Aminopeptidase-catalyzed hydrolysis of charge-flipping polymers and their applications

Abstract

The present invention discloses a charge reversal polymer catalyzed by aminopeptidase and its application. The polymer contains an aminopeptidase response primitive, and the aminopeptidase response primitive structure is shown in formula Ⅰa or Ⅰb: #imgabs0# wherein R1 is one of hydrogen, alkyl, aromatic, and halogen atoms; and R2 is one of hydrogen, alkyl, aromatic, carbonyl, and halogen atoms. The present invention is used as an anti-tumor drug carrier, which can effectively prolong the blood circulation time of the drug, and hydrolyzes to produce amino groups under the catalysis of aminopeptidase highly expressed on the surface of tumor cells, realizing the conversion of charge from negative or neutral to positive, so that the positively charged polymer is quickly endocytosed by tumor cells, intracellularly transported, and then excreted from tumor cells, greatly improving the therapeutic effect of the drug on tumors, and has important significance for anti-tumor treatment in the medical field.

Description

Technical Field

[0001] The invention relates to the technical field of drug carrier production, and in particular to a charge-reversal polymer catalyzed by aminopeptidase and hydrolyzed and used thereof. Background Art

[0002] Efficient treatment options for malignant tumors have always been a major need in my country. At present, the theoretical basis for the development of nanomedicines is still to utilize the permeability and retention effect (Enhanced Permeability and Retention, EPReffect) of the tumor site. However, a large number of studies in recent years have shown that the permeability of tumor capillaries in clinical patients is much weaker than that in animal models, and is highly heterogeneous (varying with tumor stage, site, cancer type and patient). At the same time, nanomedicines are large in size and have a very small diffusion coefficient, while tumor tissues have pathological characteristics such as high cell density, dense matrix and high intratumoral pressure. Therefore, clinical tumors have weak EPR effects and strong heterogeneity, and the large size of nanomedicines makes it very difficult for them to diffuse within the tumor. After leaking out of the blood vessels, they cannot diffuse to cells far away from the blood vessels to complete drug delivery, resulting in low efficacy. This is also the main reason for the failure of many newly developed nanomedicine clinical trials (Kohli AG, S,Tiffany MR,Szoka FC,Improving the distribution of in the tumor matrix by depletion of tumorhyaluronan. Journal of controlled release, 2014, 191: 105-114; Scott L, Yao J, Benson A, et al. A phase II study of pegylated-camptothecin (pegamotecan) in the treatment of locally advanced and metastatic gastric and gastro-oesophagealjunction adenocarcinoma. Cancer chemotherapy and pharmacology, 2009, 63(2):363-370).

[0003] Studies have found that tumor cells (especially active tumor cells around tumor capillaries) secrete a large amount of enzymes (Choi KY, Swierczewska M, Lee S, Chen XY, Protease-activated drug development. Theranostics, 2012, 2(2): 156-178). For example, tumor cells of renal cancer, liver cancer, pancreatic cancer and invasive breast cancer secrete a large amount of γ-glutamyl transpeptidase. Polymer drug carriers based on charge reversal caused by γ-glutamyl transpeptidase-catalyzed hydrolysis are hydrolyzed by γ-glutamyl transferase overexpressed in tumor capillary endothelial cells to produce amino groups and carry positive charges. They are then adsorbed and trigger endothelial cell endocytosis, intracellular transport and contralateral exocytosis (transcytosis), and the positively charged polymers are delivered to the outside of the blood vessels (EPR-independent vascular extravasation). In the tumor, the positively charged polymers are also adsorbed on tumor cells to trigger their transcytosis process, and through this iterative endocytosis-exocytosis effect, they achieve cross-cellular delivery and active penetration of tumor tissues (Zhou Q, Shao SQ, Wang JQ, et al. Enzyme-activatable polymer-drug conjugate augments tumour penetration and treatment efficacy. Nature Nanotechnology, 2019, 14(8): 799-809).

[0004] Therefore, it is urgent to develop polymer drug carriers with charge reversal caused by enzyme-catalyzed hydrolysis, which have a long blood circulation time and can realize the charge conversion from negative or neutral to positive under the action of enzymes highly expressed in tumors, so that the positively charged polymer triggers a fast and efficient transcytosis process. Since the transcytosis process is an active transport process in which cells consume energy, factors such as high osmotic pressure and high cell density in the tumor that hinder traditional passive diffusion have no effect on this active transport process. Therefore, the carrier has a strong intratumor penetration ability and can deliver drugs to every cell, thus having a high anti-tumor efficacy.

[0005] Aminopeptidase is a transmembrane enzyme on the cell membrane surface and plays an important role in many physiological and pathological processes (Fukasawa K, Fujii H, Saitoh Y, et al. Aminopeptidase N (APN / CD13) is selectively expressed in vascular endothelial cells and plays multiple roles in angiogenesis. Cancer Letters, 2006, 243 (1): 135-143; Mina-Osorio P, The moonlighting enzyme CD13: old and new functions to target. Trends in Molecular Medicine, 2008, 14 (8): 361-371). Taking aminopeptidase as a biological target, by inhibiting its normal function, many diseases can be treated. For example, by inhibiting the activity of aminopeptidase, the formation and metastasis of tumor blood vessels can be effectively prevented (Guzman-Rojas L, Rangel R, Salameh A, et al. Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109 (5): 1637-1642). In addition, studies have shown that due to the high expression of aminopeptidase in tumor cells, aminopeptidase-responsive diagnostic agents and therapeutic agents can be designed and prepared, thereby realizing tumor diagnostic agent treatment under enzyme catalysis (Xiao M, Sun W, Fan J, et al. Aminopeptidase-N-activated theranostic prodrug for NIR tracking of local tumor chemotherapy. Advanced Functional Materials, 2018, 28 (47): 1805128). Summary of the invention

[0006] The purpose of the present invention is to provide a polymer that undergoes charge reversal by aminopeptidase-catalyzed hydrolysis and its application, which can be used as an anti-tumor drug carrier. It can be hydrolyzed to produce amino groups under the catalysis of aminopeptidase highly expressed in tumors, and the charge reversal is positively charged, so that the positively charged polymer is quickly internalized by tumor cells, transported intracellularly, and then excreted from tumor cells. Through this iterative and continuous internalization-excretion effect, cross-cellular transmission is achieved and effective penetration into deep tumor tissues is achieved. This process is an active transport process in which cells consume energy, which solves the problem of low passive diffusion and penetration efficiency of traditional polymer drug carriers in tumors.

[0007] The technical solution adopted by the present invention to solve its technical problem is:

[0008] An aminopeptidase-catalyzed hydrolysis-induced charge flipping polymer, the polymer contains an aminopeptidase-responsive motif, the aminopeptidase-responsive motif structure is as shown in formula Ⅰa or Ⅰb:

[0009]

[0010] Among them, R1 is one of hydrogen, alkyl, aromatic group, and halogen atom; R2 is one of hydrogen, alkyl, aromatic group, carbonyl group, and halogen atom.

[0011] Aromatic groups include benzene, naphthalene, anthracene, fluorene, pyridine, benzothiophene, benzofuran, indole, quinoline, etc. Alkyl groups include methyl, ethyl, propyl, propyl, butyl, pentyl, Hexyl , octyl, decyl, dodecyl, octadecyl, etc.

[0012] Preferably, the aminopeptidase-catalyzed hydrolysis-induced charge-flipping polymer is obtained by direct polymerization of monomers containing aminopeptidase-responsive motifs. The polymerization method is an existing polymerization method.

[0013] Preferably, the monomer containing the aminopeptidase response motif is composed of an aminopeptidase response motif portion and a functional group portion containing polymerization ability, and its structural formula is shown in Formula IIa or IIb:

[0014]

[0015] Wherein, X is a functional group part of the monomer having polymerization ability; R1 is one of hydrogen, alkyl, aromatic, and halogen atoms. The functional group having polymerization ability is one of acrylamide group, acrylate group, methacrylamide group, and methacrylate group.

[0016] Preferably, the monomers containing aminopeptidase response motifs include acrylamide monomers containing aminopeptidase response motifs, acrylate monomers containing aminopeptidase response motifs, methacrylamide monomers containing aminopeptidase response motifs, and methacrylate monomers containing aminopeptidase response motifs.

[0017] A drug carrier prepared by using the aminopeptidase-catalyzed hydrolysis of the charge-reversal polymer, wherein the general structural formula of the drug carrier is shown as IIIa or IIIb:

[0018]

[0019] Among them, M1 is a monomer containing an aminopeptidase-responsive motif, and M2 is a monomer loaded with drugs;

[0020] Formula IIIa represents the random copolymer structure formed by M1 and M2, wherein x is the proportion of M1 monomer, x=0.01-0.99, and n is the degree of polymerization, n=3-300;

[0021] Formula IIIb represents the block copolymer structure formed by M1 and M2, wherein m and n are the number of structural units, m=3 to 500, n=1 to 500. M2 is an acrylamide monomer, acrylate monomer, methacrylamide monomer, or methacrylate monomer loaded with drugs.

[0022] Preferably, the drug carrier is prepared by random copolymerization or block copolymerization of a monomer containing an aminopeptidase responsive unit and a monomer carrying a drug.

[0023] Preferably, the loaded drug is an anti-tumor drug, which includes doxorubicin, camptothecin, 7-ethyl-10-hydroxycamptothecin, 7-ethyl-camptothecin, paclitaxel, platinum drugs, curcumin, irinotecan, methotrexate, matrine, salvianolic acid, anti-tumor proteins, anti-tumor polypeptides, anti-tumor DNA molecules, and anti-tumor RNA molecules.

[0024] Preferably, the polymer drug carrier is a random copolymer, and its specific structural formula is shown in one of IIIc:

[0025]

[0026] Among them, x=0.01~0.99, n=3~300.

[0027] Preferably, the polymer drug carrier is a block copolymer, and its specific structural formula is shown in one of IIId:

[0028]

[0029] Among them, m=3~300, n=3~300.

[0030] Application of aminopeptidase-catalyzed hydrolysis-induced charge-flipping polymers in the preparation of drug carriers.

[0031] The beneficial effects of the present invention are:

[0032] (1) The tumor inhibition results of the mouse tumor-bearing model showed that compared with zwitterionic drug carriers that cannot achieve charge reversal, the charge reversal carriers synthesized by the present invention can quickly achieve positive charge conversion under the action of cell surface aminopeptidase, so the rate of entry into cells is greatly improved, overcoming the disadvantage of similar carriers that they enter tumor cells slowly, greatly improving the concentration of anti-tumor drugs in cells, and improving the utilization rate of drugs in tumor cells.

[0033] (2) The polymer provided by the present invention that is hydrolyzed by aminopeptidase to cause charge reversal is sensitive, efficient and specific in response to aminopeptidase. The polymer carrier can be dissolved in water to form an emulsion, nano-sized micelles or vesicles. The required solution dispersion form can be obtained by using the preparation methods commonly used in the art, which is easy to implement.

[0034] (3) The aminopeptidase-catalyzed hydrolysis-induced charge reversal polymer synthesized by the present invention has good water solubility and biocompatibility as a drug carrier, and can circulate in the body for a long time, so that it is easy to be enriched in tumor areas with irregular and sparse blood vessels, thereby reducing the enrichment of drugs in normal tissues, reducing the toxic and side effects of drugs on normal tissues, improving biocompatibility, and significantly improving the pharmaceutical effects of drugs.

[0035] (4) The aminopeptidase-catalyzed hydrolysis of the synthesized polymer causing charge reversal as a drug carrier has a unique advantage over drug carriers designed based on the EPR principle in that it relies on the transcytosis process for tumor penetration, which is an active transport process that consumes energy for cells. Therefore, factors such as high osmotic pressure and high cell density in the tumor that hinder traditional passive diffusion have no effect on this active transport process. Therefore, the carrier has a strong ability to penetrate into the tumor.

[0036] The present invention proposes to use aminopeptidase-catalyzed charge reversal polymers as anti-tumor drug carriers, providing a new type of carrier for anti-tumor drug delivery: it can have a long circulation time in the blood and can achieve the charge conversion from negative or neutral to positive at the periphery of the tumor, so that the positively charged polymer can effectively penetrate deep into the tumor tissue and quickly enter the tumor cells, greatly improving the drug's tumor treatment effect, which is of great significance to anti-tumor treatment in the medical field.

[0037] In addition, when the polymer that is hydrolyzed by aminopeptidase to cause charge reversal provided by the present invention is used as a charge reversal drug carrier, if the drug carrier is chemically labeled, then the drug carrier will simultaneously have the function of treating diseases, preventing diseases, carrying therapeutic genes, or tracing lesion sites, that is, the polymer has the potential to become a molecular precursor with the function of treating diseases, preventing diseases, carrying therapeutic genes, or tracing lesion sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1The hydrogen nuclear magnetic resonance spectrum of the aminopeptidase-catalyzed hydrolysis monomers M-Me, M-Et, M-iBu, and M-Bn prepared in Example 1 and the hydrogen nuclear magnetic resonance spectrum of the aminopeptidase-catalyzed hydrolysis monomers M(P)Me, M(P)Et, M(P)iBu, and M(P)Bn with a protecting group are shown in FIG.

[0039] Figure 2 The HPLC chromatogram of the aminopeptidase-catalyzed hydrolysis of monomers M-Me, M-Et, M-iBu and M-Bn by aminopeptidase in Example 2, wherein the upper curve represents the monomers after hydrolysis and the lower curve represents the monomers before hydrolysis.

[0040] Figure 3 This is the gel permeation chromatography (GPC) chart of the aminopeptidase-responsive polymers HP-Me, HP-Et, HP-iBu and HP-Bn prepared in Example 3.

[0041] Figure 4 This is the GPC chart of the aminopeptidase-responsive block polymers BP-Me, BP-Et, BP-iBu and BP-Bn prepared in Example 4.

[0042] Figure 5 This is the GPC chart of the aminopeptidase-responsive random copolymers RP-Me, RP-Et, RP-iBu and RP-Bn prepared in Example 5.

[0043] Figure 6 The graph shows the change of Zeta potential over time of each polymer prepared in Examples 3-5 under the action of aminopeptidase.

[0044] Figure 7 These are the cytotoxicity experimental diagrams of RP-Me, RP-Et, RP-iBu, RP-Bn, BP-Me, BP-Et, BP-iBu and BP-Bn on the aminopeptidase-positive cell lines HepG2 and CT26 in Application Example 1, wherein the left figure is the cytotoxicity experimental diagram of RP-Ala, RP-Et, RP-iBu, RP-Bn, BP-Ala, BP-Et, BP-iBu and BP-Bn on the aminopeptidase-positive cell line HepG2; the right figure is the cytotoxicity experimental diagram of RP-Ala, RP-Et, RP-iBu, RP-Bn, BP-Me, BP-Et, BP-iBu and BP-Bn on the aminopeptidase-positive cell line CT26.

[0045] Figure 8 For Application Example 1 Cy5 RP-Me, Cy5 RP-Et, Cy5 RP-iBu, Cy5 RP-Bn, Cy5 BP-Me, Cy5 BP-Et,Cy5 BP-iBu and Cy5 Laser confocal microscopy images of BP-Bn in aminopeptidase-positive cell lines HepG2 and CT26, of which the left column is Cy5 RP-Me, Cy5 RP-Et, Cy5 RP-iBu, Cy5 RP-Bn, Cy5 BP-Me, Cy5 BP-Et, Cy5 BP-iBu and Cy5 Laser confocal microscopy photos of BP-Bn in the aminopeptidase-positive cell line HepG2. Cy5 RP-Me, Cy5 RP-Et, Cy5 RP-iBu, Cy5 RP-Bn, Cy5 BP-Me, Cy5 BP-Et, Cy5 BP-iBu and Cy5 Laser confocal microscopy images of BP-Bn in the aminopeptidase-positive cell line CT26.

[0046] Fig. 9 This is a working curve diagram of the plasma clearance experiment of BP-iBu, BP-Bn and the control drug CPT-11 in mice in Application Example 1.

[0047] Fig.10 For Application Example 1 Cy5 BP-iBu and Cy5 Comparison of BP-Bn penetration in ear tumor model.

[0048] Fig.11 These are the tumor growth curves in the inhibition test of BP-iBu, BP-Bn and the control drug CPT-11 on HepG2 human liver cancer cell-bearing nude mice tumors and CT26 mouse colon cancer cell-bearing white mice tumors in Application Example 1. The left figure is the tumor growth curve in the inhibition test of BP-iBu, BP-Bn and the control drug CPT-11 on HepG2 human liver cancer cell-bearing nude mice tumors, and the right figure is the tumor growth curve in the inhibition test of BP-iBu, BP-Bn and the control drug CPT-11 on CT26 mouse colon cancer cell-bearing white mice tumors. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described in detail below through specific embodiments.

[0050] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0051] In the present invention, the monomers containing aminopeptidase response motifs include acrylamide monomers containing aminopeptidase response motifs, acrylate monomers containing aminopeptidase response motifs, methacrylamide monomers containing aminopeptidase response motifs, and methacrylate monomers containing aminopeptidase response motifs.

[0052] As examples of methacrylate monomers containing aminopeptidase-responsive motifs, M-Me, M-Et, M-iBu, and M-Bn, their structures are shown below:

[0053]

[0054]

[0055] M(P)Me, M(P)Et, M(P)iBu, and M(P)Bn are monomers with protective groups, wherein the amino group contains a tert-butyloxycarbonyl group and the carboxyl group contains a tert-butyloxy group, and their structures are shown below:

[0056]

[0057] The aminopeptidase-catalyzed hydrolysis-induced charge reversal polymer is prepared by using the above-mentioned M(P)Me, M(P)Et, M(P)iBu and M(P)Bn monomers by utilizing the existing polymerization method and the protective group removal method.

[0058] As an example, the methacrylate monomers M(P)Me, M(P)Et, M(P)iBu, and M(P)Bn containing aminopeptidase responsive motifs are polymerized by reversible addition-fragmentation chain transfer (RAFT) and deprotected by trifluoroacetic acid to prepare aminopeptidase-catalyzed hydrolysis-induced charge reversal polymers: HP-Me, HP-Et, HP-iBu, and HP-Bn, the structural formulas of which are shown below:

[0059]

[0060] Wherein, n is the degree of polymerization, which can be controlled by the feed ratio, n=3-300.

[0061] The methacrylate monomer containing the aminopeptidase response unit and the drug-loaded monomer are used to prepare a polymer drug carrier that is hydrolyzed by aminopeptidase to cause charge reversal by using the existing polymerization method.

[0062] As an example, the methacrylate monomers M(P)Me, M(P)Et, M(P)iBu, and M(P)Bn containing aminopeptidase response motifs are subjected to RAFT random copolymerization with the methacrylate monomer MSN38 loaded with the anti-tumor drug 7-ethyl-10-hydroxycamptothecin (SN38), and deprotected by trifluoroacetic acid to prepare aminopeptidase-catalyzed hydrolysis-induced charge reversal polymer drug carriers: RP-Me, RP-Et, RP-iBu, and RP-Bn, the composition of which can be regulated by the ratio of the two monomers, and the structural formula is shown in IIIc:

[0063]

[0064]

[0065] Among them, x=0.01~0.99, n=3~300.

[0066] As an example, the methacrylate monomers M(P)Me, M(P)Et, M(P)iBu, and M(P)Bn containing aminopeptidase responsive motifs are sequentially RAFT-copolymerized with the methacrylate monomer MSN38 loaded with the anti-tumor drug SN38, and deprotected by trifluoroacetic acid to prepare aminopeptidase-catalyzed hydrolysis-induced charge reversal polymer drug carriers: BP-Me, BP-Et, BP-iBu, and BP-Bn, the composition of which can be regulated by the ratio of the two monomers, and the structural formula is shown in IIId:

[0067]

[0068]

[0069] Among them, m=3~300, n=3~300.

[0070] The mechanism of polymer drug carriers with charge reversal caused by aminopeptidase-catalyzed hydrolysis for anti-tumor treatment is as follows: the terminal glutamyl group is a zwitterionic pair structure, which provides the polymer drug carrier with good water solubility and enables it to circulate in the blood for a long time. When it penetrates into the tumor site, the aminopeptidase highly expressed on the surface of peripheral cells of the tumor catalyzes the hydrolysis of the glutamyl group to produce amino groups and cause charge reversal. The positively charged polymer drug carrier can carry the drug quickly into the cell, transport it intracellularly, and then excrete it from the tumor cells. Through this iterative endocytosis-excretion effect, it achieves cross-cellular delivery and cancer cell killing effects.

[0071] Example 1: Synthesis of aminopeptidase-responsive monomers M-Me, M-Et, M-iBu and M-Bn

[0072] (1) N-(tert-Butyloxycarbonyl)ethanolamine (9.0 g, 55.1 mmol) and triethylamine (TEA, 70.0 mmol) were dissolved in 200 mL of anhydrous dichloromethane (DCM), and the solution was cooled in an ice bath. Methacryloyl chloride (6.7 mL, 66.1 mmol) was dissolved in 100 mL of DCM and added dropwise to the solution. After stirring for 12 hours, the organic phase was washed three times with NaHCO3 (1 M) and HCl (0.5 M), respectively, and then concentrated to a yellow solid. It was then recrystallized from ethyl acetate / n-hexane to obtain a white solid 1 with a yield of 89%.

[0073] (2) N-tert-Butyloxycarbonyl-L-alanine (4.0 g, 20.7 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 4.9 g, 24.8 mmol) were dissolved in 150 mL DCM, and then N-hydroxysuccinimide (NHS, 2.6 g, 22.8 mmol) was added and stirred at room temperature for 2 h to obtain NHS-activated N-tert-Butyloxycarbonyl-L-alanine ester (Boc-Me-NHS) solution. 5 g of compound 1 (22 mmol) was stirred in a solution of equal volume ratio of dichloromethane and trifluoroacetic acid for 1 h, and a precipitant of ether and n-hexane in a volume ratio of 1:1 was added for precipitation, and then dissolved in 50 mL DCM and 25 mmol of TEA was added; the solution was mixed with the above Boc-Me-NHS solution, stirred for 2 h, and then washed three times with 0.5 M HCl and saturated NaCl aqueous solution, respectively, and concentrated in vacuo. Compound 2 was further purified by silica gel chromatography using ethyl acetate / n-hexane in a volume ratio of 1:3 as the eluent, with a yield of 74%. Compounds 3-5 were synthesized using the same method.

[0074] (3) tert-Butyloxycarbonyl-L-glutamic acid-5-tert-butyl ester (5.8 g, 20.7 mmol) and EDC·HCl (4.9 g, 24.8 mmol) were dissolved in 150 mL DCM, and then NHS (2.6 g, 22.8 mmol) was added and stirred at room temperature for 2 h to obtain NHS-activated tert-butoxycarbonyl-L-glutamic acid-5-tert-butyl ester (Boc-Glu(OtBu)-NHS) solution. 3 g of compound 2 (10 mmol) was stirred in a solution of equal volume ratio of dichloromethane and trifluoroacetic acid for 1 h, ether was added for precipitation, and then dissolved in 50 mL DCM, and 15 mmol of TEA was added; the solution was mixed with the above Boc-Glu(OtBu)-NHS solution, stirred for 2 h, and washed three times with 0.5 M HCl and saturated NaCl aqueous solution, respectively, and concentrated in vacuo. M-Me was purified by silica gel chromatography using ethyl acetate / n-hexane in a volume ratio of 1:1 as the eluent with a yield of 58%. The synthesis methods of M-Et, M-iBu and M-Bn were the same.

[0075] Depend on Figure 1 From the NMR images, we can see that we have obtained a monomer with high purity.

[0076] Example 2: Aminopeptidase-catalyzed hydrolysis of small molecules M-Me, M-Et, M-iBu and M-Bn (1) Before the hydrolysis experiment, M-Me, M-Et, M-iBu and M-Bn were stirred in a solution of equal volume ratio of dichloromethane and trifluoroacetic acid for 1 hour, ether was added for precipitation, and the mixture was dried in a vacuum oven for 48 hours.

[0077] (2) M-Me, M-Et, M-iBu and M-Bn were dissolved in a buffer solution (50 mM Tris, pH 7.0), and then aminopeptidase was added. The final test concentration was 1 μM monomer concentration and 0.5 μg / mL aminopeptidase concentration. After shaking at 37°C for 20 hours, each monomer solution was placed in an ice bath for cooling and the monomer concentration was detected by high performance liquid chromatography. The test conditions were 35°C, H2O / methanol = 7 / 3, and absorbance at 220 nm. The peak integrated area of ​​the monomer was recorded as S T The monomer solution without aminopeptidase treatment was used as the control, and its peak area was recorded as S C . T and S C Substitute into the monomer standard curve to obtain the concentration, which is recorded as C T and C c The hydrolysis ratio of the monomer can be calculated by the following formula:

[0078]

[0079] Depend on Figure 2It can be seen that the hydrolysis activity of aminopeptidase on M-Me, M-Et, M-iBu and M-Bn increases in sequence.

[0080] Example 3: Synthesis of aminopeptidase-responsive polymers HP-Me, HP-Et, HP-iBu and HP-Bn

[0081] (1) The synthesis method of aminopeptidase-responsive polymer is as follows: an initiator (AIBN, 1.10 mg, 0.0067 mmol), a chain transfer agent (2-phenyl-2-propylbenzodisulfide, CTA, 5.62 mg, 0.02 mmol) and a monomer (0.80 mmol, target degree of polymerization 40, molar ratio of monomer / CTA / AIBN 40 / 1 / 0.3) are added to a round-bottom flask, nitrogen is bubbled to remove oxygen in the solution, and the solution is heated to 65 °C under a nitrogen atmosphere for 12 hours. After the reaction is completed, air is introduced to terminate the polymerization, and ether is added to the solvent. The solution is dissolved and precipitated three times to obtain a polymer.

[0082] (2) Before further analysis, the polymer was deprotected in a mixed solvent of DCM / TFA (1:1) for 2 hours. After deprotection, the polymer was transferred to a 3.5 kDa dialysis bag for dialysis, and the final product was obtained by lyophilization.

[0083] Depend on Figure 3 It can be seen that we obtained a polymer with uniform distribution and a number average molecular weight of about 13,000 Daltons before deprotection.

[0084] Example 4: Synthesis of aminopeptidase-responsive block polymers BP-Me, BP-Et, BP-iBu and BP-Bn (1) According to the literature (Zhao H, Rubio B, Sapra P, et al. Novel prodrugs of SN38 using multiarm poly(ethylene glycol) linkers. Bioconjugate Chemistry, 2008, 19(4): 849-859), tert-butyloxycarbonyl-protected SN38 was synthesized. SN38 (2 g, 5 mmol) was suspended in 200 mL of anhydrous DCM, di-tert-butyl dicarbonate (1.4 g, 6.5 mmol) and 10 mL of anhydrous pyridine were added, and stirred at room temperature overnight. The organic layer was washed three times with HCl (0.5 M) and NaHCO3 (1 M), respectively, and dried over anhydrous MgSO4. The solvent was removed under vacuum to obtain Boc-protected SN38 with a yield of 95%.

[0085] (2) 2,2'-Dithioethanol (3.1 g, 20 mmol) was dissolved in 200 mL of anhydrous tetrahydrofuran (THF). After adding 25 mmol of TEA, the solution was placed in an ice bath and allowed to stand for 30 minutes, then stirred vigorously, and methacryloyl chloride (2 mL, 20 mmol) dissolved in 50 mL of anhydrous THF was added dropwise. The solution was stirred and monitored by thin layer chromatography (TLC) until the reaction was completed. The product was filtered and concentrated under vacuum to obtain a crude product. Compound 6 was purified by silica gel chromatography using a mixed solvent of ethyl acetate / n-hexane in a volume ratio of 1 / 3 as the eluent, with a yield of 70%.

[0086]

[0087] (3) Synthesize SN38 drug monomer (MMA-SN38) according to the literature (Sun R et al, Supramolecular PEGylation of camptothecin for cancer therapy. Materials Today Nano, 2021, 14; Sun R et al, Supramolecular engineering of polymeric nanodrugs for antitumor chemotherapy. Chemical Engineering Journal, 2021, 416). Dissolve triphosgene (300 mg, 1 mmol) and 4-dimethylamine pyridine (810 mg, 6.5 mmol) in 50 mL of anhydrous DCM, stir for 30 minutes under nitrogen protection, then add dropwise Boc-protected SN38 (1 g, 2.0 mmol) dissolved in 150 mL of anhydrous DCM. After stirring for 2 hours, slowly add compound 6 (4.0 mmol) dissolved in 50 mL of anhydrous DCM, and continue stirring for 12 hours. The solution was washed with HCl (0.5 M) and saturated NaCl aqueous solution and concentrated in vacuo. The crude product was purified by silica gel chromatography using ethyl acetate / n-hexane = 1 / 1 as the eluent to obtain MMA-SN38 with a yield of 68%.

[0088] (4) The synthesis scheme of aminopeptidase-responsive block copolymers is as follows: AIBN (1.65 mg, 0.01 mmol), HP-Me (335 mg, 0.03 mmol, or HP-Et, HP-iBu, HP-Bn) and MMA-SN38 monomer (133 mg, 0.18 mmol; target degree of polymerization 6) were added to a round-bottom flask, nitrogen was bubbled to remove oxygen in the solution, and the solution was heated to 65 °C under a nitrogen atmosphere for 12 hours. After the reaction was completed, air was introduced to terminate the polymerization; samples were taken before and after the reaction, and the monomer conversion rate was calculated by nuclear magnetic resonance spectroscopy; ether was added to the polymerization reaction solution, and the solution was dissolved and precipitated repeatedly for 3 times to obtain a block polymer.

[0089] (5) Before further analysis, the block polymer was deprotected in a DCM / TFA (1:1) mixed solvent for 2 h. After deprotection, the block polymer was transferred to a 3.5 kDa dialysis bag for dialysis, and the final product was obtained by lyophilization.

[0090] Depend on Figure 4 It can be seen that we obtained a block polymer with uniform distribution and a number average molecular weight of about 17,000 before deprotection.

[0091]

[0092] Example 5: Synthesis of aminopeptidase-responsive random copolymers RP-Me, RP-Et, RP-iBu and RP-Bn (1) The synthesis scheme of aminopeptidase-responsive random copolymers is as follows: AIBN (1.1 mg, 0.0067 mmol), monomer and MMA-SN38 (target degree of polymerization 40, monomer / MMA-SN38 / CTA / AIBN molar ratio of 34 / 6 / 1 / 0.3) are added to a round-bottom flask, and nitrogen is bubbled for 30 minutes, followed by reaction at 65°C for 20 hours. After the reaction is completed, air is introduced to terminate the polymerization; samples are taken before and after the reaction, and the monomer conversion rate is calculated by nuclear magnetic hydrogen spectrum analysis; ether is added to the polymerization reaction solution, and the precipitate is dissolved and dissolved repeatedly for 3 times to obtain a random copolymer.

[0093] (2) Before further analysis, the random copolymer was deprotected in a mixed solvent of DCM / TFA (1:1) for 2 hours. After deprotection, the random copolymer was transferred to a 3.5 kDa dialysis bag for dialysis, and the final product was obtained by lyophilization.

[0094] Depend on Figure 5 It can be seen that we obtained a random copolymer with uniform distribution and a number average molecular weight of about 19,000 Daltons before deprotection.

[0095] Example 6: Aminopeptidase-catalyzed polymer charge reversal experiment

[0096] 43nM aminopeptidase was added to a polymer solution containing 2mg / mL (50mM Tris, pH 7.0) and shaken at 37°C. 20μL of the polymer solution was taken out from the dialysis bag at regular intervals and diluted to 500μL with PBS (pH=7.40). The corresponding Zeta potential value was measured and the average value was taken after three measurements. The polymer solution without APN was used as a control.

[0097] Depend on Figure 6 It can be seen that under the catalysis of aminopeptidase, each polymer has a potential change, the potential of the block polymer can be transformed from negative to positive, while the charge reversal ability of the random copolymer is slightly weaker, and the charge reversal ability of the homopolymer is the weakest.

[0098] Example 7: Synthesis of fluorescently labeled aminopeptidase-responsive polymer

[0099] 20 mg of RP-Me, RP-Et, RP-iBu, RP-Bn, BP-Me, BP-Et, BP-iBu and BP-Bn were dissolved in 1 mL of deionized water, and the pH was adjusted to 8.0 with sodium bicarbonate. Then, 0.1 mg of water-soluble Cy5-NHS was added to the above solution, stirred overnight at room temperature, dialyzed, and freeze-dried to obtain the fluorescently labeled products. Cy5 RP-Me, Cy5 RP-Et, Cy5 RP-iBu, Cy5 RP-Bn, Cy5 BP-Me, Cy5 BP-Et, Cy5 BP-iBu and Cy5 BP-Bn, and then a microplate reader was used to detect and quantify the fluorescence intensity of the modified Cy5.

[0100] Application Example 1: Application of random copolymers RP-Me, RP-Et, RP-iBu, RP-Bn and block copolymers BP-Me, BP-Et, BP-iBu, BP-Bn as anti-tumor drugs

[0101] (1) Preparation of RP-Me, RP-Et, RP-iBu, RP-Bn, BP-Me, BP-Et, BP-iBu and BP-Bn injections According to the corresponding dosage concentrations calculated with the same SN38 equivalent concentration, the RP-Me, RP-Et, RP-iBu, RP-Bn, BP-Me, BP-Et, BP-iBu and BP-Bn prepared in Example 4 and Example 5 were dissolved in physiological saline buffer or PBS and sonicated to obtain clear and transparent homogeneous injection solutions, which were stored at -20°C to avoid repeated freezing and thawing.

[0102] Subsequent experiments were carried out using RP-Me, RP-Et, RP-iBu, RP-Bn, BP-Me, BP-Et, BP-iBu and BP-Bn solutions prepared by the above methods as examples, and the detection methods all adopted conventional methods in the art.

[0103] (2) Cytotoxicity assay

[0104] The in vitro cytotoxicity of RP-Me, RP-Et, RP-iBu, RP-Bn, BP-Me, BP-Et, BP-iBu, BP-Bn and the camptothecin small molecule prodrug SN38 was compared. The results showed that the in vitro anti-aminopeptidase positive cell line mouse colon cancer cell line CT26 and the aminopeptidase positive cell line human liver cancer cell line HepG2 were Figure 7 As shown in the figure, it can be seen from the results that BP-iBu and BP-Bn have stronger cytotoxicity than random copolymers and block copolymers with slow aminopeptidase hydrolysis rate. This result shows that BP-iBu and BP-Bn can be used as anti-tumor drugs.

[0105] (3) Cell phagocytosis assay of random copolymers RP-Me, RP-Et, RP-iBu, RP-Bn and block copolymers BP-Me, BP-Et, BP-iBu, BP-Bn CT26 or HepG2 cells were evenly plated in confocal dishes, with 100,000 cells per dish. After overnight culture, each dish was added with a final concentration of 10 μg / mL. Cy5 RP-Me, Cy5 RP-Et, Cy5 RP-iBu, Cy5 RP-Bn, Cy5 BP-Me, Cy5 BP-Et, Cy5 BP-iBu and Cy5 BP-Bn, after incubation for 1 hour, was washed three times with PBS, the cell nucleus was stained with Hoechst33342, and the cellular endocytosis of the polymer was observed using a laser confocal microscope.

[0106] CT26 or HepG2 cells were evenly plated in a 12-well plate, 100,000 per well. After overnight culture, each well was added with a final concentration of 10 μg / mL. Cy5 RP-Me, Cy5 RP-Et, Cy5 RP-iBu, Cy5 RP-Bn, Cy5 BP-Me, Cy5 BP-Et, Cy5 BP-iBu and Cy5BP-Bn, after incubation for 1 h, were washed three times with PBS, trypsinized, resuspended in PBS, and the intracellular fluorescence intensity was detected by flow cytometry.

[0107] The results are as follows Figure 8 As shown, this experiment shows Cy5 BP-iBu and Cy5 The cellular endocytosis rate of BP-Bn was significantly faster than that of other random copolymers and block copolymers with slow aminopeptidase hydrolysis rate.

[0108] (4) Plasma clearance experiment

[0109] ICR mice weighing about 20g were randomly divided into 3 groups, 3 mice in each group, and the same SN38 equivalent of BP-iBu, BP-Bn and control group CPT-11 were injected into the tail vein. 50μL of blood was collected from the orbits of each group of mice at the set time point, 50μL of NaOH (0.1M) was added to each sample, and incubated at 37℃ overnight after ultrasound. 100μL of HCL (0.1M) was added to acidify, and then 800μL of acetonitrile was added to extract. After centrifugation, the supernatant was taken, and the drug content in the blood of each group at different time points was measured by HPLC and the plasma clearance working curve was drawn. The results are shown in Fig. 9 As shown, the experiment showed that polymers BP-iBu and BP-Bn had longer in vivo circulation time than CPT-11.

[0110] (5) Tumor penetration experiment

[0111] 200,000 HepG2 cells were inoculated subcutaneously in the ears of Balb / c nude mice and divided into 4 groups. After the tumor was formed, each group was injected with Cy5 BP-iBu and Cy5 BP-Bn, after the mice were anesthetized, the penetration of the polymer into the tumor and surrounding blood vessels in the mouse ears was observed using a laser confocal microscope at different time points; the results are shown in Fig.10 As shown, the experiment shows that Cy5 BP-iBu and Cy5 BP-Bn undergoes charge reversal under the hydrolysis of aminopeptidase, achieving rapid penetration from blood vessels to tumor lesions.

[0112] (6) Tumor inhibition experiment

[0113] Balb / c nude mice weighing about 20 g were divided into 4 groups, with 6 mice in each group. Two million HepG2 cells were inoculated subcutaneously on the right posterior flank. When the tumor grew to 100 mm, 3The drug was given once every three days for a total of five times. The four groups were blank control group, CPT-11, BP-iBu of the present invention and BP-Bn. The dosage was 10 mg / kg of SN38 equivalent. The tumor size was measured and the body weight of nude mice was weighed each time the drug was administered. The nude mice were observed continuously after drug withdrawal. When the tumor size of the blank group reached the ethical value, the nude mice were killed, the tumors of all nude mice were separated and the wet weight of the tumors in each group was weighed.

[0114] Similarly, 20 g Balb / c mice were divided into 4 groups, 6 mice in each group, and 500,000 CT26 cells were inoculated subcutaneously on the right posterior flank. 3 The drug was given once every three days for a total of five times. The four groups were blank control group, CPT-11, BP-iBu of the present invention and BP-Bn, and the dosage was 10 mg / kg of SN38 equivalent.

[0115] The results are as follows Fig.11 As shown, the experiment showed that compared with the blank group and CPT-11 group, BP-iBu and BP-Bn had more significant anti-tumor effects in HepG2 and CT26 models, among which BP-iBu had the best tumor inhibition effect.

[0116] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. An aminopeptidase-catalyzed hydrolysis of a charge-reversal polymer, characterized in that: The polymer contains an aminopeptidase responsive motif, and the aminopeptidase responsive motif structure is shown in formula Ⅰa or Ⅰb: Wherein, R1 is one of methyl and ethyl; R2 is carbonyl; The aminopeptidase-catalyzed hydrolysis-induced charge-flipping polymer is obtained by direct polymerization of monomers containing aminopeptidase-responsive motifs; The monomers containing aminopeptidase response primitives include acrylamide monomers containing aminopeptidase response primitives, acrylate monomers containing aminopeptidase response primitives, methacrylamide monomers containing aminopeptidase response primitives, and methacrylate monomers containing aminopeptidase response primitives.

2. A drug carrier prepared by using the aminopeptidase-catalyzed hydrolysis of the charge-reversal polymer according to claim 1, characterized in that: The general structural formula of the drug carrier is shown in IIIa or IIIb: ； Wherein, M1 is the monomer containing the aminopeptidase response motif as claimed in claim 1, and M2 is a monomer loaded with a drug; Formula IIIa represents the random copolymer structure formed by M1 and M2, wherein x is the proportion of M1 monomer, x=0.01-0.99, and n is the degree of polymerization, n=3-300; Formula IIIb represents the structural formula of the block copolymer formed by M1 and M2, wherein m and n are the number of structural units, m=3-500, n=1-500.

3. The drug carrier according to claim 2, characterized in that The drug carrier is prepared by random copolymerization or block copolymerization of a monomer containing an aminopeptidase response unit and a monomer loaded with a drug.

4. The drug carrier according to claim 2 or 3, characterized in that The drug is an anti-tumor drug, which includes doxorubicin, camptothecin, 7-ethyl-10-hydroxycamptothecin, 7-ethyl-camptothecin, paclitaxel, platinum drugs, curcumin, irinotecan, methotrexate, matrine, salvianolic acid, anti-tumor proteins, anti-tumor polypeptides, anti-tumor DNA molecules, and anti-tumor RNA molecules.

5. A drug carrier, characterized in that The drug carrier is a random copolymer, and its specific structural formula is shown in one of IIIc: Ⅲc Among them, x=0.01~0.99, n=3~300.

6. A drug carrier, characterized in that The drug carrier is a block copolymer, and its specific structural formula is shown in one of IIId: Ⅲd Among them, m=3~300, n=3~300.

7. Use of the aminopeptidase-catalyzed hydrolysis-induced charge-flipping polymer as claimed in claim 1 in the preparation of a drug carrier.

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