Polypeptide micelle inhibitor as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide micelle inhibitor as well as a preparation method and application thereof, and particularly relates to an ApoE peptide modified polypeptide micelle inhibitor. According to the micelle disclosed by the invention, the stability of the micelle is remarkably improved through the hydrophobic interaction of phenylboronic acid groups in the polypeptide and the small-molecule inhibitor, B-N coordination and pi-pi accumulation; through the action of the ApoE and the low-density lipid protein, the ApoE can efficiently penetrate through a blood brain barrier and enter glioblastoma cells in a targeted endocytosis manner, so that the growth of in-situ glioblastoma is remarkably inhibited, and the lifetime of tumor-bearing mice is greatly prolonged. The polypeptide micelle based on ApoE peptide modification disclosed by the invention has the advantages of being convenient to prepare, high in stability, capable of penetrating through a blood-brain barrier and killing glioblastoma in a targeted manner, and the like, and has relatively large transformation potential in the aspect of jointly and actively delivering small-molecule inhibitors with different molecular targets; the compound shows a huge application prospect in the aspect of synergistic treatment of brain glioma.

Description

Technical Field

[0001] The present invention relates to a technology for delivering drugs using polypeptides, which is a technology for targeted drug delivery. Specifically, it relates to a method for preparing an ApoE peptide-modified polypeptide micelle inhibitor, and the application of this micelle inhibitor in synergistic targeted anti-glioblastoma. Background Art

[0002] With the continuous progress of molecular biology and genomics and the gradual deepening of the understanding of the tumor pathogenesis mechanism, molecular targeted therapy has attracted great interest. As an important part of the molecular targeted drug system, small molecule inhibitors have the characteristics of strong specificity, significant efficacy, and low toxicity and side effects, and are increasingly used in the clinical treatment of malignant tumors. Although small molecule inhibitors can specifically target tumor cells and show clinical efficacy, most drugs face problems such as low bioavailability, insufficient accessibility to the target, cross-talk, compensation and feedback mechanisms between signaling pathways, and intrinsic or acquired drug resistance. Glioblastoma is the most malignant primary brain tumor, with high invasiveness and high inter-tumor and intra-tumor heterogeneity. Its five-year survival rate is only 5%. The emergence of highly specific and low off-target toxicity targeted inhibitors has significantly promoted the progress of glioblastoma treatment. However, the therapeutic effect of small molecule drugs on brain tumors is usually limited by low bioavailability and the blood-brain barrier, and more than 98% of small molecule drugs cannot effectively penetrate into the brain. Summary of the Invention

[0003] Aiming at the problems existing in existing small molecule drugs, especially the technical obstacles for glioblastoma, the present invention discloses a technology for delivering drugs using polypeptides. Small molecule drugs are encapsulated with polypeptides carrying phenylboronic acid groups to achieve proportional encapsulation and responsive release of small molecule inhibitors, improve the therapeutic effect of small molecule drugs on tumors, especially showing excellent blood-brain barrier penetration ability and being efficiently taken up by glioblastoma cells.

[0004] To achieve the above invention purpose, the technical solution adopted by the present invention is as follows: A polypeptide micelle inhibitor, comprising a polypeptide carrying a phenylboronic acid group and its encapsulated small molecule drug.

[0005] In the present invention, the small molecule drug includes a PLK1 inhibitor and / or a BCL-2 / xL inhibitor; preferably, the inhibitor is two or more inhibitors, preferably two different inhibitors; as an example, the inhibitor includes the PLK1 inhibitor volasertib and the BCL-2 / xL inhibitor navitoclax.

[0006] The present invention discloses a polypeptide for a micelle inhibitor, comprising a polypeptide carrying a phenylboronic acid group.

[0007] In the present invention, the polypeptide micelle inhibitor is conjugated with a targeting molecule.

[0008] In the present invention, the micelle inhibitor is conjugated with a targeting molecule using a polypeptide.

[0009] Preferably, the targeting molecule includes a peptide targeting molecule, for example, the targeting molecule is an ApoE peptide.

[0010] In the present invention, the raw materials for preparing the polypeptide with a phenylboronic acid group include a macroinitiator, 4-boron-L-phenylalanine, and L-tyrosine.

[0011] In the present invention, the amino acids include 4-boron-L-phenylalanine and L-tyrosine.

[0012] Preferably, the molecular weight of the macroinitiator is 0.5 kg / mol to 5 kg / mol; more preferably, the average molecular weight of the macroinitiator is 2 kg / mol.

[0013] Preferably, the molar ratio of 4-boron-L-phenylalanine to L-tyrosine is (2 to 5):1.

[0014] In the present invention, when the polypeptide micelle inhibitor is conjugated with a targeting molecule or the micelle inhibitor is conjugated with a targeting molecule using a polypeptide, the macroinitiator includes a macro molecule with a targeting molecule linking group and a macro molecule without a targeting molecule linking group, and the obtained polypeptide with a phenylboronic acid group is a polypeptide with a phenylboronic acid group and conjugated with a targeting molecule linking group.

[0015] In the present invention, the macroinitiator is a hydrophilic polymer, such as polyethylene glycol. Specifically, the macro molecule with a targeting molecule linking group is polyethylene glycol with a targeting molecule linking group, such as maleimide-polyethylene glycol; the macro molecule without a targeting molecule linking group is polyethylene glycol, such as methoxy polyethylene glycol.

[0016] The present invention discloses a method for preparing the above-mentioned polypeptide micelle inhibitor, which includes the following steps: mixing a solution of the polypeptide with a phenylboronic acid group and a solution of a small molecule drug, and then performing dialysis to obtain the polypeptide micelle inhibitor; or mixing a solution of the polypeptide with a phenylboronic acid group, a solution of the polypeptide with a phenylboronic acid group and conjugated with a targeting molecule linking group, a solution of a small molecule drug, and a solution of a targeting molecule, and then performing dialysis to obtain the polypeptide micelle inhibitor, which is a polypeptide micelle inhibitor conjugated with a targeting molecule.

[0017] In the present invention, a poly-polypeptide solution with a phenylboronic acid group and a small molecule drug solution are mixed in a buffer solution, and then dialyzed to obtain a poly-polypeptide micelle inhibitor; or a poly-polypeptide solution with a phenylboronic acid group, a poly-polypeptide solution with a phenylboronic acid group and a targeting molecule linking group, and a small molecule drug solution are mixed in a buffer solution, then a targeting molecule solution is added and mixed, and then dialyzed to obtain a poly-polypeptide micelle inhibitor, which is a poly-polypeptide micelle inhibitor conjugated with a targeting molecule.

[0018] In the present invention, the molar ratio of the poly-polypeptide with a phenylboronic acid group to the poly-polypeptide with a phenylboronic acid group and a targeting molecule linking group is (2 - 20):1; preferably, the molar ratio of the poly-polypeptide with a phenylboronic acid group to the poly-polypeptide with a phenylboronic acid group and a targeting molecule linking group is (4 - 19):1.

[0019] In the present invention, when there are two inhibitors, the mass ratio of the two inhibitors is (0.1 - 10):1; preferably, the mass ratio of the two inhibitors is (0.2 - 5):1; more preferably, the mass ratio of the two inhibitors is (0.5 - 2):1.

[0020] The present invention discloses the application of the above poly-polypeptide micelle inhibitor in the preparation of drugs.

[0021] The present invention discloses the application of the poly-polypeptide used for the above micelle inhibitor in the preparation of drugs.

[0022] In the present invention, the drug is a small molecule drug; preferably, the drug is a small molecule inhibitor drug.

[0023] In the present invention, the drug includes drugs for treating tumors. Preferably, the tumors include brain tumors, such as glioblastoma.

[0024] As an example, taking the ApoE peptide as the targeting molecule, 4-boron-L-phenylalanine and L-tyrosine as amino acids, polyethylene glycol as the macromolecular initiator, and volasertib and navitoclax as small molecule drugs, the examples of the present invention are as follows.

[0025] A poly-polypeptide micelle inhibitor modified based on the ApoE peptide, the ApoE peptide sequence is LRKLRKRLLLRKLRKRLLC, and the ApoE peptide reacts with the maleimide group in maleimide-polyethylene glycol- b -poly(4-boron-L-phenylalanine- co -L-tyrosine) through the thiol group at its C-terminus, thereby realizing the modification of the micelle and endowing the micelle with targeting function.

[0026] The poly-polypeptide includes polyethylene glycol- b -poly(4-boron-L-phenylalanine- co-L-tyrosine) and maleimide-polyethylene glycol- b -poly(4-boron-L-phenylalanine- co -L-tyrosine); As an example, the molecular weight of polyethylene glycol is 2.0 kg / mol, and the molar ratio of 4-boron-L-phenylalanine to L-tyrosine in the chain segment is 3.4:1, ensuring that the polypeptide has good solubility, self-assembly performance, and the ability to interact with small molecule inhibitors.

[0027] The preparation method of the above-mentioned ApoE peptide-modified polypeptide micelle inhibitor includes the following steps: adding the polypeptide and the small molecule inhibitor solvent to a buffer solution, then adding the ApoE peptide solution, and then dialyzing to obtain the ApoE peptide-modified polypeptide micelle inhibitor. As an example, the molecular weight cut-off of the dialysis bag is 3500 Da, the dialysis time is 8 hours, the dialysis solution is a phosphate buffer at pH 7.4, and the dialysis medium is changed every 1 hour to effectively remove organic solvents and free drugs.

[0028] In the present invention, using methoxy-polyethylene glycol-amine as an initiator, ring-opening polymerization of 4-boron-L-phenylalanine-N-carboxyanhydride and L-tyrosine-N-carboxyanhydride to obtain polyethylene glycol- b -poly(4-boron-L-phenylalanine- co -L-tyrosine) polypeptide; using maleimide-polyethylene glycol-amine as an initiator, ring-opening polymerization of 4-boron-L-phenylalanine-N-carboxyanhydride and L-tyrosine-N-carboxyanhydride to obtain maleimide- b -poly(4-boron-L-phenylalanine- co -L-tyrosine) polypeptide.

[0029] The present invention discloses the application of the above-mentioned ApoE peptide-modified polypeptide micelle inhibitor in targeted co-delivery of nano-inhibitors; the nano-inhibitor is an anti-tumor small molecule inhibitor; the anti-tumor is against glioblastoma; the small molecule inhibitor has a synergistic effect.

[0030] The present invention discloses the preparation method of the above-mentioned ApoE peptide-modified polypeptide micelle inhibitor, including the following steps: mixing the solution of polyethylene glycol- b -poly(4-boron-L-phenylalanine- co -L-tyrosine) and maleimide-polyethylene glycol- b -poly(4-boron-L-phenylalanine- co -L-tyrosine) copolymer with the solutions of volasertib and navitoclax and dropping them into a buffer solution. After the dropping is completed, add the ApoE polypeptide solution and react overnight in a constant temperature shaking water bath. After the reaction is completed, dialyze to obtain.

[0031] The ApoE peptide-modified poly-polypeptide micelle inhibitor of the present invention exhibits excellent blood-brain barrier penetration ability, is efficiently taken up by glioblastoma cells, and co-delivers PLK1 and BCL-2 / xL small molecule inhibitors in proportion for the synergistic treatment of glioblastoma. In the present invention, the phenylboronic acid group in the poly-polypeptide forms B-N coordination and π-π stacking interactions with the small molecule inhibitor to achieve proportional encapsulation and responsive release of the small molecule inhibitor.

[0032] Preferably, when the molar ratio of polyethylene glycol- b -poly(4-borono-L-phenylalanine- co -L-tyrosine) to maleimide-polyethylene glycol- b -poly(4-borono-L-phenylalanine- co -L-tyrosine) is in the range of (4 to 19):1, the micelles formed can achieve a better comprehensive balance in terms of stability, drug loading capacity, and targeting performance. When the molar ratio is lower than 4:1, the stability of the micelles will decrease, and aggregation or drug leakage is likely to occur during storage and transportation; when the molar ratio is higher than 19:1, although the stability of the micelles may be improved, it will affect the targeting ability to tumor cells and is not conducive to subsequent treatment effects. Further preferably, the mass ratio of volasertib to navitoclax is (0.5 to 2):1.

[0033] In the present invention, the solution of the copolymer and the small molecule inhibitor is a dimethyl sulfoxide (DMSO) solution; the buffer solution is a phosphate (PB) buffer solution.

[0034] The present invention discloses the application of the above-mentioned ApoE peptide-modified poly-polypeptide micelle inhibitor in the preparation of or as an anti-glioblastoma nanomedicine. Preferably, the small molecule inhibitors have a synergistic effect.

[0035] Due to the application of the above technical solutions, the present invention has the following advantages compared with the prior art: 1. The ApoE peptide-modified poly-polypeptide micelle inhibitor disclosed in the present invention has the characteristics of simple preparation method, repeatability and controllability, and at the same time has good enzymatic degradability; after ApoE peptide modification, it still retains high stability and responsive release characteristics; in particular, the two co-loaded small molecule inhibitors in the present invention can achieve proportional encapsulation and release.

[0036] 2. The ApoE peptide-modified poly-polypeptide micelle inhibitor disclosed in the present invention has high efficiency in crossing the blood-brain barrier and targeting tumor cells. The micelles effectively cross the blood-brain barrier through the transcytosis mediated by ApoE and low-density lipoprotein receptor, and bind to the overexpressed related receptors on the glioblastoma cell membrane to actively target and be taken up by tumor cells.

[0037] 3. The ApoE peptide-modified poly-polypeptide micelle inhibitor prepared by the present invention has a synergistic inhibitory effect on targets such as BCL-2, BCL-xL, and MCL-1, providing a general and effective platform for the co-active delivery of small molecule inhibitors with different molecular targets, and showing great potential in the synergistic treatment of glioblastoma. Description of the Drawings

[0038] Figure 1 Characterization of the copolymer in Example 1. (A) 1 H NMR (400 MHz, DMSO- d 6 / CD 3 OD, 5 / 1, v / v) characterization; (B) MALDI-TOF characterization.

[0039] Figure 2 Characterization of the multiple interactions, co-encapsulation and controlled release of the small molecule inhibitor and the micelle in Example 2. (A) Drug loading efficiency of Nav and Vol alone or co-encapsulated; (B) UV-visible spectra characterization of PM, VN and PMVN; (C) NMR boron spectra characterization of BPA mixed with Vol, Nav or VN; (D) Particle size distribution of PMVN and ApoE-PMVN (10 mol.% ApoE) and TEM image of ApoE-PMVN; (E) Particle size and potential of PMVN with different ApoE densities; (F) Stability of ApoE-PMVN stored at 4 °C, diluted 50-fold or in 10% FBS for 30 days; (G) Particle size change of ApoE-PMVN under the condition of proteinase K (12 units / mL); (H) In vitro drug release of ApoE-PMVN in the presence of PK; (I) Mass ratio of Vol / Nav released from PM in the presence of PK.

[0040] Figure 3 Characterization of the cellular uptake of ApoE-PM in Example 3. (A) Effect of ApoE density on the uptake of Cy5-labeled ApoE-PM by GL261-Luc cells; (B) Average fluorescence intensity in GL261-Luc cells; (C) Laser scanning confocal microscopy images of GL261-Luc cells incubated with Cy5-labeled ApoE-PM for 2 or 4 h.

[0041] Figure 4 Characterization of the blood-brain barrier penetration ability of ApoE-PM in Example 3. (A) Schematic diagram of establishing an in vitro Transwell model; (B) Uptake of bEnd.3 and (C) GL261-Luc cells incubated with PM or ApoE-PM for 2, 8 and 24 h; (D) Quantitative analysis of the average fluorescence intensity of bEnd.3 and GL261-Luc cells.

[0042] Figure 5 It is the in vitro cytotoxicity characterization of the drug in Example 4. (A) Cell viability of GL261-Luc cells after incubation with free inhibitor or micelle inhibitor; (B) Cell viability of GL261-Luc cells after incubation with different inhibitor micelles; (C) Cell viability of L929 and (D) GL261-Luc cells after incubation with ApoE-PM.

[0043] Figure 6 It is the Western blot, mitochondrial membrane potential change, apoptosis and cell cycle analysis of cells in Example 5. (A) Protein expression of PLK1, Bim, Cleaved caspase-3 and β -actin; (B) Mitochondrial membrane potential change; (C) Flow cytometry analysis of JC-1 aggregation and monomers; (D) Apoptosis analysis; (E) Cell cycle analysis.

[0044] Figure 7 It is the RNA sequencing analysis of GL261-Luc cells in Example 6. (A) Venn diagram of differentially expressed genes; (B) Volcano plot of differentially expressed genes; (C) Heat map analysis of differentially expressed genes; (D) KEGG analysis of different signaling pathways.

[0045] Figure 8 It is the in vivo biodistribution characterization of ApoE-PM in Example 7. (A) In vivo fluorescence images of GL261-Luc GBM mice in situ after intravenous injection of Cy5-labeled ApoE-PM; (B) Ex vivo fluorescence images of major organs and brain 12 h after injection; (C) Quantitative analysis of ex vivo fluorescence intensity.

[0046] Figure 9 It is the in vivo anti-GBM effect of ApoE-PMVN in Example 8. (A) Schematic diagram of treatment of GL261-Luc GBM model in situ; (B) Body weight change; (C) Survival curve. Detailed implementation manners

[0047] The therapeutic effect of small molecule drugs on brain tumors is usually limited by low bioavailability and the blood-brain barrier, and more than 98% of small molecule drugs cannot effectively penetrate into the brain. Therefore, the research direction of the treatment of malignant tumors today is to construct a polypeptide micelle inhibitor that crosses the blood-brain barrier, targets glioblastoma, and co-delivers small molecule inhibitors with different properties in proportion to act synergistically on tumor-related targets.

[0048] The present invention discloses a polypeptide micelle inhibitor, which comprises a polypeptide with a phenylboronic acid group and a small molecule drug encapsulated therein. Preferably, the targeting molecule includes a peptide targeting molecule, such as the targeting molecule being an ApoE peptide.

[0049] The present invention discloses a preparation method of the above-mentioned polypeptide micelle inhibitor, which comprises the following steps: mixing a polypeptide solution with a phenylboronic acid group and a small molecule drug solution, and then performing dialysis to obtain a polypeptide micelle inhibitor; or mixing a polypeptide solution with a phenylboronic acid group, a polypeptide solution with a phenylboronic acid group and a targeting molecule linking group, a small molecule drug solution, and a targeting molecule solution, and then performing dialysis to obtain a polypeptide micelle inhibitor, which is a polypeptide micelle inhibitor conjugated with a targeting molecule.

[0050] The present invention specifically discloses a preparation method of an ApoE peptide-modified polypeptide micelle inhibitor and its application in targeted synergistic treatment of glioblastoma.

[0051] The preparation method of the ApoE peptide-modified polypeptide micelle inhibitor disclosed by the present invention comprises the following steps: (1) Under nitrogen protection, using methoxy-polyethylene glycol-amine as an initiator, ring-opening polymerization of 4-boron-L-phenylalanine-N-carboxylic anhydride and L-tyrosine-N-carboxylic anhydride to obtain polyethylene glycol- b -poly(4-boron-L-phenylalanine- co -L-tyrosine); using maleimide-polyethylene glycol-amine as an initiator, ring-opening polymerization of 4-boron-L-phenylalanine-N-carboxylic anhydride and L-tyrosine-N-carboxylic anhydride to obtain maleimide-polyethylene glycol- b -poly(4-boron-L-phenylalanine- co -L-tyrosine); (2) Mixing the polymer solution, volasertib and navitoclax solutions uniformly, slowly and uniformly dropping the mixture into a buffer solution under stirring, continuing to stir for 3 min after the dropping is completed, then adding an ApoE solution, mixing uniformly and placing it in a constant temperature shaking incubator to react overnight; (3) After the reaction is completed, dialysis is performed with a buffer solution to obtain an ApoE peptide-modified polypeptide micelle inhibitor.

[0052] In the above technical solution, the mass ratio of methoxy-polyethylene glycol-amine, 4-boron-L-phenylalanine-N-carboxylic anhydride, and L-tyrosine-N-carboxylic anhydride is 1∶(1.35-1.5)∶(0.25-0.3), and the mass ratio of maleimide-polyethylene glycol-amine, 4-boron-L-phenylalanine-N-carboxylic anhydride, and L-tyrosine-N-carboxylic anhydride is 1∶(1.4-1.55)∶(0.28-0.35); preferably, the temperature of the ring-opening polymerization is 60-90 °C, the time is 50-100 h, and the solvent is N , N -dimethylformamide.

[0053] In the above technical solution, the small molecule co-loaded volasertib and navitoclax inhibitory micelles are prepared by the solvent displacement method. The specific steps are as follows: The poly-polypeptide and the small molecule inhibitor are dissolved in DMSO at a concentration of 10-30 mg / mL, and the ApoE polypeptide is dissolved in secondary water. Taking 10 wt.% theoretical drug loading and 10 mol.% ApoE density as an example, the polymer solution and the small molecule inhibitor solution (DMSO) are mixed evenly and slowly added dropwise to the buffer solution (PB, pH 8.0) under stirring; after the addition is completed, stirring is continued for 3 min, and then the ApoE solution is added. After mixing evenly, the reaction is carried out overnight in a constant temperature shaking incubator; after the reaction is completed, the reaction solution is loaded into a dialysis bag to remove organic solvents and free drugs, and finally the poly-polypeptide micelle ApoE-PMVN co-loaded is obtained.

[0054] The present invention further discloses the application of the ApoE peptide-modified poly-polypeptide micelle inhibitor in targeted synergistic anti-glioblastoma therapy.

[0055] The present invention discloses a poly-polypeptide micelle inhibitor, its preparation method and application, specifically an ApoE peptide-modified poly-polypeptide micelle inhibitor. As an example, it is obtained by co-loading the PLK1 inhibitor volasertib and the BCL-2 / xL inhibitor navitoclax with poly-polypeptide modified by phenylboronic acid, and then modifying with ApoE peptide. Specifically, polyethylene glycol- b -poly(4-boron-L-phenylalanine- co -L-tyrosine) solution, maleimide-polyethylene glycol- b -poly(4-boron-L-phenylalanine- co -L-tyrosine) solution and the small molecule inhibitor solution are mixed evenly and then added dropwise to the buffer solution; after the addition is completed, the ApoE polypeptide solution is added, and the reaction is carried out overnight in a constant temperature shaking incubator; after the reaction is completed, dialysis is carried out to obtain the ApoE peptide-modified poly-polypeptide micelle inhibitor. The micelles of the present invention significantly improve the stability of the micelles through the B-N coordination and π-π stacking interaction between the phenylboronic acid group in the poly-polypeptide and the small molecule inhibitor; through the endocytosis of ApoE and low-density lipoproteins, they efficiently penetrate the blood-brain barrier and target the glioblastoma tumor area, significantly inhibiting the growth of orthotopic glioblastoma and greatly prolonging the survival period of tumor-bearing mice. The ApoE peptide-modified poly-polypeptide micelle inhibitor disclosed by the present invention has the advantages of convenient preparation, high stability, and ability to target and kill glioblastoma, etc., and has great transformation potential in co-actively delivering small molecule inhibitors of different molecular targets, and shows great application prospects in the synergistic treatment of glioma.

[0056] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. The specific preparation operations and performance tests are all conventional techniques, and data statistical analysis is carried out using conventional statistical methods.

[0057] 4-boron-L-phenylalanine (BPA), L-tyrosine (Tyr), α -methoxy- ω -amino-polyethylene glycol (mPEG-NH 2 , M n = 2.0 kg / mol), α -maleimide- ω -amino-polyethylene glycol (MAL-PEG-NH 2 , M n = 2.0 kg / mol), thiolated apolipoprotein E (ApoE) (sequence: LRKLRKRLLLRKLRKRLLC, >95%), volasertib (Vol, 98.0%), navitoclax (Nav, 98.9%) are commercially available products and are used directly after purchase. Reagents without special instructions are all purchased from Sinopharm Chemical Reagent Co., Ltd. and used directly.

[0058] The proton nuclear magnetic resonance spectrum ( 1 H NMR) of the polymer uses DMSO- d 6 / CD 3 OD (5 / 1, v / v) as the solvent, with the deuterated dimethyl sulfoxide solvent signal as the standard, and is tested with a 400 MHz liquid superconducting nuclear magnetic resonance spectrometer (AVANCE NEO, Btuker). The boron nuclear magnetic resonance spectrum ( 11 B NMR) of the polymer uses DMSO- d 6 as the solvent and is determined by a 400 MHz liquid superconducting nuclear magnetic resonance spectrometer. 11 B NMR uses boron trifluoride diethyl etherate (BF 3 OEt 2 ) as the external reference. The molecular weight of the polymer is determined using trans-2-(3-(4-tert-butylphenyl)-2-methyl-2-propenylidene) malononitrile (DCTB) and sodium trifluoroacetate (CF3COONa +A mixture (9 / 1, v / v) was used as the matrix and tested with a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF, Ultraflextreme). The particle size, particle size distribution, and surface Zeta potential of the micelles were tested with a dynamic light scattering instrument (DLS, Zetasizer Nano-ZS, Malvern). The concentration of the small molecule inhibitor was tested with high performance liquid chromatography (HPLC, Waters e2695). The mobile phase was pure acetonitrile / secondary water (70 / 30, v / v), and the pH of the secondary water was adjusted to 7 with triethylamine (0.15% of the water volume) and phosphoric acid. The test conditions were a flow rate of 1 mL / min, an injection volume of 20 μL, a column temperature of 35 °C, and a Sepax BR-C18 reverse chromatographic column (4.6×250 mm, 5 μm, 120 Å); the detection wavelengths for volasertib and navitoclax were 330 nm and 301 nm, respectively. Cytotoxicity was determined with a multifunctional microplate reader (Varioskan LUX, Thermo Fisher). Apoptosis and the cell cycle were detected with a flow cytometer (FACSVerse, BD) and analyzed with Flowjo software. The uptake of nanoparticles by cells was observed with a confocal laser scanning microscope (CLSM, TCS SP5, Leica).

[0059] Example 1 Synthesis and Characterization of Copolymers The copolymer polyethylene glycol- b -poly(4-borono-L-phenylalanine- co -L-tyrosine) was prepared by ring-opening polymerization of BPA-NCA and Tyr-NCA monomers using mPEG-NH 2 ( M n = 2.0 kg / mol) as the initiator. Under a nitrogen atmosphere, a DMF solution (2.8 mL) of BPA-NCA (282 mg, 1.2 mmol) and a DMF solution (0.6 mL) of Tyr-NCA (58 mg, 0.28 mmol) were added to a 10 mL sealed reactor and stirred, and then a DMF solution (0.2 mL) of mPEG-NH 2 (200 mg, 0.1 mmol) was added. Subsequently, the mixed solution was heated and stirred at 80 °C for 72 h under nitrogen protection. After the reaction was completed, the product was added dropwise to ice ether approximately 20 times the volume of the reaction solution (~70 mL) for precipitation, centrifugation (7000 rpm, 5 min), and the supernatant was discarded. The precipitate was redissolved in anhydrous methanol, and the precipitation, centrifugation, and redissolution operations were repeated twice. After the third precipitation and centrifugation, the precipitate was placed in a vacuum drying oven at 37 °C for 48 h to finally obtain a pale yellow solid product. Yield: 81%.1 H NMR (400 MHz, DMSO- d 6 / CD 3 OD (5 / 1, v / v), δ ): 7.65, 7.17 (-C 6 H 4 B(OH) 2 ); 6.98, 6.61 (-C 6 H 4 CH 2 ); 4.53 (-COC H NH-); 3.50 (-OC H 2 C H 2 O-); 2.95 - 2.65 (-C6H4C H 2 -).

[0060] Similarly, using M n = 2.0 kg / mol of MAL-PEG-NH 2 as the initiator, ring-opening polymerization of BPA-NCA and Tyr-NCA was carried out to obtain maleimide-polyethylene glycol- b -poly(4-boron-L-phenylalanine- co -L-tyrosine), yield: 74%. 1 H NMR (400 MHz, DMSO- d 6 / CD 3 OD (5 / 1, v / v), δ ): 7.65, 7.17 (-C 6 H 4 B(OH) 2 ); 6.94, 6.60 (-C 6 H 4 CH 2 ); 6.77 (-C H =C H -); 4.53 (-COC H NH-); 3.51 (-OC H 2 C H 2 O-); 2.90 - 2.64 (-C6H 4 C H 2 -).

[0061] Table 1 Synthesis of Copolymers

[0062] a Calculated by 1 1H NMR; b Measured by MALDI-TOF; c Measured by GPC.

[0063] Copolymers obtained by ring-opening polymerization initiated by different initiators are listed in Table 1. Figure 1 Characterization of the copolymer in Example 1. (A) 1 1H NMR (400 MHz, DMSO- d 6 / CD 3 OD, 5 / 1, v / v) characterization; (B) MALDI-TOF characterization.

[0064] Example 2 Preparation and Interaction Characterization of Micelles First, dissolve the polymers mPEG- b -P(BPA- co -Tyr), MAL-PEG- b -P(BPA- co -Tyr) and the drugs Vol, Nav in DMSO at a concentration of 20 mg / mL, and dissolve the ApoE polypeptide in secondary water (10 mg / mL). Taking 10 wt.% theoretical drug loading and 10 mol.% ApoE density as an example, mix 45 μL of mPEG- b -P(BPA- co -Tyr) and 5 μL of MAL-PEG- b -P(BPA- co -Tyr) polymer solutions, 5.5 μL of Vol and 5.5 μL of Nav solution uniformly, and drop them into 940 μL of phosphate buffer (PB, pH 8.0, 10 mM). Then add 7.7 μL of ApoE solution, and place the mixture in a constant temperature shaking incubator at 120 rpm and 25 °C to react overnight. After the reaction, dialyze the reaction solution to PB 7.4 by gradient method (dialysis bag MWCO: 3500 Da), and dialyze for a total of 8 h, changing the dialysis medium every 1 h to remove organic solvents and free drugs, and finally obtain the targeted co-loaded polypeptide micelles ApoE-PMVN.

[0065] Referring to the above method, change the polymers mPEG- b -P(BPA- co -Tyr) and MAL-PEG- b-P(BPA- co -Tyr), and the amount of ApoE were used to obtain targeted co-loaded polypeptidic micelles with different ApoE ratios; 50 μL of mPEG- b -P(BPA- co -Tyr) was used, and the ApoE solution was omitted to obtain co-loaded polypeptidic micelles PMVN; The preparation methods of micelles loaded with Vol or Nav alone were similar; The drug was omitted to obtain empty micelles; According to the conventional method, Cy5 was conjugated to the micelles for tracing.

[0066] The drug loading content (DLC) and drug loading efficiency (DLE) of the drug-loaded micelles were calculated as follows: DLC (wt.%) = mass of the loaded inhibitor / mass of the inhibitor in the micelles × 100 DLE (%) = mass of the loaded inhibitor / mass of the input inhibitor × 100 Table 2 Characterization of PMV and PMN

[0067] a Determined by HPLC; b Determined by DLS (1.0 mg / mL, 25 °C).

[0068] Table 3 Characterization of ApoE-PMVN

[0069] a Measured by HPLC; b Mass ratio of Vol and Nav in the micelles; c Determined by DLS (1.0 mg / mL, 25 °C); d Determined by electrophoresis (1.0 mg / mL, 25 °C).

[0070] The small molecule inhibitors Vol and Nav can be rapidly encapsulated in PM by the nanoprecipitation method, and the formed micelles have high drug loading capacity. Even when the theoretical drug loading content (DLC) is as high as 30 wt.%, the drug loading efficiency (DLE) of Nav still exceeds 95% (Table 2). When Vol and Nav are co-encapsulated, it has little effect on the drug loading efficiency of Nav. When the theoretical DLC of Vol is 10 wt.%, the DLE is significantly increased to 64% ( Figure 2 A), and it can be maintained above 60% even at 30 wt.%.

[0071] Such as Figure 2As shown in Fig. B, an obvious red shift from 380 nm (free drug mixture) to 428 nm (PMVN) was observed in the ultraviolet spectrum, confirming the existence of π-π stacking interaction between the aromatic groups of Vol, Nav and the BPA and Tyr units. In addition, boron nuclear magnetic resonance spectroscopy ( 11 11B NMR) showed that after mixing Vol, Nav, VN (mixture of Vol and Nav) with BPA, the characteristic signal (31 ppm) of BPA shifted significantly to a higher magnetic field (20 ppm) ( Figure 2 Fig. C).

[0072] The drug-loaded micelles PMVN had a particle size of 91.3 nm, a uniform particle size distribution, a polydispersity index (PDI) of 0.12, and a surface charge close to neutral ( Figure 2 Fig. D). Increasing the ApoE density from 5 mol.% to 20 mol.% had little effect on the DLE (Table 3), with a slight increase in particle size and a change in surface charge from -3.41 mV to +2.75 mV ( Figure 2 Fig. E). Transmission electron microscopy observations showed that ApoE-PMVN presented a spherical morphology and had excellent stability. Under the conditions of storage at 4 °C for 30 days, dilution by 50 times, and containing 10% FBS, its particle size and particle size distribution hardly changed ( Figure 2 Fig. F). It is worth noting that the presence of proteinase K (PK) led to obvious swelling and decomposition of ApoE-PMVN ( Figure 2 Fig. G), and about 80% of the drug was released within 48 h ( Figure 2 Fig. H). Within 48 h, the mass ratio of Vol and Nav released from ApoE-PMVN was about 1:1, close to the ratio of the drugs encapsulated in the micelles ( Figure 2 Fig. I). In the absence of PK, ApoE-PMVN showed good stability, with less than 22% of the drug leaking ( Figure 2 Fig. H).

[0073] Example 3 ApoE-PM Cell Uptake and In Vitro Blood-Brain Barrier Penetration Ability The specific steps of the cell endocytosis experiment are as follows: Inoculate 1.8 mL of cell suspension in a 6-well plate (3×10 5 / well) and culture overnight. Add 0.2 mL of PM-Cy5 or ApoE-PM-Cy5 (Cy5 concentration: 2.0 μg / mL) to each well, and the ApoE densities are 0 mol.%, 5 mol.%, 10 mol.%, 15 mol.%, and 20 mol.%, respectively. After culturing in an incubator for 4 h, aspirate the upper culture medium, wash once with PBS, collect the cells, wash twice with PBS, then resuspend with 0.4 mL of PBS, and finally detect the Cy5 fluorescence intensity with a flow cytometer.

[0074] The specific steps of the CLSM experiment are as follows: Inoculate 1.8 mL of cell suspension in a glass-bottom culture dish (1×10 5 / well) and culture overnight. Add 0.2 mL of PM-Cy5 or ApoE-PM-Cy5 (ApoE density: 10 mol.%, Cy5 concentration: 4.0 μg / mL) to each well. After incubating in the incubator for 2 h or 4 h, aspirate the upper medium and wash 3 times with PBS. First, fix with 4% paraformaldehyde at room temperature for 15 min, then wash 3 times with PBS. Next, stain the cell nuclei with DAPI for 5 min, and finally wash 5 times with PBS and completely aspirate the PBS. Observe and photograph with CLSM.

[0075] The steps of the in vitro blood-brain barrier penetration experiment are as follows: Inoculate 200 μL of bEnd.3 cell suspension in the upper chamber of a 24-well plate equipped with a transwell insert (3×10 5 / well), and add 600 μL of medium to the bottom. Culture for 7 days, and change the fresh medium every 2 days. When the transwell monolayer transmembrane resistance is higher than 200 Ω·cm 2 , it is determined that a complete cell barrier is formed. Then inoculate the GL261-Luc cell suspension at the bottom of the well plate (1×10 5 / well). After culturing overnight, add PM-Cy5 or ApoE-PM-Cy5 (ApoE density: 10 mol.%, Cy5 concentration: 2.0 μg / mL) to the chambers respectively. After incubating for 4, 8, and 24 h, collect the bEnd.3 cells in the chamber and the GL261-Luc cells at the bottom, and detect the Cy5 fluorescence intensity with a flow cytometer.

[0076] ApoE-modified micelles promoted the uptake of micelles by GL261-Luc cells, and the cellular uptake amount depended on the density of ApoE. Among them, the micelles modified with 10 mol.% ApoE showed an uptake amount 10.2 times that of the unmodified micelles ( Figure 3 A, B), while when the ApoE density was increased to 15 mol.% and 20 mol.%, the promoting effect on cellular uptake was negligible. The results observed by laser scanning confocal microscopy showed that obvious Cy5 fluorescence could be observed after incubating ApoE-PM with GL261-Luc cells for 2 h. When the incubation time was extended to 4 h, the fluorescence intensity was stronger, and the fluorescence was almost distributed in all cells ( Figure 3 C). In contrast, weak Cy5 fluorescence was shown when the unmodified ApoE micelles PM were co-incubated with GL261-Luc cells for 4 h. This was consistent with the results detected by the flow cytometer, further indicating that ApoE-PM could be rapidly and efficiently taken up by cells.

[0077] The blood-brain barrier is a major obstacle in the treatment of glioblastoma. A blood-brain barrier model was established in vitro using bEnd.3 cells. PM or ApoE-PM was added to the transwell chambers and co-incubated with bEnd.3 cells for 4, 8, and 24 h. ApoE-PM showed efficient endocytosis in both bEnd.3 and GL261-Luc cells. At 4 and 8 h, the Cy5 fluorescence intensity of ApoE-PM in GL261-Luc cells was 4.4 and 3.4 times higher than that of PM ( Figure 4 ). These results indicate that ApoE-modified micelles can effectively penetrate the BBB and actively target GL261-Luc cells.

[0078] Unless otherwise specified, the ApoE density was 10 mol% in subsequent experiments.

[0079] Example 4 Investigation of in vitro cytotoxicity The toxicity of the drug to GL261-Luc cells was determined by the MTT method. The specific steps were as follows: 80 μL of cell suspension was inoculated into a 96-well plate (5×10 3 / well) and cultured overnight. 20 μL of the control group (PBS) or the experimental group (samples with set concentrations and drug ratios) was added to each well. After co-incubation in an incubator for 4 h, the fresh medium was replaced and incubation continued for 44 h. Subsequently, 10 μL of MTT solution (5.0 mg / mL) was added to each well and incubated in the incubator for 4 h. The upper layer of the medium was carefully aspirated, and the purple formazan crystals formed at the bottom were dissolved with 150 μL of DMSO. Finally, the OD at 570 nm was detected by a multi-functional microplate reader. Six replicates were set for each concentration group. The relative survival rate IC 50 and the combination index CI were calculated according to the following calculation formulas. The toxicity of ApoE-PM to GL261-Luc and L929 cells was also evaluated by the MTT method, and the method was the same as above, that is, the relative viability of the cells was obtained.

[0080] Cell survival rate (%) = OD of experimental group / OD of control group × 100 CI = E A / D A + E B / D B where E A and E B represent the concentrations of drug A and drug B respectively when the combination reaches IC 50 , and D A and D B represent the concentrations of single inhibitors when reaching IC 50The concentrations of drug A and drug B at that time. CI < 1, CI = 1, and CI > 1 represent synergistic, additive, and antagonistic effects of A and B, respectively, and the lower the data, the better.

[0081] Table 4 IC 50 and CI values in GL261-Luc cells for different formulations

[0082] The MTT results showed that compared with the free small molecule inhibitors, encapsulating Vol or Nav in micelles significantly enhanced their toxicity to GL261-Luc cells ( Figure 5 A). The micelle PMVN co-encapsulating these two inhibitors further increased the cytotoxicity, and the IC 50 decreased significantly (from 0.11 to 0.08 μg / mL) (Table 4), which was about 1 / 10 of the IC50 of PMV and PMN. As the ratio of Vol / Nav increased from 1 / 0.5 to 1 / 1, the IC 50 of PMVN decreased significantly, and then leveled off when the ratio was further increased to 1 / 2 ( Figure 5 B). It is worth noting that in the range of Vol / Nav mass ratio from 1 / 0.5 to 1 / 2, the CI values of PMVN were all lower than 0.5 (Table 4), confirming the synergistic cytotoxic effect of these two inhibitors in polymer micelles. Surprisingly, compared with free VN, PMVN showed stronger antitumor activity, and the IC 50 was slightly lower than the sum of PMV and PMN at the same concentration ( Figure 5 B), indicating that co-encapsulating two small molecule inhibitors in micelles can produce a better synergistic effect than separately encapsulating and then mixing them, verifying the importance of delivering two inhibitors to tumor cells simultaneously. ApoE-PMVN has better active targeting ability and shows higher antitumor cell activity. More importantly, even at a concentration as high as 100 μg / mL, the cell viability of the blank micelle PM containing 10 mol.% ApoE for normal cells (L929) and tumor cells (GL261-Luc) remained above 85% ( Figure 5 C, D), indicating that the micelles have good biocompatible safety.

[0083] Example 5 Western Blotting, Mitochondrial Membrane Potential Changes, Apoptosis, and Cell Cycle Analysis The protein expression in GL261-Luc cells after treatment with ApoE-PMVN was characterized by Western blotting. The specific steps are as follows: Inoculate 1.8 mL of cell suspension in a 6-well plate (1×10 6 / (well), after overnight incubation, 0.2 mL of PBS, ApoE-PMV, ApoE-PMN, and ApoE-PMVN (Vol: 0.2 μg / mL, Nav: 0.2 μg / mL) were added to each well. After 24 h of incubation, the cells were collected, 100 μL of cell lysate was added to lyse the cells, and then centrifuged at 12,000 rpm at 4 °C for 15 min. The supernatant was aspirated to quantify the protein concentration by BCA, and 20% (v / v) bromophenol blue loading buffer was added. The mixture was placed in a 95 °C water bath for 5 min to denature the protein. 20 μg of protein from each group was separated by 12.5% SDS-PAGE gel electrophoresis, transferred to a PVDF membrane after electrophoresis, blocked with Tris-HCl buffer / 0.1% Tween-20 (TBST) containing 5% skim milk powder at room temperature for 1 h, incubated with corresponding primary antibody solutions of different molecular weights overnight at 4 °C, then incubated with HRP-labeled secondary antibody solution at room temperature for 1.5 h, and finally developed and photographed with a chemiluminescence detection system after treatment with the developer solution.

[0084] The change of mitochondrial membrane potential was explored by JC-1 fluorescent probe. The specific steps were as follows: 1.8 mL of cell suspension was inoculated in a 12-well plate (5×10 5 / (well), then 0.2 mL of PBS, ApoE-PMV, ApoE-PMN, and ApoE-PMVN (10 mol.% ApoE) were added to each well. After the cells were incubated in an incubator for 24 h, they were centrifuged and collected, and then 500 μL of fresh medium JC-1 staining working solution was added successively. After the mixture was homogenized, it was incubated at 37 °C for 20 min, then centrifuged to discard the supernatant, washed and resuspended with JC-1 staining buffer, and the change of mitochondrial membrane potential was detected by flow cytometry.

[0085] The apoptosis of AML cells induced by PMQV was detected by double staining with Annexin V-allophycocyanin (APC) and 7-amino-actionomycin D (7-AAD) fluorescence. The specific steps were as follows: 1.8 mL of cell suspension was inoculated in a 6-well plate (4×10 5 / (well), then 0.2 mL of PBS, ApoE-PMV, ApoE-PMN, and ApoE-PMVN were added to each well. The drug concentration was the same as that in the Western blot experiment. After the cells were incubated in an incubator for 48 h, they were collected, washed with PBS, resuspended with binding buffer, 5 μL of Annexin V-APC and 10 μL of 7-AAD staining solution were added, and incubated at 37 °C in the dark for 5 min. Subsequently, the apoptosis of the cells was detected by flow cytometry.

[0086] The cell cycle arrest was detected by PI / RNase A staining method. The specific steps were as follows: 1.8 mL of cell suspension was inoculated in a 12-well plate (4×10 5 / well), and then an equal volume of 0.2 mL of PBS, ApoE-PMV, ApoE-PMN, and ApoE-PMVN was added to each well. After incubation for 24 h, the cells were collected by centrifugation, washed and resuspended with PBS, and then gradually added dropwise to 4 mL of ice-cold 95% ethanol under low-speed stirring. After mixing evenly, the cells were fixed at 4 °C for 18 h. After washing with PBS, the cells were resuspended in cell staining buffer, and 15 μL of PI staining solution and 4 μL of RNase A were added. The cells were incubated at 37 °C in the dark for 30 min, and then the cell cycle arrest was detected by flow cytometry.

[0087] Western blot analysis showed that ApoE-PMV and ApoE-PMVN significantly downregulated the expression of PLK1 in GL261-Luc cells ( Figure 6 A). In addition, cleaved caspase-3 expression was significantly increased in cells treated with ApoE-PMN, and further enhanced after treatment with co-loaded micelles ApoE-PMVN. The changes in mitochondrial membrane potential were explored using JC-1 dye. The results showed that all micellar drugs significantly reduced the "J-aggregates" and increased the JC-1 monomers in GL261-Luc cells ( Figure 6 B). Compared with the aggregate percentages of 72.3% and 69.4% after treatment with ApoE-PMV and ApoE-PMN ( Figure 6 C), the proportion of aggregates after treatment with ApoE-PMVN was less, only 24.4%, verifying that ApoE-PMVN had the best effect on disrupting the outer mitochondrial membrane. Annexin V-APC / 7-AAD staining showed that all micellar drugs induced a large number of apoptotic cells in GL261-Luc cells. Among them, treatment with ApoE-PMVN led to approximately 54.3% of the cells undergoing late apoptosis and 25.7% of the cells undergoing early apoptosis ( Figure 6 D). In addition, ApoE-PMV could effectively inhibit the expression of PLK1, a major regulator of mitosis, resulting in 71.5% of the cells being arrested in the G2 / M phase ( Figure 6 E). It is worth noting that the cells treated with ApoE-PMVN showed a higher cell cycle arrest (88.3%). These results fully demonstrated that two small molecule inhibitors were loaded and released controllably by micelles to exert a synergistic effect.

[0088] Example 6 Transcriptome Gene Analysis Sequencing Evaluating the changes in RNA of GL261-Luc cells after treatment with different nanodrugs using RNA-seq, the specific steps are as follows: Inoculate 4 mL of GL261-Luc cell suspension in a T25 culture flask (5×10 6 / well), after incubating overnight, add 1 mL of PBS, free VN, ApoE-PMV, ApoE-PMN, and ApoE-PMVN (Vol: 0.2 μg / mL, Nav: 0.2 μg / mL) to each flask, incubate for 24 h, collect the cells, add 1 mL of TRIzol, and then construct a library and analyze according to the conventional method.

[0089] Compared with free VN, ApoE-PMVN induced significantly more differentially expressed genes (757 and 503 genes respectively), which means that significant changes occurred in the transcriptome ( Figure 7 A). Volcano plot analysis further revealed the gene differences between the two groups. The abscissa represents the fold change in gene expression relative to the control group, and the larger the absolute value of log 2 FC, the greater the difference of the gene. The ordinate represents the significance level of the expression difference. Red circles represent significantly upregulated genes, green diamonds represent significantly downregulated genes, and blue squares represent genes with no difference. Among them, after incubation of ApoE-PMVN cells, compared with PBS, the expression of 844 genes was upregulated and the expression of 1555 genes was downregulated ( Figure 7 B); compared with incubation with free VN, the expression of 148 genes was upregulated and the expression of 578 genes was downregulated.

[0090] Perform cluster heatmap analysis on the significantly different genes in each group. The abscissa is different groups, and the ordinate is the gene name. The color change from red to blue represents the increase to decrease in expression level. It is worth noting that the pro-apoptotic Bcl2l11 (Bim) gene was significantly upregulated, while the key survival genes including BCL-2, PLK1, CDK4, and MCL-1 were significantly downregulated ( Figure 7 C). The downregulation of MCL-1 and the upregulation of Bim by ApoE-PMVN can prevent the resistance of MCL-1 to BCL-2 inhibitors, thus producing a synergistic cytotoxic effect. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the cells treated with ApoE-PMVN had a higher proportion in the "cancer-related pathways" (including p53 signaling pathway, apoptosis, and cell cycle regulation, etc.) ( Figure 7 D), confirming that ApoE-PMVN can have multiple effects on apoptosis-related targets simultaneously.

[0091] Example 7 In vivo biodistribution of ApoE-PM All animal experiments were approved by the Animal Care and Use Committee of Soochow University, and all experimental protocols complied with the Guide for the Care and Use of Laboratory Animals. Using a stereotaxic apparatus, 5 μL (2.5 μL of Matrigel and 2.5 μL of PBS) of GL261-Luc cell suspension (5×10 4 / mouse) was inoculated into the brains of C57BL / 6 mice. When inoculating, the anterior cranial was used as the coordinate origin, 0.5 mm anterior, 1.9 mm left, and 3.1 mm deep, and the needle was left in place for 5 min. On the 10th day after modeling, an IVIS spectrum near-infrared fluorescence imaging system was used to detect the modeling situation in vivo. The mice were randomly divided into 3 groups, and PM-Cy5 or ApoE-PM-Cy5 (ApoE density: 5 mol.% or 10 mol.%, Cy5 concentration: 50 μg / mouse) was injected via the tail vein. In vivo imaging was performed using the IVIS imaging system at 4, 8, and 12 h after injection. 10 min before imaging, the mice were intraperitoneally injected with potassium D-luciferin (75 mg / kg). At 12 h after injection, the mice were sacrificed, and the main organs and brain tissues were dissected and subjected to ex vivo imaging analysis using the IVIS imaging system.

[0092] After intravenous injection via the tail vein, obvious fluorescence of ApoE-PM was produced in the mouse brain during the entire experimental period, while only a small amount of Cy5 fluorescence was observed in the mouse brain within 12 h after injection of PM ( Figure 8 A). The ApoE concentration is an important factor affecting the enrichment of PM. The fluorescence intensity of ApoE-PM with an ApoE density of 10 mol.% was significantly higher than that of ApoE-PM with 5 mol.% at 4, 8, and 12 h ( Figure 8 B). Ex vivo imaging further confirmed that at 12 h after administration, the accumulation of ApoE-PM in the brains of mice treated with 10 mol.% ApoE was the highest, and the fluorescence intensity was approximately twice that of ApoE-PM with 5 mol.% ApoE ( Figure 8 C).

[0093] Example 8 Antitumor effect in vivo The antitumor effect of ApoE-PMVN on orthotopic GBM was investigated by modeling according to the same method as above. As Figure 9 shown in A, all mice were randomly divided into 6 groups and intravenous injection was started on the 3rd day after modeling (PBS, free VN, PMVN, ApoE-PMV, ApoE-PMN, and ApoE-PMVN), once every 3 days, for a total of 6 injections. The dosing doses of Vol and Nav were 10 mg / kg and 10 mg / kg, respectively. The body weight changes of the mice were detected during the treatment period, and the survival time was recorded.

[0094] Compared with the sharp weight loss of mice in the PBS group, the mice treated with the micellar inhibitor showed a slight increase in body weight ( Figure 9 B), indicating that the micellar inhibitor has good safety and can inhibit tumor progression to a certain extent. The Kaplan-Meier survival curves showed that the median survival times of free VN, PMVN, and ApoE-PMVN were 25.5, 27, and 36 days, respectively, and the survival rates of the mice increased in turn ( Figure 9 C). Notably, 1 / 3 of the mice survived without tumors after treatment with ApoE-PMVN, which was significantly higher than that of the micellar drugs loaded with a single inhibitor (ApoE-PMV and ApoE-PMN), further confirming the powerful efficacy of the micellar inhibitor combined in an appropriate ratio.

[0095] The present invention discloses a glioblastoma-targeted co-delivery of PLK1 inhibitor and BCL-2 / xL inhibitor based on ApoE peptide-modified polypeptidic micellar inhibitor. The present invention has the following characteristics: 1) Through the B-N coordination, π-π stacking and hydrophobic interaction between the phenylboronic acid group and the small molecule inhibitor, efficient co-loading in proportion and enzyme-responsive release of small molecule inhibitors with different physicochemical properties and molecular targets are achieved; 2) It can effectively cross the in vitro blood-brain barrier model, actively target glioblastoma cells and be efficiently taken up, showing a powerful synergistic anti-proliferation effect; 3) The ApoE-PMVN micelles co-loaded with Vol / Nav at a mass ratio of 1 / 1 have a synergistic inhibitory effect on targets such as BCL-2, BCL-xL, and MCL-1, can significantly inhibit the growth of tumors in mice with orthotopic GL261-Luc glioblastoma, and significantly prolong their survival time. In summary, the ApoE peptide-modified polypeptidic micellar inhibitor provides a general and effective platform for the co-active delivery of small molecule inhibitors with different molecular targets and shows great potential in the synergistic treatment of glioblastoma.

Claims

1. A polypolypeptide micelle inhibitor, characterized in that: It includes a polypolypeptide with a phenylboronic acid group and a small molecule drug encapsulated therein; or it includes a polypolypeptide with a phenylboronic acid group connected to a targeting molecule and a small molecule drug encapsulated therein.

2. The polypolypeptide micelle inhibitor according to claim 1, characterized in that: Small molecule drugs include PLK1 inhibitors and / or BCL-2 / xL inhibitors.

3. The polypolypeptide micelle inhibitor according to claim 1, characterized in that: The raw materials for preparing the polypeptide with phenylboronic acid groups include a macromolecular initiator, 4-boron-L-phenylalanine and L-tyrosine.

4. The polypolypeptide micelle inhibitor according to claim 3, characterized in that: The molecular weight of the macromolecular initiator is 0.5kg / mol to 5 kg / mol; the molar ratio of 4-boron-L-phenylalanine to L-tyrosine is (2 to 5): 1; when the polypolypeptide micelle inhibitor is connected with a targeting molecule, the macromolecular initiator includes a macromolecule with a targeting molecule connecting group and a macromolecule without a targeting molecule connecting group, and the obtained polypolypeptide with a phenylboronic acid group is a polypolypeptide with a phenylboronic acid group and connected with a targeting molecule connecting group.

5. A polypolypeptide for micelle inhibitor, characterized in that: It includes a polypeptide with a phenylboronic acid group or a polypeptide with a phenylboronic acid group and connected with a targeting molecule.

6. The method for preparing the polypolypeptide micelle inhibitor according to claim 1, characterized in that: The method comprises the following steps: mixing a polypolypeptide solution with phenylboronic acid groups and a small molecule drug solution, and then dialyzing to obtain a polypolypeptide micelle inhibitor; or mixing a polypolypeptide solution with phenylboronic acid groups, a polypolypeptide solution with phenylboronic acid groups and connected with a targeting molecule linking group, a small molecule drug solution, and a targeting molecule solution, and then dialyzing to obtain a polypolypeptide micelle inhibitor.

7. The method for preparing the polypolypeptide micelle inhibitor according to claim 6, characterized in that: The molar ratio of the polypeptide with phenylboronic acid group and the polypeptide with phenylboronic acid group and connected with targeting molecule linking group is (2-20):

1.

8. Use of the polypolypeptide micelle inhibitor according to claim 1 or the polypolypeptide for micelle inhibitor according to claim 5 in the preparation of drugs.

9. A drug, the active ingredient of which comprises the polypolypeptide micelle inhibitor according to claim 1.

10. The use according to claim 8 or the medicine according to claim 9, characterized in that: The drug is an anti-tumor drug.

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