Preparation method and application of injectable bismuth-based metal repairing agent

Abstract

The invention discloses a preparation method of an injectable bismuth-based metal repairing agent, which comprises the following steps of: putting weighed bismuth, indium, tin and zinc metals into a crucible, putting the crucible into a vacuum high-temperature smelting furnace, heating the mixed metals in a vacuum environment until the mixed metals are completely molten by adjusting the smelting temperature, stirring and shaking the formed molten alloy, and then cooling the molten alloy to obtain the injectable bismuth-based metal repairing agent. And ensuring that four elements of bismuth, indium, tin and zinc are uniformly distributed in the alloy, naturally cooling the fully and uniformly mixed molten alloy, and solidifying to obtain the solid injectable bismuth-based metal repairing agent. The repairing agent can be used for treating osteosarcoma, firstly, the repairing agent is injected into a medullary cavity, so that the repairing agent is solidified in situ to form an internal fixing structure, then a magnetocaloric effect is triggered through an alternating magnetic field to induce osteosarcoma immunogenic death, and integrated treatment of tumor removal and bone repair is achieved. According to the scheme, the biological safety and the osseointegration function are achieved, the magnetic-thermal conversion process is efficient, and the good treatment effect on osteosarcoma is achieved.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation method of an injectable bismuth-based metal repair agent and application thereof in treating osteosarcoma, and belongs to the technical fields of biomedical materials and tumor treatment. BACKGROUND

[0002] Osteosarcoma is the most common malignant bone tumor in adolescents, and its clinical treatment faces severe challenges. At present, the standard treatment relies on surgical resection combined with adjuvant chemotherapy. This treatment has the following shortcomings: on the one hand, surgery is difficult to completely remove the infiltrating tumor cells, and the local recurrence rate is as high as 30-50%, and about 40% of patients will develop resistance to methotrexate, cisplatin and other chemotherapeutic drugs, resulting in a long-term stagnation of the 5-year survival rate at 60-70%; on the other hand, the repair of large bone defects after surgery still needs to rely on autologous bone transplantation or metal prosthesis implantation. Among them, autologous bone transplantation has the defects of complications in the donor area and insufficient bone mass. The use of metal prosthesis implantation is prone to aseptic loosening due to the mismatch between the mechanical modulus of titanium alloy and the natural bone tissue, and the secondary revision rate within 10 years is more than 35%; more importantly, the existing repair materials completely lack the anti-tumor function and cannot solve the problem of recurrence caused by micro-residual lesions.

[0003] In recent years, it has been found that mild magnetic hyperthermia (42-45℃) can specifically induce immunogenic death (ICD) of tumor cells, activate dendritic cells to present antigens and initiate CD8⁺T cell immune response through exposure of calreticulin (CRT) and release of ATP / HMGB1 and other damage-associated molecular patterns (DAMPs), and this mechanism is particularly effective for osteosarcoma cells. Based on this, the use of alternating magnetic field (AMF) to stimulate the heat generation of magnetic materials provides a new idea for the treatment of osteosarcoma in the form of killing tumor cells. However, the traditional magnetic material (such as Fe3O4 nanoparticles) has low specific absorption rate, and the penetration depth in bone tissue is insufficient, and high-dose use is prone to iron overload oxidative damage, and the use of gold nanorods has sufficient penetration depth, but there is a risk of liver accumulation toxicity.

[0004] Gallium-based and bismuth-based low-melting-point injectable metal materials have gradually replaced pure magnetic materials due to their minimally invasive implantation characteristics, and have attracted more attention. However, the traditional gallium-based alloy is in a liquid state at room temperature and is easy to inject and implement, but it is easy to migrate to non-target areas, and the released Ga 3+ ions can significantly inhibit the differentiation of osteoblasts; and the traditional bismuth-based alloy (for example, Wood's alloy) has a melting point of about 70℃, but contains more than 10% of the highly toxic cadmium (Cd) and more than 25% of lead (Pb), and long-term implantation will induce chronic inflammation and neurotoxicity of bone tissue.

[0005] In view of the above, the present case proposes an injectable bismuth-based metal repair agent with high-efficiency magnetic heat conversion, biological safety, and bone integration function. SUMMARY

[0006] The purpose of the present application is to solve the above problems, provide a preparation method and use of an injectable bismuth-based metal repair agent to improve the application effect of fluid metal in the magnetic heat treatment of osteosarcoma.

[0007] The technical solution of the present application is: a preparation method of an injectable bismuth-based metal repair agent, comprising the following steps:

[0008] Step S1: Put the weighed bismuth, indium, tin, and zinc metals into a crucible and place it in a vacuum high-temperature smelting furnace. Adjust the smelting temperature to heat the mixed metals to a molten state in a vacuum environment;

[0009] Step S2: After the mixed metals in step S1 are completely melted, stir and shake the formed molten alloy to ensure that the four elements of bismuth, indium, tin, and zinc are uniformly distributed in the alloy, obtaining a molten system with uniform composition;

[0010] Step S3: The fully mixed molten alloy obtained in step S2 is naturally cooled, and after solidification, a solid injectable bismuth-based metal repair agent is obtained.

[0011] The present application also provides the use of the injectable bismuth-based metal repair agent prepared by the above method, i.e. it is applied to the treatment of osteosarcoma. The main application process is: first inject the bismuth-based metal repair agent into the medullary cavity, allowing it to solidify in situ to form an internal fixation structure, then trigger the magnetic heat effect through an alternating magnetic field (AMF) to induce immunogenic cell death (ICD) of osteosarcoma, achieving integrated treatment of tumor removal and bone repair.

[0012] Preferably, when the alternating magnetic field (AMF) is clinically transformed, a nano material-AMF synergistic design method can be used, which requires matching the frequency and intensity of the corresponding AMF with the Curie temperature and specific absorption rate characteristics of the magnetic nanoparticles used. When using a Curie temperature nano material with self-regulating temperature control function (such as a two-dimensional ferromagnetic material or a nanocrystalline alloy), the corresponding AMF intensity also needs to be heated to its Curie point. In addition, by setting local coils to match the tumor dissection position, such as solenoid coils or "saddle-shaped" coils for arms or legs, the magnetic field energy can be highly concentrated on the affected area, minimizing the impact on the whole body; the output power of the AMF can be adjusted according to the size, depth of the tumor, and the concentration of the nanoparticles, while the temperature and time of the heat dose are precisely controlled to ensure effective induction of immunogenic cell death, so as to effectively improve the compatibility of the alternating magnetic field (AMF) with the treatment needs.

[0013] Compared with the prior art, the melting point of the injectable bismuth-based metal repair agent obtained after the technical scheme is adopted is about 50 DEG C, the injectable bismuth-based metal repair agent can be injected into the medullary cavity where the tumor is located in a fluid state by heating a syringe, and can be solidified into a solid stent at 37 DEG C body temperature in only 10 seconds, perfectly fits the medullary cavity morphology and avoids migration, and further improves the treatment effect in combination with the magnetic heating effect generated under an alternating magnetic field (AMF), the ICD immune response is triggered at a self-limiting temperature of about 45 DEG C. Moreover, the technical scheme is lead and cadmium free, completely avoids the neurotoxicity and osteonecrosis risk of traditional alloys, and finally can safely and integrally realize tumor immunotherapy and bone structure reconstruction, and the technical effect is very remarkable. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 A magnetic heating temperature image of a bismuth-based fluid metal in the injectable bismuth-based metal repair agent of the same volume of the present application within 60s under different magnetic field strengths;

[0015] Figure 2 A schematic diagram of the relationship between the cell viability percentage (Cvi%) of the examples and comparative examples 1-3 under the evaluation method of the present application;

[0016] Figure 3 A schematic diagram of the relationship between the proportion of apoptotic cells (including early apoptosis and late apoptosis) in the total cells in the examples and comparative examples 1-3 under the flow cytometry evaluation method of the present application. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical scheme and advantages of the present application more clear and explicit, the present application will be further described below in combination with specific examples, but should not be understood as limiting the protection scope of the above-mentioned subject matter of the present application to the following examples. The cell model involving mouse osteosarcoma cells (K7M2) is a commonly used model generally recognized by those skilled in the art, and the present application has no special requirements therefor.

[0018] According to the technical scheme of the present application, the present application provides a preparation method of an injectable bismuth-based metal repair agent for treating osteosarcoma, comprising the following steps: placing weighed bismuth, indium, tin and zinc metals into a crucible and placing them in a vacuum high-temperature smelting furnace, adjusting the smelting temperature to heat the mixed metals to a molten state in a vacuum environment; after the mixed metals are completely molten, stirring and shaking the formed molten alloy to ensure that the four elements of bismuth, indium, tin and zinc are uniformly distributed in the alloy, and a molten system with uniform composition is obtained; the fully mixed molten alloy is naturally cooled, and after solidification, the injectable bismuth-based metal repair agent in a solid state at room temperature is obtained.

[0019] Further, in the process of selecting the metal raw materials, each metal raw material with a purity of not less than 99.99% is selected, and bismuth (Bi) is 34.0%, indium (In) is 49.6%, tin (Sn) is 15.9%, and zinc (Zn) is 0.5% by mass percentage; the component ratio is a specific embodiment obtained by the inventors after several studies, and the effect is more ideal.

[0020] In the smelting process, the smelting temperature is set to 300-350℃; after the mixed metal is completely melted, the stirring speed is 200-400 N / RPM, the shaking frequency is 100-150 RPM, and the shaking amplitude is 20-30 mm during the stirring and shaking treatment. Preferably, the stirring speed is 300 N / RPM, the shaking frequency is 120 RPM, and the shaking amplitude is 25 mm.

[0021] In the natural cooling process, the cooling time is 1-2 h, preferably 1.5 h; and during or after the cooling process, the injectable bismuth-based metal repair agent (LM) is analyzed and verified by using a scanning electron microscope and an energy spectrum to confirm whether the overall chemical composition is consistent with the design or standard. If it meets the ASTM and ISO standards, it can be used for experiments.

[0022] As shown in Figure 1 , the bismuth-based fluid metal in the injectable bismuth-based metal repair agent prepared in the present case is obviously affected by the field strength of the alternating magnetic field. The present application uses mouse osteosarcoma cells (K7M2) as a cell model, and performs osteosarcoma treatment intervention through different examples and comparative examples, and then evaluates the killing effect of different examples and comparative examples 1-3 on K7M2 osteosarcoma cells through CCK8 and flow cytometry evaluation methods, and the specific cases are as follows.

[0023] Example

[0024] The injectable bismuth-based metal repair agent (LM) prepared is injected into K7M2 cells, and an alternating magnetic field (AMF) intervention is applied.

[0025] Comparative Example 1

[0026] K7M2 cells are routinely cultured without any intervention.

[0027] Comparative Example 2

[0028] Only the injectable bismuth-based metal repair agent (LM) is injected into K7M2 cells without alternating magnetic field (AMF) intervention.

[0029] Comparative Example 3

[0030] Only an alternating magnetic field (AMF) intervention is applied to K7M2 cells without injecting the injectable bismuth-based metal repair agent (LM).

[0031] I. CCK8 evaluation method

[0032] The specific steps are as follows: K7M2 cells in the logarithmic growth phase are inoculated in a 96-well plate at a certain density (1X10 4 Cells) and cultured until the cells adhere, then the intervention experiment is performed on the examples and comparative examples 1-3. After the intervention, an appropriate amount of CCK8 reagent is added to each well, incubated at 37°C for 2 hours, and the absorbance value at 450nm wavelength is detected using an enzyme label instrument. The cell survival rate of each group is calculated according to the absorbance.

[0033] As shown in Figure 2 , the vertical coordinate of the graph is the percentage of cell viability (Cvi%). The cell viability of comparative example 1 is maintained at a high level, and the cell viability of comparative examples 2 and 3 is slightly lower than that of comparative example 1, but the difference is not significant; the cell viability of the example is significantly reduced and is significantly lower than that of the other three comparative examples. This experiment shows that the alternating magnetic field combined with the injectable bismuth-based metal repair agent has a significant killing effect on K7M2 osteosarcoma cells, and at the same time verifies the effectiveness of the magnetic heating effect in inducing tumor cell death.

[0034] II. Flow cytometry evaluation method

[0035] The specific steps are as follows: K7M2 cells after intervention of the examples and comparative examples 1-3 are collected, washed with PBS for 2 times, and stained according to the Annexin V-FITC / PI apoptosis detection kit instructions, that is, after adding Annexin V-FITC and PI dye solution and incubating in the dark for 15 min, the apoptosis of the cells is detected by BD C6 Plus flow cytometry, and the proportion of apoptotic cells (including early and late apoptosis) in the total cells is analyzed.

[0036] As shown in Figure 3 , the Q2 quadrant represents late apoptotic cells, the Q3 quadrant represents viable cells, and the Q4 quadrant represents early apoptotic cells. The proportion of viable cells (Q3) in comparative example 1 is high, and the proportion of apoptotic cells is extremely low; the proportion of apoptotic cells in comparative examples 2 and 3 is slightly higher than that in comparative example 1, but the change is not significant; the proportion of early and late apoptotic cells in the example is significantly increased, and the proportion of viable cells is significantly reduced, further confirming that the magnetic heating effect of the injectable bismuth-based metal repair agent triggered by the alternating magnetic field can effectively induce osteosarcoma cell apoptosis and efficiently play an anti-tumor role.

[0037] In the technical scheme of the present application, the key technology is to inject a new injectable bismuth-based metal repair agent and then apply an alternating magnetic field. The injectable bismuth-based metal repair agent is injected into the medullary cavity, solidified in situ to form an internal fixation structure, and the magnetic heat effect is triggered by the alternating magnetic field (AMF) to induce immunogenic cell death (ICD) of osteosarcoma. For the CCK8 evaluation method and the flow cytometry evaluation method, a person skilled in the art can perform routine operations according to the prior art, and the present application does not have special requirements for the selection and use of reagent samples and the like.

[0038] As can be seen from the above description, compared with the prior art, the melting point of the injectable bismuth-based metal repair agent obtained by the technical scheme of the present application is about 50℃, which can be injected into the medullary cavity where the tumor is located in a fluid state by heating the syringe. At 37℃, it only takes 10 seconds to solidify into a solid-state scaffold, perfectly fitting the medullary cavity morphology and avoiding migration. In combination with the magnetic heat effect generated under the alternating magnetic field (AMF), the temperature is self-limited to about 45℃ to trigger ICD immune response, further improving the treatment effect. Moreover, the present application does not contain lead and cadmium, completely avoiding the neurotoxicity and osteonecrosis risk of traditional alloys, and finally achieving safe and integrated tumor immunotherapy and bone structure reconstruction, with very significant technical effects.

[0039] The technical scheme, working process and implementation effect of the present application have been described in detail above. It should be noted that the described is only a typical example of the present application, and in addition to this, the present application can have other various specific implementation manners. Any technical scheme formed by equivalent replacement or equivalent transformation falls within the scope of the present application.

Claims

1. A method for the preparation of an injectable bismuth-based metal restorative characterized in that The method comprises the following steps: Step S1: Put the weighed bismuth, indium, tin and zinc metals into a crucible and place it in a vacuum high-temperature smelting furnace, and heat the mixed metals to a molten state under a vacuum environment by adjusting the smelting temperature; Step S2: After the mixed metals in step S1 are completely melted, the molten alloy formed is subjected to stirring and shaking treatment to ensure that the four elements of bismuth, indium, tin and zinc are uniformly distributed in the alloy, and a molten system with uniform composition is obtained; Step S3: The molten alloy uniformly mixed in step S2 is naturally cooled, and a solid-state injectable bismuth-based metal repair agent is obtained after solidification.

2. The method of claim 1, wherein: In step S1, each metal raw material with a purity of not less than 99.99% is accurately weighed according to the mass percentage: bismuth is 34.0%, indium is 49.6%, tin is 15.9%, and zinc is 0.5%, and then high-temperature smelting is performed.

3. The method of claim 1, wherein: In step S1, the smelting temperature is set to 300-350℃.

4. The method of claim 1, wherein: In step S2, the stirring speed is 200-400 N / RPM, the shaking oscillation frequency is 100-150 RPM, and the shaking amplitude is 20-30 mm.

5. The method of claim 1, wherein: In step S3, the cooling time is 1-2 h.

6. The method of claim 1, wherein: In step S3 or thereafter, the injectable bismuth-based metal repair agent is analyzed and verified using a scanning electron microscope and an energy spectrum to confirm whether the overall chemical composition is consistent with the design or standard.

7. Use of the injectable bismuth-based restorative agent prepared according to any one of claims 1 to 6, characterized in that: When it is applied to treat osteosarcoma, the bismuth-based metal repair agent is first injected into the medullary cavity to form an internal fixation structure in situ, and then the magnetic heating effect is triggered by an alternating magnetic field to induce immunogenic death of osteosarcoma, achieving integrated treatment of tumor removal and bone repair.

8. Use of an injectable bismuth-based metal restorative according to claim 7, characterized in that: When the alternating magnetic field is clinically transformed, a nano material-AMF collaborative design method is used, and the frequency and intensity of the corresponding AMF need to match the Curie temperature and specific absorption rate characteristics of the magnetic nanoparticles used.

9. Use of an injectable bismuth-based metal restorative according to claim 7, characterized in that: The coil of the alternating magnetic field is set to match the tumor anatomical position.

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