Polymer prodrug and preparation method and application of TPP-DOX-loaded nano-micelle of polymer prodrug

Abstract

According to the invention, a polymer prodrug is designed and prepared, and a dasatinib (DAS) and triphenylphosphine-adriamycin (TPP-DOX, TD) co-loaded nano-micelle is constructed; the preparation method has the advantages that 1) the optimal preparation process of the polymer micelle is determined by a single factor investigation method, namely the optimal nano-micelle is prepared by using a dialysis method and taking DMF (Dimethyl Formamide) as a solvent according to the mass ratio of a prodrug material to a carried drug being 15: 1, and the preparation method is simple and clear and is easy to industrialize; 2) the particle size is small and uniform, so that the nanoparticles can be enriched at a tumor part through an EPR effect; 3) the amphiphilic micromolecule prodrug improves the water solubility of DAS, improves the drug stability, has high drug loading capacity, and avoids adverse reactions possibly caused by carriers and auxiliary materials; and 4) the polymer nano-micelle co-loaded with the DAS and the TPP-DOX has good MMP-2 sensitive responsiveness, enhances the drug targeting delivery capability, has good blood compatibility and safety, improves the drug curative effect, reduces the toxic and side effects, and has a good tumor MDR reversing effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations and new dosage forms, and particularly relates to a matrix metalloproteinase 2 (MMP-2) sensitive amphiphilic block polymer prodrug, a preparation method thereof, and a construction method and application of a TPP-DOX co-loaded nanomicelle thereof. Background Art

[0002] Cancer is a major disease threatening human health, and its clinical treatment mainly relies on chemotherapy. However, long-term chemotherapy not only causes serious toxic and side effects, but also triggers other tissue lesions in the patient's body, and long-term medication is extremely likely to cause tumor cells to develop multidrug resistance (MDR) to therapeutic drugs, resulting in the failure of tumor treatment. Most cancer treatment drugs have problems such as extremely poor solubility, low stability, and certain toxicity, making it difficult for the drugs to play their due anti-cancer role in clinical treatment.

[0003] For example, doxorubicin (DOX) is an antitumor antibiotic with strong antitumor activity and has been widely used in the treatment of various malignant tumors. However, it lacks selectivity, has large toxic and side effects, and has problems such as low solubility and low bioavailability. Therefore, the clinical application of DOX is greatly restricted. Dasatinib (DAS) is a potent multi-target tyrosine kinase inhibitor (TKI) that can specifically act on cytokines such as BCR-ABL, Src, c-Kit, and VEGFR, block downstream cell pathways, inhibit tumor neovascularization, migration, and promote tumor vessel normalization. The metabolism and its function of mitochondria are indispensable in the occurrence and development of tumors, which makes the metabolism and its function of mitochondria gradually become a reasonable target for anti-cancer treatment. At the same time, MMP-2, also known as gelatinase A, can exhibit different degrees of effects under physiological or pathological conditions. Therefore, designing some substrate peptides that respond to MMP-2 in human tissue cells in the nano-drug carrier can enhance the excellent characteristics of targeted drug release of the nano-drug carrier.

[0004] Therefore, based on the characteristics of the site-specific drug release of MMP-2 sensitive peptides, the anti-tumor angiogenesis effect of DAS, and the mitochondrial targeting function of triphenylphosphine-doxorubicin (TPP-DOX, TD), on the basis of the previously synthesized material mPEG-DCA amphiphilic block copolymer, using MMP-2 sensitive peptides as a bridge, dasatinib was grafted through an esterification reaction to construct an MMP-2 sensitive amphiphilic block copolymer prodrug mPEG-DCA-SA-peptide-DAS (MDC-p-D). The MMP-2 responsiveness of the polymeric prodrug was investigated, and the nano-micelles loaded with DAS and TPP-DOX, TPP-DOX / MDC-p-D, were prepared and their in vitro and in vivo activities were studied. This invention is of great necessity, feasibility and innovative significance for improving the therapeutic effect of tumors, achieving a better effect of reversing tumor MDR and anti-tumor activity, etc.

[0005] Based on this, this application was developed. Summary of the Invention

[0006] The object of the present invention is to overcome the defects of the prior art and provide an MMP-2 sensitive amphiphilic block polymer prodrug (mPEG-DCA-SA-peptide-DAS, abbreviated as MDC-p-D). The prodrug is prepared into an MMP-2 sensitive polymeric nano-micelle co-loaded with DAS and loaded with the mitochondrial targeting drug TPP-DOX, so as to realize the combined use of the two drugs, enhance the targeted delivery of the drugs, and have the effects of high drug loading, good stability and low toxicity and side effects, thereby reducing tumor multi-drug resistance.

[0007] The present invention designed and synthesized a polymeric prodrug carrier (MDC-p-D) with MMP-2 sensitivity, constructed a nano-micelle drug delivery system (TPP-DOX / MDC-p-D) co-loaded with DAS and loaded with a highly hydrophobic mitochondrial targeting anti-cancer drug (TPP-DOX), investigated the stability, hemolysis rate, MMP-2 sensitivity and in vitro release rate of the micelles, and conducted in vitro and in vivo anti-tumor studies on the nano-micelles co-loaded with DAS and TPP-DOX, investigated their biosafety, and compared the drug resistance, cytotoxicity, mitochondrial targeting and anti-tumor activity of different nano-micelles.

[0008] The present invention realizes the above object through the following technical solutions: A polymeric prodrug, which comprises an amphiphilic block copolymer PEG-DCA-SA-peptide-DAS (MDC-p-D) with MMP-2 sensitivity and a polymeric prodrug PEG-DCA-SA-DAS (MDCD) without an MMP-2 sensitive response peptide. The structural formula is as follows: ; In the formula, R = CH2-CH2 or CH2-CH2-COO-peptide, and the sequence of the peptide is: Gly-Pro-Leu-Gly-Ala, Gly-Leu-Gly-Cys, Pro-Leu-Ser-Ala, Gly-Pro-Gly-Val-Lys, His-Gly-Ile-Ala, or Pro-Leu-Gly-Val-Gln.

[0009] In the present invention, abbreviations and chemical names of some compounds are as follows: HOBt: 1-hydroxybenzotriazole; EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; DMAP: 4-dimethylaminopyridine. DMF refers to N,N-dimethylformamide.

[0010] As one option, the preparation method of the MMP-2 sensitive polymer prodrug (PEG-DCA-SA-peptide-DAS, MDC-p-D) includes the following steps: 1) Under an inert gas atmosphere, dissolve PEG-DCA-SA (MDC) and HOBt in redistilled DMF, and stir and react at room temperature for 0.5 - 2 h; under ice bath conditions, add the DMF solution containing peptide and EDCI, then add triethylamine (Et3N), and continue to react at room temperature for 24 - 48 h. After the reaction is completed, dialyze and freeze-dry to obtain a white solid product PEG-DCA-SA-peptide (MDC-p); 2) Under an inert gas atmosphere, dissolve the synthesized PEG-DCA-SA-peptide and DMAP in redistilled DMF, and stir and activate at room temperature for 0.5 - 2 h; under ice bath conditions, add the DMF solution containing the drug and EDCI, then add triethylamine, and raise the temperature to 35 - 60 °C and continue to react for 24 - 48 h. After the reaction is completed, dialyze and freeze-dry to obtain a white solid product PEG-DCA-SA-peptide-DAS (MDC-p-D); The drug is dasatinib, gilteritinib, suvoratinib, ibrutinib, acalabrutinib, etc.

[0011] Specifically, in step 1), the molar ratio of PEG-DCA-SA, HOBt, peptide, and EDCI is 1:1-5:0.1-5:1-5; for every 0.1 mmol of PEG-DCA-SA, 0.1-0.5 mL of triethylamine is added. In step 2), the molar ratio of PEG-DCA-SA-peptide, DMAP, drug, and EDCI is 1:1-5:0.5-5:1-5; for every 0.1 mmol of PEG-DCA-SA-peptide, 0.1-0.5 mL of triethylamine is added.

[0012] As another option, the polymer prodrug is a control polymer prodrug without MMP-2 sensitive peptide (PEG-DCA-SA-DAS, MDCD), and it may include the following steps: Under an inert gas atmosphere, dissolve PEG-DCA-SA and DMAP in redistilled DMF, stir and activate at room temperature for 0.5-2 h; add a redistilled DMF solution containing the drug and EDCI under ice bath conditions, then add triethylamine, and continue to react at room temperature for 24-48 h. After the reaction is completed, dialyze and freeze-dry to obtain a white solid product PEG-DCA-SA-DAS (MDCD); The drug is dasatinib, gilteritinib, sunvozertinib, ibrutinib, acalabrutinib, etc.

[0013] Specifically, the molar ratio of PEG-DCA-SA, DMAP, drug, and EDCI is 1:1-5:0.2-2:1-5, and for every 0.1 mmol of PEG-DCA-SA, 0.1-0.5 mL of triethylamine is added.

[0014] The synthetic routes of the above MMP-2 sensitive polymer prodrug (PEG-DCA-SA-peptide-DAS, MDC-p-D) and the control polymer prodrug without MMP-2 sensitive peptide (PEG-DCA-SA-DAS, MDCD) are as shown below: 。

[0015] The present invention provides a preparation method of polymer nanomicelles co-loaded with DAS and loaded with mitochondrial-targeted drug triphenylphosphine-doxorubicin (TPP-DOX, TD). The screening of the nanomicelle preparation method includes ① preparing micelles by the thin film dispersion method, ② preparing micelles by the solvent evaporation method, and ③ preparing micelles by the dialysis method; after comparison, the dialysis method is selected as the method for preparing micelles; it uses the synthesized MMP-2 sensitive polymer prodrug as a nanocarrier to prepare drug-loaded micelles, which specifically includes the following steps: Preparation method of TPP-DOX / MDC-p-D (i.e., TD / MDC-p-D) drug-loaded nanomicelles: Weigh 10 - 20 mg of the MMP-2 sensitive polymeric prodrug MDC-p-D, and another 1 - 5 mg of TPP-DOX. Add the two into 1 - 3 mL of an organic solvent for dissolution, and under high-speed stirring (1600 r / min), dropwise add the solution into a bottle containing 2 - 6 mL of ultrapure water. After stirring for 10 - 20 min, transfer it into a dialysis bag with a molecular weight cut-off not exceeding 1000, and use ultrapure water as the dialysis medium to dialyze for 12 - 24 h to remove the organic solvent in the preparation, thus obtaining the required micelle solution.

[0016] Preparation method of MDC-p-D prodrug nanomicelles: Weigh 10 - 20 mg of the MMP-2 sensitive polymeric prodrug MDC-p-D, and add it into 1 - 3 mL of an organic solvent for dissolution. Under the condition of high-speed stirring (1600 r / min), slowly dropwise add the solution into an eggplant-shaped flask containing 2 - 6 mL of ultrapure water. After high-speed stirring for 10 - 20 min, transfer the solution into a dialysis bag with a molecular weight cut-off not exceeding 1000, and use ultrapure water as the dialysis medium to dialyze for 12 - 24 h to remove the organic solvent in the preparation, finally obtaining the required micelle solution.

[0017] Furthermore, the organic solvent used can be any one of N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone (CH3COCH3), etc. Among them, after screening, DMF is used as the optimal solvent for preparing nanomicelles.

[0018] As a preference, the dialysis method is used for the preparation of the MMP-2 sensitive polymeric nanomicelles co-loaded with DAS and the mitochondrial-targeted drug TPP-DOX. Using DMF as the solvent, the optimal nanomicelles are prepared according to the mass ratio of the MMP-2 sensitive polymeric prodrug to the drug-loading material of 15:1.

[0019] The present invention provides polymeric nanomicelles co-loaded with DAS and the mitochondrial-targeted drug TPP-DOX prepared by the above method.

[0020] The present invention also provides the application of the above polymeric prodrug or the polymeric nanomicelles co-loaded with DAS and the mitochondrial-targeted drug TPP-DOX in a drug delivery system or the preparation of anti-tumor drugs.

[0021] The present invention also provides the application of the above polymeric prodrug or the polymeric nanomicelles co-loaded with DAS and the mitochondrial-targeted drug TPP-DOX in systems such as injection administration, oral administration, or local administration.

[0022] In the present invention, the raw material PEG-DCA-SA used is synthesized through the following steps: Under an inert gas atmosphere, PEG-DCA, succinic anhydride, and DMAP were dispersed in DMF and reacted at room temperature for 12 - 24 h. After the reaction, DMF was removed by rotary evaporation. The substrate was dissolved in dichloromethane, first extracted with 0.1 - 0.5 mol / L hydrochloric acid - dichloromethane solution to remove the aqueous phase, then extracted with saturated brine, dried over anhydrous magnesium sulfate, the solvent was concentrated, precipitated with ether, filtered by suction, and dried in vacuo at 37 °C to obtain the product PEG-DCA-SA.

[0023] In the above preparation process of PEG-DCA-SA, the molar ratio of PEG-DCA, succinic anhydride, and DMAP is 1:1 - 5:1 - 5. Among them, PEG-DCA refers to polyethylene glycol - deoxycholic acid, and its synthesis method can be referred to the description in the Chinese patent "Preparation Method and Application of a Polyethylene Glycol - Deoxycholic Acid and Its Derivatives" (Patent No. ZL 201711223766.6).

[0024] The present invention discloses a polymeric prodrug and its preparation method. The prodrug is realized in two structural forms: one is an MMP-2 sensitive amphiphilic block copolymer prodrug PEG-DCA-SA-peptide-DAS (MDC-p-D), and the other is a control polymeric prodrug PEG-DCA-SA-DAS (MDCD) without MMP-2 sensitive peptide. The preparation methods are respectively based on the successful synthesis of the amphiphilic copolymer polyethylene glycol - deoxycholic acid PEG-DCA grafted with succinic anhydride (PEG-DCA-SA) in the early stage of the laboratory. By controlling different feeding ratios, an MMP-2 sensitive peptide is grafted as a linker to carry out the reaction of grafting drugs such as dasatinib onto PEG-DCA-SA to synthesize the polymeric prodrug PEG-DCA-SA-peptide-DAS (MDC-p-D) containing MMP-2 sensitive peptide; using drugs such as dasatinib as the substrate and DMAP as the catalyst to react with PEG-DCA-SA to synthesize PEG-DCA-SA-DAS (MDCD). This prodrug system has the advantages of tumor-targeted release characteristics and structural stability. By using the previously synthesized amphiphilic block copolymer PEG-DCA to improve the water solubility and bioavailability of drugs such as dasatinib, and at the same time, taking advantage of the different characteristics of MMP-2 under different physiological or pathological conditions, the MMP-2 sensitive peptide is designed as a linker in the nano-drug carrier, which weakens its first-pass effect in the liver while enhancing its anti-tumor activity in vivo and improving the therapeutic effect.

[0025] The polymer prodrug of the present invention was first prepared, and the DAS and TPP-DOX co-loaded nanomicelles were first discovered and constructed. The advantages of this nano-drug delivery system are as follows: (1) Through the single-factor investigation method, the optimal preparation process of the polymer micelles was determined to be the dialysis method, using DMF as the solvent, and preparing the optimal nanomicelles according to the mass ratio of the prodrug material to the loaded drug of 15:1. The production method is simple and clear, and is easy to industrialize; (2) The particle size is small and uniform, which is beneficial for the nanoparticles to accumulate in the tumor site through the EPR effect; (3) The amphiphilic small molecule prodrug improves the water solubility of DAS, enhances the drug stability, has a high drug loading capacity, and avoids the adverse reactions that may be caused by the carrier and excipients; (4) The MMP-2 sensitive polymer nanomicelles co-loaded with DAS and TPP-DOX have good MMP-2 sensitive responsiveness, enhance the targeted drug delivery, have good blood compatibility and safety, improve the drug efficacy and reduce the toxic and side effects, and have a good effect of reversing tumor MDR.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. Designed and synthesized an amphiphilic block polymer prodrug, and the synthesis method is simple and feasible; 2. Through comparison and screening, it was finally determined to use the dialysis method, using DMF as the solvent, and preparing the optimal nanomicelles according to the mass ratio of the prodrug material to the drug-loading material of 15:1. The particle size is uniform and small, which improves the drug loading capacity and drug stability, and improves the hemolytic property and biological safety of the drug; 3. Prepared polymer nanomicelles co-loaded with DAS and TPP-DOX. The preparation method is simple and feasible. The released TPP-DOX and DAS play a synergistic effect, and the inhibitory effect of the material MD on extracellular efflux enhances the anti-tumor effect of the drug; 4. The polymer nanomicelles co-loaded with DAS and TPP-DOX utilize the bond-breaking mechanism of the MMP-2 sensitive peptide in the environment with high expression of MMP-2 to achieve the co-delivery of the two drugs, which not only improves the bioavailability of the tumor treatment drug but also reduces its toxic and side effects; 5. The nanomicelles have good MMP-2 sensitive responsiveness and mitochondrial targeting. The MMP-2 sensitive TPP-DOX / MDC-p-D micelles are depolymerized under the action of MMP-2 highly expressed in the tumor extracellular matrix. The released TPP-DOX and DAS play a synergistic effect, and with the assistance of the material MD, the drug uptake by cells increases, and it has a good effect of reversing tumor MDR. Description of the Drawings

[0027] Figure 1 1H NMR of PEG-DCA-SA-peptide prepared in Example 1 1 1H NMR; Figure 2 1H NMR of PEG-DCA-SA-peptide-DAS prepared in Example 1 1 1H NMR Figure 3 1H NMR of PEG-DCA-SA-DAS prepared in Example 2 1 1H NMR Figure 4 FT-IR spectra of PEG-DCA-SA-peptide (left) and PEG-DCA-SA-peptide-DAS (right) prepared in Example 1 of the present invention Figure 5 FT-IR spectrum of PEG-DCA-SA-DAS (MDCD, labeled as MDC-DAS in the figure) prepared in Example 2 Figure 6 Particle size distributions of micelles MDCD, MDC-p-D, TD / MDCD, and TD / MDC-p-D Figure 7 Zeta potential map of micelle MDCD Figure 8 Zeta potential map of micelle MDC-p-D Figure 9 Zeta potential map of micelle TD / MDCD Figure 10 Zeta potential map of micelle TD / MDC-p-D Figure 11 Particle size changes of different polymeric micelles stored at room temperature and 4 °C for 35 days Figure 12 In vitro release of DAS from drug-loaded micelles TD / MDCD and TD / MDC-p-D Figure 13 In vitro release of TPP-DOX from drug-loaded micelles TD / MDCD and TD / MDC-p-D Figure 14 In vitro release of DAS from prodrug micelles MDCD and MDC-p-D Figure 15 Cell viability of different drugs on human breast cancer cells MCF-7 after 24 h (left) and 48 h (right) of treatment Figure 16 Cell viability of different drugs on human breast cancer drug-resistant cells MCF-7 / ADR after 24 h (left) and 48 h (right) of treatment Figure 17 Tumor volume changes in tumor-bearing mice treated with different drugs Detailed implementation manners

[0028] The technical solution of the present invention will be further introduced in detail below in combination with embodiments, but the protection scope of the present invention is not limited thereto.

[0029] In the following embodiments, the raw materials used are all ordinary commercially available products that can be directly purchased or can be prepared by using conventional techniques in the art.

[0030] Room temperature refers to 25 ± 5°C.

[0031] In the following embodiments, the raw material PEG-DCA-SA is synthesized through the following steps: Weigh 0.7 mmol of PEG-DCA, 3.5 mmol of succinic anhydride, and 0.8 mmol of DMAP into a three-necked flask containing 40 mL of DMF. Under a nitrogen atmosphere at a rotation speed of 600 r / min, react at room temperature for 18 h. After the reaction is completed, remove DMF by rotary evaporation under reduced pressure; then dissolve the substrate with 3 mL of dichloromethane. First, extract twice with 0.1 mol / L hydrochloric acid-dichloromethane solution to remove the aqueous phase, and then extract twice with saturated brine. Dry the organic phase with anhydrous magnesium sulfate, concentrate the solvent, precipitate with ether, filter by suction, and dry in vacuo at 37°C to obtain the product PEG-DCA-SA.

[0032] The preparation method of 0.1 mol / L hydrochloric acid-dichloromethane solution is specifically as follows: Measure about 9 mL of concentrated hydrochloric acid (concentration about 12 mol / L), slowly inject it into 900 mL of water, shake well, and then dilute with water to 1000 mL.

[0033] Among them, PEG-DCA refers to polyethylene glycol-deoxycholic acid, and its synthesis method can be referred to the description in the Chinese patent "A Preparation Method and Application of Polyethylene Glycol-Deoxycholic Acid and Its Derivatives" (Patent No. ZL 201711223766.6).

[0034] Example 1 The synthesis method of the MMP-2 sensitive polymer prodrug (PEG-DCA-SA-peptide-DAS, MDC-p-D) provided in this example includes the following steps: 1) Weigh 0.2 mmol of PEG-DCA-SA (MDC) and 0.4 mmol of HOBt and place them in a three-necked flask. Add 4 mL of redistilled DMF and stir to dissolve at 600 r / min at room temperature. Then stir and react for 0.5 h. Weigh 0.4 mmol of MMP-2 sensitive peptide (Gly-Pro-Leu-Gly-Ala) and 0.4 mmol of EDCI in a vial. After adding 5 mL of DMF and fully dissolving, slowly add the aforementioned reaction solution dropwise under ice bath conditions. Finally, add 0.2 mL of triethylamine (Et3N) to the reaction system, keep the rotation speed unchanged, and continue to react at room temperature for 48 h. Nitrogen protection is carried out throughout the experiment. After the reaction is completed, transfer the reaction solution into a dialysis bag (cut-off molecular weight MW = 2000) for dialysis. First, dialyze with DMF: distilled water (volume ratio about 1:1) for 24 h, then dialyze with pure distilled water for 48 h. Then put the dialyzed reaction solution into the refrigerator, pre-freeze overnight, and freeze-dry the next day to obtain a white solid product PEG-DCA-SA-peptide (MDC-p).

[0035] 2) Weigh 0.1 mmol of the above-synthesized PEG-DCA-SA-peptide and 0.3 mmol of DMAP in an eggplant-shaped flask. Add 4 mL of redistilled DMF and stir to activate at 600 r / min at room temperature for 30 min while carrying out nitrogen protection. After 0.5 h, weigh 0.3 mmol of dasatinib (DAS) and 0.3 mmol of EDCI in a vial. After adding 5 mL of DMF and fully dissolving, slowly add the aforementioned reaction solution dropwise at 0 °C under ice bath conditions. Finally, add 0.2 mL of Et3N dropwise, raise the temperature to 35 °C and continue to react for 48 h. Nitrogen protection is carried out throughout the experiment. After the reaction is completed, transfer the reaction solution into a dialysis bag (cut-off molecular weight MW = 2000) for dialysis. First, dialyze with DMF: distilled water (volume ratio about 1:1) for 24 h, then dialyze with pure distilled water for 48 h. Then put the dialyzed reaction solution into the refrigerator, pre-freeze overnight, and freeze-dry the next day to finally obtain a white solid product PEG-DCA-SA-peptide-DAS (MDC-p-D), as follows.

[0036] Example 2 This example provides a synthesis method of a control polymer prodrug (PEG-DCA-SA-DAS, MDCD) without MMP-2 sensitive peptide, including the following steps: Weigh 0.1 mmol of PEG-DCA-SA (MDC) and 0.2 mmol of DMAP into a 25 mL eggplant-shaped flask, add 4 mL of redistilled DMF and dissolve it under nitrogen protection. Stir and activate at a speed of 600 r / min at room temperature for 30 min. Weigh 0.3 mmol of DAS and 0.2 mmol of EDCI into a vial in sequence, add 6 mL of redistilled DMF and dissolve it fully, then add it dropwise to the aforementioned eggplant-shaped flask containing the reaction solution under an ice bath. Finally, add 0.2 mL of Et3N, keep the rotation speed unchanged, and continue the reaction at room temperature for 48 h. Nitrogen protection is carried out throughout the experiment. After the reaction is completed, transfer the reaction solution into a dialysis bag (cut-off molecular weight MW = 2000) for dialysis. First, dialyze with DMF: distilled water (volume ratio about 1:1) for 24 h, then dialyze with pure distilled water for 48 h. Then, put the dialyzed reaction solution into the refrigerator, pre-freeze overnight, and freeze-dry the next day to finally obtain a pale yellow solid product PEG-DCA-SA-DAS (MDCD), as follows.

[0037] Example 3 The preparation method of the MMP-2 sensitive polymer nanomicelles co-loaded with DAS and TPP-DOX provided in this example uses the synthesized MMP-2 sensitive polymer MDC-p-D as a nanocarrier to prepare drug-loaded micelles. Using the dialysis method and DMF as a solvent, the optimal nanomicelles are prepared according to the mass ratio of the prodrug material to the drug-loading material of 15:1. The specific steps are as follows: The preparation method of TPP-DOX / MDC-p-D (i.e., TD / MDC-p-D) drug-loaded nanomicelles: Weigh precisely 15 mg of the MMP-2 sensitive polymer prodrug MDC-p-D, and weigh another 1 mg of TPP-DOX. Add the two to 1 mL of the organic solvent DMF for dissolution. Under the condition of high-speed stirring (1600 r / min), slowly drop it into an eggplant-shaped flask containing 5 mL of ultrapure water. After high-speed stirring for 10 min, transfer it to a dialysis bag (cut-off molecular weight MW = 1000), and use ultrapure water as the dialysis medium to dialyze for 24 h to remove the organic solvent in the preparation. Finally, the required micelle solution is obtained.

[0038] Meanwhile, referring to the above preparation process, the polymer prodrug MDC-p-D was replaced with MDCD, and TPP-DOX / MDCD (i.e., TD / MDCD) drug-loaded nanomicelles were prepared.

[0039] Preparation method of MDC-p-D prodrug nanomicelles: Weigh 15 mg of MMP-2 sensitive polymer prodrug MDC-p-D precisely, and add 1 mL of DMF for dissolution. Under the condition of high-speed stirring (1600 r / min), slowly drop it into an eggplant-shaped flask containing 5 mL of ultrapure water. After high-speed stirring for 10 min, transfer the solution into a dialysis bag (MW = 1000), use ultrapure water as the dialysis medium, and dialyze for 24 h to remove the organic solvent in the preparation, finally obtaining the required micelle solution.

[0040] Meanwhile, referring to the above preparation process, replace the polymer prodrug MDC-p-D with MDCD, and prepare MDCD prodrug nanomicelles.

[0041] Related tests The 1H NMR spectra of the products PEG-DCA-SA-peptide (MDC-p) and PEG-DCA-SA-peptide-DAS (MDC-p-D) prepared in the above examples are as Figure 1 、 Figure 2 shown. In the 1H NMR spectrum of MDC-p, the peaks at positions n, p, and s are the characteristic proton peaks of the amide bond in the MMP-2 sensitive peptide. m and o are the two methyl peaks in the sensitive peptide. The positions of d, e, and f are the characteristic methyl proton peaks in PEG-DCA-SA. The newly emerged proton peak at l is the amide bond proton peak formed by the bonding of the MMP-2 sensitive peptide and PEG-DCA-SA. Moreover, the carboxyl peak (δ 12.45 ppm) at m in PEG-DCA-SA disappears. Therefore, it can be considered that MDC-p is successfully synthesized. In the MDC-p-D spectrum, the peaks at positions b´, c´, d´, u, v, and y are the characteristic proton peaks on the heterocycle in dasatinib. The x and z peaks are the characteristic proton peaks of the amide group on dasatinib. It can be seen from the final spectrum that the characteristic methyl proton peaks such as d, e, f, and o on MDC-p all appear. The peak at t (δ 3.65 ppm) is the methylene proton peak formed by the bonding of MDC-p and dasatinib. Moreover, the characteristic hydroxyl (OH) peak at l (δ 4.5 ppm) in the original dasatinib spectrum disappears significantly. The above proves that PEG-DCA-SA-peptide-DAS (MDC-p-D) is successfully synthesized.

[0042] Figure 3Comparing with the proton nuclear magnetic resonance spectrum of dasatinib, it can be found that at positions m, n, q, t, u, and v are some specific proton peaks on the heterocycle in dasatinib. The positions of p and r are the characteristic peaks of secondary amino group and amide group on dasatinib respectively. The chemical shifts of these characteristic peaks remain unchanged in the product spectrum. While at positions l, o, and s are the methylene and methyl peaks on dasatinib respectively, and their chemical shifts remain unchanged in the product spectrum. Moreover, the hydroxyl peak (-OH) at position l (δ 4.5 ppm) in the original dasatinib spectrum and the carboxyl peak at position m (δ 12.45 ppm) in the PEG-DCA-SA spectrum disappear significantly. Not only that, the peaks of the four-membered ring on PEG-DCA-SA such as g, h, i, j, etc. are all presented in the final spectrum. The above indicates that PEG-DCA-SA-DAS (MDCD) is successfully synthesized.

[0043] The infrared spectra of PEG-DCA-SA-peptide (MDC-p) and PEG-DCA-SA-peptide-DAS (MDC-p-D) prepared in the above examples are as Figure 4 shown. The stretching vibration peaks at 3307 cm -1 and 1664.5 cm -1 are respectively the v -CH2-NH2 , v -CONH stretching vibration peaks in the MMP-2 sensitive peptide. Among them, the stretching vibration peak of the amino group on the peptide ( v -CH2-NH2 , 3307 cm -1 ) disappears significantly in the product; while the stretching vibration peaks at 1200 cm -1 and 1735.4 cm -1 are respectively the v -CN on the sensitive peptide and the v -C=O stretching vibration peak of the keto carbonyl group on the carboxyl or amide group. The stretching vibration peaks at 2952.6 cm -1 and 2881.6 cm -1 are respectively the asymmetric stretching vibration peak of the methyl group ( v -C-H ) and the symmetric stretching vibration peak of the methylene group ( v -CH2- ) in MDC-p, and they are all presented in the infrared spectrum of the product, which fully proves the synthesis of PEG-DCA-SA-peptide. Then, from the spectrum of dasatinib grafted with PEG-DCA-SA-peptide, it can be seen that not only the relevant characteristic peaks on PEG-DCA-SA-peptide appear in the product spectrum, but also the 1500.6 cm -1 and 779.4 cm on the benzene ring of dasatinib-1 The characteristic peaks at and the δ -C-H Bending vibration peak (1200 cm -1 ) is also present, and the symmetrical stretching vibration peak of the hydroxyl group ( v -OH ) also disappeared, which proved the successful synthesis of the amphiphilic polymer prodrug PEG-DCA-SA-peptide-DAS (MDC-pD).

[0044] The infrared spectrum of PEG-DCA-SA-DAS (MDCD) prepared in the above example is as follows: Figure 5 As shown, 2952.6cm -1 、2881.6 cm -1 、1735.4 cm -1 They are the asymmetric stretching vibration peaks of the methyl group in MDC ( v -C-H ), the symmetric stretching vibration peak on the methylene group ( v -CH2- ) and carboxyl or ketone carbonyl on ester bond v -C=O stretching vibration peak, and 1500.6 cm -1 、779.4cm -1 、1194.9 cm -1 、1614.7 cm -1 They are the characteristic peaks of the benzene ring on dasatinib and the bending vibration peaks on the methyl group (δ -C-H ) and the β-NH in-plane bending vibration peaks on the amino or amide groups. These characteristic peaks all appear at the same or similar positions in the FT-IR spectrum of the product, and the hydroxyl group on dasatinib (3214.4 cm -1 ) also disappeared obviously, indicating that PEG-DCA-SA-DAS (MDCD) has been successfully synthesized.

[0045] Particle size and distribution of nanomicelles Take an appropriate amount of the above-prepared MDCD, MDC-pD, TD / MDCD and TD / MDC-pD micelle solutions and place them in a particle size colorimetric cell. Use a laser particle size Zeta potential instrument to measure their particle size and potential. Before using the instrument, turn on the instrument and preheat it for 0.5h, then open the corresponding measurement software on the computer, set the measurement temperature and other detailed parameters, and finally click the start button to measure and record the data. Results are shown in Figures 6 to 10 .

[0046] The particle size and Zeta potential of micelle MDCD, MDC-pD, TD / MDCD and TD / MDC-pD are shown in Figure 2. Figure 6 , 7As shown in Figures 8, 9, and 10. It can be seen from the results in the figures that the particle sizes of MDCD, MDC-p-D, TD / MDCD, and TD / MDC-p-D micelles are approximately 86 nm, 93 nm, 155 nm, and 166 nm respectively. The particle sizes are all relatively small, and the PDI of the particle sizes is less than 0.3, indicating that the particle size distribution is relatively uniform; the Zeta potentials are approximately 7.28 mV, 6.62 mV, 11.3 mV, and 10.6 mV respectively. The micelles are all positively charged, and it is speculated that this may be related to the structure of the dasatinib drug itself. The positive charges carried by the micelles indicate that there is a strong repulsive force between the nano-micelle molecules, and it is speculated that the micelles will have good stability.

[0047] Investigation on the stability of nano-micelles Storage stability The MDCD, MDC-p-D, TD / MDCD, and TD / MDC-p-D micelle solutions were aliquoted into 20 mL vials, with three replicates for each, and stored at 4 °C and room temperature for 35 days. Samples were taken at regular intervals during this period to measure the change in their particle size Size and record it. The results are shown in Figure 11 .

[0048] The particle size changes of MDCD, MDC-p-D, TD / MDCD, and TD / MDC-p-D micelles measured after storing at 4 °C and room temperature for 0, 1, 3, 6, 9, 12, 15, 21, 28, and 35 days are as shown in Figure 11 As shown. It can be seen from the results in the figure that the particle size changes of the four micelles after storing at 4 °C for 22 days are all less than 15 nm; the particle size changes after storing at room temperature for 22 days also fluctuate within 20 nm; this may be due to the fact that the polymer prodrug has a low critical micelle concentration (CMC), so that the four micelles have good stability for a period of time after preparation.

[0049] In vitro release of nano-micelles (1) Preparation of 0.01 M Tris Buffered Saline (TBS): First, weigh 11.1 g of TBS buffer salts and place them in a 1 L volumetric flask. Add ultrapure water to dissolve and make up to the mark to obtain a 0.01 M TBS solution, and adjust the pH to 7.4.

[0050] (2) Prepare 250 mL of 0.01 M TBS buffer with MMP-2 (matrix metalloproteinase-2) concentrations of 0, 1.25 μg / mL, 2.5 μg / mL, and 5 μg / mL respectively as the four release media for the micelles. Then take 20 mL of each and place them in 150 mL high-foot beakers, and reserve the remaining release media. There are 3 parallel samples for each concentration at the four concentrations. Pipette 1 mL of each of the prepared MDCD, MDC-p-D, TD / MDCD, and TD / MDC-p-D polymer micelle solutions into dialysis bags (MW = 2000 Da), tie the two ends of the dialysis bag with a thin string, and then place the dialysis bag in the aforementioned high-foot beaker containing 20 mL of release medium, and quickly place it in a constant temperature incubation shaker at 37 °C with an oscillation speed of 100 rpm for incubation for 48 h. Start timing after putting it in, and take samples at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, and 48 h respectively. The sampling volume each time is 1 mL, and 1 mL of the same fresh release medium is added in time to keep the total volume of the release medium unchanged. Finally, inject the samples into an HPLC instrument to measure the drug content, and finally calculate the cumulative release amounts of DAS and TPP-DOX of the micelles at different MMP-2 concentrations. The results are shown in Figure 12 、 13 and 14.

[0051] Figure 12 In vitro release of DAS from drug-loaded micelles TD / MDCD and TD / MDC-p-D; Figure 13 In vitro release of TPP-DOX from drug-loaded micelles TD / MDCD and TD / MDC-p-D; Figure 14In vitro release of prodrug micelles MDCD and MDC-p-D of DAS. As can be seen from the results in the figure, within 48 h, as time extended, for the polymer drug-loaded nanomicelles TD / MDC-p-D in an environment with MMP-2 enzyme concentrations of 2.5 μg / mL and 5 μg / mL, the cumulative release amounts of drug DAS at 48 h could reach 50% and over 90% respectively, and the cumulative release amounts of drug TPP-DOX could reach 55% and over 90% respectively; in an environment with a low MMP-2 concentration of 1.25 μg / mL, the 48 h cumulative release amounts of DAS and TPP-DOX were only about 30%; when under the condition of no MMP-2, the 48 h cumulative release amounts did not exceed 20%; while for the TD / MDCD micelles, the cumulative release amounts under all conditions were less than 25%. This shows that the release of the drug-loaded micelles TD / MDC-p-D is positively correlated with the MMP-2 concentration, indicating that it has strong MMP-2 sensitive responsiveness, while the release of the drug-loaded micelles TD / MDCD is hardly affected by the MMP-2 concentration, and this result is also consistent with the MMP-2 sensitivity conclusion of the previous nanomicelles. Similarly, in the release results of the polymer prodrug MDCD and MDC-p-D micelles, we found that the MDC-p-D micelles also have good MMP-2 sensitive responsiveness. In an environment with MMP-2 enzyme concentrations of 2.5 μg / mL and 5 μg / mL, the 48 h cumulative release amounts of DAS were both over 50%; when under the conditions of a low MMP-2 concentration of 1.25 μg / mL and no MMP-2, the release behavior of the MDC-p-D micelles was similar to the release results of its drug-loaded micelles, and the release results of the control MDCD micelles were also not much different from the release results of its drug-loaded micelles. This fully shows that the MDC-p-D micelles and their drug-loaded micelles prepared in this study have good MMP-2 sensitive responsiveness, which is consistent with the expected drug release behavior, laying a foundation for DAS and TPP-DOX to target tumors and play an anti-tumor role more efficiently in the later stage.

[0052] Based on the above results, we speculate that in the later in vivo experiments, when the polymer drug-loaded nanomicelles TD / MDC-p-D reach normal tissues, due to the low MMP-2 enzyme concentration in normal tissues, the polymer drug-loaded nanomicelles are released relatively slowly, and most of DAS and TPP-DOX are encapsulated inside the micelles, reducing the damage of anti-cancer drugs to normal tissues. When the polymer drug-loaded nanomicelles reach tumor tissues through blood circulation, due to the high-concentration MMP-2 environment at the tumor site, the polymer drug-loaded nanomicelles depolymerize, and a large amount of drugs are released. Coupled with the pro-angiogenic normalization effect of DAS, it can increase the infiltration amount of TPP-DOX into tumor cells, thus achieving the anti-tumor effect.

[0053] Cytotoxicity Human breast cancer cells MCF-7 in logarithmic growth phase and human breast cancer doxorubicin-resistant cells MCF-7 / ADR cells were evenly seeded into 96-well plates at a density of 6×10 3 cells / well, and cultured in a constant temperature incubator at 37°C and 5% CO2 for 24 h. The original RPMI-1640 medium was aspirated and discarded, and drug-containing media with a series of gradient concentrations (1.9, 3.8, 7.6, 15.2, 30.4 μg / mL) of DAS, TPP-DOX, MDCD, MDC-p-D, TD / MDCD, and TD / MDC-p-D were added, with a volume of 0.1 mL per well. At the same time, a medium without drugs with the same volume was used as a negative control, and a fresh medium without cells was used as a blank control. Six replicate wells were set for each concentration. After incubation in the cell constant temperature incubator for 24 h and 48 h respectively, the old medium was aspirated and discarded, 10 μL of MTT solution (5 mg / mL) was added, and incubation was continued in the incubator. After 4 h, the supernatant was discarded, 0.1 mL of DMSO was added to each well, and the mixture was shaken vigorously to completely dissolve the crystals. Then, the OD value of each well at 570 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate of each group was calculated. The results are shown in Figure 15 and 16 .

[0054] As Figure 15 and 16As shown, it presents the cell survival rates after the action of free drug DAS solutions and free drug TPP-DOX solutions at different concentrations, TD / MDC-p-D and TD / MDCD drug-loaded micelle solutions, MDCD and MDC-p-D prodrug micelle solutions on MCF-7 and MCF-7 / ADR cells for 24 h and 48 h. From the results in the figure, it can be seen that the toxic effects of each group of drugs on MCF-7 and MCF-7 / ADR cells gradually increase with the increase in drug concentration. Among them, after the action of the TPP-DOX free drug group on MCF-7 and MCF-7 / ADR cells for 24 h and 48 h, at a relatively high drug concentration of 30.4 μg / mL, the survival rates of MCF-7 cells are 30.50% and 22.26% respectively, and the survival rates of MCF-7 / ADR cells are 51.48% and 45.61% respectively, indicating that TPP-DOX not only has a strong toxic effect on MCF-7 cells, but also has a certain killing effect on MCF-7 / ADR cells, which can be attributed to its anti-tumor drug resistance effect. When the highest concentration of the preparation is 30.4 μg / mL, the cell survival rates of the MDC-p-D and MDCD prodrug micelle solution groups after acting on MCF-7 cells for 24 h and 48 h are 41.26%, 32.57% and 55.74%, 46.20% respectively, and the cell survival rates after acting on MCF-7 / ADR cells for 24 h and 48 h are 61.13%, 54.76% and 69.52%, 64.46% respectively. It can be clearly seen that the MDC-p-D micelle group containing MMP-2 sensitive peptide has a stronger killing effect on MCF-7 and MCF-7 / ADR cells. Similarly, for the TD / MDC-p-D and TD / MDCD drug-loaded micelle groups, the TD / MDC-p-D group containing MMP-2 sensitive peptide has a stronger toxic effect on MCF-7 and MCF-7 / ADR cells, indicating that under the action of high MMP-2 concentration in the tumor extracellular matrix, the MMP-2 sensitive peptide in the micelle breaks the bond, the micelle depolymerizes, and a large amount of drug is released to produce a killing effect on the cells.

[0055] Pharmacodynamic evaluation of nanoformulations The purchased BALB / c mice were bred to an appropriate state (body weight 18 ± 2 g) for tumor inoculation; during this period, a tube of cryopreserved 4T1 murine breast cancer cells was resuscitated, cultured to an appropriate inoculation density, and then diluted with physiological saline at a certain ratio (1.5×10 6 cell / only) to obtain a cell suspension. Then, the cell suspension was inoculated into the right anterior axilla of BALB / c mice at a dose of 0.2 mL / only, and the tumor growth of the mice was observed. When the volume of the tumor reached 100 mm 3At that time, 70 mice were randomly divided into 7 groups with 10 mice in each group, and there were no obvious differences in the physical constitution of the mice among the groups. A normal saline group was set as the negative control group, and the medicinal liquids of each group were sterilized by ultraviolet irradiation in a super-clean workbench before each use. The intravenous administration was carried out every other day. The MDCD, MDC-p-D, TD / MDCD and TD / MDC-p-D micelle groups and the DAS, TPP-DOX solution groups were administered at a dose of 2.5 mg / kg, and the negative control group was injected with the same volume of normal saline. The administration was continued for seven times. Before each administration, the body weight and tumor volume of the mice were measured regularly. After 14 days, the mice were sacrificed, and tissues such as the heart, liver, spleen, lungs, and kidneys were fixed in 4% paraformaldehyde for standby. The tumors were dissected and weighed, and the anti-tumor effects of each treatment group were analyzed. The results are shown in Figure 17 .

[0056] Note: a represents the width of the tumor; b represents the length of the tumor.

[0057] During the experiment, the changes in the tumor volume of the tumor-bearing mice were measured every other day as shown in Figure 17 the figure. It can be seen from the figure that compared with the DAS and TPP-DOX groups, the growth of the tumor volume in the nanomicelle groups was smaller. Due to the synergistic effect of DAS and TPP-DOX, the polymer nanomicelles co-loaded with TPP-DOX showed better tumor inhibition effects than the nanomicelles of the polymer prodrug. Among all the groups, the TD / MDC-p-D group had the most significant tumor inhibition effect. This may be because in the presence of the MMP-2 sensitive peptide, after the TD / MDC-p-D nanomicelles reached the tumor site, the highly expressed MMP-2 in the tumor site caused them to break the bond and depolymerize. The synergistic effect of the released TPP-DOX and DAS plus the inhibitory effect of the material PEG-DCA on efflux enhanced its killing ability against tumor cells, thereby enhancing the anti-tumor effect of the nanomicelles. This experimental result was consistent with the previous in vitro anti-tumor experimental results.

Claims

1. A polymeric prodrug, characterized in that, The structural formula is as follows: ； In the formula, R = CH2-CH2 or CH2-CH2-COO-peptide, and the sequence of the peptide is: Gly-Pro-Leu-Gly-Ala, Gly-Leu-Gly-Cys, Pro-Leu-Ser-Ala, Gly-Pro-Gly-Val-Lys, His-Gly-Ile-Ala, or Pro-Leu-Gly-Val-Gln.

2. The preparation method of the polymeric prodrug according to claim 1, characterized in that, It includes the following steps: 1) Under an inert gas atmosphere, dissolve PEG-DCA-SA and HOBt in redistilled DMF, and stir and react at room temperature for 0.5 - 2 h; add the DMF solution containing peptide and EDCI under ice bath conditions, then add triethylamine, and continue to react at room temperature for 24 - 48 h. After the reaction is completed, dialyze and freeze-dry to obtain a white solid product PEG-DCA-SA-peptide; 2) Under an inert gas atmosphere, dissolve the synthesized PEG-DCA-SA-peptide and DMAP in redistilled DMF, and stir and activate at room temperature for 0.5 - 2 h; add the DMF solution containing the drug and EDCI under ice bath conditions, then add triethylamine, and raise the temperature to 35 - 60 °C and continue to react for 24 - 48 h. After the reaction is completed, dialyze and freeze-dry to obtain a white solid product PEG-DCA-SA-peptide-DAS; The drug is dasatinib, gilteritinib, suvoratinib, ibrutinib or acalabrutinib.

3. The preparation method of the polymer prodrug according to claim 2, characterized in that, In step 1), the molar ratio of PEG-DCA-SA, HOBt, peptide, and EDCI is 1:1 - 5:0.1 - 5:1 - 5; in step 2), the molar ratio of PEG-DCA-SA-peptide, DMAP, drug, and EDCI is 1:1 - 5:0.5 - 5:1 - 5.

4. The preparation method of the polymer prodrug according to claim 1, characterized in that, It includes the following steps: Under an inert gas atmosphere, dissolve PEG-DCA-SA and DMAP in redistilled DMF, and stir and activate at room temperature for 0.5 - 2 h; add the redistilled DMF solution containing the drug and EDCI under ice bath conditions, then add triethylamine, and continue to react at room temperature for 24 - 48 h. After the reaction is completed, dialyze and freeze-dry to obtain a white solid product PEG-DCA-SA-DAS; The drug is dasatinib, gilteritinib, suvoratinib, ibrutinib or acalabrutinib.

5. The preparation method of the polymer prodrug according to claim 4, characterized in that, The molar ratio of PEG-DCA-SA, DMAP, drug, and EDCI is 1:1 - 5:0.2 - 2:1 - 5.

6. A preparation method of a polymer nanomicelle co-loading DAS and loading a mitochondrial-targeted drug TPP-DOX, characterized in that, It includes the following steps: Weigh 10 - 20 mg of the polymeric prodrug described in claim 1, and weigh another 1 - 5 mg of TPP-DOX. Add the two to 1 - 3 mL of an organic solvent to dissolve, and dropwise add it to a bottle containing 2 - 6 mL of ultrapure water while stirring. After stirring for 10 - 20 min, transfer it to a dialysis bag with a molecular weight cut-off not exceeding 1000, and dialyze with ultrapure water as the dialysis medium for 12 - 24 h to obtain the product.

7. The preparation method of the polymer nanomicelles co-loaded with DAS and the mitochondrial-targeted drug TPP-DOX according to claim 6, characterized in that, The organic solvent used is any one of N,N-dimethylformamide, dimethyl sulfoxide, and acetone.

8. A polymer nanomicelle co-loaded with DAS and loaded with the mitochondrial-targeted drug TPP-DOX prepared by the method according to claim 6 or 7.

9. Use of the polymer prodrug according to claim 1 or the polymer nanomicelle co-loaded with DAS and loaded with the mitochondrial-targeted drug TPP-DOX according to claim 8 in a drug delivery system or for preparing an anti-tumor drug.

10. Use of the polymer prodrug according to claim 1 or the polymer nanomicelle co-loaded with DAS and loaded with the mitochondrial-targeted drug TPP-DOX according to claim 8 in an injection administration, oral administration, or local administration system.

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