Preparation method and application of folic acid targeted multi-drug synergistic nano polymer prodrug

Abstract

The invention discloses a preparation method and application of a folic acid targeted multi-drug synergistic nano polymer prodrug, and belongs to the technical field of nano biomedical materials. The preparation method comprises the following steps: on the basis of a click reaction, crosslinking polyfunctional 2-methylglycerol tripropiolate with adriamycin and resveratrol to prepare a nano polymer prodrug BDR with an acid response characteristic; a folic acid targeting molecule FA-PEG-NH2 is efficiently grafted by utilizing a click reaction, so that the active targeting nano polymer prodrug BDRP with high selectivity and multi-drug synergism is prepared. Experiments find that pH-sensitive chemical bonds with tumor microenvironment dual stimulation response are introduced into poly-prodrugs, intelligent controlled release of chemotherapeutic drugs at tumor parts can be achieved, and the bioavailability is improved; fA-PEG-NH2 is further modified, so that the active targeting efficiency is improved, the poly-prodrug has better specificity, selectivity and biological safety on tumor cells, and the treatment effect on cervical cancer can be improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanobiomedical materials, in particular to a preparation method of a folate-targeted multi-drug synergistic nano-polymer prodrug and application thereof. BACKGROUND

[0002] Cervical cancer is the fourth most common cancer in women worldwide, caused by persistent infection with high-risk human papillomavirus (HPV), and is one of the malignant tumors that seriously threaten the health of women worldwide. It is a common malignant tumor that seriously threatens the health of women worldwide. At present, the commonly used methods for treating tumors in clinical practice include surgical treatment, radiotherapy, and chemotherapy. Among them, chemotherapy is still the mainstay of tumor treatment, and the commonly used chemotherapeutic drugs in clinical practice include alkylating agents, antimetabolites, and antitumor antibiotics. Doxorubicin (DOX) is a commonly used antitumor drug in clinical practice, which has a broad spectrum of antitumor activity and is used to treat solid tumors and hematological malignancies, including breast, bile duct, prostate, uterus, ovary, esophagus, stomach, and liver tumors, as well as acute myeloblasts and lymphoblasts leukemia, and nephroblastoma, etc. The mechanism of action of DOX against tumors is that after entering the cell through the channel protein, it interacts with the DNA molecule and is embedded therein, inhibits the function of TopoII, prevents the resealing of the DOX double helix, thereby blocking the replication of DNA, leading to double-stranded DNA breakage, and causing cell death. Although DOX is a recognized potent antitumor drug, it has serious toxic side effects and is prone to drug resistance, which greatly reduces the therapeutic effect of DOX. Compared with the use of a single antitumor drug, combination chemotherapy with synergistic effects can activate different signaling pathways, maximize the therapeutic effect, reduce the side effects of drugs by reducing the dosage of drugs, and overcome drug resistance.

[0003] More and more studies have shown that many natural chemicals can inhibit the proliferation and metastasis of tumor cells, have excellent anti-tumor activity, and the combination of chemotherapy drugs can enhance the anti-tumor activity, reverse the drug resistance of chemotherapy drugs, and improve the side effects of chemotherapy drugs, and is considered as a potential drug for co-administration with chemotherapy drugs in cancer treatment. Studies have shown that when resveratrol (RES) is combined with doxorubicin, it can inhibit the activity of P-gp, inhibit the level of MRP-1 protein, down-regulate the expression of NF-κB, increase the uptake of chemotherapy drugs in tumor cells, reverse the drug resistance of tumor cells to doxorubicin, inhibit tumor expansion, and reduce the damage of doxorubicin to the heart. Tian et al. studied the effect of RES on DOX-induced cardiotoxicity, and the results showed that RES can reduce DOX-induced cardiotoxicity in rats by up-regulating vascular endothelial growth factor B. However, both of them have the characteristics of rapid metabolism, poor water solubility and chemical stability, insufficient cell uptake, and poor oral absorption. The combination of resveratrol and doxorubicin can enhance the cytotoxicity of doxorubicin and reduce the side effects, and the two have good synergistic effect. In fact, the simple mixture of doxorubicin and resveratrol cannot overcome the drug resistance of doxorubicin, nor can it enhance the anti-tumor effect of doxorubicin. It is necessary to develop a new type of drug delivery system with low toxicity, high efficiency and high selectivity to ensure the synergistic effect of combined chemotherapy drugs and achieve better therapeutic effect.

[0004] With the deep cross-fusion of modern medicine and polymer chemistry, polymer prodrug (Polymer Prodrug), simply called polyprodrug, has attracted the attention of researchers. Polymer prodrug has obvious advantages: (1) passively or actively target drug release, protect the drug from inactivation before transportation to the target organs, tissues and cells; (2) increase drug solubility, stability, improve bioavailability, and improve pharmacokinetics; (3) flexible modification, controllable preparation process, and adjustable physicochemical properties. Nano-polymer prodrug not only can change the properties of the drug, reduce the dispersion of the drug in the body during circulation; respond to certain stimuli in a timely manner (such as: acid response, enzyme response, oxidation-reduction response, multiple stimulus response, etc.) and controllable release; enrich the drug at the target site and improve the bioavailability. Therefore, the research of nano-polymer prodrug is of great significance for the treatment of malignant tumors.

[0005] Nano-prodrug does not have targeting properties. The key to improving nano-polymer prodrug therapy is to modify the surface of nano-polymer prodrug to achieve active targeting therapy for a specific tumor. However, different targets based on receptor-ligand interaction have great differences in drug uptake and other aspects. It is of great significance to provide an active targeting nano-polymer prodrug with high selectivity and multi-drug synergy to provide new ideas for precise and personalized treatment plans for drug-resistant cervical cancer patients. SUMMARY

[0006] The present application aims to provide a preparation method and application of a folate-targeted multi-drug synergistic nanopolymer prodrug, to solve the problems in the prior art. The anthraquinone structure of doxorubicin is used in the synthesis technology of phenolic hydroxyl-acylalkynyl and amino-acylalkynyl click reaction, and the chemotherapeutic drugs (doxorubicin and resveratrol) are efficiently polymerized with 2-methyltriglycol tripropargylate to prepare a multi-drug synergistic passive targeting polyprodrug BDR and a modified folate active targeting polyprodrug BDRP, respectively, so as to improve the active targeting efficiency, make the polyprodrug show better selectivity to tumor cells, further improve the chemotherapy effect, and relieve the drug resistance of doxorubicin and improve the treatment effect of cervical cancer.

[0007] To achieve the above-mentioned purpose, the present application provides the following solutions.

[0008] The present application provides a multi-drug synergistic passive targeting nanopolymer prodrug, which comprises a chemotherapeutic drug and a crosslinking agent 2-methyltriglycol tripropargylate crosslinked with the chemotherapeutic drug, and the chemotherapeutic drug is doxorubicin and resveratrol.

[0009] The present application also provides a folate-targeted multi-drug synergistic nanopolymer prodrug, which comprises the multi-drug synergistic passive targeting nanopolymer prodrug and a folate-targeting molecule FA-PEG-NH2 modified on the surface of the multi-drug synergistic passive targeting nanopolymer prodrug.

[0010] In the above-mentioned folate-targeting molecule FA-PEG-NH2, folic acid (FA) is an important vitamin, also known as vitamin B9 or folic acid ester. It plays a variety of important roles in the human body, including participating in the growth and repair process of new cells, participating in the formation of red blood cells, reducing homocysteine levels, and having important significance for fetal nervous system development during pregnancy. Mammals lack the enzyme to synthesize FA and must obtain it from the outside. Most mammals absorb FA through FR-mediated endocytosis. Studies have shown that FR is overexpressed in tumor cells, and folate-targeted drugs increase the uptake of antitumor drugs by tumor cells through FR-mediated endocytosis, improve the targeting of drugs, reduce the distribution of drugs in normal tissue cells, reduce the effect on normal cells, and reduce drug adverse reactions.

[0011] Polyethylene Glycol (PEG) is non-toxic to cells and has excellent biocompatibility. It can be used as a carrier material to increase the solubility of poorly soluble drugs. The FA and amino are connected to the α-end and w-end of the PEG chain respectively to form the FA-PEG-NH2 structure, which can modify the drug through amino reaction. The use of polyethylene glycol to modify nanomedicine can reduce the adsorption capacity of nanocarriers to blood proteins, avoid the phagocytosis of the mononuclear phagocyte system to nanoparticles, improve the stability of nanomedicine, and prolong the half-life. The folate nanostructure can target the delivery of drugs to tumor cells and tumor sites by receptor-ligand mediated endocytosis, control drug release, reduce the distribution of chemotherapeutic drugs to normal tissues or cells, and improve the uptake of drugs by tumor cells.

[0012] The application also provides a preparation method of the passive targeting nanopolymer prodrug with multi-drug synergy, comprising the following steps:

[0013] (1) respectively dissolving doxorubicin and NH4HCO3 in water, and dissolving 2-methylpropanetriol tripropynyl acid ester and white resveratrol in anhydrous ethanol to prepare doxorubicin solution, NH4HCO3 solution, 2-methylpropanetriol tripropynyl acid ester solution and white resveratrol solution;

[0014] (2) after ultrasonicating and heating the water, adding the 2-methylpropanetriol tripropynyl acid ester solution to ultrasonicate, and then adding the doxorubicin solution, white resveratrol solution and NH4HCO3 solution to ultrasonicate for self-assembly to obtain the passive targeting nanopolymer prodrug with multi-drug synergy.

[0015] Preferably, in step (1), the mass-volume ratio of the doxorubicin and water is (5-6) mg: 4 mL; and / or the mass-volume ratio of the NH4HCO3 and water is (8-10) mg: 1 mL; and / or the mass-volume ratio of the 2-methylpropanetriol tripropynyl acid ester and anhydrous ethanol is (6-7) mg: 0.4 mL; and / or the mass-volume ratio of the white resveratrol and anhydrous ethanol is (2-3) mg: 0.5 mL.

[0016] Preferably, in step (2), the volume ratio of the water and 2-methylpropanetriol tripropynyl acid ester solution is 20-25: 0.4;

[0017] and / or the ultrasonic power is 100-200 W, the temperature of the water bath heating is 40-45℃, and all the solutions need to be added drop by drop when being added into the reaction conical flask.

[0018] The application also provides a preparation method of the folate targeting nanopolymer prodrug with multi-drug synergy, comprising the following steps:

[0019] (1) respectively dissolving doxorubicin, NH4HCO3 and FA-PEG-NH2 with water, dissolving 2-methylpropanetriol tripropynoate and white wine with anhydrous ethanol, to prepare doxorubicin solution, NH4HCO3 solution, FA-PEG-NH2 solution, 2-methylpropanetriol tripropynoate solution and white wine solution;

[0020] (2) adding doxorubicin solution, NH4HCO3 solution, FA-PEG-NH2 solution, 2-methylpropanetriol tripropynoate solution and white wine solution in water, and performing self-assembly by ultrasonic and water bath heating to obtain folate-targeted multi-drug synergistic nano-polymer prodrug.

[0021] Preferably, in step (1), the mass-volume ratio of doxorubicin and water is (5-6) mg: 4 mL; and / or the mass-volume ratio of NH4HCO3 and water is (8-10) mg: 1 mL; and / or the mass-volume ratio of FA-PEG-NH2 and water is (1-2) mg: 5 mL; and / or the mass-volume ratio of 2-methylpropanetriol tripropynoate and anhydrous ethanol is (6-7) mg: 0.4 mL; and / or the mass-volume ratio of white wine and anhydrous ethanol is (2-3) mg: 0.5 mL.

[0022] Preferably, in step (2), the volume ratio of water and 2-methylpropanetriol tripropynoate solution is 20-25: 0.4;

[0023] and / or the ultrasonic power is 100-200 W, and the water bath heating temperature is 40-45℃.

[0024] The application further provides the use of the multi-drug synergistic passive targeting nano-polymer prodrug or the folate-targeted multi-drug synergistic nano-polymer prodrug in the preparation of a drug for treating cervical cancer.

[0025] The application further provides the use of the folate-targeted multi-drug synergistic nano-polymer prodrug in the preparation of a drug for relieving doxorubicin resistance.

[0026] The application discloses the following technical effects:

[0027] (1) The application adopts the synthesis technology of phenolic hydroxyl-acylalkynyl and amino-acylalkynyl click reaction, and copolymerizes trifunctional 2-methyl glycerol tripropargyl ester with doxorubicin and resveratrol in a simple and efficient manner according to the specific physiological environment of tumor cell tissues, so as to realize the acid response characteristic of the nano-polymer prodrug; the system directly introduces the chemotherapeutic drug into the polymer main chain, has a high drug loading capacity, and thus overcomes the disadvantages of traditional polyprodrugs, such as low drug loading capacity caused by the introduction of auxiliary carriers or electrostatic adsorption and the like. The polyprodrug introduces the pH-sensitive chemical bond with double stimulation response of tumor microenvironment, realizes the 'intelligent' controlled release of the chemotherapeutic drug at the tumor site, improves the bioavailability, reverses the drug resistance of doxorubicin through the drug synergy of resveratrol and doxorubicin, reduces the toxic side effects, and improves the accumulation of doxorubicin in cervical cancer cells; the polyprodrug is further modified with the active targeting ligand folate-polyethylene glycol-amino, the selectivity of the polyprodrug to tumor cells is enhanced, the active targeting efficiency is improved, the polyprodrug shows better selectivity to tumor cells, the chemotherapy effect is further improved, and the multi-drug synergistic folate-targeted polyprodrug with high drug loading capacity relieves the drug resistance of doxorubicin. By changing the administration mode, the problems of traditional chemotherapeutic drugs, such as high toxic side effects, poor specificity, low drug efficacy, short blood circulation time and the like, are solved to a certain extent, the nano-polymer prodrug provides a new idea for improving the drug efficacy of conventional chemotherapeutic drugs, expanding the indications of chemotherapy and improving the drug resistance, provides a new idea for the precise and individualized treatment scheme for drug-resistant cervical cancer patients, and lays a solid foundation for the in-depth research and clinical application of the polyprodrug.

[0028] (2) The novel main chain type nano-polymer prodrug constructed based on the click reaction can realize the controllable preparation process of the polyprodrug with high drug loading capacity by directly cross-linking doxorubicin, resveratrol and 2-methyl glycerol tripropargyl ester through an acid response chemical bond, and the reaction has the advantages of mild conditions, simple and efficient, high drug loading capacity and biodegradability. The polyprodrug can be biodegraded in the tumor microenvironment, so that the chemotherapeutic drug is released specifically and the drug circulation time in the body is prolonged, the drug is efficiently accumulated at the lesion site, and the effect on cervical cancer is enhanced.

[0029] (3) The nano-polymer prodrug designed in the application can realize rapid acid-triggered degradation and release of the chemotherapeutic drugs doxorubicin and resveratrol when in the microenvironment of tumor cells, and the drugs are efficiently released through the change of the structure. The in-vitro slow-release behavior of the prepared nano-polymer prodrug is studied in the process of simulating degradation and release in an acid environment, the structure of the degradation product is identified by high-resolution liquid-mass spectrometry, the biodegradation behavior and polymerization reaction mechanism of the polyprodrug are revealed, and theoretical and experimental bases are provided for the design of the polyprodrug.

[0030] (4) The prepared nanopolymer prodrug is biologically evaluated at the cell and animal levels, and the results show that the nanopolymer prodrug can exert drug synergy, enhance the uptake of drugs by tumor cells, has small killing power to normal cells, is safe, and is suitable for drug delivery in vivo. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0032] Figure 1 Synthetic route of folate-targeted multi-drug synergistic nanopolymer prodrug and its therapeutic effect on cervical cancer (A) and synthetic route of 2-methylpropanetriol tripropargylate (B);

[0033] Figure 2 NMR hydrogen spectrum of 2-methylpropanetriol tripropargylate;

[0034] Figure 3 UV-visible absorption spectrum of different samples; (a): BDR, (b): BDRP;

[0035] Figure 4 Infrared spectrum of different samples; (a): BDR, (b): BDRP;

[0036] Figure 5 Fluorescence spectrum of different samples;

[0037] Figure 6 BDR and BDRP water particle size histogram (a) and Zeta potential diagram (b);

[0038] Figure 7 BDR and BDRP water particle size stability change diagram; (a): BDR, (b): BDRP;

[0039] Figure 8 SEM and TEM diagrams of BDR and BDRP; (a), (c) are SEM and TEM diagrams of BDR, respectively; (b), (d) are SEM and TEM diagrams of BDRP, respectively;

[0040] Figure 9 Fluorescence spectrum of DOX and RES; (a), (b) are fluorescence excitation spectrum and emission spectrum of DOX, respectively; (c), (d) are fluorescence excitation spectrum and emission spectrum of RES, respectively;

[0041] Figure 10 Photos of supernatant of BDR degradation 72h; (a): Cumulative release curves of DOX in BDR at pH 5.0 / 6.5 / 7.4, respectively; (b): Cumulative release curves of RES in BDR at pH 5.0 / 6.5 / 7.4, respectively; (c): Photos of supernatant of BDR degradation 0h at pH 5.0 / 6.5 / 7.4; (d): Photos of supernatant of BDR degradation 72h at pH 5.0 / 6.5 / 7.4;

[0042] Figure 11 MS of DOX cleavage related substances (m / z = 544.1822);

[0043] Figure 12 MS of RES cleavage related substances (m / z = 212.0197);

[0044] Figure 13 TIC of BDR degradation products;

[0045] Figure 14 Cell viability of BDR and BDRP and laser confocal fluorescence images of Calcein-AM / PI staining; (a) Cell viability of HeLa cells incubated with different drug groups (BDR, BDRP or pure drug) for 24 and 48h, (b) Cell viability of HUVEC cells incubated with different drug groups (BDR, BDRP or pure drug) for 24 and 48h (*P<0.05, **P<0.01, ***P<0.001); (c) Laser confocal fluorescence images of Calcein-AM / PI staining of HeLa cells incubated with BDR (d) Laser confocal fluorescence images of Calcein-AM / PI staining of HeLa cells incubated with BDRP; Calcein AM (green, live cells): 495~545nm (λex 488nm); PI (red, dead cells): 580~700nm (λex 552nm); Concentration 10μg / mL; Scale bar: 200μm;

[0046] Figure 15 Cell toxicity of HUVEC incubated with 2-methylpropanetriol tripropargylate for 24 and 48h;

[0047] Figure 16 In vivo anti-tumor treatment effect diagram; (a): Photos of tumor dissection of tumor-bearing nude mice in different experimental groups; (b): Tumor volume change curve; (c): Body weight change curve;

[0048] Figure 17Blood and biochemical parameters of nude mice in different experimental groups; treatments for each experimental group: A: PBS; B: RES; C: DOX; D: MIX (RES and DOX); E: BDR; F: BDRP;

[0049] Figure 18 H&E stained tissue sections of heart, liver, spleen, lung and kidney after dissection of nude mice in different experimental groups (scale bar: 100μm);

[0050] Figure 19 Effects of different experimental groups on tumor cell apoptosis in tumor-bearing nude mice (TUNEL staining); (a): PBS; (b): BDR; (c): BDRP; Image magnification 400x. Detailed Implementation

[0051] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0052] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0053] like Figure 1 As shown in Figure A, this invention presents the synthetic route of the folic acid-targeted multi-drug synergistic nanopolymer prodrug and its mechanism of action in treating cervical cancer. Specifically, it utilizes the anthraquinone structure of doxorubicin and employs a click reaction technique involving phenolic hydroxyl-acylynyl and amino-acylynyl groups to efficiently polymerize chemotherapeutic drugs (dxorubicin and resveratrol) with 2-methylpropanetriol tripropynate, respectively preparing the passively targeted multi-drug synergistic polymer prodrug BDR and the actively targeted polymer prodrug BDRP modified with folic acid. This significantly improves the active targeting efficiency of the polymer prodrug, and experiments have shown that the polymer prodrug exhibits significant therapeutic effects against cervical cancer.

[0054] Example 1: Preparation method and application of a folic acid-targeting multi-drug synergistic nanopolymer prodrug

[0055] 1. Synthesis and characterization of the crosslinking agent 2-methylpropanetriol tripropynate

[0056] 1.1 Synthesis Method

[0057] Accurately weigh 4.8 g of trimethylolethane and 16.8 g of propargyl acid, and 31.7 g of p-toluenesulfonic acid (TsOH) into a 150 mL CHCl3 solution and heat under reflux in a round-bottom flask. After cooling, filter the crude product, rotary evaporate the solvent, and purify by silica gel column chromatography using petroleum ether / ethyl acetate (6:1, v / v) as the eluent to obtain a white 2-methylpropanetriol tripropargyl ester (TMP) powder. The synthetic route is as follows: Figure 1As shown in B.

[0058] 1.2 Structural Characterization

[0059] like Figure 2 The image shown is the 1H NMR spectrum of 2-methylpropanetriol tripropynate. 1 ¹H NMR (400MHz, DMSO-d6), δ (TMS, ppm): 4.57 (s, 3H), 4.14 (s, 6H), 0.98 (s, 3H). 1.00 ppm is the signal peak of hydrogen on the methyl group, 2.04 ppm is the signal peak of alkyne hydrogen, and 4.14 ppm and 4.57 ppm are the signal peaks of hydrogen on the methylene group connected to oxygen. This proves that 2-methylpropanetriol tripropynate was synthesized.

[0060] 2. Design and synthesis of folic acid-targeted multi-drug synergistic nanopolymer prodrugs

[0061] 2.1 Preparation of nanopolymer prodrug BDR

[0062] Accurately weigh 5.8 mg DOX and dissolve it in 4.0 mL of ultrapure water, 10 mg NH4HCO3 and dissolve it in 1.0 mL of ultrapure water, 6.9 mg 2-methylpropanetriol tripropynate and 2.3 mg RES and dissolve them in 0.4 mL and 0.5 mL of anhydrous ethanol, respectively. Add 25 mL of ultrapure water to a 100 mL Erlenmeyer flask, sonicate at 100 W, and heat in a 40 °C water bath. Add the 2-methylpropanetriol tripropynate solution to the Erlenmeyer flask, sonicate for 3 min, then add the DOX, RES and NH4HCO3 solutions and sonicate to induce self-assembly. After the reaction system becomes turbid, centrifuge at high speed and discard the supernatant. Wash the reaction product BDR polymer prodrug nanoparticles with ultrapure water and anhydrous ethanol, respectively, to ensure that the nanopolymer prodrug BDR is uniformly dispersed in ultrapure water. Store in the dark.

[0063] 2.2 Preparation of folic acid-targeting nanopolymer prodrug BDRP

[0064] Accurately weigh 5.8 mg DOX and dissolve it in 4.0 mL of ultrapure water; 10 mg NH4HCO3 and dissolve it in 1.0 mL of ultrapure water; 6.9 mg 2-methylpropanetriol tripropynate and 2.3 mg RES and dissolve them in 0.4 mL and 0.5 mL of anhydrous ethanol, respectively; and 2.0 mg FA-PEG-NH2 (FBN) and dissolve it in 5.0 mL of ultrapure water. Add 25 mL of ultrapure water, 2-methylpropanetriol tripropynate, DOX, RES, NH4HCO3, and FBN solutions to an Erlenmeyer flask, and perform self-assembly under ultrasonic power of 100 W and water bath heating at 40 °C. After the reaction is complete, centrifuge and discard the supernatant. The reaction product, folic acid-targeting nanopolymer prodrug BDRP, is washed with ultrapure water and anhydrous ethanol to ensure uniform dispersion of BDRP nanoparticles in ultrapure water, and stored in the dark.

[0065] 3. Structural and morphological characterization of folic acid-targeted multi-drug synergistic nanopolymer prodrug

[0066] 3.1 Ultraviolet Spectroscopy Measurement

[0067] A certain amount of the prepared BDR and BDRP nanosuspensions were diluted, and the ultraviolet absorption spectra of the samples were measured using a Shimadzu UV-2600 UV-Vis spectrophotometer.

[0068] Depend on Figure 3 It is known that the maximum UV absorption wavelength of DOX is 233 nm, and it also has a strong absorption peak at 480 nm. The maximum UV absorption peak of RES is at 310 nm. The synthesized BDR has a shoulder peak at 240 nm, which should be due to the red shift of the DOX peak at 233 nm. Analysis shows that the crosslinking of 2-methylpropanetriol tripropynate with DOX and RES produces an alkenyl ether bond, thereby creating a large conjugated system. Due to the formation of conjugation, the peak position is red-shifted. The absorption peak of BDR at 300 nm should be the UV absorption peak of RES. This shows that BDR is crosslinked with DOX and RES, proving that the synthetic route is feasible. The maximum UV absorption wavelength of FPN is 289 nm, while BDRP molecules produce an absorption peak at 310 nm. BDRP has a shoulder peak at 237 nm, which should be the maximum absorption peak of DOX, and an absorption peak at 520 nm, which should be caused by the red shift of the absorption peak of DOX at 480 nm. Therefore, it can be inferred that 2-methylpropanetriol tripropynate underwent a cross-linking reaction with DOX, RES and FPN during the synthesis of BDRP.

[0069] 3.2 Infrared Spectroscopy Measurement

[0070] A certain amount of the prepared prodrug BDR and BDRP nanosuspension were centrifuged at high speed, the supernatant was discarded, and the products were dried in a vacuum drying oven at 40°C. The products were mixed with spectroscopically pure KBr, ground into powder, and then tableted. Using a Bruker Tensor 27 Fourier transform infrared spectrophotometer, the frequency of the collected data was selected to be between 4000 and 400 cm⁻¹. -1 Measurement.

[0071] like Figure 4 As shown, 2-methylpropanetriol tripropynate (T) contains an acynyl group, and the acynyl group has a diameter of 3333–3267 cm⁻¹. -1 The characteristic peak of the alkyne hydrogen stretching vibration is in the range of 2260–2100 cm⁻¹. -1 Characteristic peaks of stretching vibrations of the alkynyl group are present. The RES molecule contains only the conventional functional group of the phenolic hydroxyl group, lacking characteristic vibrational or rotational peaks, making it difficult to characterize using infrared spectroscopy. 2-Methylpropanetriol tripropynate (T) shows a peak at 3257 cm⁻¹. -1stretching vibration characteristic peak of acylalkynyl alkyne hydrogen; at 2119 cm -1 stretching vibration characteristic peak of carbon-carbon triple bond; the synthesis of BDR will destroy the acylalkyne bond to generate enol ether bond, thus losing the characteristic peak of acylalkynyl group. BDR has no peak at 2119 cm -1 -1,209 cm -1 -1, respectively, the characteristic peak of enol ether bond appears, indicating that crosslinking reaction occurs and acylalkyne bond is broken to generate enol ether bond. BDR has 1722 cm -1 -1, 1407 cm -1 -1, the same as DOX, indicating that it is DOX. BDR has 1737 cm -1 -1, 1418 cm -1 -1, which is red-shifted. The synthesis of BDR has the generation of enol ether bond and the appearance of the stretching vibration peak of carbon-oxygen bond on the phenolic hydroxyl group of DOX, proving that 2-methyl glycerol tripropargylate is crosslinked with DOX. BDRP has 1726 cm -1 -1, 1411 cm -1 -1, 1204 cm -1 -1, the characteristic peak of enol ether bond, and 3400 cm -1 -1, the characteristic peak of carboxylic acid, so it can be known that 2-methyl glycerol tripropargylate is crosslinked with DOX and FA-PEG-NH2 (FPN) in the synthesis of BDRP.

[0072] 3.3 Fluorescence spectrum determination

[0073] A certain amount of prepared BDR and BDRP nanosuspension was diluted respectively. Using the LS55 type fluorescence spectrophotometer of American PE company, DOX was determined with 480 nm as the excitation wavelength; RES, BDR and BDRP were determined with 370 nm as the excitation wavelength, and the slit width was 5 nm. The fluorescence spectrum of the sample was determined.

[0074] The fluorescence spectrum of DOX, RES, BDR, FPN and BDRP is shown in Figure 5The DOX excitation light wavelength is 480 nm; the RES, BDR, and BDRP excitation light wavelengths are 370 nm; and the FPN excitation light wavelength is 380 nm. The dashed line is the emission spectrum of DOX excited by BDRP at an excitation light wavelength of 480 nm. DOX has a relatively wide characteristic emission peak of 550-600 nm and a maximum emission peak at 574 nm when excited by 480 nm excitation light. FPN has a maximum characteristic emission peak at 450 nm. RES has a maximum fluorescence emission peak at 390 nm when excited by 370 nm excitation light. The peak at 411 nm on BDR is caused by red shift of the fluorescence emission peak of RES, which may be because the enol ether bond is generated on the crosslinked RES, the chemical environment of the group is changed, and the fluorescence emission peak is red-shifted. At the same time, the characteristic emission peak of DOX of 550-600 nm is visible on BDR, indicating that the crosslinking reaction of DOX, RES, and 2-methyltriglyceraldehyde tripropargylate occurs. BDRP has both the fluorescence emission peak of RES at 370 nm and the fluorescence emission peak of FPN at 440 nm, but the fluorescence emission peak of DOX is not visible, which is because the DOX peak is generated in the BDRP sample after changing the excitation wavelength (excitation by 480 nm excitation light) to avoid generating a Raman peak, indicating that the crosslinking reaction of FPN, DOX, RES, and 2-methyltriglyceraldehyde tripropargylate occurs. Based on the above, it is shown that the synthesis route of BDR and BDRP is feasible.

[0075] 3.4 Gel chromatography determination

[0076] After the BDR suspension was washed and vacuum dried, it was dissolved in DMF to prepare a 5.0 mg / mL solution. The molecular weight was determined by using DMF as the mobile phase and an Agilent 1260 gel permeation chromatograph (Refractive Index: 1.431, Flow Rate: 1.000 mL / min, Calibration Constant: 3.3926 x 10 -5 ). The molecular weight of BDR was determined to be Mn= 2.650 x 10 7 (± 29.254%) g / mol.

[0077] 3.5 Hydrated particle size and Zeta potential determination

[0078] The BDR and BDRP nanosuspensions were diluted with ultrapure water and uniformly dispersed by ultrasonic. An appropriate amount was added to a cuvette, and the hydrated particle size was determined with ultrapure water as the dispersant. In addition, a small amount of BDR and BDRP suspensions were diluted and added to a Zeta potential cell to determine the Zeta potential.

[0079] Figure 6The hydrated particle size histogram of (a) BDR and BDRP polymer prodrugs is 155.7 nm and 275.3 nm respectively, which meets the requirements of EPR effect, can enter tumor cells through the intervascular gap, promote the selective distribution of macromolecular substances in tumor tissues, can increase the drug efficacy and reduce systemic side effects. Figure 6 The Zeta potential of (b) BDR and BDRP polymer prodrugs is -30.7 mV and -39.9 mV respectively, wherein the absolute value of the Zeta potential of BDRP is greater than 30, and the greater the electrostatic repulsion between the nanoparticles, the better the physical stability of the dispersion system.

[0080] 3.6 Particle size stability determination

[0081] An appropriate amount of BDR and BDRP nanosuspension was taken respectively, and the particle size (hydrated particle size) of the nanosuspension was measured continuously for seven days to evaluate its stability.

[0082] From Figure 7 It can be seen from (a) that the particle size of BDR is relatively stable within seven days, increasing from 141.9 nm to 225.1 nm; Figure 7 (b) is the change of the particle size of BDRP in seven days, increasing from 260.5 nm to 375.9 nm. The particle size of BDR and BDRP both shows an increasing trend with time, but the particle size is less than 400 nm after seven days, showing good stability.

[0083] 3.7 Field emission scanning electron microscopy and transmission electron microscopy

[0084] BDR and BDRP nanosuspension was taken in centrifuge tubes, diluted with an appropriate amount of water, ultrasonicated to disperse uniformly, and an appropriate amount was dropped on a silicon wafer, then placed in a surface dish covered with filter paper, and after natural drying, the powder sample was evenly coated on the double-sided adhesive of the sample dish, and gold plating film treatment was performed. Finally, it was observed by field emission scanning electron microscopy (SEM). A small amount of BDR and BDRP nanoparticle suspension (1.0 mg / mL) was prepared and diluted, and 10 μL of the liquid was removed and slowly added to the carbon support film, and then naturally air-dried. Finally, the dried carbon support film containing the sample was placed under field transmission electron microscopy (TEM) for observation.

[0085] From Figure 8 It can be seen that the BDR and BDRP polymer prodrug nanoparticles are regular spherical nanoparticles with a particle size of 100-200 nm and a uniform distribution, which meets the requirements of EPR effect.

[0086] 4, Encapsulation efficiency and drug loading of nanoparticle polymer prodrug

[0087] 4.1 Standard curve of doxorubicin and resveratrol

[0088] Precisely weigh 1.0 mg DOX, 31.5 mL 43% ethanol aqueous solution as dispersant, ultrasonic dissolution and dispersion, then take a little solution in cuvette, scan the DOX solution in 200-700 nm wavelength. Precisely weigh 1.0 mg RES, 31.2 mL 2% acetonitrile and 45% ethanol aqueous solution as dispersant, scan the RES solution in 200-700 nm wavelength by UV-visible spectrophotometry.

[0089] Take appropriate amount of DOX / RES control solution in 10 mL volumetric flask containing 29.0% ethanol and 1.1% acetonitrile aqueous solution, dilute to volume, shake well, determine the maximum absorption wavelength by full wavelength scanning in 200-700 nm using UV spectrophotometer. Precisely weigh 2.5 mg DOX and RES control solution respectively in 10 mL volumetric flask, dissolve and dilute to the mark, dilute to volume, shake well, to obtain DOX and RES control solution. From which, precisely transfer 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25 mL respectively in 10 mL volumetric flask, dilute to volume, shake well. Determine the absorbance at the maximum absorption wavelength, then draw the standard curve of DOX and RES respectively.

[0090] 4.2 Determination of encapsulation efficiency and drug loading

[0091] Take the supernatant of prepared cross-linked DOX / RES nanoparticles (BDR), collect the supernatant and the solution used for washing nanoparticles in 500 mL volumetric flask, dilute to volume; determine the absorbance of DOX and RES in the solution at the maximum absorption wavelength by UV spectrophotometer for three times in parallel, take the average value, calculate the concentration of DOX and RES contained therein according to the standard curve of DOX and RES in the simulated reaction system respectively, C 游离 DOX and C 游离RES , calculate the encapsulation efficiency of DOX and RES in the prepared BDR material according to formula (1). Since RES has almost no UV absorption at the maximum absorption wavelength of DOX, it has no interference with the determination of DOX; but DOX has certain UV absorption at the maximum absorption wavelength of RES, the measured absorbance is the sum of the absorbance of the two components, so when calculating the encapsulation efficiency and drug loading of RES, the influence of DOX on the absorbance of RES should be deducted. Divide the prepared BDR suspension into four equal parts, each 2.0 mL, take one part, centrifuge and discard the supernatant, disperse with 10 mL 50% ethanol, centrifuge and discard the supernatant, dry in vacuum drying oven at 35°C, calculate the drug loading of DOX and RES in BDR according to formula (2).

[0092] Formula (1):

[0093]

[0094] Equation (2):

[0095]

[0096] m in Equation (1) is the mass of DOX or RES fed, 投料DOX / RES C is the concentration of free DOX or RES, and V is the volume of free drug solution. m in Equation (2) is the mass of nanoparticles, 游离DOX / RES m is the mass of DOX or RES drug fed. BDR 投料DOX / RES m is the mass of DOX or RES drug fed.

[0097] According to Equations (1) and (2), the encapsulation efficiency and drug loading of BDR were calculated, and the results were as follows: the encapsulation efficiency of DOX and RES in BDR was 69.4% and 35.5%, respectively, and the drug loading of DOX and RES was 50.5% and 38.4%, respectively.

[0098] 5. In vitro sustained release of nanopolymer prodrug

[0099] 5.1 Preparation of PBS buffer

[0100] Preparation of stock solution: precisely weigh Na2HPO4 13.7226 g and KH2PO4 18.2525 g, respectively, and dissolve in 500 mL of ultrapure water to obtain a stock solution.

[0101] Preparation of pH=5.0 buffer solution: take 100 mL of KH2PO4 solution, dilute to 400 mL with ultrapure water, and adjust the pH to 5.0 with Na2HPO4 solution.

[0102] Preparation of pH=6.5 buffer solution: take 68 mL of Na2HPO4 solution and 32 mL of KH2PO4 solution, dilute to 400 mL with ultrapure water, and adjust the pH to 6.5 with Na2HPO4 solution.

[0103] Preparation of pH=7.4 buffer solution: take 19 mL of Na2HPO4 solution and 81 mL of KH2PO4 solution, dilute to 400 mL with ultrapure water, and adjust the pH to 7.4 with Na2HPO4 solution.

[0104] The above solutions are all added with Tween 80 to make the content 1%.

[0105] 5.2 Establishment of standard curve

[0106] ​Accurately weigh 1.0 mg of DOX into ultrapure water, and dissolve it in a 25 mL volumetric flask, then dilute to the mark, shake well to prepare the DOX stock solution, and then dilute it to 1.0 μg / mL, 2.0 μg / mL, 5.0 μg / mL, 7.0 μg / mL, 8.0 μg / mL, 9.0 μg / mL of DOX control solution; accurately weigh 1.0 mg of RES into anhydrous ethanol in a 50 mL volumetric flask, dilute to the mark and shake well to prepare the RES stock solution. Then dilute it to 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 0.8 μg / mL of resveratrol standard solution. Using a fluorescence spectrophotometer, the fluorescence intensity of DOX solution of different concentrations was measured at an excitation wavelength (Ex) of 480 nm and an emission wavelength (Em) of 589 nm; the fluorescence intensity of RES solution of different concentrations was measured at Ex of 340 nm and Em of 380 nm. The concentration (C) was taken as the abscissa and the fluorescence intensity (F) was taken as the ordinate to draw the DOX and RES standard curve.

[0107] The fluorescence excitation and emission spectra of DOX and RES are shown in FIGS. 1 and 2, respectively. Figure 9 As shown in FIGS. 1 and 2, the DOX standard curve equation is F = 67.782C - 12.013, R = 0.9999; the RES standard curve equation is F = 995.63C + 79.789, R = 0.9997; according to the two formulas, the in vitro cumulative release curve of BDR can be calculated and drawn.

[0108] 5.3 Precision test

[0109] Prepare DOX stock solution with a concentration of 1.00 μg / mL, 5.00 μg / mL, 9.0 μg / mL, and use a fluorescence spectrophotometer to measure each concentration for 6 times continuously, and then measure again after a period of time to calculate the intra-day and inter-day precision of the determination method; prepare RES stock solution to 0.10 μg / mL, 0.50 μg / mL, 0.7 μg / mL, and measure each concentration for 6 times continuously, and then measure again after a period of time to calculate the intra-day and inter-day precision of the determination method.

[0110] The intra-day and inter-day precision of DOX and RES measured by fluorescence spectrophotometry are shown in Tables 1 and 2, respectively, indicating that the method established for determining the concentration of DOX and RES is accurate and stable, and can be used for the determination of the content of DOX and RES.

[0111] Table 1 Intra-day and inter-day precision of DOX detection (n = 6)

[0112]

[0113] Table 2 Intra-day and inter-day precision of RES detection (n = 6)

[0114]

[0115] 5.4 Reproducibility test

[0116] Under different pH conditions of BDR degradation (pH = 5.0 / 6.5 / 7.4), the sampling time was 12h, 24h, 48h, respectively. After dilution, the fluorescence intensity of DOX and RES in the sample was measured by fluorescence spectrophotometer and measured continuously for 6 times, and the reproducibility RSD (%) was calculated.

[0117] Table 3 Reproducibility of DOX in samples (n = 6)

[0118]

[0119] Table 4 Reproducibility of RES in samples (n = 6)

[0120]

[0121] Reproducibility is the consistency of the results obtained by the same operator using the same instrument under the same conditions and continuously measuring the same sample for a short period of time. It helps to verify the stability and reliability of the results. According to Table 3 and Table 4, the results measured by this method have good reproducibility and stability.

[0122] 5.5 Spiked recovery test

[0123] Under the condition of BDR degradation at pH = 6.5, samples with degradation times of 12h, 24h, 48h were taken, appropriately diluted, and the fluorescence intensity was measured. The measured value of the sample was calculated by substituting the regression equation of the standard curve. DOX and RES control solution was prepared, and a certain amount of standard was added to the sample. The fluorescence intensity was measured, and the measured value after spiking was calculated by substituting the regression equation of the standard curve. The spiked recovery rate was calculated using formula (3).

[0124] Formula (3):

[0125]

[0126] From Table 5, under the condition of pH = 6.5, the spiked recovery rate of DOX was 97.25% to 106.35%, and the spiked recovery rate of RES was 91.66% to 111.63%. The results show that this experimental method is accurate and reliable, and can meet the experimental requirements.

[0127] Table 5 Determination results of recovery test (n = 6)

[0128]

[0129] 5.6 In vitro sustained release experiment

[0130] Studies have shown that the environment of human normal tissue and tumor tissue pH exist obvious difference, the environment of human normal tissue pH is 7.4, the pH of tumor tissue is 5.0~6.8, and the pH of tumor cell lysosome is 4.0~5.0.Therefore, the PBS buffer solution with pH=7.4 is selected to simulate the environment of human normal tissue, and the PBS buffer solution with pH=5.0 and 6.5 is selected to simulate the acid environment of tumor cell to study the in vitro release performance of the prepared nanoparticles.Three 2.0mL DOX or RES suspensions are taken from the DOX / RES suspension agent, 30mL PBS buffer solution with pH=5.0 / 6.5 / 7.4 (containing 1% Tween 80) is added, and the mixture is placed in a constant temperature oscillator (37.5℃, 200rpm / min) to shake, 10mL is sampled at 0, 2, 4, 8, 12, 24, 36, 48 and 72, respectively, and after centrifugation (10000rpm, 10min), 3.0mL supernatant is taken, the nanoparticles are poured back into the original bottle, and 3.0mL PBS buffer solution with corresponding pH is added to continue shaking, and three groups are determined in parallel.

[0131] The collected supernatant is scanned by using a UV spectrophotometer, and the absorbance is determined at the maximum absorption wavelength of DOX and RES.The concentration is calculated according to the standard curve, and the cumulative release rate of BDR is calculated by formula (4).The release curves of DOX and RES are drawn by taking time (T) as the abscissa and the cumulative release rate as the ordinate.

[0132] Formula (4):

[0133]

[0134] In formula (4), DL is the drug loading of DOX or RES, C i The drug concentration (mg / mL) of the i-th released liquid, m BDR The mass (mg) of BDR, V0 The initial release liquid volume, V e The release liquid volume taken each time, and n is the sampling number.

[0135] Figure 10From (a), (b), the 72h cumulative release amount of DOX is 74.2% (pH=5.0), 31.0% (pH=6.5) and 15.52% (pH=7.4) respectively; the 72h cumulative release rate of RES is 76.6% (pH=5.0), 42.2% (pH=6.5) and 12.0% (pH=7.4) respectively. The release curves of DOX and RES in the first 12 hours are steep, and gradually flatten after 12 hours. There is a significant difference at different pH, among which the cumulative release rate is the largest at pH=5.0 (simulating the micro-acidic environment in tumor cells), followed by pH 6.5 and 7.4, indicating that the BDR prodrug can release drugs to a large extent and play a therapeutic effect in the acidic environment of tumor, while the release of DOX and RES is relatively slow under normal physiological conditions (pH=7.4), indicating that the BDR prodrug is relatively stable under normal physiological conditions. From (c), (d), the supernatant photos can be observed that the 0h BDR supernatant is light red or colorless, and after 72h degradation, the color is the deepest at pH 5.0, indicating that the prepared BDR prodrug has obvious acid responsiveness, and is stable under normal tissue environment, so the prepared BDR prodrug can achieve better directional aggregation and cell killing on tumor cells. Figure 10

[0136] 6. High-resolution liquid-mass spectrometry for structure identification of prodrug degradation products

[0137] 6.1 Preparation of ABS buffer

[0138] Accurately weigh ammonium acetate 0.7708g and acetic acid (W%=99.5) 0.6030g, respectively dissolve in 100.0mL ultrapure water, constant volume, shake well. Mix 47.4mL of ammonium acetate solution and 2.6mL of acetic acid solution uniformly, and adjust the pH of the ABS buffer to 5.0 with an acidity meter.

[0139] 6.2 High-resolution liquid-mass spectrometry determination

[0140] The washed BDR suspension was centrifuged at high speed, and the supernatant was discarded. The centrifugation product was placed in a 50ml conical flask, 30.0mL of ABS buffer solution with pH=5.0 was added to the conical flask, and it was placed in a constant temperature oscillator (37.5℃, 200rpm / min) for shaking. After complete degradation, the supernatant was taken after centrifugation, filtered through a 0.45μm water system filter membrane, appropriately diluted, and then determined by high-resolution liquid-mass spectrometry (HPLC-MS). (HPLC conditions: chromatographic column Waters C 18 Column, ​1.7 μm, 2.1 mm × 100 μm, mobile phase: acetonitrile-0.1% formic acid solution, column temperature 35 °C, injection volume 10 μL; MS conditions: TOF MS IDA positive / negative mode, scan range 40–1000 Da.

[0141] 6.3 Structural identification of degradation products

[0142] like Figures 11-13 As shown in Table 6, DOX and RES fragment ions are clearly visible and exhibit high abundance. The main components of BDR degradation products are DOX and RES, and several fragment ions with high abundance are listed. m / z = 212.0197 should be fragments related to RES cleavage; m / z = 544.1822 should be molecular ion fragments of DOX. The above indicates that the products released by the degradation of the prepared BDR prepolymer under acidic conditions at pH 5.0 are mostly DOX and RES drug molecules themselves. By identifying and analyzing the molecular structures of the products, the polymerization mechanism was clarified.

[0143] Table 6. The top seven fragment ions by abundance.

[0144]

[0145] 7. Cytotoxicity evaluation of nanopolymer prodrugs

[0146] 7.1 Cell Culture

[0147] The human cervical cancer cells HeLa and human umbilical vein endothelial cells HUVEC used in this invention were obtained from Shanghai Saibaikang Biotechnology Co., Ltd. The HUVEC and HeLa cell lines were cultured in a 37°C, 5% CO2 incubator using DMEM high-glucose medium (purchased from Zhejiang Tianhang Biotechnology Co., Ltd.) prepared with 10% fetal bovine serum and 1% penicillin-streptomycin mixture.

[0148] Place the required reagents in a clean bench and expose to UV light for 30 minutes; remove the cell culture flask from the incubator, observe it, and then place it back into the clean bench (if there are many suspended cells or the culture and color change, the medium needs to be changed); aspirate the culture medium from the flask, add 3 mL of PBS to the culture flask to wash it; add 4 mL of culture medium back into the culture flask, observe it under a microscope, and place it in a cell incubator.

[0149] Put the required reagents into the ultraclean bench under UV for 30 min; take out the cell culture bottle in the incubator, observe and put it into the ultraclean bench; aspirate the culture medium in the bottle, add 3 mL of PBS for cleaning; take 1 mL of trypsin and put it into the culture bottle, digest for 3 min in the incubator; gently blow the cells sticking to the bottle wall, and transfer all the liquid to a 15 mL centrifuge tube and centrifuge; discard the supernatant, add 4 mL of culture medium to resuspend; add 3 mL of culture medium in the culture bottle in advance to wet it, take 1 mL of the mixed and uniformly blown cell suspension and transfer it to the culture bottle, and then transfer 1 mL of the cell suspension to the culture bottle, and place it in a 37℃, 5% CO2 incubator for culture.

[0150] 7.2 Cell resuscitation and cryopreservation

[0151] Take the cryopreserved cell tube from the -80℃ refrigerator, quickly put it into the preheated 37℃ water bath, and immediately take it out after most of the solid in the cryopreserved tube is dissolved, and place it on the operating table; add 3 mL of culture medium to a 15 mL centrifuge tube, and transfer the cell suspension in the cryopreserved tube into it and mix well, and then centrifuge; discard the supernatant, add 1 mL of culture medium to resuspend; add 3 mL of culture medium in the culture bottle in advance to wet it, then transfer 1 mL of the cell suspension to the culture bottle, and place it in a 37℃, 5% CO2 incubator for culture.

[0152] Put the required reagents into the ultraclean bench under UV for 30 min; take out the cell culture bottle in the incubator, observe and put it into the ultraclean bench; aspirate the culture medium in the bottle, add 3 mL of PBS for cleaning; take 1 mL of trypsin and put it into the culture bottle, digest for 3 min in the incubator; gently blow the cells sticking to the bottle wall, and transfer all the liquid to a 15 mL centrifuge tube and centrifuge; discard the supernatant, add 1 mL of cryopreservation solution (90% fetal bovine serum + 10% DMSO ratio) to resuspend, and then aspirate the cell suspension into the cryopreserved tube after uniform blowing, seal it and store it in a -80℃ refrigerator.

[0153] 7.3 Cell counting

[0154] Put the required reagents into the ultraclean bench under UV for 30 min; take out the cell culture bottle in the incubator, observe and put it into the ultraclean bench; aspirate the culture medium in the bottle, add 3 mL of PBS for cleaning; take 1 mL of trypsin and put it into the culture bottle, digest for 3 min in the incubator; gently blow the cells sticking to the bottle wall, and transfer all the liquid to a 15 mL centrifuge tube and centrifuge; discard the supernatant, add 5 mL of culture medium to dilute the cells; take 10 μL of the diluted cell suspension to the cell counting plate, slowly add it to avoid air bubbles, and count after standing for 3 min.

[0155] 7.4 CCK-8 cytotoxicity assay

[0156] Put the required reagents into the ultraclean bench under UV for 30 min; take out the cell culture bottle in the incubator, observe and put it into the ultraclean bench; aspirate the culture medium in the bottle, add 3 mL of PBS for cleaning in the culture bottle; take 1 mL of trypsin and put it into the culture bottle, digest for 3 min in the incubator; gently blow the cells sticking to the bottle wall, and centrifuge all the liquid into a 15 mL centrifuge tube; discard the supernatant and add 5 mL of culture medium to dilute the cells; take 10 μL of the diluted cell suspension to a cell counting plate, slowly inject it (avoid air bubbles), calculate the required cell suspension volume, and then plate, while gently mixing the cell suspension to prevent cell sedimentation, add PBS to the outermost side of the 96-well plate to prevent evaporation, and continue to culture in a 37°C, 5% CO2 incubator after plating is completed.

[0157] Put the required reagents into the ultraclean bench under UV for 30 min; take out the cell culture bottle in the incubator, observe and put it into the ultraclean bench; aspirate the culture medium in the bottle, add 3 mL of PBS for cleaning in the culture bottle; take 1 mL of trypsin and put it into the culture bottle, digest for 3 min in the incubator; gently blow the cells sticking to the bottle wall, and centrifuge all the liquid into a 15 mL centrifuge tube; discard the supernatant and add 5 mL of culture medium to dilute the cells; take 10 μL of the diluted cell suspension to a cell counting plate, slowly inject it (avoid air bubbles), calculate the required cell suspension volume, and then plate, while gently mixing the cell suspension to prevent cell sedimentation, add PBS to the outermost side of the 96-well plate to prevent evaporation, and continue to culture in a 37°C, 5% CO2 incubator after plating is completed.

[0158] Plate with the required cell suspension diluted to contain 5 x 10 3 cells per 100 μL. First add 50 μL of base to each well, then slowly drop 100 μL of cell suspension into each well. When plating, resuspend the cells immediately after completing each row to prevent cell sedimentation, and do not add cell suspension to the outermost side of the 96-well plate. After plating, let stand for 10 min to allow the cells to settle, then label and continue to culture in a constant temperature incubator. Set up blank groups (CCK-8 and culture medium), control groups (containing cells, CCK-8 and culture medium), DOX groups, MIX groups (DOX + RES), BDR groups, and BDRP groups, with 5 concentration gradients (0.5, 5, 10, 15, and 25 μg / mL, with drug concentrations based on DOX concentration) for each group, and 5 replicate wells for each concentration; after the cells have completely adhered and grown to 90%, add the above drugs and continue to culture in a constant temperature incubator for 24 and 48 h.

[0159] After the incubation time, the 96-well plate was taken out, 100 μL CCK-8 reagent (90 μL DMEM medium + 10 μL CCK-8) was added to each well after suction, and incubated in the incubator; the wavelength of the enzyme marker was set to 450 nm, the absorbance value of each well after 30 min, 1 h, 2 h, and 3 h of incubation was measured, and the cell survival rate was calculated according to formula (5).

[0160] Formula (5):

[0161]

[0162] In the formula, As refers to the experimental hole (cell-containing medium, CCK-8, drug), Ac refers to the control hole (cell-containing medium, CCK-8, no drug), and Ab refers to the blank hole (medium without cells and drug, CCK-8).

[0163] 7.5 Live / dead cell double staining experiment

[0164] Cells were seeded in a confocal dish at a density of 1.0 x 10 5 cells / well, 1.0 mL of phenol red-free medium was added to a 35 mm confocal dish, and after the cells adhered, BDR and BDRP nanosuspension (DOX concentration: 10 μg / mL) were added for co-incubation for 24 h, and then the medium was removed. According to the Calcein-AM / PI cell activity and cytotoxicity detection kit instructions, the live / dead cell staining agent was prepared and added to 1.0 mL in the confocal dish, and after 37°C incubation in the dark for 15-20 min, the staining was observed using a laser confocal fluorescence microscope (Calcein-AM: λ ex = 488 nm, PI: λ ex = 552 nm).

[0165] 7.6 Analysis of cell experiment results

[0166] 7.6.1 CCK-8 toxicity determination results

[0167] As shown in Figure 14 , the CCK-8 method was used to study the cytotoxicity of each group of drugs on human cervical cancer cells HeLa (a) and human umbilical vein endothelial cells HUVCE (b). Figure 14 Figure 14 ​The concentration gradient of different groups of drugs was set in terms of the concentration of DOX, and the cell survival rates after incubation with HeLa cells and HUVCE cells for 24 h and 48 h were analyzed and compared. The results showed that with the increase of the concentration of DOX, the survival rates of HeLa cells and HUVCE cells were obviously reduced; the RES group did not show obvious cell proliferation inhibition effect on HeLa cells; compared with the DOX group, the MIX group showed stronger cell inhibition effect, which might be due to the combination of RES and DOX to enhance the cytotoxicity of DOX and enhance its inhibition effect on tumor cell proliferation; the killing power of the BDR group on tumor cells was slightly stronger than that of the MIX group, which might be because BDR could release RES and DOX rapidly in tumor cells by acid-responsive bond cleavage to achieve combination drug use; the inhibition effect of the BDR group on tumor cells was slightly weaker than that of the BDRP group, indicating that the BDRP grafted with folic acid could improve the drug uptake capacity of tumor cells and achieve the effect of targeted drug delivery; the killing power of BDR and BDRP on HUVEC was smaller than that of the MIX group and the DOX group under the same concentration gradient. In normal cells, the acid condition for cleavage of enol ether bond is not met, so the drug release is small and the cell survival rate is high.

[0168] The half-inhibitory concentration IC 50 was calculated from the survival rate obtained by the CCK-8 method, as shown in Table 7. The IC 50 of each drug group on tumor cells was enhanced with the extension of time; for tumor cells HeLa, the IC 50 of the BDR group and the BDQR group was lower than that of the DOX and MIX groups; for normal cells HUVEC, the IC 50 of the BDR and BDRP groups was higher than that of the DOX and MIX groups.

[0169] Table 7 IC 50 values (μg / mL) of BDR, BDRP and pure drugs on HeLa and HUVEC cells

[0170]

[0171]

[0172] There is a special microenvironment in the tumor site. Due to the rapid growth of blood vessels at the tumor site, the blood vessels are insufficient to supply the oxygen and energy required for the expansion of cancer cells, thereby producing metabolic products such as ATP hydrolysis products and lactic acid, resulting in a lower pH value of the tumor tissue cell microenvironment, about 6.5, while the pH value of human blood and normal tissue organs is about 7.4. The high drug loading and high selectivity of the multi-drug synergistic polyprodrug (BDR / BDRP) prepared in the application realizes the crosslinking of the carrier and the drug, so that the drug is actively released in the acidic environment of the tumor. However, due to the low efficiency of passive targeting, only increasing the dosage of the drug can improve the anti-tumor effect, but this will cause problems such as high toxicity. Therefore, the present application further grafts folic acid to prepare a targeted polyprodrug BDRP, which can accurately and effectively transport the drug to the tumor site and control the release of the drug, reduce the distribution of the chemotherapeutic drug to normal tissues or cells, improve the drug uptake capacity of tumor cells, and achieve the effect of targeted drug delivery.

[0173] 7.6.2 Live / dead cell double staining results

[0174] By the method of live / dead cell staining, Calcein-AM stains Hela into green, representing live cells, and PI stains the cells into red, representing dead cells, and then the fluorescence images are obtained by shooting under a laser confocal fluorescence microscope. As shown in FIG. 6, it can be observed that BDR (c) and BDRP (d) in the application have obvious killing effect on Hela cells, which is consistent with the results of the CCK-8 method. Figure 14 Figure 14 Figure 14

[0175] 7.6.3 Physiological safety of the crosslinking agent

[0176] HUVEC cells were co-incubated with 2-methylpropanetriol tripropargylate for 24h and 48h, and the cell survival rate was as shown in FIG. 7. The toxicity of 2-methylpropanetriol tripropargylate to normal cells was small at a high concentration of 25 μg / mL, indicating that the prepared polyprodrugs BDR and BDRP have good physiological safety. Figure 15 8. In vivo therapeutic effect evaluation of the nano-polymer prodrug

[0177] Based on the good selectivity and proliferation inhibition effect of the nano-polymer prodrugs BDR and BDRP on tumor cells in the cell experiment, 4-6 week old female BALB / C nude mice were selected as model animals to evaluate the physiological toxicity and tumor inhibition effect of different nano-polymer prodrugs.

[0178] 8.1 Animal condition

[0179]

[0180] ​​​​BALB / c female nude mice (4-6 weeks, 18-22 g, purchased from Shanghai Slek Experimental Animal Company, divided into 5 cages, and adapted for one week in the SPF level animal feeding room after purchase. All items entering the animal room were sterilized by high temperature and pressure or ultraviolet light, and water and feed were changed every two days.

[0181] 8.2 Subcutaneous tumor implantation

[0182] The Hela cells in the logarithmic growth phase were trypsinized, transferred to a 10 mL centrifuge tube for centrifugation (4°C, 1000 rpm, 5 min), the supernatant was discarded, and the cells were resuspended in serum-free DMEM medium (cell concentration 1 x 10 8 / mL) to collect HeLa cells in the logarithmic growth phase as a tumor model. The cell suspension was uniformly mixed with BD Matrigel matrix glue at 1:1 (V:V), and the bubbles in the mixed solution were completely removed by flicking the outer wall of the centrifuge tube. 0.1 mL of cell suspension containing 1.0 x 10 8 concentration of suspended cells was injected subcutaneously into the right posterior wing of each healthy nude mouse, and the Hela tumor-bearing nude mouse animal model was obtained.

[0183] 8.3 Body condition of nude mice

[0184] After planting, observation was made within two weeks, and when the tumor volume was about 80 mm 3 , the mice were randomly divided into groups, with 5 mice in each group, for subsequent experiments. The body weight of all nude mice was measured every 2 days, and the length and width of the tumor were measured with a vernier caliper. The tumor volume calculation formula (6) is as follows:

[0185] V = ab 2 / 2

[0186] wherein V represents the tumor volume, a represents the long diameter of the tumor read with a vernier caliper (unit: mm), and b represents the short diameter of the tumor (unit: mm). According to the statistical data, the body weight change curve of the nude mice and the body weight change curve were drawn.

[0187] 8.4 Tail vein injection administration

[0188] When the tumor grew to 80-100 mm 3, about 30 tumor-bearing nude mice, 5 in each group, were randomly divided into 6 groups, and injected with PBS, DOX, RES, MIX, BDR, and BDRP, respectively. The volume changes of the tumor on the back of each group of nude mice after administration were observed. Each group of nude mice was administered at a dose of 5 mg DOX / kg (the concentration was calculated based on the concentration of DOX), and the administration was performed by tail vein injection, with a dosage of about 0.2 mL, and a drug concentration of 0.5 mg / mL (about 0.1 mg). Before the tail vein injection of the nude mice, the tail of the nude mouse was soaked in warm water to make the tail vein swell properly, and if the tail cuticle was thick, it could be wiped with alcohol cotton. A No. 4 medical syringe was used for injection, and the injection was started from the tip of the tail. The needle angle was about 15°, the needle tip entered about 5 mm deep, and all the liquid was pushed at one time. After the needle was pulled out, it was pressed to stop bleeding, and then the mouse was marked and put back into the cage for further feeding. The time point of the first injection was recorded as the first day of treatment (day 1), and the same dose was injected again by tail vein injection on the third day (day 3) and the sixth day (day 6) of treatment.

[0189] 8.5 Dissection and Blood Test

[0190] After 14 days of administration, enucleation and blood collection were performed on all nude mice, and then all nude mice were executed by cervical dislocation and dissected, and internal organs (liver, lung, kidney, heart, spleen) and tumors were collected. Blood collection: after each nude mouse was fixed upside down, enucleation and blood collection were performed quickly, about 1 mL of blood was collected from each nude mouse, half of which was collected in a sterile EP tube, and the other half was collected in a vacuum blood collection tube with added sodium citrate. The collected blood was tested for the content of red blood cells, white blood cells, hemoglobin, hematocrit, platelets, creatinine, and glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase.

[0191] Dissection: The liver, lung, kidney, heart, spleen, and tumor tissues were carefully removed and quickly immersed in formalin for fixation. The internal organs of one untreated nude mouse were used as a control group.

[0192] 8.6 Tissue Sectioning

[0193] The fixative was washed away with water to terminate fixation. The tissue was then gradually dehydrated in ethanol dehydration solutions of different concentrations, followed by clearing with xylene. After treatment, the tissue blocks were placed in soft paraffin at 52–54°C for 1 hour, then in another soft paraffin solution at 52–54°C for 1 hour, and finally in hard paraffin at 56–58°C for 1 hour. The paraffin-impregnated tissue blocks were then embedded in paraffin, and the paraffin blocks were trimmed to maintain a 1–2 mm paraffin edge around the tissue, with both sides parallel. After sectioning with a microtome, the tissue sections were stained with hematoxylin and eosin (H&E) staining solutions, and observed after treatment. Formalin-fixed tumor tissue was trimmed into equal-sized tissue blocks, placed in a dehydration box, and rinsed to remove the formalin fixative. Routine dehydration and paraffin embedding were performed, followed by serial sectioning at a thickness of 3 μm.

[0194] The TUNEL in situ apoptosis detection kit (POD) was used, and the staining of cells in TUNEL tissue sections was observed under an optical microscope according to the instructions.

[0195] 8.7 Animal Experiment Results and Analysis

[0196] 8.7.1 Analysis of Tumor Suppression Effect

[0197] Using HeLa tumor-bearing nude mice as an animal model, tumor growth was assessed by injecting PBS, DOX, RES, MIX, BDR, and BDRP into the tail vein. The day of injection was designated as day 0, the time after injection was plotted on the x-axis, and the change in tumor volume was plotted on the y-axis.

[0198] Tumor volume growth in tumor-bearing nude mice, such as Figure 16 As shown in (b). The PBS group represents an equal dose of injected pH 7.4 PBS buffer as a negative control. On day 14 after injection, the tumor volume in the PBS group was the largest, with an average increase of 8.2 times. The tumor inhibition rate in the RES group was 22.96%, indicating that RES alone did not show a significant inhibitory effect on tumor growth, and the treatment effect was not obvious. The tumor inhibition rate in the DOX group was 47.24%, and the tumor inhibition rate in the MIX group was 66.02%, indicating that DOX and RES had a certain synergistic effect. The tumor inhibition rate in the BDR group was 76.22%, showing a significant tumor inhibition effect, but it can be seen that the tumor volume increased from day 0 to day 8, and then remained basically unchanged. After modifying FA-PEG-NH2, the tumor inhibition rate in the BDRP group was 83.16% using its active targeting. The changes in body weight of tumor-bearing nude mice are shown in the figure. Figure 16The average weight of each group of randomly grouped mice was in the range of 14-16 g, as shown in Fig. c. Within 14 days of treatment, the slow release of DOX in the body circulation due to the nano-polymer prodrug can reduce the systemic physiological toxicity of DOX. The body weight of each group showed a gradual upward trend, and no abnormal weight change was observed, indicating that the feeding condition was not affected. This shows that the nano-polymer prodrug prepared in this experiment reduces the systemic toxic side effects of DOX, which is beneficial to improve the quality of life.

[0199] As Figure 16 As shown in Fig. a, after the end of treatment, the tumor tissue samples obtained by dissecting the mice showed that the tumor growth of BDR and BDRP was significantly inhibited, and the treatment effect of the BDRP group modified with FA-PEG-NH2 was the best, indicating that the nano-polymer prodrug with active targeting through FA has good anti-tumor effect.

[0200] 8.7.2 Blood routine and biochemical indicators

[0201] As Figure 17 shown, 7 main blood routine indicators in the blood of nude mice were detected, including platelet count (PLT), mean corpuscular volume (MCV), hemoglobin (HGB), white blood cell count (WBC), red blood cell count (RBC), red blood cell count (RBC), mean corpuscular hemoglobin concentration (MCHC), and 2 biochemical indicators: alanine aminotransferase (ALT), aspartate aminotransferase (AST). Compared with the control group, the 7 blood indicators in the experimental group did not show obvious differences. Only in the experimental group of nude mice using DOX pure drug, the PLT index was observed to be reduced to a certain extent, which may be related to the physiological toxicity of DOX. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are mainly present in the cytoplasm and mitochondria of liver cells, respectively, and the damage state of liver cells can be evaluated by the ratio of the two. Compared with the control group, the ALT and AST indicators of each experimental group did not show obvious changes. The blood routine and biochemical analysis of each indicator was basically normal, indicating that the prepared polyprodrug itself has high physiological safety.

[0202] 8.7.3 Tissue sections

[0203] The main organs of nude mice were observed for obvious damage by H&E staining, such as Figure 18The main organs of the PBS, DOX, RES, MIX, BDR, BDRP groups of nude mice were taken for H&E tissue section observation, and there was no obvious difference in the main organs of the heart, liver, spleen, lung, kidney of each group. The myocardial cells were arranged in order, the liver cells were arranged densely and compactly, no edema was found in the spleen cells, the alveoli were uniform, the glomeruli were normal in shape without enlargement, and no pathological damage or organic lesion was observed. Defects and reduced number of nuclei were observed in tumor tissues, combined with the changes in tumor mass and volume, it can be judged that the polyprodrug BDR and the active targeting BDRP transported by the EPR effect passively enriched in tumor tissues, and did not cause damage to the mouse organs. It is shown that the prepared nano-polymer prodrug has no obvious toxic side effects on tissues and organs, has good physiological safety, and is suitable for in vivo drug delivery.

[0204] The TUNEL in situ cell apoptosis detection kit (POD) was used according to the instructions. The TUNEL tissue section cell staining was observed under an optical microscope.

[0205] The deoxyribonucleotide terminal transferase-mediated in situ gap end labeling method (Tunel) was used to stain the intact single apoptotic nuclei or apoptotic bodies in situ. The cells with brown-yellow particles in the nuclei were positive cells, i.e. apoptotic cells. The nuclei with blue color were negative cells stained with hematoxylin, i.e. tumor cells. As shown in Figure 19 , only a few apoptotic cells were observed in the PBS group (a), a small amount of apoptotic cells were observed in the BDR group (b), and a large number of apoptotic cells were observed in the BDRP group (c). Figure 19 Figure 19 Figure 19 The above results show that the BDR and BDRP groups can significantly promote the apoptosis of tumor cells in tumor-bearing nude mice.

[0206] ​​From the above examples, the present application prepared a high drug loading, multi-drug synergy, folate-targeted nanopolymer prodrug, and characterization, in vitro release and biological evaluation. Based on click reaction, 2-methyl glycerol tripropargylate is crosslinked with chemotherapeutic drugs DOX, RES respectively, and the structure of BDR and BDRP nanoparticles is determined and analyzed by UV spectrum. The structure and morphology of BDR and BDRP are further determined by TEM, particle size and Zeta structure characterization methods. The particle size of BDR and BDRP is 155.7 nm and 275.3 nm respectively, which meets the requirements of EPR effect, can enter the tumor cells through the vascular gap, promote the selective distribution of macromolecular substances in tumor tissues, and can increase the drug efficacy and reduce the systemic side effects. The Zeta potential of BDR and BDRP is-30.7 mV and-39.9 mV respectively, which has high stability. The prepared polyprodrug BDR has outstanding high drug loading and good in vitro release performance, that is, it can be biodegraded and efficiently release chemotherapeutic drugs under the acidic conditions simulating the tumor microenvironment. The degradation release behavior of BDR at pH=5.0 / 6.5 / 7.4 is quantitatively analyzed by UV spectrophotometry and fluorescence spectrophotometry, and the cumulative release curve is drawn. The results show that under the degradation condition of pH=5.0, the degradation release amount of the nanopolymer prodrug is the most, while in the normal physiological environment (pH=7.4), it shows the characteristics of low release rate. The degradation products are qualitatively analyzed by high-resolution liquid chromatography-mass spectrometry method, and it is found that most of the degradation products are drugs themselves, and only a small part is the substitution byproduct of drug and material molecules, which shows that it has high drug loading, biodegradability and obvious acid stimulation response characteristics.

[0207] Cytotoxicity experiments show that with the increase of DOX concentration, the survival rate of HeLa cells and HUVCE cells decreases significantly; compared with the DOX group, the MIX group shows strong cell inhibition, which may be due to the combination of RES and DOX, which can enhance the cytotoxicity of DOX and enhance its inhibition effect on tumor cell proliferation; the killing power of BDR group on tumor cells is slightly stronger than that of MIX group, which may be due to the fact that BDR can release RES and DOX rapidly in tumor cells through acid-responsive bond cleavage to achieve combination drug; the inhibition effect of BDR group on tumor cells is slightly weaker than that of BDRP group, which shows that the grafting of folate on BDRP can improve the drug uptake capacity of tumor cells and achieve the effect of targeted drug delivery; by Calcein-AM / PI live / dead cell staining method, it is observed that BDR and BDRP have obvious killing effect on Hela cells, which is consistent with the results of CCK-8 method. The survival rate of normal cells HUVEC shows that 2-methyl glycerol tripropargylate has small killing power on normal cells at high concentration, and has good physiological safety.

[0208] The prepared polyprodrug was biologically evaluated at the animal level using Hela tumor-bearing nude mice as an animal model. On the 14th day after injection, the tumor inhibition rate of the DOX group was 47.24%, the tumor inhibition rate of the MIX group was 66.02%, the tumor inhibition rate of the BDR group was 76.22%, the tumor inhibition rate of the BDRP group using the active targeting of the modified FA-PEG-NH2 was 83.16%; the body weight of each group showed a gradual upward trend, and no abnormal weight change was observed, the polyprodrug reduced the systemic toxic side effects of DOX, and was beneficial to improve the quality of life; the results of tissue section showed that the polyprodrugs BDR and BDRP were passively transported by the EPR effect and actively targeted, enhanced drug enrichment in tumor tissue, and did not cause damage to the mouse organs, indicating that the prepared nanopolymer prodrug had no obvious toxic side effects on tissues and organs, had good physiological safety, and was suitable for in vivo drug delivery.

[0209] The problems of traditional chemotherapy drugs such as large toxic side effects, poor specificity, low drug efficacy and short blood circulation time are solved to some extent by changing the administration mode. The system provides a new idea for improving the drug efficacy of conventional chemotherapy drugs, expanding the indications of chemotherapy and improving drug resistance, and lays a solid foundation for the in-depth research and clinical application of polyprodrugs.

[0210] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A multi-drug synergistic, passively targeted nanopolymeric prodrug, characterized in that, It comprises a chemotherapy drug and 2-methyl glycerol tripropynoate crosslinked with the chemotherapy drug, wherein the chemotherapy drug is doxorubicin and resveratrol.

2. A folate targeted multi-drug synergistic nanopolymer prodrug, characterized in that, It comprises the multi-drug synergistic passive targeting nano-polymer prodrug of claim 1 and a folate targeting molecule FA-PEG-NH2 modified to the multi-drug synergistic passive targeting nano-polymer prodrug.

3. A method for preparing a multi-drug synergistic, passively targeted nanopolymeric prodrug, characterized in that, It comprises the following steps: (1) respectively dissolving doxorubicin hydrochloride and NH4HCO3 in water, and dissolving 2-methyl glycerol tripropynoate and resveratrol in anhydrous ethanol to prepare a doxorubicin solution, an NH4HCO3 solution, a 2-methyl glycerol tripropynoate solution and a resveratrol solution; (2) after ultrasonicating and water bath heating the water, adding the 2-methyl glycerol tripropynoate solution to ultrasonicate, and then adding the doxorubicin solution, the resveratrol solution and the NH4HCO3 solution drop by drop to ultrasonicate for self-assembly to obtain the multi-drug synergistic passive targeting nano-polymer prodrug.

4. The production method according to claim 3, wherein In step (1), the mass-volume ratio of the doxorubicin and water is (5-6) mg:4 mL; and / or the mass-volume ratio of the NH4HCO3 and water is (8-10) mg:1 mL; and / or the mass-volume ratio of the 2-methyl glycerol tripropynoate and anhydrous ethanol is (6-7) mg:0.4 mL; and / or the mass-volume ratio of the resveratrol and anhydrous ethanol is (2-3) mg:0.5 mL.

5. The production method according to claim 1, wherein In step (2), the volume ratio of the water and the 2-methyl glycerol tripropynoate solution is 20-25:0.4; and / or the ultrasonic power is 100 W, and the water bath heating temperature is 40-45℃.

6. A method for preparing folate-targeted multi-drug synergistic nanopolymer prodrug, characterized in that, It comprises the following steps: (1) respectively dissolving doxorubicin, NH4HCO3 and FA-PEG-NH2 in water, and dissolving 2-methyl glycerol tripropynoate and resveratrol in anhydrous ethanol to prepare a doxorubicin solution, an NH4HCO3 solution, a FA-PEG-NH2 solution, a 2-methyl glycerol tripropynoate solution and a resveratrol solution; (2) adding the doxorubicin solution, the NH4HCO3 solution, the 2-methyl glycerol tripropynoate solution, the resveratrol solution and the FA-PEG-NH2 solution drop by drop in water, ultrasonicating and water bath heating for self-assembly to obtain the folate targeting multi-drug synergistic nano-polymer prodrug.

7. The production method according to claim 6, wherein In step (1), the mass-volume ratio of the doxorubicin and water is (5-6) mg:4 mL; and / or the mass-volume ratio of the NH4HCO3 and water is (8-10) mg:1 mL; and / or the mass-volume ratio of the FA-PEG-NH2 and water is (1-2) mg:5 mL; and / or the mass-volume ratio of the 2-methyl glycerol tripropynoate and anhydrous ethanol is (6-7) mg:0.4 mL; and / or the mass-volume ratio of the resveratrol and anhydrous ethanol is (2-3) mg:0.5 mL.

8. The production method according to claim 6, wherein In step (2), the volume ratio of the water and the 2-methyl glycerol tripropynoate solution is 20-25:0.4; and / or the ultrasonic power is 100-200 W, and the water bath heating temperature is 40-45℃.

9. Use of the multi-drug synergistic passively targeted nanopolymeric prodrug of claim 1 or the folate targeted multi-drug synergistic nanopolymeric prodrug of claim 2 for the preparation of a medicament for the treatment of cervical cancer.

10. Use of the folate targeted multi-drug synergistic nanopolymeric prodrug of claim 2 for the preparation of a medicament for multi-drug synergistic alleviation of doxorubicin resistance and enhancement of cytotoxicity against tumor cells.

Images