Aromatic sulfated ketone-modified chondroitin sulfate derivative, and preparation method and application thereof

Abstract

The application discloses a kind of aromatic thioketals modified chondroitin sulfate derivatives and preparation method and application, belong to the field of biological medicine.The aromatic thioketals solution is added to 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and reaction is carried out for 1~5 h in the dark, to obtain aromatic thioketals activation solution;Aromatic thioketals activation solution and chondroitin sulfate solution are mixed, and reaction is carried out for 12~36 h in the dark under nitrogen protection, anhydrous ether is added to reaction liquid, and precipitate is generated and filtered, and aromatic thioketals modified chondroitin sulfate derivative is obtained by drying and purifying precipitate.Aromatic thioketals modified chondroitin sulfate derivative inherits the natural bone matrix affinity and receptor-mediated targeting ability of chondroitin sulfate, integrates stimulus-responsive switch, and chondroitin sulfate is chemically modified by aromatic thioketals, can efficiently load drug to realize controllable release.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an aromatic thioketal modified chondroitin sulfate derivative, its preparation method, and its application. Background Technology

[0002] With the increasing aging of the population, bone-related diseases such as osteoporosis and bone metastases from tumors seriously threaten human health. Although various therapeutic drugs have been developed clinically, their effectiveness is limited by the special structure of bone tissue, restricting drug penetration, resulting in very small amounts of drugs reaching the lesion area, and immunosuppression. The main reasons are: the dense inorganic matrix of bone tissue and the unique bone marrow-blood barrier make it difficult for drugs circulating in the body to efficiently penetrate to the lesion area; osteoporosis and bone metastases from tumors are often accompanied by bone matrix destruction and severe inflammatory reactions, which also lead to reduced efficacy.

[0003] Drug delivery systems have become an important research area in targeted immunotherapy for diseases due to their advantages such as good targeting, controlled release, improved drug solubility and stability, and reduced toxicity. Nanocarriers can utilize the characteristics of the disease microenvironment (such as acidic pH, high reactive oxygen species, and high esterase activity) to achieve responsive drug release. Modifying materials with reactive oxygen species (ROS)-responsive structures can achieve specific drug release within tumors, helping to improve treatment efficiency and reduce systemic toxicity. Although ROS-responsive smart nanomaterials have emerged in recent years, their construction often involves complex chemical synthesis steps. The introduction of many responsive groups requires stringent reaction conditions and expensive catalysts. Existing smart materials are often difficult to automate, have poor reproducibility, and are costly to produce, severely hindering their transition from laboratory research to large-scale industrialization. Furthermore, existing delivery materials are mostly bioinert, acting only as carriers and unable to actively repair or protect the damaged bone microenvironment. This single delivery mode limits the comprehensive therapeutic effect of bone-related diseases.

[0004] In summary, current drug delivery technologies have shortcomings in terms of blood-bone marrow barrier penetration, bone lesion targeting, and bone function protection. There is an urgent need for a new type of delivery carrier that is safe, controllable, can penetrate the barrier, has strong targeting, and can also inhibit bone destruction. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention provides an aromatic thioketal modified chondroitin sulfate derivative, its preparation method and application, which inherits the natural bone matrix affinity and receptor-mediated targeting ability of chondroitin sulfate, integrates a stimulus-response switch, and after being chemically modified by aromatic thioketal, chondroitin sulfate can efficiently load drugs to achieve controlled release.

[0006] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for preparing an aromatic thioketal-modified chondroitin sulfate derivative, comprising the following steps: (1) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the aromatic thioketal solution and react in the dark for 1-5 h to obtain the aromatic thioketal activated solution. (2) Mix the aromatic thioketal activation solution and the chondroitin sulfate solution, and react under nitrogen protection in the dark for 12-36 h. Add anhydrous diethyl ether to the reaction solution to produce a precipitate and filter it. Dry and purify the precipitate to obtain the aromatic thioketal modified chondroitin sulfate derivative.

[0007] Further, the method for preparing the aromatic thioacetal is as follows: 9-fluorenone and 4-mercaptobutyric acid are mixed and dissolved, an acid catalyst is added, and the reaction is carried out at room temperature in the dark for 12-36 h. Saturated sodium bicarbonate solution is added to the reaction solution to precipitate the product, and the product is obtained by filtration, washing, and recrystallization.

[0008] Furthermore, the molar ratio of 9-fluorenone to 4-mercaptobutyric acid is 1:1 to 5, and the solvent for mixing and dissolving is dichloromethane; the acid catalyst is trifluoroacetic acid, and the molar ratio of the acid catalyst to 9-fluorenone is approximately 1:2 to 5.

[0009] Further, the mass ratio of N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and aromatic thioacetal is 1:2~5:6~15; the concentration of the aromatic thioacetal solution is 100~200 mg / mL, and the solvent is N,N-dimethylformamide.

[0010] Furthermore, the mass ratio of the aromatic thioacetal to chondroitin sulfate is 1:2~5; the concentration of the chondroitin sulfate solution is 100~1000 mg / mL, and the solvent is water.

[0011] Further, the purification method in step (2) is as follows: the dried precipitate is dissolved in water and dialyzed in a dialysis bag with a MWCO of 3500~14000 Da. The dialysate is freeze-dried to obtain an aromatic thioketal modified chondroitin sulfate derivative.

[0012] In a second aspect, the present invention provides an aromatic thioketal-modified chondroitin sulfate derivative prepared by the above-described preparation method.

[0013] In a third aspect, the present invention provides the use of the aromatic thioketal-modified chondroitin sulfate derivative in the preparation of bone repair drugs.

[0014] Furthermore, the aromatized thioketal-modified chondroitin sulfate derivative is used as a drug carrier to load triptolide. In the aromatized thioketal-modified chondroitin sulfate derivative (CST), chondroitin sulfate (CS) is a hydrophilic shell and the aromatized thioketal (AKT) is a hydrophobic segment, utilizing their self-assembly to encapsulate the drug. Triptolide (TP), as a natural small molecule compound with anti-tumor, bone loss inhibition, and immunogenic death induction (ICD) properties, can simultaneously regulate bone tumor metastases in a "tumor-bone-immune" relationship. It is a potential drug for treating TNBC bone metastases, but its extremely poor water solubility and severe hepatotoxicity and nephrotoxicity limit its clinical application. Loading triptolide onto the aromatized thioketal-modified chondroitin sulfate derivative can effectively solve this problem.

[0015] Furthermore, the method for loading triptolide is as follows: an aromatic thioketal-modified chondroitin sulfate derivative is dissolved in water to form a micelle solution; triptolide is added to the aromatic thioketal-modified chondroitin sulfate derivative solution and ultrasonicated or stirred to encapsulate it; then, unencapsulated drug is removed by centrifugation, dialysis or ultrafiltration; and finally, the aromatic thioketal-modified chondroitin sulfate derivative loaded with triptolide (TP@CST) is freeze-dried.

[0016] Furthermore, the bone repair mentioned refers to the repair of bone damage caused by osteoporosis or tumor bone metastasis; bone is a major metastatic site for many solid malignant tumors. In diseases such as breast cancer, prostate cancer, and lung cancer, more than 60% of patients with metastatic disease eventually develop bone metastases, leading to refractory skeletal-related events (SREs) and shortened survival. Triple-negative breast cancer is highly aggressive and has a strong bone tropism; bone metastases are often accompanied by severe bone matrix destruction and inflammation. Although existing bisphosphonates can inhibit bone resorption, they have drawbacks such as nephrotoxicity and limited function.

[0017] Aromaticated thioketal-modified chondroitin sulfate derivatives (CST) possess the following triple properties: (1) Dual targeting and blood-bone marrow barrier breakthrough: Due to the presence of chondroitin sulfate (CS) on the surface of CST, it exerts an EPR-like effect at the metastatic site by utilizing the optimal particle size (100-300 nm), while bypassing the size rejection barrier of healthy bone marrow, thereby minimizing bone marrow toxicity; the tight hydration layer and moderate surface negative charge of CST inhibit non-specific phagocytosis and macrophage uptake, prolonging systemic circulation; in addition, its inherent mechanical deformability helps to adapt to hemodynamic pressure and penetrate the disordered vascular system in the metastatic microenvironment; the natural affinity of CST to the bone matrix and its specific recognition ability of CD44 receptor on cancer cells; achieve long circulation, effectively break through the blood-bone marrow barrier, and achieve targeted and deep penetration of pathological bone metastatic tumor cells.

[0018] (2) Bioactive carrier and environmental responsiveness: CST is not only a carrier delivery tool, but also has the function of inhibiting osteoclasts and promoting osteoblast repair. This makes the carrier have the characteristics of "drug adjuvant" in the treatment of bone metastasis. Compared with ordinary thioacetal, aromatized thioacetal (AKT) has better hydrophobicity and specific sensitivity to ROS, ensuring that the encapsulated drug breaks down and releases the drug in a controlled manner in the high reactive oxygen environment of tumor cells.

[0019] (3) Biosafety and translational potential: CST’s superior particle potential and mechanical deformability enable it to effectively penetrate and accumulate in bone metastases. Its acute toxicity is also significantly reduced in the liver, kidneys and healthy bone tissue, demonstrating good biosafety. CST is amphiphilic and can be self-assembled. Its preparation process is simple and highly reproducible. The basic material, chondroitin sulfate, has been used clinically in some countries for a long time in the form of intramuscular or intra-articular injections, which reflects CST’s good potential for large-scale translation, biocompatibility and translational medicine.

[0020] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: (1) The invention realizes the integration of “targeting-treatment-repair”: The invention utilizes the natural affinity of CST to bone matrix and the specific recognition of CD44 receptor on cancer cells, so that the drug carried by CST actively recognizes and accumulates in the tumor cells in bone metastases. At the same time, the carrier material CST can help prevent bone destruction, which is an advantage that conventional carriers (such as liposomes, polylactic acid, etc.) do not have.

[0021] (2) It combines long-lasting blood compatibility with high blood-bone barrier penetration capability: The introduction of aromatic groups increases the stability of the thioketal structure, avoiding premature degradation in the bloodstream, but it can break down rapidly under high H2O2 conditions, achieving precise controlled release of "rapid release within the tumor and low release in normal tissues", improving efficacy while reducing side effects. Utilizing the optimal particle size, it exerts an EPR-like effect at the bone metastasis site, reducing toxicity to healthy bone marrow. The tight hydration layer and moderate surface negative charge inhibit non-specific phagocytosis and macrophage uptake, prolonging systemic circulation; its mechanical deformability allows it to penetrate the disordered vascular system in the bone metastasis microenvironment and cross the blood-bone barrier.

[0022] (3) High material safety and good biocompatibility: Compared with traditional inorganic carriers, chondroitin sulfate in this invention has been used for clinical intramuscular injection or intra-articular injection for a long time. CST is a biodegradable organic structure with no organic matter, heavy metals or other residues. It has low long-term toxicity risk and is more suitable for clinical application.

[0023] (4) The preparation method is simple and scalable: the synthesis route of the present invention has mild reaction conditions, low cost, and is easy to scale up for industrial production, making it suitable for subsequent clinical translation applications.

[0024] (5) In terms of drug delivery: the material has multiple functions such as bone protection and anti-tumor treatment; CST promotes osteoblast proliferation and inhibits osteoclast differentiation, which can reduce bone destruction. For example, combined with loaded TP, it can work synergistically with CST to achieve the integration of tumor treatment and bone disease prevention and treatment. It can also load other substances (such as small molecule compounds, nucleic acids and biomolecules) to work synergistically with CST to achieve the treatment of other diseases and the prevention and treatment of bone diseases. Attached Figure Description

[0025] Figure 1 The graphs show the particle size, potential, and mechanical properties of CST and TP@CST. In the graphs, a is the particle size distribution of CST, b is the particle size distribution of TP@CST, c is the potential characterization graph of CST and TP@CST, d is the adhesion ability graph of CST, e is the elastic modulus graph of CST, and f is the Young's modulus graph of CST. Figure 2 Here are the XPS and FT-IR spectra of CST, where a is the XPS plot and b is the FT-IR spectrum; Figure 3 The images are transmission electron microscope (TEM) images of CST with and without hydrogen peroxide, where a represents the absence of hydrogen peroxide and b represents the presence of hydrogen peroxide. Figure 4 Laser confocal micrographs of CST uptake by breast cancer cells in vitro, where a is a laser confocal micrograph of CST uptake by 4T1 cells (including a magnified view), and b is a laser confocal micrograph of CST uptake by MAD-MB-231 cells (including a magnified view). Figure 5 The images show targeted bioimaging of CST in vivo, where a is an in vivo fluorescence imaging image of mice carrying 4T1 tumors; b is a statistical diagram of in vivo fluorescence imaging results of tumor bone metastases; c is an in vitro fluorescence imaging image of major organs and tibia; and d is a statistical diagram of imaging results of major organs and tibia. Figure 6 Representative in vivo CT images of mouse bone processed with TP, CST, TP@CST, and ZA are shown, where a is a representative CT image of living mouse bone tissue, and b is a representative Micro-CT reconstruction analysis image of ex vivo bone tissue.

[0026] Figure 7The images show the efficacy validation of TP, CST, TP@CST, and ZA in vivo and in vitro. In the images, a represents the relative survival rate of 4T1 cells after treatment with TP, CST, and TP@CST; b represents the relative survival rate of MDA-MB-231 cells after treatment with TP, CST, and TP@CST; c represents the relative survival rate of MCF-10A cells after treatment with TP, CST, and TP@CST; d represents the real-time fluorescence imaging at different time points after implantation of triple-negative breast cancer cells into the tibia of mice; and e represents the statistical analysis of the real-time fluorescence imaging. Figure 8 The following diagrams illustrate the in vivo safety evaluation of TP, CST, TP@CST, and ZA. Diagram a shows the detection of ALT activity in mouse serum, b shows the detection of AST activity in mouse serum, c shows the determination of UREA activity in mice, and d shows the histological analysis of organs stained with H&E in mice. Figure 9 The pharmacokinetic evaluation diagrams of TP and TP@CST in vivo are shown below. In the diagram, a is the mean plasma concentration-time curve of TP@CST and TP, b is the tissue distribution diagram of TP@CST and CST 5 min after intravenous injection, c is the tissue distribution diagram of TP@CST and CST 30 min after intravenous injection, and d is the tissue distribution diagram of TP@CST and CST 12 h after intravenous injection. Detailed Implementation

[0027] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions.

[0029] Example 1 (1) Preparation of aromatic thioacetal (AKT): 1.50 g of 9-fluorenone and 1.71 mL of 4-mercaptobutyric acid were dissolved in 5 mL of dichloromethane, and 2.55 mL of trifluoroacetic acid was added dropwise. The mixture was stirred continuously for 24 h in the dark at room temperature (25±1℃). During the reaction, the solution gradually became turbid. The reaction solution was poured into 50 mL of saturated NaHCO3 solution and stirred rapidly. The precipitate was collected by filtration and dried in a vacuum drying oven at 45℃ for 12 h. The crude product was dissolved in methanol and recrystallized to obtain a pale yellow solid AKT.

[0030] (2) Preparation of chondroitin sulfate derivative (CST) modified with aromatized thioketal: 0.65 g AKT was dissolved in N,N-dimethylformamide to obtain an AKT solution (130 mg / mL). 200 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 73 mg N-hydroxysuccinimide (NHS) were added to the AKT solution and activated by stirring at room temperature in the dark for 2 h. 1.50 g chondroitin sulfate (CS) was dissolved in water to obtain a CS solution with a concentration of 500 mg / mL. The CS solution was added dropwise to the AKT solution, and N,N-dimethylformamide was added until the solution became transparent. The reaction system was reacted under nitrogen protection in the dark for 24 h. The reaction solution was added to 200 mL of anhydrous diethyl ether, and a large amount of precipitate was produced by rapid stirring. The precipitate was filtered and dried under vacuum at 45 °C for 24 h to obtain the crude product. The crude product was dissolved in deionized water and dialyzed in a dialysis bag with a MWCO of 3500 Da for 1 hour. d. Freeze-drying to obtain an aromatic thioacetal-modified chondroitin sulfate derivative (CST).

[0031] The chemical reaction formula for preparing the aromatic thioketal-modified chondroitin sulfate derivative (CST) is as follows: .

[0032] Example 2 2.0 mg of CST prepared in Example 1 was dissolved in PBS buffer (pH=7.4), and triptolide (TP) was added. The mass ratio of triptolide to CST was 1:4. The mixture was magnetically stirred for 12 h in an ice-water bath. After centrifugation at 12000 rpm for 15 min, the unloaded drug was removed by dialysis through a dialysis bag with a MWCO of 3500 Da. The mixture was then freeze-dried to obtain an aromatic thioacetal-modified chondroitin sulfate derivative loaded with triptolide (TP@CST).

[0033] Example 3 (1) The particle size, potential, and mechanical properties of CST prepared in Example 1 and TP@CST prepared in Example 2 were tested, and the results are as follows: Figure 1 As shown; where a is the particle size distribution of CST, b is the particle size distribution of TP@CST, c is the potential characterization of CST and TP@CST, d is the adhesion ability diagram of CST (compared to PLGA), e is the elastic modulus diagram of CST (compared to PLGA), and f is the Young's modulus diagram of CST (compared to PLGA). Figure 1The particle sizes of CST and TP@CST range from 100 to 500 nm, and the Zeta potential is -30 ± 20 mV, indicating that the prepared CST and TP@CST have good stability. Compared with the commonly used drug delivery carrier PLGA, CST has a smaller Young's modulus and elastic modulus, and a greater adhesion ability. This characteristic gives it good deformability to overcome the blood-bone marrow barrier.

[0034] (2) XPS and FT-IR spectra of CST prepared in Example 1 (with and without hydrogen peroxide, CST(+H2O2) and CST(-H2O2) are shown below. Figure 2 As shown in Figures a and b, the results indicate that the CST surface contains C, N, O, and S elements, and the CST at 721 cm⁻¹... -1 The characteristic band observed corresponds to the CS stretching vibration, indicating the formation of a ketethiol acetal bond. Under high ROS conditions, 721 cm⁻¹ -1 The breakage of the CS bond at the site released the drug, accompanied by a flow rate of 1070 to 1250 cm. -1 S=O bonds are formed at the location.

[0035] (3) CST was diluted to 0.1 mg / mL with ddH2O and added dropwise to a carbon film copper mesh. After the CST was allowed to dry naturally, 100 μL of 2% uranium acetate was added for staining for 1 min. The CST was observed and photographed using a HITACHI HT 7800 transmission electron microscope. The morphology of CST was observed using the transmission electron microscope. Transmission electron microscope images of CST with and without hydrogen peroxide are shown below. Figure 3 As shown, where a represents no hydrogen peroxide and b represents hydrogen peroxide; from Figure 3 It is evident that the amphiphilic CST can self-assemble into spherical nanoparticles with uniform particle size, exhibiting good dispersibility without particle aggregation or adhesion. CST rapidly disintegrates in H₂O₂ solution, releasing the drug, indicating good ROS response performance.

[0036] Example 4 Cell uptake experiment (1) Preparation of Cy5-CST solution: Dissolve 1 mg CST in 2 mL PBS buffer (pH=7.4), add an appropriate amount of Cy5-NHS active ester to maintain a final concentration of 200 ug / mL. The NHS ester specifically reacts with the free amino group on CST to form a stable amide bond, thereby synthesizing Cy5@CST solution that can be used for cell imaging and in vivo in mice.

[0037] (2) After digesting and counting the 4T1 cells and MDA-MB-231 cells in the logarithmic growth phase, the concentration was adjusted to 2×10⁻⁶. 5Cells / mL were seeded in laser confocal microscopy dishes (35 mm in diameter) and cultured overnight. Cell culture medium containing Cy5@CST (final concentration 50 μg / mL) was added, and incubation was performed for 1 h, 2 h, and 4 h, respectively. After discarding the supernatant, Hoechst 33342 (1 μg / mL) was added to stain the cell nuclei for 30 min, followed by washing three times with PBS. Lysosomes were stained with LysoTracker (50 nM) and incubated for 30 min, followed by washing with PBS and adding serum-free culture medium. The cells were then observed under a confocal microscope, acquiring multiple channels (DAPI / Cy5 / Green). Laser confocal micrographs of CST uptake by breast cancer cells in vitro are shown below. Figure 4 As shown, a is a laser confocal micrograph of CST uptake in 4T1 cells (including a magnified view), and b is a laser confocal micrograph of CST uptake in MAD-MB-231 cells (including a magnified view). Figure 4 It was found that 4T1 cells and MDA-MB-231 cells effectively internalized CST, and the increase was time-dependent.

[0038] Example 5 In vivo targeting validation: (1) Preparation of Cy5-CST: Dissolve 1 mg of CST in 2 mL of PBS buffer (pH=7.4), add an appropriate amount of Cy5-NHS active ester to maintain a final concentration of 200 ug / mL. The NHS ester specifically reacts with the free amino group on CST to form a stable amide bond, thereby synthesizing Cy5@CST solution that can be used for cell imaging and in vivo in mice.

[0039] (2) Animal modeling: Mice were anesthetized with isoflurane, and a 29G insulin needle was used to puncture the tibial medullary cavity, injecting 10μL containing 2×10 5 A suspension of 4T1-luc cells was used to create a model; (3) Seven days after successful modeling, 40 μL of free Cy5 (200 μg / kg) and Cy5-CST (200 μg / kg) were injected via the tail vein. In vivo fluorescence imaging was performed at 1 h, 6 h, 12 h, and 24 h. After 24 h, the mice were sacrificed, and the major organs and bones were collected for in vitro imaging to compare fluorescence distribution. The targeting bioimaging diagram of CST in vivo is shown below. Figure 5 As shown, a) is an in vivo fluorescence imaging image of mice carrying 4T1 tumors; b) is a statistical graph of in vivo fluorescence imaging results of tumor bone metastases; c) is an in vitro fluorescence imaging image of major organs and the tibia; d) is a statistical graph of imaging results of major organs and the tibia. Figure 5As shown in a and b, in mice injected with Cy5-CST, the fluorescence intensity at the tumor bone metastasis site peaked at 12 h and then gradually decreased. Mice injected with Cy5-CST still showed strong fluorescence signals 24 h after injection, while imaging of mice injected with free Cy5 showed no fluorescence signal at the tumor bone metastasis site. This indicates that Cy5-CST nanoparticles have good tumor targeting and accumulation properties in vivo. Figure 5 As shown in C and D, the Cy5 signal was detected strongly in the tumors of the Cy5-CST group, confirming its prolonged circulation and residence time, and enhanced tumor-specific targeting. Furthermore, the size of TP@CST (100-200 nm) allows it to cross the bone marrow-blood barrier structure in the bone marrow, prolonging its circulation time within the host.

[0040] Example 6 Validation of osteoprotective effect in mouse tumor bone metastasis model (1) Animal modeling: Mice were anesthetized with isoflurane, and a 29G insulin needle was used to puncture the tibial medullary cavity, injecting 10μL containing 2×10 5 A suspension of 4T1-luc cells was used to create a model; (2) Seven days after successful modeling, the effect of the drug delivery system on tumor development was tracked using in vivo imaging technology in mice. Subsequently, mice were randomly divided into five groups: control group, TP group (TP), CST group (CST), TP@CST group (TP-CST), and zoledronic acid (ZA) group. The treatment dose for the TP group was 0.2 mg / kg, the CST group was 0.8 mg / kg, and the TP@CST group (TP was 0.2 mg / kg, CST was 0.8 mg / kg) received intravenous injections every two days, 100 uL / mouse each time. The control group received an equal volume of physiological saline. The treatment dose for the ZA group was 30 ug / kg, and the treatment dose was 100 uL / mouse every two days via intraperitoneal injection. The entire treatment process lasted for 21 days.

[0041] (3) X-ray bone destruction imaging was performed on the mice one day before sacrifice. Representative CT images of the mice's living bone tissue are shown below. Figure 6 As shown in Figure a; isolated mouse femurs, tibias, and fibulas were fixed in 4% paraformaldehyde and analyzed by Micro-CT X-ray. Figure 6 Image b shows a representative Micro-CT reconstruction analysis of ex vivo bone tissue, recording the degree of tibial bone destruction as a basis for subsequent histological evaluation. Figure 6 As shown in a and b, tumors can lead to severe bone loss, fractures, and even complete loss of bone trabeculae. CST and TP@CST can alleviate bone loss and play a role in bone protection. TP@CST significantly improves bone loss and its treatment effect is significantly better than that of the CST group and the ZA group.

[0042] Example 7 In vitro efficacy verification The cytotoxicity of different concentrations of TP, CST, and TP@CST was assessed using the MTT assay. Logarithmically growing 4T1, MDA-MB-231 breast cancer cells, and MCF-10A cells were seeded into 96-well plates. After treating these cell types with different concentrations of TP, CST, and TP@CST for 48 h, MTT solution was added to the culture medium, followed by DMSO after 4 h. The absorbance of each well was measured at 490 nm using an ELISA reader, and cell viability was calculated. The relative viability of 4T1, MDA-MB-231, and MCF-10A cells after treatment with different concentrations of TP, CST, and TP@CST are shown below. Figure 7 As shown in a, b, and c, TP@CST exhibited concentration-dependent cytotoxicity against 4T1 and MDA-MB-231 cells, and significantly enhanced cell killing activity compared to the free TP group, while almost no cytotoxicity was observed in normal MCF-10A cells.

[0043] In vivo efficacy verification A mouse model of breast cancer with bone metastasis was established by intratibial injection of 4T1 luc cells (same as in Example 5). The effect of the drug delivery system on tumor development was tracked using in vivo imaging techniques in mice. Subsequently, mice were randomly divided into five groups for treatment, with the treatment process the same as step (2) in Example 6. The entire treatment process lasted 21 days, during which tumor fluorescence imaging was performed every 7 days. Real-time fluorescence imaging images at different times after intratibial implantation of triple-negative breast cancer cells in mice are shown below. Figure 7 As shown in Figure d, the statistical analysis graph of tumor signal intensity is as follows. Figure 7 As shown in Figure e, the tumor signal enhancement was strongest in the control group. Compared with other treatment groups, TP@CST treatment was more effective in reducing bioluminescent signals and inhibiting the proliferation and bone metastasis of breast cancer in vivo.

[0044] Example 8 In vivo safety evaluation In the in vivo efficacy verification experiment, the in vivo safety evaluation graph after 21 days of treatment is shown in the figure below. Figure 8As shown in the figures, a) is a graph showing the detection of alanine aminotransferase (ALT) activity in mouse serum, b) is a graph showing the detection of aspartate aminotransferase (AST) activity in mouse serum, c) is a graph showing the detection of urea nitrogen (UREA) activity in mouse blood, and d) is a graph showing the histological analysis of mouse organs stained with H&E. Biochemical analysis of mouse serum after drug administration revealed that TP treatment increased the levels of AST, ALT, and urea nitrogen. The liver and kidney serum biochemical indicators of mice treated with CST and TP@CST remained within the normal range. H&E staining of the kidneys and livers in the CST and TP@CST groups also showed good biocompatibility.

[0045] Example 9 Pharmacokinetic evaluation In in vivo efficacy validation experiments, blood, heart, liver, spleen, lung, kidney, normal tibia, and tibia with tumors were collected from mice treated with TP and TP@CST. High-performance liquid chromatography (HPLC; Agilent 1260, China) was used for detection and quantitative analysis. The in vivo pharmacokinetic evaluation graph is shown below. Figure 9 As shown in Table 1, a) is the mean plasma concentration-time curve of TP@CST and TP, b) is the tissue distribution map of TP@CST and CST 5 min after intravenous injection, c) is the tissue distribution map of TP@CST and CST 30 min after intravenous injection, and d) is the tissue distribution map of TP@CST and CST 12 h after intravenous injection. The pharmacokinetic parameters of TP@CST and TP are shown in Table 1. The concentrations of both TP@CST and TP reached their peak values ​​after administration and then showed a decreasing trend, with TP decreasing at a significantly faster rate than TP@CST. The comparison of pharmacokinetic parameters showed that TP belonged to a two-compartment model, while the TP@CST group belonged to a three-compartment model. The half-life of TP@CST in mice was prolonged to 9.71 h, and the residence time of the drug in the body was 12.32 h, while the maximum concentration C max Both the clearance rate (CL) and the concentration of TP@CST were higher than those of TP, indicating that the bioavailability of the TP@CST group was superior to that of the TP group. Compared with the TP group, TP@CST had a longer duration of action and a slower release rate in vivo, enabling it to effectively penetrate and accumulate in bone metastases. Its acute toxicity was also significantly reduced in the liver, kidneys, and healthy bone tissue.

[0046] Table 1. Pharmacokinetic parameters of TP@CST and TP The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention are included within the protection scope of the present invention.

Claims

1. A method for preparing an aromatic thioketal-modified chondroitin sulfate derivative, characterized in that, Includes the following steps: (1) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the aromatic thioketal solution and react in the dark for 1 to 5 hours to obtain the aromatic thioketal activated solution. (2) Mix the aromatic thioketal activation solution and chondroitin sulfate solution, and react under nitrogen protection in the dark for 12 to 36 hours. Add anhydrous diethyl ether to the reaction solution to produce a precipitate and filter it. Dry and purify the precipitate to obtain the aromatic thioketal modified chondroitin sulfate derivative. The method for preparing the aromatic thioacetal is as follows: 9-fluorenone and 4-mercaptobutyric acid are mixed and dissolved, an acid catalyst is added, and the reaction is carried out at room temperature in the dark for 12 to 36 hours. Saturated sodium bicarbonate solution is added to the reaction solution to precipitate the product. The product is then filtered, washed, and recrystallized to obtain the aromatic thioacetal. The mass ratio of the aromatic thioacetal to chondroitin sulfate is 1:2 to 5. The purification method in step (2) is as follows: the dried precipitate is dissolved in water and dialyzed in a dialysis bag with a MWCO of 3500 ~ 14000 Da. The dialysate is freeze-dried to obtain an aromatic thioacetal modified chondroitin sulfate derivative.

2. The method for preparing the aromatized thioketal-modified chondroitin sulfate derivative according to claim 1, characterized in that, The molar ratio of 9-fluorenone and 4-mercaptobutyric acid is 1:1 to 5, and the solvent for mixing and dissolving is dichloromethane; the acid catalyst is trifluoroacetic acid, and the molar ratio of the acid catalyst to 9-fluorenone is 1:2 to 5.

3. The method for preparing the aromatized thioketal-modified chondroitin sulfate derivative according to claim 1, characterized in that, The mass ratio of N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and aromatic thioacetal is 1:2 ~ 5:6 ~ 15; the concentration of the aromatic thioacetal solution is 100 ~ 200 mg / mL, and the solvent is N,N-dimethylformamide.

4. The method for preparing the aromatized thioketal-modified chondroitin sulfate derivative according to claim 1, characterized in that, The concentration of the chondroitin sulfate solution is 100-1000 mg / mL, and the solvent is water.

5. An aromatic thioketal-modified chondroitin sulfate derivative prepared by the preparation method according to any one of claims 1 to 4.

6. The use of an aromatic thioketal-modified chondroitin sulfate derivative prepared by the method according to any one of claims 1 to 4, or the aromatic thioketal-modified chondroitin sulfate derivative according to claim 5, in the preparation of bone repair drugs; characterized in that, The bone repair mentioned refers to the repair of bone damage caused by tumor bone metastasis.

7. The application according to claim 6, characterized in that, The aromatic thioketal-modified chondroitin sulfate derivative is used as a drug carrier to load triptolide.

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