A reduction-responsive nanodelivery system and use thereof in the preparation of a medicament for treating drug-resistant tumors

Abstract

The application provides a reduction-responsive nano delivery system and application thereof in preparation of a drug for treating drug-resistant tumors, and belongs to the pharmaceutical field. The application constructs a reduction-responsive branched copolymer functionalized by pegylation and deoxycholic acid (DA) (referred to as: pegylated branched poly(HPMA-DA)), which can effectively encapsulate small molecule drugs to form a nano delivery system. Experiments prove that the nano delivery system can be taken up by tumor cells, has good tumor targeting, and exhibits excellent anti-tumor effect in tumor xenografts (CDX) derived from non-small cell lung cancer (NSCLC) chemoresistant cell lines and patient-derived xenograft mouse models (PDX). The pegylated branched poly(HPMA-DA) provided by the application has wide application prospect in preparation of a drug carrier, and the nano delivery system provided by the application has wide application prospect in preparation of a drug for treating cancer.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a reduction-responsive nanodelivery system and its use in the preparation of drugs for treating drug-resistant tumors. Background Technology

[0002] Tumor resistance is one of the biggest challenges in the treatment of non-small cell lung cancer (NSCLC), for which there are currently no specific drugs. Increasing evidence suggests the presence of small numbers of tumor stem cells (CSCs) in various cancers, including NSCLC. CSCs, also known as tumor initiating cells (TICs), are a subtype of tumor cells capable of self-renewal and differentiation into ordinary tumor cells. Notably, the stem cell-like characteristics of CSCs may be associated with tumor recurrence and treatment failure. These cells can act as a reservoir to replenish tumor cells, contributing to tumor development and resistance to conventional chemotherapy and radiotherapy. Furthermore, studies have shown that radiotherapy and chemotherapy can induce the expression of stem cell genes in cancer cells, and the high drug-resistant stemness of CSCs ultimately leads to tumor recurrence. Therefore, targeting CSCs may be an effective way to overcome treatment resistance in NSCLC. One strategy is to correct or repair dysfunctional signaling pathways that play a crucial role in regulating CSC self-renewal and tumorigenesis.

[0003] The Janus kinase / signal transducer and activator of transcription (JAK / STAT) pathway is involved in all stages of tumor progression, including proliferation, invasion, and metastasis. In particular, STAT3, a member of the STAT family, promotes the self-renewal and differentiation of cancer cells (CSCs). STAT3 is activated in various cancers, including lung cancer, and its phosphorylation level is highly correlated with tumor progression and patient survival, making it a potential therapeutic target for CSCs. BBI608 (Napabucasin) is a small STAT3-targeting molecule that has been found in preclinical studies to effectively inhibit stem gene expression, induce cancer cell death, and suppress cancer recurrence and metastasis.

[0004] However, like many small-molecule anticancer drugs, BBI608 suffers from poor water solubility and non-specific biodistribution, leading to low bioavailability, reduced efficacy, and toxic side effects. Compared to conventional small-molecule antitumor drugs, polymer-based nanodelivery systems can, to some extent, improve drug targeting ability, control drug release rate, prolong drug circulation time, and reduce drug toxicity. Among them, poly(2-hydroxypropyl)methacrylamide (PHPMA) has attracted widespread attention as a non-toxic and non-immunogenic water-soluble polymer carrier. Shen Tong (Research on the structural regulation and biological function of 2-hydroxypropyl methacrylamide copolymer, Doctoral dissertation, Beijing University of Chemical Technology, 2016) reported a triblock copolymer based on poly(2-hydroxypropyl)methacrylamide and further synthesized a polymer-doxorubicin-bonded drug through chemical bonding (synthetic route as follows), which improved the tumor inhibition rate of doxorubicin and reduced its toxicity. However, this triblock copolymer can only bond doxorubicin through the reaction of hydrazine with ketone groups. On the one hand, this bonding method is relatively complex and has high production costs; on the other hand, this bonding method has a limited range of applicability to drugs and cannot encapsulate drugs that do not have corresponding reactive groups.

[0005]

[0006] Therefore, it is of great significance to develop a nanodelivery system that is simpler to prepare, has a wider range of applications, and has excellent targeted therapeutic effects on drug-resistant tumors. Summary of the Invention

[0007] One object of the present invention is to provide a novel polymeric drug carrier, and another object of the present invention is to provide a nanodelivery system prepared by encapsulating a drug in the polymeric drug carrier, and the use of the nanodelivery system in the preparation of a drug for treating drug-resistant tumors.

[0008] This invention provides a polymer prepared by reacting MA-SS-CTA, MA-SS-MA, HPMA, MA-SS-mPEG, MA-SS-DA and an initiator as raw materials, wherein the structures of MA-SS-CTA, MA-SS-MA, HPMA, MA-SS-mPEG and MA-SS-DA are as follows:

[0009]

[0010] n is between 20 and 100.

[0011] Further, the polymer has a weight-average molecular weight Mw of 13-14 kDa and a number-average molecular weight Mn of 15-16 kDa; preferably, the polymer has a weight-average molecular weight Mw of 13.39 kDa and a number-average molecular weight Mn of 15.19 kDa.

[0012] Further, the molar ratio of MA-SS-CTA, MA-SS-MA, HPMA, MA-SS-mPEG, MA-SS-DA, and the initiator is 1:(0.5-2):(60-80):(5-20):(10-30):(0.1-2);

[0013] And / or, the initiator is an azo initiator;

[0014] And / or, the n is 30-60.

[0015] Furthermore, the molar ratio of MA-SS-CTA, MA-SS-MA, HPMA, MA-SS-mPEG, MA-SS-DA, and the initiator is 1:1:70:10:21:0.7;

[0016] And / or, the azo initiator is azobisisobutyrazoline or a salt thereof, preferably a hydrochloride salt;

[0017] And / or, the n is 40-50.

[0018] Furthermore, the solvent for the reaction is one or a mixture of two of the following: an organic solvent and water.

[0019] And / or, the reaction time is 12-36 hours.

[0020] And / or, the temperature of the reaction is 40-60°C;

[0021] And / or, the reaction is carried out in an inert gas atmosphere;

[0022] Preferably, the organic solvent is 2,2,2-trifluoroethanol;

[0023] And / or, the reaction time is 24 hours;

[0024] And / or, the temperature of the reaction is 47°C.

[0025] The present invention also provides the use of the above polymer in the preparation of drug carriers.

[0026] The present invention also provides a nanodelivery system, which is obtained by encapsulating the drug in the above-mentioned polymer.

[0027] Furthermore, the drug is a cancer stem cell inhibitor, preferably BBI608;

[0028] And / or, the mass ratio of the polymer to the drug is (5-20):1, preferably 10:1.

[0029] The present invention also provides a method for preparing the above-mentioned nanodelivery system, the method comprising the following steps: dissolving a polymer and a drug in an organic solvent, evaporating the solvent to obtain a polymer-drug film, dissolving the polymer-drug film in water, and self-assembling to form a nanodelivery system; wherein the organic solvent is preferably one or a mixture of two of chloroform and acetonitrile.

[0030] The present invention also provides the use of the above-described nanodelivery system in the preparation of drugs for treating tumors, wherein the tumor is preferably a drug-resistant tumor, more preferably a chemotherapy-resistant tumor; the chemotherapy-resistant tumor is preferably chemotherapy-resistant lung cancer, and the lung cancer is preferably non-small cell lung cancer.

[0031] This invention constructs a reduction-responsive PEGylated and deoxycholic acid (DA)-functionalized branched copolymer (PEGylated branched N-(2-hydroxypropyl)methacrylamide (HPMA)-deoxycholic acid (DA) polymer), abbreviated as PEGylated branched polymer (HPMA-DA). PEGylated branched polymer (HPMA-DA) can effectively encapsulate BBI608 to form drug-loaded nanoparticles BBI608-NPs for targeted delivery of BBI608. Experiments have demonstrated that BBI608-NPs can be taken up by tumor cells and exhibit good tumor targeting, showing excellent anti-tumor effects in tumor xenografts (CDX) derived from chemotherapy-resistant NSCLC cell lines and patient-derived xenograft mouse models (PDX), and its anti-tumor mechanism is related to the dual inhibition of the STAT3 and Wnt signaling pathways.

[0032] The polyethylene glycol branched poly(HPMA-DA) provided by this invention can be used to encapsulate a variety of hydrophobic small molecule drugs, including BI608, with high drug selectivity and broad application prospects.

[0033] The nanodelivery system of this invention has a simple preparation method that avoids the chemical bonding step and has broad application prospects in the preparation of drugs for treating tumors.

[0034] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0035] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0036] Figure 1 BBI608 inhibits the proliferation of NSCLC cells and drug-resistant NSCLC. (A) In vitro cytotoxicity of BBI608 on four NSCLC cell lines (A549, H1975, PC-9, H226), two drug-resistant NSCLC cell lines (cisplatin (DDP) and gemcitabine (GEM) resistant A549 cells), and human lung fibroblasts (IMR-90) (after 48 hours of incubation). Images (B) and quantitative results (C) of A549 and H1975 cell colony formation after 2 weeks of treatment with 0.2 and 0.4 μM BBI608. Images (D) and quantitative results (E) of scratch assay of A549 and H1975 cells after 24 hours of treatment with BBI608. Effects of BBI608 on cell cycle (F) and apoptosis (G and H) in A549 and H1975 cells were measured by flow cytometry. (I) Expression of STAT3, p-STAT3, and Bcl-2 in H1975 cells treated with BBI608 for 48 hours. Flow cytometry results (J) and percentage changes (K) of CD44+ cells in A549 / DDP and A549 / GEM cells treated with BBI608 (0.2 μM) for 48 hours. (L) Expression of stem-related proteins (STAT3, p-STAT3, β-catenin, SOX2) in A549 / DDP cells treated with BBI608. Data are presented as mean ± SEM (n = 3), **P < 0.01.

[0037] Figure 2 Preparation, characterization, cellular uptake, and cytotoxicity of BBI608-NPs. (A) Schematic diagram of the chemical structure of the polyethylene glycol-modified branched HPMA-DA polymer and the reduction-responsive nanoparticles (NPs) formed after self-assembly. (B) Scanning electron microscopy (SEM) images of blank NPs and BBI608-NPs. (C) Size distribution measured by dynamic light scattering (DLS). In vitro cytotoxicity of BBI608-NPs (D) and blank NPs (E) to different NSCLC cells after 48 hours of incubation. (F) Cell uptake of H1975 cells after 30 minutes or 4 hours of incubation with free DiD and DiD-labeled NPs (DiD-NPs, red) (confocal microscopy images). Cell nuclei were stained with DAPI (blue). Scale bar: 10 μM.

[0038] Figure 3Real-time biodistribution and tumor targeting of DiD-NPs in NSCLC PDX models, and therapeutic efficacy and safety of BBI608-NPs in A549 / DDP CDX and NSCLC PDX models. (A) In vivo imaging of NSCLC PDX mice at different time points after intravenous injection of free DiD or DiD-NPs, and (B) ex vivo images of major organs and tumors obtained by dissecting mice 48 hours after injection. Relative tumor volume (C) and body weight changes (D) in A549 / DDP CDX models after treatment with free BBI608 or BBI608-NPs. Relative tumor volume (E) and body weight changes (F) in NSCLC PDX mice after treatment with free BBI608 or BBI608-NPs. In both animal models, BBI608 was administered orally, and BBI608-NPs were administered intravenously every other day. Mice injected with saline served as controls. At the end of the experiment, blood samples were collected from NSCLC PDX mice for (G) blood cell count and (H) routine blood biochemistry tests. (I) Histopathological examination of tumor sections from the NSCLC PDX model, including H&E, Ki67, CD44 markers (scale bar: 5 μm) and TUNEL (scale bar: 100 μm). Data are expressed as mean ± SEM, *P<0.05.

[0039] Figure 4Mechanism of action of BBI608-NPs in a PDX model of NSCLC (via RNA sequencing (RNA-seq) and Western blot analysis). (A) Volcano map of upregulated or downregulated genes between the BBI608-NPs treatment group and the control group obtained by RNA-seq analysis of tumor samples in the PDX model. Enrichment of differentially expressed genes after treatment with BBI608-NPs by KEGG (B) and GO (C). Heatmap (D) and Western blot results (E, F) of key differentially expressed genes after BBI608-NPs treatment. Heatmap (G) of genes significantly upregulated after BBI608-NPs treatment (fold change ≥10, P<0.05) and Western blot results (H, I) of PTGDS, PPARγ and p-GSK3β. Data are expressed as mean ± SEM, *P<0.05, **P<0.01. (J) Schematic diagram of BBI608-NPs targeted therapy for drug-resistant NSCLC and its potential mechanism. Upon endocytosis into tumor cells, the disulfide bonds in BBI608-NPs break in response to high intracellular GSH levels, leading to NP disintegration and the release of BBI608. BBI608 directly regulates downstream target genes by inhibiting STAT3 phosphorylation and indirectly inhibits the expression of β-catenin, a key protein in the Wnt pathway, by mitigating STAT3's inhibition of PPARγ. Therefore, BBI608-NPs can inhibit the proliferation of NSCLC cells and tumor stem cells through a "dual inhibition" mechanism.

[0040] Figure 5 .MA-SS-OH 1 1H NMR spectrum (recorded in d6-DMSO).

[0041] Figure 6 .MA-SS-OH 13 C10 NMR spectra (recorded in d6-DMSO).

[0042] Figure 7 LC-MS spectrum of .MA-SS-OH. Product peak at 1.30 min (A), single ion peak at 222.2 [M+H] + (B) (Recorded in acid).

[0043] Figure 8 The HRMS spectrum of .MA-SS-OH shows [M+H] + The ion peak is located at 222.0616 m / z.

[0044] Figure 9 .MA-SS-AE 1 1H NMR spectrum (recorded in d6-DMSO).

[0045] Figure 10 .MA-SS-mPEG(2K) 1 1H NMR spectrum (recorded in d6-DMSO).

[0046] Figure 11 MALDI-TOF-MS mass spectra of MA-SS-mPEG(2K). The average molecular weight of this compound is 2214.

[0047] Figure 12 .DA-SS-MA 1 1H NMR spectrum (recorded in d6-DMSO).

[0048] Figure 13 .DA-SS-MA 13 C10 NMR spectra (recorded in CDCl3).

[0049] Figure 14 LC-MS spectrum of DA-SS-MA. Product peak at 2.53 min (A), [M+H] + (B) has a single ion peak at 596.3 m / z (recorded in acid).

[0050] Figure 15 HRMS spectrum of DA-SS-MA, showing [M+H] + The ion peak is 596.3431 m / z, [M+Na] + The ion peak is 618.3253 m / z.

[0051] Figure 16 .MA-SS-CTA 1 1H NMR spectrum (recorded in d6-DMSO).

[0052] Figure 17 .MA-SS-CTA 13 C10 NMR spectra (recorded in CDCl3).

[0053] Figure 18 LC-MS spectrum of MA-SS-CTA. Product peak at 2.02 min (A), [M+H] + (B) has a single ion peak at 482.8 m / z (recorded in acid).

[0054] Figure 19 The HRMS spectrum of MA-SS-CTA shows the ion peak as [M+H]. + The value is 483.0929 m / z, [M+Na] + The value is 505.0715 m / z, [M+K] + It is 521.0476 m / z.

[0055] Figure 20 Polyethylene glycol-branched poly(HPMA-DA) and d6-DMSO were used as solvents. 1 H NMR spectrum.

[0056] Figure 21 .MA-SS-MA 1 1H NMR spectrum (recorded in d6-DMSO).

[0057] Figure 22 .MASS-MA 13 C10 NMR spectra (recorded in d6-DMSO).

[0058] Figure 23 LC-MS spectrum of .MA-SS-MA. Product peak at 1.50 min (A), [M+H] + (B) has a single ion peak at 289.2 m / z (recorded in acid).

[0059] Figure 24 HRMS spectrum of .MA-SS-MA, showing [M+H] + The ion peak is at 289.1044 m / z, [M+Na] + The ion peak is 311.0864 m / z.

[0060] Figure 25 In vitro cytotoxicity and IC50 of (A) cisplatin (DDP) and (B) gemcitabine (GEM) against A549 cells and their corresponding drug-resistant A549 cells (A549 / DDP and A549 / GEM) after 72 hours of incubation. 50 value.

[0061] Figure 26 The change in the percentage of SP cells in A549 / DDP and A549 / GEM cells treated with 0.2 μM BBI608 for 48 hours was analyzed by flow cytometry.

[0062] Figure 27 Particle size distribution of BBI08-NPs after incubation with PBS or PBS + 10mM GSH for 24 hours.

[0063] Figure 28 The morphology of blank NPs and BBI608-NPs observed under a transmission electron microscope (TEM). The particle size range of blank NPs is 100-120 nm and that of BBI608-NPs is 180-200 nm.

[0064] Figure 29Particle size distribution of BBI608-NPs at 0, 24 and 48 hours in PBS (A) and PBS+10%FBS (B) as measured by DLS.

[0065] Figure 30 Representative confocal micrographs of cellular uptake of DiD-NPs (red fluorescence) in A549 cells after 30 minutes or 4 hours of incubation. Cell nuclei were stained with DAPI. Scale bar: 50 μM.

[0066] Figure 31 (A) Representative TEM images of blank micelles after incubation with different concentrations (0 mM, 5 mM, 10 mM, 20 mM) of GSH. (B) Cellular uptake images of DiD-NPs in H1975 cells after incubation with different concentrations (0 mM, 10 mM, 20 mM) of GSH for 30 minutes. Cell nuclei were stained with DAPI. Scale bar: 10 μM. Detailed Implementation

[0067] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0068] Example 1: Synthesis of polyethylene glycol branched poly(HPMA-DA)

[0069] 1. Synthesis of compound MA-SS-OH:

[0070] MA-SS-Py (4.67 g, 18.36 mmol) and 2-3 mg of polymerization inhibitor 4-methoxyphenol were dissolved in a MeOH / CH3COOH (20:1, 50 mL) solution in an ice bath to obtain a reaction mixture. A 10 mL solution of 2-mercaptoethanol (2.00 mL, 28.41 mmol) in MeOH was added dropwise to the reaction mixture. The mixture was stirred in an ice bath for 0.5 hours, and then stirred for another 4 hours at room temperature. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel chromatography to obtain MA-SS-OH (3.25 g, 80.05%), a pale yellow oily liquid. 1 H NMR (400MHz, DMSO) δ8.10 (s, 1H), 5.65 (s, 1H), 5.34 (s, 1H), 4.87 (t, J = 5.4Hz, 1H), 3.62 (dd, J=12.2,6.3Hz,2H),3.39(dd,J=13.3,6.3Hz,2H),2.80(dd,J=14.0,7.2Hz,4H),1.84(s,3H)( Figure 5 ). 13C NMR (101MHz, DMSO) δ167.47, 139.73, 119.19, 59.51, 41.00, 38.45, 37.04, 18.55 ( Figure 6 LC-MS (ES) + ): m / z = 222.2[M+H] + ( Figure 7 HR-MS (ESI): C8H 15 NO2S2[M+H] + Calculated value: 222.0622, measured value: 222.0616 Figure 8 ).

[0071] 2. Synthesis of compound MA-SS-AE:

[0072] MA-SS-OH (1.25 g, 5.92 mmol), TEA (1.0 mL, 7.21 mmol), and DMAP (0.71 g, 5.81 mmol) were dissolved in anhydrous THF (50 mL), placed in an ice bath, and 2-3 mg of polymerization inhibitor 4-methoxyphenol was added to obtain the reaction mixture. A solution of 4-nitrobenzene chloroformate (2.50 g, 12.16 mmol) in anhydrous THF (20 mL) was added dropwise to the reaction mixture, and the mixture was stirred in an ice bath for 1 hour, then stirred overnight at room temperature. The suspension was filtered and the filter cake was washed with anhydrous THF (20 mL). The collected filtrate was concentrated, and the crude product was purified by silica gel chromatography to obtain product MA-SS-AE (1.00 g, 43.67%), a grayish-white solid. 1 H NMR (400MHz, CDCl3) δ8.29(d,J=9.1Hz,2H),7.40(d,J=9.1Hz,2H),6.24(s,1H),5.72(s,1H),5.36(s,1H) ,4.56(t,J=6.5Hz,2H),3.68(q,J=6.1Hz,2H),3.04(t,J=6.6Hz,2H),2.91(t,J=6.2Hz,2H),1.97(s,3H)( Figure 9 ).

[0073] 3. Synthesis of compound MA-SS-mPEG(2K):

[0074]

[0075] NH₂-mPEG-2K (3.00 g, 1.50 mmol) was added to an anhydrous DCM (50 mL) solution of MA-SS-AE (0.94 g, 2.10 mmol) and incubated in an ice bath under N₂. DMAP (0.77 g, 6.30 mmol) and 2-3 mg of the polymerization inhibitor 4-methoxyphenol were added. The mixture was stirred in an ice bath for 0.5 hours, then stirred overnight at room temperature. The organic solvent was evaporated to remove the solid and redissolved in MeOH / H₂O (1:2, 9 mL). After dialyzing with water for 48 hours, the solid was lyophilized to give MA-SS-mPEG(2K) (2.56 g) as a white solid. The molecular structure was determined by... 1 H NMR spectroscopy ( Figure 10 ) and MALDI-TOF-MS ( Figure 11 )confirm.

[0076] 4. Synthesis of compound MA-SS-DA:

[0077]

[0078] In an ice bath, DA (1.60 g, 4.08 mmol) and HATU (3.12 g, 8.21 mmol) were dissolved in anhydrous DMF (30 mL) under N2 and stirred for 20 minutes. DIPEA (2.70 mL, 16.32 mmol) and MA-SS-OH (1.00 g, 4.52 mmol) were added sequentially to the reaction mixture. 2–3 mg of polymerization inhibitor 4-methoxyphenol was added, and the reaction mixture was stirred in an ice bath for 0.5 hours, then stirred overnight at room temperature. The reaction mixture was poured into a saturated NaHCO3 aqueous solution. The aqueous layer was extracted twice with ethyl acetate (100 mL), and the combined organic layers were washed twice with saturated NaHCO3 aqueous solution, 1 M HCl, and saturated NaCl aqueous solution, and dried over Na2SO4. The organic phase was filtered and concentrated under reduced pressure. The crude product was purified by silica gel chromatography to obtain MA-SS-DA (1.50 g, 61.73%), a colorless oily liquid. 1H NMR(400MHz, DMSO)δ8.09(t,J=5.5Hz,1H),5.65(s,1H),5.36–5.30(m,1H),4.46(d,J=4.3Hz,1H),4.2 3(t,J=6.3Hz,2H),4.20(d,J=4.1Hz,1H),3.78(d,J=3.2Hz,1H),3.39(dd,J=13.1,6.2Hz,3H),2.96(t, J=6.3Hz,2H),2.83(t,J=6.9Hz,2H),2.33(ddd,J=14.7,9.3,5.1Hz,1H),2.21(dt,J=15.7,7.9Hz,1H), 1.84(s,3H),1.80–1.41(m,11H),1.39–1.14(m,13H),0.91(d,J=6.3Hz,3H),0.84(s,3H),0.59(s,3H)( Figure 12 ). 13 C NMR (101MHz, DMSO) δ173.16,167.45,139.72,119.45,139.72,1191,61.60,54.87,47.42,46.11,45.97,41.57,38.31,37.03,36.38,36. 26,35.61,35.16,34.87,33.78,31,34.87,33.78,32.88,30.61,30.19,28.55,27.15,26.95,26.08,23.46,23.06,18.54,16.81,12.38( Figure 13 LC-MS (ES) + ): m / z = 596.3 [M+H] + ( Figure 14 HR-MS (ESI): C 32 H 53 NO5S2[M+H] + Calculated value: 596.3433, measured value: 596.3431 Figure 15 ).

[0079] 5. Synthesis of compound MA-SS-CTA:

[0080]

[0081] A solution of MA-SS-OH (0.25 g, 1.13 mmol) in DMF (10 mL) was added to anhydrous DCM (30 mL) containing CTA (0.30 g, 1.07 mmol) and DIC (0.50 mL, 3.25 mmol), and the mixture was placed in an ice bath under N2. Catalytic amounts of DMAP (14 mg, 0.11 mmol) and 2-3 mg of polymerization inhibitor 4-methoxyphenol were added. The reaction solution was stirred in an ice bath for 0.5 h, then stirred for another 2 h at room temperature. The reaction mixture was poured into water and extracted three times with DCM (50 mL). The combined organic layers were washed twice with saturated NaHCO3 aqueous solution, 1 M (HCl), and saturated NaCl aqueous solution, and dried over Na2SO4. The organic phase was filtered, concentrated under reduced pressure, and the crude product was purified by silica gel chromatography to obtain MA-SS-CTA (0.28 g, 53.85%), a reddish-brown oily liquid. 1 HNMR (400MHz, DMSO) δ8.10(t,J=5.4Hz,1H),7.97–7.84(m,2H),7.69(t,J=7.4Hz,1H),7.51(t,J=7.9Hz,2H),5.66(s,1H),5.39–5.25(m, 1H),4.29(t,J=6.3Hz,2H),3.43–3.34(m,4H),2.98(t,J=6.3Hz,2H),2.83(t,J=6.9Hz,2H),2.71–2.61(m,2H),1.92(s,3H),1.84(s,3H)( Figure 16 ). 13 C NMR (101MHz, CDCL3) δ171.41,168.52,1333.52,139.77,133.13,128.64,126.72,120 .00,118.51,62.84,45.75,39.30-38.59,38.20,36.73,33.34,29.79,24.20,18.68( Figure 17 LC-MS(ES) + ): m / z = 482.8 [M+H] + ( Figure 18 HR-MS (ESI): C 21 H 26 N₂O₃S₄[M+H] + Calculated value: 483.0905, Measured value: 483.0929 Figure 19 ).

[0082] 6. Synthesis of branched polyethylene glycol (HPMA-DA):

[0083]

[0084] MA-SS-CTA (10.3 mg, 0.021 mmol), MA-SS-MA (6.1 mg, 0.021 mmol), HPMA (210.0 mg, 1.47 mmol), MA-SS-mPEG(2K) (467.5 mg, 0.21 mmol), MA-SS-DA (260.1 mg, 0.44 mmol), and VA-044 initiator (azobisisobutyrazoline hydrochloride, 4.6 mg, 0.014 mmol) were dissolved in 2,2,2-trifluoroethanol (3.6 mL) and water (0.7 mL), protected from light, and purged with argon in an ice bath for 30 minutes. The reaction mixture was stirred at 47 °C for 24 hours, and then quenched in liquid nitrogen. The copolymer was purified by precipitation in anhydrous diethyl ether to obtain the crude product. After dialysis with water for 48 hours, the aqueous solution was freeze-dried to obtain the final product (320 mg), a light pink solid, named polyethylene glycol branched poly(HPMA-DA). 1 H NMR ( Figure 20 The structure and molecular weight of the final product were verified by GPC (Table 1).

[0085] Table 1. GPC results of the synthesized polymers

[0086]

[0087] a) Mw: weight-average molecular weight; b) Mn: number-average molecular weight.

[0088] Example 2: Preparation of drug-loaded nanoparticles supported on BBI608: BBI608-NPs

[0089] BBI608 was loaded into polyethylene glycol-branched poly(HPMA-DA) using a rotary evaporation method. The specific procedure was as follows: BBI608 (1 mg) and polyethylene glycol-branched poly(HPMA-DA) (10 mg) were dissolved in 10-15 mL of a chloroform / acetonitrile mixture (1:1 volume ratio). After rotary evaporation, a polymer-drug film was obtained. The polymer-drug film was redissolved in 5 mL of deionized water and stirred overnight to allow the polymer to self-assemble into drug-loaded nanoparticles. Unloaded drug was removed by filtration through a 0.45 μm filter, and the product was lyophilized to obtain a solid powder, named BBI608-NPs.

[0090] The following experimental examples demonstrate the beneficial effects of the preparation method of this invention.

[0091] Experimental materials: BBI608 was purchased from APExBIO (Houston, USA). CD44-FITC and Hoechst 33342 were purchased from BDBiosciences (New York, USA). Verapamil hydrochloride was purchased from Solarbio (Beijing, China). 4,6-Diamidinyl-2-phenylindole (DAPI, blue), 1,10-octadecyl-3,3,30,30-tetramethylindole dicarboxylic acid perchlorate (DiD), and chemiluminescence (ECL) reagents were purchased from Thermo Fisher Scientific (Massachusetts, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Tokyo, Japan). Annexin V-FITC / PI apoptosis detection kit was purchased from 4ABiotech (Beijing, China). Cell cycle detection kit was purchased from KeyGen Biotech (Nanjing, China). All antibodies were purchased from Cell Signaling Technology (Boston, USA). Bicinchoninic acid (BCA) protein assay and 10% SDS-polyacrylamide gel electrophoresis were purchased from Beyotime (Shanghai, China). PVDF membranes were purchased from Bio-Rad (California, USA).

[0092] Experiment Example 1: Testing the effect of BB608 on NSCLC cells

[0093] 1. Experimental Methods

[0094] 1.1 Cell Culture

[0095] A549, H1975, and H226 cells were purchased from the Cell Bank of the Center for Type Culture Collection, Chinese Academy of Sciences (Shanghai, China). PC-9, A549 / cisplatin-resistant cells (A549 / DDP), and A549 / gemcitabine-resistant cells (A549 / GEM) were kindly provided by Professor Zhou Qinghua of West China Hospital, Sichuan University. Cells were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin at 37°C under a 5% CO2 atmosphere.

[0096] 1.2 Cytotoxicity assay and colony formation

[0097] Cells were seeded at a density of 3000-3500 cells / well in 96-well plates and incubated at 37°C for 24 hours. 100 μL of different concentrations of BBI608 or BBI608-NPs were added to the plates and incubated for 48 hours. Cell viability was determined by CCK8 assay. 20 μL of CCK8 solution was added to each well and incubated at 37°C for 1 hour. Absorbance at 450 nm and 600 nm was measured using a microplate reader (Epoch, BioTek, USA). Cell viability = [(OD(sample) - OD(blank)) / (OD(control) - OD(blank)) × 100%].

[0098] For colony formation assays, cells were seeded at a density of 200 cells / well in 6-well plates. After 24 hours, fresh medium containing 0.5% FBS (control) or drug-containing medium containing 0.2 μM or 0.4 μM BBI608 was added, and the cells were grown for 2 weeks. Cell clones were fixed with methanol for 10 minutes, stained with Giemsa, and then rinsed three times with tap water to remove excess dye. The number of colonies was counted under a microscope.

[0099] 1.3 Cell scratches

[0100] A549 and H1975 cells were incubated overnight and treated with BBI608 in a manner similar to that described in Section 1.3. Scratches were made using the tip of a 1 mL pipette, and images were taken immediately after treatment (0 h) and at 24 h. The migration distance of the monolayer cells was measured, and the result is expressed as the migration index: (Dt0 - Dtt) / Dt0 × 100%. Dt0 is the distance measured immediately after scratching (0 h), while Dtt is the distance migrated by cells at hour t after treatment.

[0101] 1.4 Apoptosis and Cell Cycle

[0102] For apoptosis assays, cells were prepared at a concentration of 3 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates. After 24 hours, the medium was replaced with fresh medium containing different concentrations of BBI608. After 48 hours of incubation, cells were collected and washed three times with cold physiological saline, then resuspended in annexin binding buffer (5 × 10⁶ / mL). Cells were then stained with 5 μL Annexin-V-FITC for 5 minutes and 10 μL propidium iodide (PI) (20 μg / mL) for 1 minute, and analyzed by flow cytometry (CytoFLEX, Beckman, USA).

[0103] For cell cycle analysis, cells were digested, washed, and fixed overnight in 70% cold ethanol at 4°C. After fixation, cells were washed with cold physiological saline, then stained with PI for 30 minutes in a buffer containing 100 μg / ml RNase A, 20 μg / ml PI, and 0.1% Triton X-100, and then analyzed by flow cytometry (CytoFLEX, Beckman Coulter, USA).

[0104] 1.5 Effects of BBI608 on CD44+ cells and side population (SP) cells

[0105] A549 / DDP and A549 / GEM cells were divided into 5×10⁻⁶ cells. 5 Cells were seeded at a density of 10 cells / well in 6-well cell culture plates and treated with different concentrations of BBI608 for 48 hours. For CD44+ cell detection, cells were digested and seeded at 1 x 10⁻⁶ cells / well. 6 Cells were resuspended at a density of 100 cells / mL in cold physiological saline and stained with an appropriate concentration of anti-CD44-FITC antibody. After incubation at room temperature for 20 minutes, the cells were washed three times and resuspended in 500 μL of cold physiological saline. Cell analysis was performed on a flow cytometer (CytoFLEX, Beckman, USA). For SP cell identification, after collecting and resuspending the cells, they were stained with Hoechst 33342 (5 mg / mL in physiological saline) at 37°C with or without the addition of 100 mM verapamil hydrochloride for 120 minutes with intermittent shaking. The cells were then centrifuged at 1000 rpm for 5 minutes at 4°C, resuspended in 500 μL of ice-cold physiological saline, and stained with PI at a concentration of 1 mg / mL to distinguish dead cells. Cell analysis was performed on a flow cytometer (MoFlo Astrios EQ, Beckman, USA).

[0106] 2. Experimental Results

[0107] 2.1 BB608 effectively inhibits the proliferation, colony formation, and migration of NSCLC cells.

[0108] Table 2. IC50 of BBI608-NPs or free BBI608 on different cell lines after 48 hours of incubation. 50 (μM) value

[0109]

[0110]

[0111] This experiment evaluated the in vitro cytotoxicity of BBI608 in a group of NSCLC cell lines (A549, H1975, H226, and PC-9) using CCK8. The results showed that BBI608 effectively inhibited the proliferation of all cell lines in a concentration-dependent manner. Figure 1 A) The IC50 of these cell lines 50 The values ​​ranged from 0.2 to 1.1 μM (Table 2). In contrast, BBI608 showed significantly lower cytotoxicity against the IMR-90 cell line (normal lung fibroblasts) than against the NSCLC cell line. This may be due to the presence of a functional inhibitor of cytokine signaling 3 (SOCS3) in normal cells, which thereby inhibits excessive STAT3 activation through feedback. Figure 1 A). The effect of BBI608 on NSCLC cell proliferation was further investigated using colony formation assays. For example... Figure 1 As shown in B and C, BBI608 significantly inhibited colony formation of NSCLC cells compared to the control group (P<0.01). It has been reported that aberrantly activated STAT3 participates in the degradation and resynthesis of the extracellular matrix (ECM) by regulating the expression of protease genes such as MMP-2 and MMP-9, thereby leading to tumor metastasis. Therefore, a scratch assay was performed to investigate the effect of BBI608 on NSCLC cell migration. Figure 1 As shown in D and E, compared with the control group, BBI608 treatment reduced the migration rates of A549 cells and H1975 cells by 8% and 13%, respectively.

[0112] 2.2 BBI608 arrests the cell cycle and induces apoptosis

[0113] The effects of BBI608 on the cell cycle and apoptosis of NSCLC were analyzed by flow cytometry. Figure 1 As shown in Figure F, BBI608 treatment led to an increase in the number of cells in the G2 / M phase, an increase that was particularly noticeable in H1975 cells, indicating cell cycle arrest in these cells after BBI608 treatment. Furthermore, BBI608 treatment significantly induced apoptosis in both A549 and H1975 cells. Figure 1 G and H). Western blot results showed that BBI608 inhibited STAT3 phosphorylation and the expression of the anti-apoptotic protein Bcl-2. Figure 1 I). These results indicate that BBI608 can significantly inhibit the proliferation of NSCLC cells by inducing cell cycle arrest and apoptosis.

[0114] 2.3 BBI608 inhibits the proliferation of chemotherapy-resistant NSCLC cells by killing CSCs

[0115] Considering the large number of chemotherapeutic stem cells (CSCs) in drug-resistant tumor cells, A549 / cisplatin-resistant cells (A549 / DDP) and A549 / gemcitabine-resistant cells (A549 / GEM) were selected to study the inhibitory effect of BB6608 on chemotherapeutic resistant cells. The resistance index (RI) was calculated to assess the degree of resistance in the two cell lines. Figure 25 As shown, the RI values ​​for A549 / DDP cells were 3.96 and for A549 / GEM cells were 5.56, indicating that both cell lines exhibited drug resistance, consistent with previous findings. In vitro cytotoxicity results showed that BBI608 effectively inhibited the proliferation of A549 / DDP and A549 / GEM cells, with an IC50 value of [missing value]. 50 The values ​​were 0.19 μM and 0.17 μM, respectively. Figure 1 A).

[0116] Cellular stem cells (CSCs) can be distinguished by specific cell phenotypes. For example, CD44-positive cells exhibit high tumorigenicity both in vitro and in vivo, and the cell surface marker CD44 has been widely used to identify CSCs in various cancer tissues. Furthermore, a small subset of tumor cells, known as "side populations" (SPs), can excrete Hoechst 33342 dye and exhibit characteristics of high invasiveness and drug resistance, typical of CSCs. Therefore, in this study, CSC subsets of two drug-resistant A549 cell lines were detected using anti-CD44 antibody or Hoechst 33342 staining, and the inhibitory effect of BBI608 on these CSC subsets (CD44+ and SP cells) was detected by flow cytometry. The results showed that the proportion of CD44+ cells was high in both A549 / DDP and A549 / GEM cells (>30%), and BBI608 treatment significantly reduced the proportion of CD44+ cells in both cell types (<3%, P<0.01). Figure 1 J and K). BBI608 treatment also significantly reduced the percentage of SP cells in A549 / DDP and A549 / GEM cells (J and K). Figure 26 This indicates that BBI608 can effectively inhibit CSCs. Furthermore, Western blot experiments were used to investigate the expression of genes related to CSC self-renewal, including β-catenin and SOX-2. The results showed that BBI608 treatment significantly reduced the expression of p-STAT3, β-catenin, and SOX-2 in A549 / DDP cells. Figure 1 These results indicate that BBI608 can inhibit the proliferation of lung CSCs by suppressing STAT3 phosphorylation and the expression of downstream stemness-related proteins.

[0117] Example 2: Characterization of Polyethylene Glycol Branched Polymer (HPMA-DA)

[0118] 1. Experimental Methods

[0119] Characterization methods: DMSO-d6 and D2O were used as solvents for different products, and data were obtained using a 400MHz Bruker Advanced Spectrometer. 1 ¹H NMR spectral data. Liquid chromatography-mass spectrometry (LC-MS) spectra were obtained on an Agilent 1200 Series 6120 mass spectrometer with electrospray ionization in a Waters×Bridge C18 column (50 mm × 4.6 mm × 3.5 μm) at a flow rate of 2.0 mL / min and a column temperature of 40 °C. The mobile phase consisted of water (0.01 mol / L NH₄HCO₃) and acetonitrile. The elution gradient was from 5% to 95% acetonitrile over 1.6 min, with a hold time of 1.4 min. High-resolution mass spectra (HRMS) were recorded on a Bruker Daltonics Bio ESI-TOF mass spectrometer. The molecular weight (MW) and polydispersity (PDI) of polymers were measured by size exclusion chromatography (SEC) on a Superose 6HR10 / 30 column at GE Healthcare. The mobile phase was 0.35 M LiCl solution containing 65% N,N-dimethylformamide, and the flow rate was 0.6 mL / min (40 °C).

[0120] Size, zeta potential, and morphology: The hydrodynamic dimensions and zeta potential of the polymer were measured using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, England). Scanning electron microscopy (SEM) (S-4800, Hitachi, Japan) and transmission electron microscopy (TEM, FEI Tecnai GF 20S-TWIN, USA) were used to further characterize the morphology and size of the nanoparticles. Drug loading (DL) and encapsulation efficiency (EE) were measured by high-performance liquid chromatography (HPLC) (1260 Infinity II, Agilent Technologies, USA). All measurements were performed at room temperature.

[0121] In vitro reduction-responsive degradation: Reduction-responsive biodegradation was analyzed in McIlvaine buffer (50 mM citrate / 0.1 M phosphate, 2 mM EDTA, pH 5.4). The polymer (3 mg / mL) was dissolved in McIlvaine buffer containing 2 mM GSH, and the solution was incubated in a 37°C water bath. The degradation was determined by SEC. Degradation products were analyzed on a Superose 6HR10 / 30 column using a system (Cytiva, USA). Sodium acetate buffer / acetonitrile (7:3, pH 6.5) was used as the mobile phase at a flow rate of 0.4 mL / min.

[0122] Cytotoxicity assay: The method is the same as in Experiment 1.

[0123] Preparation of blank NPs: The method of Example 2 was followed, except that BBI608 was not added, to prepare blank NPs.

[0124] 2. Experimental Results

[0125] Table 3. Biodegradation of polyethylene glycol branched poly(HPMA-DA) (in 2.0 mM GSH)

[0126]

[0127]

[0128] a) M w Weight-average molecular weight.

[0129] Table 4. Physicochemical properties of blank NPs and BBI608-NPs

[0130]

[0131] Size: Particle size; PDI: Polydispersity index; Zeta Potential: Zeta potential; DL: Drug loading; EE: Encapsulation efficiency.

[0132] To investigate the degradation of the polymer under reducing conditions, polyethylene glycol-branched polymer (HPMA-DA) was incubated with 2 mM GSH in McIlvaine buffer, and the products at different time points were analyzed by size exclusion chromatography (SEC). As shown in Table 3, due to its reduction-responsive characteristics, the macromolecular copolymer degraded into smaller molecular weight (7.4 kDa) fragments in a high GSH environment, which could be completely eliminated by the kidneys with a renal threshold <50 kDa. Furthermore, the particle size of the NPs became dispersed after incubation with 10 mM GSH, further demonstrating its reduction-responsive characteristics. Figure 27 BBI608 was successfully loaded into polyethylene glycol branched polymer (HPMA-DA) to obtain drug-loaded nanoparticles (BBI608-NPs). Table 4 shows the high drug loading (DL) of 8.4% and the encapsulation efficiency (EE) of 84.3%. The morphology of the nanoparticles was observed under scanning electron microscopy (SEM) and transmission electron microscopy (TEM). It can be seen that the blank NPs and BBI608-NPs are well dispersed and spherical. Figure 2 B and Figure 28 The particle size of BBI608-NPs in aqueous medium was 230.1 nm (PDI 0.234) (DLS measurement). Compared with blank nanoparticles (177.9 nm, PDI 0.294), the particle size increased slightly due to successful drug encapsulation. Figure 2 (C and Table 4). The sizes of blank and BBI608-loaded NPs in the SEM images are 80-100 nm and 100-120 nm, respectively. Figure 2 B), they are slightly smaller than the DLS measurements. This may be due to the different sample preparation methods: SEM samples are prepared in a dry state, while the copolymer samples used for DLS measurements are dispersed in an aqueous medium, which will cause some swelling. After incubation with PBS and PBS + 10% FBS, the changes in the size and PDI of BBI608-NPs are negligible, indicating that the formed NPs are stable under physiological conditions. Figure 29 Furthermore, neutrally charged surfaces (Table 4) can protect NPs from elimination by reticuloendothelial system (RES) and macrophage uptake, thereby prolonging circulation time.

[0133] Consistent with the therapeutic effects of the free drug, in vitro cytotoxicity results showed that BBI608-NPs could inhibit various NSCLC cell lines in a dose-dependent manner, with an IC50 concentration of [missing information]. 50 The value range is 0.2 μM to 1.3 μM. Figure 2 (D and Table 2). But the IC of BBI608-NPs 50 The value was slightly higher than that of free drug, possibly because small molecule drugs can rapidly diffuse into tumor cells, while NPs need to be translocated to lysosomes via endocytosis and then slowly release the encapsulated drug. Furthermore, blank NPs showed no significant toxicity to any cell line, indicating that the polyethylene glycol branched poly(HPMA-DA) of this invention has excellent biocompatibility. Figure 2 E).

[0134] Experimental Example 3: Study on Cellular Uptake and Biodistribution in Vivo

[0135] 1. Experimental Methods

[0136] Female BALB / c nude mice and NOD-SCID mice (6–8 weeks old) were purchased from Vital River Laboratory Animal Technology (Beijing, China). All animals were kept under pathogen-free conditions and allowed to acclimatize for at least 3 days prior to any experiment, in accordance with AAALAC guidelines. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of West China Hospital, Sichuan University.

[0137] Fresh tumor specimens were obtained from patients diagnosed with non-small cell lung cancer at West China Hospital of Sichuan University, and the procedure was approved by the hospital's ethics committee. The excised tumor tissue (P) was cut into 2mm pieces. 3 Around 1000 mm², NOD-SCID mice (P0) were implanted to establish a patient-derived xenograft mouse model (PDX) of NSCLC. When the tumor size reached 1000 mm²... 3 Then, it was subsequently passed on to other nude mice (P1).

[0138] A549 and H1975 cells were grown at 3 x 10 cells per well. 5 Cells were seeded at a density of 100 μg / mL in 6-well plates and incubated for 24 h. Free DiD or DiD-NPs at a concentration of 100 μg / mL were added to each well. After 0.5 h and 4 h, the cells were washed three times, fixed with 4% paraformaldehyde, and stained with DAPI. Cell uptake was observed under a confocal laser scanning microscope (IX83, Olympus, Japan). Cell uptake was also assessed in the presence of GSH. DiD and DiD-NPs at a concentration of 100 μg / mL were added to RPMI-1640 medium with GSH concentrations of 0, 10, or 20 mM and incubated with tumor cells at 37 °C for 0.5 h. Cells were then fixed, stained, and observed.

[0139] Nude mice with lung cancer PDX were injected intravenously with DiD or DiD-NPs at a concentration of 0.5 mg / mL, and scans were performed at different time points using an IVIS imaging system (Lumina III, PerkinElmer, Germany). Animals were sacrificed 48 hours after injection, and tumors and major organs were removed and imaged.

[0140] 2. Experimental Results

[0141] To detect cellular uptake and in vivo biodistribution, the hydrophobic near-infrared dye DiD (with strong fluorescence and tissue penetration) was encapsulated in micelles as a drug substitute. Intracellular uptake and localization of free DiD and DiD-loaded NPs (DiD-NPs) in H1975 and A549 cells were investigated using confocal fluorescence microscopy. After 0.5 h of incubation, strong fluorescence signals appeared in the cytoplasm of cells treated with free DiD, while the fluorescence signals in cells treated with DiD-NPs were weaker. With prolonged incubation, the intracellular fluorescence intensity of DiD-NPs gradually increased, and after 4 h of incubation, it was comparable to that of free DiD. Figure 2 F and Figure 30 This result indicates that nanoparticles can effectively enter cancer cells, and they can gradually degrade to achieve sustained, slow drug release.

[0142] To further verify the reduction-responsiveness of the nanoparticles, the NPs were incubated with different concentrations of GSH and then subjected to TEM imaging. Figure 31 As shown in Figure A, with increasing GSH concentration, disulfide bonds gradually broke, and the structure of NPs became irregular and their size decreased, indicating the disintegration of reduction-responsive NPs. Furthermore, the release of DiD-NPs in NSCLC cells with and without GSH pretreatment was observed under confocal microscopy. The results showed that, compared with untreated cells, GSH treatment led to a concentration-dependent increase in the fluorescence signal of DiD-NPs in H1975 cells. Figure 31 (B) This may be because, in the presence of high concentrations of GSH, DiD-NPs may rapidly dissociate when a critical number of disulfide bonds are broken. These results suggest that disulfide bonds in NPs can be reduced by GSH, leading to rapid degradation of the copolymer and stable drug release in a tumor-reducing microenvironment.

[0143] Real-time in vivo biodistribution of NPs was tracked using near-infrared fluorescence (NIRF) optical imaging. Free DiD and DiD-NPs were intravenously injected into mice with NSCLC PDX. In vivo optical imaging showed that after injection of free DiD, the fluorescence signal of DiD rapidly disappeared from the circulation and was almost undetectable in the tumor. In contrast, DiD-NPs had a longer circulation time and preferentially accumulated at the tumor site due to the EPR effect. Figure 3 A). In vitro images 48 hours after injection, compared to normal organs, further confirmed the accumulation of DiD-NPs in tumor tissue. Relatively strong fluorescence signals were also detected in the lungs and liver. Figure 3 B) This may be due to the non-specific clearance of NPs by RES.

[0144] Experimental Example 4: Study on the antitumor efficacy and mechanism of action of BBI608-NPs in chemotherapy-resistant CDX and PDX models of NSCLC

[0145] 1. Experimental Methods

[0146] A cell line-derived tumor xenograft (CDX) model of drug-resistant NSCLC was established by subcutaneously implanting A549 / DDP cells into the right side of nude mice. When the tumor xenograft volume reached 50-100 mm², a tumor xenograft model was established. 3Treatment began on day 0. Mice were randomly assigned to three groups (n=4): (i) a control group receiving intravenous saline, (ii) a group receiving free BBI608 orally (BBI608 dissolved in a 5:95 (v / v) mixture of DMSO and corn oil) at a dose of 10 mg / kg, and (iii) a group receiving BBI608-NPs intravenously at a dose of 10 mg / kg. Treatment was initiated on day 0. Mice were administered the medication intravenously or orally every other day for a total of ten doses. Tumor volume was measured twice weekly and calculated using the formula (L x W). 2 ) / 2, where L is the longest tumor diameter and W is the shortest tumor diameter. Mouse body weight was measured twice weekly to monitor drug toxicity.

[0147] A patient-derived lung cancer xenograft PDX mouse model was established using tumor tissue from NSCLC patients. Once the tumor volume reached approximately 50 to 100 mm... 3 The mice were randomly divided into two groups (n=5) and administered either saline or BBI608-NPs intravenously every other day at a dose of BBI608 = 10 mg / kg. On day 21, the animals were sacrificed, and blood samples were collected to determine blood cell counts and serum chemical parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine. In addition, tumors were excised for histopathological examination.

[0148] RNA sequencing (RNA-seq): Tumors were rapidly cryoexcised using liquid nitrogen, and total RNA was isolated and extracted. RNA sequencing of each sample yielded billions of reads, performed on the Illumina HiSeq2000 platform. Differentially expressed genes were identified by comparing them with the control and BBI608-NPs-treated groups; a 2-fold change with a p-value <0.05 was considered the threshold for downregulation or upregulation. All sequences were purchased from Shanghai Meiji Biotechnology Co., Ltd., and data were analyzed on the online platform Majorbio I-Sanger.

[0149] Western blotting: Cells or frozen tumor tissue were mixed with RIPA lysis buffer and lysed at 4°C for 15 min. The lysate was then centrifuged at 15,000 g for 15 min at 4°C, and the supernatant was collected. The total protein concentration was determined by the BCA protein assay. Equal volumes of protein were separated by 10% SDS-polyacrylamide gel electrophoresis (Beyotime, China) and transferred to a PVDF membrane (0.22 μm). The membrane was incubated with primary antibody overnight at 4°C, followed by incubation with secondary antibody at room temperature for 1 h. The membrane was washed three times, analyzed using enhanced chemiluminescence (ECL) reagent, and analyzed using a chemiluminescence imaging system (ChemiScope 6000Touch, Clinx, China).

[0150] 2. Experimental Results (2.1) In vivo antitumor drug efficacy results

[0151] To evaluate the in vivo therapeutic effect of BBI608-NPs on cancer cells (CSCs) of the present invention, a CDX model of drug-resistant NSCLC was established by subcutaneously implanting A549 / DDP cells into the right side of nude mice. Surprisingly, compared with the saline control, oral administration of free BBI608 did not significantly inhibit tumor growth, while intravenous injection of BBI608-NPs (10 mg / kg) significantly inhibited tumor growth (P < 0.05). Figure 3 C). Furthermore, there was no significant difference in weight between the treatment groups. Figure 3 D). The improved anti-tumor therapeutic effect of this nano-formulation can be attributed to the increased water solubility of BBI608, the prolonged circulation time of NPs, and the large accumulation of BBI608 in tumor tissue.

[0152] Recent studies have shown that cell line-derived tumor xenografts (CDXs) cannot accurately mimic the three-dimensional growth of tumor cells in vivo and their interactions with surrounding tissues because long-term culture and repeated passages of cell lines in culture dishes can induce genetic mutations and other irreversible changes. Compared to conventional CDXs, PDX models created by implanting fresh patient tumor fragments into immunodeficient mice are considered to preserve the histological and genetic characteristics of human tumor tissue. Therefore, the anticancer effects of BBI608-NPs were further evaluated using a PDX model of NSCLC. Compared to the control group, intravenous injection of BBI608-NPs (10 mg / kg) effectively inhibited tumor growth (…). Figure 3 E). Furthermore, no significant changes in mouse body weight were observed, and blood cell counts and routine biochemical indicators remained within the normal range, indicating that BBI608-NPs were well-tolerated in PDX mice. Figure 3(F to H). At the end of the experiment, tumor tissue was removed for histopathological examination. H&E staining showed that tumor cells in the BBI608-NPs-treated group exhibited typical apoptotic features, such as cytoplasmic condensation and nuclear pyknosis. TUNEL assays further confirmed the increase in apoptotic cells after treatment with BBI608-NPs. Figure 3 I). Simultaneously, immunohistochemical results showed a significant reduction in Ki67 and CD44 positive cells in the BBI608-NPs treatment group, indicating that BBI608-NPs can effectively kill lung cancer cells and CSCs (I). Figure 3 I). These results demonstrate that by effectively delivering BBI608 to tumor cells, BBI608-NPs exhibit excellent anti-tumor therapeutic effects in both drug-resistant NSCLC and PDX models, suggesting that BBI608-NPs have the potential to become a candidate drug for the treatment of drug-resistant NSCLC.

[0153] (2.2) Mechanism of antitumor action in vivo

[0154] It has been reported that STAT3 activation leads to tumorigenesis by inducing proliferation, angiogenesis, invasion, metastasis, and promoting the formation of tumor sclerosis cells (CSCs). To investigate the role of STAT3 in lung cancer development and the therapeutic mechanism of BBI608-NPs in a PDX model of NSCLC, transcriptome sequencing analysis was performed on collected tumor tissues using RNA sequencing (RNA-seq). The results showed that after treatment with BBI608-NPs, a total of 636 downregulated genes and 929 upregulated genes were identified. Figure 4 A). KEGG analysis showed that these differentially expressed genes were mainly enriched in arachidonic acid (AA) metabolism, JAK-STAT, Wnt, and ERBB pathways. Figure 4 B). GO enrichment analysis also indicated that these genes are mainly involved in AA metabolism and the aforementioned cancer-related pathways (B). Figure 4 C). Heatmap results showed that treatment with BBI608-NPs downregulated the expression of STAT3-activating genes, such as the anti-apoptotic gene Bcl-2, while upregulating the expression of anti-tumor genes and pro-apoptotic genes (such as P21 and BAX). These results were further validated by Western blotting experiments. Figure 4 (D to F). Furthermore, the expression of stemness-related genes such as β-catenin, C-myc, and SOX2 was analyzed. The results showed that the mRNA and protein levels of these genes were downregulated, indicating that BBI608-NPs can effectively inhibit lung CSCs in NSCLC PDX mice. Figure 4 (D to F).

[0155] It is important to note that β-catenin is not only a protein expressed by CSCs, but it also acts as a key transcriptional coactivator, activating target genes in the canonical Wnt pathway by transmitting extracellular signals. When the Wnt pathway is activated by specific ligands, one of the components of the β-catenin disruption complex, glycogen synthase kinase-3β (GSK3β), can be phosphorylated and degraded, leading to the dissociation of β-catenin from the disruption complex. Accumulated in the cytoplasm, β-catenin can enter the nucleus and activate the Wnt transcriptional program. Since phosphorylation of GSK3β (p-GSK3β) may play a role in alleviating β-catenin inhibition, p-GSK3β expression was first detected by Western blotting. Figure 4 As shown in H and I, treatment with BBI608-NPs resulted in increased phosphorylation levels of GSK3β (P < 0.05). However, the expression level of β-catenin in the cytoplasm remained very low. Figure 4 The results (D to F) indicate that β-catenin may be inhibited by other proteins. Peroxisome proliferator-activated receptor γ (PPARγ) is one of the downstream receptors in the AA metabolic pathway. It is a ligand-activated transcription factor that inhibits tumors by regulating immunity, inflammation, and oxidative stress. Studies have shown that after activation by upstream signals (such as fatty acid derivatives like PGD2), PPARγ heterodimerizes with the retinoid X receptor (RXR) and then binds to the PPAR response element (PPRE) to regulate ROS production, thereby inhibiting the accumulation of cytoplasmic β-catenin. Notably, after BBI608-NPs treatment, GO and KEGG analyses revealed that differentially expressed genes were mainly enriched in AA metabolism. Furthermore, prostaglandin d2 synthase (PTGDS), a key gene in AA metabolism, was significantly upregulated after BBI608 treatment (fold change ≥10, P<0.05). Figure 4 (G to H). Since the protein encoded by PTGDS is involved in catalyzing the conversion of PGH2 to PGD2, this may stimulate the expression of the downstream protein PPARγ; therefore, PPARγ expression was further examined. As expected, PPARγ protein expression was shown to be upregulated. Figure 4 H and I)(P<0.05). Therefore, BBI608-NPs treatment can directly regulate downstream target genes by inhibiting STAT3 phosphorylation and indirectly inhibit the expression of β-catenin, a key protein in the Wnt pathway, by reducing the expression of STAT3-inhibited PPARγ. Therefore, BBI608-NPs treatment can simultaneously inhibit the activation of the JAK-STAT and Wnt pathways through "dual inhibition," providing a new strategy for the treatment of NSCLC. Figure 4 J).

[0156] In summary, this invention provides a reduction-responsive nanodelivery system and its use in the preparation of drugs for treating drug-resistant tumors. This invention constructs a reduction-responsive polyethylene glycol-branched poly(HPMA-DA) that can effectively encapsulate small molecule drugs to form a nanodelivery system. Experiments have demonstrated that this nanodelivery system can be taken up by tumor cells, exhibits good tumor targeting, and demonstrates excellent anti-tumor effects in tumor xenografts (CDX) derived from chemotherapy-resistant non-small cell lung cancer (NSCLC) cell lines and patient-derived xenograft mouse models (PDX). The polyethylene glycol-branched poly(HPMA-DA) provided by this invention can be used to encapsulate various hydrophobic small molecule drugs, including BI608, with high drug selectivity and broad application prospects. The preparation method of the nanodelivery system of this invention is simple, avoiding the chemical bonding step, and has broad application prospects in the preparation of drugs for treating tumors.

Claims

1. A polymer, characterized in that, It is prepared by reacting MA-SS-CTA, MA-SS-MA, HPMA, MA-SS-mPEG, MA-SS-DA and an initiator as raw materials. The molar ratio of MA-SS-CTA, MA-SS-MA, HPMA, MA-SS-mPEG, MA-SS-DA and the initiator is 1:(0.5-2):(60-80):(5-20):(10-30):(0.1-2). The structures of MA-SS-CTA, MA-SS-MA, HPMA, MA-SS-mPEG and MA-SS-DA are shown below: ； n is between 20 and 100.

2. The polymer according to claim 1, characterized in that, The polymer has a weight-average molecular weight Mw of 13-14 kDa and a number-average molecular weight Mn of 15-16 kDa.

3. The polymer according to claim 2, characterized in that, The polymer has a weight-average molecular weight Mw of 13.39 kDa and a number-average molecular weight Mn of 15.19 kDa.

4. The polymer according to claim 1, characterized in that, The initiator is an azo initiator; And / or, the n is 30-60.

5. The polymer according to claim 4, characterized in that, The molar ratio of MA-SS-CTA, MA-SS-MA, HPMA, MA-SS-mPEG, MA-SS-DA, and the initiator is 1:1:70:10:21:0.7; And / or, the azo initiator is azobisisobutyrazoline or a salt thereof; And / or, the n is 40-50.

6. The polymer according to claim 5, characterized in that, The salt is a hydrochloride salt.

7. The polymer according to any one of claims 1-6, characterized in that, The solvent for the reaction is one or a mixture of two of the following: an organic solvent and water. And / or, the reaction time is 12-36 hours. And / or, the temperature of the reaction is 40-60°C; And / or, the reaction is carried out in an inert gas atmosphere.

8. The polymer according to claim 7, characterized in that, The organic solvent is 2,2,2-trifluoroethanol; And / or, the reaction time is 24 hours; And / or, the temperature of the reaction is 47°C.

9. Use of the polymer according to any one of claims 1-8 in the preparation of a drug carrier.

10. A nanodelivery system, characterized in that, It is obtained by encapsulating the drug in the polymer according to any one of claims 1-8.

11. The nanodelivery system according to claim 10, characterized in that, The drug is a cancer stem cell inhibitor; And / or, the mass ratio of the polymer to the drug is (5-20):

1.

12. The nanodelivery system according to claim 11, characterized in that, The drug in question is BBI608; And / or, the mass ratio of the polymer to the drug is 10:

1.

13. A method for preparing the nanodelivery system according to any one of claims 10-12, characterized in that, The method includes the following steps: dissolving the polymer and drug in an organic solvent, evaporating the solvent to obtain a polymer-drug film, dissolving the polymer-drug film in water, and self-assembling to form a nanodelivery system.

14. The method according to claim 13, characterized in that, The organic solvent is one or a mixture of two of chloroform and acetonitrile.

15. Use of the nanodelivery system according to any one of claims 10-12 in the preparation of a medicament for treating tumors.

16. The use according to claim 15, characterized in that, The tumor in question is a drug-resistant tumor.

17. The use according to claim 16, characterized in that, The tumor in question is a chemotherapy-resistant tumor.

18. The use according to claim 17, characterized in that, The chemotherapy-resistant tumor is chemotherapy-resistant lung cancer.

19. The use according to claim 18, characterized in that, The lung cancer in question is non-small cell lung cancer.

Images