis bag used for dialysis has a molecular weight cut-off of 1500-7000.

4. The method of claim 2, wherein the prepolymer is a zwitterionic polyamidoamine dendrimer. 5 The S2 specifically comprises the following steps: S21. 3,3'-dithiodipropionic acid and paclitaxel are dissolved in dichloromethane, stirred at room temperature for 1 h, then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine are added, and reflux reaction is carried out at room temperature for 48 h; after the reaction is completed, column chromatography purification is carried out and the product is collected to obtain dithiodipropionate paclitaxel, denoted as PTX-S-S-PTX; S22. PTX-S-S-PTX and dithiothreitol are dissolved in dichloromethane, stirred at room temperature for 1 h, then triethylamine is added, and stirring reaction is carried out under N2 atmosphere for 12 h; after the reaction is completed, the organic phase is collected, and then washing, drying, concentration treatment is carried out, and the product is collected by centrifugation after precipitation using petroleum ether to obtain the PTX-SH; S23. mixing the M-M and PTX-SH in a molar ratio of 1:1-15, dissolving in methanol, stirring under N2 protection at room temperature for 24-48 h to obtain a second reaction solution; dialyzing the second reaction solution in a dialysis medium of methanol at a frequency of replacement every 6 h, collecting the dialysate after dialysis for 3-6 times, and vacuum drying to obtain the M-MP; In S23, the dialysis bag used for dialysis has a molecular weight cut-off of 2000-7000.

5. The method of claim 2, wherein the preparation of the prodrug molecule based on the zwitterionic polyamido-amine dendrimer is characterized by, The S3 specifically comprises the following steps: mixing the M-MP and c(RGDfC) in a molar ratio of 1:1-10, dissolving in a mixed solvent of methanol and water in a volume ratio of 10:1, stirring under N2 protection at room temperature for 24 h to obtain a third reaction solution; dialyzing the third reaction solution in a dialysis medium of methanol at a frequency of replacement every 6 h, collecting the dialysate after dialysis for 4 times, and vacuum drying to obtain the M-MPR; In S3, the dialysis bag used for dialysis has a molecular weight cut-off of 2000-7000.

6. The method of claim 2, wherein the preparation of the prodrug molecule based on the zwitterionic polyamido-amine dendrimer is characterized by, The S4 specifically comprises the following steps: S41. mixing mercaptoethylamine hydrochloride and triphenylmethyl chloride, dissolving with trifluoroacetic acid, stirring at room temperature for 2 h to obtain a fourth reaction solution; rotary evaporation, washing, collecting the organic phase, and drying and concentrating to obtain 2-(triphenylmethylthio)ethylamine; S42. N α - N τ - Triphenylmethylhistamine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were mixed and dissolved in dichloromethane, then triethylamine was added, stirred at room temperature for 2 h, then the 2-(triphenylmethylsulfenyl)ethylamine was added, stirred at room temperature for 12 h; after the reaction was completed, the white solid by-product was filtered, the filtrate was collected, concentrated by rotary evaporation, purified by column chromatography, the product was collected and dried under vacuum to obtain N'-(2-(triphenylmethylsulfenyl)ethyl)-N α - tert-Butyloxycarbonyl-N τ - Triphenylmethylhistamine amide; S43. N'-(2-(tritylsulfenyl)ethyl)-N α - tert-butyloxycarbonyl-N τ - triphenylmethylhistidinamide is mixed with triisopropylsilane, after adding trifluoroacetic acid, stirring in ice bath for 2 h, after the reaction is completed, most of the trifluoroacetic acid is removed by rotary evaporation, then precipitated with cold ether, after centrifugation, the precipitate is dissolved in water, the insoluble matter is filtered off, and the filtrate is freeze-dried to obtain the HIS-SH; S44. mixing the M-MPR and HIS-SH in a molar ratio of 1:1-30, dissolving in methanol, stirring under N2 protection at room temperature for 24-48 h to obtain a fifth reaction solution; dialyzing the fifth reaction solution in a dialysis medium of methanol at a frequency of replacement every 6 h, collecting the dialysate after dialysis for 3-6 times, and vacuum drying to obtain the M-MPRH; In S44, the dialysis bag used for dialysis has a molecular weight cut-off of 2000-7000.

7. The use of the prodrug molecule based on the zwitterionic polyamidoamine dendrimer in the field of nanomedicine delivery systems according to claim 1.

8. A nanocarrier micelle, characterized by, The nanomedicine-loaded micelles comprise the prodrug molecule based on the zwitterionic polyamidoamine dendrimer according to claim 1 to encapsulate a hydrophobic anticancer drug.

9. The method for preparing nano-drug-loaded micelles as described in claim 8, characterized in that, The preparation method comprises using PAMAM as a carrier, hydrophobic antitumor drug PTX-SH as a hydrophobic end, c(RGDfC) capable of specifically recognizing α V β3 integrin overexpressed by tumor cells and HIS-SH with pH sensitivity as a hydrophilic end, physically embedding another hydrophobic anticancer drug after self-assembly of the prodrug molecule based on the amphoteric ionized polyamide-amine dendrimer to obtain the nano drug-loaded micelles.

10. The method for preparing drug-loaded nanomicelles according to claim 9, characterized in that, The preparation method comprises: S5. encapsulating a hydrophobic anticancer drug with the M-MPRH to prepare the nanomedicine-loaded micelles; The S5 specifically comprises the following steps: S51. dissolving the hydrophobic anticancer drug in methanol to prepare a hydrophobic anticancer drug methanol solution; S52. dissolving the M-MPRH in methanol to prepare a M-MPRH methanol solution; S53. mixing the M-MPRH methanol solution and the hydrophobic anticancer drug methanol solution in a mass ratio of 5-20:1, stirring at room temperature in the dark for 2-6 h to obtain a sixth reaction solution; S54. dialyzing the sixth reaction solution in water, the dialysis medium is water, the frequency of replacement is every 6 h, the dialysis is performed for 3 times, and the dialysate is collected after the dialysis to obtain the nanomedicine-loaded micelles.

11. The method of claim 10, wherein the drug-loaded nanomicelle is prepared by the process comprising the steps of: In S52, the hydrophobic anticancer drug comprises at least one of doxorubicin, camptothecin, fluorouracil, and chlorin e6.

12. The method for preparing nano-drug-loaded micelles according to claim 10, characterized in that, In the S54, the mass ratio of the reaction solution to water is 1:0.5-3, and the dropping speed into the water is 0.5-1 mL / min.

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