Application of PSMA targeted nano prodrug constructed by multi-arm polyethylene glycol in preparation of drug for inhibiting prostatic cancer

Abstract

The invention provides an application of a PSMA targeted nano prodrug constructed by multi-arm polyethylene glycol in preparation of drugs for inhibiting prostatic cancer, and belongs to the technical field of biological medicines. The preparation method of the PSMA targeted nano prodrug constructed by multi-arm polyethylene glycol comprises the following steps: adding DCL-Mal into an N, N-dimethylformamide solution containing 4armPEG-SH under the protection of N2 to obtain a reaction solution, and carrying out stirring reaction at room temperature; after the reaction is finished, cooling, quenching and dialyzing the reaction solution, and freeze-drying to obtain solid 4armPEG-D-SH; trifluoroacetic acid is added into an N, N-dimethylformamide solution containing the 4armPEG-D-SH and PySS-DTX, a reaction mixture is obtained, and the reaction mixture is stirred at the room temperature for a reaction; and after the reaction is finished, dropwise adding the reaction mixture into diethyl ether to obtain a solid reactant, filtering, washing with ether, dialyzing, and freeze-drying to obtain the 4armPEG-DD. The 4armPEG-DD constructed by the invention can be used as a good candidate drug for clinical treatment of the PSMA positive prostate cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of a PSMA-targeted nano-prodrug constructed with multi-arm polyethylene glycol in the preparation of a drug for inhibiting prostate cancer. Background Art

[0002] Prostate cancer (PCa) is a common disease in men over 60 years old. Prostate-specific membrane antigen (PSMA) is a membrane protein that is expressed at high levels in prostate cancer. It is estimated that 80-90% of PCa are PSMA-positive. Therefore, PMSA-based diagnostic or therapeutic drugs or drug delivery systems have been applied to the diagnosis and treatment of PCa, especially for metastatic or recurrent PCa. The FDA has approved the clinical use of radionuclide diagnosis of prostate cancer based on PSMA targets, in which PMSA antibodies (mAb 7E11) are related to indium-111 and are more accurate in diagnosing metastatic prostate cancer than current computed tomography (CT) or magnetic resonance imaging (MRI). In addition, PMSA-specific chimeric antigen receptor (CAR) T cells have begun testing as a treatment in clinical trials for metastatic castration-resistant prostate cancer (mCRPC).

[0003] Androgen receptor (AR) is the driving force that determines the development of PCa, so AR inhibition (ARI) can be used as a golden treatment for this type of disease. However, patients with metastatic castration-resistant PCa (mCRPC) with acquired or genetic resistance have little effect on ARI treatment and can only seek other treatments, such as immunotherapy and chemotherapy. Immunotherapy is a common personalized treatment and is quite expensive, so chemotherapy has become one of the preferred strategies for treating prostate cancer. Docetaxel (DTX) is an FDA-approved chemotherapy drug that binds to b-tubulin to inhibit microtubule dynamics, leading to mitotic arrest. In addition, studies have shown that DTX inhibits mCRPC by downregulating AR protein levels. In general, DTX is used clinically to treat mCRPC, and even for castration-sensitive PCa patients, DTX+ARI can effectively prolong patient survival compared to ARI alone.

[0004] DTX, like most anti-tumor small molecules, has problems such as poor water solubility, lack of in vivo targeting ability, low bioavailability, short half-life, toxicity (fatigue, diarrhea, anemia or other side effects). In recent years, the construction of nanoprodrugs (nanoprodrugs) by loading drugs and other functional components (such as targeting ligands) onto various hydrophilic carriers has brought hope to overcome the above difficulties. It is worth noting that the ideal carrier should have basic characteristics such as biological inertness, non-immunogenicity, simple preparation, and clear chemical structure. Polyethylene glycol (PEG) can meet these requirements. For a long time, PEGylation has great application potential for the development of new powerful diagnostic and therapeutic drugs. However, the commonly used linear polyethylene glycol has several disadvantages: low drug loading effect, only two limited terminal coupling active sites; excessive flexibility leads to loose and instability of nanostructures.

[0005] Multi-arm PEG has more terminal active sites, increases loading capacity, and can be modified in a variety of ways, such as introducing targeting groups. In addition, the branched configuration of multi-arm polyethylene glycol can give the corresponding nanostructure enough rigidity, making it more compact and stable, thereby better protecting the loaded drug. Therefore, the technical problem to be solved in the present invention is to develop a nano drug that can load small molecule drugs into multi-arm polyethylene glycol, and quickly release the drug when reaching the tumor mass to enhance drug efficacy and reduce toxicity. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a PSMA-targeted nano-prodrug constructed with multi-arm polyethylene glycol, and the prepared 4armPEG-DD can be used as a good candidate drug for the clinical treatment of PSMA-positive prostate cancer.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a PSMA-targeted nano-prodrug constructed with multi-arm polyethylene glycol, comprising the following steps:

[0009] (1) Under N2 protection, DCL-Mal was added to the N,N-dimethylformamide solution containing 4armPEG-SH to obtain a reaction solution, and the reaction was stirred at room temperature;

[0010] (2) After the reaction solution is cooled, quenched, and dialyzed, the material in the dialysis bag is freeze-dried to obtain solid 4armPEG-D-SH;

[0011] (3) adding trifluoroacetic acid to the N,N-dimethylformamide solution containing the 4armPEG-D-SH and PySS-DTX to react to obtain a reaction mixture, and stirring the reaction at room temperature;

[0012] (4) The reaction mixture is dropped into ether to obtain a solid reactant, which is filtered and washed with ether. The material in the dialysis bag is dialyzed and freeze-dried to obtain 4armPEG-DD.

[0013] Preferably, in step (1), the molar ratio of the 4armPEG-SH to the DCL-Mal is 1-2:1-2, and the mass volume ratio of the 4armPEG-SH to the N,N-dimethylformamide is 300-500:2-4 mg / mL.

[0014] Preferably, the reaction in step (1) is carried out under continuous stirring at 20-25° C. for 10-20 h.

[0015] Preferably, the cooling in step (2) is performed by cooling to -12 to -8°C in an ice-salt bath, and the quenching is performed by adding deionized water at -10 to 0°C.

[0016] Preferably, the dialysis in step (2) is performed in deionized water for 20 to 28 hours using a dialysis bag with a specification of MWCO: 7.0 to 9.0 kDa.

[0017] Preferably, in step (3), the molar ratio of the 4armPEG-D-SH and PySS-DTX is 1:5-7, the mass volume ratio of the 4armPEG-D-SH and the N,N-dimethylformamide is 150-250:1-2 mg / mL, and the volume ratio of the trifluoroacetic acid to the N,N-dimethylformamide is 1:15-25.

[0018] Preferably, the reaction mixture in step (3) is obtained by stirring the reaction at 20-25° C. for 40-50 hours.

[0019] Preferably, in step (4), the volume ratio of the reaction mixture to diethyl ether is 1.5-2.3:28-32, the washing times are 2-4 times, and during the dialysis, the washed solid reactant is first dissolved in deionized water, and a dialysis bag with a specification of MWCO: 7.0-9.0 kDa is selected to dialyze in deionized water for 20-28 hours.

[0020] The present invention also provides a PSMA-targeted nano-prodrug constructed with multi-arm polyethylene glycol obtained by the preparation method.

[0021] The present invention also provides an application of the PSMA-targeted nano-prodrug constructed with multi-arm polyethylene glycol in the preparation of a drug for inhibiting prostate cancer.

[0022] The nano prodrug (4armPEG-DD) prepared by the present invention combines DTX with 4-arm polyethylene glycol through a reduction-sensitive disulfide bond and is modified by (((S)-5-amino-1-carboxypentyl)carbamoyl)-L-glutamic acid ((2S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid, DCL, PSMA targeting ligand). In vitro and in vivo experiments show that this nano prodrug specifically recognizes PSMA-positive prostate tumor cells, successfully locates DTX into prostate cancer cells, quickly releases DTX, and shows considerable toxicity to PSMA-positive prostate tumors. In addition, compared with small molecule DTX alone, 4armPEG-DD has improved safety. Therefore, 4armPEG-DD can be used as a good candidate drug for the clinical treatment of PSMA-positive prostate cancer.

[0023] The novel tumor subtype-selective nanoprodrug constructed by the present invention provides a safe and robust treatment method for PSMA-positive prostate cancer in animal models. Therefore, this nanoprodrug has the potential to achieve clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the DCL-Mal in Example 1 1 H NMR spectrum (recorded in d6-DMSO);

[0025] Figure 2 is the DCL-Mal in Example 1 13 CNMR spectra (recorded in d6-DMSO);

[0026] Figure 3 LC-MS spectrum of DCL-Mal in Example 1, product peak at 0.27min, [M+H] + 471.2 m / z, [M+Na] + 493.2m / z;

[0027] Figure 4 is the MAIDI-HRMS spectrum (negative ion mode) of DCL-Mal in Example 1,

[0028] [M-H2O-H] - is 451.1475m / z, [MH]- is 469.1570m / z, [M+Cl]- is 525.2214m / z;

[0029] Figure 5 is the PySS-DTX in Example 1 1 HNMR spectrum (recorded in d6-DMSO);

[0030] Figure 6 is the PySS-DTX in Example 1 13 C NMR spectrum (recorded in d6-DMSO);

[0031] Figure 7 is the LC-MS spectrum of PySS-DTX in Example 1, the product peak is at 2.09 min;

[0032] Figure 8 is the MAIDI-HRMS spectrum of PySS-DTX in Example 1, [M+H] + 1005.3509 m / z, [M+Na] + 1027.3351m / z, [M+K] + It is 1043.3063m / z.

[0033] Fig. 9 is the 4armPEG-D-SH in Example 1 1 HNMR spectrum (recorded in d6-DMSO)

[0034] Fig.10 For the chemical characterization of 4armPEG-DD in Example 1, (A) 1 HNMR spectrum (recorded in d6-DMSO); (B) DTX loading rate (13.65%, measured by UV); (C) and (D) particle size and morphology of the polymer (ca.110 nm and nearly spherical, detected by DLS and TEM, respectively); (E) surface potential of the polymer, 0 mV, measured by DLS); (F) DTX release profiles of 4armPEG-DD under different reducing environments: 10 mM or 10 μM DTT in PBS, pH 7.4, 37 °C;

[0035] Fig.11 The anticancer activity of 4armPEG-DD on PCa cells in Example 1 depends on its recognition of PSMA; (A) Effects of different concentrations of 4armPEG-DD, 4armPEG-D-SH and DTX on the cell viability of PCa cells; (B) Colony formation assay of CWR22Rv1 and PC3 cells treated with 10nM 4armPEG-DD or without drug carrier to monitor cell growth; Top, representative images; Bottom, statistical analysis; (C) After CWR22Rv1 cells were treated with 10nM 4armPEG-DD or without drug carrier for 24 hours, their cytoskeleton structure was monitored by F-actin staining. Scale bar: 5 mm; (D) PSMA staining of CWR22Rv1 cells treated with or without 10nM4armPEG-DD for 4 hours; Scale bar: 5 mm;

[0036] Fig.12 In Example 1, 4armPEG-DD inhibited the cell proliferation of PMSA-positive PCa and triggered cell apoptosis; (A) Flow cytometry was used to detect the apoptosis of CWR22Rv1 and PC3 cells after treatment with 10nM 4armPEG-DD or drug-free 4armPEG-D-SH for 24 hours; the left figure is a representative image of flow cytometry; right, statistical analysis; (B) Western blotting was used to detect the level of cleaved PARP in CWR22Rv1 cells treated with 10nM 4armPEG-DD or 4armPEG-D-SH for 24 hours; (C) CWR22Rv1 cells treated with 10nM 4armPEG-DD for 24 hours were stained with Edu to detect cell proliferation; DAPI was used to monitor cell nuclei; scale bar: 50mm; (D) After treatment with 10nM 4armPEG-DD for 24 hours, cell proliferation was detected by Edu staining; DAPI was used to monitor cell nuclei; scale bar: 50mm.

[0037] Fig.13 The 4armPEG-DD in Example 1 inhibits the cell migration and cell invasion of PSMA-positive PCa; (A) Wound healing assay detects the cell migration ability of CWR22Rv1 (left) and PC3 cells (right) after treatment with 10nM 4armPEG-DD or drug-free 4armPEG-D-SH for 24 hours; left, representative images; (B) Transwell invasion assay detects the cell invasion ability of CWR22Rv1 (top) and PC3 cells (bottom) after treatment with 10nM 4armPEG-DD or drug-free 4armPEG-D-SH for 24 hours; left, representative images; right, statistical analysis; (C) Western blotting detects EMT-related genes in CWR22Rv1 cells treated with 10nM 4armPEG-DD or drug-free 4armPEG-D-SH for 24 hours; GAPDH is used as control; (D) Real-PCR detects 10nM EMT-related genes in CWR22Rv1 cells treated with 4armPEG-DD or drug-free 4armPEG-D-SH for 24 h; the expression of target genes was normalized to GAPDH mRNA;

[0038] Fig.14The 4armPEG-DD treatment in Example 1 resulted in proteasome-mediated AR degradation; (A) Western blot analysis of AR protein levels in CWR22Rv1 cells treated with 10 nM 4armPEG-DD or 4armPEG-D-SH for 24 hours; GAPDH was the normal control; (B) Real-time fluorescence quantitative PCR detection of AR target gene expression levels in CWR22Rv1 cells after 10 nM 4armPEG-DD treatment for 24 hours; GAPDH mRNA was used as the internal control; (C) Before and after 4armPEG-DD treatment, protein synthesis was inhibited by cycloheximide (CHX), and AR degradation was detected by western blotting; (D) The protein degradation curve of AR was drawn; (E) 4armPEG-DD-mediated AR degradation was proteasome-dependent. Pretreatment with 10 μM proteasome inhibitor MG132 could block 4armPEG-DD-induced AR degradation, while treatment with 50 mM lysosomal inhibitor Chloroquine had no effect on 4armPEG-DD-induced AR degradation; (F) Ubiquitination assay of AR with or without 4armPEG-DD treatment; (G) Autodock predicted that DTX was a potential AR binding molecule.

[0039] Fig.15 This is a preclinical study on the use of 4armPEG-DD to inhibit the progression of PMSA-positive PCa in Example 1; (A) Tumor growth curve after administration of 4armPEG-D-SH, 4armPEG-DD or DTX; (B) and (C) are tumor images and tumor weights after treatment, respectively; (D) Body weight curve after administration of 4armPEG-D-SH, 4armPEG-DD or DTX; (E) ALT and AST levels in plasma of 4-armPEG-D-SH, 4-armPEG-DD or DTX-administered mice; (F) HE staining of the liver after administration of 4-armPEG-D-SH, 4-armPEG-DD or DTX; Scale bar: 200 mm; (G) IHC staining of AR and Ki67 in PCa tumors after administration of 4-armPEG-D-SH, 4-armPEG-DD or DTX; Scale bar: 200 mm. DETAILED DESCRIPTION

[0040] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] Example 1

[0042] 1.1 Materials

[0043] All commercial reagents met the experimental requirements and were used directly without additional purification unless otherwise specified. tert-Butyl l-DCL, docetaxel, Mal-OSu, PySS-COOH, 4armPEG-SH, DCC, and DMAP were purchased from TCI (Japan).

[0044] 1.2 Synthesis of Nanoprodrug (4armPEG-DD)

[0045] The PSMA targeting group (2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid, DCL) and drug molecules (DTX) were functionalized and covalently linked to a carrier material (4armPEG-SH) to construct a drug delivery system.

[0046] 1.2.1 Synthesis of 4armPEG-D-SH

[0047] Under N2 protection (i.e., the reaction system was protected by nitrogen to isolate oxygen and moisture in the air), DCL-Mal (9.4 mg, 0.02 mmol) was added to a solution of 4armPEG-SH (400 mg, 0.02 mmol) in N,N-dimethylformamide (3 mL), and the mixture was stirred continuously overnight at room temperature to obtain a reaction solution. The reaction solution was cooled to -10°C with an ice-salt bath, and deionized water (30 mL) was added to quench the solution, during which the temperature was controlled below 0°C. After quenching, the solution was dialyzed with deionized water (dialysis bag specification MWCO: 8.0 kDa) for 24 h, and the substance in the dialysis bag was lyophilized to obtain 4armPEG-D-SH (374 mg, yield 91.4%) as a white solid.

[0048] 1.2.2 Synthesis of 4armPEG-DD

[0049] Under N2 protection, 0.1 mL of trifluoroacetic acid was added to a solution of 4armPEG-D-SH (205 mg, 0.01 mmol) and PySS-DTX (60 mg, 0.06 mmol) obtained in 1.2.1 above in N, N-dimethylformamide (2 mL), and the mixture was stirred at room temperature for 48 h to obtain a reaction mixture. The reaction mixture was dropped into ether (30 mL) to produce a solid, which was filtered through a sand core funnel and washed three times with ether (30 mL). The solid was then dissolved in deionized water (10 mL), dialyzed in deionized water (MWCO: 8.0 kDa) for 24 h, and the material in the dialysis bag was lyophilized to obtain 4armPEG-DD (188 mg, yield 80.0%). After entering the water environment of human body fluids, 4armPEG-DD self-assembled into spherical nanostructures with a particle size of 110 nm and a zeta potential of 0 mV.

[0050] 1.3 Determination of DTX content

[0051] The DTX content in 4armPEG-DD synthesized in 1.2.2 was determined by ultraviolet spectrophotometer (METASH, Shanghai) at an absorption wavelength of 230 nm. The standard curve (y = 44.378x-6.49, R 2 =0.9993), and the DTX content in 4armPEG-DD was determined.

[0052] result

[0053] First, DCL-Mal was obtained. DCL was prepared by amidation reaction of (S)-di-tert-butyl 2-(3-(S)-6-amino-1-(tert-butyl)-1-oxohexyl-2-yl)ureidoglutarate (tert-butyl-DCL) with 3-maleimidopropionic acid succinimidyl ester (Mal-OSu) to generate a maleimido modified intermediate (tert-butyl-DCL-mal). The chemical structure was analyzed by nuclear magnetic resonance ( 1 H NMR and 13 C mass spectrometry analysis confirmed) LC-MS and MAIDI-HRMS, and then, under acidic conditions CF3COOH / DCM, the tert-butyl ester on the tert-butyl-DCL-Mal was removed to expose three carboxyl groups to obtain the maleimide-functionalized PSMA targeting group (DCL-Mal), whose chemical structure was also confirmed by NMR analysis ( 1 H NMR and 13 C mass spectrometry (LC-MS and MAIDI-HRMS) Figure 1-4 ), DCL-Mal can be introduced onto a thiol-containing hydrophilic carrier (4armPEG-SH) via an efficient thiol-maleimide click reaction.

[0054] Secondly, docetaxel reacted with PySS-COOH under mild esterification conditions (DCC / DMAP, ambient temperature 20-25°C) to obtain PySS-functionalized docetaxel (PySS-DTX) with a high yield (60.6% after purification). 1 H NMR, 13 C NMR, LC-MS and MAIDI-HRMS confirmed the correctness of the chemical structure of PySS-DTX ( Figure 5-8 ), Figure 5 for 1H NMR spectrum of PySS-DTX. The two hydrogens (q, z2, z3) on the methylene group have different chemical shifts due to their different chemical environments. Multi-arm PEG is selected as the carrier. The molecular weight and the number of arms directly determine the properties of the final nanodrug. In terms of molecular weight, too large will affect the drug loading, and too small will lead to insufficient water solubility. In addition, due to steric hindrance, too many arms will affect the reaction efficiency, resulting in poor monodispersity of nanodrugs, while too few arms will weaken the structural rigidity of nano-dds, resulting in poor stability. By balancing the above factors, a four-arm polyethylene glycol with a MW of 20KDa (4armPEG-SH) was finally selected as a hydrophilic carrier. First, the PSMA targeting ligand DCL was introduced into the carrier through the efficient thiol-maleimide click reaction of DCL-Mal and 4armPEG-SH to form 4armPEG-D-SH. In this reaction, the molar ratio of DCL-Mal to 4armPEG-SH was controlled at 1:1, which not only ensured the targeting effect but also the subsequent high drug loading. 1 H NMR spectroscopy ( Fig. 9 ), the hydrogen signals on the carboxyl, amide and carbonyl amide of DCL are clearly visible, proving the correctness of its chemical structure. The DTX prodrug (PySS-DTX) is covalently bound to the carrier through a thiol-disulfide exchange reaction, and the molar number of PySS-DTX needs to exceed that of the carrier by more than three times to achieve maximum drug introduction. After necessary purification, its chemical structure, drug loading capacity and self-assembly behavior were characterized. 1 H NMR spectrum ( Fig.10 A), the hydrogen signal of DTX is visible. UV analysis shows that the DTX loading capacity of 4armPEG-DD is 13.65% ( Fig.10 B). In addition, 4armPEG-DD is amphiphilic (both hydrophilic and lipophilic) and self-assembles into nearly spherical nanostructures in an aqueous environment with a particle size of 110 nm ( Fig.10 C, D), the surface potential is 0mV ( Fig.10 E), has the ability of long circulation in the body and good passive targeting to tumor tissues.

[0055] 1.4 In vitro release of DTX

[0056] The reduction-sensitive release of DTX by 4armPEG-DD was studied by simulating an in vitro physiological reduction environment (tumor cells: 10mM DTT in PBS, normal cells: 10μM DTT in PBS). The release of DTX from the polymer in vitro was studied by incubating 4armPEG-DD (2.17mg, containing 300μg DTX) with DTT (10mM) or DTT (10μM) in 3.0mL release medium (PBS, pH 7.4) (n=3 per group). After incubation for a certain time (0h, 1h, 2h, 4h, 8h, 8h, 12h, 24h, 48h), 200μL of the incubation solution was extracted and mixed with 400μL of methanol, and the mixed solution was divided into 3 portions. Finally, the drug release behavior of the DTX-containing polymer was detected by HPLC.

[0057] result

[0058] Compared with the normal physiological environment, the ultra-high reducibility of tumors enables the release of smart drugs within tumors. We simulated two different reducing environments in vitro (10mM or 10μM DTT in PBS, pH 7.4, 37°C) and studied the release behavior of DTX from 4armPEG-DD. As expected, the results at 48 hours showed that the release rate of DTX in the high reducing environment (about 69%) was significantly higher than that in the low reducing environment (about 21%) ( Fig.10 F), indicating that the nanoprodrug is stable under normal physiological conditions and maintains long circulation, while it can be rapidly degraded and release the drug in tumor cells due to the cleavage of the reduction-sensitive disulfide bond.

[0059] 1.5 Cell culture

[0060] C4-2, CWR22Rv1, and PC3 cells were obtained from Xiamen Immune Cell Biotechnology Co., Ltd. (Xiamen, Fujian, China) and cultured at 37°C in 5% CO2. Cells were maintained in RPMI supplemented with 10% fetal bovine serum (VivaCell, Shanghai, China) supplemented with 2 mM-glutamine, 100 IU / mL penicillin, and 100 ug / ml streptomycin.

[0061] result

[0062] The anticancer activity of 4armPEG-DD on PCa cells depends on its recognition of PSMA

[0063] Prostate cancer cell lines (CWR22Rv1, C4-2, and PC3) were exposed to different concentrations of 4armPEG-DD and drug-free 4armPEG-D-SH. After 48 hours, the cell viability was measured by MTT assay. Fig.11The data showed that 4armPEG-DD could inhibit the viability of CWR22Rv1 and C4-2 cells (PSMA positive), but had little effect on PC3 cells (PSMA negative), suggesting that the anticancer activity of 4armPEG-DD against PCa depends on PMSA. As a control, the small molecule DTX significantly inhibited the viability of all PCa cells, but had no PMSA selectivity ( Fig.11 A). Consistent with this, 4armPEG-DD significantly inhibited the colony formation ability of CWR22Rv1 cells, while this inhibitory effect was minimal in PC3 cells ( Fig.11 B) These results clearly demonstrate that this nanoprodrug possesses surprising PCa subtype selectivity.

[0064] As an important clinical drug for patients with metastatic prostate cancer, DTX binds to b-tubulin and disrupts the microtubule network. Therefore, we investigated whether 4armPEG-DD has an effect on the cytoskeletal structure of PCa cells. Fig.11 As shown in C, 4armPEG-DD severely damaged the multi-branched microtubule network of CWR22Rv1 cells, while 4armPEG-D-SH without drug loading had low bioefficiency. To detect the specific recognition of PSMA antigen by 4armPEG-DD, we used its antibody to stain PSMA antigen on the surface of CWR22Rv1 cells in the presence or absence of 4armPEG-DD. As expected, the intensity of PSMA antigen was much weaker in the presence of 4armPEG-DD ( Fig.11 D), indicating that 4armPEG-DD specifically recognizes the PMSA antigen of PCa cells, thereby hindering the recognition of antibodies. In summary, these data indicate that 4armPEG-DD specifically recognizes PSMA-positive PCa cells, benefits from the good targeting ability of DCL, and limits cell growth due to the antitumor effect of DTX.

[0065] 4armPEG-DD inhibits PMSA-positive PCa cell proliferation and triggers cell apoptosis

[0066] Cell growth is the net result of cell proliferation and apoptosis. To this end, we investigated the potential effects of 4armPEG-DD on proliferation and apoptosis of PSMA-positive CWR22Rv1 cells. After treating PCa cells with 4armPEG-DD for 48 hours, the cells were collected for AnnexinV / flow cytometry assay, and we found that 4-armPEG-DD significantly increased the apoptosis rate of CWR22Rv1 cells (19.5%-2.3%) compared with drug-free 4armPEG-D-SH. As a control, in PSMA-negative PC3 cells, the apoptosis induction rate of 4armPEG-DD was minimal (3.7%-5.6%) ( Fig.12A). Immunoblotting analysis also confirmed the effect of 4armPEG-DD on apoptosis of CWR22Rv1 cells, showing that after 4armPEG-DD treatment, the apoptosis marker, cleaved PARP, increased significantly ( Fig.12 B). Similarly, drug-free 4armPEG-D-SH also failed to promote PARP cleavage in CWR22Rv1 cells. Next, we continued to investigate whether 4armPEG-DD had an effect on the proliferation of PCa cells. As cells proliferate, EdU is incorporated into the synthesized DNA and can be easily monitored. After PCa cells were treated with 4armPEG-DD for 48 hours, the cells were fixed with 4% paraformaldehyde and EdU staining was performed. Fig.12 As shown in C, the EdU staining positive rate of CWR22Rv1 cells exposed to 4armPEG-DD was lower than that of the control group treated with 4armPEG-D-SH (17.3% vs. 30.9%), which means that 4armPEG-DD treatment reduced the proliferation rate of PCa cells. In contrast, the cell proliferation inhibition mediated by 4armPEG-D-SH was not obvious ( Fig.12 D). The above results further confirmed that DCL endowed 4armPEG-DD with good targeting to PSMA-positive PCa, and the biological activity of the nanoprodrug mainly came from DTX, while the carrier itself contributed little. Taken together, these findings suggest that 4armPEG-DD inhibits the growth of PCa cells at least by regulating cell apoptosis and cell proliferation.

[0067] Effects of 4armPEG-DD on migration and invasion of PSMAPCa cells

[0068] Cell migration and cell invasion are two hallmarks of metastatic cancer cells. After treating PCa cells with 4armPEG-DD for 48 hours, the cells were plated in a 6-well plate at a density of 80%. The next day, a wound was made in the middle of the cells with a yellow gun tip to observe the wound healing. The results showed that compared with 4armPEG-D-SH, 4armPEG-DD significantly slowed down the migration ability of CWR22Rv1 cells ( Fig.13 A, left). In contrast, 4armPEG-DD had no significant effect on the migration ability of PC3 ( Fig.13 A, right), further supporting that the effect of 4armPEG-DD is dependent on PSMA. In addition, after treatment with 4armPEG-DD, the invasiveness of CWR22Rv1 cells was significantly reduced, while that of PC3 cells was not significantly reduced ( Fig.13B). Mesenchymal-epithelial transition (EMT) is a key step in cancer metastasis, and the EMT status can be reflected by detecting some genes, such as e-cadherin (epithelial marker) and n-cadherin (mesenchymal marker). After PCa cells were treated with 4armPEG-DD for 48 hours, the cells were lysed and western blot experiments were performed to detect the expression of EMT-related proteins E-cadherin, N-cadherin and Snail. As expected, western blot analysis showed that 4armPEG-DD treatment led to upregulation of e-cadherin and reduction of n-cadherin and Snail in CWR22Rv1 cells ( Fig.13 C). At the same time, after PCa cells were treated with 4armPEG-DD for 48 hours, RNA was extracted for reverse transcription, and the expression of EMT-related genes was detected by real-time fluorescence PCR. Changes in the mRNA levels of EMT markers, such as upregulation of E-cadherin and downregulation of N-cadherein, also indicated that 4armPEG-DD blocked the progression of EMT ( Fig.13 D). In summary, this indicates that 4armPEG-DD has an inhibitory effect on the migration and invasion of PSMA-positive PCa cells.

[0069] 4armPEG-DD treatment leads to proteasome-mediated AR degradation

[0070] The significance of AR signaling in the development of PCa prompted us to investigate whether 4armPEG-DD treatment is involved in this important pathway. To this end, we treated CWR22Rv1 cells with different concentrations of 4armPEG-DD and the corresponding vehicle (4armPEG-D-SH) for 48 h and then lysed the cells for Western blot analysis. The results showed that 4armPEG-DD slightly reduced the protein levels of AR and its variants in CWR22Rv1 cells, while the vehicle had no effect on AR expression ( Fig.14 A). AR, as a transcription factor, enters the cell nucleus through androgen binding and regulates gene expression. Consistent with the reduction of AR, the AP activity of PSA, TMPRSS2, and FKBP5 was significantly inhibited by 4armPEG-DD treatment ( Fig.14 B), further supporting the view that 4armPEG-DD treatment leads to AR signaling inhibition. Next, after CWR22Rv1 cells were treated with 4armPEG-DD for 48 hours, the cells were plated in 6 empty plates at a density of 80%, and protein synthesis was inhibited using 10 μM cycloheximide (CHX). The degradation rate of AR under 4armPEG-DD treatment was detected by protein introduction experiment. Fig.14As shown in C, AR degradation in CWR22Rv1 cells exposed to 4armPEG-DD was much faster than that in cells exposed to the vector, indicating that 4armPEG-DD caused AR to become less stable and then degraded. Protein degradation occurs in lysosomes or proteasomes. The data demonstrated that 4armPEG-DD-mediated AR degradation is proteasome-dependent, and MG132 (proteasome inhibitor) but not chloroquine (lysosome inhibitor) ablated the nanomedicine and reduced AR levels ( Fig.14 D). Therefore, compared with 4armPEG-D-SH, 4armPEG-DD treatment resulted in more ubiquitin-bound AR ( Fig.14 F), indicating that in the presence of 4armPEG-DD, AR undergoes the ubiquitin-proteasome degradation pathway. Interestingly, the DTX-AR docking procedure showed that DTX may physically bind to AR with a free binding energy of -4.96 Kcal / mol and lead to its degradation ( Fig.14 G). Taken together, these data strongly support that the anticancer activity of 4armPEG-DD is at least partially dependent on its inhibition of AR signaling.

[0071] In vivo animal models

[0072] Male nude mice aged 6 to 8 weeks were purchased from Chongqing Tengxin Experimental Animal Co., Ltd. (Chongqing, China). CWR22Rv1 cells (1×10 6 ) was mixed with growth factor-reducing matrix (356230, Corning) and implanted subcutaneously on the right side of mice (100 μl / mouse), and randomly divided into groups. Drug-containing 4armPEG-DD, blank vector 4armPEG-D-SH and DTX (drug delivery, drug delivery control, and DTX drug alone, 6 mice in each group) were injected into mice every other day. The effective concentration of DTX is 5 mg / kg. According to the loading dose of 5 mg / kg DTX, 4armPEG-DD and 4armPEG-D-SH were converted according to the loading amount of DTX. The length (a) and width (b) of the tumor were recorded every week. The formula for calculating the tumor volume is V=ab 2 / 2(cm 3 ). Tumor weight was monitored and measured at the end of treatment, and photographs were taken.

[0073] result

[0074] Data such as Fig.15 As shown, Fig.15AC, 4armPEG-DD and DTX administration significantly inhibited the growth of PCa tumors, supporting that 4armPEG-DD has similar biological activity to DTX in vivo. Since biosafety is an important parameter we need to consider, body weight and liver damage were detected by histological and molecular analysis. Although DTX effectively inhibited the growth of PCa tumors, it caused a significant loss of body weight ( Fig.15 D). However, mice given 4armPEG-DD did not lose significant weight ( Fig.15 D), which means that the designed nanodrug was successfully delivered to the PCa area with less side effects on other organs. Importantly, ALT level (alanine transaminase level) and AST level (aspartate transaminase) were also increased in the serum plasma of DTX-treated mice ( Fig.15 E), while the 4 groups of armPEG-DD had little effect on the secretion of ALT and AST ( Fig.15 E); Histological analysis by HE staining also confirmed that 4armPEG-DD mice had less liver damage and showed increased immune cell infiltration compared with the DTX-treated group ( Fig.15 F). In summary, compared with the traditional chemotherapy drug DTX, 4armPEG-DD is less toxic to PSMA-positive PCa tumors.

[0075] In vitro cell model studies have shown that 4armPEG-DD treatment leads to a slow growth of PCa cells and a reduction in AR. In vivo mouse models have also shown that after 4armPEG-DD administration, immunohistochemical staining of Ki-67 (a proliferation marker) and AR levels showed that 4armPEG-DD significantly reduced the levels of Ki-67 and AR ( Fig.15 G, brown). Thus, 4armPEG-DD can precisely and effectively inhibit the progression of PMSA-positive prostate cancer in vivo.

[0076] In summary, the present invention prepares a nano prodrug 4armPEG-DD for the treatment of PSMA-positive prostate cancer. Due to the ingenious chemical regulation and the rigidity imparted by the branched structure, 4armPEG-DD exhibits bidirectionality, thereby forming a stable near-spherical self-assembled nanoaggregate with a particle size of 110nm and a surface potential of 0mV in an aqueous environment. In the nanoaggregate, the hydrophobic drug DTX is wrapped in the inner layer, and the hydrophilic targeting ligand DCL is arranged in the outer layer together with PEG. First, its excellent nanomorphology enables the nano prodrug to avoid the harsh attack in the blood, obtain a longer in vivo circulation, and then achieve high distribution of tumors through the EPR effect. Second, the specific binding ability of DCL to PSMA makes the nano prodrug highly selective for the PSMA-positive subtype of prostate cancer. Third, the ultra-high reducibility of tumor cells causes the disulfide bond between DTX and the carrier to break, resulting in the rapid release of active drug molecules. In addition, the toxic and side effects of the nano prodrug in vivo are lower than those of DTX. The results show that 4armPEG-DD greatly improves the in vitro and in vivo therapeutic effects of PSMA-positive prostate cancer, while having good biocompatibility.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a PSMA-targeted nano-prodrug constructed with multi-arm polyethylene glycol, characterized in that: The steps include: (1) Under N2 protection, DCL-Mal was added to the N,N-dimethylformamide solution containing 4armPEG-SH to obtain a reaction solution, and the reaction was stirred at room temperature; (2) After the reaction solution is cooled, quenched, and dialyzed, the material in the dialysis bag is freeze-dried to obtain solid 4armPEG-D-SH; (3) adding trifluoroacetic acid to the N,N-dimethylformamide solution containing the 4armPEG-D-SH and PySS-DTX to react to obtain a reaction mixture, and stirring the reaction at room temperature; (4) The reaction mixture is dropped into ether to obtain a solid reactant, which is filtered and washed with ether. After dialysis, the material in the dialysis bag is lyophilized to obtain 4armPEG-DD.

2. The preparation method according to claim 1, characterized in that: In step (1), the molar ratio of the 4armPEG-SH to the DCL-Mal is 1-2:1-2, and the mass volume ratio of the 4armPEG-SH to the N,N-dimethylformamide is 300-500:2-4 mg / mL.

3. The preparation method according to claim 2, characterized in that: The reaction in step (1) is carried out under continuous stirring at 20-25° C. for 10-20 hours.

4. The preparation method according to claim 3, characterized in that: The cooling in step (2) is performed by cooling to -12 to -8°C using an ice-salt bath, and the quenching is performed by adding deionized water at -10 to 0°C.

5. The preparation method according to claim 4, characterized in that: The dialysis in step (2) is carried out using a MWCO specification of: The 7.0-9.0 kDa dialysis bag was dialyzed in deionized water for 20-28 h.

6. The preparation method according to claim 5, characterized in that: In step (3), the molar ratio of 4armPEG-D-SH to PySS-DTX is 1:5-7, the mass volume ratio of 4armPEG-D-SH to N,N-dimethylformamide is 150-250:1-2 mg / mL, and the volume ratio of trifluoroacetic acid to N,N-dimethylformamide is 1:15-25.

7. The preparation method according to claim 6, characterized in that: The reaction in step (3) is carried out by stirring at 20-25° C. for 40-50 hours.

8. The preparation method according to claim 7, characterized in that: In step (4), the volume ratio of the reaction mixture to ether is 1.5-2.3:28-32, the washing times are 2-4 times, and during the dialysis, the washed solid reactant is first dissolved in deionized water, and a dialysis bag with a specification of MWCO: 7.0-9.0 kDa is selected to dialyze in deionized water for 20-28 hours.

9. A PSMA-targeted nano-prodrug constructed with multi-arm polyethylene glycol obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the PSMA-targeted nano-prodrug constructed with multi-arm polyethylene glycol according to claim 9 in the preparation of drugs for inhibiting prostate cancer.

Images