PH-sensitive adriamycin prodrug of amphiphilic star polymer containing benzaldehyde group as well as preparation method and application of pH-sensitive adriamycin prodrug

Abstract

The invention discloses a pH-sensitive adriamycin prodrug of a benzaldehyde group-containing amphiphilic star polymer as well as a preparation method and application of the pH-sensitive adriamycin prodrug. The preparation method comprises the following steps: firstly, preparing a star-shaped macromolecular initiator 2-bromine-2-methyl propionyloxy group modified cyclodextrin based on cyclodextrin; then preparing a hydroxyl-containing random copolymerization type star polymer; a random copolymerization type star polymer containing a benzaldehyde group; the invention relates to a pH sensitive adriamycin prodrug of an amphiphilic star polymer containing a benzaldehyde group. And finally, preparing the pH-sensitive doxorubicin prodrug nano-micelle. The nano-micelle can effectively inhibit the growth of tumor cells, the carrier material shows excellent biocompatibility, and a reliable guarantee is provided for clinical application. The pH-sensitive doxorubicin polymer prodrug nano-micelle with high stability and good biological safety is successfully prepared through a simple and efficient preparation route. The method has the advantages of wide raw material source and easiness in large-scale production, and has wide application prospects in the fields of tumor treatment and precise drug delivery.

Description

Technical Field

[0001] The invention belongs to the technical field of polymer prodrug preparation, and particularly relates to a pH-sensitive doxorubicin prodrug of an amphiphilic star polymer containing a benzaldehyde group, and a preparation method and application thereof. Background Art

[0002] Cancer is one of the most deadly diseases in the world. It is characterized by high metastasis and poor prognosis and has become one of the main causes of death. Chemotherapy is one of the most widely used cancer treatment strategies. Doxorubicin (DOX) is a widely used anthracycline anticancer drug that destroys the stability of genetic material by embedding into the double helix of DNA and induces cancer cell death. It has a significant inhibitory effect on many types of cancer and is usually used as a second-line anticancer drug in clinical practice.

[0003] However, as a hydrophobic small molecule, doxorubicin has limited solubility in water. After administration, doxorubicin is non-selectively distributed throughout the body through the blood circulation, causing damage to normal tissues, among which cardiotoxicity is one of the most serious side effects. In addition, in order to maintain effective therapeutic concentrations, doxorubicin often needs to be administered repeatedly, which can easily induce tumor resistance, thereby significantly reducing the anti-cancer effect.

[0004] The preparation of doxorubicin nano drug delivery system is one of the key ways to optimize its administration, reduce toxic side effects, and improve chemotherapy effects. Nano drug delivery systems usually achieve drug loading through physical encapsulation or chemical bond coupling. The complex formed by loading the drug onto a polymer carrier without pharmacological activity through chemical bond coupling is called polymer prodrug or polymer prodrug. Polymer prodrugs are usually composed of hydrophilic polymer chains and hydrophobic drug units, and self-assemble in aqueous solution to form nano micelles with a hydrophilic shell and a hydrophobic core. In recent years, nano drug delivery systems based on polymer prodrugs have attracted widespread attention in the field of doxorubicin delivery.

[0005] Anaerobic glycolysis of tumor cells causes their tissues to exhibit a unique pH gradient: normal tissues and blood are neutral (pH = 7.4), tumor tissues are weakly acidic (pH < 6.5), and the intracellular environment of tumor cells (pH = 5.0-6.0) and lysosomes (pH = 4.0-5.0) exhibit lower pH values. In view of this acidic microenvironmental characteristic, the design of pH-sensitive polymer prodrugs has become an important research direction for drug delivery systems. pH-sensitive polymer prodrugs remain stable in neutral and alkaline environments to avoid premature release of drugs during delivery. In the acidic environment of tumor tissues or lysosomes, their pH-sensitive chemical bonds are hydrolyzed and broken, triggering drug release. This mechanism effectively increases the concentration of drugs at the target site, which is beneficial to improving the therapeutic effect of tumors.

[0006] pH-sensitive acylhydrazone bonds and imine bonds are often used to bind doxorubicin to polymer drug carriers containing hydrazide or aldehyde groups, thereby preparing pH-sensitive doxorubicin polymer prodrugs. However, such doxorubicin polymer prodrugs usually rely on the preparation of new monomers containing hydrazide or aldehyde groups, and the carrier polymers mostly adopt block copolymer structures. The preparation of block polymer prodrugs with ideal structures faces great challenges due to the need for precise control of the molecular weight of each component. In contrast, random copolymer amphiphilic polymers have a simpler preparation route, but such polymers are generally difficult to form structurally stable nanomicelles. These problems have, to a certain extent, limited the further development of pH-sensitive doxorubicin polymer prodrug nanomicelles.

[0007] Therefore, designing a random copolymer polymer prodrug that can form structurally stable nanomicelles without relying on the preparation of new monomers has important development potential. Summary of the invention

[0008] In view of the above-mentioned problems existing in the pH-sensitive doxorubicin polymer prodrug nano-delivery system, the present invention aims to provide a preparation method and application of a random copolymerized pH-sensitive doxorubicin polymer prodrug which can form structurally stable nano-micelles without relying on the preparation of new monomers.

[0009] The technical solution of the present invention is as follows:

[0010] The present invention discloses a pH-sensitive doxorubicin prodrug of an amphiphilic star polymer containing a benzaldehyde group, referred to as DOX@SPPH, and has the structural formula:

[0011]

[0012] In the structural formula, CD is α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin, the value of m is 3-9, the ratio of x to y is in the range of x / y=0.5-2, and the sum of the polymerization degree values ​​of HEMA and PEGMA (x+y) is in the range of 10-100.

[0013] The present invention discloses a method for preparing a pH-sensitive doxorubicin prodrug of an amphiphilic star polymer containing a benzaldehyde group, comprising the following steps:

[0014] (1) A star-shaped macromolecular initiator based on cyclodextrin (CD) and modified with 2-bromo-2-methylpropionyloxy group (CD-BIB) was prepared. The preparation route thereof is as follows, comprising the following steps:

[0015] Under nitrogen atmosphere, cyclodextrin was dissolved in anhydrous N-methylpyrrolidone (NMP), triethylamine (TEA) was added, and then cooled to 0°C in an ice bath;

[0016] Dissolve 2-bromo-2-methylpropionyl bromide (BIBB) in anhydrous N-methylpyrrolidone (NMP), drip into the cyclodextrin solution dropwise under stirring, and react in an ice bath for 0.5-5 h, and then react at room temperature for 12-48 h;

[0017] After the reaction is completed, the mixture is concentrated, diluted with dichloromethane (DCM), washed with water, and precipitated in n-hexane or petroleum ether. The collected white precipitate is dried in a vacuum for 12-24 h to obtain a cyclodextrin-based star-shaped macroinitiator (CD-BIB);

[0018] CD is α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin;

[0019]

[0020] (2) Preparation of a hydroxyl-containing random copolymer star polymer (SP (PEGMA-co-HEMA), referred to as SPPH), the preparation route of which is as follows, comprising the following steps:

[0021] Dissolve 2-hydroxyethyl methacrylate (HEMA), polyethylene glycol methyl ether methacrylate (PEGMA), 10-phenylphenothiazine (PTH) and the CD-BIB obtained in step (1) in N,N-dimethylacetamide (DMA) in a reaction container;

[0022] After fully dissolved, seal the reaction vessel, introduce high-purity argon gas to deoxygenate for 20-30 minutes, transfer to a UV lamp and stir at room temperature for reaction for 2-12 hours;

[0023] After the reaction is completed, the reaction solution is precipitated in diethyl ether, dissolved in tetrahydrofuran (THF), and then dripped into diethyl ether again, and the precipitate is dried to obtain SPPH;

[0024] In the structural formula of SPPH, the value of m is 3 to 9, the ratio of x to y is in the range of x / y = 0.5 to 2, and the sum of the polymerization degree values ​​of HEMA and PEGMA (x + y) is in the range of 10 to 100;

[0025]

[0026] (3) Preparation of a random copolymer star polymer containing benzaldehyde groups (SP[PEGMA-co-(HEMA-FBA)], referred to as SPPH-COH), the preparation route of which is as follows, comprising the following steps:

[0027] At room temperature and nitrogen atmosphere, the SPPH prepared in step (2) was dissolved in ultra-dry THF, and 4-formylbenzoic acid (FBA) and 4-dimethylaminopyridine (DMAP) were added, and the mixture was cooled to 0°C;

[0028] Dissolve N,N′-dicyclohexylcarbodiimide (DCC) in ultra-dry THF and add dropwise, stirring at room temperature for 20-48 hours;

[0029] The reaction solution was filtered and concentrated, diluted with N,N-dimethylformamide (DMF), transferred to a dialysis bag, dialyzed in DMF for one day, then dialyzed with water for two days, and freeze-dried to obtain a random copolymer star polymer SPPH-COH containing benzaldehyde groups;

[0030]

[0031] (4) Preparation of a pH-sensitive doxorubicin prodrug of an amphiphilic star polymer containing benzaldehyde groups (SP[PEGMA-co-(HEMA-DOX)], referred to as DOX@SPPH). The preparation route is as follows, comprising the following steps:

[0032] Dissolve doxorubicin hydrochloride (DOX·HCl) in DMSO, add triethylamine (TEA), and stir for 2-12 hours in the dark to perform dehydrochlorination treatment;

[0033] The star polymer SPPH-COH prepared in step (3) is dissolved in DMF, and the mixture is added dropwise to the doxorubicin solution after dehydrochlorination treatment, and reacted at room temperature in the dark for 24-72 hours;

[0034] The reaction solution was then transferred to a dialysis bag, dialyzed in DMF for one day, and then dialyzed in water for two days, and freeze-dried to obtain a pH-sensitive star polymer prodrug (DOX@SPPH) lyophilized powder;

[0035]

[0036] (5) Preparing pH-sensitive doxorubicin prodrug nanomicelles, comprising the following steps:

[0037] Take a certain amount of DOX@SPPH obtained in step (4), dissolve it in THF, DMF or DMSO, slowly add a certain amount of water while stirring, stir for 10-60 minutes, and dialyze with water to remove THF, DMF or DMSO used as a solvent to obtain a pH-sensitive doxorubicin prodrug nanomicelle aqueous solution.

[0038] Furthermore, the concentration of cyclodextrin dissolved in anhydrous N-methylpyrrolidone in step (1) is 0.05 to 0.2 mol·L -1 ;

[0039] The concentration of triethylamine (TEA) dissolved in anhydrous N-methylpyrrolidone is 3-6 mol·L -1 ;

[0040] The concentration of 2-bromo-2-methylpropionyl bromide (BIBB) dissolved in anhydrous N-methylpyrrolidone is 1 to 6 mol·L -1 ;

[0041] The molar ratio of CD, TEA and BIBB is 1:(20-100):(20-100);

[0042] Wash with saturated NaHCO3 aqueous solution and deionized water in sequence.

[0043] Furthermore, in step (2), the molar ratio of CD-BIB, HEMA, PEGMA and PTH is 1: (200-1000): (250-2000): (0.5-2); wherein the molar concentration of the initiator CD-BIB is 0.5-10 mmol·L -1 .

[0044] Furthermore, in step (3), the mass volume concentration of SP (PEGMA-co-HEMA) is 0.05 to 0.5 g·mL -1 ;

[0045] The molar ratio of FBA, DMAP and DCC is 1:(0.2-1):(1-2);

[0046] The molar concentration of DCC dissolved in ultra-dry THF is 0.5-2 mol·L -1 ;

[0047] The molecular weight cutoff of the dialysis bag used in dialysis is 3500Da;

[0048] Freeze-drying conditions are: -30°C, sublimation time is 24 to 72 hours.

[0049] Furthermore, in step (4), the molar concentration of doxorubicin hydrochloride (DOX·HCl) dissolved in DMSO is 0.02 to 0.2 mol·L -1 ;

[0050] The molar ratio of DOX·HCl to triethylamine (TEA) is 1:(1-5);

[0051] The mass volume concentration of SPPH-COH dissolved in ultra-dry DMF solution is 0.02-0.2 g·mL -1 ; The molecular weight cut-off of the dialysis bag used is 3500Da;

[0052] Freeze-drying conditions are: -30°C, sublimation time is 24 to 72 hours.

[0053] Furthermore, in step (5), the mass volume concentration of the pH-sensitive star polymer prodrug (DOX@SPPH) dissolved in THF, DMF or DMSO is 1 to 10 g·L -1 ;

[0054] The volume ratio of the organic solvent (THF or DMF or DMSO) to water is 1:(5-50);

[0055] The particle size of the nanomicelles obtained after dialysis in water is 10 to 100 nm.

[0056] The present invention also discloses a pH-sensitive doxorubicin prodrug prepared by the above method, wherein the nano-micelle particle size of the prodrug ranges from 10 to 100 nm, which not only enhances its stability in blood circulation, but also improves its penetration ability in solid tumor tissue, thereby helping the drug to penetrate deeper into the tumor more effectively and improving the therapeutic effect.

[0057] The present invention also discloses the use of the pH-sensitive doxorubicin prodrug prepared by the above method in preparing a drug for treating tumors. Furthermore, the tumor is lung cancer.

[0058] The main advantages of the present invention are:

[0059] (1) In order to solve the problem of complex process for preparing structurally stable pH-sensitive doxorubicin polymer prodrug nanomicelles, the present invention innovatively designs and prepares an amphiphilic star polymer containing benzaldehyde groups as a doxorubicin carrier, and develops a simple and efficient preparation method, which can significantly improve the structural stability of pH-sensitive doxorubicin polymer prodrug nanomicelles.

[0060] (2) The polymer prodrug of the present invention can self-assemble in aqueous solution to form single-molecule nanomicelles, which have excellent stability and can effectively avoid the rupture of micelles during the delivery process, thereby significantly improving its enrichment efficiency in tumor tissues and tumor cells, providing a guarantee for precise drug delivery.

[0061] (3) The polymer prodrug of the present invention is combined with doxorubicin through an imine bond, which can effectively avoid the premature release of the drug during the delivery process, and can respond to the low pH conditions of the tumor microenvironment to quickly trigger the release of doxorubicin, thereby enhancing the killing effect on cancer cells. In addition, the carrier material is safe and non-toxic within a reasonable concentration range, ensuring the biological safety of the treatment and achieving efficient and accurate treatment of cancer.

[0062] (4) The pH-sensitive doxorubicin prodrug nanomicelles of the present invention have a stable nanostructure, slow drug release rate and extremely low drug release amount under neutral physiological conditions; in an acidic environment, the drug release rate and drug release amount are significantly increased. The nanomicelles can effectively inhibit the growth of tumor cells, and the carrier material exhibits excellent biocompatibility, providing a reliable guarantee for clinical application.

[0063] (5) The polymer prodrug of the present invention adopts a random copolymer structure, and the preparation process is simple, and no new monomers need to be prepared. The raw materials required for the preparation process are abundant, and it is easy to achieve large-scale production, showing its great potential in drug delivery systems, and has important clinical application value and industrialization prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The hydroxyl-containing random copolymer star polymer SPPH prepared in Example 1 1 H-NMR spectrum, the solvent is CDCl3.

[0065] Figure 2 The random copolymer star polymer SPPH-COH containing benzaldehyde groups prepared in Example 1 1 H-NMR spectrum, the solvent is CDCl3.

[0066] Figure 3 The pH-sensitive doxorubicin prodrug DOX@SPPH of the amphiphilic star polymer containing benzaldehyde groups prepared in Example 1 1 H-NMR spectrum, the solvent is CDCl3.

[0067] Figure 4 It is the UV-visible absorption spectra of SPPH-COH, DOX@SPPH, and free doxorubicin (DOX)X prepared in Example 1.

[0068] Figure 5 This is the particle size distribution test result of the pH-sensitive doxorubicin prodrug nanomicelles in Example 2.

[0069] Figure 6 These are the Zeta potential test results of the pH-sensitive doxorubicin prodrug nanomicelles and free doxorubicin (DOX)X in Example 2.

[0070] Figure 7 This is an in vitro drug release curve of the pH-sensitive doxorubicin prodrug nanomicelles in Example 4 at different pH values.

[0071] Figure 8 This is a graph showing the cytotoxicity of the pH-sensitive doxorubicin prodrug nanomicelles and free doxorubicin (DOX)X in Example 5 to human large cell lung cancer cells (H460).

[0072] Fig. 9 This is a flow cytometric quantitative uptake result of human large cell lung cancer cells (H460) in Example 6 for pH-sensitive doxorubicin prodrug nanomicelles and free doxorubicin (DOX).

[0073] Fig.10 This is the cytotoxicity test result of the pH-sensitive doxorubicin prodrug carrier material in Example 7 on human large cell lung cancer cells (H460). DETAILED DESCRIPTION

[0074] The technical solution of the present invention is further described below by way of embodiments, which are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0075] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0076] Example 1

[0077] Preparation of a pH-sensitive doxorubicin prodrug of an amphiphilic star polymer containing benzaldehyde groups:

[0078] (1) Preparation of CD-BIB:

[0079] Under nitrogen atmosphere, β-CD (2 g, 1.76 mmol) was first dissolved in 20 mL of anhydrous NMP, TEA (12.1 g, 120 mmol) was added, and then cooled to 0 °C in an ice bath;

[0080] BIBB (27.6 g, 120 mmol) was dissolved in anhydrous NMP (10 mL), and the mixture was added dropwise into the β-CD solution through a constant pressure dropping funnel under stirring. After stirring in an ice bath for 2 h, the nitrogen was stopped, and the reaction was continued at room temperature for 24 h;

[0081] After the reaction, the mixture was concentrated, diluted with DCM, and washed 5 times with saturated NaHCO3 aqueous solution (80 mL) and deionized water (80 mL). The organic phase was dehydrated with anhydrous Na2SO4 and concentrated, then precipitated in n-hexane and dried to obtain white powder β-CD-BIB.

[0082] (2) Preparation of hydroxyl-containing random copolymer star polymer (SPPH):

[0083] β-CD-BIB (0.01 g, 0.0023 mmol), HEMA (0.14 g, 1.08 mmol), and PEGMA (molecular weight: M) prepared in step (1) were added to a Schlenk reaction tube.w =475 g / mol, 0.85 g, 1.79 mmol), PTH (0.8 mg, 0.0029 mmol) and N,N-dimethylacetamide (DMA, 2.5 mL) were fully dissolved;

[0084] After full dissolution, the Schlenk reaction tube was sealed, and high-purity argon was bubbled in to deoxygenate for 30 min, and then the tube was transferred to a UV lamp and stirred at room temperature for reaction for 5 h;

[0085] After the reaction, the reaction solution was precipitated in ether, dissolved with THF and then dropped into ether again, and the precipitate was dried to obtain SPPH. Figure 1 This is the hydrogen NMR spectrum of SPPH. From the figure, it can be seen that the hydrogen signal of the methoxy group in the PEGMA repeating unit appears at 3.3ppm, and the hydrogen signal of the hydroxyl group in the HEMA repeating unit appears at 4.3ppm, indicating that SPPH was successfully prepared.

[0086] (3) Preparation of random copolymer star polymer containing benzaldehyde group (SPPH-COH):

[0087] At room temperature and nitrogen atmosphere, SPPH (0.66 g) prepared in step (2) was dissolved in ultra-dry THF (4 mL), and FBA (0.21 g, 1.4 mmol) and DMAP (0.027 g, 0.22 mmol) were added, and the mixture was placed in an ice bath and cooled to 0°C;

[0088] A mixed solution of DCC (0.59 g, 2.86 mmol) and ultra-dry THF (3 mL) was added dropwise while stirring. After the addition was complete, the mixture was stirred in an ice bath for 30 min, then the ice bath was removed and the reaction was continued at room temperature for 24 h;

[0089] The reaction solution was filtered and concentrated, and then diluted with DMF (7 mL) and transferred to a dialysis bag with a molecular weight cutoff of 3500 Da. The solution was dialyzed in 100 mL of DMF for 24 h (DMF was changed every 4 h), and then dialyzed in 500 mL of water for 48 h (water was changed every 6 h), and freeze-dried at -30 °C.

[0090] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of SPPH-COH. Compared with the spectrum of SPPH, new characteristic peaks appear at 8.0ppm, 8.2ppm and 10.3ppm, which are the signals of hydrogen on the benzaldehyde group, indicating that SPPH-COH was successfully prepared.

[0091] (4) Preparation of pH-sensitive doxorubicin prodrug (DOX@SPPH) based on amphiphilic star polymers containing benzaldehyde groups:

[0092] DOX·HCl (98.6 mg, 0.17 mmol) and TEA (0.086 g, 0.85 mmol) were dissolved in ultra-dry DMSO (2.5 mL) and stirred for 5 h in the dark for dehydrochlorination treatment;

[0093] The SPPH-COH (0.1 g) prepared in step (3) was dissolved in ultra-dry DMF (4.3 mL), and the mixture was added dropwise into the DOX solution after dehydrochlorination, and reacted at room temperature in the dark for 48 h;

[0094] The resulting mixture was dialyzed in DMF and water in turn, with a molecular weight cutoff of 3500 Da in the dialysis bag, and finally freeze-dried at -30 °C;

[0095] like Figure 3 In the H NMR spectrum, characteristic peak signals of DOX appeared at chemical shifts of 14.0 ppm and 13.2 ppm, indicating that DOX was successfully coupled with SPPH-COH; Figure 4 This was also confirmed by the UV-visible spectrum in . DOX had a characteristic absorption signal at 400-570 nm (with the maximum absorption intensity at 480 nm), SPPH-COH had no absorption signal in the same wavelength range, and DOX@SPPH showed an absorption signal similar to that of DOX at 400-570 nm, confirming the successful coupling of DOX and SPPH-COH.

[0096] (5) Preparation of pH-sensitive doxorubicin prodrug nanomicelles:

[0097] The DOX@SPPH (10 mg) prepared in step (4) was dissolved in 1 mL THF. After it was completely dissolved, 10 mL of water was slowly added dropwise while stirring. The mixed solution was dialyzed in water for 2 days to obtain pH-sensitive doxorubicin prodrug nanomicelles.

[0098] Example 2

[0099] Particle size distribution and Zeta potential of pH-sensitive doxorubicin prodrug nanomicelles: The pH-sensitive doxorubicin prodrug nanomicelle aqueous solution prepared in Example 1 was diluted to a micelle solution with a concentration of 0.1 mg / mL, and the size and Zeta potential of the micelles were measured in a cuvette with light transmittance on all four sides and a potential tank, respectively. Figure 5 This is the particle size distribution diagram of pH-sensitive doxorubicin prodrug nanomicelles, and the particle size is about 25 nm; Figure 6 The Zeta potential values ​​of free doxorubicin and pH-sensitive doxorubicin prodrug nanomicelles are shown. The potential of free doxorubicin is +7.5mV, while the potential of pH-sensitive doxorubicin prodrug nanomicelles is -6mV. The negative Zeta potential is beneficial for the nanomicelles to avoid protein adsorption in the blood and increase their circulation stability.

[0100] Example 3

[0101] Drug loading of pH-sensitive doxorubicin prodrug: Weigh 3.5 mg of the DOX@SPPH lyophilized powder prepared in Example 1, add an appropriate amount of DMF to dissolve it, and use a UV spectrophotometer to measure the absorbance at 480 nm. The concentration of DOX in the DOX@SPPH polymer prodrug is calculated by the concentration-absorbance standard curve of DOX at 480 nm. The drug loading is calculated by the formula. As shown in Table 1, the pH-sensitive doxorubicin prodrug nanomicelles have a high drug loading capacity.

[0102] Table 1 shows the results of drug loading, micelle size and Zeta potential.

[0103]

[0104] The calculation method of drug loading is as follows:

[0105] Drug loading (%) = the mass of doxorubicin in the doxorubicin polymer prodrug ÷ the mass of the doxorubicin polymer prodrug × 100%.

[0106] Example 4

[0107] In vitro drug release behavior of pH-sensitive doxorubicin prodrug nanomicelles: The pH-sensitive doxorubicin prodrug nanomicelle aqueous solution (0.2 mL, DOX concentration of 14 μg / mL) prepared in Example 1 was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, immersed in containers containing 40 mL of different pH buffers (pH of 7.4, 5.0 and 4.0, respectively), and shaken on a constant temperature shaker (37°C, 120 rpm). 3 mL of dialysate was collected at a set time point and 3 mL of fresh buffer corresponding to the pH value was added. The absorbance of the collected dialysate was measured at 480 nm, the cumulative drug release was calculated, and the drug release curve was drawn. Figure 7 The figure shows the drug release curves at different pH values. In a buffer solution with a pH value of 7.4, the cumulative release of doxorubicin is less than 10% at 90 hours. In a buffer solution with a pH value of 5.0, the release rate of doxorubicin is significantly accelerated, and the release rate of doxorubicin reaches 40% at 90 hours. At a pH value of 4.0, the release rate of doxorubicin is further accelerated, and the cumulative release rate of doxorubicin reaches about 60% at 90 hours. Therefore, the drug release behavior of the pH-sensitive doxorubicin prodrug nanomicelles has significant pH responsiveness.

[0108] Example 5

[0109] Cytotoxicity of pH-sensitive doxorubicin prodrug nanomicelles: Human large cell lung cancer cells (H460) were inoculated in 96-well plates. After culturing for 24 hours, the culture medium was removed and 100 μL of culture medium containing pH-sensitive doxorubicin prodrug nanomicelles was added to each well (6 replicate wells were set for each drug concentration), and the plates were placed in an incubator at 37°C containing 5% CO2 and incubated for 48 hours. After removing the culture medium, 150 μL of culture medium solution containing 10% CCK8 reagent was added to each well, and the plates were incubated for another 3 hours. The absorbance was measured at 450 nm using a microplate reader to further calculate the cell survival rate. Figure 8 The figure is a cytotoxicity curve of pH-sensitive doxorubicin prodrug nanomicelles. As shown in the figure, the doxorubicin prodrug nanomicelles can significantly inhibit the growth of tumor cells H460.

[0110] Example 6

[0111] Cellular uptake of pH-sensitive doxorubicin prodrug nanomicelles: Human large cell lung cancer cells (H460) were inoculated in a 6-well plate, and the culture medium was removed after culturing for 24 hours. Then 2 mL of fresh culture medium containing free DOX or the pH-sensitive doxorubicin prodrug nanomicelles prepared in Example 1 (the concentration of doxorubicin was 8 μg / mL) was added, and the plates were incubated in an incubator at 37°C containing 5% CO2 for 4 hours; the old culture medium was discarded, and after washing with PBS, 1 mL of PBS solution containing 0.25% trypsin was added to each well for digestion. After 2 minutes, the plates were centrifuged (1000 rpm, 5 minutes) and the supernatant was discarded. 200 μL of PBS solution was added to disperse the cells at the bottom, and the doxorubicin fluorescence intensity in the cells was detected by flow cytometry. Fig. 9 This is the quantitative uptake result of H460. After the cells were incubated with the drug-loaded micelles for 4 h, an obvious DOX fluorescence signal was detected in the cells, indicating that the doxorubicin prodrug nanomicelles were successfully taken up by H460 cells.

[0112] Example 7

[0113] Biocompatibility of pH-sensitive doxorubicin prodrug carrier material: The random copolymer star polymer (SPPH-COH) containing benzaldehyde groups prepared in Example 1 was dissolved in the cell culture medium to obtain a culture medium solution containing different SPPH-COH concentrations. Human large cell lung cancer cells (H460) were inoculated in a 96-well plate, and the culture medium was removed after culturing for 24 hours. 100 μL of culture medium containing SPPH-COH was added to each well (6 replicate wells were set for each concentration), and the plate was placed in a 37°C incubator containing 5% CO2 for 48 hours; after removing the culture medium, 150 μL of culture medium containing SPPH-COH was added to each well.

[0114] μL of culture medium solution containing 10% CCK8 reagent was added, and the incubation was continued for 3 h. The absorbance was measured at 450 nm using an enzyme reader to further calculate the cell survival rate. Fig.10This is the cytotoxicity test result of pH-sensitive doxorubicin prodrug carrier material. As shown in the figure, within the concentration range of 0 to 500 μg / mL, the carrier material SPPH-COH has almost no cytotoxicity to H460 cells, indicating that the carrier material has good biocompatibility.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of the present invention. Although the above embodiments have explained the present invention in detail, those skilled in the art should understand that within the technical scope disclosed in the present invention, the above embodiments can still be modified in different forms in terms of solutions and details. These modifications do not deviate from the spirit and scope of the claims of the present invention.

Claims

1. A pH-sensitive doxorubicin prodrug of an amphiphilic star polymer containing benzaldehyde groups, characterized in that: The pH-sensitive doxorubicin prodrug is referred to as DOX@SPPH, and its structural formula is: In the structural formula, CD is α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin, the value of m is 3-9, the ratio of x to y is in the range of x / y=0.5-2, and the sum of the polymerization degree values ​​of HEMA and PEGMA (x+y) is in the range of 10-100.

2. The method for preparing a pH-sensitive doxorubicin prodrug according to claim 1, characterized in that: The preparation route of the pH-sensitive doxorubicin prodrug is as follows: wherein CD is α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin; In the structural formula of SPPH, the value of m is 3 to 9, the ratio of x to y is in the range of x / y = 0.5 to 2, and the sum of the polymerization degree values ​​of HEMA and PEGMA (x + y) is in the range of 10 to 100; The preparation method comprises the following steps: (1) A star-shaped macroinitiator 2-bromo-2-methylpropionyloxy-modified cyclodextrin (CD-BIB) based on cyclodextrin (CD) was prepared. The preparation route is as follows, comprising the following steps: Under nitrogen atmosphere, cyclodextrin was dissolved in anhydrous N-methylpyrrolidone (NMP), triethylamine (TEA) was added, and then cooled to 0°C in an ice bath; Dissolve 2-bromo-2-methylpropionyl bromide (BIBB) in anhydrous N-methylpyrrolidone (NMP), drip into the cyclodextrin solution dropwise under stirring, and react in an ice bath for 0.5-5 h, and then react at room temperature for 12-48 h; After the reaction is completed, the mixture is concentrated, diluted with dichloromethane (DCM), washed with water, and precipitated in n-hexane or petroleum ether. The collected white precipitate is dried in a vacuum for 12-24 h to obtain a cyclodextrin-based star-shaped macroinitiator (CD-BIB); (2) Preparation of a hydroxyl-containing random copolymer star polymer (SP (PEGMA-co-HEMA), referred to as SPPH), the preparation route of which is shown in route (a), comprising the following steps: Dissolve 2-hydroxyethyl methacrylate (HEMA), polyethylene glycol methyl ether methacrylate (PEGMA), 10-phenylphenothiazine (PTH) and the CD-BIB prepared in step (1) in N,N-dimethylacetamide (DMA) in a reaction container; After fully dissolved, seal the reaction vessel, introduce high-purity argon gas to deoxygenate for 20-30 minutes, transfer to a UV lamp and stir at room temperature for reaction for 2-12 hours; After the reaction is completed, the reaction solution is precipitated in diethyl ether, dissolved in tetrahydrofuran (THF), and then dripped into diethyl ether again, and the precipitate is dried to obtain SPPH; (3) Preparation of a random copolymer star polymer containing benzaldehyde groups (SP[PEGMA-co-(HEMA-FBA)], referred to as SPPH-COH), the preparation route of which is shown in route (b), comprising the following steps: At room temperature and nitrogen atmosphere, the SPPH prepared in step (2) was dissolved in ultra-dry THF, and 4-formylbenzoic acid (FBA) and 4-dimethylaminopyridine (DMAP) were added, and the mixture was cooled to 0°C; Dissolve N,N′-dicyclohexylcarbodiimide (DCC) in ultra-dry THF and add dropwise, stir and react at room temperature for 20-48 hours; filter and concentrate the reaction solution, dilute with N,N-dimethylformamide (DMF), transfer to a dialysis bag, dialyze in DMF for one day, and then dialyze with water for two days, freeze-dry, and obtain a random copolymer star polymer SPPH-COH containing benzaldehyde groups; (4) Preparation of a pH-sensitive doxorubicin prodrug containing a benzaldehyde group-containing amphiphilic star polymer (SP[PEGMA-co-(HEMA-DOX)], referred to as DOX@SPPH), the preparation route of which is shown in route (c), comprising the following steps: Dissolve doxorubicin hydrochloride (DOX·HCl) in DMSO, add triethylamine (TEA), and stir for 2-12 hours in the dark to perform dehydrochlorination treatment; The star polymer SPPH-COH prepared in step (3) is dissolved in DMF, and the mixture is added dropwise to the doxorubicin solution after dehydrochlorination treatment, and reacted at room temperature in the dark for 24-72 hours; The reaction solution was then transferred to a dialysis bag, dialyzed in DMF for one day, and then dialyzed in water for two days, and freeze-dried to obtain the pH-sensitive star polymer prodrug DOX@SPPH freeze-dried powder; (5) Preparing pH-sensitive doxorubicin prodrug nanomicelles, comprising the following steps: Take a certain amount of DOX@SPPH prepared in step (4), dissolve it in THF, DMF or DMSO, slowly add a certain amount of water while stirring, stir for 10-60 minutes, and dialyze with water to remove THF, DMF or DMSO used as a solvent to obtain a pH-sensitive doxorubicin prodrug nanomicelle aqueous solution.

3. The method for preparing a pH-sensitive doxorubicin prodrug according to claim 2, characterized in that: The concentration of CD dissolved in anhydrous NMP in step (1) is 0.05 to 0.2 mol·L -1 ; The concentration of TEA dissolved in anhydrous NMP is 3-6 mol·L -1 ; The concentration of BIBB dissolved in anhydrous NMP is 1-6 mol·L -1 ; The molar ratio of CD, TEA and BIBB is 1:(20-100):(20-100); Wash with saturated NaHCO3 aqueous solution and deionized water in sequence.

4. The method for preparing a pH-sensitive doxorubicin prodrug according to claim 2, characterized in that: In step (2), the molar ratio of CD-BIB, HEMA, PEGMA and PTH is 1: (200-1000): (250-2000): (0.5-2); wherein the molar concentration of the initiator CD-BIB is 0.5-10 mmol·L -1 .

5. The method for preparing a pH-sensitive doxorubicin prodrug according to claim 2, characterized in that: The mass volume concentration of SP (PEGMA-co-HEMA) in step (3) is 0.05 to 0.5 g·mL -1 ; The molar ratio of FBA, DMAP and DCC is 1:(0.2-1):(1-2); The molar concentration of DCC dissolved in ultra-dry THF is 0.5-2 mol·L -1 ; The molecular weight cutoff of the dialysis bag used in dialysis is 3500Da; Freeze-drying conditions are: -30°C, sublimation time is 24 to 72 hours.

6. The method for preparing a pH-sensitive doxorubicin prodrug according to claim 2, characterized in that: The molar concentration of DOX·HCl dissolved in DMSO in step (4) is 0.02 to 0.2 mol·L -1 ;The molar ratio of DOX·HCl to TEA is 1:(1-5); The mass volume concentration of SPPH-COH dissolved in ultra-dry DMF solution is 0.02-0.2 g·mL -1 ; The molecular weight cut-off of the dialysis bag used is 3500Da; Freeze-drying conditions are: -30°C, sublimation time is 24 to 72 hours.

7. The method for preparing a pH-sensitive doxorubicin prodrug according to claim 2, characterized in that: The mass volume concentration of the pH-sensitive star polymer prodrug (DOX@SPPH) dissolved in THF, DMF or DMSO in step (5) is 1 to 10 g·L -1 ; The volume ratio of the organic solvent THF or DMF or DMSO to water is 1: (5-50).

8. The pH-sensitive doxorubicin prodrug prepared by the method according to any one of claims 2 to 7, characterized in that: The nano-micelle particle size of the prodrug is 10 to 100 nm, which can improve its penetration ability in solid tumor tissue and help the drug penetrate deeper into the tumor more effectively.

9. Use of the pH-sensitive doxorubicin prodrug prepared according to the method of any one of claims 2 to 7 in the preparation of a drug for treating tumors.

10. The use according to claim 9, characterized in that: The tumor is lung cancer.

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