Micellar nano-drug as well as preparation method and application thereof

Abstract

The invention discloses a micelle nano-drug as well as a preparation method and application thereof. The micelle nano-drug is a polymer micelle co-loaded photosensitizer and inhibitor nano-drug. Recurrence and metastasis of cancer are closely related to tumor stem cells (CSCs), and great challenges are brought to treatment. The micelle provided by the invention can stably load two drugs, and simultaneous ablation of tumor cells and CSCs can be realized through combination of photothermal therapy (PTT) and stem cell inhibition, so that the proportion of CSCs in cancer cells can be significantly reduced, and formation of tumor spheres can be inhibited. Targeted micelles are obtained by modifying ligands, and the curative effect is further enhanced. Experiments prove that the medicine disclosed by the invention shows obvious survival benefits in a tumor model and a postoperative model, and the expression of CSC related biomarkers is obviously reduced. The micelles are combined with NIR irradiation, a promising strategy is provided for simultaneously ablating cancer cells and inhibiting CSCs, and rapid tumor elimination and recurrence prevention can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of drug delivery, relates to nano-drugs, and specifically relates to a micelle nano-drug, a preparation method thereof, and an application thereof. Background Art

[0002] Cancer stem cells (CSCs) are responsible for drug resistance and cancer recurrence and play a crucial role in tumor recurrence and metastasis [Cancer stem cell in prostate cancer progression, metastasis and therapy resistance]. Their existence poses a major challenge to tumor treatment. In addition, the stemness of tumor cells is closely related to the activation of epithelial-mesenchymal transition. High epithelial-mesenchymal transition can promote the formation of cancer stem cells, and tumor-associated fibroblasts can also induce the formation of cancer stem cells through multiple pathways, including promoting the secretion of TGF-β and IL-6, thereby promoting cancer cell metastasis. Compared with ordinary tumor cells, cancer stem cells have stronger self-renewal ability and tumor-forming ability [Tumor microenvironment for cancer stem cells], which makes them a key target for treatment strategies. Researchers have explored various strategies to inhibit CSCs, such as targeting ATP-binding cassette transporters, DNA damage repair mechanisms, autophagy, ferroptosis, and immunosuppression, etc. [Targeting cancer stem cell pathways for cancer therapy, Signal Transduct; Targeting cancer stem cells for reversing therapy resistance: mechanism, signaling, and prospective agents, Signal Transduct]. Various differentiating agents and inhibitors have been developed for CSC pathways such as Hedgehog, STAT3, Wnt, Notch, and NF-κB, and some of them have entered the clinical trial stage. However, due to their strong ability to renew and promote metastasis, there are few effective methods to eliminate CSCs. CSCs reside in hypoxic niches and are in a quiescent state, which can evade conventional treatments, resulting in treatment resistance, tumor recurrence, and metastasis. In fact, after most treatments, the proportion of CSCs usually increases significantly [Role of prostate cancer stem-like cells in the development of antiandrogen resistance].Although small molecule inhibitors have been shown to have the potential to inhibit CSCs in solid tumors, despite some progress, there are still few treatment options that can simultaneously eliminate large solid tumors and CSCs [Elimination of SOX2 / OCT4-Associated Prostate Cancer StemCells Blocks Tumor Development and Enhances Therapeutic Response]. Summary of the Invention

[0003] The self-renewal and differentiation characteristics of cancer stem cells (CSCs) can lead to chemotherapy resistance in cancer. Although many drugs targeting CSCs have been developed, their delivery and accumulation efficiency at the lesion site are always relatively low, limiting the effect of the drugs. The present invention discloses a micellar nano-drug, its preparation method and application. The active ingredients include a photosensitizer and a cancer stem cell inhibitor, which can respond to near-infrared (NIR) to generate a photothermal effect, and deliver the inhibitor to inhibit cancer stem cells (CSCs). This strategy can efficiently treat solid tumors such as prostate cancer and pancreatic cancer.

[0004] The present invention adopts the following technical solutions.

[0005] A micellar nano-drug, comprising a polymeric micelle, a photosensitizer, and an inhibitor; wherein, the polymer forms a polymeric micelle to co-load the photosensitizer and the inhibitor.

[0006] In the present invention, the polymer comprises a hydrophilic segment and a hydrophobic segment, and forms a polymeric micelle by self-assembly of the polymer. Preferably, the polymeric micelle is conjugated with a targeting molecule.

[0007] In the present invention, the polymer for assembling the polymeric micelle comprises an amphiphilic block polymer, or the polymer for assembling the polymeric micelle comprises an amphiphilic block polymer and a targeting amphiphilic block polymer.

[0008] In the present invention, the amphiphilic block polymer comprises a hydrophilic segment and a hydrophobic segment; the targeting amphiphilic block polymer comprises a hydrophilic segment, a hydrophobic segment and a targeting molecule.

[0009] Preferably, in the amphiphilic block polymer and the targeting amphiphilic block polymer, the hydrophobic segment comprises a DTC (dithiacyclopentane trimethylene carbonate) monomer repeating unit; more preferably, the hydrophobic segment comprises a DTC monomer repeating unit and an ester monomer repeating unit, denoted as P(ester monomer unit-DTC).

[0010] Preferably, the ester monomer includes a cyclic ester monomer, a cyclic carbonate monomer, etc., such as trimethylene carbonate monomer TMC, lactide monomer LA, caprolactone monomer CL, etc.

[0011] Preferably, the polymer composed of a hydrophilic segment and a hydrophobic segment is hydrophilic segment - P(ester monomer unit - DTC), which is an existing polymer; the polymer composed of a targeting molecule, a hydrophilic segment, and a hydrophobic segment is targeting molecule - hydrophilic segment - P(ester monomer unit - DTC), which is an existing polymer.

[0012] Among them: P(ester monomer unit - DTC) is a hydrophobic segment, which is a polymerization segment of an ester monomer and a DTC monomer.

[0013] The preferred hydrophilic segment is a polyethylene glycol segment (PEG); the molecular weight of the hydrophilic segment is 1000 - 15000 Da, the molecular weight of the hydrophobic segment is 0.1 - 5 times that of the hydrophilic segment; the molecular weight of the PDTC segment is 10% - 70% of the total molecular weight of the hydrophobic segment.

[0014] Preferably, the molecular weight of PEG is 1000 - 12000 Da, and further preferably, the molecular weight of PEG is 1500 - 8000 Da.

[0015] Preferably, the molecular weight of the hydrophobic segment is 0.2 - 5 times that of the PEG molecular weight. Preferably, the molecular weight of the hydrophobic segment is 0.2 - 3 times that of the PEG molecular weight. Further preferably, the molecular weight of the hydrophobic segment is 0.5 - 2 times that of the PEG molecular weight, such as 0.75 times, 1 time, 1.5 times, 1.75 times or any multiple within the range.

[0016] Preferably, the molecular weight of PDTC is 30 - 60% of the molecular weight of the hydrophobic segment. Preferably, the molecular weight of PDTC is 40 - 60% of the molecular weight of the hydrophobic segment, such as 45%, 50%, 55% or any ratio within the range.

[0017] In the present invention, the photosensitizer includes one or more of an inorganic photosensitizer, a metal photosensitizer, and an organic photosensitizer. Preferably, it is an organic photosensitizer, and preferably a near - infrared organic photosensitizer, such as indocyanine green, etc.

[0018] In the present invention, the inhibitor includes a cancer stem cell inhibitor. Preferably, it is a cancer stem cell inhibitor; further preferably, it is a cancer stem cell pathway inhibitor; such as a STAT3 pathway inhibitor, a Hedgehog pathway inhibitor, a Wnt pathway inhibitor, a Notch pathway inhibitor, an NF - kB pathway inhibitor. Preferably, it is a small molecule inhibitor of the STAT3 pathway.

[0019] The present invention discloses a preparation method of the above - mentioned micelle nano - drug, which includes the following steps: mixing raw materials in a solution to obtain the micelle nano - drug; the raw materials include a polymer, a photosensitizer, and an inhibitor.

[0020] In the present invention, raw materials are mixed in a solution, and polymers are assembled to form micelles and loaded with photosensitizers and inhibitors to obtain micelle nano-drugs.

[0021] In the present invention, the solution includes a solvent and a buffer; wherein, the solvent includes an organic solvent.

[0022] In the present invention, the molar ratio of the photosensitizer to the inhibitor is (0 - 5):1, excluding 0; preferably, the molar ratio of the photosensitizer to the inhibitor is (0.5 - 5):1; preferably, the molar ratio of the photosensitizer to the inhibitor is (0.6 - 4):1; preferably, the molar ratio of the photosensitizer to the inhibitor is (0.7 - 3):1; for example; preferably, the molar ratio of the photosensitizer to the inhibitor is 0.75:1, 1:1, 1.5:1, 2:1 or any ratio within the range.

[0023] In the present invention, the polymers (assembled into polymer micelles) preferably include amphiphilic block polymers and targeted amphiphilic block polymers. Among them, the molar ratio of the amphiphilic block polymer to the targeted amphiphilic block polymer is (70 - 100 excluding 100):(0 - 30 excluding 0), preferably, the molar ratio of the amphiphilic block polymer to the targeted amphiphilic block polymer is (80 - 99):(1 - 20), preferably, the molar ratio of the amphiphilic block polymer to the targeted amphiphilic block polymer is (90 - 97):(3 - 10); for example 90:10, 92:8, 95:5, 96:4 or other ratios within the range.

[0024] The present invention discloses the application of the above-mentioned micelle nano-drugs in the preparation of anti-tumor drugs.

[0025] The present invention discloses the application of the above-mentioned micelle nano-drugs in the preparation of multi-modal anti-tumor drugs. Preferably, the multi-modal includes one or more of photothermal therapy and chemotherapy.

[0026] Preferably, the present invention discloses the application of the above-mentioned micelle nano-drugs in the preparation of synergistic anti-tumor drugs. The micelle nano-drugs of the present invention can be used in combination with other anti-tumor drugs to enhance the therapeutic effect.

[0027] An anti-tumor drug, the active ingredient of which includes the above-mentioned micelle nano-drugs; it may also include other anti-tumor drugs.

[0028] Among them, the tumor is a solid tumor, especially prostate cancer, particularly advanced prostate cancer, and can also effectively treat pancreatic cancer.

[0029] Cancer stem cells (CSCs) are a small but lethal subpopulation within the tumor mass, possessing strong self-renewal ability and differentiation potential. CSCs reside in the hypoxic niche, remain in a quiescent state, and can evade conventional therapies, leading to treatment resistance, tumor recurrence, and metastasis. In fact, after most treatments, the proportion of CSCs usually increases significantly. Existing therapies for prostate cancer (PCa), such as androgen deprivation therapy, chemotherapy, and radiotherapy, although somewhat effective for patients with early-diagnosed PCa, have little effect on patients with advanced PCa. The challenge is the difficulty in eradicating cancer stem cells (CSCs), which play a crucial role in tumor recurrence and metastasis. Researchers have explored various strategies to inhibit CSCs in prostate cancer, such as targeting ATP-binding cassette transporters, DNA damage repair mechanisms, autophagy, ferroptosis, and immunosuppression; various differentiating agents and inhibitors have been developed against CSC pathways such as Hedgehog, STAT3, Wnt, Notch, and NF-κB. Although some progress has been made, there are few treatment regimens that can simultaneously eliminate large solid tumors and CSCs. As an example, the present invention discloses micelles co-loaded with indocyanine green and nabumetone for photothermal ablation of prostate cancer tumor cells and eradication of tumor stem cells.

[0030] Photothermal therapy (PTT) can ablate tumors in various tumor models by local heating (48 - 60 °C), but it also faces the problems of recurrence and metastasis. Indocyanine green (ICG) has been approved by the FDA, and its photothermal conversion performance has been widely studied, but it has the problems of rapid clearance in vivo and lack of targeting; different from other drugs (such as paclitaxel, docetaxel, and gemcitabine), Nap encounters great challenges in drug use and formulation due to its stronger hydrophobicity and high crystallinity. According to the embodiments of the present invention, a micellar nanodrug (mICG - Nap) co - loaded with indocyanine green (ICG) and the small - molecule STAT3 inhibitor nabumetone (Nap) is disclosed, which can generate a photothermal effect in response to near - infrared (NIR) and deliver Nap to inhibit cancer stem cells (CSCs), and this strategy can effectively treat prostate cancer. The present invention can efficiently and stably co - load ICG and Nap through a disulfide - cross - linkable biodegradable micelle of polyethylene glycol - poly(ε - caprolactone - co - dithiolane trimethylene carbonate) (PEG - P(CL - DTC)); further, mICG - Nap is modified with an Acupa ligand (Acupa - mICG - Nap), which can specifically target RM1 - PSMA cells and tumors with high expression of PSMA. According to the technical solution of the present invention, under NIR irradiation, Acupa - mICG - Nap shows significant survival benefits in both subcutaneous RM1 - PSMA tumor models and postoperative models by simultaneously eliminating prostate cancer cells and CSCs. Experiments have confirmed that Acupa - mICG - Nap + L significantly inhibits tumor growth (****), no lung metastasis was observed on the 17th day, and in the refractory postoperative model, the median survival time (MST) of mice was significantly extended to 24 days (**). Brief Description of the Drawings

[0031] Figure 1 For the synthesis and characterization of Acupa - PEG - P(CL - DTC). (A) Synthesis route. (B) NHS - PEG - P(CL - DTC) and (C) 1 1H NMR spectra (400 MHz, DMSO - d6).

[0032] Figure 2Characterization of mICG-Nap. (A) Particle size distribution diagrams of mICG-Nap and Acupa-mICG-Nap, with micelles loaded with ICG (mICG) and empty micelles (Ms) as controls, and the particle size distribution of mICG-Nap after incubation in PB containing 10% FBS for 12 h (polymer concentration: 1 mg / mL). (B) Simulation of the intermolecular interaction between ICG and Nap using the AutoDock Vina program (molar ratio: 1 / 1). (C) UV-visible spectrum of mICG-Nap (ICG concentration: 10 μg / mL). Fluorescence spectra of mICG-Nap in (D) PB and (E) DMF (excitation wavelength: 780 nm, ICG concentration: 20 μg / mL). (F) Temperature increase of different formulations under near-infrared irradiation (300 μL, ICG concentration: 25 μg / mL, n = 3). (G) Representative thermal imaging pictures and average temperature of tumors in RM-1-PSMA tumor-bearing mice 1 h after intravenous injection of mICG-Nap (6.46 mg ICG / kg) (n = 3). (H) Semi-quantitative analysis of the ROS level produced by mICG-Nap-treated RM-1-PSMA cells using DCF as a marker by Image J analysis (ICG concentration: 10 μg / mL, n = 3). mICG, free ICG-Nap, and free ICG were used as control groups. For F, G, and H, the near-infrared irradiation conditions were: 808 nm, 1 W / cm 2 , 5 min. *p < 0.05, **p < 0.01.

[0033] Figure 3 Characterization and photothermal properties of mICG-Nap and mICG (n = 3). (A) Changes in particle size and particle size distribution of mICG-Nap before and after storage at -80 °C for 3 months. (B) Cytotoxicity of mICG-Nap against RM1-PSMA cells (before and after storage for 3 months). (C) Physical pictures of different formulations. (D) Temperature increase of different formulations at an ICG concentration of 6.25 μg / mL. (E) Temperature increase of mICG-Nap with different ICG concentrations under near-infrared irradiation (near-infrared conditions: 808 nm, 1 W / cm 2 , 5 min). (F) Temperature increase of mICG-Nap under irradiation with different laser power densities (ICG concentration: 20 μg / mL).

[0034] Figure 4Establishment of RM1-PSMA tumor spheroids to enrich CSCs. (A) Schematic diagram of three-dimensional tumor spheroid culture. (B) Photos of RM1-PSMA tumor spheroids rich in CSCs at days 0, 3, 5, and 7 (scale bar: 400 μm). (C) Representative flow cytometry plots of CD44 + CD133 + cell populations in RM1-PSMA cells and S-RM1-PSMA cells (stem cell-like cells obtained from tumor spheroids).

[0035] Figure 5 Inhibitory effect of mICG-Nap on the stemness of S-RM1-PSMA cells. (A) Flow cytometry analysis of the viable cell population (7-AAD - )(n = 3) of S-RM1-PSMA cells after incubation with mICG-Nap for 48 h. (B) Flow cytometry analysis of CD44 + CD133 + cell populations of S-RM1-PSMA cells after incubation with mICG-Nap for 48 h (n = 3). (C) Images and numbers of tumor spheroids (>50 μm) formed after incubation of single-cell suspensions with mICG-Nap for 2 days (scale bar: 200 μm, n = 4). (D) Images and numbers of remaining tumor spheroids (>100 μm) after incubation of tumor spheroids with mICG-Nap for 2 days (scale bar: 200 μm, n = 4). (E) Images and numbers of remaining tumor spheroids (>200 μm) after treatment with mICG-Nap for 1 h, near-infrared (808 nm, 1 W / cm 2 2, 5 min) irradiation and continued incubation in a hypoxic environment for 5 days. Different concentrations of mICG+L and mICG-Nap were used as control groups (scale bar: 200 μm, n = 3).

[0036] Figure 6 Effects of mICG-Nap and mICG-Nap+L on colony formation, cytotoxicity, and apoptosis of S-RM1-PSMA cells. (A) Colony formation of S-RM1-PSMA cells after incubation with mICG-Nap for 8 days. (B) Cytotoxicity of S-RM1-PSMA cells incubated with mICG-Nap for 1 h under near-infrared irradiation and further cultured for 47 h measured by CCK8 assay (n = 4). (C) Apoptosis of S-RM1-PSMA cells after incubation with mICG-Nap for 1 h under near-infrared irradiation and continued culture for 23 h. In experiments B and C, L: 808 nm, 1 W / cm2, 5 min. Different concentrations of mICG+L and mICG-Nap or free ICG-Nap were used as control groups.

[0037] Figure 7Antitumor efficacy of mICG-Nap+L combination therapy in the RM1-PSMA mouse model. (A) Treatment flow chart. On the 7th day after inoculation, mICG-Nap (mICG-Nap i.v. +L, ICG / Nap dose: 6.46 / 2 mg / kg) was intravenously injected on the 7th, 9th, and 11th days respectively, or mICG-Nap i.t +L, ICG / Nap dose: 1.6 / 0.5 mg / kg) was intratumorally injected on the 7th and 19th days. One hour after each injection, the tumor was irradiated with near-infrared laser (L: 808 nm, 5 min, 1 W / cm 2 ). (B) Mean tumor growth curve and individual tumor growth curves; (C) Relative body weight change; (D) Mouse survival curve (n = 4). (E) H&E staining pictures of lung tissues of mice in the treatment group (day 35) and PBS group (day 13). Scale bar: 500 μm. **p < 0.01.

[0038] Figure 8 Antitumor efficacy of mICG-Nap+L and Acupa-mICG-Nap+L combination therapies in the RM1-PSMA mouse model. (A) Experimental design flow chart. ICG dose: 6.46 mg / kg, Nap dose: 2 mg / kg (ICG / Nap molar ratio is 1 / 1); near-infrared laser (L: 808 nm, 5 min, 1 W / cm 2 ). (B) Mean tumor growth curve, individual tumor growth curves, and tumor inhibition rate on the 12th day; (C) Relative body weight change; (D) Mouse survival curve (n = 5). PBS, mICG-Nap, and mICG+L were used as control groups. (E) Representative infrared thermal imaging pictures of mice and the temperature at the tumor site at the first treatment (n = 3). (F) H&E staining images of lung tissue sections (scale bar: 100 μm). *p < 0.05, **p < 0.01.

[0039] Figure 9Cell experiments and pharmacokinetic studies of Acupa-mICG-Nap. (A) Flow cytometry was used to determine the expression of PSMA on the surface of RM1-PSMA, RM1, LNCaP, and PC3 cells. The uptake of Cy5-labeled Acupa-mICG-Nap containing 1.25%, 2.5%, 5%, or 10% Acupa in (B) RM1-PSMA cells and (C) the uptake of 5% Acupa-mICG-Nap by RM1 cells. The toxicity of Acupa-mICG-Nap+L and Acupa-mICG-Nap to (D) RM1-PSMA cells and (E) RM1 cells (n = 3). (F) The toxicity of empty micelles to RM1-PSMA cells (n = 6). (G) Pharmacokinetics of Acupa-mICG-Nap, mICG-Nap, and the free mixture of ICG-Nap in RM1-PSMA tumor-bearing mice were measured using IVIS and HPLC, respectively (ICG: 6.46 mg / kg, Nap: 2 mg / kg, n = 3). Parameters (t 1 / 2 , β, and AUC) were calculated by PK Solver.

[0040] Figure 10 Antitumor efficacy of Acupa-mICG-Nap+L in the postoperative RM1-PSMA model. (A) Experimental schedule for establishing and treating the postoperative model. ICG dose: 6.46 mg / kg, Nap dose: 2 mg / kg (ICG / Nap molar ratio is 1 / 1); near-infrared laser (1 h after injection, 808 nm, 1 W / cm 2 , 5 min). (B) Mean growth curve and individual growth curves of recurrent tumors; (C) relative body weight changes; (D) mouse survival curve (n = 6). (E) H&E staining pictures of lung sections on day 17. (F) Representative in vivo thermal imaging pictures of mice and the temperature at the tumor site on day 9 (n = 6). (G) Representative HIF images of the expression of OCT4, SOX2, CD133, and pSTAT3 in primary tumors (day 7) and recurrent tumors (day 15) sections. ***p < 0.001, ****p < 0.0001.

[0041] Figure 11 Antitumor efficacy of Acupa-mDTX and mDTX. (A) Particle size and particle size distribution. Toxicity (48 h, n = 3) and (C) apoptosis-promoting ability (24 h, DTX: 2 mg / mL, n = 3) to RM1-PSMA cells. (D) Treatment regimen for RM1-PSMA tumor-bearing mice and (E) tumor volume, (F) relative body weight, and (G) survival curve after treatment (DTX dose: 7.5 mg / kg, i.v. n = 3). *p < 0.05, ***p < 0.001.

[0042] Figure 12 Anti-cancer efficacy of mICG-Nap+L combination therapy in RM1-PSMA tumor-bearing mice. (A) Experimental protocol (mICG-Nap i.v. +L, ICG / Nap dose: 6.46 / 2 mg / kg; mICG-Nap i.t. +L, ICG / Nap dose: 1.6 / 0.5 mg / kg). (B) Average tumor growth curve; (C) Mouse survival rate; (D) Relative body weight (n = 3). (E) H&E staining images of lung sections (day 30). Scale bar: 100 μm. **p < 0.001.

[0043] Figure 13 In vivo efficacy of mICG-Nap+L in RM1 subcutaneous tumor-bearing mice. (A) Schematic diagram of treatment protocol and tumor rechallenge experiment design. (B) Tumor site temperature; (C) Subcutaneous tumor volume; (D) Measurement of lymphatic metastatic tumor volume; (E) Relative body weight change; (F) Kaplan-Meier survival curve of RM1 mice after different treatments (n = 6). (G) Tumor volume; (H) Spleen mass; (I) Cytotoxicity of spleen cells against RM1 cells in vitro.

[0044] Figure 14 For KC 15 -mICG-Nap+L and mICG-Nap+L in vivo anti-tumor efficacy against PC. (A) Schematic diagram of the treatment process of PANC-1 tumor-bearing mice. Temperature at the tumor site (B), tumor growth curve (C), survival curve (D), and relative body weight curve (E) after different treatment methods (n = 6). (F) H&E staining pictures of the liver of PANC-1 tumor-bearing mice in the mICG-Nap+L group and PBS group 25 days after treatment. (G) Schematic diagram of the treatment process of SW1990 tumor-bearing mice. Temperature at the tumor site (H), tumor growth curve (I), survival curve (J), and relative body weight curve (K) after different treatments (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0045] Figure 15 Design and scheme of the present invention. (A) Simple preparation of Acupa-mICG-Nap. (B) Strategy for combined PTT and CSCs eradication to treat prostate cancer and prevent its recurrence and metastasis. Detailed implementation manners

[0046] The development of cancer, especially the recurrence and metastasis of advanced prostate cancer, is closely related to cancer stem cells (CSCs), posing a great challenge to treatment. Existing technologies have explored various strategies to inhibit CSCs in prostate cancer and developed various differentiating agents and inhibitors. Although certain progress has been made, there are still few treatment regimens that can simultaneously eliminate large solid tumors and CSCs. The present invention discloses a micellar nanodrug, comprising a polymeric micelle, a photosensitizer, and an inhibitor; wherein, the polymer forms a polymeric micelle to co-load the photosensitizer and the inhibitor; the present invention uses the polymeric micelle formed by the polymer to co-load the photosensitizer and the inhibitor as the micellar nanodrug, and the micellar nanodrug ablates tumor cells and eradicates cancer stem cells through photothermal ablation. As an example, the present invention discloses a micelle co-loading indocyanine green and nabumetone for photothermal ablation of prostate cancer tumor cells and eradication of cancer stem cells; specifically, the present invention discloses a micelle (mICG-Nap) co-loading indocyanine green and nabumetone, which simultaneously ablates tumor cells and CSCs through photothermal therapy (PTT) combined with stem cell inhibition to treat prostate cancer. mICG-Nap can stably load the two drugs, has a suitable particle size, can significantly reduce the proportion of CSCs in RM1-PSMA mouse prostate cancer cells, and inhibits the formation of tumor spheres. Compared with free ICG, mICG-Nap exhibits a stronger photothermal effect under near-infrared (NIR) irradiation and completely eliminates tumor spheres. By modifying the Acupa ligand to obtain Acupa-mICG-Nap, it can specifically target RM1-PSMA cells and tumors with high PSMA expression, and the therapeutic effect is further enhanced. The increased uptake of Acupa-mICG-Nap in tumor cells results in significant survival benefits in both subcutaneous RM1-PSMA tumor models and postoperative models. Tumor analysis shows that Acupa-mICG-Nap significantly downregulates the expression of CSC-related biomarkers (such as OCT4, SOX2, CD133, and pSTAT3) and PSMA. The combination of this micelle and NIR irradiation provides a highly promising strategy for simultaneously ablating cancer cells and inhibiting CSCs, enabling rapid tumor elimination and preventing recurrence.

[0047] In the present invention, the molar ratio of indocyanine green to nabumetone is preferably (0.5-2.5):1; for example; preferably, the molar ratio of indocyanine green to nabumetone is 0.75:1, 1:1, 1.5:1, 2:1, or any ratio within the range.

[0048] In the present invention, the polymers (assembled into polymeric micelles) preferably include amphiphilic block polymers and targeting amphiphilic block polymers. Among them, the mass ratio of the amphiphilic block polymer to the targeting amphiphilic block polymer is (70 to 100 excluding 100):(0 to 30 excluding 0). Preferably, the mass ratio of the amphiphilic block polymer to the targeting amphiphilic block polymer is (80 to 99):(1 to 20). Preferably, the mass ratio of the amphiphilic block polymer to the targeting amphiphilic block polymer is (90 to 97):(3 to 10); such as 90:10, 92:8, 95:5, 96:4 or other ratios within the range.

[0049] In the present invention, the targeting molecule is a reagent that can target tumors, including small molecule targeting molecules, macromolecule targeting molecules, etc., such as ligands that target receptor proteins on the tumor cell membrane.

[0050] The following illustrates the technical progress of the present invention through specific experiments. In the present invention, the materials, instruments, cells, and animals are all conventional products or technologies. The amphiphilic block polymer PEG-P(CL-DTC) and the targeting polymer Acupa-PEG-P(CL-DTC) are synthesized according to the methods of the prior art. The molecular weight of PEG-P(CL-DTC) is 2 - 1.2 - 1.1 kg / mol, and the molecular weight of NHS-PEG-P(CL-DTC) is 3.4 - 1.7 - 1 kg / mol. The targeting molecules Acupa and KC 15 (KTLLPTPKRGDFKKC) are existing products. Nabumetone (Nap), indocyanine green (ICG), methoxypolyethylene glycol (PEG 350), RPMI 1640 medium, DMEM medium, FBS, cell proliferation and cytotoxicity detection kit (CCK-8), etc. are directly used after purchase; the cells, animals, and models involved are of the prior art. The human PSMA-negative PC3, human PSMA-positive LNCaP, and mouse RM1 prostate cancer cell lines are all purchased from the Cell Bank of the Chinese Academy of Sciences; the RM1-PSMA cells with positive PSMA are existing products and can be transfected according to conventional methods; male Balb / c nude mice (5 weeks old, 18 - 20 g, Beijing Vital River Laboratory Animal Technology Co., Ltd.) are housed in a specific pathogen-free experimental animal room. All animal experiments are carried out in accordance with the "Experimental Animal Management Measures of Soochow University" and are approved by the Animal Care and Use Committee of Soochow University.

[0051] All data are expressed as mean ± standard deviation (SD). Statistical differences between groups are evaluated by one-way analysis of variance and Tukey's multiple comparison test. Survival analysis is evaluated by log-rank (Mantel-Cox). The p-values are shown as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0052] Synthesis Example

[0053] The copolymer and its synthesis are common methods. The synthesis steps of Acupa-mICG-Nap are briefly described as follows: At 37 °C under nitrogen, a DMF solution of NHS-PEG-P(CL-DTC) (216 mg, 0.04 mmol, 2.2 mL) and a DMF solution of Acupa (64 mg, 0.2 mmol, 0.6 mL DMF) were mixed and stirred. A precipitate appeared, and then the polymer was obtained after filtration and vacuum drying. Yield: 65%. 1 The 1H NMR (DMSO-d6) spectrum showed characteristic peaks of Acupa (3.8 and 6.3 ppm) and no NHS peak (2.8 ppm). According to the end-group analysis, the functionality of Acupa was approximately 85% ( Figure 1 ). Example 1 mICG-Nap and Acupa-mICG-Nap

[0054] Prepare PEG350 solutions of Acupa-PEG-P(CL-DTC), PEG-P(CL-DTC), and ICG (with concentrations of 50 mg / mL, 300 mg / mL, and 50 mg / mL in sequence) and a DMSO solution of Nap (10 mg / mL). ICG, Nap, PEG-P(CL-DTC), and Acupa-PEG-P(CL-DTC) were pre-mixed evenly according to the drug ratios in Table 1 (ICG / Nap: 0.75 / 1, 1 / 1, 1.5 / 1, and 2 / 1) and the theoretical drug loadings (Theo.DLC, Nap: 1.4 wt.%; ICG: 3.4 wt.%, 4.6 wt.%, 6.8 wt.%, and 9.0 wt.%). At this time, the volume was 100 μL, and 900 μL of phosphate buffer (PB, pH 7.4, 10 mM) was added, and self-assembled into micelles (mICG-Nap).

[0055] Acupa-mICG-Nap with a drug ratio of 1:1 was obtained by replacing PEG-P(CL-DTC) with a mixture of 95 mol% PEG-P(CL-DTC) and 5 mol% Acupa-PEG-P(CL-DTC) in the same method. Referring to the above method, by changing the ICG / Nap molar ratio, (targeted) micelle nanodrugs with different drug ratios were prepared.

[0056] Similarly, according to a similar method, using ICG, micelle drugs loaded with only ICG (mICG) can be prepared; omitting ICG and Nap, unloaded micelles (Ms) were prepared; used as a control.

[0057] Mix the solutions of ICG and Nap in a molar ratio of 1:1 to prepare free ICG-Nap, which is used as a control.

[0058] The particle size and particle size distribution of each micelle were measured by DLS, and the concentrations of ICG and Nap were measured by ultraviolet-visible spectrophotometer (UV-vis) and high performance liquid chromatography (HPLC) respectively for calculating the drug loading (Dete.DLC) and loading efficiency (DLE) of ICG and Nap. By tracking the change of particle size, the stability of mICG-Nap was evaluated under storage at -80 °C and in the presence of 10% serum.

[0059] Table 1 Characterization of mICG-Nap and Acupa-mICG-Nap micelles

[0060]

[0061]

[0062] a Determined by ultraviolet-visible spectrophotometer; b Determined by HPLC; c Measured by DLS in PB (pH 7.4, 10 mM, 1.0 mg / mL, 25 °C); d An unstable nanopreparation with precipitation occurring 1 h after preparation. e Precipitation occurred immediately after preparation, and no nanopreparation was obtained.

[0063] Referring to the above method, by changing the molar ratio of Acupa-PEG-P(CL-DTC) and PEG-P(CL-DTC), targeted micelle nanodrugs with different targeted molecule densities were prepared, where the drug ratio was 1:1. The characterization is shown in Table 2, where Acupa was absent and the 5% density refers to Table 1.

[0064] Table 2 Characterization of Acupa-mICG-Nap with different targeted molecule densities

[0065]

[0066] When ICG / Nap = 1 / 1, the drug loading efficiency of mICG-Nap is high and stable (DLE ICG : 91%, DLE Nap : 81%), the particle size is small and the distribution is uniform (about 23 nm, PDI = 0.15) (Table 1, Figure 2 A). Unless otherwise specified, the micelle preparation with ICG / Nap = 1 / 1 was used in subsequent studies. The size of mICG-Nap changed little after storage at -80 °C for three months, showing good stability ( Figure 3 A), and the inhibitory effect on tumor cells also hardly changed (Figure 3 B). mICG-Nap also maintains stable particles and good particle size distribution in the buffer containing 10% serum ( Figure 2 A). Further adding 5% of Acupa-PEG-P(CL-DTC) can obtain the micelle Acupa-mICG-Nap that specifically targets PSMA ( Figure 13 A), Acupa-mICG-Nap has the same physicochemical properties as mICG-Nap (Table 1, Figure 2 A). The synergistic binding force between Nap and ICG provides the high stability of mICG-Nap ( Figure 2 B), which effectively inhibits the crystallization and precipitation of Nap from the micelles and solves the problem that Nap is difficult to be loaded into the preparation.

[0067] Pre-mix the solutions of PEG-P(CL-DTC)-Cy5, Acupa-PEG-P(CL-DTC) and PEG-P(CL-DTC) (concentrations are 10, 50 and 300 mg / mL respectively) uniformly at the molar ratios of 1 / 0 / 99, 1 / 1.25 / 97.75, 1 / 2.5 / 96.5, 1 / 5 / 94 and 1 / 10 / 89 to prepare Cy5-labeled Acupa-functionalized micelles with Acupa densities of 0%, 1.25%, 2.5%, 5% or 10%.

[0068] Refer to the above method, use KC 15 to replace Acupa and prepare KC 15 -mICG-Nap.

[0069] Example 2 Photothermal and photodynamic properties of mICG-Nap (molar ratio of ICG / Nap: 1 / 1)

[0070] Add 300 μL of different samples into a 96-well plate: mICG-Nap (ICG concentrations: 0, 6.25, 12.5, 25, 50 μg / mL), mICG (ICG concentrations: 6.25, 25 μg / mL), free ICG-Nap (ICG concentrations: 6.25, 25 μg / mL), free ICG (ICG concentrations: 6.25, 25 μg / mL), Ms (polymer concentration: 0.54 mg / mL) and PBS, irradiate with a near-infrared laser (808 nm, 1 W / cm 2 , 5 min), and measure the solution temperature every minute (n = 3). Add mICG-Nap (300 μL, ICG concentration: 20 μg / mL) into a 96-well plate and irradiate with near-infrared lasers (808 nm, 5 min) of different laser intensities (0.33, 0.67, 1.0 and 1.33 W / cm 2 ) and measure the solution temperature every minute (n = 3).

[0071] RM1-PSMA cells were seeded in 24-well plates (5×10 5 / well), and after overnight culture, mICG-Nap, mICG, free ICG, free Nap, or a mixture of free ICG-Nap (ICG concentration in the wells: 10 μg / mL, Nap concentration in the wells: 3.1 μg / mL) was added and the cells were treated for 2 h (PBS served as the control). After adding DCFH-DA (10 μM), the cells were irradiated with a near-infrared laser (808 nm, 1 W / cm 2 , 5 min). The cells were immediately washed twice, fixed, stained with DAPI (0.5 μg / mL, 5 min), and observed and photographed under a fluorescence microscope. The fluorescence intensity of DCF was analyzed using Image J software, and semi-quantitative analysis of the generation of intracellular ROS was performed (n = 3).

[0072] Spectral analysis showed that compared with free ICG and the mixture of free ICG-Nap, both mICG-Nap and micelles loaded with ICG alone (mICG) exhibited significant red shifts at the maximum absorption peak (from 779 nm to 803 nm)( Figure 2 C). In mICG-Nap and mICG, the fluorescence of ICG was almost completely quenched( Figure 2 D, 3C), while the fluorescence was restored after adding DMF( Figure 2 E). Under near-infrared laser irradiation (808 nm, 1 W / cm 2 , 5 min), mICG-Nap and mICG showed stronger and more persistent photothermal effects than free ICG( Figure 2 F, 2D). The photothermal effect of mICG-Nap depended on the ICG concentration and irradiation power( Figure 3 E, F). In the RM1-PSMA prostate tumor model, intravenous injection (i.v.) of mICG-Nap led to a more significant increase in tumor temperature than free ICG-Nap( Figure 2 G), indicating enhanced accumulation of mICG-Nap in tumors. In addition, mICG-Nap induced more reactive oxygen species (ROS) generation in RM1-PSMA cells( Figure 2 H).

[0073] Example 3 Inhibitory effect of mICG-Nap and mICG-Nap+L on the stemness of S-RM1-PSMA cells

[0074] A three-dimensional (3D) tumor spheroid model with a regular structure was established to enrich the CSC population in RM1-PSMA cells. Specifically, forty thousand RM1-PSMA cells were cultured in 4 mL of serum-free medium in an anaerobic incubator( Figure 4A), when cultured until the 7th day, the maximum diameter of the tumor spheroids reached approximately 570 μm, among which the proportion of CD44 + CD133 + CSCs was significantly enriched to 5.30%, which was significantly higher than 0.84% in two-dimensional (2D) RM1-PSMA cells ( Figure 4 B, C). By flow cytometry (FC) detection, the survival rate of the cells digested from the tumor spheroids (denoted as S-RM1-PSMA) was 94.6%, and the CSC proportion was 4.05% ( Figure 5 A, B).

[0075] mICG-Nap (Nap concentration: 0.1, 0.2 μg / mL) was added to the tumor spheres. After two days of culture, the tumor spheroids were digested into single-cell suspensions with accutase, and then stained with 7-AAD to exclude dead cells, and stained with FITC-CD44 and PE-CD133 antibodies followed by flow cytometry measurement to determine the content of CD44 + CD133 + CSCs (n = 3). S-RM1-PSMA cells (200 / well) were cultured in serum-free medium together with mICG-Nap (Nap concentration: 0.1 and 0.2 μg / mL). After 48 h, the tumor spheres were observed and counted with an optical microscope (>50 μm, n = 4). mICG-Nap (Nap concentration: 0.1 and 1 μg / mL) was added to the tumor spheres (>50 μm), and after 2 days of culture, the tumor spheres were observed and counted with a microscope (>100 μm, n = 4). After treatment with mICG-Nap at a Nap concentration of 0.2 μg / mL, both the survival rate and the CSC proportion (1.74%) of S-RM1-PSMA were significantly reduced ( Figure 5 A, B).

[0076] In the cell cloning experiment, S-RM1-PSMA cells were seeded in 6-well plates (100 / well), incubated overnight at 37 °C to adhere, and mICG-Nap (Nap concentration: 0.1, 0.2, and 0.4 μg / mL) was added and incubated for 4 h. The medium was removed and replaced with fresh drug-free DMEM medium (denoted as day 0), and the medium was changed on the 2nd, 4th, and 6th days. On the 8th day, the cells were fixed with 4% paraformaldehyde for 10 minutes, stained with crystal violet for 10 minutes, and then washed with pure water and photographed. mICG-Nap could significantly inhibit the formation of clones and tumor spheroids and disrupt the existing tumor spheroids ( Figure 5 C, D, 6A), which was consistent with the important role of CSCs in clone formation and proliferation.

[0077] mICG-Nap (Nap concentration: 0.2 μg / mL, ICG concentration: 6.46 and 32.3 μg / mL) was added to tumor spheres (>50 μm) and incubated for 1 h, followed by irradiation with near-infrared laser (808 nm, 1 W / cm 2 , 5 min). mICG+L, mICG-Nap, and PBS were used as controls. Cells were cultured in an anaerobic incubator for 5 days, and tumor spheres were observed and counted daily using a microscope (>200 μm, n = 3). The results showed that mICG-Nap+L significantly reduced the number and size of tumor spheres and disrupted the structural integrity, with its effect being significantly better than that of the control groups mICG+L (ICG concentration: 6.46 μg / mL and 32.3 μg / mL) and mICG-Nap (Nap concentration: 0.2 μg / mL) (****, Figure 5 E). Specifically, when the ICG concentration was 32.3 μg / mL, the treatment with mICG-Nap+L could raise the temperature to 57.7 °C, and the PTT effect and Nap could produce a strong synergistic effect, almost completely eliminating the tumor spheres on the 3rd day. As the treatment time extended, the morphology of the tumor spheres gradually changed, becoming irregular and showing a divergent sponge-like shape, which helped mICG-Nap penetrate into the interior of the spheres, thus promoting the destruction of the tumor structure and the eradication of CSCs.

[0078] S-RM1-PSMA cells were seeded in 96-well plates (3×10 3 / well) and treated with mICG-Nap, mICG, ICG-Nap, ICG, or Nap at a concentration of 0.1 - 1 μg / mL for 48 h under anaerobic conditions. After adding 10 μL of CCK8 solution and culturing for 2 h, the absorbance at 450 nm was measured using a microplate reader (n = 4). The cell viability was calculated by comparing with the absorbance of the PBS group (100% cell viability), and the curve of cell viability versus drug concentration was fitted and plotted to obtain the half-maximal inhibitory concentration (IC 50 ), and the combination index (CI) was calculated based on the IC 50 of ICG and Nap according to the conventional method. CI values <, =, or > 1 indicate synergistic, additive, or antagonistic effects of the two drugs, respectively. The results of the cytotoxicity experiment of S-RM1-PSMA showed that compared with mICG-Nap and mICG+L alone, mICG-Nap+L showed a significantly reduced half-maximal inhibitory concentration (IC 50 ), indicating a moderate synergistic effect (combination index of 0.8) ( Figure 6 B), and no obvious photothermal effect (temperature increase <7 °C) was observed when ICG ≤ 3.23 μg / mL.

[0079] S-RM1-PSMA cells were seeded in 6-well plates (5×105 / well) and cultured overnight. Add 100 μL of mICG-Nap or mICG (Nap: 0.4 and 0.8 μg / mL; ICG: 1.3, 2.6 and 13 μg / mL) and incubate for 24 h. The mICG-Nap+L group was irradiated with laser (808 nm, 1 W / cm 2 ²) for 5 min 1 h after adding the samples. Wash the cells with PBS and treat them with Annexin V-APC / 7-AAD apoptosis staining reagent, and detect them by flow cytometry. See Figure 6 C, when ICG = 2.6 μg / mL, the total apoptosis level of the mICG-Nap+L group was higher than that of the mICG-Nap or mICG+L control group; while when ICG = 13 μg / mL, mICG-Nap+L induced the temperature to rise to 44.9 °C and caused the apoptosis level to increase more than twice. It should be noted that compared with the effect of heat on S-RM1-PSMA cells, the effect of Nap on late apoptosis was more significant.

[0080] Example 4 Efficacy of mICG-Nap+L on RM1-PSMA tumor-bearing mice

[0081] RM1-PSMA cells (5×10 5 / mouse, 50 μL, PBS suspension containing 30% matrigel) were subcutaneously inoculated above the right hind limb of male BALB / c nude mice to establish an RM1-PSMA subcutaneous tumor mouse model, and the inoculation day was set as day 0. To study the efficacy of mICG-Nap+L by intravenous injection (i.v.) and intratumoral injection (i.t.), on the 7th day after inoculation (when the tumor volume reached 150-200 mm 3 ³), the mice were randomly divided into three groups: mICG-Nap i.v. +L, mICG-Nap i.t. +L and PBS control group (n = 4). The mICG-Nap i.v. +L group was intravenously injected with 200 μL of mICG-Nap (ICG: 6.46 mg / kg, Nap: 2 mg / kg) on the 7th, 9th and 11th days; the mICG-Nap i.t. +L group was intratumorally injected with 50 μL of mICG-Nap (ICG: 1.6 mg / kg, Nap: 0.5 mg / kg) on the 7th and 19th days. Both groups were irradiated with near-infrared laser (808 nm, 1 W / cm 2 ²) for 5 min 1 h after injection. Mice injected with PBS three times intravenously were used as controls. The tumor volume and body weight of the mice were monitored every two days. When the tumor volume of the mice reached 2000 mm 3Death was determined at that time. One mouse was randomly selected from each group for euthanasia (on day 35 in the two treatment groups; on day 13 in the PBS control group), and the lung metastasis was evaluated by hematoxylin-eosin (H&E) staining.

[0082] The therapeutic effects of intratumoral injection (i.t.) or intravenous injection (i.v.) of mICG-Nap+L on nude mice bearing subcutaneous RM1-PSMA tumors are shown in Figure 7 . The tumors in the PBS control group grew rapidly and reached 2000 mm 3 (determined as death) in about 13 days. The tumors in the i.t. treatment group disappeared on day 9 and recurred on day 13 ( Figure 7 B, C), and received a second intratumoral injection on day 19. The median survival time (MST) of the mice was 31.5 days (**, Figure 7 D). In addition, tumor lung metastasis was observed in this group ( Figure 7 E); the mice receiving intravenous injection of mICG-Nap+L maintained a smaller tumor volume for a longer time, with a median survival time reaching 67 days, and no obvious metastasis was observed on day 35. The good results indicate the role of mICG-Nap of the present invention in targeting and eliminating local CSCs and escaping CSCs in tumors.

[0083] Example 5 Treatment of RM1-PSMA tumor-bearing mice with mICG-Nap+L

[0084] The modeling method was the same as above. On day 7, the tumor-bearing mice were randomly divided into 4 groups (n = 5): mICG-Nap+L, mICG+L, mICG-Nap, and PBS. On days 7, 9, and 11, the micelle preparations mICG-Nap and mICG (ICG: 6.46 mg / kg, Nap: 2 mg / kg) were intravenously injected. The groups that required photothermal therapy were irradiated with an 808 nm laser (1 W / cm 2 ) on the tumor site for 5 min 1 h after injection, and the temperature of the tumor site was monitored. Tumor-bearing mice injected with PBS three times at the same time were used as controls. The tumor volume and body weight of the mice were monitored every two days. When the tumor volume of the mice reached 2000 mm 3 , death was determined. The lungs of one mouse from each group were stained with H&E to evaluate lung metastasis ( Figure 8 A).

[0085] The tumor growth curve showed that it was almost impossible to inhibit the rapid growth of tumors by eliminating CSCs only through three i.v. injections of mICG-Nap ( Figure 8 B). The PTT effect of only using mICG+L showed significant tumor inhibition in the first 3 weeks and did not cause weight loss, but the tumor recurred subsequently ( Figure 8B, C), which is relatively common in PTT ablation therapy. In contrast, the mICG-Nap+L combination therapy exhibited significantly enhanced tumor suppression, achieving 80% tumor elimination within 45 days and extending the MST of tumor-bearing mice to 55 days, significantly superior to 43 days in the mICG+L group (*, Figure 8 D), although both showed similar tumor temperatures ( Figure 8 E). In addition, histological analysis showed that, different from the thickening of the alveolar wall in the mICG+L group, mICG-Nap+L treatment did not cause damage to the lung structure ( Figure 8 F). In the prior art, high doses of Nap are required to inhibit tumor growth, which showed tumor suppression only when administered at a dose of 40 mg / kg (i.p.) every other day in a glioblastoma mouse model or at a dose of 20 mg / kg (i.p.) every three days in a pancreatic cancer mouse model. In the technical solution disclosed in the present invention, the mICG-Nap+L strategy with a Nap dose of 2 mg / kg (total 3 doses) demonstrated excellent anti-tumor efficacy, highlighting the strong synergistic effect between PTT-mediated tumor ablation and in vivo selective elimination of CSCs.

[0086] Notably, in mice bearing RM1-PSMA, the tumor temperature in the Acupa-mICG-Nap+L treatment group reached 51.5 °C ( Figure 8 E), 1.5 °C higher than that in the mICG+L and mICG-Nap+L groups, indicating a higher enrichment amount in the tumor. Compared with the mICG-Nap+L (55 days) and mICG+L (43 days) groups, Acupa-mICG-Nap+L significantly extended the MST of mice to 87 days (*, Figure 8 D). In addition, the lung structure of the mice in this group was in a healthy state without tumor lung metastasis ( Figure 8 F). These results demonstrated the in vivo active targeting and survival benefits of Acupa-mICG-Nap+L.

[0087] Example 6 Effects of Acupa-mICG-Nap+L on RM1-PSMA and Tumor-Bearing Mice

[0088] To study cell uptake, 100 μL of the above-mentioned preparation was added to the well plates respectively and incubated with RM1-PSMA or RM1 cells (5×10 5 / well) for 2 h, then digested with trypsin, centrifuged, washed and detected by flow cytometry. The PSMA expression levels of RM1-PSMA cells, RM-1, PC-3 and LNCaP cells are shown in Figure 9A. In RM1-PSMA cells, the cellular uptake of Cy5-labeled Acupa-mICG-Nap containing 5% Acupa was the highest, approximately 2.3 times that of mICG-Nap( Figure 9 B), and in RM1 cells, the uptake of mICG-Nap and Acupa-mICG-Nap was similar( Figure 9 C).

[0089] mICG-Nap and Acupa-mICG-Nap (10 μL, ICG concentration: 0.32 - 323 μg / mL; Nap concentration: 0.1 - 100 μg / mL) were added to RM1-PSMA or RM1 cells (3×10 3 / well, 90 μL). After culturing for 48 h, CCK8 was added to the cells, and the absorbance at a wavelength of 450 nm was measured using a microplate reader to calculate the cell viability (n = 3). One hour after adding the nanoplatforms, the cells were irradiated with near-infrared laser (L: 808 nm, 1 W / cm 2 , 5 min) for the combination group. After continuing to culture for 1 h, the fresh drug-free medium was replaced and the cells were further cultured for 46 h, and then the cells were detected by CCK8 to calculate the cell viability (n = 3). The effects of empty micelles Ms and Acupa-Ms (polymer concentration: 0.25, 0.5, 1, and 2 mg / mL) on the viability of RM1-PSMA cells were also determined by the CCK8 method (co-incubated for 48 h, n = 6). The MTT results showed that Acupa-mICG-Nap and Acupa-mICG-Nap+L caused more RM1-PSMA cell death, but there was no difference in RM-1 cells, and the empty micelles were non-toxic at a micelle concentration up to 2 mg / mL( Figure 9 D - F).

[0090] Male Balb / c nude mice were injected with Acupa-mICG-Nap, mICG-Nap, and free ICG-Nap (ICG: 6.46 mg / kg, Nap: 2 mg / kg, n = 3) via the tail vein. Blood was collected from the orbital vein at the predetermined time points, and 20 μL of plasma was centrifuged and mixed with 980 μL of acetonitrile, and ICG and Nap were extracted by vortexing. After measuring the fluorescence intensity of the samples using IVIS, the acetonitrile was completely evaporated and then re-dissolved in 200 μL of acetonitrile, and Nap was quantified by HPLC. Plasma was mixed with ICG and Nap solutions with known concentrations to construct the standard curves of ICG and Nap. The parameters (t 1 / 2，β and AUC) were calculated by PK Solver.

[0091] Pharmacokinetic studies showed that compared with free drugs, the in vivo circulation times of ICG and Nap were significantly prolonged after delivery by Acupa-mICG-Nap and mICG-Nap, and the half-lives (t 1 / 2 ) increased by 8.3 to 54 times respectively ( Figure 9 G). Compared with mICG-Nap, the distribution of Acupa-mICG-Nap in RM1-PSMA tumors was significantly enhanced at 3 h and 6 h after injection in tumor-bearing mice, and its ex vivo imaging at 24 h also showed higher fluorescence intensity than mICG-Nap (*), while there was no significant difference in the major organs.

[0092] Example 7 Treatment of RM1-PSMA postoperative model mice with Acupa-mICG-Nap+L

[0093] An RM1-PSMA subcutaneous tumor mouse model was established according to the above method. On the 7th day, the subcutaneous tumor was surgically removed to establish a postoperative model. When the tumor recurred or metastasized, the mice were randomly divided into two groups (n = 6). The treatment group was intravenously injected with Acupa-mICG-Nap (ICG: 6.46 mg / kg, Nap: 2 mg / kg) on the 9th, 11th, and 13th days, and the recurrent tumor site was irradiated with near-infrared laser (808 nm, 1 W / cm 2 , 5 min) 1 h after each injection. The mice in the control group were intravenously injected with PBS three times on the 9th, 11th, and 13th days. The methods for measuring the temperature, tumor volume, body weight, and survival rate of the tumor sites in mice were the same as above. On the 17th day, one mouse in each group was euthanized, and the lung condition was evaluated by H&E staining.

[0094] It has been reported that surgical intervention may prompt residual tumor cells to transform into a cancer stem cell-like state, thus promoting recurrence and distant metastasis. Prostate cancer patients often experience recurrence and lung or bone metastasis after surgery. To evaluate the efficacy of Acupa-mICG-Nap+L therapy in the postoperative setting, a postoperative RM1-PSMA model was established by removing the tumor on the 7th day, and treatment was started two days later ( Figure 10 A). The tumors of the mice in the PBS group recurred rapidly, and the tumor volume reached 1300 mm 3 within 6 days after surgery, and the body weight remained stable ( Figure 10 B, C). Its MST was slightly prolonged compared with that of non-surgically treated mice, being 15 days (13 days for non-surgically treated mice, Figure 10 D), and lung metastasis was observed in the H&E staining images on the 17th day ( Figure 10(E), indicating that surgery promoted metastasis. Notably, Acupa-mICG-Nap+L caused the temperature of the recurrent tumor to rise to 51.8 °C, indicating the enrichment of micelles in the recurrent tumor. Acupa-mICG-Nap+L significantly inhibited tumor growth (****), and no lung metastasis was observed until day 17. Figure 10 (B-F), and significantly prolonged the MST of mice to 24 days (***) in this refractory postoperative model.

[0095] To investigate the effect of Acupa-mICG-Nap+L therapy on the expression of tumor stemness-related biomarkers in vivo, Acupa-mICG-Nap and Acupa-mICG monotherapies were used as control groups, and the RM1-PSMA postoperative model was treated according to a similar treatment procedure as above. On day 15, tumors were collected and sectioned for immunofluorescence staining, and stained with SOX2, CD133, OCT4, pSTAT3, and PSMA antibodies respectively, and then treated with Alexa Fluor 488 or Alexa Fluor 647 and observed and photographed. Compared with the primary tumor without surgery, sections of recurrent tumors in the PBS group showed significantly increased levels of SOX2 and CD133. Figure 10 (G). In contrast, the expression of OCT4, SOX2, and CD133 in tumors in the Acupa-mICG+L group was significantly increased, confirming the enhanced tumor stemness tendency after PTT treatment alone, while these markers were decreased in the Acupa-mICG-Nap group. In sharp contrast, in the sections of the Acupa-mICG-Nap+L group, the above three cancer stem cell markers decreased sharply, and the tumor cell density decreased significantly, which confirmed the inhibitory ability of this therapy on tumor cells and cancer stem cells. Unexpectedly, compared with the high levels of pSTAT3 and its main localization in the tumor cell nucleus in the Acupa-mICG+L and control groups, the Acupa-mICG-Nap+L group showed a strong ability to inhibit pSTAT3 and reduce the co-localization of pSTAT3 with the cell nucleus. Figure 10 (G). This indicates the effective intracellular delivery of the micelles of the present invention and its effective inhibition of the STAT3 signaling pathway, thereby preventing the transmission of transcriptional signals to the cell nucleus. In particular, Acupa-mICG-Nap+L treatment significantly reduced PSMA expression.

[0096] Example VIII Docetaxel (DTX) micelles, a first-line clinical drug for treating prostate cancer

[0097] The preparation methods of Acupa-mDTX and mDTX are similar to that of Acupa-mICG-Nap. Briefly, at 37 °C, 100 μL of a premixed solution containing PEG-P(CL-DTC) (14.1 mg) and DTX (0.85 mg) was added dropwise to 900 μL of PB and mixed to obtain mDTX. The PEG-P(CL-DTC) was replaced with a mixture of 95 mol% PEG-P(CL-DTC) and 5 mol% Acupa-PEG-P(CL-DTC) to obtain Acupa-functionalized micelles (Acupa-mDTX). The particle size and particle size distribution were measured using DLS. After incubation for 48 and 24 hours (the drug-containing medium was removed at 2 h and replaced with fresh medium), the toxicity and apoptosis induction of Acupa-mDTX and mDTX on RM1-PSMA cells were determined (n = 3). Modeling was performed according to the above method to investigate the efficacy of Acupa-mDTX and mDTX on RM1-PSMA tumor-bearing mice. On the 5th, 7th, 9th, 11th, and 13th days (n = 3), Acupa-mDTX, mDTX (DTX: 7.5 mg / kg), and PBS were injected via the tail vein, and the tumor volume and body weight were monitored every two days. Death was determined when the mice died or the tumor volume reached 2000 mm 3 3. Compared with mDTX, Acupa-mDTX had a similar size (22 nm) and stronger cytotoxicity and apoptosis induction ability ( Figure 11 A-C). In RM1-PSMA tumor-bearing mice, Acupa-mDTX extended the MST to 26 days ( Figure 11 D-F). Compared with classical chemotherapeutic drugs, the present invention designed a polymer micelle co-loaded with a photosensitizer and an inhibitor, and utilized the synergistic effect of the photothermal / inhibition pathway to unexpectedly achieve significant technological progress, significantly extending the MST of mice from 13 days to 87 days, beyond people's imagination.

[0098] Example 9 mICG-Nap + L treatment of RM1 tumor-bearing C57 mouse model

[0099] RM1 cells (5×10 5 6 cells / mouse, 50 μL, PBS suspension containing 30% matrigel) were subcutaneously inoculated above the right hind limb of male C57 mice to establish an RM subcutaneous tumor mouse model, and the inoculation day was set as the -12th day.

[0100] The efficacy of mICG-Nap + L in RM1 tumor-bearing mice was evaluated by two routes: intratumoral administration and intravenous administration ( Figure 12 A), mICG-Nap i.v. + L, ICG / Nap dose: 6.46 / 2 mg / kg, mICG-Nap i.t.+L, ICG / Nap dose: 1.6 / 0.5 mg / kg. The tumors in the PBS control group (n = 3) progressed rapidly and reached 2000 mm 3 at the ethical endpoint volume on days 12, 13, and 14 respectively Figure 12 B). During the one-month observation period, both the intravenous injection group and the intratumoral injection group showed significant tumor suppression. However, in the i.t. group, one animal had tumor recurrence and reached the endpoint volume on day 36, while the second mouse was euthanized on day 65. The third mouse treated with i.t. remained tumor-free during the 200-day monitoring period. All mice treated with intravenous injection did not relapse during the entire 200-day observation period Figure 12 B, C). Body weight monitoring continued until the PBS group reached the endpoint. A slight increase in body weight was observed in the PBS group mice, while the body weights of mice in other groups remained stable Figure 12 D). After histopathological analysis of randomly selected mice on day 60, obvious lung metastases occurred in the i.t. group, which may be due to the lack of systemic Nap administration. In contrast, the intravenous injection group showed effective inhibition of circulating tumor cells, and no obvious lung metastases were observed Figure 12 E).

[0101] Figure 13 A is the photothermal combination therapy experiment of mICG-Nap+L in mice bearing RM1 tumors. During the first treatment, an infrared thermal imager was used to record the tumor surface temperature, and the ability of this tumor cumulative preparation to heat the tumor mass with local 808 nm laser irradiation (power intensity of 1 W / cm 2 , for 5 min) was measured. Thermal imaging analysis showed that after intravenous injection of mICG-Nap or mICG (ICG: 6.46 mg / kg; Nap: 2 mg / kg) into C57 mice bearing RM1 tumors, the tumor temperature rapidly increased to ≈59.5 °C, which was 3 °C higher than that in the ICG-Nap+L group (****), indicating that micelle-encapsulated ICG has a higher photothermal conversion efficiency and its superior in vivo pharmacokinetic properties, including extended circulation and tumor-specific accumulation. Nap had no effect on temperature Figure 13 B). mICG-Nap+L was the most effective in inhibiting tumor growth. By monitoring the tumor volume curves and survival curves of mice in different treatment groups, no obvious lymph node metastases and tumor recurrence were found in 6 / 6 RM1 tumor-bearing mice within 127 days after combination treatment. In sharp contrast, 4 / 6 mice in the PBS control group had varying degrees of lymph node metastases at the end of the experiment, and the anti-tumor effect of mICG-Nap treatment was almost negligible Figure 13 C, D, F). At the same time, during the entire treatment process, these treatment methods had little effect on the body weight of miceFigure 13 E), indicating that mICG-Nap has good biocompatibility at the tested doses.

[0102] The importance of STAT3 in cancer immunotherapy is well known. Therefore, these tumor-free mice were subcutaneously inoculated with the same RM1 tumor on day 127 to study the efficacy of the combination therapy in inducing immune memory to reject tumor recurrence in a tumor rechallenge mouse model ( Figure 13 A). It was found that on day 13 after inoculation, the re-invaded RM1 tumors in the treated mice began to grow ( Figure 13 G), while the tumors in the control group mice grew significantly. On day 140, these mice were sacrificed, and plasma, spleen, and lymph nodes were collected for flow cytometry. The good immune effect could be preliminarily seen from the weight of the spleen and the cytotoxicity of splenocytes against RM1 cells ( Figure 13 H, I). Flow cytometry analysis (FCM) showed that the treatment with mICG-Nap + L was most effective in promoting CD3 + CD8 + and CD3 + CD4 + T cells. The frequencies of effector memory T cells (T EM , CD44 high CD62L low ), central memory T cells (T CM , CD44 high CD62L high ) in the plasma, spleen, and lymph nodes of the mice treated with mICG-Nap-assisted PTT and Nap-mediated STAT3 inhibition immunotherapy were much higher than those in the mICG + L single PTT group and the control group.

[0103] Example Ten: Combining Photothermal Therapy with Stem Cell Inhibition for the Treatment of Compact Pancreatic Cancer

[0104] 1. KC 15 -mICG-Nap + L or mICG-Nap + L (targeting density 5%, drug ratio 1:1) efficacy on a subcutaneous mouse model of pancreatic cancer

[0105] The in vivo antitumor efficacy of mICG-Nap + L treatment was evaluated using a PANC-1 tumor mouse model. PANC-1 tumor tissue (50 mg) was subcutaneously transplanted and inoculated above the right hind limb of female nude mice (5 weeks old) to establish a PANC-1 subcutaneous tumor mouse model, and the inoculation day was set as day -14. As Figure 14 shown in A. Compared with the mICG-Nap and ICG-Nap + L control groups, the tumor temperatures of both the mICG-Nap + L group and the mICG + L group were higher ( Figure 14B), which is also the reason for the best tumor suppression effect after 3 times of repeated administration and stimulation. Figure 14 C). In addition, although the early tumor suppression effects were the same, the median survival time of the mice in the mICG-Nap+L group was 132.5 days, 39 days more than the 93.5 days in the mICG+L group. Figure 14 D). It is worth noting that all treatments had no significant effect on the body weight of the mice. Figure 14 E). In addition, fewer liver metastases were observed in the liver sections of the mICG-Nap+L group. Figure 14 F), indicating its ability to inhibit tumor metastasis.

[0106] The SW1990 model is a metastatic PC model with high expression of plectin-1, and plectin-1 is also a biomarker for invasive and metastatic PC. SW1990 tumor tissue (20 mg) was subcutaneously transplanted above the right hind limb of female nude mice (5 weeks old) to establish a SW1990 subcutaneous tumor mouse model, and the inoculation day was set as day -21. Using mICG-Nap+L, mICG+L, mICG-Nap and PBS as controls, the anti-tumor effect of KC 15 -mICG-Nap+L on SW1990 subcutaneous tumors was studied. KC 15 -mICG-Nap+L group, the tumor temperature rose to 50.1 °C, while the tumor temperatures of the mICG-Nap+L group and the mICG+L group were only 48.6 °C and 48.8 °C respectively (*p, Figure 14 H). Since KC 15 -mICG-Nap+L had a higher accumulation in the tumor, its phototherapy effect in vivo was better than that of mICG-Nap+L. It is worth noting that KC 15 -mICG-Nap+L and mICG-Nap+L successfully inhibited the growth of tumors in SW1990 mice, while mICG+L and other control groups had limited inhibitory effects on tumors. Figure 14 I). After 30 days, 45 days and 56 days, the tumor volumes of the mice treated with PBS, mICG-Nap and mICG+L gradually increased to 1000 mm 3 (the death criterion for statistical survival) Figure 14 J). KC 15 -mICG-Nap+L significantly prolonged the survival time of SW1990 mice. Compared with mICG-Nap+L (survival time: 76 days) and other control groups (survival time: 30, 56 days), the survival time of mICG-Nap+L was as long as 111 days. Interestingly, two months after treatment, KC 15The survival rates of the -mICG-Nap+L group and the mICG-Nap+L group were 100% and 80% respectively, while the survival rates of the mICG+L, mICG-Nap, and control groups were only 40%, 0%, and 0% respectively ( Figure 14 J). The body weights of SW1990 tumor-bearing mice during treatment further confirmed this finding. In the PBS group, without effective treatment, the body weight decreased due to tumor burden and metastasis. In contrast, 15 the body weight of the KC Figure 14 -mICG-Nap+L group did not change significantly, and with the increase of weeks of age, the body weight increased slightly ( 15 K). These results strongly verified the active targeting ability and safety of KC 15 -mICG-Nap in PC mice, as well as the powerful efficacy of KC

[0107] 2. Inhibitory effect of mICG-Nap+L on tumor stemness and metastasis of pancreatic cancer in mice

[0108] The survival time of mice treated with mICG-Nap+L was further evaluated by H&E and tissue immunofluorescence. Given the high possibility of SW1990 cell metastasis to the liver, the livers of mice in different treatment groups were examined. In the PBS or mICG+L control groups, diffuse distribution of tumor cells was observed. In contrast, the number of metastatic tumor cells in the mICG-Nap+L group was significantly reduced. The presence of PCSCs would reduce the efficacy and lead to recurrence after treatment. To further verify whether CSCs in tumors were effectively cleared after various in vivo treatments, the expression of stem cell-related proteins OCT4 and SOX2 was evaluated by immunofluorescence (HIF) staining. Compared with the PBS group, the expression of these two PCSC markers in the tumors and livers of the mICG+L group was higher, which was consistent with other reports that PTT promoted the metastasis of PC. Notably, compared with the mICG+L group, the expression of PCSCs in the mICG-Nap+L treatment group of mice decreased sharply and was extremely low. In summary, these results confirmed the excellent ability of mICG-Nap+L to effectively target and eliminate CSCs in vivo, and prevent recurrence and metastasis after treatment.

[0109] The present invention designs a polymer micelle co-loaded with a photosensitizer and an inhibitor. Taking the micelle nanodrug (mICG-Nap) co-delivering indocyanine green (ICG) and nabumetone (Nap) as an example, under near-infrared (NIR) irradiation, it is used for local ablation of tumors and targeted clearance of cancer stem cells (CSCs). Figure 15)。mICG-Nap significantly reduced the CSCs in the tumor microenvironment (TME) and inhibited the formation of CSC-driven tumor spheres. mICG-Nap + L synergistically reduced the size and number of tumor spheres, with a better therapeutic effect than single therapy. In the tumor model, mICG-Nap + L effectively inhibited tumor growth, highlighted a strong synergistic anti-tumor effect, further significantly prolonged the survival of the model, and also showed excellent therapeutic effects in the postoperative tumor model. Overall, by combining local tumor PTT ablation and CSC-targeted clearance, the ICG-Nap micelle therapy of the present invention solves the severe challenges of tumor recurrence and metastasis faced by traditional therapies, demonstrating broad prospects for treating various solid tumors.

Claims

1. A micellar nanodrug, characterized in that, It includes polymeric micelles, photosensitizers, and inhibitors.

2. The micellar nanodrug according to claim 1, wherein The polymeric micelles are formed by the assembly of polymers; the polymers include amphiphilic block polymers, or the polymers assembled into polymeric micelles include amphiphilic block polymers and targeted amphiphilic block polymers; among the amphiphilic block polymers and targeted amphiphilic block polymers, the hydrophobic segment includes DTC monomer repeating units.

3. The micellar nano-drug according to claim 2, wherein In the amphiphilic block polymer or targeted amphiphilic block polymer, the molecular weight of the hydrophilic segment is 1000 - 15000 Da, and the molecular weight of the hydrophobic segment is 0.1 - 5 times that of the hydrophilic segment.

4. The micellar nano-drug according to claim 1, wherein The photosensitizers include one or more of inorganic photosensitizers, metal photosensitizers, and organic photosensitizers; the inhibitors include cancer stem cell inhibitors.

5. The micellar nanodrug according to claim 4, wherein The photosensitizers include near-infrared organic photosensitizers; the inhibitors include cancer stem cell pathway inhibitors.

6. The preparation method of the micelle nanodrug according to claim 1, comprising the following steps: mixing the raw materials in a solution to obtain the micelle nanodrug; the raw materials include polymers, photosensitizers, and inhibitors.

7. The preparation method of the micelle nanodrug according to claim 6, wherein, The molar ratio of the photosensitizer to the inhibitor is (0 - 5)∶1, excluding 0.

8. An antitumor drug, the active ingredient of which includes the micelle nanodrug according to any one of claims 1 to 5.

9. Use of the micelle nanodrug according to any one of claims 1 to 5 and the antitumor drug according to claim 8 in the preparation of an antitumor drug.

10. Use of the micelle nanodrug according to any one of claims 1 to 5 and the antitumor drug according to claim 8 in the preparation of a multi-modal antitumor drug.

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