Functional polymer micelle for co-delivery of chemotherapeutic drug and immunostimulant as well as preparation method and application of functional polymer micelle

Abstract

The invention relates to a functional polymer micelle for co-delivery of a chemotherapeutic drug and an immunostimulant as well as a preparation method and application of the functional polymer micelle, and belongs to the field of medicines. The invention relates to a functional polymer micelle for co-delivery of a chemotherapeutic drug and an immunostimulant, the micelle takes a folic acid-hydroxyethyl starch-epirubicin polymer prodrug as a carrier, and the immunostimulant is entrapped in the carrier. The micelle is formed by self-assembly of a folic acid-hydroxyethyl starch-epirubicin prodrug and an immunologic stimulant. According to the co-delivered functional polymer micelle, a chemical therapy and an immunotherapy have a synergistic effect, and compared with an existing single treatment mode, the co-delivered functional polymer micelle can more effectively reduce the density of solid tumors of rats, induce macrophages to be converted from M2 type to M1 type, reduce organ injury and realize'attenuation and synergism 'of perfusion treatment of the non-muscular invasive bladder cancer at the same time.

Description

Technical Field

[0001] The invention relates to a functional polymer micelle for co-delivering a chemotherapeutic drug and an immunostimulant, a preparation method and application thereof, and belongs to the field of medicine. Background Art

[0002] Bladder cancer is one of the most common and most expensive malignant tumors in the urinary system. Among the three types of bladder cancer, most cases are transitional cell carcinoma, also known as urothelial carcinoma, of which about 75% are non-muscle invasive bladder cancer, which has the characteristics of high recurrence rate and low mortality rate, and has a high prevalence. Clinically, one of the main standard treatments for non-muscle invasive bladder cancer is adjuvant intravesical chemotherapy or immunotherapy after transurethral resection of bladder tumors. However, traditional intravesical instillation therapy lacks tumor tissue selectivity, and drug concentration decreases with urine elimination, resulting in frequent administration and severe local irritation symptoms. Therefore, a targeted drug delivery system is needed to prolong the retention time and enhance the penetration of drugs at the tumor site rather than the entire bladder wall, thereby minimizing side effects. Summary of the invention

[0003] The present invention provides a functional polymer micelle for co-delivery of chemotherapeutic drugs and immunostimulants, and a preparation method and application thereof. The functional micelle can expose folic acid targeting ligands, target cancer cells in the tumor microenvironment, and acid-sensitively release chemotherapeutic drugs for chemotherapy, and the released immunostimulants can repolarize M2 macrophages to M1 macrophages, creating a tumor microenvironment that is not conducive to the survival of tumor cells, thereby achieving "reducing toxicity and increasing efficacy" in the perfusion treatment of non-muscle-invasive bladder cancer.

[0004] In the first aspect of the present invention, a functional polymer micelle for co-delivering chemotherapeutic drugs and immunostimulants is provided, which has the characteristics of active targeting and pH-sensitive drug release, and achieves the treatment of bladder cancer by combining immunotherapy and chemotherapy.

[0005] The invention discloses a functional polymer micelle for co-delivering a chemotherapeutic drug and an immunostimulant. The micelle uses a folic acid-hydroxyethyl starch-epirubicin polymer prodrug as a carrier and the immunostimulant is encapsulated in the micelle.

[0006] The hydrophilic shell of the micelle of the present invention is a folic acid-hydroxyethyl starch-epirubicin polymer prodrug, and the hydrophobic core is an immunostimulant.

[0007] Furthermore, the micelle is formed by self-assembly of folic acid-hydroxyethyl starch-epirubicin prodrug and immunostimulant.

[0008] Preferably, the immunostimulant is imiquimod, resiquimod or resiquimod.

[0009] Furthermore, the drug loading of the immunostimulant is 0% to 30%, excluding 0. Furthermore, the drug loading of the immunostimulant is 0.5% to 30%.

[0010] In the polymer micelle of the present invention, in the folic acid-hydroxyethyl starch-epirubicin polymer prodrug, hydroxyethyl starch is connected to epirubicin via a pH-sensitive chemical bond, and the grafting rate of epirubicin is 0% to 30%, excluding 0. Preferably, the grafting rate of the chemotherapeutic drug is 10% to 30%.

[0011] The carrier material hydroxyethyl starch of the functional polymer micelle for co-delivery of chemotherapeutic drugs and immunostimulants provided by the present invention is connected to the chemotherapeutic drugs through pH-sensitive chemical bonds, and the pH-sensitive chemical bonds include hydrazones, oximes, imines, acetals, orthoester bonds and vinyl ethers, and have the characteristics that when the micelles are located in normal tissues and blood environments, the micelles do not release or hardly release drugs, and when the micelles are located in tumor tissues or tumor cell lysosomes, the cumulative release amount of the drugs is significantly increased. Preferably, the pH-sensitive chemical bonds are imine bonds and hydrazone bonds. Further, the pH-sensitive chemical bonds are hydrazone bonds.

[0012] In the polymer micelle of the present invention, folic acid is connected to hydroxyethyl starch via an ester bond, and the grafting rate of folic acid is 10% to 50%; further, the grafting rate of folic acid is 20% to 40%.

[0013] Preferably, the carrier material is hydroxyethyl starch, and its average molecular weight is 20kDa to 1000kDa, and its molar substitution is 0.2 to 0.8. Further preferably, the average molecular weight of hydroxyethyl starch is 100kDa to 300kDa, and its molar substitution is 0.3 to 0.6. Further, the average molecular weight of hydroxyethyl starch is 110kDa to 150kDa, and its molar substitution is 0.37 to 0.45.

[0014] The micelles of the present invention are spherical, with an average particle size of ≤200.0 nm and a PDI of ≤0.30. The particle size and PDI do not change significantly after being diluted 10 to 100 times.

[0015] Another object of the present invention is to provide a method for preparing the functional polymer micelles for co-delivering the above-mentioned chemotherapeutic drugs and immunostimulants.

[0016] A method for preparing functional polymer micelles for co-delivering chemotherapeutic drugs and immunostimulants comprises synthesizing a folic acid-hydroxyethyl starch-epirubicin prodrug; dissolving the obtained prodrug and immunostimulant in an organic solvent to obtain a prodrug solution and an immunostimulant solution, respectively, and then mixing the two to obtain a drug solution; adding the drug solution to water, heating and stirring, so that the prodrug encapsulates the immunostimulant and self-assembles to form micelles.

[0017] In the above technical solution, the mass volume ratio of the prodrug to the organic solvent is 1:100-500; the mass volume ratio of the immunostimulant to the organic solvent is 1:500-1000; the volume ratio of the prodrug solution to the immunostimulant solution is 1:1-5

[0018] In the above technical solution, the organic solvent includes one of dimethylacetamide (DMAC), N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), and dimethyl sulfoxide (DMSO), preferably DMSO.

[0019] In the above technical solution, the ratio of the organic solvent to water is 1:5 to 1:1; and the heating and stirring temperature is 20 to 45°C.

[0020] Preferably, the ratio of organic solvent to water is 1:5, and the heating and stirring temperature is 45°C.

[0021] In the above technical scheme, the folic acid-hydroxyethyl starch-epirubicin prodrug is prepared according to the following method:

[0022] 1. Take an appropriate amount of hydroxyethyl starch and prepare an aqueous solution with a concentration of 80 g / L, add a sodium periodate aqueous solution thereto, stir and react overnight at room temperature in the dark to prepare formaldehyde hydroxyethyl starch, the mass volume ratio of sodium periodate to water is 1:100-150, and the mass ratio of hydroxyethyl starch to sodium periodate is 1:50-60. After the reaction is completed, dialyze and freeze-dry to obtain formaldehyde hydroxyethyl starch;

[0023] 2. Folic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are mixed in DMSO, and the mixture is stirred at 30°C for 30 minutes to activate the terminal carboxyl groups, wherein the mass ratio of folic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1-10:2.5-1.5:1, and then a DMSO solution of formaldehyded hydroxyethyl starch is added and stirred at room temperature for 48 hours to obtain folic acid-formaldehyded hydroxyethyl starch, wherein the mass ratio of hydroxyethyl starch to folic acid is 1:0.1-2, preferably 1:0.1-1. After the reaction is completed, the precipitate is removed by filtration, and the filtrate is dialyzed, centrifuged and freeze-dried to obtain folic acid-formaldehyded hydroxyethyl starch;

[0024] 3. Mix folic acid-formaldehyde-hydroxyethyl starch and adipic acid dihydrazide in DMSO, and react at 80° C. for 36 hours to obtain a folic acid-hydroxyethyl starch-adipic acid dihydrazide reaction solution, wherein the mass ratio of folic acid-formaldehyde-hydroxyethyl starch to adipic acid dihydrazide is 1:0.1-1;

[0025] 4. Take epirubicin hydrochloride and dissolve it in DMSO, add diethylamine to prepare epirubicin free base, wherein the mass volume ratio of epirubicin hydrochloride to diethylamine should be not less than 0.25:1, then add it to the folic acid-hydroxyethyl starch-adipic acid dihydrazide reaction solution generated in the previous step, stir and react for 72 hours at 55°C in the dark to obtain folic acid-hydroxyethyl starch-epirubicin prodrug solution, wherein the mass ratio of epirubicin hydrochloride to the feed mass of folic acid-aldehyded hydroxyethyl starch in the previous step should be not less than 1:1, add the reaction solution to methanol with a volume 10 times that of the reaction solution, centrifuge, discard the supernatant, wash the precipitate, and dry to prepare folic acid-hydroxyethyl starch-epirubicin prodrug.

[0026] Another object of the present invention is to provide the use of the functional polymer micelles for co-delivering chemotherapeutic drugs and immunostimulants in the preparation of drugs for treating bladder cancer.

[0027] Furthermore, the invention provides the use of the functional polymer micelles for co-delivering the chemotherapeutic drug and the immunostimulant in the preparation of bladder instillation drugs for treating bladder cancer.

[0028] The beneficial effects of the present invention are as follows: the present invention provides a functional polymer micelle for co-delivery of chemotherapeutic drugs and immunostimulants. After administration by bladder perfusion, the preparation specifically aggregates at the tumor site where folate receptors are highly expressed. At the same time, since the pH value of the tumor tissue is lower than that of the normal tissue and blood environment, the pH-sensitive chemical bonds in the micelles are protonated, so the micelles cannot maintain their original shape and size, and then release the chemotherapeutic drugs and immunostimulants. The co-delivery micelle enables chemotherapy and immunotherapy to work synergistically. Compared with the existing single treatment method, it can more effectively reduce the density of rat solid tumors at the same time, induce macrophages to transform from M2 type to M1 type, reduce organ damage, and achieve "reducing toxicity and increasing efficacy" in the perfusion treatment of non-muscle-invasive bladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The preparation process of the active targeted hydroxyethyl starch epirubicin imine bonded drug (FA-HES-EPI) in Example 1 is shown;

[0030] Figure 2 The preparation process of the active targeted hydroxyethyl starch epirubicin hydrazone bonded drug (FA-HES-EPI) in Example 2 is shown;

[0031] Figure 3 The FA-HES-EPI / IMQ polymer micelle samples were prepared using different organic solvents in Example 3, and the particle size distribution and PDI results were detected by DLS method;

[0032] Figure 4The FA-HES-EPI / IMQ polymer micelle samples were prepared using different ratios of organic solvent to water in Example 4, and the particle size distribution and PDI results were detected by DLS method;

[0033] Figure 5 The FA-HES-EPI / IMQ polymer micelle samples were prepared at different temperatures in Example 5, and the particle size distribution and PDI results were detected by DLS method;

[0034] Figure 6 The morphology of FA-HES-EPI / IMQ polymer micelles observed by TEM in Example 6 is shown;

[0035] Figure 7 The particle size and PDI results of FA-HES-EPI / IMQ polymer micelles detected by DLS method in Example 6 are shown;

[0036] Figure 8 The particle size and PDI results of FA-HES-EPI / IMQ polymer micelles at different dilution times in Example 6 are shown;

[0037] Fig. 9 The cumulative release amount of EPI in FA-HES-EPI / IMQ polymer micelles under different pH conditions in Example 6 is shown;

[0038] Fig.10 The cumulative release amount of IMQ in FA-HES-EPI / IMQ polymer micelles under different pH conditions in Example 6 is shown;

[0039] Fig.11 (A) and (B) show the visual size, tumor volume and weight of bladder tumors in female SD rats with orthotopic bladder cancer in Example 7, which were perfused with physiological saline, EPI solution, FA-HES-EPI micelles and FA-HES-EPI / IMQ micelles;

[0040] Fig.12 (A) and (B) show the results of immunofluorescence staining and fluorescence intensity of bladder tumor sections of SD female rats with orthotopic bladder cancer perfused with physiological saline, EPI solution, FA-HES-EPI micelles and FA-HES-EPI / IMQ micelles in Example 7;

[0041] Fig.13 The expression of tumor-associated macrophage markers in female SD rats with orthotopic bladder cancer in Example 7 is shown;

[0042] Fig.14The weight changes of female SD rats with orthotopic bladder cancer in Example 7 were shown, which were perfused with physiological saline, EPI solution, FA-HES-EPI micelles and FA-HES-EPI / IMQ micelles into the bladder;

[0043] Fig.15 The results of H&E staining of the heart, liver, spleen, lung and kidney of female SD rats with orthotopic bladder cancer in Example 7 were shown by bladder perfusion of physiological saline, EPI solution, FA-HES-EPI micelles and FA-HES-EPI / IMQ micelles. DETAILED DESCRIPTION

[0044] The following non-limiting embodiments may enable a person skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0045] The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0046] Example 1 Preparation of Active Targeting Hydroxyethyl Starch Epirubicin Imine Bonded Drug (FA-HES-EPI)

[0047] Take 8.0 g of hydroxyethyl starch (HES) (130 kDa / molar substitution 0.4) and dissolve it in 100 mL of water, take 0.1460 g of sodium periodate (NaIO4) and dissolve it in 20 mL of water, then add the NaIO4 solution to the HES aqueous solution, stir and react overnight, then dissolve 0.2512 g of sodium bisulfite in 3 mL of water and add it to the reaction system to terminate the reaction, and transfer the reaction solution to a dialysis bag, dialyze against water for 12 hours to remove small molecular impurities, and freeze-dry to obtain aldehyded hydroxyethyl starch (HES-CHO).

[0048] 0.1160 g of folic acid (FA), 0.0537 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.0320 g of 4-dimethylaminopyridine (DMAP) were mixed in 24 mL of anhydrous DMSO and stirred at room temperature for 2 h; then 0.0843 g of HES-CHO was dissolved in anhydrous DMSO (10 mL) and mixed with the above solution, and stirred at room temperature for 48 h to obtain folic acid-aldehyded hydroxyethyl starch (FA-HES-CHO); after the reaction was completed, the precipitate was filtered out, the filtrate was transferred to a dialysis bag, and dialyzed with phosphate buffered saline (PBS) for 3 days to remove impurities. After the dialysis was completed, the solution was freeze-dried to obtain a FA-HES-CHO freeze-dried product for standby use.

[0049] Prescription 1: Weigh epirubicin (EPI) and FA-HES-CHO so that the molar ratio of EPI to the aldehyde group in O is 5:1, and react in a sodium acetate / acetic acid (NaAc / HAc) buffer system at 55°C and pH 5.5 for 72 hours to prepare FA-HES-EP. At this time, FA-HES-CHO and EPI are connected by an imine bond. The content of EPI in FA-HES-EPI is determined by UV-visible spectrophotometry, and the grafting rate of EPI is calculated to be 0.10%.

[0050] Prescription 2: Weigh EPI and FA-HES-CHO so that the molar ratio of EPI to the aldehyde group in FA-HES-CHO is 5:1, react in DMSO at 55°C for 24 hours to prepare FA-HES-EPI, at which time FA-HES-CHO and EPI are connected by an imine bond. The content of EPI in FA-HES-EPI is determined by UV-visible spectrophotometry, and the grafting rate of EPI is calculated to be 2.20%.

[0051] Prescription 3: Weigh EPI and FA-HES-CHO so that the molar ratio of EPI to the aldehyde group in FA-HES-CHO is 5:1, react in DMSO at 80°C for 24 hours to prepare FA-HES-EPI, at which time FA-HES-CHO and EPI are connected by an imine bond. The content of EPI in FA-HES-EPI is determined by UV-visible spectrophotometry, and the grafting rate of EPI is calculated to be 5.06%.

[0052] Prescription 4: Weigh EPI and FA-HES-CHO so that the molar ratio of EPI to the aldehyde group in FA-HES-CHO is 10:1, react in DMSO at 80°C for 24 hours to prepare FA-HES-EPI, at which time FA-HES-CHO and EPI are connected by an imine bond. The content of EPI in FA-HES-EPI is determined by UV-visible spectrophotometry, and the grafting rate of EPI is calculated to be 4.45%.

[0053] Prescription 5: Weigh EPI and FA-HES-CHO so that the molar ratio of EPI to the aldehyde group in FA-HES-CHO is 20:1, react in DMSO at 80°C for 24 hours to prepare FA-HES-EPI. At this time, FA-HES-CHO and EPI are connected by an imine bond. The content of EPI in FA-HES-EPI is determined by UV-visible spectrophotometry, and the grafting rate of EPI is calculated to be 9.60%. The preparation process is as follows Figure 1 shown.

[0054] Example 2 Preparation of Active Targeting Hydroxyethyl Starch Epirubicin Hydrazone Bonded Drug (FA-HES-EPI)

[0055] Take 2 g of HES (130 / 0.4) and dissolve it in 25 mL of water. Take 0.037 g of NaIO4 and dissolve it in 4 mL of water. Then add the NaIO4 solution to the HES aqueous solution and stir the reaction overnight. Then add 5 mL of glycerol to the reaction solution to terminate the reaction. The reaction solution is transferred to a dialysis bag, dialyzed against water for 24 hours to remove small molecular impurities, and freeze-dried to obtain HES-CHO.

[0056] FA (500 mg), EDC (118.5 mg) and DMAP (52.2 mg) were mixed in 24 mL of anhydrous DMSO and stirred at 30°C for 30 min; then HES-CHO (1.0217 g) was dissolved in anhydrous DMSO (50 mL) and mixed with the above solution, stirred at room temperature for 48 h to obtain FA-HES-CHO; after the reaction, the precipitate was removed by filtration, and the filtrate was transferred to a dialysis bag, dialyzed with PBS for 3 days to remove impurities, and then dialyzed against distilled water for 2 days to remove salt; after the dialysis was completed, the unreacted FA was removed by centrifugation; the solution was freeze-dried to obtain a FA-HES-CHO freeze-dried product for use. The FA content in FA-HES-CHO was determined by UV-visible spectrophotometry, and the FA grafting rate was calculated to be 23.01%

[0057] FA-HES-CHO (56.2 mg) and adipic acid dihydrazide (ADH) (10 mg) were weighed and dissolved in DMSO (19 mL), and reacted at 80°C for 36 h to obtain a FA-HES-ADH reaction solution, which was added dropwise to methanol, centrifuged, and the supernatant was discarded to remove unreacted ADH. The precipitate was repeatedly washed with methanol, and the methanol was removed by vacuum drying (40°C, 12 h) to obtain a FA-HES-ADH precipitate.

[0058] Weigh epirubicin hydrochloride (EPI·HCl) (117 mg), dissolve it in DMSO (8 mL), add 30 μL diethylamine to prepare epirubicin free base (EPI), add it to the FA-HES-ADH reaction solution generated in the previous step, stir the reaction at 55°C for 72 hours to obtain FA-HES-EPI prodrug solution, add the reaction solution dropwise to methanol 10 times the volume of the reaction solution, centrifuge, discard the supernatant, and remove the unreacted EPI. The precipitate is repeatedly washed with methanol, and the methanol is removed by vacuum drying (40°C, 12 hours) to prepare FA-HES-EPI. At this time, FA-HES-ADH and EPI are connected by a hydrazone bond. The content of EPI in FA-HES-EPI is determined by UV-visible spectrophotometry, and the grafting rate of EPI is calculated to be 13.81%. The preparation process is as follows Figure 2 shown.

[0059] Example 3 Investigation of organic solvent types for the preparation of FA-HES-EPI / IMQ polymer micelles

[0060] FA-HES-EPI prodrug (10.2 mg) was weighed and dissolved in DMSO (1 mL), and IMQ (2 mg) was weighed and dissolved in DMSO (1 mL) (the mass ratio of drug to carrier was about 1:5). The DMSO solutions of the two were mixed, stirred at 40°C for 5 minutes, and then added to 10 mL of water and stirred at 45°C for 10 minutes to allow the prodrug carrier to self-assemble and encapsulate imiquimod (IMQ) to form micelles. DMSO was removed by dialysis, and PBS solution with pH 7.4 was used as the elution solvent. The micelles were passed through a dextran gel-filled column to remove free IMQ, and functional polymer micelles (FA-HES-EPI / IMQ) for co-delivery of chemotherapeutic drugs and immunostimulants were prepared, and the particle size and polydispersity index (PDI) were detected by dynamic light scattering (DLS).

[0061] Keeping the other experimental conditions unchanged, the organic solvent was changed to dimethylacetamide (DMAC), N,N-dimethylformamide (DMF), and N-methylpyrrolidone (NMP) to prepare FA-HES-EPI / IMQ polymer micelles, and the particle size and PDI were detected by DLS method. The results are shown in Figure 3 shown.

[0062] Example 4 Preparation of FA-HES-EPI / IMQ polymer micelles: Investigation of the ratio of organic solvent to water

[0063] Weigh FA-HES-EPI prodrug (10.2 mg) and dissolve it in DMSO (1 mL). Weigh IMQ (2 mg) and dissolve it in DMSO (1 mL) (the drug and carrier mass ratio is about 1:5). Mix the DMSO solutions of the two, stir at 40°C for 5 min, add them to 10 mL of water, and stir at 45°C for 10 min to allow the prodrug carrier to self-assemble and encapsulate IMQ to form micelles. Dialysis was performed to remove DMSO, and PBS with pH 7.4 was used as the elution solvent. The micelles were passed through a dextran gel column to remove free IMQ to obtain FA-HES-EPI / IMQ polymer micelles. The particle size and PDI were detected by DLS.

[0064] Keeping the other experimental conditions unchanged, the ratio of organic solvent to water was changed to 1:2 and 1:1 respectively to prepare FA-HES-EPI / IMQ polymer micelles, and the particle size and PDI were measured by DLS method. The results are as follows Figure 4 shown.

[0065] Example 5 Temperature Investigation of Preparation of FA-HES-EPI / IMQ Polymer Micelle

[0066] Weigh FA-HES-EPI prodrug (10.2 mg) and dissolve it in DMSO (1 mL). Weigh IMQ (2 mg) and dissolve it in DMSO (1 mL) (the drug and carrier mass ratio is about 1:5). Mix the DMSO solutions of the two and stir them at 40°C for 5 minutes. Then add them to 10 mL of water and stir them at 20°C and 45°C for 10 minutes respectively to allow the prodrug carrier to self-assemble and encapsulate IMQ to form micelles. Dialysis was used to remove DMSO. PBS with pH 7.4 was used as the elution solvent. The micelles were passed through a dextran gel-filled column to remove free IMQ to obtain FA-HES-EPI / IMQ polymer micelles. The particle size and PDI were detected by DLS method. The results are as follows: Figure 5 shown.

[0067] Example 6 Preparation and Characterization of FA-HES-EPI / IMQ Polymer Micelle

[0068] Take 2g of HES (130 / 0.4) and dissolve it in 25mL of water, take 0.037g of NaIO4 and dissolve it in 4mL of water, then add sodium periodate solution into the HES aqueous solution, stir and react overnight; then add 5mL of glycerol to the reaction solution to terminate the reaction, transfer the reaction solution to a dialysis bag, dialyze against water for 24h to remove small molecular impurities, and freeze-dry to obtain HES-CHO.

[0069] FA (500 mg), EDC (118.5 mg) and DMAP (52.2 mg) were mixed in 24 mL of anhydrous DMSO and stirred at 30°C for 30 min. HES-CHO (1.0217 g) was then dissolved in anhydrous DMSO (50 mL) and mixed with the above solution. The mixture was stirred at room temperature for 48 h to obtain FA-HES-CHO. After the reaction, the precipitate was removed by filtration, and the filtrate was transferred to a dialysis bag and dialyzed with PBS at 37°C for 3 days to remove impurities, followed by dialysis against distilled water for 2 days to remove salt. After the dialysis was completed, the unreacted FA was removed by centrifugation. The solution was freeze-dried to obtain FA-HES-CHO.

[0070] FA-HES-CHO (56.2 mg) and ADH (10 mg) were weighed and dissolved in DMSO (19 mL), and reacted at 80°C for 36 h to obtain a FA-HES-ADH reaction solution, which was added dropwise to methanol, centrifuged, and the supernatant was discarded to remove unreacted ADH. The precipitate was repeatedly washed with methanol, and the methanol was removed by vacuum drying to obtain a FA-HES-ADH precipitate.

[0071] Weigh EPI·HCl (117 mg), dissolve it in DMSO (8 mL), add 30 μL of diethylamine to prepare EPI, add it to the FA-HES-ADH reaction solution generated in the previous step, stir and react at 55°C in the dark for 72 hours to obtain FA-HES-EPI prodrug solution, add the reaction solution to methanol 10 times the volume of the reaction solution, centrifuge, discard the supernatant, remove the unreacted EPI. The precipitate is repeatedly washed with methanol, and the methanol is removed by vacuum drying to prepare FA-HES-EPI.

[0072] FA-HES-EPI prodrug (10.2 mg) was weighed and dissolved in DMSO (1 mL), and IMQ (2 mg) was weighed and dissolved in DMSO (1 mL) (the drug and carrier mass ratio was about 1:5). The DMSO solutions of the two were mixed, stirred at 40°C for 5 minutes, and then added to 10 mL of water and stirred at 45°C for 10 minutes to allow the prodrug carrier to self-assemble and encapsulate IMQ to form micelles. DMSO was removed by dialysis, and free IMQ was removed by passing the micelles through a dextran gel-filled column using PBS at pH 7.4 as the elution solvent to obtain FA-HES-EPI / IMQ polymer micelles.

[0073] FA-HES-EPI / IMQ polymer micelles were observed by TEM and showed a spherical appearance ( Figure 6 The particle size of FA-HES-EPI / IMQ micelles measured by DLS was 95.31±1.690nm and PDI was 0.136±0.01( Figure 7 ).

[0074] The EPI drug loading of FA-HES-EPI / IMQ micelles was determined by UV-visible spectrophotometry to be 20.5±2.50%, the IMQ drug loading was determined by HPLC to be 0.98±0.34%, and the encapsulation efficiency was 8.8±1.2%.

[0075] FA-HES-EPI / IMQ micelles were diluted with PBS. The results showed that the particle size and PDI did not change significantly after the micelles were diluted 10 times, 60 times, and 100 times ( Figure 8 ), indicating that they all have good anti-dilution ability.

[0076] The in vitro release of FA-HES-EPI / IMQ micelles in PBS release media at pH 5.0, pH 6.5, and pH 7.4 was studied by dialysis. Fig. 9 and Fig.10It can be seen that the cumulative release of EPI and IMQ in pH 5.0 and pH 6.5 media is significantly higher than that in pH 7.4 media. In summary, it can be proved that FA-HES-EPI / IMQ micelles have good pH responsiveness.

[0077] Example 7 Antitumor effect of FA-HES-EPI / IMQ polymer micelles in a rat orthotopic bladder cancer model and its effect on the repolarization of M2 macrophages

[0078] Female SD rats with orthotopic bladder cancer were randomly divided into 4 groups (n=5) and bladder instilled with normal saline, EPI solution, FA-HES-EPI micelles and FA-HES-EPI / IMQ micelles (the instillation dose was the bladder filling volume, and the EPI equivalent dose was 4 mg / kg), respectively; the bladder instillation was performed once a week for a total of 6 times.

[0079] The effect of micelles on tumor growth inhibition was evaluated by measuring and comparing the size of tumor tissue. One week after the sixth administration, all rats were killed, the bladders were removed and the tumors were dissected out, photographed, and the tumor size was measured and weighed. The representative bladder tumors of the control group and the experimental group are shown in Figure 2. Fig.11 As shown in A (black circle), saline had no inhibitory effect on tumor growth, EPI solution could slightly inhibit tumor growth, and FA-HES-EPI / IMQ showed the strongest inhibitory effect on tumor growth among all groups. Fig.11 As shown in B, the tumor volume and weight of the FA-HES-EPI / IMQ group were significantly reduced, which further verified the relatively superior in vivo antitumor effect of FA-HES-EPI / IMQ.

[0080] 24 hours after the sixth administration, tumor tissues were collected, fixed, dehydrated, and sliced; EPI signals were detected using a 488 / 590 nm excitation / emission filter. Fig.12 As shown in A, immunofluorescence staining of the entire tumor section showed that strong and uniform EPI fluorescence signals were observed in FA-HES-EPI and FA-HES-EPI / IMQ; Fig.12 As shown in Figure B, the average fluorescence intensities of the FA-HES-EPI group and the FA-HES-EPI / IMQ group were 1.45 times and 1.51 times that of the free EPI group, respectively. That is, due to the interaction between FA ligands and receptors, the intratumoral retention time of FA-HES-EPI and FA-HES-EPI / IMQ micelles was prolonged, while free EPI escaped into the urine after administration, indicating that the targeting of FA-modified micelles to tumors was enhanced.

[0081] M0 macrophage markers include CD68 and F4 / 80; M1 macrophage markers include inos, CD86, and CD80; M2 macrophage markers include CD163 and CD206. Therefore, by observing the number of macrophage markers, the proportion of tumor-associated macrophages can be examined, and then the effect of micelles on macrophage repolarization can be evaluated. The results of flow cytometry showed that ( Fig.13 ), the pink fluorescence of the FA-HES-EPI / IMQ group was enhanced and the red fluorescence was weakened, showing a trend of increased expression of M1 macrophages and decreased expression of M2 macrophages. This is due to the immunomodulatory effect of the immunostimulant IMQ, which causes M2 macrophages to transform into M1 macrophages, increases the density of M1-like macrophages in the tumor microenvironment, and activates the macrophage-mediated anti-tumor immune effect, thereby achieving a better tumor inhibition effect.

[0082] According to the results of previous studies, EPI will enter the blood circulation during perfusion therapy, so there is a possibility of systemic toxicity, and the in vivo safety needs to be evaluated at the end of treatment. Therefore, the in vivo safety of FA-HES-EPI / IMQ micelles can be evaluated by the results of H&E staining of mouse body weight and major organs. The results showed that the body weight of rats treated with FA-HES-EPI micelles and FA-HES-EPI / IMQ micelles had a certain upward trend, which shows that the drug-loaded micelles have a certain safety ( Fig.14 ). H&E staining results of major organs can be seen ( Fig.15 ), compared with the control group, the liver tissue in the free EPI group was disordered, inflammatory cells infiltrated, liver cells showed necrosis, edema and nuclear membrane fragmentation; and the kidney tissue in the free EPI group showed glomerular necrosis, atrophy and basement membrane destruction, indicating that the free drug has certain hepatotoxicity; while no obvious organ damage was observed in the FA-HES-EPI and FA-HES-EPI / IMQ groups, indicating that the micelle preparation has good biocompatibility. This is due to the fact that the prodrug micelle can reduce the plasma exposure of the drug.

Claims

1. A functional polymer micelle for co-delivery of chemotherapeutic drugs and immunostimulants, characterized in that: The micelle uses folic acid-hydroxyethyl starch-epirubicin polymer prodrug as a carrier, and the immunostimulant is encapsulated in the micelle.

2. The micelle according to claim 1, characterized in that: The micelle is formed by self-assembly of folic acid-hydroxyethyl starch-epirubicin prodrug and immunostimulant.

3. The micelle according to claim 1 or 2, characterized in that: The immunostimulant is imiquimod, resiquimod or resiquimod.

4. The micelle according to claim 1, characterized in that: The drug loading of the immunostimulant is 0% to 30%, excluding 0.

5. The micelle according to claim 1, characterized in that: In the folic acid-hydroxyethyl starch-epirubicin polymer prodrug, hydroxyethyl starch is connected to epirubicin via a pH-sensitive chemical bond, and the grafting rate of epirubicin is 0% to 30%, excluding 0; folic acid is connected to hydroxyethyl starch via an ester bond, and the grafting rate of folic acid is 10% to 50%.

6. The micelle according to claim 1, characterized in that: The micelle is spherical, has an average particle size of ≤200.0 nm, and a PDI of ≤0.

30.

7. The method for preparing the functional polymer micelles for co-delivery of chemotherapeutic drugs and immunostimulants according to claim 1, characterized in that: Synthesize folic acid-hydroxyethyl starch-epiracin prodrug; dissolve the obtained prodrug and immunostimulant in organic solvents to obtain prodrug solution and immunostimulant solution respectively, and then mix the two to obtain drug solution; add the drug solution to water, heat and stir, so that the prodrug encapsulates the immunostimulant and self-assembles to form micelles.

8. Use of the functional polymer micelles for co-delivering chemotherapeutic drugs and immunostimulants according to claim 1 in the preparation of drugs for treating bladder cancer.

9. Use of the functional polymer micelles for co-delivering chemotherapeutic drugs and immunostimulants according to claim 1 in the preparation of bladder instillation drugs for treating bladder cancer.

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