A polyamino acid-based small molecule inhibitor nanoparticle, its preparation method and application

Abstract

The present invention discloses a small molecule inhibitor nanoparticle based on polyamino acid, which is composed of polyethylene glycol b ‑Poly(4‑boron‑L‑phenylalanine‑ What Specifically, polyethylene glycol- b ‑Poly(4‑boron‑L‑phenylalanine‑ What A solution of a β-L-tyrosine (L-tyrosine) copolymer and a drug solution were added dropwise to a buffer solution. After the addition was complete, dialysis was performed to produce polyamino acid-based small molecule inhibitor nanoparticles. These nanoparticles exhibited excellent drug co-encapsulation and stability, and rapidly released the drug in an acidic / hydrogen peroxide / enzyme environment, achieving synergistic killing of leukemia cells and significantly inhibiting leukemia cell infiltration in organs such as the bone marrow, spleen, and liver. The nanomedicine designed in this invention exhibits high drug loading efficiency, excellent stability, and trigger-responsiveness. Furthermore, this intelligent nanomedicine boasts a simple structure, adjustable drug combinations, high safety, and strong synergistic efficacy, making it readily applicable to cancer treatment.

Description

Technical Field

[0001] The present invention belongs to polymer drug delivery technology, and specifically relates to a polyamino acid-based phenylboronic acid functionalized polymer and small molecule inhibitor nanoparticles, and a preparation method and application thereof. Background Art

[0002] Compared to chemotherapeutic drugs, small molecule inhibitors offer advantages such as high specificity, low off-target toxicity, and significant anticancer effects. However, single-agent inhibitors generally exhibit suboptimal therapeutic effects, necessitating their combination with other agents to enhance efficacy. However, inhibitor combinations often require continuous and repeated dosing due to their inherently poor bioavailability and susceptibility to metabolism. Polypeptides, with their excellent biocompatibility, have gained widespread application in drug delivery and other fields. While polypeptide-based nanomedicines can improve drug solubility and pharmacokinetics to some extent, they still suffer from limitations such as poor stability and uncontrollable drug release (i.e., premature drug release during circulation and slow drug release at the tumor site). Furthermore, polypeptide-based nanocarriers suffer from uncontrollable drug ratios and low drug loading efficiency when co-encapsulating two or more drugs. Therefore, the development of polypeptide nanomedicines with excellent stability, responsive drug release (e.g., pH-responsive, ROS-responsive, or enzyme-responsive), controllable drug loading ratios, and high drug loading efficiency has become a research hotspot. Summary of the Invention

[0003] This invention uses polyamino acid-based nanoparticles to effectively co-load and responsively release multiple small molecule inhibitors. The invention constructs phenylboronic acid-functionalized nanoparticles co-loaded with small molecule inhibitors for synergistic tumor treatment. Results demonstrate that the polyamino acid-based nanoparticles achieve efficient loading and delivery of drugs, including small molecule inhibitors, and, in particular, enhance the synergistic therapeutic effect of nanoparticles against tumors.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] A small molecule inhibitor nanoparticle based on polyamino acid, composed of polyethylene glycol- b -Poly(4-borono-L-phenylalanine- co -L-tyrosine) copolymer loaded with small molecule inhibitors. Specifically, it includes the following steps: b -Poly(4-borono-L-phenylalanine- co A solution of a β-L-tyrosine (β-L-tyrosine) copolymer and a drug solution are added dropwise to a buffer solution. After the addition is complete, the solution is dialyzed to obtain polyamino acid-based small molecule inhibitor nanoparticles. Preferably, the drug solution contains one or more drugs. Preferably, the drug is a small molecule inhibitor, including one or more of a BCL2 inhibitor, an MCL1 inhibitor, and a PLK1 inhibitor.

[0006] In the present invention, polyethylene glycol- b -Poly(4-borono-L-phenylalanine- co -L-tyrosine) copolymer has the structure of formula I:

[0007]

[0008] Formula I

[0009] Wherein, n is 70-210, m is 17-55, and x is 11-42; preferably, n is 90-150, m is 24-40, and x is 16-27.

[0010] The present invention discloses the use of the above-mentioned small molecule inhibitor nanoparticles based on polyamino acids in the preparation of anti-tumor nanomedicines or in the preparation of nanomedicines with synergistic effects.

[0011] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0012] The polyethylene glycol designed and prepared by the present invention b -Poly(4-borono-L-phenylalanine- co -L-tyrosine) copolymers exhibit excellent biocompatibility and enzymatic degradation, and are simple to prepare with reproducible controllability. The designed polypeptide nanoparticles exhibit controllable particle size (79-148 nm), a narrow size distribution, excellent stability, and responsive release of drugs (acids / reactive oxygen species / enzymes). They achieve synergistic killing of leukemia cells, significantly inhibiting leukemia cell infiltration in organs such as the bone marrow, spleen, and liver. The nanodrug designed in this invention exhibits high drug loading efficiency, excellent stability, and trigger-responsiveness. Furthermore, this intelligent nanodrug boasts a simple structure, adjustable drug combinations, high safety, and strong synergistic efficacy. It can be easily applied to cancer treatment, providing a simple strategy for improving drug efficacy and enabling effective and safe combined synergistic treatments for different cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The polymer PEG- b -P(BPA- co -Tyr) characterization (Table 1, No. 1). (A) 1 H NMR (400 MHz, DMSO- d 6 / CD3OD- d 4, 5 / 1, v / v) characterization; (B) MALDI-TOF characterization;

[0014] Figure 2The following are the physical and chemical characterizations of the polymer in Example 1 and the micelles in Example 2. (A) Particle size distribution and TEM image of NPAT; (B) Particle size and PDI changes of NPAT (1.0 mg / mL) under long-term storage. (C) Particle size and PDI changes of NPAT (1.0 mg / mL) in 10% FBS and 100-fold dilution; (D) Particle size changes of NPAT under the action of pH 5.5, 100 μM H2O2 and proteinase K (PK, 12 U / mL); In vitro drug release behavior of NPAT under the action of (E) acidic (pH 5.5), (F) H2O2 (100 μM), and (G) PK (12 U / mL); (H) Particle size changes of NPAV over time under the action of proteinase K (PK, 12 U / mL); (I) In vitro drug release behavior of NPAV under the action of PK (12 U / mL);

[0015] Figure 3 is the cytotoxicity of empty nanoparticles incubated with (A) L929, (B) MOLM-13-Luc, and (C) MV-411 cells for 48 h;

[0016] Figure 4 Figure 3: Hemocompatibility and cytotoxicity of NPAT in Example 3: (A) Representative images of erythrocytes co-treated with free ABT199, TW37, ABT199 / TW37, NPA, NPT, and NPAT (low concentration: 100 μg / mL, high concentration: 200 μg / mL). 1% Triton X-100 and 0.9% NaCl were used as positive and negative controls, respectively; (B) Hemolysis rate of erythrocytes after treatment with different samples; Cytotoxicity of different samples co-incubated with (CE) MOLM-13-Luc and (FH) MV-411 cells for 48 h;

[0017] Figure 5 Figure 4 shows the protein expression and apoptosis induced by free ABT199, TW37, ABT199 / TW37 (A:T = 1:1), NPA, NPT, and NPAT (A:T = 1:1) in MOLM-13-Luc (ABT199: 10 ng / mL, TW37: 10 ng / mL) and MV-411 (ABT199: 60 ng / mL, TW37: 60 ng / mL) cells. (A) Western blot analysis was used to test the protein expression induced by the nanomedicines. (B) The apoptosis-inducing ability of the free and (C) nanomedicines was determined by annexin V-APC / 7-AAD double staining.

[0018] Figure 6This is the in vivo anti-tumor experiment of NPAT in Example 5. In vivo treatment of mice bearing orthotopic MOLM-13-Luc AML with NPAT (n = 4): Mice were intravenously injected with PBS, NPT, NPA, or NPAT on days 0, 3, 6, and 9. Leukemic cell infiltration was assessed using bioluminescence imaging on days 5, 8, 11, and 14. (A) Experimental design; (B) Bioluminescence imaging; (C) Quantitative analysis of bioluminescence changes over time; (D) Mouse spleen weight on day 14; (E) Body weight change.

[0019] Figure 7 This is the in vivo anti-tumor experiment of NPAT in Example 5. (A) Analysis of MOLM-13-Luc leukemia cell infiltration in bone marrow (BM), lung (Lu), liver (Li), spleen (Sp), and peripheral blood (PB); (B) Quantitative analysis of leukemia cell infiltration (n = 3);

[0020] Figure 8 This is the in vivo anti-tumor study of NPAT in Example 5. Micro-CT analysis of the femur and tibia of mice in different treatment groups. (A) Micro-CT images; (B) Quantitative analysis of various indicators: bone mineral density (BMD), bone volume / tissue volume (BV / TV), trabecular separation / spacing (Tb.Sp), trabecular thickness (Tb.Th), trabecular number (Tb.N), and bone surface area / tissue volume (BS / TV) (n = 3).

[0021] Figure 9 This is the in vivo anti-tumor study of NPAT in Example 5. Analysis of femurs and tibias of mice in different treatment groups. (A) H&E staining; (B) Analysis of osteoclasts by TRAP staining. Scale bar: 100 μm.

[0022] Figure 10 This is the in vivo anti-tumor experiment of NPAT in Example 5. Complete blood count and blood biochemistry tests (n = 3). Blood was collected from the celiac artery on day 14 for analysis of white blood cells (WBC), red blood cells (RBC), platelets (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and urea.

[0023] Figure 11 This is the in vivo anti-tumor experiment of NPAT in Example 5. H&E staining analysis of the spleen (Sp), kidney (Ki), liver (Li), heart (He), and lung (Lu) of orthotopic MOLM-13-LucAML-bearing mice 14 days after administration. Scale bar: 100 μm.

[0024] Figure 12This is the in vivo anti-tumor experiment with NPAT in Example 5. The in vivo therapeutic effect of NPAT on orthotopically MV-411 AML-bearing mice (n = 4). PBS, NPT, NPA, or NPAT were intravenously injected on days 0, 3, 6, and 9, and leukemic cell infiltration was assessed by bioluminescence imaging on days 5, 8, 11, and 14. (A) Experimental design; (B) MV-411 cell infiltration in BM, Lu, Li, Sp, and PB; (C) Quantitative analysis of cell infiltration; (D) Time-dependent changes in MV-411 cell counts in the peripheral blood of mice in the PBS group; (E) Body weight changes; (F) Spleen weights of mice on day 17 after receiving different drug treatments. DETAILED DESCRIPTION

[0025] The present invention constructs a polyamino acid small molecule inhibitor nanomedicine, specifically relates to nanoparticles prepared from polyethylene glycol and a polyamino acid material based on 4-boron-L-phenylalanine and L-tyrosine, which encapsulate the small molecule inhibitor drug and its application in tumor treatment.

[0026] The preparation method of the polyamino acid-based small molecule inhibitor nanoparticles disclosed in the present invention comprises the following steps:

[0027] (1) Under nitrogen conditions, polyethylene glycol with a single amino end was used as an initiator to obtain polyethylene glycol- b -Poly(4-borono-L-phenylalanine- co -L-tyrosine) copolymer PEG- b -P(BPA- co -Tyr);

[0028] (2) While stirring, add polyethylene glycol- b -Poly(4-borono-L-phenylalanine- co A mixed solution of a poly(amino acid-containing) copolymer and a drug is added dropwise to a buffer solution, and after the addition is complete, dialyzed to obtain small molecule inhibitor nanoparticles based on poly(amino acid).

[0029] In the above technical solution, in step (1), the mass ratio of polyethylene glycol with an amino group at one end, 4-boron-L-phenylalanine-N-carboxylic anhydride, and L-tyrosine-N-carboxylic anhydride is 1:0.62~1.23:0.32~0.95, preferably 1:1.23:0.32, the temperature of the ring-opening polymerization is room temperature~80°C, and the time is 60~80 hours; preferably, the ring-opening polymerization is carried out in a solvent, and the solvent is preferably DMF.

[0030] The above polyethylene glycol- b -Poly(4-borono-L-phenylalanine- coThe specific reaction steps of the preparation scheme of the copolymer of tyrosine and L-tyrosine can be exemplified as follows:

[0031] Polyethylene glycol (PEG-NH2) with a single amino group at one end was used as a macroinitiator to initiate the ring-opening polymerization (ROP) of 4-boron-L-phenylalanine-N-carboxylic anhydride (BPA-NCA) and L-tyrosine-N-carboxylic anhydride (Tyr-NCA) to prepare polyethylene glycol- b -Poly(4-borono-L-phenylalanine- co -L-tyrosine) copolymer. Under nitrogen atmosphere, BPA-NCA, Tyr-NCA and PEG-NH2 DMF solution were mixed and reacted for three days. After precipitation in excess ether, the crude product was further purified by redissolving in dichloromethane and precipitating in ether three times. The obtained precipitate was vacuum dried to obtain polyethylene glycol- b -Poly(4-borono-L-phenylalanine- co -L-tyrosine).

[0032] The above preparation scheme can be expressed as follows:

[0033]

[0034] As one of the specific technical solutions of the present invention, when the drugs are BCL2 inhibitor ABT199 and MCL1 inhibitor TW37, the nano drug NPAT that simultaneously encapsulates ABT199 and TW37 is prepared by polyethylene glycol- b -Poly(4-borono-L-phenylalanine- co The mixed solution of ABT199 solution and TW37 solution (DMSO) was prepared by solvent replacement method. The specific process was as follows: Under stirring, the calculated amount of ABT199 solution and TW37 solution (DMSO) were mixed with polyethylene glycol- b -Poly(4-borono-L-phenylalanine- co -L-tyrosine) solution (DMSO) was mixed and dispersed in HEPES buffer (pH 7.4); then placed in a dialysis bag (MWCO = 3500Da), dialyzed in HEPES buffer for 4 hours to remove unloaded drugs and organic solvents, and then dialyzed in PBS buffer (pH 7.4) for 2 hours to replace the buffer, and the buffer medium was replaced every hour; finally, the nanodrug was obtained.

[0035] As another specific technical solution of the present invention, when the drugs are BCL2 inhibitor ABT199 and PLK1 inhibitor volasertib, the nano drug NPAV that simultaneously encapsulates ABT199 and volasertib is polyethylene glycol- b -Poly(4-borono-L-phenylalanine- coThe mixed solution of ABT199 solution and volasertib solution (DMSO) was prepared by solvent replacement method. The specific process was as follows: Under stirring, the calculated amount of ABT199 solution and volasertib solution (DMSO) were mixed with polyethylene glycol- b -Poly(4-borono-L-phenylalanine- co -L-tyrosine) solution (DMSO) was mixed and dispersed in HEPES buffer (pH 7.4); then placed in a dialysis bag (MWCO = 3500 Da), dialyzed in HEPES buffer for 4 hours to remove unloaded drugs and organic solvents, and then dialyzed in PBS buffer (pH 7.4) for 2 hours to replace the buffer, and the buffer medium was replaced every hour; finally, the nanodrug was obtained.

[0036] The present invention further discloses the use of the polyamino acid-based phenylboronic acid functionalized nanoparticles in the preparation of anti-tumor nanomedicines. Preferably, the nanomedicines are used in the synergistic treatment of acute myeloid leukemia.

[0037] α-Methoxy-ω-amino-polyethylene glycol (mPEG-NH2, M n: 5.0 kg / mol, ≥ 95%, Xiamen Sinobond Biotechnology Co., Ltd.), L-tyrosine (Tyr-OH, Jier Biochemical Shanghai Co., Ltd.), 4-borono-L-phenylalanine (BPA, Beijing Myrida Technology Co., Ltd.), proteinase K (PK, > 40 U / mg, Shanghai Thermo Fisher Scientific Co., Ltd.), TW37 (Med Chem Express), volasertib (Med Chem Express), and ABT199 (Venetoclax, Med Chem Express) were purchased and used directly. Triphosgene (BTC, Shanghai Aladdin Biochemical Technology Co., Ltd.) was recrystallized from ethyl acetate before use. Tetrahydrofuran (THF) and petroleum ether (boiling point 60-90°C) were purified using a solvent purification system (Innovative Technology, USA) and used directly. N,N-dimethylformamide (DMF) was purified using a solvent purification system, dried over anhydrous magnesium sulfate overnight, and distilled under reduced pressure before use. All other reagents, unless otherwise specified, were purchased from Sinopharm Chemical Reagent Co., Ltd. and used directly.

[0038] Polymer H NMR spectroscopy ( 1 H NMR) with DMSO- d 6 / CD3OD- d4 (5 / 1, v / v) as solvent, was used for determination using a Unity Inova-400 MHz superconducting nuclear magnetic resonance spectrometer (Agilent), with chemical shifts standardized to the solvent signal. Polymer molecular weight was determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS, Daltonics Ultraflex II, Bruker) using a mixture of trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) and sodium trifluoroacetic acid (CF3COONa+) (9 / 1, v / v) as the matrix. Polymer molecular weight distribution was determined using a Waters 1515 gel permeation chromatography (GPC). Nanoparticle size and size distribution were determined using a dynamic light scattering instrument (Zetasizer Nano-ZS, Malvern Instruments, USA). Cytotoxicity assays were performed using a multifunctional microplate reader (Varioskan LUX, Thermo Fisher). The micromorphology of the micelles was characterized by transmission electron microscopy (TEM, Tecnai G220, 200 kV, USA). Flow cytometry (Becton Dickinson, FACSVerse, USA) was used to investigate the infiltration of leukemic cells into the bone marrow and various organs. Western blots (WB) were performed on PDVF membranes incubated with developer and then imaged using an ultrasensitive chemiluminescence imager (GE Amersham Imager 600). Hematoxylin and eosin (H&E) staining images were captured using an inverted fluorescence microscope (Nikon Eclipse Ti). The drug concentrations in the drug loading and in vitro release experiments of TW37, volasertib, and ABT199 were determined by ProStar LC240 high-performance liquid chromatography (Waters Alliance HPLC). HPLC was performed using a Sepax BR-C18 reversed-phase column (size: 4.6×250 (mm); particle size: 5 μm; pore size: 120 Å). The test conditions were: flow rate of 0.8 mL / min, injection volume of 10 μL, UV detection wavelength of 300 nm, and the mobile phase was pure acetonitrile / secondary water (containing 0.05% phosphoric acid) (80 / 20, v / v).

[0039] The present invention is further described below with reference to the accompanying drawings and examples. The specific experimental operations and performance tests are conventional techniques.

[0040] Example 1 Polyethylene glycol- b -Poly(4-borono-L-phenylalanine- co -L-tyrosine) synthesis

[0041] Use PEG-NH2 ( Mn = 5.0 kg / mol) was used to initiate the ring-opening polymerization of BPA-NCA and Tyr-NCA monomers in anhydrous DMF to obtain PEG- b -P(BPA- co -Tyr) copolymers. b -P(BPA- co -Tyr)( M For example, a solution of PEG-NH2 (350 mg, 0.07 mmol) in DMF was added to a solution of BPA-NCA (430 mg, 1.83 mmol) and Tyr-NCA (111 mg, 0.57 mmol) in DMF under nitrogen. The mixture was then reacted at 80°C for 72 h. The product was then precipitated with 20 times the volume of glacial ether, centrifuged at 4000 rpm for 5 min, and the precipitate collected. The product was then reconstituted with methanol and precipitated twice under the same conditions. The solid was collected by centrifugation and placed in a vacuum oven for 72 h to remove any residual organic solvent, yielding a white product with a yield of 83%. 1 H NMR (400 MHz, DMSO- d 6 / CD3OD- d 4,5:1, v / v, δ): 7.68 and 7.22 (-C6H4B(OH)2), 6.95 and 6.61 (-C6H4OH), 4.47 and 4.37 (-COCHNH-), 3.52 (-OCH2CH2O-), 2.82 and 2.63 (-C6H4CH2-).

[0042]

[0043] a Calculated by 1H NMR; b Calculated by MALDI-TOF; c Measured by GPC

[0044] The composition ratio of BPA and Tyr in the polymer was adjusted by changing the NCA feed ratio. The results are listed in Table 1. Figure 1 The polymer PEG- b -P(BPA- co -Tyr) characterization (Table 1, Entry 1). (A) 1 H NMR (400 MHz, DMSO- d 6 / CD3OD- d 4, 5 / 1, v / v) characterization; (B) MALDI-TOF characterization.

[0045] Example 2 Preparation and Characterization of Nanoparticles

[0046] TW37 and ABT199 were loaded into nanoparticles by solvent replacement method. b -P(BPA- co A DMSO solution of β-Tyr (20 mg / mL, 50 μL), TW37 (20 mg / mL, 5.6 μL), and ABT199 (20 mg / mL, 5.6 μL) was mixed and added dropwise to 940 μL of HEPES buffer (pH 7.4, 10 mM). The mixture was dialyzed against HEPES buffer for 4 hours and then against PBS (pH 7.4, 150 mM) for another 2 hours to obtain the final nanodrug, NPAT. NPAV (co-loaded with ABT199 and volasertib) was prepared using a similar method. If only one drug was used, the resulting nanodrugs were NPT (loaded with TW37), NPA (loaded with ABT199), and NPV (loaded with volasertib), respectively.

[0047] Encapsulated TW37, ABT199, and volasertib were quantified using high-performance liquid chromatography (HPLC) at absorbances of 300 nm, 289 nm, and 320 nm, respectively. Drug loading capacity (DLC) and drug loading efficiency (DLE) were calculated according to the following formula:

[0048] DLC (wt.%) = drug loading mass / (mass of empty micelles + drug loading mass) × 100%

[0049] DLE (%) = loaded drug mass / total drug mass × 100%

[0050] The nanodrug (1.0 mg / mL) was placed for 30 days, dispersed in PBS solution (containing 10% FBS) or diluted 100 times, and the particle size changes of the nanodrug were observed using DLS detection. The responsiveness of NPAT was investigated by DLS analysis of particle size changes under four conditions: (i) PBS (150 mM, pH 7.4), (ii) PBS (150 mM, pH 5.5), (iii) PBS (150 mM, pH 7.4) containing proteinase K (PK, 12 U / mL), and (iv) PBS (150 mM, pH 7.4) containing 100 μM H2O2. The drug release behavior of NPAT under these four conditions was also investigated. Specifically, 0.5 mL of NPAT solution was placed in a release bag (MWCO: 30 kDa) and placed in 25 mL of different release media solutions. At pre-set time points, 5 mL of the media was aspirated and an equal volume of fresh media was added. After all samples were lyophilized, they were reconstituted with 0.3 mL of acetonitrile / secondary water (5 / 1, v / v). The released TW37 and ABT199 were measured by HPLC. Three parallel groups were set up for each group, and the cumulative drug release was calculated according to the following formula:

[0051]

[0052] Where: E r : Cumulative release of TW37 and ABT199 (%); V e : replacement volume of release medium (5.0 mL); V0: total volume of release medium (25 mL); C i : Concentrations of TW37 and ABT199 in the release medium at the time of sampling (μg / mL); m drug : the total amount of TW37 and ABT199 in NPAT used for release (μg); n: the number of times the medium was replaced.

[0053]

[0054] a Determined by HPLC; b Determined by DLS (PBS 7.4, 25 °C); c The results were determined by electrophoresis in PBS solution (PBS 7.4, 25 °C).

[0055] Table 3 Characterization of NPAV

[0056]

[0057] a Determined by HPLC; b Determined by DLS (PBS 7.4, 25 °C);c Measured by electrophoresis in PBS solution (PBS7.4, 25 °C)

[0058] Empty nanoparticles (NPs) were synthesized by PEG- b -P(BPA- co -Tyr) self-assembled. Select polymer PEG- b -P(BPA- co -Tyr)( M The drug loading capacity of NPAT and NPAV nanoparticles was significantly improved by increasing the loading capacity of ABT199. The excellent co-loading capacity of the two drugs in NPAT was beneficial for adjusting the ratio between the nanoparticles.

[0059] See also Figure 2 The particle size distribution and morphology of NPAT were characterized by TEM, revealing a spherical structure. All drug-loaded NPs (NPT, NPA, and NPAT) exhibited a monodisperse distribution, compared to the polydisperse distribution of empty NPs. Even after a 100-fold dilution, 10% FBS, and 30 days of storage, the particle size and distribution of NPAT remained minimal, demonstrating the high stability of NPAT under normal physiological conditions. This further confirms that the interaction between the drug and the polypeptide segment contributes to the stability of the nanomedicine. The nanoparticles exhibited trigger-responsiveness in acidic or H2O2 environments. Incubation for 6 hours in either pH 5.5 or 100 μM H2O2 resulted in significant swelling and dissociation of the NPs. Furthermore, proteinase K (PK) can degrade the polypeptide segment, and results showed that NPAT also exhibited responsiveness after incubation for 6 hours in the presence of 12 U / mL PK. Consistently, in the environments of pH 5.5, H2O2 (100 μM), and PK (12 U / mL), NPAT released 66%, 80%, and 82% of the total drug, respectively, after 48 h, while in the environment of pH 7.4, the total drug released by NPAT was less than 20%.

[0060] Example 3 Blood compatibility and in vitro cytotoxicity experiments of NPAT

[0061] Cytotoxicity of nanoparticles: First, PEG- b -P(BPA- co-Tyr) to form empty nanoparticles (NPs). Equal volumes of NPs at different concentrations (1 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL) were then incubated with L929, MOLM-13-Luc, and MV-411 cells for 48 hours. A blank control group was treated with an equal volume of PBS. After 48 hours, 10 μL of CCK8 solution was added to each sample well and the cells were incubated at 37°C for approximately 3 hours. Finally, the absorbance (OD) at 450 nm was measured using a multifunctional microplate reader. Cell viability was calculated according to the following formula:

[0062] Cell viability (%) = OD of experimental group / OD of PBS control group × 100%

[0063] Blood from healthy mice was collected in anticoagulant blood collection tubes and then centrifuged at 2500 rpm at 4°C for 10 minutes to remove the upper serum layer. A 0.9% NaCl solution was added and centrifuged again. This operation was repeated several times until the supernatant was colorless, resulting in a 2% red blood cell suspension. Equal volumes of 0.9% NaCl, 1% Triton X-100, TW37, ABT199, TW37 / ABT199, NPT, NPA, and NPAT solutions were then mixed with the red blood cell suspension. The mixture was incubated in a 37°C water bath for 20 minutes. After centrifugation, the absorbance of the supernatant was measured at 570 nm. The 0.9% NaCl solution group served as a negative control, and the 1% Triton X-100 group served as a positive control. The hemolysis rate (HR) was calculated using the following formula:

[0064]

[0065] AS, ANC and APC represent the UV absorbance of the sample, negative control and positive control at 570 nm, respectively.

[0066] The toxicity of drugs to MOLM-13-Luc and MV-411 cells was determined by CCK8 assay. First, MOLM-13-Luc or MV-411 cells (2×10 4 Each well was then added with equal volumes of TW37, ABT199, Volasertib, TW37 / ABT199, Volasertib / ABT199, NPT, NPA, NPV, NPAT, and NPAV at varying concentrations and drug ratios. The cells were incubated for 48 hours using the same testing method as above. The drug synergy index (CI) was calculated as follows:

[0067]

[0068] in,S A and S T Represent the IC of NPA (or ABT199) and NPT (or TW37, Volasertib, NPV), respectively. 50 , C A and C T Represents the IC of ABT199 and TW37 in NPAT (or TW37 / ABT199) respectively 50 Generally speaking, 0.2≤CI<0.4 indicates strong synergy, while higher values indicate weak synergy.

[0069] The toxicity of empty NPs to cancer cells (MOLM-13-Luc, MV-411 cells) and normal cells (L929 cells) was analyzed by CCK8 assay. The results showed that even after incubation with cells at a concentration of 200 μg / mL for 48 h, the cell survival rate was still above 90%. Figure 3 AC), indicating that the polypolypeptide carrier has good biocompatibility and high safety.

[0070] Blood compatibility is one of the important indicators for evaluating the biocompatibility of polymer nanomedicines. Hemolysis experiments were conducted to investigate the hemolysis of drug-loaded nanomicelles. The experimental results showed that the hemolysis rates of free ABT199 at different concentrations were all less than 5%. In the low-concentration free TW37 (100 μg / mL) group, there was little red blood cell sedimentation after centrifugation, and the hemolysis rate was 55%. As the concentration of TW37 increased to 200 μg / mL, there was basically no red blood cell sedimentation after centrifugation, and the hemolysis rate reached over 75%. All nanomedicine groups had significant red blood cell sedimentation and could be redispersed in the corresponding medium. There was no red blood cell aggregation, and the hemolysis rate was less than 5% ( Figure 4 AB). This fully demonstrates that the nanomicelles co-loaded with TW37 and ABT199 have good blood compatibility and also provide greater possibilities for intravenous administration.

[0071] Cytotoxicity results showed that NPT and NPA had higher cytotoxicity than free drugs in both MOLM-13-Luc and MV-411 cells ( Figure 4 Co-loading of NPAT further increased the toxicity, and the IC value of NPAT (ABT199 / TW37, 1:1, w / w) against MOLM-13-Luc cells was 0. 50 The concentration of TW37 in NPAT was 1.15 ng / mL, which was about 3-fold and 222-fold lower than that in NPA and NPT, respectively (Table 4).50 Slightly lower, but the TW37 IC 50 When the amount of TW37 in NPAT was reduced to 1:0.5 (w / w), the IC of ABT199 50 Although NPAT has a higher IC for MV-411 cells than for MOLM-13-Luc cells, 50 However, the nanodrug NPAT (ABT199 / TW37, 1:1, w / w) had a similar synergistic index of about 0.35 for MOLM-13-Luc and MV-411 cells (Tables 4 & 5), showing a strong synergistic effect.

[0072]

[0073]

[0074] The results of cytotoxicity experiments showed that NPV exhibited certain cytotoxicity in MOLM-13-Luc cells, and NPAV further increased this toxicity. The IC value of NPAV (A:V = 1:1, w / w) on MOLM-13-Luc cells was 50 The IC values of NPA and NPV for MOLM-13-Luc cells were 1.7 ng / mL. 50 The synergistic effect of NPAV on MOLM-13-Luc cells and the IC value were 2.5 to 6 times that of NPAV (Table 6), with a CI value of 0.53, indicating a moderate synergistic effect. Further increasing the amount of volasertib in NPAV increased the synergistic index, indicating that the synergistic effect was weakened. 50 Both are worse than NPAT.

[0075]

[0076] Example 4 Protein Expression and Apoptosis in Leukemia Cells

[0077] Western blot experiments were performed to investigate the effect of NPAT on the expression of BCL2, MCL1, and Bim proteins in MOLM-13-Luc and MV-411 cells. 5Cells were plated in 12-well plates, and 200 μL of NPT, NPA, and NPAT (TW37: 10 ng / mL, ABT199: 10 ng / mL) were added, respectively. The cells were incubated at 37°C for 48 h, and then the cells were lysed to extract protein samples. The protein concentration was determined using a BCA protein quantification kit, and the proteins were denatured. The denatured proteins were loaded onto SDS-PAGE gels (10%) and electrophoresed. After electrophoresis, the proteins were transferred to PVDF membranes and blocked in blocking buffer at room temperature for 1.5 h. Different locations of the membranes were incubated with primary antibody solutions for BCL2, MCL1, Bim, and β-actin at 4°C overnight, and then incubated with the corresponding secondary antibody solutions at room temperature for 1.5 h. The membranes were developed using a chemiluminescence detection system. The determination of protein expression in MV-411 cells was similar to that in MOLM-13-Luc cells, except that the cells were incubated with higher concentrations of drugs (TW37: 60 ng / mL, ABT199: 60 ng / mL).

[0078] Annexin V-allophycocyanin (APC) and 7-amino-actinomycin D (7-AAD) double staining techniques were used to further investigate the ability of NPAT to induce apoptosis in MOLM-13-Luc or MV-411 cells using flow cytometry. 5 Cells were incubated in 12-well plates with equal volumes of different drugs (TW37: 10 ng / mL, ABT199: 10 ng / mL) for 48 hours. After washing with PBS and resuspending, Annexin V-APC and 7-AAD staining solutions were added sequentially. The cells were incubated in the dark for 5 minutes at room temperature before analysis by flow cytometry. The apoptosis assay for MV-411 cells was similar to that for MOLM-13-Luc cells, except that higher concentrations of drugs (TW37: 60 ng / mL, ABT199: 60 ng / mL) were used.

[0079] Western blot analysis (WB) revealed that both BCL2 and MCL1 proteins were overexpressed in MOLM-13-Luc and MV-411 cells, and TW37 could significantly reduce the expression of MCL1. ABT199 can occupy the hydrophobic groove of BCL2 protein, resulting in the loss of BCL2's anti-apoptotic function. In both AML cells, the nanoformulation of ABT199 had little effect on BCL2 protein expression ( Figure 5A). Annexin V-APC / 7-AAD double staining technique was used to evaluate the ability of drugs to induce apoptosis in MOLM-13-Luc and MV-411 cells. The results showed that the ability of NPA, NPT, and NPAT to induce apoptosis was much greater than that of free drugs ( Figure 5 NPAT exhibited significant pro-apoptotic activity, inducing 44.3% late apoptosis in MOLM-13-Luc cells, with apoptosis-inducing capacity 1.8 and 5.7 times that of NPA and NPT, respectively.

[0080] Example 5 In vivo anti-tumor effect experiment

[0081] All experimental animal operations were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Soochow University and approved by the Animal Ethics Committee of Soochow University. To study the antitumor effect of nanoparticles in AML mice, a PBS suspension of MOLM-13-Luc cells (5 × 10 5The MOLM-13-Luc AML tumor was established by injection of 10 mg / kg (100 mg / kg) into B-NDG mice via the tail vein. On day 3 post-inoculation, MOLM-13-Luc AML tumor-bearing mice were randomly divided into four groups (PBS, NPT, NPA, and NPAT). Dosing began on day 3 post-inoculation, with the first dose designated as day 0. All groups received tail vein administration (TW37: 10 mg / kg, ABT199: 10 mg / kg) on days 0, 3, 6, and 9. The proliferation of MOLM-13-Luc cells in tumor-bearing mice was assessed using an IVIS imaging system on days 5, 8, 11, and 14. Each mouse was injected with luciferin saline (1.5 mg / mouse) prior to imaging. On day 14, peripheral blood (PB) was collected from the orbital cavity and placed in PBS (containing 1% v / v FBS). Blood was then collected from the celiac aorta for biochemical and routine blood analysis. In addition, the lungs, liver, spleen, and one leg bone of the mice were dissected and then ground to obtain cells. Red blood cell lysis buffer was added for 15 minutes, and leukemic cells were labeled with APC-anti-human-CD45 antibody for 20 minutes before analysis by flow cytometry. Leukemic cell infiltration into major organs and bone marrow (BM) was visualized using H&E staining and inverted fluorescence microscopy. The extent of femoral and tibia destruction was analyzed by staining osteoclasts with tartrate-resistant acid phosphatase (TRAP). Micro-CT was used to evaluate bone mineral density (BMD), bone mass, and morphology and structure in the femur and tibia. To further investigate the in vivo antitumor effect of NPAT in AML mice, an MV-411 AML mouse model was established. Tumor progression was tracked by measuring leukemic cell infiltration in peripheral blood during the experimental period (days 3, 6, 9, 12, 15, and 17). MV-411 AML mice were sacrificed on day 17, and leukemic cell infiltration in the BM and major organs of the AML mice was observed. At the end of the experiment, the spleen of the mice was weighed, and the body weight of the tumor-bearing mice was measured every 3 days.

[0082] To investigate the in vivo anti-AML activity of NPAT, an orthotopic MOLM-13-Luc AML mouse model was established by injecting MOLM-13-Luc cells into the tail vein of B-NDG mice ( Figure 6 A). Imaging results of MOLM-13-Luc tumor cells in mice showed that the disease progressed rapidly in the PBS group. Fluorescence began to appear locally on the 8th day, became more pronounced on the 11th day, and spread throughout the entire body by the 14th day. The mice also became paralyzed in their hind legs and had whitened ears. Compared to the PBS group, the NPT and NPA groups delayed the infiltration of leukemia cells in the mice, especially within the first 11 days. NPAT almost completely inhibited the infiltration of MOLM-13-Luc cells in the mice until the end of the experiment ( Figure 6 BC). After NPAT treatment, the weight of the mouse spleen was similar to that of healthy mice, in sharp contrast to the enlarged spleen in the mice in the PBS, NPA, and NPT groups ( Figure 6 D). These results preliminarily indicate that NPAT potently inhibits the infiltration of MOLM-13-Luc tumor cells in mice. No significant weight loss was observed in any group of mice during and after treatment, indicating that NPAT has good safety. Figure 6 E).

[0083] On the 14th day after the first administration, peripheral blood, liver, spleen, lung and hind leg bones were collected, ground and then APC-anti-human-CD45 antibody was added to label leukemia cells. The infiltration of leukemia cells in various organs of mice was detected by flow cytometry. The results showed that a large number of leukemia cells infiltrated the bone marrow (BM), lung (Lu), liver (Li), spleen (Sp), and peripheral blood (PB) of mice in the PBS group, with the proportion of leukemia cells being 35.2%, 39.9%, 80.5%, 86.0% and 15.4%, respectively ( Figure 7 AB), indicating that MOLM-13-Luc cells have invaded and infiltrated multiple organs. The NPA, NPT, and NPAT groups significantly inhibited the infiltration of leukemia cells in various organs. The NPAT group demonstrated the best inhibitory ability, with leukemia cell infiltration rates in the bone marrow and other organs less than 0.4% and 2%, respectively. Although the NPA and NPT groups also reduced the infiltration of leukemia cells in mice on day 14, the infiltration rates in the bone marrow, liver, and spleen had already exceeded 15%. The results indicate that the NPAT dual-drug combination significantly increased the therapeutic effect in the in situ MOLM-13-Luc AML mouse model, effectively inhibiting the proliferation of leukemia cells in mice and improving the quality of life of mice.

[0084] Micro-CT images showed that the hind leg bones of mice in the PBS group had severe osteolysis and a large number of trabeculae were lost. After NPAT treatment, the osteolytic lesions of the mice were significantly improved ( Figure 8 A). Further quantitative analysis showed that the femur and tibia bone mineral density (BMD) of mice in the PBS group was the lowest, while NPA, NPT, and NPAT significantly increased BMD. The BMD of the NPAT group was approximately 2.8 times that of the PBS group ( Figure 8B). Compared with the PBS group, mice in the NPAT group had significantly increased bone mass, with bone surface area / tissue volume (BS / TV) and bone volume / tissue volume (BV / TV) increasing approximately twofold, respectively. In the PBS group, trabecular spacing (Tb.Sp) increased, trabecular thickness (Tb.Th) decreased, and trabecular number (Tb.N) decreased, indicating that AML-induced osteolytic lesions lead to significant bone damage. NPAT-induced trabecular bone spatial morphology and structure were normal, with Tb.Sp, Tb.Th, and Tb.N similar to those in healthy mice.

[0085] HE analysis of the femur and tibia of mice showed that a large number of leukemia cells were distributed in the bone marrow cavity of mice in the PBS group, with only a small number of hematopoietic cells. However, the infiltration of MOLM-13-Luc leukemia cells in the bone marrow cavity was significantly reduced in the NPA, NPT and NPAT groups ( Figure 9 A). Leukemic cells were virtually absent in the bone marrow of mice treated with NPAT. Osteoclasts and osteoblasts exist in a dynamic equilibrium in the bones of healthy mice. An increase in osteoclasts often leads to osteolytic lesions, ultimately leading to osteoporosis and osteolysis. Tartrate-resistant acid phosphatase (TRAP), a primary marker of osteoclasts, was detected by staining for TRAP, revealing the presence of numerous osteoclasts in the leg bones of mice in the PBS group. However, treatment with NPA, NPT, and NPAT significantly reduced osteoclast levels, reaching levels comparable to those in healthy mice.

[0086] Blood biochemistry and routine blood analysis showed that ( Figure 10 ), mice in the PBS, NPT, and NPA groups showed decreased red blood cells and platelets, and increased white blood cells. However, mice in the NPAT group showed essentially no differences in these parameters compared to healthy mice, consistent with the bone H&E results. Notably, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and urea levels in mice treated with all nanomedicines (NPA, NPT, and NPAT) were similar to those in healthy mice, demonstrating the remarkable safety of the nanomedicines.

[0087] H&E staining results showed that in the liver, spleen, and kidneys of mice, a large number of MOLM-13-Luc leukemia cells were infiltrated in the PBS group, a small number of leukemia cells were infiltrated in the NPT and NPA groups, but no leukemia cells were infiltrated in the NPAT group. In addition, the cell morphology of each organ in NPAT was normal and uniform, with liver cells arranged tightly and orderly, abundant red pulp in the spleen, and dense distribution of immune cells ( Figure 11 ).

[0088] The therapeutic effect of NPAT was further evaluated in orthotopic MV-411 tumor-bearing B-NDG mice ( Figure 12A). In the PBS group, MV-411 leukemia cells rapidly infiltrated the mice. On day 17, the percentages of leukemia cells in BM, Lu, Li, Sp, and PB were 14.2%, 40.0%, 24.7%, 15.0%, and 4.9%, respectively ( Figure 12 BC). NPA, NPT, and NPAT can all inhibit the infiltration of leukemia cells in major organs, and NPAT has the best ability to inhibit the infiltration of leukemia cells in the bone marrow (< 2%), which verifies the strong synergistic effect of NPAT combined with dual drugs. Increasing the drug dose of ABT199 and TW37 from 10 mg / kg to 15 mg / kg can further improve the treatment results and show better inhibitory ability. The proportion of leukemia cell infiltration in the bone marrow is reduced by about 3.4 times (0.36% vs 1.21%). Compared with NPAT, the number of MV-411 cells infiltrating in the spleen and lungs of mice in the NPAT-H group was reduced by about 1.9 times and 3.4 times, respectively. It can also be seen that the number of leukemia cells in the peripheral blood of mice in the PBS group increased rapidly with time ( Figure 12 D), indicating that the MV-411 leukemia model was successfully established and progressed rapidly. The spleen weights of mice in the PBS, NPA, and NPT groups were significantly greater than those in healthy mice. The spleen weights after NPAT treatment were similar to those in healthy mice ( Figure 12 E). Importantly, NPA, NPT, and NPAT induced minimal changes in body weight during treatment, whereas mice in the PBS group lost approximately 7% of their body weight, primarily due to the rapid infiltration of leukemia cells at a later stage, which worsened the condition of the mice ( Figure 12 F). These results collectively demonstrate that the NPAT dual-drug combination significantly enhanced the therapeutic efficacy in the orthotopic AML mouse model, effectively inhibiting the proliferation of leukemia cells in mice and their infiltration into organs.

[0089] The present invention discloses a polyamino acid functionalized nanocarrier, specifically polyethylene glycol- b -Poly(4-borono-L-phenylalanine- co -L-tyrosine) copolymers, to prepare polyamino acid phenylboronic acid functionalized co-loaded small molecule inhibitor nanoparticles for the synergistic treatment of tumors. The polyethylene glycol- b -Poly(4-borono-L-phenylalanine- co-L-tyrosine) copolymers have good biocompatibility and enzymatic degradation, and are simple to prepare and reproducibly controllable. The designed and constructed polypeptide nanoparticles have controllable particle size (79-148 nm), narrow particle size distribution, good stability, and the ability to responsively release (acid / reactive oxygen / enzyme) drugs, achieving efficient encapsulation of anti-tumor small molecule drugs. In in vitro cytotoxicity experiments, they showed significant cytotoxicity against MOLM-13-Luc and MV-411 AML cells. At an ABT199 / TW37 weight ratio of 1:1, the IC 50 The concentrations of NPAT in the MOLM-13-Luc and MV-411 AML models were 1.15 and 7.45 ng / mL, respectively. Furthermore, NPAT significantly inhibited leukemia cell infiltration in the bone marrow, lungs, liver, spleen, and peripheral blood, and significantly improved mouse survival. Therefore, phenylboronic acid-functionalized smart nanomedicines offer advantages such as simple structure, adjustable drug combinations, high safety, and strong synergistic efficacy, and can be easily extended to the treatment of various cancers.

Claims

1. A small molecule inhibitor nanoparticle based on polyamino acid, characterized in that: The polyamino acid-based small molecule inhibitor nanoparticles are obtained by loading a small molecule inhibitor with a polyethylene glycol-b-poly (4-boron-L-phenylalanine-co-L-tyrosine) copolymer; the small molecule inhibitor is ABT199 and TW37 in a mass ratio of 1:0.5, or the small molecule inhibitor is ABT199 and TW37 in a mass ratio of 1:1, or the small molecule inhibitor is ABT199 and TW37 in a mass ratio of 1:2, or the small molecule inhibitor is ABT199 and volasertib in a mass ratio of 1:0.5, or the small molecule inhibitor is ABT199 and volasertib in a mass ratio of 1:1, or the small molecule inhibitor is ABT199 and volasertib in a mass ratio of 1:2; the polyethylene glycol-b-poly (4-boron-L-phenylalanine-co-L-tyrosine) copolymer has a structure of Formula I: ； Formula I; Among them, n is 70 to 210, m is 17 to 55, and x is 11 to 42.

2. The method for preparing the polyamino acid-based small molecule inhibitor nanoparticles according to claim 1, characterized in that: The polyethylene glycol-b-poly(4-boron-L-phenylalanine-co-L-tyrosine) copolymer solution and the small molecule inhibitor solution were added dropwise to the buffer solution, and then dialyzed to obtain small molecule inhibitor nanoparticles based on polyamino acids.

3. The method for preparing small molecule inhibitor nanoparticles based on polyamino acids according to claim 2, characterized in that: Polyethylene glycol-b-poly(4-boron-L-phenylalanine-co-L-tyrosine) copolymer was synthesized by ring-opening polymerization of 4-boron-L-phenylalanine-N-carboxylic anhydride and L-tyrosine-N-carboxylic anhydride using polyethylene glycol as initiator.

4. Use of polyethylene glycol-b-poly (4-boron-L-phenylalanine-co-L-tyrosine) copolymer in the preparation of the polyamino acid-based small molecule inhibitor nanoparticles according to claim 1, characterized in that: The polyethylene glycol-b-poly(4-boron-L-phenylalanine-co-L-tyrosine) copolymer has the structure of Formula I: ； Formula I; Among them, n is 70 to 210, m is 17 to 55, and x is 11 to 42.

5. The use of the polyamino acid-based small molecule inhibitor nanoparticles according to claim 1 in the preparation of nanomedicines, characterized in that: The nanomedicine is an anti-tumor drug.

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