Photodynamic combined active oxygen response type tripterine polymer micelle as well as preparation method and application thereof

Abstract

The invention provides a photodynamic combined active oxygen response type tripterine polymer micelle as well as a preparation method and application thereof, and belongs to the technical field of medicines. According to the present invention, the photodynamic therapy is combined, the photosensitizer Ce6 and the antitumor drug Cela are co-entrapped in the reactive oxygen species (ROS)-responsive liver targeting polymer micelle, and the prepared drug-loaded micelle has characteristics of small particle size and high stability. When the photosensitizer absorbs exogenous visible-near infrared light energy, ROS free radicals can be generated, the ROS concentration of a tumor site is improved, the breaking speed of ketal thiol bonds in the micelle is accelerated, and drug release is accelerated. The polymer micelle drug-loading system is good in biological safety, has the ROS response type drug release characteristic, gives full play to the synergistic effect in combination with the photodynamic therapy, prolongs the residence time in the body, enhances the targeting to liver tumors, improves the bioavailability and the anti-tumor effect of Cela, reduces the toxic and side effects on healthy tissues and visceral organs, and has a good application prospect. And a better anti-tumor synergistic treatment effect is exerted.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a photodynamic combined with active oxygen responsive type tripterygium wilfordii polymer micelle, and a preparation method and use thereof. Background Art

[0002] Primary liver cancer is the sixth most commonly diagnosed cancer worldwide and the third leading cause of cancer death worldwide 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 bile duct carcinoma (10%-15%), as well as other rare types.

[0003] Celastrol is a triterpenoid compound with biological activity extracted from the root of Tripterygium wilfordii Hook.F. Currently, a large number of studies have shown that Celastrol's excellent anti-liver cancer effect provides a promising anti-cancer drug for cancer patients.

[0004] Although tripterygium wilfordii has shown unique advantages in the treatment of liver cancer, its clinical application is limited by its low water solubility, poor bioavailability, narrow dosage window, and easy to cause toxic side effects. The poor solubility of tripterygium wilfordii results in its oral bioavailability of only 17.06%, and poor systemic absorption after administration. In addition, the side effects and narrow therapeutic window of tripterygium wilfordii are also problems that need to be faced in its clinical application. The narrow therapeutic range indicates that tripterygium wilfordii can easily reach toxic concentrations when there is a slight change in blood concentration. At low doses, tripterygium wilfordii can cause serious adverse reactions, including hepatotoxicity, nephrotoxicity, hematopoietic system toxicity, cardiotoxicity, teratogenicity, etc.

[0005] Chinese invention patent application CN 115554240 A discloses a tripterygium wilfordii / glycyrrhetinic acid-carboxymethyl chitosan-thioketal-rhein micelle. However, the micelle releases drugs based on the level of active oxygen in the tumor site, and the level of active oxygen varies in different tumors and at different stages of tumor development. It is difficult to control the release of drugs, and the efficacy of tumor treatment needs to be improved, and the toxicity of drugs needs to be further reduced.

[0006] Therefore, researching new products, accelerating the release of tripterygium wilfordii at the tumor site, giving full play to the therapeutic potential of tripterygium wilfordii, improving its anti-liver cancer efficacy and safety, and reducing toxic side effects have become major issues that need to be urgently addressed in the clinical application of tripterygium wilfordii in the treatment of liver cancer. Summary of the invention

[0007] The purpose of the present invention is to provide a micellar preparation that can accelerate the release of tripterygium wilfordii at the tumor site, exert the therapeutic potential of tripterygium wilfordii, improve its anti-liver cancer efficacy and safety, and reduce toxic side effects. The micelle has strong targeting and is particularly suitable as a drug for the treatment of liver cancer, providing a new option for clinical practice.

[0008] The present invention provides a polymer micelle, characterized in that it is a micelle formed by tripterygium wilfordii, a photosensitizer and a carrier; the carrier is a glycyrrhetinic acid-modified carboxymethyl chitosan-thioketal-rhein conjugate; in the micelle, the mass ratio of tripterygium wilfordii, the photosensitizer and the carrier 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, in the micelle, the mass ratio of tripterygium wilfordii, photosensitizer and carrier is 2:1.7:2.4.

[0010] Preferably, the micelle is prepared using tripterygium wilfordii, a photosensitizer and a carrier as raw materials, and the feed ratio of tripterygium wilfordii, the photosensitizer and the carrier is 9:(1-9):12.5.

[0011] The feed ratio of tripterygium wilfordii, photosensitizer and carrier is 9:9:12.5.

[0012] Preferably, the polymer micelles are obtained by adding a solution containing tripterygium wilfordii and a photosensitizer dropwise into a solution of glycyrrhetinic acid-modified carboxymethyl chitosan-thioketal-rhein conjugate under stirring, stirring, ultrasonicating in an ice-water bath, dialyzing, ultrasonicating in an ice-water bath again, centrifuging, filtering, and freeze-drying.

[0013] Preferably, the photosensitizer is dihydrochlorin e6; and / or, the glycyrrhetinic acid-modified carboxymethyl chitosan-thioketal-rhein conjugate is a micelle formed by modified carboxymethyl chitosan, and the modified carboxymethyl chitosan is a carboxymethyl chitosan substituted with the following two substituents:

[0014]

[0015] Preferably, the modified carboxymethyl chitosan is obtained by connecting rhein to one end of thioketal, and grafting glycyrrhetinic acid and the thioketal onto the carboxymethyl chitosan respectively.

[0016] and / or, the molar ratio of rhein to thioketal 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 thioketal is 1:1.2 to 1:2.4;

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

[0020] Preferably, the rhein is connected to the carboxyl group at one end of the thioketal through a polyamino compound, and the polyamino compound is preferably selected from one of ethylenediamine, 1,3-propylenediamine or 1,4-butylenediamine; 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 particle 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 micelle 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) dissolving triptertium wilfordii and a photosensitizer in a solvent to form a solution containing triptertium wilfordii and the photosensitizer;

[0024] (2) weighing the GCTR conjugate and dissolving it to form a GCTR conjugate solution;

[0025] (3) under stirring conditions, the solution containing celastrol and photosensitizer is added dropwise to the glycyrrhetinic acid-modified carboxymethyl chitosan-thioketal-rhein conjugate solution, stirred, ultrasonicated in an ice-water bath, dialyzed, ultrasonicated in an ice-water bath again, centrifuged, filtered, and freeze-dried.

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

[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) comprises: adding the solution obtained in step (1) dropwise into the solution obtained in step (2) under vigorous stirring at 1500 rpm, stirring at high speed at 1000 rpm for 20 min, and then immediately ultrasonicating the mixture in an ice water bath for 20 min at an ultrasonic power of 220 W; dialyzing; ultrasonicating the dialyzed mixture in an ice water bath for 20 min at an ultrasonic power of 220 W, centrifuging at 3500 rpm / min for 10 min, filtering with a filter membrane, and freeze-drying.

[0030] The present invention also provides a use of the mixed micelle in preparing a drug for treating cancer; preferably, the drug for treating cancer is a drug for treating liver cancer.

[0031] The experimental results show that the present invention combines photodynamic therapy to co-encapsulate the photosensitizer Ce6 and the anti-tumor drug Cela in ROS-responsive liver-targeted polymer micelles, and the prepared drug-loaded micelles have a small particle size and high stability. When the photosensitizer absorbs exogenous visible near-infrared light energy, it can generate ROS free radicals, increase the ROS concentration at the tumor site, accelerate the breaking rate of ketalthiolate bonds in the micelles, and accelerate drug release. The polymer micelle drug-loading system has good biological safety and the characteristics of ROS-responsive drug release. It fully exerts the synergistic effect in combination with photodynamic therapy, prolongs the retention time in the body, enhances the targeting of liver tumors, improves the bioavailability and anti-tumor effect of Cela, reduces the toxic and side effects on other tissues and organs, and exerts a better anti-tumor synergistic therapeutic effect.

[0032] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0033] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

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

[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 light treatment.

[0036] Figure 3 Particle size distribution (A) and dispersion coefficient (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 The particle size of Cela / Ce6 / GCTR micelles mixed with 0.9% sodium chloride injection and 5% glucose injection at different time periods.

[0039] Figure 6 The cytotoxicity of Ce6 or different micelles with or without NAC pretreatment and with or without infrared light irradiation on 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 Body weight changes of tumor-bearing mice during treatment (n=8).

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

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

[0044] Fig.11 Treatment effect diagram of tumor-bearing mice (n=8)

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

[0046] Fig.13 Blood concentration-time curves of Cela (A) and Ce6 (B) after injection of each preparation into the tail vein of mice (n=6) DETAILED DESCRIPTION

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

[0048] The GCTR conjugate of the present invention is the GCTR conjugate described in Patent No.: ZL 202110753678.7, Patent Name: A glycyrrhetinic acid-carboxymethyl chitosan-thioketal-rhein conjugate, its preparation method and use thereof. Specifically, the GCTR conjugate is synthesized by the following method:

[0049] Step 1: 0.1mmol of rhein (R) was heated and dissolved with 1% NaHCO3, and 0.12mmol of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) was added under stirring, and reacted at room temperature for 20 minutes, and then 0.12mmol of N-hydroxysuccinimide (NHS) was added and reacted at room temperature for 10 minutes to obtain R-NHS. 0.15mmol of thioketal (EDA) was added and dissolved in water, and R-NHS was added dropwise to the EDA aqueous solution, and reacted for 6 hours to obtain thioketal-rhein conjugate (R-EDA).

[0050] Step 2: 0.15 mmol thioketal (TK) was dissolved in N,N-dimethylformamide (DMF), 0.18 mmol EDC·HCl was added and reacted at room temperature for 20 min, then 0.18 mmol NHS was added and reacted at room temperature for 10 min, and then added to the R-EDA solution and reacted at room temperature to obtain TER.

[0051] Step 3: 0.083mmol O-carboxymethyl chitosan (CMCS) was dissolved in water. The TER solution was centrifuged and the supernatant was taken. 0.12mmol EDC·HCl was added to react at room temperature for 20min, and then 0.12mmol NHS was added to react at room temperature for 10min, and then added to the CMCS aqueous solution and reacted for 24h to obtain CTR. Step 4: 0.12mmol GA was dissolved in DMF, 0.14mmol EDC·HCl was reacted at room temperature for 20min, 0.14mmol NHS was reacted at room temperature for 10min, and then added to the CTR solution and reacted at room temperature for 24h. The reaction solution was placed in a dialysis bag (MWCO 3500), dialyzed with distilled water for 72h, probe ultrasonicated for 20min, and freeze-dried to obtain the product GCTR conjugate.

[0052] 1. Abbreviations

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

[0054] 2. The HPLC conditions used in the following experiments of the present invention are:

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

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

[0057] Weigh 9 mg Cela and 9 mg Ce6 and dissolve them in 0.45 μL DMSO. Weigh 12.5 mg GCTR conjugate. Add 2.5 mL ultrapure water and perform ultrasonic treatment at 220 W for 15 min in an ice-water bath to make it dispersed evenly and completely dissolved. Under vigorous stirring at 1500 rpm, add the DMSO solution of Cela and Ce6 dropwise into the GCTR conjugate solution. After high-speed stirring at 1000 rpm for 20 min, immediately perform ultrasonic treatment at 220 W for 20 min in an ice-water bath. Then, place the mixed solution in a dialysis bag (MWCO 3500) and dialyze with 2 L distilled water for 12 h to remove the organic solvent. The dialyzed mixed solution is ultrasonicated at 220 W for 20 min in an ice-water bath, centrifuged at 3500 rpm / min for 10 min, and the supernatant is filtered through a 0.8 μm filter membrane and freeze-dried to obtain Cela / Ce6 / GCTR micelles.

[0058] When the dosage ratio of the Cela / Ce6 / GCTR micelle group 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 beneficial effects of the present invention are demonstrated by experimental examples below.

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

[0061] 1. Experimental Methods

[0062] (I) Study on the optimal ratio of Cela / Ce6 / GCTR micelles

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

[0064] Weigh the mass ratio of Cela and Ce6 in Table 1 and dissolve them in 0.45μLDMSO. Weigh 12.5mg GCTR conjugate, add 2.5mL ultrapure water and perform probe ultrasonication for 15min in an ice-water bath to make it evenly dispersed and completely dissolved. Under vigorous stirring at 1500rpm in a high-speed stirrer, add the DMSO solution of Cela and Ce6 dropwise into the GCTR conjugate solution, stir at 1000rpm for 20min, and then immediately perform probe ultrasonication at 220W for 20min in an ice-water bath. Then, place the mixed solution in a dialysis bag (MWCO 3500) and dialyze with 2L distilled water for 12h to remove the organic solvent. The dialyzed mixed solution is subjected to probe ultrasonication at 220W for 20min in an ice-water bath, centrifuged at 3500rpm / min for 10min, and the supernatant is filtered through a 0.8μm filter membrane and freeze-dried to obtain 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 in a 5 mL volumetric flask, destroy the micelle structure with methanol and dilute to the mark. Measure under HPLC condition 1, record the peak area, and calculate the Cela and Ce6 contents. The drug loading (DL) and encapsulation efficiency (EE) of Cela and Ce6 were calculated based on the drug content and the total mass of Cela / Ce6 / GCTR micelles, respectively. The calculation formula is 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, dispersed evenly by ultrasonication for 15 min, and filtered through a 0.8 μm filter membrane. DLS was used to determine the particle size, PDI, and Zeta potential of Cela / Ce6 / GCTR micelles.

[0071] 2. Experimental Results

[0072] 1. Effect of different Cela, Ce6 and GCTR conjugate feed ratios 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 can be seen from Table 1, after the dosage of the fixed GCTR conjugate was 12.5 mg and the dosage of Cela was 9 mg, the encapsulation efficiency of Cela increased with the increase of the Ce6 feeding amount, reaching (95.93±8.21)% when the Cela to Ce6 feeding 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 increasing trend, which were (26.43±2.29)% and (73.94±5.21)% respectively when the Cela to Ce6 feeding ratio was 1:1. The Cela / Ce6 / GCTR micelles obtained at a feed ratio of 1:1 had the lowest particle size, (163.63±4.46) nm, a PDI of 0.15±0.01, and a potential of (-23.63±0.25) mV. The micelles were more tightly packed and had a more stable structure, so they were smaller in size and had a higher absolute value of Zeta. This shows that the addition of Ce6 increased the contact between Cela and Ce6 and the GCTR conjugate micelles, which was beneficial for the GCTR conjugate micelles to better encapsulate Cela and Ce6. In summary, when the feed ratio of Cela to Ce6 was 1:1, the Cela / Ce6 / GCTR micelles had a small particle size and uniform distribution, a large drug loading of Cela and Ce6, and a high encapsulation rate. After comprehensive consideration, the Cela, Ce6, and GCTR conjugate feed ratio of 9 mg:9 mg:12.5 mg was selected as the optimal feed ratio of Cela, Ce6, and GCTR conjugates.

[0075] Table 1 Effect of different Cela, Ce6 and GCTR conjugate feed ratios 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] Cela: Ce6: GCTR feed amount (w / w / w) Actual ratio of Cela / Ce6 / GCTR micelles 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] The experimental results show that when the dosage of GCTR conjugate and Cela is fixed, the GCTR conjugate is 12.5 mg, Cela is 9 mg, and the mass ratio of Cela and Ce6 is in the range of 1:0 to 1:1, when the Cela:Ce6 (w / w) feed ratio is 1:1 (i.e., Cela and Ce6 are both 9 mg), the Cela / Ce6 / GCTR micelles have small particle size and uniform distribution, and the drug loading of Cela and Ce6 is large and the encapsulation efficiency is high. After comprehensive consideration, the optimal feed ratio of Cela, Ce6 and GCTR conjugate is determined to be 9:9:12.5. The actual drug loading ratio of Cela / Ce6 / GCTR micelles is 2:1.7:2.4

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

[0081] 1. Experimental Methods

[0082] Cela powder, Ce6 powder, GCTR conjugate powder, physical mixed powder of Cela and Ce6 and GCTR conjugate, and Cela / Ce6 / GCTR micelle powder with the best feed ratio were thermodynamically characterized by differential scanning calorimetry. In an argon atmosphere, the temperature was raised from 40°C to 500°C at a heating rate of 10K / min, and the scanning curve was recorded.

[0083] 2. Experimental Results

[0084] The DSC characterization results of Cela, Ce6, GCTR conjugate, mixture of Cela and Ce6 and GCTR conjugate, and Cela / Ce6 / GCTR micelle are shown in Figure 1 As shown. Cela has an obvious exothermic peak at around 150°C and an obvious absorption peak at around 210°C, Ce6 has an obvious exothermic peak at around 170°C, while the GCTR conjugate has no obvious exothermic peak and absorption peak at 25°C-500°C. The exothermic peak and absorption peak of Cela and Ce6 still exist in the physical mixture of the three. There is no obvious absorption peak and exothermic peak in Cela / Ce6 / GCTR micelles, indicating that there is no free Cela and Ce6. The DSC characterization results show that Cela and Ce6 are successfully encapsulated into the GCTR conjugate.

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

[0086] 1. Experimental Methods

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

[0088] The present invention observes the morphology of Cela / Ce6 / GCTR micelles through transmission electron microscopy (TEM) to determine that the Cela / Ce6 / GCTR micelles are ROS-sensitive.

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

[0090] Prepare a 0.5 mg / mL Cela / Ce6 / GCTR micelle solution, add H2O2 solution to make the final H2O2 concentration of 0.1 mmol / L, place it in a 37°C shaker, take it out at different time points to measure the particle size, and observe the change in the particle size of Cela / Ce6 / GCTR micelles in the ROS environment.

[0091] Diphenylisobenzofuran (DPBF) was used as a reactive oxygen species (ROS) probe to detect the photodynamic properties of Cela / Ce6 / GCTR micelles. Weigh 2 mg of DPBF and add 10 mL of DMF to completely dissolve it. Dissolve the Cela / Ce6 / GCTR micelles in ultrapure water to a concentration of 0.05 mg / mL, and add H2O2 solution to make the final concentration of H2O2 0.1 mmol / L. Take 160 μL of DPBF solution and add it to the Cela / Ce6 / GCTR micelle solution to make a total volume of 3.5 mL. Place the Cela / Ce6 / GCTR micelle solution in a 37°C shaker and illuminate the solution with a 660 nm laser at an illumination intensity of 0.3 W / cm 2 , determine the absorbance value of DPBF at 415nm during different illumination time periods.

[0092] 2. Experimental Results

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

[0094] The TEM results of Cela / Ce6 / GCTR micelles are shown in Figure 2 As shown in the figure, Cela / Ce6 / GCTR micelles are spherical with a particle size of about 150nm, which is consistent with the particle size results measured by DLS. After H2O2 treatment, the micelle boundaries are blurred, the micelles are destroyed, the particle size increases, and after near-infrared light irradiation, there are almost no spherical micelles in the system, and the particle size changes more. The results show that Cela and Ce6 are encapsulated in the GCTR conjugate to form spherical polymer micelles. After H2O2 treatment, the ROS content in the solution increases, the TK bond in the GCTR conjugate breaks, the micelle structure is destroyed, and the drug is released. After Ce6 absorbs near-infrared light energy, it will produce ROS free radicals, which increases the ROS content in the solution, further promotes the destruction of the Cela / Ce6 / GCTR micelle structure in the solution, and the volume becomes larger and the shape becomes irregular, thus proving that Cela / Ce6 / GCTR micelles are ROS sensitive.

[0095] (II) The thioketal (TK) in the GCTR conjugate is a ROS-sensitive material. When ROS exists in the environment, TK breaks and the micelles depolymerize. The experiment simulated the high ROS environment in the tumor site by adding H2O2 solution. The particle size change results of Cela / Ce6 / GCTR micelles in H2O2 solution are shown in Figure 2. Figure 3 When Cela / Ce6 / GCTR micelles were in contact with H2O2, they were evenly dispersed in the first 2 hours, and the particle size began to increase after 4 hours, and the dispersion coefficient became larger, indicating that Cela / Ce6 / GCTR micelles can disaggregate in the high ROS microenvironment of the tumor site and are ROS sensitive.

[0096] DPBF is a singlet oxygen (ROS) scavenger with a characteristic absorption peak at 415nm. When ROS binds to DPBF, the absorption of DPBF at 415nm decreases, which can be used to observe the generation of ROS in Cela / Ce6 / GCTR micelles in vitro. Figure 4 It can be seen that in H2O2 solution, after the drug-loaded micelles were irradiated by 660nm laser, as the irradiation time increased, the absorption intensity of DPBF at 415nm weakened and the absorbance value decreased. When the H2O2 solution was not irradiated with laser and there was no H2O2 in the environment, the absorbance value of DPBF remained basically unchanged. This shows that Ce6 will produce singlet oxygen under 660nm laser irradiation, which will increase ROS in the system, and thus make Cela / Ce6 / GCTR micelles better for photodynamic therapy.

[0097] The experimental results show that Cela / Ce6 / GCTR micelles are ROS-sensitive, and Ce6 will produce singlet oxygen under laser irradiation, which increases the ROS in the system, and the Cela / Ce6 / GCTR micelles have better photodynamic therapy effects.

[0098] Experimental Example 4: Study on the stability of Cela / Ce6 / GCTR micellar infusion

[0099] 1. Experimental Methods

[0100] 0.9% sodium chloride injection and 5% glucose injection were used to investigate the compatibility stability of Cela / Ce6 / GCTR micelles. Appropriate amount of Cela / Ce6 / GCTR micelles were weighed and added to the above two injections respectively. They were placed at room temperature away from light, and the particle size and polydispersity coefficient were measured by 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 with 0.9% sodium chloride injection, and each concentration was repeated three times, and the pH value was measured with a pH acidometer.

[0101] 2. Experimental Results

[0102] The particle size change was used as an indicator to investigate the compatibility stability of Cela / Ce6 / GCTR micelles with 0.9% sodium chloride injection and 5% glucose injection. Figure 5 As shown, Cela / Ce6 / GCTR micelles had no precipitation or flocculation in the three solutions during the test time, and the particle size did not change significantly and was evenly distributed. Among them, the particle size was the smallest in 0.9% sodium chloride injection, so it was selected as the compatible solvent for clinical intravenous injection.

[0103] The results of pH measurement after Cela / Ce6 / GCTR micelles were dissolved in 0.9% sodium chloride injection are shown in Table 3. The pH of 0.9% sodium chloride injection is 5.0-7.0. The pH values ​​of Cela / Ce6 / GCTR micelle freeze-dried preparation solutions with different concentrations were similar to the pH value of 0.9% sodium chloride injection, which meets the requirements of human intravenous administration preparations.

[0104] Table 3 pH values ​​of Cela / Ce6 / GCTR micellar freeze-dried preparations at different concentrations

[0105]

[0106]

[0107] The experimental results show that the infusion stability results indicate that after 0.9% sodium chloride injection dissolves Cela / Ce6 / GCTR micelles, the micelle particle size is small and stable, and the pH value of the solution meets the pH requirements of the injection. Therefore, 0.9% sodium chloride injection is selected as the compatible solvent for intravenous injection of Cela / Ce6 / GCTR micelles.

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

[0109] Human hepatoma cell HepG2 was purchased from the Institute of Hepatocyte and Regenerative Medicine, Chinese Academy of Sciences, and human hepatoma cell BEL-7402 and normal hepatocyte L-02 were purchased from Cybio Biotech Co., Ltd.

[0110] 1. Experimental Methods

[0111] (I) ROS sensitivity detection

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

[0113] Cytotoxicity experiments were performed by adding the antioxidant N-acetyl L-cysteine ​​(NAC), and the ROS sensitivity of Cela / Ce6 / GCTR micelles was investigated by comparing the cytotoxicity of drugs to cells with and without ROS. Using HepG2 cells as a model, 20mmol / L NAC was added 2h in advance to inhibit the production of intracellular ROS, and the corresponding drugs were added and under the corresponding environmental conditions (Cela / GCTR micelles, Ce6, Cela / Ce6 / GCTR micelles, Ce6 illuminated by near-infrared light, and Cela / Ce6 / GCTR micelles illuminated by near-infrared light), and the cells were cultured for 24h. The absorbance of each well was measured by the MTT method to investigate the ROS sensitivity of Cela / Ce6 / GCTR micelles.

[0114] (II) Cytotoxicity experiments on HepG2, BEL-7402, and L-02 cells

[0115] The experiment used human liver cancer cell HepG2 cells, human liver cancer cell BEL-7402 cells and normal liver cell L-02 cells as cell models, and used MTT method to investigate the effects of free Cela, Ce6 and its solvent DMSO, GCTR conjugate, Cela / GCTR micelles, Cela / Ce6 / GCTR micelles on the survival rate of the three cells under illumination and non-illumination conditions, and to evaluate the cytotoxicity of each drug. 2 Irradiation. The test solution was incubated with the cells for 24h, 48h, and 72h, and the cell survival rate was detected. The Ce6 concentration was 1μg / mL, and the concentrations of other drugs were equivalent to the Ce6 concentration. (Cela: 1.2μg / mL, GCTR conjugate: 1.68μg / mL, Cela / GCTR micelle: 2.67μg / mL, Cela / Ce6 / GCTR micelle: 3.33μg / mL)

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

[0117] Ce6 itself has fluorescence, with an excitation wavelength of 660nm and an emission wavelength of 670nm, which can directly label cells. The uptake of Cela / 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 the logarithmic growth phase were taken and 2×10 5 The cells were inoculated in laser confocal culture dishes. After the cells adhered to the wall, the drug solution was discarded, Ce6 and Cela / Ce6 / GCTR micelles were incubated with the cells for 6 hours, the drug solution was discarded, and the cells were washed 3 times with PBS. They were fixed with 4% paraformaldehyde for 20 minutes, and then washed 3 times with PBS. They were stained with Hoechst33258 nuclear staining solution for 15 minutes, washed 3 times with PBS, and 200μL PBS was added. The laser confocal culture dish was placed under a laser confocal microscope to observe and take pictures of the cell uptake.

[0118] 2. Experimental Results

[0119] (I) Results of sensitivity test 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] The experimental results are as follows Figure 6As shown. Under normal circumstances, each group of drugs has a certain degree of cytotoxicity to HepG2 cells. The survival rates of HepG2 cells after Cela / GCTR micelles, Ce6, Cela / Ce6 / GCTR micelles, Ce6 irradiated with near-infrared light, and Cela / Ce6 / GCTR micelles irradiated with near-infrared light 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 It can be seen that the survival rate of HepG2 cells was significantly improved after NAC pretreatment, which was 1.26, 1.14, 1.35, 1.35, and 1.79 times higher than that of the group without NAC pretreatment, respectively, with significant differences (P < 0.01). This result shows that the ROS environment affects the killing effect of drug-loaded micelles on tumors, and the high ROS environment of tumors is conducive to the rapid release of drugs from micelles to produce cytotoxicity.

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

[0124] The experimental results are as follows Figure 7 As shown. Under the experimental concentrations, the solvent DMSO and the GCTR conjugate had no effect on the survival rates of the three cell types, proving that the GCTR conjugate would not cause damage to the cells and had good safety.

[0125] Free Cela has a significant inhibitory effect on the proliferation of the three types of cells, and the proliferation inhibition effect is obvious as time goes by, and the cell survival rate decreases, indicating that free Cela has no specific selection for cells and has cytotoxic effects on both liver cancer cells and normal liver cells. Free Ce6 has a certain cytotoxic effect on HepG2 and BEL-7402 liver cancer cells, and the cell survival rate decreases, but has no significant effect on the survival rate of L-02 cells.

[0126] The experimental results of HepG2 cells showed that Cela / GCTR micelles had an inhibitory effect on the proliferation of HepG2 cells, which was stronger than that of free Cela, and the inhibitory effect became more obvious as time went on. This may be because after Cela / GCTR micelles were taken up by cells, due to the presence of certain ROS in the cells, the TK bond of the ROS-sensitive material in the GCTR conjugate was broken, releasing Cela, rhein, and glycyrrhetinic acid. When free Ce6 was exposed to near-infrared light, the cell survival rate decreased, and the cell survival rates at 24h, 48h, and 72h were 1.45, 2.24, and 2.00 times lower than those without near-infrared light. Cela / Ce6 / GCTR micelles had a significant inhibitory effect on cell growth. After near-infrared light irradiation, the cell survival rate decreased by 1.22, 1.62, and 4.80 times at 24h, 48h, and 72h, which was 1.21, 1.39, and 3.22 times lower than that of the Cela / GCTR micelle group. The cell proliferation inhibition effect was significant. The results showed that the photosensitizer Ce6 can produce a large amount of ROS under near-infrared light irradiation, thereby promoting the breakage of the TK bond in the Cela / Ce6 / GCTR micelles, and Cela is released in large quantities, which has a 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 had cytotoxic effects on cells. Cela / GCTR micelles and Cela / Ce6 / GCTR micelles had obvious inhibitory effects on cell growth, and the growth inhibitory effect became stronger with the passage of time. After near-infrared light irradiation, the inhibitory effect of free Ce6 and Cela / Ce6 / GCTR micelles on cell proliferation increased, and the cell survival rate decreased significantly. At 24h, 48h, and 72h, the survival rate of cells in the Cela / Ce6 / GCTR micelle group decreased by 1.24, 1.71, and 3.09 times, which was 1.36, 1.83, and 2.08 times lower than that of Cela / GCTR micelles.

[0128] The experimental results of 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 rate, indicating that Cela encapsulated in GCTR conjugates has liver targeting and can effectively reduce its damage to normal cells. After near-infrared light irradiation, the cell survival rate of free Ce6 and Cela / Ce6 / GCTR micelle groups decreased, but they were all above 80%. This may be due to the presence of trace ROS in normal cells. A small amount of Ce6 and Cela / Ce6 / GCTR micelles act on normal cells. After near-infrared light irradiation, the small amount of Ce6 and Cela / Ce6 / GCTR micelles taken in play a role and produce weak cytotoxicity.

[0129] The experimental results show that under the same experimental concentration and time conditions, the toxic effect of Cela / Ce6 / GCTR micelles combined with photodynamic therapy on liver cancer cells is stronger than that of free Cela and Cela / GCTR micelles, and combined with photodynamic therapy can effectively improve the proliferation inhibition of liver cancer cells. In normal liver cells, Cela / Ce6 / GCTR micelles have no significant effect on the survival rate of normal cells and cause less damage to normal cells. The results of ROS sensitivity test show that Cela / Ce6 / GCTR micelles will not kill cells in the absence of ROS, but Cela / Ce6 / GCTR micelles release drugs in the high ROS environment of tumor cells, causing 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 in Figure 2, the blue fluorescent signal is the cell nucleus stained with Hoechst 33342, and the green fluorescent signal is emitted by Ce6.

[0132] HepG2 cells and BEL-7402 cells can absorb Ce6 dissolved in DMSO, and show green fluorescence after being excited. Ce6 is mainly distributed in the cytoplasm after being absorbed. After Cela / Ce6 / GCTR micelle treatment, blue and green fluorescence also appeared in the cells, indicating that the cells can absorb Cela / Ce6 / GCTR micelles.

[0133] In normal liver cells L-02, only blue nuclei were observed, with very little green fluorescence, indicating that normal liver cells would not take up Ce6 and Cela / Ce6 / GCTR micelles, indicating that Cela / Ce6 / GCTR micelles were targeted to liver tumor cells and had low toxic side effects on normal liver tissues.

[0134] The experimental results show that the Cela / Ce6 / GCTR micelles of the present invention have high safety, will not produce toxic effects on cells, and Cela / Ce6 / GCTR micelles are ROS-sensitive. Liver cancer cells have a good ability to absorb Ce6 and Cela / Ce6 / GCTR micelles, and normal liver cells will not absorb Ce6 and Cela / Ce6 / GCTR micelles. The safety of Cela / Ce6 / GCTR micelles is good and can reduce the damage of Cela to normal liver cells. After near-infrared light irradiation, Ce6 can produce a large amount of ROS, increase the ROS content in the tumor site, and accelerate the disaggregation of Cela / Ce6 / GCTR micelles to release drugs. Cela / Ce6 / GCTR micelles can effectively combine photodynamic and chemotherapeutic effects to improve the anti-tumor efficacy of drugs.

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

[0136] 1. Experimental Methods

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

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

[0139] H-22 cells were revived under sterile conditions and incubated in a cell culture incubator for 1 day. The cells were washed 2-3 times with sterile saline and collected into centrifuge tubes. The cells were resuspended with sterile saline and injected intraperitoneally into ICR mice for proliferation. After 7 days of feeding, the mouse ascites was extracted under sterile conditions, washed 2-3 times with sterile saline and diluted to a cell concentration of 2×10 7 Each mouse was injected with 0.1 mL of H-22 cell suspension under the right forelimb armpit. When the tumor volume grew to 100-150 mm 3 Left and right, randomly divided into groups, and carried out experiments.

[0140] (II) Grouping and dosing cycle

[0141] Tumor-bearing mice were randomly divided into groups of 8 mice each, and each drug preparation was injected into the tail vein once every two days for a total of 5 times. Two days after the last administration, the mice's eyeballs were removed for blood collection, and the mouse organs and tumors were dissected. The experimental groups were 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 micelle group (Cela / GCTR PMs) (containing Cela 2 mg / kg, GCTR conjugate: 2.6 mg / kg)

[0146] (5) Cela / Ce6 / GCTR micelle group (Cela / Ce6 / GCTR PMs): (containing Cela 2 mg / kg, Ce6 1.7 mg / kg, GCTR conjugate: 2.4 mg / kg) (6) Cela / Ce6 / GCTR micelle (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 , 5min) group (Cela / Ce6 / GCTR PMs+NIR)

[0147] (III) In vivo pharmacodynamic evaluation

[0148] 1. Mouse body weight changes and organ index measurements

[0149] The survival status of mice was observed every day, mice were weighed at fixed time, the weight of mice was recorded, and the weight change curve of mice was drawn. After the experiment, the organs of mice were weighed, and the organ index was calculated to analyze the toxic and side effects of each drug. The organ index calculation formula is as follows:

[0150] Organ index = organ weight (g) / mouse weight (g) (Formula 3)

[0151] 2. Evaluation of tumor inhibition effect

[0152] The long and short diameters of the mouse tumors were measured and recorded at a fixed time every day, the tumor volume was calculated, and the tumor volume growth curve was drawn. After the experiment, the weight of the 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 calculation formulas for 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 of the drug-treated group, and Wsaline is the tumor weight of the saline-treated group, unit: g.

[0157] 3. Serum liver function test

[0158] The eyeballs of mice were removed to collect blood, which was centrifuged at 3500r / min for 10min, and the upper serum was collected and stored in a -20℃ refrigerator for later use. According to the steps of mouse aspartate aminotransferase (AST), mouse alanine aminotransferase (ALT), and mouse glutamyl transpeptidase (GGT) ELISA kits, the OD values ​​of serum ALT, AST, and GGT of each group of mice were detected by automatic microplate reader, and the activity units of ALT, AST, and GGT were calculated to reflect the severity of liver cell damage after treatment with the preparations in each group.

[0159] 2. Experimental Results

[0160] 1. Weight changes and organ indexes

[0161] 1. Weight changes

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

[0163] The free Cela group began to experience diarrhea on the 7th day, which may be caused by Cela irritating the gastrointestinal tract. Figure 8 The weight of mice in the control group gradually increased, and the final weight increased by 20% compared with that before administration. This may be due to the fact that the mice did not receive treatment and the tumor gradually spread and grew. The mice in the free Cela group showed negative growth, and their weight decreased by 6% compared with that before administration. This may be due to the toxic side effects of Cela, which caused the mice to have decreased physical functions and weight loss.

[0164] The body weights of mice in the other groups increased to varying degrees. The body weights of mice in the positive drug oxaliplatin group, Cela / GCTR micelle group, Cela / Ce6 / GCTR micelle group and near-infrared light irradiation group increased by 8%, 9%, 13% and 16% respectively compared with before administration, indicating 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] The damage of drugs to various organs can be judged by calculating the organ index. The organ index results of tumor-bearing mice after tail vein injection of various drugs are as follows: Fig. 9 shown.

[0167] Compared with the Control group, each drug-loaded micelle had no significant effect on the various organs of mice. In the positive drug oxaliplatin group, the liver index and spleen index were significantly reduced, the lung index was slightly reduced, and atrophy or other degenerative changes occurred, indicating that oxaliplatin was damaging to the liver, spleen, and lungs. Free Cela had obvious damage to the liver of mice, which was manifested by a significant reduction in the liver index. The results show that the Cela / Ce6 / GCTR micelles of the present invention can improve the damage of Cela to organs, reduce the toxic and side effects of Cela, and play a certain protective role on various organs.

[0168] (II) Evaluation of tumor inhibition effect

[0169] 1. Changes in tumor volume

[0170] Tumor volume change curve of tumor-bearing mice Fig.10 As shown in the figure, since the administration, the tumor in the Control group grew rapidly, and the tumor volume was 6.75 times that before the administration.

[0171] The other treatment groups all showed certain tumor treatment effects. Oxaliplatin group, free Cela group, Cela / GCTR micelle group, Cela / Ce6 / GCTR micelle group and Cela / Ce6 / GCTR micelle group (containing Cela 2mg / kg, Ce6 1.7mg / kg) and 660nm near-infrared light irradiation (0.5W / cm 2 The tumor volumes of the 5-min and 3-min groups were 3.69, 4.50, 4.11, 3.06 and 2.22 times those before administration, respectively, which were significantly different from those of the Control group (P<0.001). The positive drug Oxaliplatin had a better inhibitory effect on tumor growth than free Cela. When Cela was encapsulated in GCTR micelles, its anti-tumor effect was stronger than that of free Cela (P<0.05).

[0172] The tumor growth of mice in the Cela / Ce6 / GCTR micelle group was slow, and the tumor growth was relatively slow 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 was stronger than that of Cela / GCTR micelles at the same dose (P < 0.05). The results showed that after Cela and Ce6 were encapsulated in GCTR micelles, combined with photodynamic therapy, they could show better anti-tumor effects.

[0173] 2. Tumor weight and tumor inhibition rate

[0174] After the experiment, the mice were killed, the tumors were dissected and weighed, the tumor inhibition rate was calculated, and the dissected tumors were photographed. The treatment effect diagram is shown in Fig.11 As shown. Fig.11 From the tumor photos, after the treatment, the tumor size of the tumor-bearing mice in each drug-treated group was smaller than that in the Control group, and the tumor in the Cela / Ce6 / GCTR micelle group was significantly smaller than that in the Control group, showing a good tumor inhibition effect. Cela / Ce6 / GCTR PMs+NIR has a very significant tumor inhibition effect.

[0175] The results of tumor weight and tumor inhibition rate of tumor-bearing mice showed that ( Fig.12), the tumor weight of the Control group was (2.07±0.34) g. Compared with the Control group, the tumor weight of each preparation 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 anti-tumor effect of Cela / GCTR micelle was close to that of the positive drug. Compared with the Cela / GCTR micelle group, the tumor weight of the Cela / Ce6 / GCTR micelle group decreased, and the tumor weight decreased significantly after near-infrared light irradiation to (0.71±0.12) g. The tumor inhibition rate was the highest, at (66.95±4.84)%, indicating that chemotherapy combined with photodynamic therapy can effectively improve the anti-tumor effect of the drug and achieve better therapeutic effects.

[0176] (III) Serum liver function test

[0177] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are sensitive markers of liver cell damage. When the liver is damaged, the concentrations of ALT and AST in the serum will increase abnormally. When the liver is inflamed or cancerous, the patient's serum glutamyl transpeptidase (GGT) level will increase. Therefore, this experiment evaluated the liver function of tumor-bearing mice after treatment with each group of preparations by detecting the levels of ALT, AST, and GGT in the serum of tumor-bearing mice.

[0178] The experimental results show (Table 4) that the negative control group reflects the levels of ALT, AST and GGT in normal mice, and the tumor-bearing mice in the Control group have more severe liver damage, which is manifested by higher levels of ALT, AST and GGT. The liver function of the free Cela group was severely impaired, and the levels of AST, ALT and GGT were high, indicating that Cela may be hepatotoxic. The AST, ALT and GGT values ​​of the other drug-treated groups were lower than those of the saline group and the free Cela group, indicating that the degree of liver damage in the tumor-bearing mice in these groups was lighter than that of the positive control group and the free Cela group to varying degrees.

[0179] Table 4 Effects of various therapeutic drugs on liver function of 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 degree of liver damage caused by Cela / GCTR micelle group and positive drug Oxaliplatin group was similar, and the liver damage of tumor-bearing mice was much lower than that of Control group, indicating that both have similar liver protection effects. Cela / Ce6 / GCT micelle group showed significant liver protection effect, and ALT and AST values ​​were significantly reduced compared with Control group (P < 0.05); after near-infrared light irradiation, ALT and AST values ​​were smaller (P < 0.01), and compared with free Cela, Cela / CGTRPMs group greatly reduced liver damage (P < 0.01).

[0186] Compared with the free Cela group, the GGT level in the Cela / Ce6 / GCTR micelle group was reduced (P < 0.05), and significantly reduced after near-infrared light irradiation (P < 0.001), showing a better liver protection effect than the Cela / GCTR micelle group (P < 0.05). It can be seen that Cela and Ce6 co-encapsulated in GCTR micelles can play a better liver protection role, and the liver protection effect is more significant after near-infrared light irradiation.

[0187] The experimental results show that the Cela / Ce6 / GCTR micelle group of the present invention has the best effect compared with other anti-tumor drugs. When the Cela / Ce6 / GCTR micelles of the present invention are irradiated with 660nm near-infrared light (0.5W / cm2, 5min), the anti-tumor effect is further increased, and 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 than the Cela / CGTR PMs group.

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

[0189] 1. 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 free Cela group (containing Cela 2 mg / kg), Cela / GCTR micelle group (containing Cela 2 mg / kg, GCTR conjugate: 2.6 mg / kg), Cela / Ce6 / GCTR micelle group (containing Cela 2 mg / kg, Ce6 1.7 mg / kg, GCTR conjugate: 2.4 mg / kg), 6 mice in each group, fasting but not water before the experiment.

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

[0193] Take 200 μL of plasma, add 10 μL of 5 μg / mL Tan IIA solution, vortex mix, add 3 mL of ethyl acetate for extraction, vortex oscillation for 5 minutes, centrifuge at 3500 r / min for 10 minutes in a high-speed centrifuge, take the supernatant to a 5 mL centrifuge tube, evaporate to dryness at 45°C, add 100 μL of methanol, vortex oscillation for 5 minutes to re-dissolve, centrifuge at 14000 r / min for 10 minutes, take 70 μL of supernatant for UPLC-MS / MS analysis. Calculate the concentrations of Cela and Ce6 in plasma, and draw the blood drug-concentration time curve with time as the horizontal axis and concentration as the vertical axis.

[0194] 2. Experimental Results

[0195] (I) Pharmacokinetic experiments in mice

[0196] 1. Blood drug concentration-time curve

[0197] After the mice were injected with saline solution of Cela, Cela / GCTR micelles and Cela / Ce6 / GCTR micelles, the blood concentration-time curves of Cela and Ce6 were shown in Figure 2. Fig.13 Pharmacokinetic data showed that after administration, each Cela preparation mainly had a metabolic elimination stage in the body. At the initial time point, the Cela concentrations were similar, which were 2330.98±198.16, 2553.73±189.69, and 2426.69±191.58 ng / mL in the Cela group, Cela / GCTR micelles, and Cela / Ce6 / GCTR micelles, respectively. After that, the Cela concentration in plasma dropped significantly.

[0198] The plasma concentration of free Cela group was 64.96±22.74ng / mL at 24h and 46.31±8.63ng / mL at 48h, and Cela was almost completely metabolized and cleared. The Cela blood concentration of Cela / GCTR micelle and Cela / Ce6 / GCTR micelle groups began to decline slowly from 2h, and the release curve remained stable until 48h, maintaining a high Cela concentration in plasma. The Cela concentration of Cela / Ce6 / GCTR micelle group (675.87±44.35ng / mL) was slightly higher than that of Cela / GCTR micelle group (554.59±36.47ng / mL), and was always higher than that of free Cela group.

[0199] The Ce6 concentration in the plasma of the Cela / Ce6 / GCTR micelle group showed the same trend, with a concentration of 1811.05±206.58 ng / mL at 5 minutes. After 2 hours, the blood concentration decreased slowly and remained at a higher concentration. At 48 hours, the blood concentration was 527.51±97.92 ng / mL.

[0200] Experimental results show that after GCTR micelles are encapsulated with Cela and Ce6, they can avoid direct contact between the encapsulated drugs and the complex physiological environment of the blood. After entering the blood, the impact of the blood on the drugs is reduced, thereby improving the stability and activity of the drugs. They can also avoid being quickly cleared, prolonging the time the drugs circulate in the blood and increasing the drug retention time, thereby achieving the purpose of sustained release, long-term effect and stability.

[0201] The present invention successfully prepared Cela / Ce6 / GCTR micelles, encapsulated hydrophobic drugs Cela and Ce6 in polymer micelles to form a stable spherical structure, and the drug-loaded micelles had small particle size, high drug loading and encapsulation efficiency. The present invention also selected 0.9% sodium chloride injection as the cela / Ce6 / GCTR micelle infusion compatibility solution.

[0202] The combination of Cela / Ce6 / GCTR micelles and photodynamic therapy improves the bioavailability of Cela, improves Cela's liver and tumor targeting, enhances the anti-liver cancer effect, increases drug retention time, and has good safety. Photodynamic therapy combined with ROS-responsive polymer micelles to deliver anti-tumor drugs increases the content of reactive oxygen species in the tumor site and releases drugs in an intelligent responsive manner. Compared with single photodynamic therapy, it increases the stability of the photosensitizer and improves the targeting of the photosensitizer, overcomes the limitations of single therapy, and makes full use of synergistic therapy to improve the anti-tumor effect of chemotherapy drugs. It has a high-efficiency, low-toxicity therapeutic effect. Achieve better tumor treatment effects and provide new strategies for emerging tumor treatment methods.

Claims

1. A polymer micelle, characterized in that It is a micelle formed by celastrol, a photosensitizer and a carrier; the carrier is a glycyrrhetinic acid-modified carboxymethyl chitosan-thioketal-rhein conjugate; in the micelle, the mass ratio of celastrol, the photosensitizer and the carrier 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).

2. The polymer micelle according to claim 1, characterized in that In the micelle, the mass ratio of tripterygium wilfordii, photosensitizer and carrier is 2:1.7:2.

4.

3. The polymer micelle according to claim 1, characterized in that The micelle is prepared by taking tripterygium wilfordii, photosensitizer and carrier as raw materials, and the feed ratio of tripterygium wilfordii, photosensitizer and carrier is 9:(1-9):12.

5.

4. The polymer micelle according to claim 3, characterized in that The feed ratio of tripterygium wilfordii, photosensitizer and carrier is 9:9:12.

5.

5. The polymer micelle according to claim 3 or 4, characterized in that The polymer micelle is prepared by dropping a solution containing tripterygium wilfordii and a photosensitizer into a glycyrrhetinic acid-modified carboxymethyl chitosan-thioketal-rhein conjugate solution under stirring, stirring, ultrasonicating in an ice-water bath, dialysis, ultrasonicating in an ice-water bath again, centrifuging, filtering, and freeze-drying.

6. The polymer micelle according to any one of claims 1 to 5, characterized in that The photosensitizer is dihydrochlorin e6; and / or, the glycyrrhetinic acid-modified carboxymethyl chitosan-thioketal-rhein conjugate is a micelle formed by modified carboxymethyl chitosan, and the modified carboxymethyl chitosan is a carboxymethyl chitosan substituted with the following two substituents:

7. A drug for treating cancer, characterized in that: The invention relates to a solution prepared by mixing the polymer micelles described in any one of claims 1 to 6 with 0.9% sodium chloride injection or 5% glucose injection.

8. A method for preparing the polymer micelle according to any one of claims 1 to 6, characterized in that: The preparation steps are as follows: (1) dissolving triptertium wilfordii and a photosensitizer in a solvent to form a solution containing triptertium wilfordii and the photosensitizer; (2) weighing the GCTR conjugate and dissolving it to form a GCTR conjugate solution; (3) under stirring conditions, the solution containing celastrol and photosensitizer is added dropwise to the glycyrrhetinic acid-modified carboxymethyl chitosan-thioketal-rhein conjugate solution, stirred, ultrasonicated in an ice-water bath, dialyzed, ultrasonicated in an ice-water bath again, centrifuged, filtered, and freeze-dried.

9. The method for preparing the polymer micelle according to claim 8, characterized in that: In step (1), the solvent is an organic solvent, preferably DMSO; In step (2), the GCTR conjugate is dissolved in water to form a GCTR conjugate solution; Step (3) comprises: adding the solution obtained in step (1) dropwise into the solution obtained in step (2) under vigorous stirring at 1500 rpm, stirring at high speed at 1000 rpm for 20 min, and then immediately ultrasonicating the mixture in an ice water bath for 20 min at an ultrasonic power of 220 W; dialyzing; ultrasonicating the dialyzed mixture in an ice water bath for 20 min at an ultrasonic power of 220 W, centrifuging at 3500 rpm / min for 10 min, filtering with a filter membrane, and freeze-drying.

10. Use of the mixed micelle according to any one of claims 1 to 6 in the preparation of a drug for treating cancer; preferably, the drug for treating cancer is a drug for treating liver cancer.

Images