An amorphous calcium phosphate composite hydrogel for adjuvant therapy after osteosarcoma surgery and its preparation method

Abstract

This invention provides an amorphous calcium phosphate composite hydrogel for adjuvant therapy after osteosarcoma surgery and its preparation method, belonging to the field of biomedical materials. In this composite material, epigallocatechin gallate (EGCG) is loaded onto amorphous calcium phosphate (ACP) via co-precipitation to form an EGCG-ACP complex, which is then coated with hyaluronic acid to obtain a HA / (EGCG-ACP) precursor material. Next, a hydrogel precursor solution is constructed using chitosan and sodium β-glycerophosphate. Combining these two components yields the composite hydrogel. Utilizing its temperature-sensitive properties and the injectable nature of the precursor solution, a temperature-sensitive adjuvant therapy system for osteosarcoma surgery is formed. This composite hydrogel can effectively promote the repair of bone defects while clearing residual osteosarcoma cells and inhibiting tumor metastasis.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, and in particular to a method for preparing HA / (EGCG-ACP) and an adjuvant therapy material for osteosarcoma surgery, and its application. Background Technology

[0002] Osteosarcoma is a highly malignant bone tumor that occurs during the rapid growth phase of bone and commonly affects children and adolescents. Current treatment options include neoadjuvant chemotherapy, surgical removal, and adjuvant chemotherapy. However, postoperative metastasis of osteosarcoma cells leads to a poor prognosis and low long-term survival rates. Current research indicates that the osteosarcoma microenvironment is acidic, with a significant increase in the production of mineralized extracellular matrix. This matrix is ​​denser and more invasive, easily causing bone defects that are difficult to heal spontaneously. Furthermore, this microenvironment acts as a biological barrier, hindering the penetration of chemotherapy drugs, resulting in poorer efficacy and greater harm to the body from postoperative adjuvant chemotherapy.

[0003] Chemotherapy drugs commonly used include high-dose methotrexate, doxorubicin, and cisplatin. However, traditional chemotherapy drugs suffer from significant side effects and high toxicity to normal cells. Furthermore, due to a lack of targeted therapy, these drugs are dispersed throughout the body, resulting in low drug concentrations at the tumor site and affecting efficacy. Effective polyphenols from plants offer unique advantages in cancer treatment. For example, tea polyphenols, a collective term for polyphenolic substances in tea leaves, include catechins, phenolic acids, flavonoids, and anthocyanins, which possess anti-tumor and anti-inflammatory effects. Epigallocatechin gallate (EGCG), a major catechin component, has the potential to promote bone regeneration and inhibit osteosarcoma in bone tissue engineering. However, polyphenolic drugs suffer from low bioavailability.

[0004] Amorphous calcium phosphate (ACP) is an important precursor for bone mineralization, possessing extremely high osteogenic activity. Its particle size distribution, ranging from tens to hundreds of nanometers, facilitates penetration into the tumor microenvironment. Its loose structure is advantageous for loading large doses of drugs while maintaining drug stability and activity, thus improving bioavailability. However, ACP exhibits poor stability, with rapid solubility and degradation under acidic conditions. Despite its broad application potential in the biomedical field, these drawbacks significantly limit its application. The literature *Injectable and body temperature sensitive hydrogels based on chitosan and hyaluronic acid for pH sensitive drug release* (Wei Zhang, Xin Jin, Heng Li, Run-run Zhang, Cheng-wei Wu, Carbohydrate Polymers, 2018, 186: 82-90) describes the development of an injectable temperature-sensitive hydrogel based on chitosan (CS), hyaluronic acid (HA), and sodium β-glycerophosphate (GP) for pH-responsive drug delivery. The system is an injectable solution at room temperature, transforms into a gel at body temperature (37°C), and triggers drug (such as doxorubicin DOX) release in an acidic environment (such as the tumor microenvironment, pH 4.00). However, the system's component design lacks functionality, the application scenarios of the composite components are unclear, it does not take into account the dual needs of tumor treatment and tissue repair, it is not precisely designed for specific tumor types, and it uses the highly toxic chemotherapy drug doxorubicin.

[0005] Therefore, there is a need for a composite hydrogel material that can be used as an adjunct therapy to remove residual tumor cells, inhibit tumor cell metastasis, and promote bone defect repair after osteosarcoma surgery. Summary of the Invention

[0006] In view of the shortcomings of existing materials and technologies, the purpose of this invention is to provide a method for preparing HA / (EGCG-ACP) material and an amorphous calcium phosphate composite hydrogel for adjuvant treatment after osteosarcoma surgery, as well as its application.

[0007] The objective of this invention is achieved through the following technical solution: A HA / (EGCG-ACP) material is prepared by the following steps: Epigallocatechin gallate (EGCG) was pre-mixed with a phosphorus solution to obtain an EGCG-phosphorus solution. Calcium solution was slowly added dropwise to the EGCG-phosphorus solution, and after stirring and mixing, the mixture was quickly filtered or centrifuged and freeze-dried to obtain EGCG-ACP. EGCG-ACP was then added to an aqueous hyaluronic acid solution, mixed thoroughly, and freeze-dried to obtain HA / (EGCG-ACP).

[0008] Preferably, the phosphorus solution is a soluble orthophosphate solution, such as diammonium hydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, or potassium dihydrogen phosphate; the concentration of the phosphorus solution is 10 mM-30 mM.

[0009] Preferably, the calcium solution is a soluble calcium salt solution, such as anhydrous calcium chloride or calcium nitrate; the concentration of the calcium solution is 10mM-30mM.

[0010] Preferably, the concentration of EGCG added is 1~8 mg / ml.

[0011] Preferably, EGCG is pre-mixed with the phosphorus solution until homogeneous, and the mixing time is greater than or equal to 10 minutes.

[0012] Preferably, the calcium solution is added dropwise to the mixed solution of EGCG and phosphorus solution.

[0013] Preferably, the volume ratio of calcium solution to EGCG-phosphorus solution is 0.5 to 2.

[0014] Preferably, the mixing time between the calcium solution and the EGCG-phosphorus solution is 30 seconds to 5 minutes.

[0015] Preferably, all the above mixing and reaction processes are carried out in an ice-water bath.

[0016] Preferably, the freeze-drying time is 12h to 48h, and the temperature is -60℃ to -80℃.

[0017] Preferably, the mass ratio of the EGCG-ACP particles to the hyaluronic acid solution is (10~80):(10~30).

[0018] More preferably, the freeze-drying time is 24h~48h, and the temperature is -60℃~-80℃.

[0019] The HA / (EGCG-ACP) prepared by the above method.

[0020] A method for preparing an amorphous calcium phosphate composite hydrogel for adjuvant therapy after osteosarcoma surgery includes the following steps: HA / (EGCG-ACP) and sodium β-glycerophosphate were dissolved in water to obtain a mixed solution of ACP. Chitosan was dissolved in acetic acid solution. The ACP mixed solution was added dropwise to the chitosan acetic acid solution and stirred slowly until homogeneous to obtain the precursor solution of the composite hydrogel.

[0021] Preferably, the mass of HA / (EGCG-ACP) is 10mg~120mg.

[0022] Preferably, the mass concentration of sodium β-glycerophosphate is 0.2~0.6 g / ml.

[0023] Preferably, the concentration of the acetic acid solution is 0.08M~0.1M. The mass concentration of chitosan is 0.01~0.03g / ml.

[0024] Further preferred chitosans have a degree of deacetylation ≥95% and a viscosity of 100~200 mpa.s.

[0025] Preferably, the volume ratio of chitosan acetate solution to ACP mixed solution is 4:1.

[0026] Preferably, all the solutions are pre-cooled at 0℃~4℃. The mixing process is carried out at 0℃~4℃, and an ice-water bath is used to control the temperature to ensure stability.

[0027] Preferably, the stirring time between the ACP mixed solution and the chitosan acetate solution is 1-2 hours.

[0028] Preferably, the composite hydrogel precursor solution is in a liquid state at 4℃~24℃, and transforms into a gel solid state when heated to 25℃~40℃.

[0029] The amorphous calcium phosphate composite hydrogel obtained by the above preparation method.

[0030] The HA / (EGCG-ACP) and composite hydrogel obtained by the above preparation method are used in the adjuvant treatment and bone tissue repair after osteosarcoma surgery, and can be used to prepare drugs or materials related to adjuvant treatment and bone tissue repair after osteosarcoma surgery or other tumor surgery.

[0031] Compared with the prior art, the present invention has the following beneficial effects: (1) The HA / (EGCG-ACP) and amorphous calcium phosphate composite hydrogel provided by the present invention utilizes its temperature sensitivity and injectable precursor solution to adapt to bone defect sites and fill irregular wounds.

[0032] (2) The HA / (EGCG-ACP) and amorphous calcium phosphate composite hydrogel provided by the present invention forms a drug library in situ after tumor surgery, which releases drugs in a long-term sustained manner, continuously clears residual tumor cells and effectively inhibits tumor cell metastasis, reducing the damage caused by the inability to accurately locate the tumor cells in traditional chemotherapy.

[0033] (3) ACP has extremely high osteogenic activity, effectively assisting in the repair of bone defects. As a drug carrier, it improves the bioavailability of EGCG and protects the activity of EGCG.

[0034] (4) The present invention has the advantages of simple preparation process and low cost. Attached Figure Description

[0035] Figure 1 The image shows the XRD patterns of ACP, EGCG-ACP, and HA / (EGCG-ACP) in Embodiment 1 of the present invention.

[0036] Figure 2 This is a schematic diagram of an inverted vial of an amorphous calcium phosphate composite hydrogel in an embodiment of the present invention; wherein, a and a' are composite hydrogels of Example 2, a is in a fluid state at 4°C, and a' is in a solid state after incubation at 37°C; b and b' are composite hydrogels of Example 3, b is in a fluid state at 4°C, and b' is in a solid state after incubation at 37°C.

[0037] Figure 3 The rheological properties of the composite hydrogels in the embodiments of the present invention are shown below; wherein (a) shear thinning of the hydrogel, (b) temperature sensitivity of the blank hydrogel, (c) temperature sensitivity of the EGCG-ACP hydrogel in Embodiment 3 of the present invention, and (d) temperature sensitivity of the HA / (EGCG-ACP) hydrogel in Embodiment 2 of the present invention.

[0038] Figure 4 The image shows the infrared spectrum of the composite hydrogel in Example 2 of this invention.

[0039] Figure 5 These are scanning electron microscope images of the composite hydrogels of the present invention; where (a) is an image of the composite hydrogel of Example 2 and (b) is an image of the composite hydrogel of Example 3.

[0040] Figure 6 The image shows the cytotoxicity of the composite hydrogel to mouse mesenchymal stem cells (BMSCs) and osteosarcoma cells 143B in Example 2 of this invention, where (a) represents BMSC cells and (b) represents 143B cells.

[0041] Figure 7 This is an image of alizarin red staining of calcium nodules in the composite hydrogel of Example 2 of the present invention after 21 days.

[0042] Figure 8 The rheological properties of the composite hydrogel in Example 4 of this invention are shown in the diagram; where (a) represents shear properties and (b) represents temperature-sensitive properties.

[0043] Figure 9This is a cytotoxicity diagram of the composite hydrogel on mouse mesenchymal stem cells (BMSCs) and osteosarcoma cells 143B in Example 5 of the present invention; where (a) represents BMSC cells and (b) represents 143B cells. Detailed Implementation

[0044] The specific implementation of the present invention will be described below with reference to examples. The implementation and protection of the present invention are not limited thereto; if any process is not specifically described in detail below, those skilled in the art can refer to existing technology for implementation or understanding. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0045] Example 1 A method for preparing HA / (EGCG-ACP) material includes the following steps: Dissolve 1.11 g of anhydrous calcium chloride in 500 mL of deionized water to prepare a 20 mM calcium solution. Dissolve 1.32 g of diammonium hydrogen phosphate in 500 mL of deionized water to prepare a 20 mM phosphorus solution. Pre-cool both the calcium and phosphorus solutions. Place 10 mL of the phosphorus solution in a polytetrafluoroethylene beaker, weigh 20 mg of EGCG and dissolve it in the phosphorus solution, and stir at 1400 r / min for 20 min. After mixing thoroughly, add the same volume of calcium solution dropwise. After the calcium solution has been completely added, react for 30 seconds, then immediately transfer the solution to a vacuum filtration device. Freeze-dry the filter residue to obtain EGCG-ACP particles.

[0046] Weigh 30 mg of hyaluronic acid and dissolve it in 20 ml of water to obtain a hyaluronic acid solution. Take 10 mg of EGCG-ACP and disperse it evenly in the hyaluronic acid solution. The mass ratio of EGCG-ACP to HA is 10:30. Freeze-dry the solution to obtain HA / (EGCG-ACP) and characterize the material by XRD.

[0047] In this embodiment, the XRD pattern of the prepared HA / (EGCG-ACP) is as follows: Figure 1 As shown in the figure, under these preparation conditions, the obtained powder exhibits a diffuse diffraction peak similar to that of ACP at around 30°, indicating that the material synthesized by this precipitation method is an amorphous phase. The materials obtained after loading EGCG and after coating EGCG-ACP with hyaluronic acid also remain amorphous phases.

[0048] Example 2 A method for preparing an amorphous calcium phosphate composite hydrogel for adjuvant therapy after osteosarcoma surgery includes the following steps: Take 40 mg of the HA / (EGCG-ACP) material obtained in Example 1 and 1.2 g of sodium β-glycerophosphate, dissolve them in 2 ml of deionized water to obtain an ACP mixed solution, and pre-cool at 4°C. Dilute 2.87 ml of glacial acetic acid to 500 ml to obtain a 0.1 M acetic acid solution, and dissolve 0.24 g of chitosan in 8 ml of the 0.1 M acetic acid solution to obtain a chitosan-acetic acid solution, and pre-cool at 4°C. Under an ice-water bath environment, add the ACP mixed solution dropwise to the chitosan-acetic acid solution and stir at a uniform speed for 2 hours to obtain a hydrogel precursor solution at low temperature.

[0049] The solution was transferred to a glass vial. This hydrogel is liquid below 25°C and solid at 37°C. Figure 2 As shown. The rheological properties of the hydrogel were tested, and the results are as follows. Figure 3 As shown. Among them, Figure 3 (a) shows the shear performance test of the blank hydrogel and Examples 2 and 3. The results show that the hydrogel can be thinned by shearing and has injectable properties. Figure 3 The results in (b) indicate that the sol-gel transition temperature of the blank hydrogel is 40.15℃, and the gel transition point of Example 3 is 37.61℃. Figure 3 As shown in (c) in the figure. Figure 3 The results in (d) indicate that Example 2 underwent a sol-gel transition at 29.39°C. These results demonstrate that hydrogels possess temperature-sensitive properties, and the addition of hyaluronic acid lowers the gel transition temperature. The solidified hydrogel was then treated with liquid nitrogen, freeze-dried, and subjected to infrared spectroscopy. Figure 4 As shown, after chitosan dissolves in an acidic solution, -NH3 appears. + The characteristic peaks of the hydrogel showed amino protonation. The addition of HA-containing materials shifted the position of the characteristic peaks, indicating that hydrogen bonds formed between the carboxyl groups in hyaluronic acid molecules and the amino groups in chitosan. Scanning electron microscopy revealed that... Figure 5 As shown in (a), the hydrogel with added HA / (EGCG-ACP) has a porous structure with micron-sized pores, roughly distributed in the range of 30-40 μm, and the pore distribution and pore size are uniform.

[0050] Liquid hydrogel was evenly spread into the wells of a 96-well plate, incubated at 37°C for solidification, and then sterilized. Mouse mesenchymal stem cells (BMSCs) and human osteosarcoma cells (143B) were cultured and seeded at a density of 5000 cells / well into the treated 96-well plates. After culturing at 37°C with 5% CO2 for 72 hours, the cell viability of BMSCs and 143B cells after co-culturing with the hydrogel was detected by the CCK8 assay. Figure 6As shown, after 3 days of co-culture, the activity of BMSC cells remained basically unchanged compared with the blank control group, while the activity of 143B cells decreased significantly. The composite hydrogel in this embodiment has almost no cytotoxicity to normal cells, but has a significant inhibitory effect on osteosarcoma cells.

[0051] After the hydrogel solidified, it was extracted with culture medium for 3 days, and the extract was filtered and sterilized. Third-generation mouse mesenchymal stem cells (mBMSCs) were then introduced at a concentration of 1 × 10⁻⁶. 4 Cells were seeded at a density of [insert density here] into 48-well plates and cultured until 80% confluence. The culture medium was then replaced with osteogenic medium and incubated at 37°C with 5% CO2 for 21 days. After removing the medium, the cells were washed with PBS and stained with Alizarin Red. The nodules were then observed and photographed using a stereomicroscope. Figure 7 As shown, the composite hydrogel material in this embodiment has good osteogenic properties, forming a large number of calcium nodules within a 21-day cycle.

[0052] Example 3 The preparation method was basically the same as in Example 2, except that an equal amount of EGCG-ACP prepared in Example 1 was added to replace HA / (EGCG-ACP). Rheological property tests revealed that, compared to Example 2, the composite hydrogel obtained in Example 3 was shear-thinner, could be applied by injection, and also exhibited temperature-sensitive properties, but its sol-gel transition temperature was higher, requiring curing at around 37°C. Scanning electron microscopy observation of Example 3 revealed… Figure 5 As shown in (b), this hydrogel has a porous structure, but the pore size is uneven and some pore structures are not fully intact. Figure 6 As shown, Example 3 still exhibits high antitumor activity. However, Figure 7 The osteogenic experimental results show that Example 3 exhibits weaker osteogenic capacity compared to Example 2, with fewer calcium nodules formed. Compared to Example 2, Example 3 has poorer temperature sensitivity; the increased gel transition temperature limits its temperature response in the human body. The non-uniform structure of Example 3 leads to poorer stability, and its osteogenic properties are somewhat reduced.

[0053] Example 4 The preparation method was basically the same as in Example 2, except that the mass of HA / (EGCG-ACP) added was 10 mg. The same rheological property tests were performed on Example 4, and the results are as follows: Figure 8As shown in the figure. Similar to Example 2, Example 4 also exhibits injectable properties through shear thinning and is temperature-sensitive, transitioning to a gel at higher temperatures. However, unlike Example 2, Example 4 has a higher transition temperature, requiring approximately 38.43°C to cure. This indicates that the content of HA / (EGCG-ACP) has a significant impact on the rheological properties of the composite material. A decrease in HA / (EGCG-ACP) content increases the sol-gel transition temperature of the composite material, even exceeding human body temperature, thus limiting its applications.

[0054] Example 5 The preparation method was basically the same as in Examples 1 and 2, except that 80 mg of EGCG-ACP was dispersed in 30 mg of hyaluronic acid solution, and the mass ratio of EGCG-ACP to HA was 80:30. The biocompatibility of Example 5 was tested, and the results are as follows: Figure 9 As shown. Compared to Example 2, Example 5 exhibits poorer biocompatibility. Although it demonstrates excellent killing power against 143B cells, Example 5 shows high cytotoxicity against normal mouse mesenchymal stem cells (BMSCs). This indicates that higher concentrations of EGCG-ACP may conceal toxicity issues, thus requiring strict control of its dosage.

Claims

1. A method for preparing HA / (EGCG-ACP) material, characterized in that, Includes the following steps: EGCG was pre-mixed with a phosphorus solution to obtain an EGCG-phosphorus solution. Calcium solution was slowly added dropwise to the EGCG-phosphorus solution. After stirring and mixing, the mixture was quickly filtered or centrifuged and freeze-dried to obtain EGCG-ACP. EGCG-ACP was added to an aqueous hyaluronic acid solution, mixed thoroughly, and freeze-dried to obtain HA / (EGCG-ACP).

2. The method for preparing HA / (EGCG-ACP) according to claim 1, characterized in that, The calcium solution is a soluble calcium salt solution with a concentration of 10mM-30mM; the phosphorus solution is a soluble phosphate solution with a concentration of 10mM-30mM.

3. The method for preparing HA / (EGCG-ACP) according to claim 1, characterized in that, The concentration of EGCG added is 1~8 mg / ml, and the mass ratio of the EGCG-ACP particles to the hyaluronic acid solution is (10~80):(10~30).

4. The method for preparing HA / (EGCG-ACP) according to claim 1, characterized in that, The volume ratio of calcium solution to EGCG-phosphorus solution is 0.5~2, and the mixing time is 30s~5min.

5. HA / (EGCG-ACP) prepared by the preparation method according to any one of claims 1 to 4.

6. A method for preparing an amorphous calcium phosphate composite hydrogel for adjuvant treatment after osteosarcoma surgery, characterized in that, Includes the following steps: Chitosan is dissolved in acetic acid solution, and sodium β-glycerophosphate and the HA / (EGCG-ACP) of claim 5 are dissolved in water to obtain ACP solution. The ACP solution is added dropwise to the chitosan solution and slowly mixed evenly to obtain the precursor solution of the composite hydrogel.

7. The method for preparing amorphous calcium phosphate composite hydrogel according to claim 6, characterized in that, The acetic acid solution has a concentration of 0.1M, the chitosan has a mass concentration of 0.01~0.03g / ml, the sodium β-glycerophosphate has a mass concentration of 0.2~0.6g / ml, and the HA / (EGCG-ACP) has a mass of 10mg~120mg.

8. The method for preparing amorphous calcium phosphate composite hydrogel according to claim 6, characterized in that, The composite hydrogel precursor solution is in a liquid state at 4℃~24℃, and transforms into a gel solid state when heated to 25℃~40℃.

9. The amorphous calcium phosphate composite hydrogel obtained by the preparation method according to any one of claims 6 to 8.

10. The use of the HA / (EGCG-ACP) of claim 5 or the amorphous calcium phosphate composite hydrogel of claim 9 in the preparation of drugs or materials related to adjuvant therapy and bone tissue repair after osteosarcoma surgery or other tumor surgery.

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