A photodynamic combination active oxygen response type tripterine polymer micelle and a preparation method and use thereof

Abstract

This invention provides a photodynamic therapy combined with reactive oxygen species (ROS)-responsive triptolide polymer micelles, its preparation method, and its applications, belonging to the field of pharmaceutical technology. This invention combines photodynamic therapy with the co-encapsulation of the photosensitizer Ce6 and the antitumor drug Cela in ROS-responsive liver-targeting polymer micelles. The prepared drug-loaded micelles have small particle size and high stability. When the photosensitizer absorbs external visible-near-infrared light energy, it generates ROS free radicals, increasing the ROS concentration at the tumor site, accelerating the breaking rate of ketithiothiols in the micelles, and speeding up drug release. This polymer micelle drug-loaded system has good biocompatibility and ROS-responsive drug release characteristics. Combined with photodynamic therapy, it fully leverages the synergistic effect, prolongs the retention time in vivo, enhances the targeting of liver tumors, improves the bioavailability and antitumor effect of Cela, reduces toxic side effects on healthy tissues and organs, and achieves a superior synergistic antitumor therapeutic effect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a photodynamic-coupled reactive oxygen species-responsive triptolide polymer micelle, its preparation method, and its uses. Background Technology

[0002] Primary liver cancer is the sixth most frequently diagnosed cancer worldwide and the third leading cause of cancer death globally in 2020, with approximately 906,000 new cases and 830,000 deaths. Primary liver cancer includes hepatocellular carcinoma (HCC) (accounting for 75%-85% of cases) and intrahepatic cholangiocarcinoma (10%-15%), as well as other rare types.

[0003] Celastrol is a bioactive triterpenoid compound extracted from the root of the Tripterygium wilfordii Hook.F. plant. Numerous studies have demonstrated that celastrol's excellent anti-liver cancer activity offers a promising anti-cancer drug for cancer patients.

[0004] Although triptolide exhibits unique advantages in treating liver cancer, its clinical application is limited by its low water solubility, poor bioavailability, narrow therapeutic window, and tendency to cause toxic side effects. The poor solubility of triptolide results in an oral bioavailability of only 17.06%, and its systemic absorption after administration is also poor. Furthermore, the side effects and narrow therapeutic window of triptolide are also problems that need to be addressed in its clinical application. The narrow therapeutic range means that triptolide can easily reach toxic concentrations with even small changes in blood concentration. At low doses, triptolide can cause serious adverse reactions, including hepatotoxicity, nephrotoxicity, hematopoietic system toxicity, cardiotoxicity, and teratogenicity.

[0005] Chinese invention patent application CN 115554240 A discloses a triptolide / glycyrrhetinic acid-carboxymethyl chitosan-ketothiol-rheic acid micelle. However, this micelle releases the drug based on the reactive oxygen species (ROS) level at the tumor site. Since ROS levels vary among different tumors and at different stages of tumor development, the release of the drug is difficult to control, the efficacy of tumor treatment needs to be improved, and the drug toxicity needs to be further reduced.

[0006] Therefore, researching new products to accelerate the release of triptolide at the tumor site, unleash its therapeutic potential, improve its efficacy and safety in treating liver cancer, and reduce its toxic side effects have become major issues that urgently need to be addressed in the clinical application of triptolide in the treatment of liver cancer. Summary of the Invention

[0007] The purpose of this invention is to provide a micelle formulation that can accelerate the release of triptolide at the tumor site, exert the therapeutic potential of triptolide, improve its efficacy and safety in treating liver cancer, and reduce toxic side effects. This micelle has strong targeting and is particularly suitable as a drug for treating liver cancer, providing a new option for clinical practice.

[0008] This invention provides a polymer micelle, characterized in that it is a micelle formed from triptolide, a photosensitizer, and a carrier; the carrier is a carboxymethyl chitosan-ketithiolide-rheic acid coupling compound modified with glycyrrhetinic acid; and the mass ratio of triptolide, photosensitizer, and carrier in the micelle is (7.40±0.41~8.64±0.75):(0.77±0.04~6.65±0.47):(9.91±1.60~11.07±0.57).

[0009] Preferably, the mass ratio of triptolide, photosensitizer and carrier in the micelles is 2:1.7:2.4.

[0010] Preferably, the micelles are prepared from triptolide, photosensitizer and carrier as raw materials, and the ratio of triptolide, photosensitizer and carrier is 9:(1~9):12.5.

[0011] The ratio of triptolide, photosensitizer, and carrier was 9:9:12.5.

[0012] Preferably, the polymer micelles are obtained by adding a solution containing triptolide and a photosensitizer dropwise to a solution of glycyrrhetinic acid-modified carboxymethyl chitosan-ketithiol-rheic acid coupling agent under stirring conditions, followed by stirring, sonication in an ice-water bath, dialysis, sonication in an ice-water bath again, centrifugation, filtration, and freeze-drying.

[0013] Preferably, the photosensitizer is dihydroporphyrin E6; and / or, the glycyrrhetinic acid-modified carboxymethyl chitosan-ketithiolide-rheic acid coupling compound is a micelle formed from modified carboxymethyl chitosan, wherein the modified carboxymethyl chitosan is a carboxymethyl chitosan substituted with the following two substituents:

[0014]

[0015] Preferably, the modified carboxymethyl chitosan is obtained by attaching rhein to one end of a ketethiocarbamate and grafting glycyrrhetinic acid and the ketethiocarbamate onto the carboxymethyl chitosan.

[0016] And / or, the molar ratio of rhein to ketithiolide is 1:1 to 1:2;

[0017] And / or, the molar ratio of carboxymethyl chitosan to glycyrrhetinic acid is 1:1 to 1:2;

[0018] And / or, the molar ratio of carboxymethyl chitosan to ketithiolide is 1:1.2 to 1:2.4;

[0019] And / or, the molar ratio of rhein, ketithiolide, carboxymethyl chitosan and glycyrrhetinic acid is 1.2:1.8:1:1.5.

[0020] Preferably, the rhein is linked to the carboxyl group at one end of the ketithiolide via a polyamino compound, wherein the polyamino compound is preferably selected from ethylenediamine, 1,3-propylenediamine, or 1,4-butyldiamine; and / or, the carboxymethyl chitosan is selected from O-carboxymethyl chitosan; and / or, the molecular weight of the carboxymethyl chitosan is 10,000 to 100,000; and / or, the micelle size is 140 to 190 nm.

[0021] The present invention also provides a drug for treating cancer, characterized in that it is a solution of the polymer micelles described herein mixed with 0.9% sodium chloride injection or 5% glucose injection.

[0022] The present invention also provides a method for preparing the polymer micelles, characterized in that the preparation steps are as follows:

[0023] (1) Dissolve triptolide and photosensitizer in a solvent to form a solution containing triptolide and photosensitizer;

[0024] (2) Weigh out the GCTR conjugate and dissolve it to form a GCTR conjugate solution;

[0025] (3) Under stirring conditions, add the solution containing triptolide and photosensitizer dropwise to the solution of glycyrrhetinic acid modified carboxymethyl chitosan-ketithiol-rheic acid coupling compound, stir, sonicate in an ice-water bath, dialyze, sonicate in an ice-water bath again, centrifuge, filter, and freeze dry.

[0026] The method for preparing the polymer micelles is characterized in that:

[0027] In step (1), the solvent is an organic solvent, preferably DMSO;

[0028] In step (2), the GCTR conjugate is dissolved in water to form a GCTR conjugate solution;

[0029] Step (3) is as follows: under vigorous stirring at 1500 rpm, the solution obtained in step (1) is added dropwise to the solution obtained in step (2), and after stirring at 1000 rpm for 20 min, it is immediately sonicated in an ice-water bath for 20 min with an ultrasonic power of 220 W; dialysis; the dialyzed mixture is sonicated in an ice-water bath for 20 min with an ultrasonic power of 220 W, centrifuged at 3500 rpm / min for 10 min, filtered through a filter membrane, and then freeze-dried.

[0030] The present invention also provides the use of the mixed micelles in the preparation of a medicament for treating cancer; preferably, the medicament for treating cancer is a medicament for treating liver cancer.

[0031] Experimental results show that this invention, combined with photodynamic therapy, co-encapsulates the photosensitizer Ce6 and the antitumor drug Cela in ROS-responsive liver-targeting polymer micelles, resulting in drug-loaded micelles with small particle size and high stability. When the photosensitizer absorbs external visible and near-infrared light energy, it generates ROS free radicals, increasing the ROS concentration at the tumor site, accelerating the breaking rate of ketithiothiol bonds in the micelles, and speeding up drug release. This polymer micelle drug-loaded system exhibits good biocompatibility and ROS-responsive drug release characteristics. Combined with photodynamic therapy, it fully leverages synergistic effects, prolongs the retention time in vivo, enhances targeting to liver tumors, improves the bioavailability and antitumor efficacy of Cela, reduces toxic side effects on other tissues and organs, and achieves superior synergistic antitumor therapeutic effects.

[0032] 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.

[0033] 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

[0034] Figure 1 The DSC characterization spectra are as follows: a: Cela; b: Ce6; c: GCTR conjugate; d: physical mixture of Cela + Ce6 + GCTR conjugate; e: optimal feed ratio of Cela / Ce6 / GCTR micelles.

[0035] Figure 2 TEM images of Cela / Ce6 / GCTR micelles: a: Cela / Ce6 / GCTR micelles; b: Cela / Ce6 / GCTR micelles after H2O2 treatment; c: Cela / Ce6 / GCTR micelles after H2O2 and near-infrared irradiation treatment.

[0036] Figure 3 Particle size distribution (A) and dispersion factor (B) of Cela / Ce6 / GCTR micelles in 0.1 mmol / L H2O2 solution (`x±s, n=3).

[0037] Figure 4 Photodynamic characteristics of Cela / Ce6 / GCTR micelles (A); absorbance value of DPBF at 415 nm (B).

[0038] Figure 5 Particle size of Cela / Ce6 / GCTR micelles in combination with 0.9% sodium chloride injection and 5% glucose injection at different time points.

[0039] Figure 6 The cytotoxicity of Ce6 cells with and without NAC pretreatment, or different micelles, and with and without infrared irradiation to HepG2 cells (n=6, `x±s) Note: **P<0.01.

[0040] Figure 7 Cytotoxicity of different drugs on HepG2 cells (A), BEL-7402 cells (B), and L-02 cells (C)

[0041] Figure 8 Changes in body weight during treatment in tumor-bearing mice (n=8).

[0042] Figure 9 Organ index of tumor-bearing mice (n=8), Note: *P<0.05, **P<0.01.

[0043] Figure 10 Changes in tumor volume in tumor-bearing mice (n=8), Note: *P<0.05, ***P<0.001.

[0044] Figure 11 Treatment efficacy in tumor-bearing mice (n=8)

[0045] Figure 12 Tumor weight (A) and tumor inhibition rate (B) in tumor-bearing mice (n=8) Note: *P<0.05, **P<0.01, ***P<0.001

[0046] Figure 13 Blood concentration-time curves of Cela(A) and Ce6(B) after tail vein injection in mice (n=6) Detailed Implementation

[0047] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0048] The GCTR conjugate of this invention is the GCTR conjugate described in patent number ZL 202110753678.7, patent title: A glycyrrhetinic acid-carboxymethyl chitosan-ketothiol-rheic acid conjugate, its preparation method and uses. Specifically, the GCTR conjugate is synthesized by the following method:

[0049] Step 1: Dissolve 0.1 mmol of rhein (R) in 1% NaHCO3 by heating. Add 0.12 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) with stirring. After reacting at room temperature for 20 min, add 0.12 mmol of N-hydroxysuccinimide (NHS) and react at room temperature for 10 min to obtain R-NHS. Dissolve 0.15 mmol of ketothiols (EDA) in water. Add R-NHS dropwise to the EDA aqueous solution and react for 6 h to obtain the ketothiols-rhein conjugate (R-EDA).

[0050] Step 2: Dissolve 0.15 mmol ketethiol (TK) in N,N-dimethylformamide (DMF), add 0.18 mmol EDC·HCl and react at room temperature for 20 min, then add 0.18 mmol NHS and react at room temperature for 10 min, then add to R-EDA solution and react at room temperature to obtain TER.

[0051] Step 3: Dissolve 0.083 mmol O-carboxymethyl chitosan (CMCS) in water. Centrifuge the TER solution and collect the supernatant. Add 0.12 mmol EDC·HCl and react at room temperature for 20 min. Then add 0.12 mmol NHS and react at room temperature for 10 min. Finally, add the supernatant to the CMCS aqueous solution and react for 24 h to obtain CTR. Step 4: Dissolve 0.12 mmol GA in DMF. React with 0.14 mmol EDC·HCl at room temperature for 20 min, and with 0.14 mmol NHS at room temperature for 10 min. Add the supernatant to the CTR solution and react at room temperature for 24 h. Dialyze the reaction solution in a dialysis bag (MWCO 3500) with distilled water for 72 h, sonicate with a probe for 20 min, and freeze-dry to obtain the product GCTR conjugate.

[0052] 1. Abbreviations

[0053] Cela; glycyrrhetinic acid-modified carboxymethyl chitosan-ketothiolate-rheic acid conjugate (GCTR conjugate); photosensitizer dihydroporphyrin e6 (Ce6); dimethyl sulfoxide (DMSO); polydispersity index (PDI); reactive oxygen species (ROS); ketothiolate (TK);

[0054] 2. The high-performance liquid chromatography (HPLC) conditions used in the following experiments of this invention.

[0055] Chromatographic column: Inertsil ODS-SP (4.6 mm × 250 mm, 5 μm), mobile phase: acetonitrile-5% phosphoric acid water (Cela: 87:13, v / v; Ce6: 75:25, v / v), flow rate: 1 mL / min, column temperature: 30 ℃, injection volume: 20 μL, detection wavelengths for Cela and Ce6 were 425 nm and 401 nm, respectively.

[0056] Example 1: Preparation of Cela / Ce6 / GCTR micelles of the present invention

[0057] 9 mg of Cela and 9 mg of Ce6 were weighed and dissolved together in 0.45 μL of DMSO. 12.5 mg of the GCTR conjugate was weighed, and 2.5 mL of ultrapure water was added. The mixture was then sonicated at 220 W for 15 min in an ice-water bath to ensure uniform dispersion and complete dissolution. With vigorous stirring at 1500 rpm, the Cela and Ce6 DMSO solution was added dropwise to the GCTR conjugate solution. After stirring at 1000 rpm for 20 min, the mixture was immediately sonicated at 220 W for 20 min in an ice-water bath. The mixture was then placed in a dialysis bag (MWCO 3500) and dialyzed with 2 L of distilled water for 12 h to remove the organic solvent. The dialyzed mixture was then sonicated at 220 W for 20 min in an ice-water bath, centrifuged at 3500 rpm for 10 min, and the supernatant was filtered through a 0.8 μm filter and freeze-dried to obtain the Cela / Ce6 / GCTR micelles.

[0058] When the dosage ratio of Cela / Ce6 / GCTR micelles is 9:9:12.5, the ratio of Cela / Ce6 / GCTR in the Cela / Ce6 / GCTR micelles prepared by the process is 2:1.7:2.4.

[0059] The following experimental examples demonstrate the beneficial effects of the present invention.

[0060] Experimental Example 1: Screening of Cela / Ce6 / GCTR micelles

[0061] I. Experimental Methods

[0062] (I) Investigation into the optimal ratio of Cela / Ce6 / GCTR micelles

[0063] 1. Preparation of Cela / Ce6 / GCTR micelles with different mass ratios

[0064] The Cela and Ce6 molecules, as shown in Table 1, were dissolved together in 0.45 μL DMSO. 12.5 mg of the GCTR conjugate was weighed, and 2.5 mL of ultrapure water was added. The mixture was then sonicated in an ice-water bath for 15 min to ensure uniform dispersion and complete dissolution. Under vigorous stirring at 1500 rpm, the Cela and Ce6 DMSO solution was added dropwise to the GCTR conjugate solution. After stirring at 1000 rpm for 20 min, the mixture was immediately sonicated at 220 W for 20 min in an ice-water bath. The mixture was then placed in a dialysis bag (MWCO 3500) and dialyzed with 2 L of distilled water for 12 h to remove the organic solvent. The dialyzed mixture was then sonicated at 220 W for 20 min in an ice-water bath, centrifuged at 3500 rpm for 10 min, and the supernatant was filtered through a 0.8 μm filter and freeze-dried to obtain the Cela / Ce6 / GCTR micelles.

[0065] 2. Determination of drug loading and encapsulation efficiency

[0066] Weigh 4 mg of drug-loaded micelles and dissolve them in 4 mL of ultrapure water. Take 0.5 mL of this solution and transfer it to a 5 mL volumetric flask. Disrupt the micelle structure with methanol and dilute to the mark. Perform high-performance liquid chromatography (HPLC) under conditions 1, record the peak areas, and calculate the contents of Cela and Ce6. Calculate the drug loading (DL) and encapsulation efficiency (EE) of Cela and Ce6 based on the drug content and the total mass of the Cela / Ce6 / GCTR micelles, respectively. The calculation formulas are as follows:

[0067]

[0068]

[0069] 3. Determination of Cela / Ce6 / GCTR micelle size, PDI, and Zeta potential

[0070] 4 mg of Cela / Ce6 / GCTR micelles were weighed and dissolved in 4 mL of ultrapure water. The mixture was sonicated for 15 min to disperse it evenly. After passing through a 0.8 μm filter membrane, the particle size, PDI, and Zeta potential of the Cela / Ce6 / GCTR micelles were determined by DLS.

[0071] II. Experimental Results

[0072] 1. Effects of different feed ratios of Cela, Ce6, and GCTR conjugates on the drug loading capacity of GCTR conjugates

[0073] The effects of different feed ratios on the ability of GCTR conjugate micelles to encapsulate Cela and Ce6 were investigated using drug loading, encapsulation efficiency, particle size, PDI, and potential as indicators. The results are shown in Table 1.

[0074] As shown in Table 1, after fixing the dosage of GCTR conjugate to 12.5 mg and Cela to 9 mg, the encapsulation efficiency of Cela increased with the increase of Ce6 dosage, reaching (95.93±8.21)% when the Cela to Ce6 dosage ratio was 1:1. At this time, the drug loading of Cela was (34.33±3.51)%. The drug loading and encapsulation efficiency of Ce6 also showed an overall increasing trend, reaching (26.43±2.29)% and (73.94±5.21)% respectively when the Cela to Ce6 dosage ratio was 1:1. Furthermore, the Cela / Ce6 / GCTR micelles with the lowest particle size (163.63±4.46) nm, PDI (0.15±0.01), and potential (-23.63±0.25) mV) obtained at a feed ratio of 1:1 showed tighter micelle encapsulation and a more stable structure, resulting in smaller particle size and a higher absolute Zeta value. This indicates that the addition of Ce6 increased the contact between Cela and Ce6 and the GCTR conjugate micelles, which is beneficial for better encapsulation of Cela and Ce6 by the GCTR conjugate micelles. In summary, a feed ratio of 1:1 for Cela and Ce6 results in smaller and more uniformly distributed Cela / Ce6 / GCTR micelles, higher drug loading, and higher encapsulation efficiency. Considering all factors, a feed ratio of 9 mg:9 mg:12.5 mg for the Cela, Ce6, and GCTR conjugate was selected as the optimal feed ratio.

[0075] Table 1. Effect of different feed ratios of Cela, Ce6, and GCTR conjugates on the drug loading capacity of GCTR conjugates ( n=3)

[0076]

[0077] Table 2 Actual drug loading ratio of Cela / Ce6 / GCTR micelles obtained with different feed ratios.

[0078] 9:1.8:12.5 7.40±0.41:0.77±0.04:11.07±0.57 9:3.6:12.5 8.08±0.42:2.47±0.02:9.93±1.23 9:5.4:12.5 8.39±0.32:3.79±0.09:10.39±0.14 9:7.2:12.5 8.56±0.62:5.82±0.47:9.86±0.76 9:9:12.5 8.64±0.75:6.65±0.47:9.91±1.60

[0079] Experimental results showed that, with fixed amounts of GCTR conjugate and Cela (12.5 mg GCTR conjugate, 9 mg Cela), and a Cela / Ce6 mass ratio within the range of 1:0 to 1:1, when the Cela:Ce6 (w / w) feed ratio was 1:1 (i.e., both Cela and Ce6 were 9 mg), the Cela / Ce6 / GCTR micelles exhibited small and uniform particle size distribution, higher drug loading, and higher encapsulation efficiency. Considering all factors, the optimal feed ratio of Cela, Ce6, and GCTR conjugate was determined to be 9:9:12.5. The actual drug loading ratio of the Cela / Ce6 / GCTR micelles was found to be 2:1.7:2.4.

[0080] Experimental Example 2: Characterization of Cela / Ce6 / GCTR micelles

[0081] I. Experimental Methods

[0082] Thermodynamic characterization of Cela powder, Ce6 powder, GCTR conjugate powder, physically mixed powders of Cela and Ce6 with GCTR conjugate, and Cela / Ce6 / GCTR micelle powder with the optimal feed ratio was performed using differential scanning calorimetry. Under an argon atmosphere, the temperature was increased from 40 °C to 500 °C at a heating rate of 10 K / min, and the scanning curves were recorded.

[0083] II. Experimental Results

[0084] DSC characterization results of Cela, Ce6, GCTR conjugates, mixtures of Cela and Ce6 with GCTR conjugates, and Cela / Ce6 / GCTR micelles are as follows: Figure 1 As shown, Cela exhibits a distinct exothermic peak around 150℃ and an absorption peak around 210℃, while Ce6 shows a distinct exothermic peak around 170℃. The GCTR conjugate, however, shows no distinct exothermic or absorption peaks between 25℃ and 500℃. The exothermic and absorption peaks of Cela and Ce6 are still present in the physical mixture of the three. The absence of distinct absorption and exothermic peaks in the Cela / Ce6 / GCTR micelles indicates the absence of free Cela and Ce6. DSC characterization results show that Cela and Ce6 were successfully encapsulated within the GCTR conjugate.

[0085] Experimental Example 3: Cela / Ce6 / GCTR micelle ROS sensitivity

[0086] I. Experimental Methods

[0087] (I) Observation of Cela / Ce6 / GCTR micelle morphology using transmission electron microscopy (TEM)

[0088] This invention uses transmission electron microscopy (TEM) to observe the morphology of Cela / Ce6 / GCTR micelles and determine that Cela / Ce6 / GCTR micelles are ROS sensitive.

[0089] (II) In vitro ROS sensitivity assay of Cela / Ce6 / GCTR micelles

[0090] A 0.5 mg / mL Cela / Ce6 / GCTR micelle solution was prepared, and H2O2 solution was added to make the final H2O2 concentration 0.1 mmol / L. The solution was placed in a shaker at 37℃, and the particle size was measured at different time points to observe the particle size change of Cela / Ce6 / GCTR micelles under ROS environment.

[0091] Diphenylisobenzofuran (DPBF) was used as a reactive oxygen species (ROS) probe to detect the photodynamic properties of Cela / Ce6 / GCTR micelles. 2 mg of DPBF was weighed and added to 10 mL of DMF until completely dissolved. The Cela / Ce6 / GCTR micelles were dissolved in ultrapure water to a concentration of 0.05 mg / mL, and H₂O₂ solution was added to bring the final H₂O₂ concentration to 0.1 mmol / L. 160 μL of DPBF solution was added to the Cela / Ce6 / GCTR micelle solution, making the total volume 3.5 mL. The Cela / Ce6 / GCTR micelle solution was placed in a shaker at 37 °C and irradiated with a 660 nm laser at an intensity of 0.3 W / cm². 2 The absorbance of DPBF at 415 nm was measured during different illumination periods.

[0092] II. Experimental Results

[0093] (I) TEM results of Cela / Ce6 / GCTR micelles

[0094] TEM results of Cela / Ce6 / GCTR micelles are as follows: Figure 2 As shown, the Cela / Ce6 / GCTR micelles are spherical with a particle size of approximately 150 nm, consistent with the particle size results measured by DLS. After H2O2 treatment, the micelle boundaries became blurred, the micelles were disrupted, and the particle size increased. Furthermore, after near-infrared irradiation, almost no spherical micelles were present in the system, and the particle size variation was even greater. These results indicate that Cela and Ce6 are incorporated into the GCTR conjugate to form spherical polymer micelles. H2O2 treatment increases the ROS content in the solution, breaks the TK bonds in the GCTR conjugate, disrupts the micelle structure, and releases the drug. Ce6 absorbs near-infrared light energy and generates ROS free radicals, increasing the ROS content in the solution and further promoting the disruption of the Cela / Ce6 / GCTR micelle structure, resulting in larger volume and irregular shape. Therefore, this demonstrates that the Cela / Ce6 / GCTR micelles are ROS sensitive.

[0095] (II) The ketithial thiol (TK) in the GCTR conjugate is a ROS-sensitive material. When ROS is present in the environment, TK cleavage causes micelle depolymerization. The experiment simulated a high-ROS environment at the tumor site by adding H2O2 solution. The particle size changes of the Cela / Ce6 / GCTR micelles in H2O2 solution are shown below. Figure 3 As shown, when Cela / Ce6 / GCTR micelles come into contact with H2O2, they disperse uniformly for the first 2 hours, and the particle size begins to increase after 4 hours, with the dispersion coefficient also increasing. This indicates that Cela / Ce6 / GCTR micelles can depolymerize in the high ROS microenvironment of tumor sites and are ROS sensitive.

[0096] DPBF is a singlet oxygen (ROS) scavenger with a characteristic absorption peak at 415 nm. When ROS binds to DPBF, the absorption of DPBF at 415 nm decreases, allowing observation of ROS generation in Cela / Ce6 / GCTR micelles in vitro. Figure 4 It was observed that in H2O2 solution, after irradiation with a 660nm laser, the absorption intensity of DPBF at 415nm decreased, and the absorbance value decreased with prolonged irradiation time. However, in H2O2 solution without laser irradiation and in the absence of H2O2 in the environment, the absorbance value of DPBF remained essentially unchanged. This indicates that Ce6 generates singlet oxygen under 660nm laser irradiation, increasing ROS in the system, thus allowing Cela / Ce6 / GCTR micelles to be better utilized for photodynamic therapy.

[0097] Experimental results show that Cela / Ce6 / GCTR micelles are ROS sensitive, and Ce6 generates singlet oxygen under laser irradiation, which increases the ROS in the system, resulting in better photodynamic therapy effects of Cela / Ce6 / GCTR micelles.

[0098] Experimental Example 4: Stability Study of Cela / Ce6 / GCTR Micellar Infusion

[0099] I. Experimental Methods

[0100] The compatibility and stability of Cela / Ce6 / GCTR micelles were investigated using 0.9% sodium chloride injection and 5% glucose injection. Appropriate amounts of Cela / Ce6 / GCTR micelles were weighed and added to both injection solutions, respectively. The solutions were stored at room temperature in the dark, and the particle size and polydispersity index were measured using a laser particle size analyzer at 0, 2, 4, 6, and 8 hours. A series of Cela / Ce6 / GCTR micelle solutions with concentrations of 0.5, 1, and 2 mg / mL were prepared using 0.9% sodium chloride injection, with each concentration in triplicate. The pH value was measured using a pH meter.

[0101] II. Experimental Results

[0102] The compatibility stability of Cela / Ce6 / GCTR micelles with 0.9% sodium chloride injection and 5% glucose injection was investigated using particle size change as an indicator. The results are as follows: Figure 5 As shown, Cela / Ce6 / GCTR micelles showed no precipitation or flocculation in any of the three solutions within the measurement time, and the particle size remained unchanged and was uniformly distributed. Among them, the particle size was smallest in 0.9% sodium chloride injection solution, therefore it was selected as the compatible solvent for clinical intravenous injection.

[0103] The pH values ​​of Cela / Ce6 / GCTR micelles after dissolving in 0.9% sodium chloride injection are shown in Table 3. The pH values ​​of 0.9% sodium chloride injection range from 5.0 to 7.0. The pH values ​​of lyophilized Cela / Ce6 / GCTR micelle formulations of different concentrations are similar to those of 0.9% sodium chloride injection, which meets the requirements for intravenous administration of formulations to humans.

[0104] Table 3. pH values ​​of different concentrations of Cela / Ce6 / GCTR micelle lyophilized formulations

[0105]

[0106]

[0107] Experimental results show that the infusion stability results indicate that the micelles of Cela / Ce6 / GCTR are small and stable after being dissolved in 0.9% sodium chloride injection, and the pH value of the solution meets the pH requirements of the injection. Therefore, 0.9% sodium chloride injection was selected as the compatibility solvent for intravenous injection of Cela / Ce6 / GCTR micelles.

[0108] Experimental Example 5: In vivo pharmacokinetic and tissue distribution study of Cela / Ce6 / GCTR micelles

[0109] Human hepatocellular carcinoma cells HepG2 were purchased from the Innovation Institute of Hepatocellular and Regenerative Medicine, Chinese Academy of Sciences, while human hepatocellular carcinoma cells BEL-7402 and normal hepatocytes L-02 were purchased from Cybio Biotechnology Co., Ltd.

[0110] I. Experimental Methods

[0111] (I) ROS Sensitivity Detection

[0112] N-acetyl-L-cysteine ​​(NAC) is a commonly used specific ROS inhibitor. Pretreatment of cells with NAC can effectively inhibit intracellular ROS.

[0113] Cytotoxicity experiments were conducted by adding the antioxidant N-acetyl-L-cysteine ​​(NAC) to compare the cytotoxicity of the drug to cells under ROS conditions, and to investigate the ROS sensitivity of Cela / Ce6 / GCTR micelles. Using HepG2 cells as a model, 20 mmol / L NAC was added 2 h in advance to inhibit intracellular ROS production. The cells were then cultured for 24 h under the corresponding drug and environmental conditions (Cela / GCTR micelles, Ce6, Cela / Ce6 / GCTR micelles, Ce6 under near-infrared light illumination, and Cela / Ce6 / GCTR micelles under near-infrared light illumination). The absorbance of each well was measured using the MTT assay to investigate the ROS sensitivity of Cela / Ce6 / GCTR micelles.

[0114] (II) Cytotoxicity assays of HepG2, BEL-7402, and L-02

[0115] Human hepatocellular carcinoma HepG2 cells, human hepatocellular carcinoma BEL-7402 cells, and normal hepatocytes L-02 cells were used as cell models in this experiment. The effects of free Cela, Ce6, and their solvents DMSO, GCTR conjugates, Cela / GCTR micelles, and Cela / Ce6 / GCTR micelles on the viability of the three cell types under light and no light conditions were investigated using the MTT assay to evaluate the cytotoxicity of each drug. In the light-exposed group, the cells were exposed to a 660nm laser at 100mW / cm² for 6 hours after drug administration. 2 Irradiation. Cells were co-incubated with the test drug solution for 24 h, 48 h, and 72 h, and cell viability was measured. The Ce6 concentration was 1 μg / mL, and the concentrations of other drugs were equivalent to the Ce6 concentration. (Cela: 1.2 μg / mL, GCTR conjugate: 1.68 μg / mL, Cela / GCTR micelles: 2.67 μg / mL, Cela / Ce6 / GCTR micelles: 3.33 μg / mL)

[0116] (III) HepG2, BEL-7402, L-02 cell uptake assay

[0117] Ce6 itself is fluorescent, with an excitation wavelength of 660 nm and an emission wavelength of 670 nm, and can be directly labeled into cells. The uptake of Ce1 / Ce6 / GCTR micelles by HepG2, BEL-7402, and L-02 cells was investigated using laser confocal microscopy. HepG2, BEL-7402, and L-02 cells in logarithmic growth phase were collected and analyzed at a concentration of 2 × 10⁻⁶ cells / cells. 5 Cells were seeded per laser confocal microscopy dish. After cell adhesion, the drug solution was discarded, and Ce6 and Cela / Ce6 / GCTR micelles were co-incubated with the cells for 6 hours. The drug solution was then discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 20 minutes, followed by three washes with PBS. The cells were stained with Hoechst 33258 nuclear staining solution for 15 minutes, washed three times with PBS, and 200 μL of PBS was added. The laser confocal microscopy dish was then placed under a laser confocal microscope to observe and photograph cell uptake.

[0118] II. Experimental Results

[0119] (I) Results of sensitivity testing of different drugs to ROS

[0120] The survival rate of HepG2 cells after NAC pretreatment was (101.48±2.69)%, and the addition of NAC did not affect cell viability.

[0121] Experimental results are as follows Figure 6As shown. Under normal circumstances, each group of drugs exhibits a certain degree of cytotoxicity towards HepG2 cells. The survival rates of HepG2 cells after near-infrared irradiation of Cela / GCTR micelles, Ce6, Cela / Ce6 / GCTR micelles, Ce6, and Cela / Ce6 / GCTR micelles were (74.22±2.15)%, (84.65±3.87)%, (71.21±3.75)%, (64.02±4.05)%, and (52.02±8.11)%, respectively.

[0122] from Figure 6 The results showed that NAC pretreatment significantly improved the survival rate of HepG2 cells, increasing it by 1.26, 1.14, 1.35, 1.35, and 1.79 times compared to the untreated group, respectively (P < 0.01). This indicates that the ROS environment affects the tumor-killing effect of drug-loaded micelles, and the high ROS environment in tumors facilitates the rapid release of drugs from micelles, leading to cytotoxicity.

[0123] (II) Results of HepG2, BEL-7402, and L-02 Cytotoxicity Assays

[0124] Experimental results are as follows Figure 7 As shown in the figure, at the experimental concentrations, the solvent DMSO and the GCTR conjugate had no effect on the viability of the three cell types, demonstrating that the GCTR conjugate does not damage the cells and has good safety.

[0125] Free Ce6 significantly inhibited the proliferation of all three cell types, with the inhibitory effect becoming more pronounced over time, leading to decreased cell viability. This indicates that free Ce6 does not specifically select for cells and has cytotoxic effects on both hepatocellular carcinoma cells and normal hepatocytes. Free Ce6 exhibited some cytotoxicity against HepG2 and BEL-7402 hepatocellular carcinoma cells, reducing cell viability, but had no significant effect on the viability of L-02 cells.

[0126] Experimental results on HepG2 cells showed that Cela / GCTR micelles inhibited HepG2 cell proliferation, with a stronger inhibitory effect than free Cela. This inhibitory effect became more pronounced over time. This may be because after Cela / GCTR micelles are taken up by cells, the presence of ROS within the cell causes the TK bonds of the ROS-sensitive material in the GCTR conjugate to break, releasing Cela, rhein, and glycyrrhetinic acid. When free Ce6 was exposed to near-infrared light, cell viability decreased; the cell viability at 24h, 48h, and 72h was 1.45, 2.24, and 2.00 times lower than that without near-infrared light, respectively. Cela / Ce6 / GCTR micelles significantly inhibited cell growth. After near-infrared light irradiation for 24h, 48h, and 72h, cell viability decreased by 1.22, 1.62, and 4.80 times, respectively, compared to the Cela / GCTR micelle group, which decreased by 1.21, 1.39, and 3.22 times, indicating a significant inhibitory effect on cell proliferation. These results suggest that the photosensitizer Ce6 can accept near-infrared light irradiation and generate a large amount of ROS, thereby promoting the breakage of TK bonds in the Cela / Ce6 / GCTR micelles, leading to the large-scale release of Cela and its killing effect on tumor cells.

[0127] The experimental results of BEL-7402 cells were basically consistent with those of HepG2 cells. Both free Cela and Ce6 exhibited cytotoxic effects on cells. The growth-inhibiting effects of Cela / GCTR micelles and Cela / Ce6 / GCTR micelles were significant, and the growth-inhibiting effects became stronger over time. After near-infrared light irradiation, the cell proliferation inhibition effect of free Ce6 and Cela / Ce6 / GCTR micelles was enhanced, and the cell survival rate decreased significantly. At 24h, 48h, and 72h, the cell survival rate of the Cela / Ce6 / GCTR micelle group decreased by 1.24, 1.71, and 3.09 times, respectively, which was 1.36, 1.83, and 2.08 times that of the Cela / GCTR micelle group.

[0128] Experimental results on L-02 cells showed that free Cela had a significant cytotoxic effect on normal L-02 cells, while Cela / GCTR micelles and Cela / Ce6 / GCTR micelles had no significant effect on cell survival. This indicates that Cela encapsulated in GCTR conjugates has liver-targeting properties and can effectively reduce its damage to normal cells. After near-infrared light irradiation, the cell survival rates of the free Ce6 and Cela / Ce6 / GCTR micelle groups decreased, but remained above 80%. This may be because trace amounts of ROS are also present in normal cells. Small amounts of Ce6 and Cela / Ce6 / GCTR micelles act on normal cells, and after near-infrared light irradiation, the small amount of Ce6 and Cela / Ce6 / GCTR micelles that are taken up exert their effects, producing weak cytotoxicity.

[0129] Experimental results showed that, under the same experimental concentration and time conditions, Cela / Ce6 / GCTR micelles combined with photodynamic therapy exhibited stronger toxicity to liver cancer cells than free Cela and Cela / GCTR micelles alone. Combined with photodynamic therapy, this effectively enhanced the inhibitory effect on liver cancer cell proliferation. However, in normal liver cells, Cela / Ce6 / GCTR micelles had no significant effect on cell survival and caused minimal damage. ROS sensitivity testing indicated that Cela / Ce6 / GCTR micelles did not kill cells in the absence of ROS, but in the high ROS environment of tumor cells, the release of drugs from Cela / Ce6 / GCTR micelles caused strong toxicity to liver cancer cells.

[0130] (III) Results of HepG2, BEL-7402, and L-02 cell uptake experiments

[0131] The results are as follows Figure 8 As shown, the blue fluorescent signal is from Hoechst 33342 stained cell nuclei, and the green fluorescent signal is emitted by Ce6.

[0132] HepG2 and BEL-7402 cells were able to take up Ce6 dissolved in DMSO, exhibiting green fluorescence upon excitation. After takeup, Ce6 was mainly distributed in the cytoplasm. After treatment with Cela / Ce6 / GCTR micelles, blue and green fluorescence also appeared in the cells, indicating that the cells were able to take up Cela / Ce6 / GCTR micelles.

[0133] In normal hepatocytes L-02, only blue nuclei were observed, with very little green fluorescence, indicating that normal hepatocytes do not take up Ce6 and Cela / Ce6 / GCTR micelles. This suggests that Cela / Ce6 / GCTR micelles have targeting properties for liver tumor cells and low toxicity to normal liver tissue.

[0134] Experimental results show that the Cela / Ce6 / GCTR micelles of this invention have high safety and do not produce cytotoxic effects. The Cela / Ce6 / GCTR micelles are ROS-sensitive. Liver cancer cells exhibit good uptake of Ce6 and Cela / Ce6 / GCTR micelles, while normal liver cells do not. The Cela / Ce6 / GCTR micelles demonstrate good safety and can reduce the damage of Cela to normal liver cells. Near-infrared light irradiation can generate a large amount of ROS in Ce6, increasing the ROS content at the tumor site and accelerating the depolymerization and drug release from the Cela / Ce6 / GCTR micelles. The Cela / Ce6 / GCTR micelles can effectively combine photodynamic and chemotherapeutic effects, enhancing the antitumor efficacy of the drug.

[0135] Experimental Example 6: In vivo pharmacodynamic evaluation of Cela / Ce6 / GCTR micelles for injection

[0136] I. Experimental Methods

[0137] (I) Establishment of a tumor-bearing mouse model by subcutaneous transplantation of H-22 mice

[0138] ICR mice, SPF grade, male, weighing (18-22)g were selected.

[0139] H-22 cells were resuscitated under aseptic conditions and incubated in a cell culture incubator for 1 day. The cells were then washed 2–3 times with sterile saline and collected into centrifuge tubes. The cells were resuspended in sterile saline and injected intraperitoneally into ICR mice for expansion. After 7 days of feeding, ascites fluid was extracted from the mice under aseptic conditions, washed 2–3 times with sterile saline, and diluted to a cell concentration of 2 × 10⁻⁶. 7 H-22 cell suspension was injected subaxillarily into the right forelimb of each mouse, at a rate of 0.1 mL / mL. Tumors were allowed to grow to 100–150 mm in size. 3 The participants were randomly divided into left and right groups for the experiment.

[0140] (II) Grouping and Dosing Cycle

[0141] Tumor-bearing mice were randomly divided into groups of eight. Each group received a tail vein injection of the drug formulation every two days for a total of five administrations. Two days after the last administration, the mice were enucleated to collect blood, and their organs and tumors were dissected. The experimental groups are as follows:

[0142] (1) Control group: 0.9% sodium chloride injection

[0143] (2) Positive control group (Oxaliplatin group): Oxaliplatin (2.5 mg / kg)

[0144] (3) Free Cela group: Cela (2 mg / kg)

[0145] (4) Cela / GCTR micelles (Cela / GCTR PMs) (containing Cela 2 mg / kg, GCTR conjugate: 2.6 mg / kg)

[0146] (5) Cela / Ce6 / GCTR micelles (Cela / Ce6 / GCTR PMs): (containing Cela 2 mg / kg, Ce6 1.7 mg / kg, GCTR conjugate: 2.4 mg / kg) (6) Cela / Ce6 / GCTR micelles (containing Cela 2 mg / kg, Ce6 1.7 mg / kg, GCTR conjugate: 2.4 mg / kg) and irradiated with 660 nm near-infrared light (0.5 W / cm²).2 (5 min) group (Cela / Ce6 / GCTR PMs+NIR)

[0147] (III) In vivo pharmacodynamic evaluation

[0148] 1. Measurement of mouse body weight changes and organ indexes

[0149] The mice were observed daily for their survival status, weighed at fixed times, and their weight was recorded and plotted as a curve showing weight changes. At the end of the experiment, the mice's organs were weighed, and organ indices were calculated to analyze the toxic side effects of each drug. The formula for calculating organ indices is as follows:

[0150] Organ index = Organ weight (g) / Mouse body weight (g) (Formula 3)

[0151] 2. Evaluation of tumor suppression effect

[0152] The long and short diameters of mouse tumors were measured and recorded at fixed times daily. Tumor volume was calculated, and tumor volume growth curves were plotted. After the experiment, the weight of mouse tumor tissue was recorded, the tumor inhibition rate of each drug was calculated, and the tumor inhibition effect of each drug was evaluated. The formulas for calculating tumor volume and tumor inhibition rate are as follows:

[0153] Tumor volume: Vtumor = (a² × b) / 2 (Formula 4)

[0154] Note: a is the short diameter of the tumor, b is the long diameter of the tumor, unit: mm.

[0155] Tumor inhibition rate: IR=(Wsaline-Wdrug) / Wsaline×100% (Formula 5)

[0156] Note: Wdrug is the tumor weight in the drug administration group, and Wsaline is the tumor weight in the saline group. Unit: g.

[0157] 3. Serum liver function tests

[0158] Blood was collected from the eyes of mice, centrifuged at 3500 rpm for 10 min, and the supernatant serum was collected and stored at -20℃ for later use. Following the procedures of the mouse aspartate aminotransferase (AST), mouse alanine aminotransferase (ALT), and mouse gamma-glutamyl transferase (GGT) ELISA kits, the OD values ​​of serum ALT, AST, and GGT in each group of mice were measured using an automated microplate reader. The activity units of ALT, AST, and GGT were calculated to reflect the severity of hepatocyte damage after treatment with each formulation.

[0159] II. Experimental Results

[0160] (I) Weight Changes and Organ Index

[0161] 1. Weight changes

[0162] During the tumor growth process in mice, a decrease in the mice's activity level was observed. After the tail vein injection of each drug preparation, the mice gradually recovered their vitality and were in good condition, indicating that each preparation had a therapeutic effect.

[0163] The free Cela group began experiencing diarrhea on day 7, possibly due to Cela irritating the gastrointestinal tract. The mouse weight change curve is shown below. Figure 8 As shown, the mice in the Control group gradually increased in weight, eventually reaching a 20% increase compared to pre-treatment weight. This may be due to the lack of treatment, allowing the tumors to gradually spread and enlarge. The mice in the free Cela group showed negative growth, with a 6% decrease in weight compared to pre-treatment weight. This may be due to the toxic side effects of Cela, which impaired the mice's bodily functions and caused weight loss.

[0164] The weight of mice in the other groups increased to varying degrees. The weight of mice in the positive control group (oxaliplatin), the Cela / GCTR micelle group, the Cela / Ce6 / GCTR micelle group, and the group exposed to near-infrared light increased by 8%, 9%, 13%, and 16% respectively compared with before administration. This indicates that the above preparations played a role in tumor treatment, the mice were treated, their physical condition improved, and their weight gradually increased.

[0165] 2. Organ Index

[0166] Organ indices can be calculated to determine the extent of drug damage to various organs. The organ indices of tumor-bearing mice after tail vein injection of various drugs are shown below. Figure 9 As shown.

[0167] Compared with the control group, the drug-loaded micelles had no significant effect on any organ in mice. The oxaliplatin positive control group showed significantly decreased liver and spleen indices, and a slight decrease in lung indices, with atrophy or other degenerative changes, indicating that oxaliplatin damages the liver, spleen, and lungs. Free Cela caused significant liver damage in mice, manifested as a significant decrease in liver indices. These results demonstrate that the Cela / Ce6 / GCTR micelles of this invention can improve Cela-induced organ damage, reduce Cela's toxic side effects, and provide some protection to various organs.

[0168] (II) Evaluation of Tumor Suppression Effect

[0169] 1. Changes in tumor volume

[0170] The tumor volume change curve of tumor-bearing mice is shown in the figure. Figure 10 As shown in the figure. From the start of drug administration, the tumors in the Control group grew rapidly, with the tumor volume increasing to 6.75 times that before drug administration.

[0171] All other treatment groups showed some tumor treatment effect. The Oxaliplatin group, free Cela group, Cela / GCTR micelle group, Cela / Ce6 / GCTR micelle group, and Cela / Ce6 / GCTR micelles (containing Cela 2 mg / kg and Ce6 1.7 mg / kg) were also treated with 660 nm near-infrared light irradiation (0.5 W / cm²). 2 The tumor volumes in the control group (after 5 minutes) were 3.69, 4.50, 4.11, 3.06, and 2.22 times that before drug administration, respectively, which were significantly different from those in the control group (P < 0.001). The positive control drug Oxaliplatin showed better tumor growth inhibition than free Cela. When Cela was encapsulated in GCTR micelles, its antitumor effect was stronger than that of free Cela (P < 0.05).

[0172] Tumor growth in mice in the Cela / Ce6 / GCTR micelle group was slow, and tumor growth was relatively slowed after near-infrared light irradiation. The tumor inhibition effect was significantly different from that of free Cela at the same dose (P < 0.001), and stronger than that of Cela / GCTR micelles at the same dose (P < 0.05). The results demonstrate that Cela and Ce6 encapsulated in GCTR micelles, combined with photodynamic therapy, exhibit superior antitumor effects.

[0173] 2. Tumor weight and tumor inhibition rate

[0174] After the experiment, the mice were euthanized, the tumors were dissected and weighed, the tumor inhibition rate was calculated, and the dissected tumors were photographed. The treatment effect is shown in the figure below. Figure 11 As shown. From Figure 11 Tumor images showed that after treatment, the tumor size in all treatment groups was smaller than that in the Control group. The tumor in the Cela / Ce6 / GCTR micelle group was significantly smaller than that in the Control group, demonstrating a good tumor inhibition effect. The Cela / Ce6 / GCTR PMs+NIR showed a very significant tumor-suppressing effect.

[0175] The results of tumor weight and tumor inhibition rate in tumor-bearing mice showed that ( Figure 12The tumor weight in the Control group was (2.07±0.34) g. Compared with the Control group, the tumor weight of each formulation group was reduced, and the tumor inhibition rate was high, with statistically significant differences (P<0.05). The tumor weight and tumor inhibition rate of the oxaliplatin group and the Cela / GCTR micelle group were similar, and the antitumor effect of Cela / GCTR micelles was close to that of a positive control drug. Compared with the Cela / GCTR micelle group, the tumor weight of the Cela / Ce6 / GCTR micelle group was reduced. After near-infrared light irradiation, the tumor weight decreased significantly to (0.71±0.12) g, and the tumor inhibition rate was the highest at (66.95±4.84)%, indicating that chemotherapy combined with photodynamic therapy can effectively improve the antitumor effect of drugs and achieve better therapeutic results.

[0176] (III) Serum liver function tests

[0177] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are sensitive markers of hepatocellular damage; serum ALT and AST concentrations are abnormally elevated when the liver is damaged. When the liver is inflamed or cancerous, serum gamma-glutamyl transferase (GGT) levels are elevated. Therefore, this experiment evaluated the liver function of tumor-bearing mice after treatment with different formulations by detecting serum ALT, AST, and GGT levels.

[0178] The experimental results (Table 4) showed that the negative control group reflected the levels of ALT, AST, and GGT in normal mice. The tumor-bearing mice in the control group exhibited severe liver damage, as evidenced by elevated levels of ALT, AST, and GGT. The free Cela group showed severe liver dysfunction, with elevated levels of AST, ALT, and GGT, indicating potential hepatotoxicity of Cela. The AST, ALT, and GGT values ​​in the remaining treatment groups were lower than those in the saline and free Cela groups, indicating that the degree of liver damage in these tumor-bearing mice was less severe to varying degrees than in the positive control and free Cela groups.

[0179] Table 4. Effects of various therapeutic drugs on liver function in tumor-bearing mice.

[0180]

[0181] Note: Compared with the negative control group: *P<0.05; **P<0.01; ***P<0.001;

[0182] Compared with the Control group: #P < 0.05; ##P < 0.01; ###P < 0.001;

[0183] Compared with the free Cela group: &P<0.05; &&P<0.01; &&&P<0.001;

[0184] Compared with Cela / CGTR PMs: $P < 0.05.

[0185] The Cela / GCTR micelle group and the positive control drug Oxaliplatin group caused similar levels of liver damage, but the liver damage in tumor-bearing mice was significantly reduced compared to the Control group, indicating that both groups have similar hepatoprotective effects. The Cela / Ce6 / GCT micelle group showed significant hepatoprotective effects, with significantly lower ALT and AST values ​​compared to the Control group (P < 0.05); after near-infrared light irradiation, the ALT and AST values ​​were even lower (P < 0.01), and compared to free Cela, the Cela / GCTRPMs group significantly reduced liver damage (P < 0.01).

[0186] Compared with the free Cela group, the Cela / Ce6 / GCTR micelle group showed a decrease in GGT levels (P < 0.05), which was significantly reduced after near-infrared light irradiation (P < 0.001), demonstrating a superior hepatoprotective effect compared to the Cela / GCTR micelle group (P < 0.05). This indicates that the co-encapsulation of Cela and Ce6 in GCTR micelles can exert a superior hepatoprotective effect, and the hepatoprotective effect is more significant after near-infrared light irradiation.

[0187] Experimental results show that the Cela / Ce6 / GCTR micelle group of the present invention has the best effect compared with other antitumor drugs. When the Cela / Ce6 / GCTR micelles of the present invention are irradiated with 660nm near-infrared light (0.5W / cm2, 5min), the antitumor effect is further increased. Moreover, the Cela / Ce6 / GCTR micelles of the present invention have a better liver protection effect, especially after near-infrared light irradiation, the liver protection effect is more significant, and it also has a better protective effect compared with the Cela / CGTR PMs group.

[0188] Experimental Example 7: In vivo pharmacokinetic study of Cela / Ce6 / GCTR micelles

[0189] I. Experimental Methods

[0190] (I) Pharmacokinetic Experiments in Mice

[0191] SPF-grade ICR mice, half male and half female, weighing (22-25) g. ICR mice were randomly divided into three groups: free Cela group (containing 2 mg / kg Cela), Cela / GCTR micelle group (containing 2 mg / kg Cela, 2.6 mg / kg GCTR conjugate), and Cela / Ce6 / GCTR micelle group (containing 2 mg / kg Cela, 1.7 mg / kg Ce6, 2.4 mg / kg GCTR conjugate), with 6 mice in each group. The mice were fasted but allowed free access to water before the experiment.

[0192] After mice were injected with different drugs via the tail vein, blood was collected by enucleation at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, and 48 h. The blood samples treated with heparin sodium were centrifuged at 3500 r / min for 10 min, and the supernatant plasma was transferred to a new EP tube and stored at -20℃ in the dark for later use.

[0193] Take 200 μL of plasma, add 10 μL of 5 μg / mL Tan IIA solution, vortex to mix, then add 3 mL of ethyl acetate for extraction, vortex for 5 min, centrifuge at 3500 rpm for 10 min, collect the supernatant into a 5 mL centrifuge tube, evaporate to dryness at 45℃, add 100 μL of methanol, vortex for 5 min to reconstitute, centrifuge at 14000 rpm for 10 min, and collect 70 μL of the supernatant for UPLC-MS / MS analysis. Calculate the concentrations of Cela and Ce6 in plasma, and plot the drug-concentration time curve with time on the x-axis and concentration on the y-axis.

[0194] II. Experimental Results

[0195] (I) Pharmacokinetic Experiments in Mice

[0196] 1. Blood drug concentration-time curve

[0197] Following tail vein injection of physiological saline solutions of Cela, Cela / GCTR micelles, and Cela / Ce6 / GCTR micelles into mice, the plasma concentration-time curves of Cela and Ce6 are shown below. Figure 13 As shown in the figure. Pharmacokinetic data show that after administration, each Cela formulation mainly undergoes a metabolic elimination phase in vivo. At the initial time point, the Cela concentrations are similar: 2330.98±198.16, 2553.73±189.69, and 2426.69±191.58 ng / mL for the Cela group, Cela / GCTR micelles, and Cela / Ce6 / GCTR micelles, respectively. Subsequently, the plasma Cela concentration decreases significantly.

[0198] At 24 h, the plasma concentration of free Cela was 64.96±22.74 ng / mL, and at 48 h, it was 46.31±8.63 ng / mL, indicating that almost all Cela was metabolized and cleared. The plasma concentrations of Cela in the Cela / GCTR micelle and Cela / Ce6 / GCTR micelle groups decreased gradually from 2 h, and the release curve remained stable until 48 h, maintaining a high Cela concentration in the plasma. The Cela concentration in the Cela / Ce6 / GCTR micelle group (675.87±44.35 ng / mL) was slightly higher than that in the Cela / GCTR micelle group (554.59±36.47 ng / mL), but consistently higher than that in the free Cela group.

[0199] The Ce6 concentration in plasma of the Cela / Ce6 / GCTR micelle group showed the same trend, with a concentration of 1811.05±206.58 ng / mL at 5 min. After 2 h, the plasma concentration decreased gradually and remained at a high level, with a plasma concentration of 527.51±97.92 ng / mL at 48 h.

[0200] Experimental results show that GCTR micelles encapsulating Cela and Ce6 can prevent the encapsulated drugs from directly contacting the complex physiological environment of blood. After entering the bloodstream, this reduces the impact of blood on the drugs, improves drug stability and activity, and prevents them from being rapidly cleared, prolonging the time the drugs circulate in the blood and increasing drug retention time, thus achieving the goals of sustained release, long-lasting effect, and stability.

[0201] This invention successfully prepared Cela / Ce6 / GCTR micelles, encapsulating the hydrophobic drugs Cela and Ce6 within polymer micelles to form a stable spherical structure. These drug-loaded micelles have small particle sizes, high drug loading capacity, and high encapsulation efficiency. This invention also selected 0.9% sodium chloride injection as the infusion solution for the Cela / Ce6 / GCTR micelles.

[0202] The combination of Cela / Ce6 / GCTR micelles and photodynamic therapy improves the bioavailability of Cela, enhances its liver and tumor targeting, strengthens its anti-hepatocellular carcinoma efficacy, increases drug retention time, and exhibits good safety. Photodynamic therapy combined with ROS-responsive polymer micelle delivery of antitumor drugs increases the reactive oxygen species content at the tumor site and intelligently releases the drug in a responsive manner. Compared with photodynamic therapy alone, this increases the stability and targeting of the photosensitizer, overcomes the limitations of monotherapy, and fully utilizes synergistic therapy to enhance the antitumor effect of chemotherapy drugs, resulting in highly effective and low-toxicity treatment. This achieves better tumor treatment outcomes and provides a new strategy for emerging tumor treatment methods.

Claims

1. A polymer micelle, characterized in that, It is a micelle formed by triptolide, dihydroporphyrin E6 and a support; the support is a carboxymethyl chitosan-ketithiolide-rheic acid coupling modified with glycyrrhetinic acid; in the polymer micelle, the mass ratio of triptolide, dihydroporphyrin E6 and the support is (7.40±0.41~8.64±0.75):(0.77±0.04~6.65±0.47):(9.91±1.60~11.07±0.57); the polymer micelle is prepared from triptolide, dihydroporphyrin E6 and the support as raw materials, and the feed ratio of triptolide, dihydroporphyrin E6 and the support is 9:9:12.5; The glycyrrhetinic acid-modified carboxymethyl chitosan-ketothiol-rheic acid coupling is a carboxymethyl chitosan substituted with the following two substituents: and .

2. The polymer micelles according to claim 1, characterized in that, The polymer micelles are obtained by adding a solution containing triptolide and dihydroporphyrin E6 dropwise to a solution of glycyrrhetinic acid-modified carboxymethyl chitosan-ketithiolide-rheic acid coupling under stirring, followed by stirring, sonication in an ice-water bath, dialyzing, sonication in an ice-water bath again, centrifugation, filtration, and freeze-drying.

3. A drug for treating cancer, characterized in that: It is a solution of polymer micelles as described in any one of claims 1 to 2 mixed with 0.9% sodium chloride injection or 5% glucose injection.

4. A method for preparing polymer micelles according to any one of claims 1 to 2, characterized in that: The preparation steps are as follows: (1) Dissolve triptolide and dihydroporphyrin E6 in a solvent to form a solution containing triptolide and dihydroporphyrin E6; (2) Weigh out the glycyrrhetinic acid-modified carboxymethyl chitosan-ketothiolate-rheic acid coupling compound and dissolve it to form a solution of glycyrrhetinic acid-modified carboxymethyl chitosan-ketothiolate-rheic acid coupling compound. (3) Under stirring conditions, add the solution containing triptolide and dihydroporphyrin E6 dropwise to the solution of carboxymethyl chitosan-ketithiol-rheic acid coupling modified with glycyrrhetinic acid, stir, sonicate in an ice-water bath, dialyze, sonicate in an ice-water bath again, centrifuge, filter, and freeze dry.

5. The method for preparing polymer micelles according to claim 4, characterized in that: In step (1), the solvent is an organic solvent; In step (2), the glycyrrhetinic acid-modified carboxymethyl chitosan-ketithiolide-rheic acid coupling compound is dissolved in water to form a solution of glycyrrhetinic acid-modified carboxymethyl chitosan-ketithiolide-rheic acid coupling compound. Step (3) is as follows: under vigorous stirring at 1500 rpm, the solution obtained in step (1) is added dropwise to the solution obtained in step (2), and after stirring at 1000 rpm for 20 min, it is immediately sonicated in an ice-water bath for 20 min with an ultrasonic power of 220 W; dialysis; the mixture after dialysis is sonicated in an ice-water bath for 20 min with an ultrasonic power of 220 W, centrifuged at 3500 rpm / min for 10 min, filtered through a filter membrane and then freeze-dried.

6. The method for preparing polymer micelles according to claim 5, characterized in that: In step (1), the solvent is DMSO.

7. Use of the polymer micelles according to any one of claims 1 to 2 in the preparation of a medicament for treating liver cancer.

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