Rhein-diselenide-hyaluronic acid (RaeHA) conjugate, tripterine redox response type polymeric micelle as well as preparation method and application thereof

Abstract

The invention provides a rhein-diselenide-hyaluronic acid (RaeHA) conjugate, which is characterized in that selenocystamine (diselenide, Se-Se) is used as a connecting arm, small molecular rhein (R) is grafted to low molecular weight hyaluronic acid (HA), and a redox dual-response conjugate (RaeHA conjugate) which can be self-assembled into micelles in water is synthesized. The invention also provides a preparation method of the conjugate and a micelle, and the micelle is formed by entrapping a hydrophobic compound by the rhein-selenium-hyaluronic acid (RaeHA) conjugate. According to the invention, the antitumor drug Cela is entrapped in the redox response type tumor targeting polymer micelle, and the prepared micelle is small and uniform in particle size and good in stability; the bioavailability of the Cela is improved; the residence time of Cela in vivo is prolonged; the tumor targeting property is enhanced, and the toxic and side effects on non-targeted organs are reduced; the diselenide bond in the micelle is broken by high-level reactive oxygen species (ROS) and glutathione (GSH) environment in tumor cells, so that the drug release is accelerated, and the anti-tumor curative effect is further enhanced.

Description

Technical Field

[0001] The present invention relates to a rhein-diselenide-hyaluronic acid (RSeHA) conjugate, a tripterygium wilfordii redox-responsive polymer micelle, and a preparation method and application thereof. Background Art

[0002] Celastrol (Cela) is a triterpenoid compound and the first active ingredient extracted from the root of the traditional Chinese medicine Tripterygium wilfordii. Celastrol has been shown to possess a variety of biological activities, including anticancer, antifungal, antifibrotic, anti-inflammatory, and antioxidant activities, as well as anti-neurodegenerative activities. Celastrol has demonstrated significant inhibitory effects on a variety of tumors, including breast, liver, and colorectal cancers. Studies have shown that Celastrol inhibits the viability of breast cancer MCF-7 cells in an AMP-activated protein kinase (AMPK)-dependent manner. Its anti-tumor mechanism was first reported in 2006. The strongly reactive C2 and C6 aromatic ketone rings in Celastrol's structure form covalent bonds with the amino group at the nitrogen terminus of threonine on the proteasome subunit 5, inhibiting the activity of proteasome chymotrypsin and thereby inducing apoptosis. Further research has revealed that Celastrol can exert its anticancer effects by modulating multiple signal transduction pathways and oncogenic molecular targets. Celastrol also induces elevated levels of reactive oxygen species (ROS), leading to AMPK phosphorylation. Protein kinase C (PKC) zeta has also been shown to play a role in triptolide-induced ROS generation. Furthermore, triptolide increased the phosphorylation of the pro-apoptotic effector p53. AMPK inhibition blocked triptolide-mediated p53 phosphorylation. Furthermore, Celastrol increased the expression of the tumor suppressor polo-like kinase 2 (PLK-2) in a p53-dependent manner. These results suggest that triptolide may exert its antitumor effects on MCF-7 cells through the AMPK-induced p53 and PLK-2 pathways. Triptolide has an inhibitory effect on breast cancer cells and may intervene in the development of cancer at three stages: tumor cell proliferation, angiogenesis, and cancer cell metastasis, thereby exerting its antitumor effects. Triptolide significantly reduced the mRNA expression of core therapeutic targets for breast cancer and significantly inhibited the proliferation of MCF-7 cells by downregulating the PI3K / AKT pathway. It inhibits breast cancer cell migration and invasion by upregulating E-cadherin; inhibits angiogenesis and biological processes in breast cancer cells through the DEGS1 / S1P signaling pathway; and inhibits IL-6 by inactivating the NF-κB pathway and IL-1β by inhibiting the mitogen-activated protein kinase / ERK-dependent pathway, thereby inhibiting breast cancer cell invasion. Celastrol can also reverse breast cancer drug resistance.

[0003] Although triptolide has significant anti-breast cancer activity, its poor water solubility, lack of specific selectivity, and significant toxic side effects limit its clinical application. Although there are studies on its structural modification and preparation into nanoparticles, micelles, nanosuspensions, etc. (Song J, He G, Dai L. A comprehensive review on celastrol, triptolide and triptonide: Insights on their pharmacological activity, toxicity, combination therapy, new dosage form and novel drug delivery routes [J]. Biomed Pharmacother. 2023, 162: 114705). Bi Chen, You Qinghui. Synthesis of triptolide-glucosamine conjugates and their toxicity-reducing and lipid-regulating effects [J]. Chinese Herbal Medicine. 2024, 55(4): 1-9., there is still no marketed preparation. In addition, the preparation disclosed in this document is an oral preparation. The tripterine-glucosamine amide conjugate is a small molecule. Tripterine and the small molecule glucosamine are coupled together through an amide bond, thereby increasing the water solubility of tripterine. This conjugate reduces toxicity to liver tumor cells and is used to resist lipid oxidation. It is slowly released in the blood and released more rapidly in gastrointestinal fluid. There is no mention of a targeting effect. Therefore, how to overcome the drug's shortcomings, ensure its anti-breast cancer efficacy, and reduce toxic side effects has become an urgent problem to be solved in the clinical application of tripterine in the treatment of breast cancer.

[0004] Most anti-tumor drugs are poorly soluble in water and have significant toxic and side effects. To address the shortcomings of current anti-tumor drugs, researchers have developed a variety of nanoformulations to address these issues. Nanoformulations are drugs delivered to target cells, target organs, target tissues, or intracellular structures with the help of carriers, antibodies, or ligands. On the one hand, they can increase drug solubility, and on the other hand, they can aggregate the drug to the target site through active targeting, passive targeting, or physicochemical targeting, thereby reducing toxic and side effects. Nanoformulations have a variety of dosage forms, including nanocapsules, nanospheres, nanoemulsions, liposomes, micelles, etc., and can be flexibly designed according to the characteristics of the drug. For example, polymer nanoparticles or micelles are used as drug carriers, and their advantages such as targeting and stimulus-responsive release have effectively improved the therapeutic effect of breast cancer. Polymeric micelles (PMs) are a new type of nano drug delivery system that has developed rapidly in recent years. The hydrophobic core of polymer micelles can encapsulate hydrophobic anti-tumor drugs, increasing the solubility of anti-tumor drugs. Different materials that make up polymer micelles can make them have the characteristics of tumor cell targeting and tumor environment response, improve drug bioavailability, prolong drug blood circulation time, and be more conducive to the release of drugs in tumor tissues. Wang Meifang, research on the use of diselenide cross-linked dual redox responsive nanohydrogels for tumor treatment, Jilin University, June 2020, disclosed a diselenide cross-linked hyaluronic acid nanohydrogel, loaded with oxaliplatin, and conducted in vitro anti-tumor studies. It is a nanohydrogel prepared by hyaluronic acid-diselenide-hyaluronic acid cross-linking, with a very low drug loading (8.42±0.43)%, and is used to treat colon cancer. The choice of micelle material and the properties of the encapsulated drug have an impact on the drug loading, encapsulation efficiency, and efficacy of the prepared micelles. Summary of the Invention

[0005] The technical solution of the present invention is to provide a rhein-diselenide-hyaluronic acid (RSeHA) conjugate. Another technical solution of the present invention is to provide the conjugate encapsulating tripterygium wilfordii to prepare micelles.

[0006] The present invention provides a rhein-diselenide-hyaluronic acid (RSeHA) conjugate, which uses selenocystamine (diselenide, Se-Se) as a linker arm to graft a small molecule of rhein (R) onto a low molecular weight hyaluronic acid (HA) to synthesize a redox dual-responsive conjugate (RSeHA conjugate) that can self-assemble into micelles in water;

[0007] The structural formula of the conjugate is:

[0008]

[0009] The molar ratio of rhein (R), Se-Se (calculated as selenocystamine dihydrochloride), and hyaluronic acid (HA) is 1-1.5:1-2:1.

[0010] More preferably, the molar feed ratio of rhein (R), Se-Se (calculated as selenocystamine dihydrochloride), and hyaluronic acid (HA) is 1:1.2:1.

[0011] Among them, the RSeHA micelle particle size is (193.48±21.61) nm, the PDI is 0.19±0.04, and the potential is (-31.24±2.30) mV; the R and Se-Se substitution degrees are (9.59±1.48)% and (9.09±1.08)%, respectively.

[0012] The present invention provides a method for preparing a rhein-diselenide-hyaluronic acid (RSeHA) conjugate, and the synthesis route is:

[0013]

[0014] It includes the following steps:

[0015] a. Preparation of R-Se-Se: Dissolve R in 1% NaHCO3, add EDC·HCl and react for 20 min, then add NHS and react for 10 min; dissolve selenocystamine dihydrochloride in DMSO completely, add to the R solution, and stir to obtain the product;

[0016] b. Preparation of HA-NHS: Dissolve HA in ultrapure water, stir on a constant temperature magnetic stirrer, and heat at 60°C until completely dissolved. Add EDC·HCl and react for 20 minutes, then add NHS and react for 10 minutes to obtain HA-NHS.

[0017] The R-Se-Se prepared in step a was added dropwise to HA-NHS and reacted for 24 h to obtain a product mixture;

[0018] c. Add 95% ethanol to the product mixture and allow to settle for 3 hours. Filter with suction, and re-dissolve the residue in distilled water. Use ultrasonic dispersion with a probe until the solution is clear. Centrifuge at 3900 rpm for 15 minutes, place in a dialysis bag (MWCO = 3500), and dialyze in distilled water for 3 days.

[0019] The dialyzed product solution was probed again for 15 min, centrifuged at 3900 r / min for 15 min, filtered through a 0.8 μm filter membrane, pre-frozen for 24 h, and freeze-dried for 24 h to obtain the product RSeHA conjugate.

[0020] The invention provides a micelle, which is prepared by encapsulating a hydrophobic compound in the rhein-diselenide-hyaluronic acid (RSeHA) conjugate.

[0021] Wherein, the hydrophobic compound is Celacotene (Cela); the mass ratio of Celacotene (Cela) to rhein-diselenide-hyaluronic acid (RSeHA) conjugate is 1:0.8-1.6; and the concentration of the micellar carrier (RSeHA conjugate) is 3-7 mg / mL.

[0022] Among them, the mass ratio of the tripterygium wilfordii (Cela) to the rhein-diselenide-hyaluronic acid (RSeHA) conjugate is 1:1.4; the concentration of the micelle carrier (RSeHA conjugate) is 4 mg / mL; the particle size of the Cela / RSeHA micelles is (159.61±8.33) nm, the PDI is 0.07±0.02; the drug loading is (36.57±1.66)%, and the encapsulation efficiency is (68.39±0.75)%.

[0023] The present invention provides a method for preparing the micelle, which comprises the following steps:

[0024] a. Weigh the RSeHA conjugate, add distilled water, and ultrasonicate in an ice-water bath until uniformly dispersed;

[0025] b. Weigh Cela powder and dissolve it in DMSO. Then add it dropwise to the vigorously stirred RSeHA conjugate solution and continue stirring for 30 minutes. Then, disperse it by ultrasonic probe in an ice-water bath and dialyze it in distilled water in a dialysis bag for 24 hours. Then, ultrasonic probe in an ice-water bath for 20 minutes, centrifuge at 3500 rpm for 10 minutes, remove the supernatant, filter it through a 0.8 μm filter membrane, and lyophilize the filtrate to obtain Cela / RSeHA micelle lyophilized powder.

[0026] The present invention provides use of the micelle in preparing medicine for treating breast cancer.

[0027] The present invention synthesizes a polymer carrier material (with hyaluronic acid as the skeleton material) to physically encapsulate tripterine, which has an anti-breast cancer effect. The encapsulation and delivery of the polymer carrier material increases the water solubility of tripterine and its toxicity to breast cancer cells; the material is used for injection; it is slowly released in the blood and quickly releases tripterine in the tumor microenvironment, thereby increasing the anti-breast cancer efficacy. Due to the targeted delivery effect and redox-responsive drug release characteristics of the carrier material, the toxic side effects on normal tissues are reduced.

[0028] This invention encapsulates the anti-tumor drug Cela in redox-responsive tumor-targeting polymer micelles. The resulting micelles are small, uniform, and stable. This improves Cela's bioavailability, prolongs its in vivo retention time, enhances its tumor targeting, and reduces toxic side effects on non-targeted organs. High levels of reactive oxygen species (ROS) and glutathione (GSH) within tumor cells rupture diselenide bonds in the micelles, accelerating drug release and further enhancing anti-tumor efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Synthesis route of RSeHA conjugates;

[0030] Figure 2 Full wavelength scanning spectrum;

[0031] Figure 3 RSeHA micelle size distribution;

[0032] Figure 4 FT-IR spectra (Note: a: R; b: Se-Se; c: HA; d: RSeHA conjugate);

[0033] Figure 5 1 H-NMR spectra (Note: a: RSeHA conjugate; b: HA; c: Se-Se; d: R);

[0034] Figure 6 DSC graph (Note: a: R; b: Se-Se; c: HA; d: physical mixture of R+Se-Se+HA; e: RSeHA conjugate);

[0035] Figure 7 TEM images of RSeHA micelles (Note: a: RSeHA micelles; b: RSeHA conjugate treated with GSH+H2O2);

[0036] Figure 8 I 333 / I 338 The relationship curve with the logarithm of RSeHA conjugate concentration;

[0037] Figure 9 HPLC profile of Cela (a: Cela reference solution; b: Cela / RSeHA micelle sample; c: blank solvent (acetonitrile));

[0038] Figure 10 Cela / RSeHA micelle size and distribution;

[0039] Figure 11TEM images of Cela / RSeHA micelles (Note: a: Cela / RSeHA micelles; b: Cela / RSeHA micelles after GSH+H2O2 treatment);

[0040] Figure 12 DSC characterization spectra (Note: a: RSeHA conjugate; b: Cela; c: Cela+RSeHA physical mixture; d: Cela / RSeHA micelles);

[0041] Figure 13 Tumor volume change curve of tumor-bearing mice (n=8) (Note: * P < 0.05; ** P < 0.01; *** P < 0.001);

[0042] Figure 14 Figure 3 Treatment effects of tumor-bearing mice (n=8);

[0043] Figure 15 Tumor weight and tumor inhibition rate of tumor-bearing mice (n=8) (Note: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001);

[0044] Figure 16 Figure 3 Body weight changes of tumor-bearing mice during treatment (n=8);

[0045] Figure 17 Figure 3. Effect of organ index on tumor-bearing mice (n=8);

[0046] Figure 18 Sections of organs and tumor tissues of tumor-bearing mice (×400). DETAILED DESCRIPTION

[0047] Example 1 Synthesis and Characterization of Rhein-Diselenide-Hyaluronic Acid (RSeHA) Conjugate

[0048] The redox-sensitive material selenocystamine (diselenium, Se-Se) was used as a linker to graft the small molecule rhein (R) onto hyaluronic acid (HA) to synthesize the amphiphilic RSeHA conjugate. The results were analyzed by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy ( 1 The structure of the RSeHA conjugate was characterized by H-NMR and differential scanning calorimetry (DSC). The critical micelle concentration (CMC) for the self-assembly of the RSeHA conjugate into micelles was determined using pyrene as a probe. The size and morphology of the RSeHA micelles were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM).

[0049] 1 Experimental Materials

[0050] 1.1 Experimental Instruments

[0051]

[0052] 1.2 Drugs and Reagents

[0053]

[0054]

[0055] 2 Experimental methods

[0056] 2.1 Synthesis of RSeHA Conjugates

[0057] RSeHA conjugates were synthesized in three steps:

[0058] (1) R was dissolved in 2 mL of 1% NaHCO3, and 1.5 mmol of EDC·HCl was added and reacted for 20 min. Then, 1.5 mmol of NHS was added and reacted for 10 min. Selenocysteine ​​dihydrochloride was completely dissolved in 2 mL of DMSO and added to the R solution. The mixture was stirred for 24 h to obtain R-Se-Se.

[0059] (2) HA was dissolved in 16 mL of ultrapure water, stirred on a constant temperature heating magnetic stirrer, and heated at 60°C until completely dissolved. 1.5 mmol of EDC·HCl was added and reacted for 20 min, followed by 1.5 mmol of NHS and reacted for 10 min to obtain HA-NHS. R-Se-Se was added dropwise to HA-NHS and reacted for 24 h to obtain a product mixture.

[0060] (3) The product mixture was added with 95% ethanol and precipitated for 3 hours. The mixture was then filtered and the residue was re-dissolved in distilled water. The solution was dispersed by ultrasonic probe until the solution was clear. The solution was then centrifuged at 3900 r / min for 15 minutes. The solution was placed in a dialysis bag (MWCO = 3500) and dialyzed in distilled water for 3 days. The dialyzed product solution was again ultrasonically probed for 15 minutes, centrifuged at 3900 r / min for 15 minutes, filtered through a 0.8 μm filter membrane, pre-frozen for 24 hours, and freeze-dried for 24 hours to obtain the RSeHA conjugate.

[0061] 2.2 Determination of Se-Se Substitution Degree in RSeHA Conjugates

[0062] The degree of substitution of Se-Se on RSeHA conjugate was determined by UV spectrophotometry.

[0063] A Se-Se methanol solution of a certain concentration was prepared, and UV-visible light wavelength scanning was performed in the range of 200-600 nm to determine that the Se-Se UV detection wavelength was 303 nm.

[0064] Accurately weigh 2 mg of Se-Se into a 5 mL volumetric flask, dissolve it in methanol and dilute to the mark to prepare a 400 μg / mL Se-Se stock solution. Using methanol as the solvent, prepare Se-Se solutions with mass concentrations of 80, 70, 60, 50, 40, and 30 μg / mL, respectively. Measure the absorbance (A) at a wavelength of 303 nm with methanol as the blank solution. The concentration of Se-Se (C Se-Se ) as the horizontal axis and the absorbance value (A) as the vertical axis, draw the standard curve of Se-Se, perform linear regression, and obtain the regression equation.

[0065] Weigh 4 mg of the RSeHA conjugate, dissolve it in water, and then add 0.8 mL to a 10 mL volumetric flask. Dilute to the mark with methanol to obtain the RSeHA conjugate solution, and measure its absorbance by UV spectrophotometry. Substitute the absorbance value (A) at 303 nm into the Se-Se standard curve to obtain the Se-Se concentration. Calculate the mass of Se-Se in the RSeHA micelles. Calculate the molar substitution (DS, mol%) of Se-Se on the RSeHA conjugate according to formula 1-1:

[0066]

[0067] In the formula, m Se-Se is the mass of Se-Se grafted on RSeHA conjugate (g); M Se-Se is the molecular weight of Se-Se; m RSeHA is the mass of RSeHA conjugate (g); m RSe is the mass of R-Se-Se grafted on RSeHA conjugate (g); M HA is the average molecular weight of HA (g).

[0068] 2.3 Determination of the degree of substitution of R in RSeHA micelles

[0069] The degree of substitution of R in the RSeHA conjugate was determined by UV spectrophotometry.

[0070] Prepare a certain concentration of R methanol solution, perform UV-visible light wavelength scanning in the range of 200-600nm, and determine that the R UV detection wavelength is 439nm.

[0071] Accurately weigh 2 mg of R and place it in a 10 mL volumetric flask. Dissolve it in methanol and dilute to the mark to prepare a 200 μg / mL R stock solution. Using methanol as the solvent, prepare R solutions with mass concentrations of 23, 20, 17, 14, 11, and 8 μg / mL, respectively.

[0072] The absorbance was measured at 439 nm with methanol as blank solution. R ) as the horizontal axis and the absorbance value (A) as the vertical axis, draw the standard curve of R, perform linear regression, and obtain the regression equation.

[0073] Weigh 4 mg of the RSeHA conjugate, dissolve it in water, and then add 0.8 mL to a 10 mL volumetric flask. Dilute to the mark with methanol to obtain the RSeHA conjugate solution, and measure its absorbance by UV spectrophotometry. Substitute the absorbance value (A) at 439 nm into the R standard curve to obtain the concentration of R. Calculate the mass of R in the carrier micelles, and calculate the molar substitution (DS, mol%) of R on the RSeHA conjugate according to formula 1-2:

[0074]

[0075] In the formula, m R is the mass of R grafted on the RSeHA conjugate (g); M R is the molecular weight of R; m RSeHA is the mass of RSeHA conjugate (g); m RSe is the mass of R-Se-Se grafted on RSeHA conjugate (g); M HA is the average molecular weight of HA (g).

[0076] 2.4 Particle size distribution and potential determination

[0077] Take an appropriate amount of RSeHA conjugate and dilute it with water to obtain a 1 mg / mL RSeHA micelle solution. The particle size, PDI and Zeta potential of the RSeHA micelles were determined by DLS.

[0078] 2.5 Single factor investigation of synthesis process

[0079] The molar substitution, particle size and PDI of R and Se-Se were used as indicators to investigate the effect of the feed amount of R and Se-Se on the synthesis.

[0080] 2.5.1 Investigating the effect of Se-Se feed amount on synthesis

[0081] The molar feed amounts of HA and R and the reaction time were fixed at 24 h, and the molar feed amount of Se-Se was changed. Experiments were conducted according to R:Se-Se:HA=1:(1, 1.2, 1.5 and 2):1 to investigate the effect of the feed amount of Se-Se on the synthesis of RSeHA conjugates.

[0082] 2.5.2 Investigating the effect of R feed amount on synthesis

[0083] The molar feed amounts of HA and Se-Se and the reaction time were fixed at 24 h, and the molar feed amount of R was changed to make R:Se-Se:HA = (1, 1.2 and 1.5):1.2:1. The effect of the feed amount of R on the synthesis of RSeHA conjugates was investigated.

[0084] Characterization of 2.5RSeHA Conjugates

[0085] 2.5.1 FT-IR characterization

[0086] Take appropriate amounts of HA, R, Se-Se and RSeHA conjugates, add appropriate amounts of KBr respectively, grind and mix under infrared light, press into tablets, perform FT-IR detection, and record FT-IR spectra.

[0087] 2.5.2 1 H-NMR characterization

[0088] Take appropriate amount of HA, R, Se-Se and RSeHA conjugates and dissolve them in appropriate deuterated solvents. Detect them by NMR spectroscopy at 500 MHz and 20 °C. Record the results. 1 H-NMR spectrum.

[0089] 2.5.3DSC Characterization

[0090] The thermodynamic characteristics of RSeHA conjugates were determined by differential calorimetry. -1 The heating rate is from 40℃ to 500℃, and the scanning curve is recorded.

[0091] 2.6 CMC value determination

[0092] The CMC value of RSeHA micelles was determined by pyrene fluorescence probe method. The specific method is as follows: acetone was used as solvent to prepare a 6×10 -6 mol / L pyrene solution. Take 1 mL of each and add it to a 10 mL brown stoppered test tube, and place it in a vacuum desiccator to evaporate the acetone. Weigh an appropriate amount of RSeHA conjugate and add an appropriate amount of ultrapure water to prepare RSeHA micelle solutions with final concentrations of 2000, 1000, 500, 250, 100, 50, 20, 10, 5, 1, 0.5 and 0.2 μg / mL, respectively. Take 1 mL of RSeHA micelle solution of each concentration and add it to the test tube containing pyrene (so that the final concentration of pyrene is 6×10 -6 mol / L), the above solution was placed in a water bath in the dark and ultrasonic for 30 min, and then placed in a 37°C water bath for incubation overnight. The excitation spectrum of pyrene in the RSeHA micelle solution was measured by a microplate reader, and the emission wavelength was λ em =390nm, and record the fluorescence values ​​of each solution at 338nm and 333nm, with I 333 / I 338The logarithmic concentration of RSeHA micelles was plotted and the CMC value was calculated.

[0093] 2.7 Morphological studies

[0094] TEM was used to observe the morphology of RSeHA micelles. TEM sample solution preparation: An appropriate amount of RSeHA polymer micelle solution was dripped onto a copper grid for 2-3 minutes. The solution was then negatively stained with 2.0% phosphotungstic acid and dried at room temperature. The micelle morphology was then observed by TEM. Separate RSeHA micelle solution was treated with a solution containing 0.1 mmol / L H₂O₂ and 2 mmol / L GSH for 6 hours before TEM observation using the same method.

[0095] 3 Results

[0096] 3.1 Synthesis process of RSeHA conjugate

[0097] like Figure 1 As shown, the redox-sensitive material diselenide is used as a connecting arm to graft the hydrophobic R onto the low molecular weight HA to synthesize the RSeHA conjugate. Synthesis route ( Figure 1 ) as shown.

[0098] 3.2 Establishment of the determination method of R and Se-Se substitution degree

[0099] 3.2.1 Determination of R and Se-Se Measurement Wavelengths

[0100] The UV spectra of R and Se-Se are as follows Figure 2 As shown, the R reference solution has a maximum absorption at 230 nm and a certain absorption at 250 nm. Se-Se has a maximum absorption at 211 nm. In order to eliminate the interference of R on the determination of Se-Se substitution degree as much as possible, 439 nm is selected as the determination wavelength of R; 303 nm is selected as the determination wavelength of Se-Se.

[0101] 3.2.2R standard curve and linear range

[0102] Analyze under the conditions described in "2.4" and plot a standard curve by linearly regressing the UV absorbance values ​​of different R concentrations against their concentrations. The linear regression equation for R at 430 nm is A = -0.0819C + 0.7249, r = 0.9996. Good linearity is observed within the concentration range of 8 to 23 μg / mL.

[0103] 3.2.3R precision and recovery

[0104] The intra-day RSDs for low, medium, and high RSDs at 430 nm were 1.35%, 1.43%, and 1.76% for RSDs, respectively. The inter-day RSDs for RSDs were 1.47%, 1.68%, and 1.95%, respectively. All RSDs were less than 2%, indicating good precision. The recoveries were 100.15%, 99.85%, and 98.94%, respectively, with RSDs less than 2%, indicating good recovery.

[0105] 3.2.4Se-Se standard curve and linear range

[0106] Analyze under the conditions described in "2.4" and perform a linear regression of the UV absorbance values ​​of different Se-Se concentrations against their concentrations to create a standard curve. The linear regression equation for Se-Se at 303 nm is A = -0.0905C + 0.7896, r = 0.9996, and the linear range is 80 to 300 μg / mL, indicating a good linear relationship.

[0107] 3.2.5Se-Se precision and recovery

[0108] At 303 nm, the RSDs for intra-day precision were 1.01%, 1.22%, and 0.98% for low, medium, and high values, respectively; the RSDs for inter-day precision were 1.07%, 1.38%, and 1.25%; all RSDs were less than 2%, indicating good precision. The recoveries were 101.05%, 98.97%, and 99.84%, respectively, with RSDs less than 2%, indicating good recoveries.

[0109] 3.3 Results of the synthesis process of RSeHA conjugates

[0110] The particle size and the molar substitution of PDI, R and Se-Se were used as the evaluation indicators for the synthesis process of RSeHA conjugates. The test results of RSeHA conjugates synthesized with different feed ratios are shown in Table 1.

[0111] When the molar loading of R and HA was fixed at 1 mmol and the molar loading of Se-Se was varied (1-2) mmol (calculated as selenocystamine dihydrochloride), the R and Se-Se substitution values ​​of the synthesized products remained similar across batches, with no discernible pattern. However, the particle size showed a trend of first decreasing and then increasing. When the ratio of R:Se-Se:HA = 1:1.2:1, the micelle size was the smallest and most uniformly distributed, indicating an optimal degree of substitution.

[0112] On the basis of Se-Se feeding amount of 1.2mmol, the feeding amount of HA was fixed and the feeding amount of R was changed (1~1.5:1.2)mmol. With the increase of R feeding amount, the substitution degree of R with Se-Se decreased and the particle size increased.

[0113] Another experiment was conducted with the ratio of R:Se-Se:HA set to 1.2:1:1. The R and Se-Se of the obtained product were relatively low, and the particle size was relatively large.

[0114] Taking into account the substitution degree of R and Se-Se, particle size, PDI and other factors, the optimal synthesis feed ratio was determined to be R:Se-Se:HA=1:1.2:1.

[0115] Table 1 Effect of different molar feed ratios on the synthesis product ( n=3)

[0116]

[0117] The final synthesis process of RSeHA conjugate was determined as follows:

[0118] (1) Weigh 1 mmol of R, dissolve it in 2 mL of 1% NaHCO3, add 1.5 mmol of EDC·HCl and react for 20 min, then add 1.5 mmol of NHS and react for 10 min; weigh 1.2 mmol of selenocystamine dihydrochloride, completely dissolve it in 2 mL of DMSO, add it to the R solution, and stir for 24 h to obtain R-Se-Se.

[0119] (2) Weigh 1 mmol of HA and dissolve it completely in 16 mL of ultrapure water. Add 1.5 mmol of EDC·HCl and react for 20 min. Then add 1.5 mmol of NHS and react for 10 min to obtain HA-NHS. Then, add R-Se-Se dropwise to HA-NHS and react for 24 h to obtain a product mixture.

[0120] (3) The product mixture was added with 95% ethanol and precipitated for 3 hours. The mixture was then filtered and the residue was re-dissolved in distilled water. The solution was dispersed by ultrasonic probe until the solution was clear. The solution was then centrifuged at 3900 r / min for 15 minutes. The solution was placed in a dialysis bag (MWCO = 3500) and dialyzed in distilled water for 3 days. The dialyzed product solution was again ultrasonically probed for 15 minutes, centrifuged at 3900 r / min for 15 minutes, filtered through a 0.8 μm filter membrane, pre-frozen for 24 hours, and freeze-dried for 24 hours to obtain the RSeHA conjugate.

[0121] The RSeHA conjugate synthesized by this process self-assembled into RSeHA micelles in water with a particle size of (193.48±21.61) nm (see Figure 3 ), and the batch-to-batch differences were small and the distribution was relatively uniform; the R molar substitution was (9.59±1.48)%, the Se-Se molar substitution was (9.09±1.08)%, and the Zeta potential was (-31.24±2.30) mV.

[0122] 3.4 Structural Characterization of RSeHA Conjugates

[0123] 3.4.1 FT-IR characterization analysis

[0124] The FT-IR spectra of RSeHA conjugate and each synthetic raw material are shown in Figure 4 As shown in the figure, a is the FT-IR spectrum of R, 1693cm -1 The peak at 784 cm is the vibration peak of -COOH; b is the FT-IR spectrum of Se-Se; -1 The Se-Se characteristic peak appears at 552 cm -1 The C-Se stretching vibration peak appears at 3385 cm; c is the FT-IR spectrum of HA, -1 The -OH absorption peak is at 1614 cm -1 , 1407cm -1 -COOH antisymmetric and symmetric stretching vibration peaks appear at 1151, 1078, 1046 and 946 cm -1 The characteristic absorption peaks of sugar are shown in Figure d. The FT-IR spectrum of RSeHA is 1646 and 1628 cm -1 An amide band appears at 2930 cm -1 The absorption peak at is the CH stretching vibration of the attached -CH2- saturated hydrocarbon, indicating that the diselenide bond has been connected and an amidation reaction has occurred.

[0125] 3.4.2 1 H-NMR characterization analysis

[0126] R, Se-Se, HA, RSeHA conjugate 1 The H-NMR spectrum is shown in Figure 5. 1 H-NMR, one of the R characteristic peaks, the proton peaks of -CH and -OH on the benzene ring, appeared at δ8.38, δ7.88, and δ7.86 ppm. b is Se-Se 1 H-NMR, -NH appears at δ8.20ppm, and the methylene adjacent to the diselenide bond at δ3.15ppm; c is the 1 H-NMR, the peak at δ1.93ppm corresponds to the H on the acetylamino-OCCH3 group, and the peak at δ3-4ppm corresponds to the H on the HA sugar ring; d is the peak of RSeHA conjugate 1 H-NMR, the presence of the acetylamino-OCCH3 group at δ1.93 ppm confirmed the retention of the HA skeleton in the product, and the presence of the -CONH- group at δ3-4 ppm proved that HA and R were successfully grafted onto Se-Se to synthesize the RSeHA conjugate.

[0127] 3.4.3DSC Characterization Analysis

[0128] Differential Scanning Calorimetry (DSC) is a thermal analysis method. The curve obtained by scanning is called DSC curve, which can be used to determine various thermodynamic and kinetic parameters, such as reaction heat, transition heat, phase diagram, etc. The DSC curves of each sample are shown in Figure 6 As shown in the figure, HA has an endothermic peak at 239.0°C; Se-Se has an exothermic peak and an endothermic peak at 182.8 and 268.6°C, respectively; R has an obvious exothermic peak at 329.7°C; and the RSeHA conjugate has an obvious exothermic peak at 208.4°C, which are inconsistent with the endothermic and exothermic peak positions of R, Se-Se, HA and the physical mixture, indicating that the RSeHA conjugate is a new compound.

[0129] 3.5 Morphological studies

[0130] The morphology of RSeHA micelles was observed by TEM. Figure 7 The vast majority of RSeHA micelles were spherical, with a particle size between 200 and 220 nm. Their size distribution was relatively uniform, with no aggregation, and the particle size was generally consistent with the DLS analysis results. The spherical morphology of the RSeHA micelles was essentially eliminated after treatment with H₂O₂ and GSH, indicating that the RSeHA micelles had disaggregated in the redox environment, thus demonstrating the redox responsiveness of the RSeHA micelles.

[0131] 3.6 CMC value determination results

[0132] With LogC as the horizontal axis, the fluorescence intensity ratio of pyrene at 333 nm and 338 nm (I 333 / I 338 ) is the vertical axis, and the result is as follows Figure 8 As shown in the figure, the concentration at the inflection point of the curve is the CMC value of the RSeHA conjugate to form micelles. The results show that at low concentrations, the RSeHA conjugate has not yet formed micelles in water, so I 333 and I 338 The fluorescence ratio of the polymer micelles is small and almost constant. When the concentration of the polymer micelles increases to the CMC value, the RSeHA conjugate begins to self-assemble to form micelles. At this time, the pyrene molecules enter the hydrophobic part of the micelle core, so I 333 / I 338 The ratio increases sharply. From the LogC concentration at the inflection point of the curve in the figure, it can be seen that the CMC value of the RSeHA conjugate to form micelles is 3.75μg / mL.

[0133] 4 Discussions

[0134] During the carrier synthesis process, other synthesis techniques were also attempted. For example, HA was weighed and completely dissolved in 16 mL of ultrapure water. 1.5 mmol of EDC·HCl was added and reacted for 20 minutes. Then, 1.5 mmol of NHS was added and reacted for 10 minutes to obtain HA-NHS. 1.2 mmol of Se-Se was weighed and completely dissolved in 2 mL of DMSO. This solution was then added to the HA-NHS solution to obtain an HA-Se-Se solution. This solution was then added dropwise to the fully dissolved R and reacted for 24 hours to obtain a product mixture. The product mixture was then added to 95% ethanol and allowed to precipitate for 3 hours. The mixture was then filtered and the residue was reconstituted in distilled water. Ultrasonic dispersion was performed using a probe until the solution was clear. The solution was then centrifuged at 3900 rpm for 15 minutes, placed in a dialysis bag (MWCO = 3500), and dialyzed against distilled water for 3 days. The product solution after dialysis was probed again for 15 minutes, centrifuged at 3900r / min for 15 minutes, passed through a 0.8μm filter membrane, pre-frozen for 24 hours, and freeze-dried for 24 hours to obtain the product RSeHA conjugate. The particle size was measured using a particle size analyzer and it was found that compared with the conjugate synthesized by the aforementioned optimized synthesis process, the particle size of the micelles formed by self-assembly in water by the conjugate synthesized by this process was significantly larger, and the degree of substitution of R and Se-Se was extremely low. Therefore, this study chose to first dissolve R, add Se-Se to obtain R-Se-Se, and then add HA to react overnight to synthesize the RSeHA conjugate.

[0135] During the synthesis process investigation, it was found that as the amount of R added increased, ultrasonic dispersion of the carrier material became increasingly difficult and required increasingly longer times. This is likely due to the hydrophobic nature of R, and excessive amounts of R reduced the water solubility of the RSeHA conjugate. Only an appropriate amount of R added can achieve an appropriate degree of substitution (DOR) on the RSeHA conjugate, forming a micellar structure with good water solubility.

[0136] Because hyaluronic acid is a polyelectrolyte, its rheological properties in aqueous solution are also affected by ionic strength, pH, and temperature: as these factors increase, the viscosity of hyaluronic acid decreases significantly and the interaction between polymer chains weakens. Therefore, the carrier material was synthesized using near-neutral distilled water dissolved between 55-66°C.

[0137] During the synthesis process, since Se-Se has amino groups at both ends, they have a certain probability of reacting with the carboxyl group of R. Further reactions only require an intermediate product with one amino group attached to R and the other retaining an amino group. Byproducts with both amino groups attached to R are removed by dialysis. The organic solvent used in the reaction is also removed during dialysis. Unreacted R, due to its poor solubility in water, can be removed by centrifugation and membrane filtration.

[0138] 5. Summary

[0139] Using Se-Se as the connecting arm, R was grafted onto HA to synthesize RSeHA conjugate. The feed ratio of each reactant was investigated by single factor and the synthesis process was optimized. The reaction conditions of this process are mild and easy to operate.

[0140] The RSeHA conjugate had good solubility, with a particle size of (193.48±21.61) nm, a PDI of 0.19±0.04, and a potential of (-31.24±2.30) mV. The molar substitution of R and Se-Se was determined by UV spectrophotometry to be (9.59±1.48)% and (9.09±1.08)%, respectively. FT-IR, 1 H-NMR, DSC and other characterization methods confirmed that the RSeHA conjugate was successfully synthesized; its CMC value was 3.75 μg·mL -1 ; The RSeHA micelles observed under TEM are nearly spherical in shape, with small and uniform particle size; and have redox responsiveness.

[0141] Example 2 Preparation and Characterization of Cela / RSeHA Conjugates

[0142] Celastrol (Cela) has significant anti-breast cancer effects, but its clinical application is limited by its poor water solubility, low bioavailability, and significant toxic side effects. Polymer micelles can encapsulate poorly soluble drugs with their unique shell-core structure, increasing their water solubility and bioavailability. Through the special design of their carrier materials, they can actively target and release drugs, thereby increasing drug efficacy and reducing toxic side effects. Using RSeHA conjugates as carrier materials, Cela-loaded RSeHA micelles (Cela / RSeHA micelles) were prepared in the hope of achieving the effects of solubilizing, enhancing efficacy, and reducing toxicity of Cela.

[0143] Dialysis was used as the drug loading method. The drug loading process was optimized by investigating the effects of drug-to-carrier ratio and carrier concentration on Cela / RSeHA micelles. The Cela / RSeHA micelles were characterized by DSC, TEM and other means.

[0144] 1 Materials and Instruments

[0145] 1.1 Experimental Instruments

[0146]

[0147] 1.2 Experimental Materials

[0148]

[0149] 2 Methods

[0150] 2.1 Establishment of Cela assay method

[0151] 2.1.1 Chromatographic conditions

[0152] Chromatographic column: Inertsil ODS-SP (4.6×150 mm, 5 μm); mobile phase: acetonitrile-formic acid solution (95%:5%), flow rate: 1 mL / min, column temperature: 30°C, detection wavelength: 425 nm, injection volume: 20 μL.

[0153] 2.1.2 Linear Relationship

[0154] Preparation of Cela test solution: Accurately weigh 3 mg of Cela into a 10 mL volumetric flask, dissolve to the mark with methanol, and shake to obtain a stock solution with a concentration of 300 μg / mL. Dilute this stock solution with methanol to prepare Cela solutions with concentrations of 70, 60, 45, 30, 15, and 1.5 μg / mL. Measure according to the chromatographic conditions in "2.1.1." Record the peak area corresponding to each Cela concentration. Plot a standard curve using peak area (A) as the ordinate and concentration (C, μg / mL) as the abscissa, and calculate the regression equation.

[0155] 2.1.3 Recovery rate experiment

[0156] Accurately measure three replicates of Cela solutions at high, medium, and low concentrations: 70, 30, and 1.5 μg / mL. Measure the solution according to the chromatographic conditions in “2.1.1.” Record the peak area corresponding to each Cela concentration and calculate the recovery at high, medium, and low concentrations.

[0157] 2.1.4 Precision experiment

[0158] Accurately measure Cela solutions at high, medium, and low concentrations of 70, 30, and 1.5 μg / mL. Measure the solution according to the chromatographic conditions in "2.1.1." Inject the solution six times on the same day, record the peak area, and calculate the intra-day precision. Inject the solution three days later under the same conditions, record the peak area, and calculate the inter-day precision.

[0159] 2.2 Determination of encapsulation efficiency and drug loading

[0160] Place an appropriate amount of Cela / RSeHA micelle solution in a volumetric flask. Add methanol to disrupt the micelle structure and dissolve Cela. Dilute to volume and measure using the chromatographic conditions described in "2.1.1." Record the peak area and calculate the Cela content. Calculate the drug loading (DL) and encapsulation efficiency (EE) using formula (2-1) and formula (2-2) as follows.

[0161]

[0162] 2.3 Investigation of drug delivery technology by dialysis

[0163] Cela / RSeHA micelles were prepared using dialysis. The following steps were performed: Weigh the RSeHA conjugate, add an appropriate amount of distilled water, and sonicate in an ice-water bath until uniformly dispersed. A 10mg volume of Cela powder was dissolved in DMSO and then added dropwise to the vigorously stirred RSeHA conjugate solution, stirring continuously for 30 minutes. The mixture was then dispersed by sonication in an ice-water bath and dialyzed in distilled water in a dialysis bag for 24 hours. The mixture was then sonicated in an ice-water bath for another 20 minutes, centrifuged at 3500 rpm for 10 minutes, and the supernatant was filtered through a 0.8μm filter membrane. The filtrate was lyophilized to obtain the Cela / RSeHA micelle lyophilized powder.

[0164] An appropriate amount of Cela / RSeHA micelles was taken and dissolved in distilled water to prepare a Cela / RSeHA micelle solution with a final concentration of 0.5 mg / mL. The particle size and distribution of Cela / RSeHA micelles were measured by DLS.

[0165] Taking drug loading, encapsulation efficiency, particle size and PDI as indicators, the effects of drug loading ratio and carrier concentration on the encapsulation of Cela in RSeHA micelles were investigated.

[0166] 2.3.1 Drug-to-carrier ratio (drug-to-carrier ratio)

[0167] The drug Cela concentration was fixed at 20 mg / mL, and DMSO was used as the Cela solvent. The effects of Cela to different carrier mass ratios (1:0.8, 1:1, 1:1.2, 1:1.4 and 1:1.6) on the drug loading capacity of RSeHA micelles were investigated.

[0168] 2.3.2 Carrier concentration

[0169] The drug-to-carrier ratio was fixed at 1:1.4, DMSO was used as Cela solvent, and the Cela concentration was 20 mg / mL. The effect of carrier concentration (3, 4, 5, 6, and 7 mg / mL) on the drug loading capacity of RSeHA micelles was investigated.

[0170] 2.4 TEM characterization

[0171] The Cela / RSeHA conjugate micelle solution prepared by the optimal process and the Cela / RSeHA micelle solution treated with 10 mmol (GSH+H2O2) solution were dropped onto a copper grid covered with a carbon support film, left for 2-3 minutes, and the excess solution was absorbed with filter paper. 2% phosphotungstic acid solution was added for negative staining for 30 seconds, and the mixture was naturally dried and observed under a transmission electron microscope (TEM).

[0172] 2.5DSC characterization

[0173] The thermodynamic characteristics of Cela, RSeHA conjugates, physical mixtures of Cela and RSeHA conjugates, and Cela / RSeHA micelles were determined using differential calorimetry. Under an Ar atmosphere, the samples were heated from 40°C to 500°C at a rate of 10°C / min, and scan curves were recorded.

[0174] 3 Results

[0175] 3.1 Establishment of Cela assay method

[0176] 3.1.1 Standard curve and linear range

[0177] Analyze according to the chromatographic conditions under "2.1.1". The HPLC chromatogram of Cela is shown in Figure 9 As shown in the figure, the solvent acetonitrile and the RSeHA conjugate had no effect on the Cela peak. Linear regression was performed by plotting peak area (A) against concentration (C, μg / mL), yielding the following regression equation: A = 34283C - 2459 (r = 0.9995). This indicates a good linear relationship for Cela concentrations within the range of 1.5 to 75 μg / mL.

[0178] 3.1.2 Recovery and precision

[0179] The recoveries at high, medium, and low concentrations were 109.8±0.06%, 101.99±0.01%, and 101.65±0.03%, respectively. The intra-day RSDs for precision were 0.05%, 0.01%, and 0.02%, respectively; and the inter-day RSDs for precision were 0.02%, 0.03%, and 0.06%, respectively. Both the recovery and precision were good and met the requirements of the assay methodology.

[0180] 3.2 Results of drug delivery process investigation

[0181] 3.2.1 Drug loading ratio

[0182] Table 2 shows the effect of the Cela / RSeHA conjugate mass ratio on the drug loading capacity of RSeHA micelles. Within the experimentally determined drug loading ratio, both drug loading and encapsulation efficiency began to decline when the ratio increased to 1:1.6, indicating that the amount of RSeHA conjugate was excessive and the hydrophobic core of the RSeHA micelles was not fully utilized. A drug loading ratio of 1:1.4 achieved the highest encapsulation efficiency and good drug loading. At a drug loading ratio of 1:1.2, although the Cela / RSeHA micelles had the highest drug loading capacity, their particle size was relatively large. Taking all factors into consideration, 1:1.4 was selected as the optimal drug loading ratio.

[0183] Table 2 Effects of different drug loading ratios on drug loading capacity of RSeHA micelles ( n=3)

[0184]

[0185]

[0186] 3.2.2 Carrier concentration

[0187] Table 3 shows the effect of carrier concentration on the drug loading capacity of RSeHA micelles. A carrier concentration of 3 mg / mL achieved the highest encapsulation efficiency and drug loading, but the Cela / RSeHA micelle particle size was relatively large. A carrier concentration of 4 mg / mL resulted in the smallest particle size, a good encapsulation efficiency, and a relatively low drug loading among the studied groups. However, a drug loading of over 30% is considered high. Therefore, after comprehensive consideration, 4 mg / mL was selected as the optimal drug loading concentration.

[0188] Table 3 Effects of different carrier concentrations on drug loading capacity of RSeHA micelles ( n=3)

[0189]

[0190] 3.3 Optimal drug delivery technology

[0191] Through single-factor investigation, the optimal drug loading process for dialysis was as follows: 14 mg of RSeHA conjugate was weighed, added to 3.5 mL of distilled water, and ultrasonicated in an ice-water bath until uniformly dispersed. 10 mg of Cela powder was weighed, dissolved in 0.5 mL of DMSO solvent, and then added dropwise to the vigorously stirred RSeHA conjugate solution, stirring continuously for 30 minutes. The solution was then ultrasonically dispersed in an ice-water bath and dialyzed in distilled water in a dialysis bag for 24 hours. The solution was ultrasonically dispersed in an ice-water bath for another 20 minutes, centrifuged at 3500 rpm for 10 minutes, and the supernatant was filtered through a 0.8 μm filter membrane. The filtrate was lyophilized to obtain Cela / RSeHA micelle lyophilized powder.

[0192] The optimal drug loading process was repeated for 3 batches, and the verification results are shown in Table 4. The prepared Cela / RSeHA micelles have small particle size and uniform distribution (see Figure 10 ), large drug loading capacity, high encapsulation efficiency, and high repeatability, indicating that the process is reliable and stable.

[0193] Table 4 Optimal drug loading process verification results ( n=3)

[0194]

[0195] 3.4TEM characterization

[0196] TEM characterization results of Cela / RSeHA micelles are shown in Figure 2. Figure 11As shown, the Cela / RSeHA micelles are spherical with a particle size of approximately 160 nm, consistent with the particle size measured by DLS. After treatment with GSH and H₂O₂, the micelle boundaries clearly become blurred, and the micelles swell and become disrupted. These results indicate that Cela is encapsulated within the RSeHA micelles, forming spherical polymer micelles. The GSH and H₂O₂ conditions cleave the Se-Se bonds within the RSeHA micelle structure, disrupting the micelle structure and promoting the rapid release of Cela.

[0197] 3.5 DSC characterization

[0198] The DSC patterns of RSeHA conjugate, Cela, physical mixture of Cela and RSeHA, and Cela / RSeHA micelles are shown in Figure 12 As shown in the figure, the RSeHA conjugate has an exothermic peak at 208.87°C, and Cela has an exothermic peak and an endothermic peak at 150.76°C and 221.28°C, respectively. The physical mixture shows that the peaks of the Cela and RSeHA conjugates are present; however, in the DSC curve of the Cela / RSeHA micelles, both peaks disappear, indicating that Cela has been encapsulated in the RSeHA micelles. However, there is a weaker endothermic peak at 201.86°C in the Cela / RSeHA micelles, which may be due to the interaction between Cela and the carrier RSeHA, generating a new endothermic peak.

[0199] 4 Discussions

[0200] During the drug loading process, as the feed amount of RSeHA conjugate increased, the drug loading of Cela gradually increased, and the particle size continued to decrease. When the drug-loading ratio was 1:1.4, the particle size was the smallest, and the drug loading and encapsulation efficiency were both good. This may be because as the hydrophobic drug continued to enter the hydrophobic core of the micelle, the interaction between the drug and the hydrophobic core caused the micelle particle size to decrease; when the carrier concentration gradually increased within the experimental concentration range, the drug loading was good, and the drug loading, encapsulation efficiency and particle size did not show a certain pattern, which may be due to factors such as the uncertainty of the interaction between the drug and the carrier. Finally, the carrier concentration of 4 mg / mL was selected. At this time, Cela had a good drug loading, a moderate encapsulation efficiency, and a minimum particle size.

[0201] During the drug loading process, Cela not encapsulated in the RSeHA micelles was removed through centrifugation and membrane filtration to ensure that the resulting Cela / RSeHA micelles were free of free Cela. The organic solvent used for drug loading was removed through dialysis to ensure drug safety in subsequent animal experiments.

[0202] 5. Summary

[0203] The optimal drug loading process of RSeHA micelles was determined by single factor investigation using drug loading capacity, encapsulation efficiency, particle size and PDI as evaluation indicators.

[0204] Results showed that the optimal drug-loading ratio of Cela to RSeHA was 1:1.4, and the carrier concentration was 4 mg / mL. The prepared Cela / RSeHA micelles had a particle size of (159.61±8.33) nm, a PDI of 0.07±0.02, a drug loading of (36.57±1.66)%, and an encapsulation efficiency of (68.39±0.75)%. TEM images revealed spherical Cela / RSeHA micelles, and DSC confirmed the encapsulation of Cela within the RSeHA micelles.

[0205] The beneficial effects of the present invention are demonstrated by specific pharmacodynamic tests below.

[0206] Experimental Example 1 In vitro release study of Cela / RSeHA micelles

[0207] In vitro stability studies in release media demonstrated that Cela / RSeHA micelles were highly stable at physiological and blood pH levels. Furthermore, the particle size increased significantly in release media consisting of pH 5.0 PBS + 10 mmol / L H2O2, pH 5.0 PBS + 10 mmol / L GSH, pH 5.0 PBS + 10 mmol / L (H2O2 + GSH), and pH 5.0 PBS + 10 mM (H2O2 + GSH + hyaluronidase). In vitro release results demonstrated that the cumulative release rate of free Cela solution reached over 70% over 12 hours at pH 7.4, while Cela / RSeHA micelles exhibited slow drug release in release media at pH 7.4 and pH 5.0, with cumulative release rates of approximately 30% and 23%, respectively. Under the conditions of pH 5.0+10mmol / LH2O2, the cumulative release rate of Cela / RSeHA micelles in 24 hours reached more than 80%; under the conditions of pH 5.0+10mmol / LGSH, the cumulative release rate of Cela / RSeHA micelles in 48 hours reached about 90%; under the conditions of pH 5.0+10mmol / L(H2O2+GSH), the cumulative release rate of Cela / RSeHA micelles in 60 hours reached about 92%; under the conditions of pH 5.0+10mmol / L(H2O2+GSH+hyaluronidase), the cumulative release rate of Cela / RSeHA micelles in 48 hours reached about 79%.

[0208] This shows that compared with free Cela, Cela / RSeHA micelles have a certain sustained release effect in the blood pH environment and have high redox responsiveness.

[0209] Test Example 2 Safety Evaluation of Cela / RSeHA Micelles

[0210] Safety testing results showed that when the hemolysis rate of Cela / RSeHA micelles is less than 5%, they are unlikely to cause coagulation or erythrocyte agglutination, demonstrating good blood compatibility. Tissue sections from rabbit ear vein injection sites showed no significant pathological changes, indicating that Cela / RSeHA micelles are minimally irritating to blood vessels. Cela / RSeHA micelles exhibit good safety and blood compatibility for intravenous injection.

[0211] Experimental Example 3 In vitro antitumor study of Cela / RSeHA micelles

[0212] Cell experiments showed that the RSeHA conjugate was non-toxic to MCF-7 and 4T1 cells within the experimental concentration range, demonstrating a good safety profile. Both free Cela and Cela / RSeHA micelles had a time- and concentration-dependent cytotoxic effect on MCF-7 and 4T1 cells, with Cela / RSeHA micelles exhibiting a stronger cytotoxic effect than free Cela.

[0213] Cell uptake experiments showed that MCF-7 and 4T1 cells had a good cellular uptake effect on (P4+Cela) / RSeHA micelles. The fluorescence intensity of (P4+Cela) / RSeHA micelles pre-saturated with HA was lower than that of (P4+Cela / RSeHA) micelles, indicating that after saturating the CD44 receptors on the cells, the uptake of (P4+Cela) / RSeHA micelles by MCF-7 and 4T1 cells decreased, further demonstrating that Cela / RSeHA micelles have strong tumor cell targeting ability through HA.

[0214] Experimental Example 4 In vivo pharmacokinetics study of Cela / RSeHA micelles

[0215] Pharmacokinetic results showed that Cela / RSeHA micelles followed a two-compartment distribution model in rats; they altered the in vivo pharmacokinetic characteristics of Cela and significantly increased the AUC (0-t) , extended the MRT (0-t) 、T 1 / 2 , reducing V and CL, improving the bioavailability of Cela, and prolonging Cela's circulation in the body and duration of action.

[0216] Experimental Example 5: Study on the Tissue Distribution of Cela / RSeHA Micelles

[0217] Tissue distribution and in vivo imaging results showed that after administration, Cela / RSeHA micelles were mostly distributed in the liver and tumor sites, which improved the tumor targeting of Cela and reduced the distribution in other non-target organs outside the liver, which was beneficial to reduce the toxic side effects on non-target organs.

[0218] Experimental Example 6 In vivo pharmacodynamic study of Cela / RSeHA micelles

[0219] BALB / c mice were used to establish a 4T1 subcutaneous transplant tumor model, and a commercially available formulation was used. As a positive control, the tumor growth curve and tumor inhibition rate were used as the main evaluation indicators. At the same time, by observing the weight changes, organ indexes, and HE staining sections of pathological tissues of mice, the CA15-3 breast cancer tumor marker and serum liver and kidney function index results were further analyzed to investigate the in vivo effectiveness and safety of Cela / RSeHA micelles.

[0220] 1 Instruments and Materials

[0221] 1.1 Instrument

[0222]

[0223] 1.2 Drugs and Reagents

[0224]

[0225]

[0226] 1.3 Animals and cell lines

[0227] Animals: Female SPF BALB / c mice, weighing 18 ± 2 g, were purchased from Wu's Experimental Animal Trading Co., Ltd. (License No.: SCXK (Beijing) 2019-0008, Certificate No.: 110322231103492013) and housed in the Animal Experiment Center of Fujian University of Traditional Chinese Medicine.

[0228] 2 Methods

[0229] 2.1 Tumor inoculation in mice

[0230] A BALB / c tumor-bearing mouse model was established using 4T1 mouse breast cancer cells. Female mice were 4 to 6 weeks old and weighed 16 ± 2 g. 4T1 cells were collected in the logarithmic growth phase in vitro and washed twice with 0.9% sodium chloride injection. The cell suspension concentration was adjusted to 1 × 10 7 / mL. 0.2mL was subcutaneously inoculated under the right forelimb of the mouse. After 7 days, the tumor grew to 100-250mm. 3 Start dosing when

[0231] 2.2 Dosage design and administration cycle

[0232] 2.2.1 Grouping and Dosage Design

[0233] (1) Control group: 0.9% sodium chloride solution;

[0234] (2) Positive control group: (2mg / kg);

[0235] (3) Free Cela group: 2 mg / kg;

[0236] (4) Cela / RSeHA micelle group: 2 mg / kg.

[0237] 2.2.2 Dosage cycle

[0238] Mice with relatively consistent tumor volumes were randomly divided into 4 groups, with 8 mice in each group. The first administration was marked as day 0. According to the above grouping and dosage, the drugs were injected into the tail vein on days 0, 2, 4, 6, and 8, respectively, for a total of 5 doses. On the 10th day, the mice were killed, blood was collected from their orbits, and the organs and tumor tissues were dissected.

[0239] 2.3 Evaluation of tumor inhibition effect

[0240] The first administration was marked as day 0. The long and short diameters of the tumor were measured at a fixed time every day. The tumor volume was calculated according to formula 3-1, and the tumor growth volume-time curve was drawn.

[0241] V tumor =(a 2 ×b) / 2 (3-1)

[0242] Note: a is the short diameter of the tumor, b is the long diameter of the tumor, unit: mm

[0243] On the 10th day, the tumor-bearing mice were sacrificed, the tumor tissues were dissected and weighed, and the tumor inhibition rate was calculated according to formula 3-2 to evaluate the therapeutic effect and perform mathematical statistics.

[0244] IR(%)=(W saline -W drug ) / W saline ×100% (3-2)

[0245] Among them, IR is the tumor inhibition rate, W drug is the tumor weight of the drug-treated group, W saline Tumor weight of the control group (normal saline group), unit: g

[0246] 2.4 Mouse weight assessment

[0247] Starting from day 0, the weight of tumor-bearing mice was measured at the same time every day, and the weight change curve of mice was drawn to measure the toxic effects of the drug on mice.

[0248] 2.5 Organ Index

[0249] After dosing, the weight of tumor-bearing mice was measured at a fixed time daily, and a weight change curve was plotted. On day 10, mice were weighed and sacrificed. The heart, liver, spleen, lungs, kidneys, and tumor were immediately removed and weighed. The weights were recorded and the organ index was calculated according to Equation 3-3. Changes in the organ index were used to assess the toxic and side effects of each drug group on the organ.

[0250] Organ index = organ mass (g) / mouse body weight (g) × 100% (3-3)

[0251] 2.6 Morphological observation of tissue sections by HE staining

[0252] The collected tissues (heart, liver, spleen, lung, kidney, tumor) were immediately fixed with fixative for 24 hours, removed, trimmed, dehydrated and immersed in wax, and the wax-immersed tissues were embedded in an embedding machine. After the wax solidified, the tissues were removed and the wax blocks were trimmed. After freezing at -20℃ overnight, the slices were transferred to glass slides, dried in an oven, stained, sealed, and examined under a microscope to collect images.

[0253] 2.7 Serum biochemical index detection

[0254] Blood was collected from the mouse eyeball into an EP tube, centrifuged at 14000 r / min for 5 min, and the upper serum was stored in a -20°C refrigerator for later use.

[0255] 2.7.1 Tumor Marker CA15-3 Detection

[0256] Serum carbohydrate antigen 15-3 (CA15-3) is a highly sensitive and specific breast cancer tumor marker. A CA153 ELISA kit was used according to the kit instructions. The absorbance (OD) was measured at 450 nm using a microplate reader. The concentration of mouse breast cancer antigen (CA15-3) in the sample was calculated using a standard curve.

[0257] 2.7.2 Liver and kidney function tests

[0258] The study aimed to investigate whether tail vein injection of Cela / RSeHA micelles caused functional damage to mouse organs. Liver detection indicators included alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and kidney detection indicators included blood urea nitrogen (BUN) and creatinine (CRE). The serum of each group was tested using a fully automatic biochemical analyzer to determine the concentrations of ALT, AST, BUN, and CRE in the serum of each group, reflecting the degree of damage to liver and kidney cells after drug treatment in each group.

[0259] 3 Results

[0260] 3.1 Tumor growth curve

[0261] In order to explore the tumor inhibition effect of Cela / RSeHA micelles in vivo, BALB / c mice inoculated with 4T1 cells were used as tumor models, free Cela was used as a comparison, and commercially available As a control, the anti-tumor effect of Cela / RSeHA micelles in tumor-bearing mice was investigated. Each group of preparations was injected into the tail vein, and the tumor volume change curves of each group of tumor-bearing mice were shown in Figure 2. Figure 13 As shown in Figure 2, the tumor volume of tumor-bearing mice on day 10 was 3.69 times larger than that on day 0. The results showed that the growth of tumors in the treatment groups was inhibited to varying degrees compared with the control group, and the positive drug The tumor growth inhibition effect is higher than that of free Cela; when Cela is encapsulated into RSeHA micelles, the anti-tumor effect is stronger than that of free Cela.

[0262] The tumor volumes of tumor-bearing mice on day 10 were compared. The Cela group and the commercially available The tumor volume of Cela / RSeHA micelle group was significantly different from that of Control group (P﹤0.001). The inhibitory effect of Cela / RSeHA micelle group on tumor growth was better than that of Cela group (P﹤0.01). (P﹤0.05). The above results preliminarily indicate that Cela / RSeHA micelles effectively enhance the anti-tumor effect of Cela.

[0263] 3.2 Tumor weight and tumor inhibition rate

[0264] On the 10th day after administration, the mice were killed and the tumors were removed. The tumors in each group are shown in the figure below. Figure 14 As shown, the tumors were weighed and the tumor inhibition rate was calculated. The results are shown in Figure 15 From the perspective of tumor weight, the Control group, Cela group, The average tumor weights of the Cela / RSeHA micelle group and the Cela / RSeHA micelle group were (0.56±0.16) g, (0.38±0.06) g, (0.32±0.05) g, and (0.21±0.05) g, respectively. Compared with the Control group, the tumor weights of all preparation groups were reduced, and all preparation groups were able to inhibit tumor growth to a certain extent. Among them, the tumor weight of the Cela / RSeHA micelle group decreased significantly compared with the Control group, with a significant difference (P﹤0.001). Compared with the Cela group, The tumor weight of the Cela / RSeHA micelle group was also reduced to varying degrees, and the therapeutic effect was relatively high.

[0265] In terms of tumor inhibition rate, compared with the Control group, the Cela group, The tumor inhibition rates of the Cela / RSeHA micelle group and the Cela / RSeHA micelle group were (29±10.61)%, (39.81±9.90)% and (59.45±12.64)%, respectively. The tumor inhibition effect of Cela / RSeHA micelle group was better than that of Cela group and There were significant differences between the two groups (P﹤0.001; P﹤0.01).

[0266] The above results indicate that each formulation significantly inhibited tumor growth and demonstrated a significant therapeutic effect on breast cancer. Encapsulating Cela in RSeHA micelles can enhance Cela's anti-tumor effect.

[0267] 3.3 Weight changes

[0268] After tail vein injection, the body weight of mice was recorded every day. Figure 16 As can be seen from the graph, the final weight of the Control group increased by 6.93% compared to before administration, which may be due to the fact that the mice did not receive timely treatment and the tumor gradually spread. The figure shows that the weight of the mice in the free Cela group increased and decreased, and the mice had diarrhea during the injection period. The average weight on the 10th day showed a negative growth compared to the average weight on the 0th day, and decreased by 2.7% compared to the weight before administration. This may be due to the toxic side effects of Cela, which caused damage to the mice's body functions, affected their appetite, and caused weight loss. The average body weight of the group and Cela / RSeHA micelle group increased by 5.27% and 7.53% respectively compared with before administration, indicating that Cela / RSeHA micelles exerted a tumor therapeutic effect without obvious side effects. The mice accepted it well, their physical condition improved, and their weight gradually increased.

[0269] 3.4 Organ Index

[0270] By calculating the organ index, we can judge the effect of drugs on the organs of mice or other degenerative changes. Increased organ index indicates congestion, edema or hyperplasia. Figure 17 Compared with the Control group, the positive drug The Cela / RSeHA micelle group and the Cela / RSeHA micelle group had no significant effects on various organs in the mice. The Cela group showed increased liver and spleen indices (P < 0.01 and P < 0.05), indicating that the drug caused congestion, edema, and hypertrophy of the liver and spleen. In summary, Cela / RSeHA micelles can ameliorate Cela-induced organ damage, reduce Cela's toxic side effects, and protect various organs.

[0271] 3.5 Morphological observation of tissue sections by HE staining

[0272] To further confirm the safety of Cela / RseHA micelles in vivo, histological analysis was performed on the heart, liver, spleen, lung, kidney and tumor of BALB / c mice bearing 4T1 after administration to observe whether there was inflammation or tissue lesions in each tissue. Figure 18 As shown, Control group, positive drug In the sections of the heart, liver, spleen, lung, and kidney of the Cela / RSeHA micelle group and the Cela / RSeHA micelle group, it was observed that the cell nuclear structure was complete and clear, the nuclear-cytoplasmic contrast was clear, and the cells were closely arranged. There was no cell inflammation or necrosis. This indicates that the positive drug The Cela / RSeHA micelle group and the Cela / RSeHA micelle group had almost no toxicity to various organs. In the liver and spleen sections of the Cela group, it was observed that the liver cells and spleen cells were loosely arranged, disordered, the cell nuclei were ablated, the cell structure was damaged, and the cells were partially atrophied. However, in the sections of organs such as the heart, lungs, and kidneys, the cells were tightly arranged and the cell nuclear structure was intact, with no obvious toxic side effects. From the above results, it can be further inferred that RSeHA micelles can reduce the distribution of Cela in the liver and spleen tissues by encapsulating Cela in the micelle core and targeting tumors, which can significantly reduce the toxic side effects of Cela on the liver and spleen.

[0273] The tumor sections of the tumor-bearing mice showed that the tumor tissue cells in the control group were tightly arranged, with a small amount of cell necrosis and good cell condition. The Cela and Cela groups showed loosely arranged tumor tissue, with some cells undergoing ablation and necrosis. In contrast, the Cela / RSeHA micelle group showed a significant increase in nuclear ablation and necrosis, indicating that Cela / RSeHA micelles have a stronger cytotoxic effect on tumor cells. These results suggest that, compared with free Cela, Cela / RSeHA micelles can carry Cela to the tumor site, reducing its toxic side effects in other organs and tissues, and enhancing its therapeutic efficacy on tumor tissue.

[0274] The enzyme-linked immunosorbent assay was used to measure the activity values ​​of ALT, AST, BUN, and CRE in the serum of each group of mice to investigate the degree of damage to the liver and kidneys of the mice. As shown in Table 5, among the liver function indicators, there was no significant difference in the ALT activity values ​​of each group; among the AST indicators, the positive drug activity values ​​were significantly higher than those of the control group. There was a significant difference between the two groups (P<0.01); compared with the Cela / RSeHA micelle group, the AST value of the Cela group was significantly increased (P<0.01). Among the renal function indicators, the CRE activity values ​​of each group were not statistically significant. The BUN activity values ​​of the Control group were greater than those of each drug-treated group. Among them, there was a significant difference between the Cela group and the Control group (P<0.01); there was a significant difference between the Cela / RSeHA micelle group and the Control group (P<0.001); the BUN activity value of the Cela / RSeHA micelle group was significantly lower than that of the positive drug The difference between the two groups was significant (P < 0.01). These results indicate that the Cela group had higher AST activity and some degree of liver damage, likely due to solvent toxicity. This also suggests that encapsulating Cela in RSeHA micelles can reduce drug damage to the liver and kidneys, leading to enhanced liver and kidney protection.

[0275] Although the aforementioned tissue distribution showed that Cela / RSeHA micelles were more distributed in the liver, the liver index, liver tissue sections, and ALT and AST values ​​in mouse serum indicated that Cela / RSeHA micelles had no effect on the liver.

[0276] The CA15-3 level in the serum of each group of mice was detected by using a mouse breast cancer marker CA15-3 ELISA kit. As shown in Table 5, after tail vein administration, the CA15-3 level of mice in each treatment group decreased by 1.29 to 10.04 ng / mL, compared with the control group. CA15-3 levels were significantly reduced in both the Cela / RSeHA micelle group and the Cela / RSeHA micelle group (P < 0.001); CA15-3 levels in the Cela / RSeHA micelle group were significantly lower than those in the free Cela group (P < 0.001). Compared with the control group, the CA15-3 concentration in the Cela / RSeHA micelle group was (11.68 ± 0.81) ng / mL, 67.48% of the control group. These results suggest that Cela / RSeHA micelles have a therapeutic effect on breast cancer.

[0277] Table 5 Effects of each group of drug treatment on breast cancer markers CA15-3, liver function (ALT, AST) and renal function (BUN, CRE) in tumor-bearing mice ( n=8)

[0278]

[0279] Note a: Compared with the Control group, *＊* P < 0.001; ** P < 0.01;

[0280] Note b: Compared with the free Cela group: ### P < 0.001; ## P < 0.01;

[0281] Note c: With positive drugs Group comparison: && P < 0.01; &&& P<0.001.

[0282] In vivo pharmacodynamic results showed that on the tenth day of treatment, tumor volume increased by 3.69-fold and 2.60-fold in the control and Cela / RSeHA micelle groups, respectively (P < 0.001); body weight increased by 6.93% and 7.53%, respectively; and tumor weights were (0.56 ± 0.16) g and (0.21 ± 0.05) g, respectively (P < 0.001). The tumor inhibition rate in the Cela / RSeHA micelle group was (59.45 ± 12.64)%. There were no significant differences in organ indices between the control group and the Cela / RSeHA micelle group. The liver and spleen indices of free Cela were increased (P < 0.01, P < 0.05). Tissue sections in the Cela / RSeHA micelle group showed densely packed cells and intact tissue structure. Serum CRE, ALT, and AST activity levels in the Cela / RSeHA micelle group were not statistically different from those in the control group. BUN activity was significantly different between the Cela / RSeHA micelle group and the control group (P < 0.001), indicating that Cela / RSeHA micelles had no significant toxic side effects on mice. The breast cancer marker CA15-3 level in the Cela / RSeHA micelle group was significantly decreased. Compared with the control group, the CA15-3 concentration in the Cela / RSeHA micelle group was (11.68 ± 0.81) ng / mL, 67.48% of that in the control group. This suggests that Cela / RSeHA micelles have a potent anti-tumor effect against breast cancer.

Claims

1. A rhein-diselenide-hyaluronic acid (RSeHA) conjugate, characterized in that: It uses selenocystamine (diselenium, Se-Se) as a linker arm to graft the small molecule rhein (R) onto low molecular weight hyaluronic acid (HA) to synthesize a redox-responsive conjugate (RSeHA conjugate) that can self-assemble into micelles in water; The structural formula of the conjugate is:

2. The rhein-diselenide-hyaluronic acid (RSeHA) conjugate according to claim 1, wherein: The molar feed ratio of rhein (R), Se-Se (calculated as selenocystamine dihydrochloride) and hyaluronic acid (HA) is 1-1.5:1-2:

1.

3. The rhein-diselenide-hyaluronic acid (RSeHA) conjugate according to claim 2, wherein: The molar feed ratio of rhein (R), Se-Se (calculated as selenocystamine dihydrochloride), and hyaluronic acid (HA) is 1:1.2:

1.

4. The rhein-diselenide-hyaluronic acid (RSeHA) conjugate according to any one of claims 1 to 3, characterized in that: The RSeHA micelle particle size was (193.48±21.61) nm, the PDI was 0.19±0.04, and the potential was (-31.24±2.30) mV. The R and Se-Se substitution degrees were (9.59±1.48)% and (9.09±1.08)%, respectively.

5. The method for preparing the rhein-diselenide-hyaluronic acid (RSeHA) conjugate according to any one of claims 1 to 4, characterized in that: The synthetic route is: It includes the following steps: a. Preparation of R-Se-Se: Dissolve R in 1% NaHCO3, add EDC·HCl for reaction, then add NHS and react for 10 min; dissolve selenocystamine dihydrochloride completely in DMSO, add to R solution, and stir to obtain the product; b. Preparation of HA-NHS: Dissolve HA in ultrapure water, stir on a constant temperature magnetic stirrer, and heat at 60°C until completely dissolved. Add EDC·HCl for reaction, and then add NHS to obtain HA-NHS. The R-Se-Se prepared in step a was added dropwise to HA-NHS and reacted for 24 h to obtain a product mixture; c. Add 95% ethanol to the product mixture and allow to settle for 3 hours. Filter with suction, and re-dissolve the residue in distilled water. Use ultrasonic dispersion with a probe until the solution is clear. Centrifuge at 3900 rpm for 15 minutes, place in a dialysis bag (MWCO = 3500), and dialyze in distilled water for 3 days. The dialyzed product solution was probed again for 15 min, centrifuged at 3900 r / min for 15 min, filtered through a 0.8 μm filter membrane, pre-frozen for 24 h, and freeze-dried for 24 h to obtain the product RSeHA conjugate.

6. A micelle, characterized in that: The hydrophobic compound is encapsulated by the rhein-diselenide-hyaluronic acid (RSeHA) conjugate described in any one of claims 1 to 4.

7. The micelle according to claim 6, characterized in that: The hydrophobic compound is Celacotene (Cela); the mass ratio of Celacotene (Cela) to the rhein-diselenide-hyaluronic acid (RSeHA) conjugate is 1:0.8-1.6; and the concentration of the micellar carrier (RSeHA conjugate) is 3-7 mg / mL.

8. The micelle according to claim 7, characterized in that: The mass ratio of the tripterygium wilfordii (Cela) to the rhein-diselenide-hyaluronic acid (RSeHA) conjugate is 1:1.4; the concentration of the micelle carrier (RSeHA conjugate) is 4 mg / mL; the particle size of the Cela / RSeHA micelles is (159.61±8.33) nm, the PDI is 0.07±0.02; the drug loading is (36.57±1.66)%, and the encapsulation efficiency is (68.39±0.75)%.

9. A method for preparing the micelle according to claim 7 or 8, characterized in that: It includes the following steps: a. Weigh the RSeHA conjugate, add distilled water, and ultrasonicate in an ice-water bath until uniformly dispersed; b. Weigh Cela powder and dissolve it in DMSO. Then add it dropwise to the vigorously stirred RSeHA conjugate solution and continue stirring for 30 minutes. Then, disperse it by ultrasonic probe in an ice-water bath and dialyze it in distilled water in a dialysis bag for 24 hours. Then, ultrasonic probe in an ice-water bath for 20 minutes, centrifuge at 3500 rpm for 10 minutes, remove the supernatant, filter it through a 0.8 μm filter membrane, and lyophilize the filtrate to obtain Cela / RSeHA micelle lyophilized powder.

10. Use of the micelle according to claim 7 or 8 in preparing a drug for treating breast cancer.

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