CN115006539A

CN115006539A - Composite porous scaffold and preparation method and application thereof

Info

Publication number: CN115006539A
Application number: CN202210507637.4A
Authority: CN
Inventors: 马立敏; 张余; 程实; 周洁龙
Original assignee: Guangdong General Hospital
Current assignee: Guangdong General Hospital
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2022-09-06
Anticipated expiration: 2042-05-11
Also published as: CN115006539B

Abstract

The invention provides a composite porous scaffold and a preparation method and application thereof. By coupling the composite nano particles with the microwave heat sensitization characteristic, the scaffold material integrating the functions of chemotherapy, immunotherapy and reconstruction is constructed. After the composite nano particles enter tumor cells, the microwave response releases chemotherapeutic drugs and immunosuppressants, the microwave heat sensitization characteristic of ZIF-8 and the chemotherapeutic drugs are utilized to kill osteosarcoma cells and release immunogenic substances, and the immunosuppressants remove the immunosuppressive effect, so that the aims of activating the anti-tumor immunity of an organism to kill local residual tumor cells and potential micrometastasis in the whole body and inhibiting tumor recurrence through the immune memory effect are fulfilled, and meanwhile, the mechanical adaptation microenvironment of the stent and ZIF-8 degradation are utilized to release zinc ions to promote osseointegration.

Description

Composite porous scaffold and preparation method and application thereof

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to a composite porous scaffold and a preparation method and application thereof.

Background

Osteosarcoma is the most common primary malignant bone tumor, is better to be developed in children and teenagers, has high malignancy degree and extremely high amputation rate and death rate in natural disease course, and has great influence on families and society. With the development and application of artificial prosthesis, imaging, chemotherapy and other technologies, a triple comprehensive treatment mode of new adjuvant chemotherapy, limb protection surgery and adjuvant chemotherapy, instead of amputation surgery, becomes the mainstream, more than 90% of osteosarcoma patients can receive limb protection treatment, but the 5-year survival rate of the patients is only 60%, and the development is not progressed in nearly 50 years. Current osteosarcoma limb-protecting treatments face a dilemma in both near term and long term: 1. surgery is difficult to ensure complete clearance of tumor cells, and may leave microscopic lesions. The postoperative intravenous delivery chemotherapy drugs are difficult to accumulate to effective concentration in an operation area and have higher toxic and side effects, and although the implant material can be used as a first battleline for resisting tumors in vivo, the clinically used implant material generally has no anti-tumor performance, so that local recurrence of the tumors in a short period is caused, and the life cycle of a patient is remarkably reduced. 2. The titanium alloy is the most commonly used implant material in clinic at present, although the titanium alloy has good biological suitability, the titanium alloy has poor mechanical suitability and no biological activity with a bone defect focus part after tumor resection, stress shielding effect is easy to cause, osseointegration is poor, and the implant body is easy to lose effect due to long-term implant material loosening. Therefore, in order to improve the treatment effect of the implant after osteosarcoma operation, the two problems of local tumor recurrence and osseointegration need to be solved at the same time, however, the current implant lacks the effects of inhibiting osteosarcoma recurrence and promoting in-situ regeneration of bone tissues, so that the development of the functional adaptive titanium alloy implant material with anti-tumor and osteogenesis functions has important significance for osteosarcoma treatment.

Disclosure of Invention

The present invention has been made to solve at least one of the above-mentioned problems occurring in the prior art. Therefore, the invention provides a composite nanoparticle with microwave heat sensitization property, which can realize the triple effects of microwave heat treatment, chemotherapy and immunotherapy.

The second aspect of the invention provides a preparation method of composite nanoparticles with microwave heat sensitization characteristics.

A third aspect of the invention provides a composite porous scaffold.

The fourth aspect of the invention provides a preparation method of the composite porous scaffold.

The fifth aspect of the invention provides application of the composite nano particles or the composite porous scaffold in preparing bone defect repair materials and/or anti-osteosarcoma implant materials.

According to the first aspect of the invention, the composite nanoparticle with microwave heat sensitization characteristic is provided, wherein the composite nanoparticle is of a core-shell structure and comprises a ZIF-8 nanoparticle, a chemotherapeutic agent and an immunosuppressant; the ZIF-8 nano particles wrap the chemotherapeutic drugs, and the immunosuppressants are loaded on the surfaces of the ZIF-8 nano particles.

In some embodiments of the present invention, the composite nanoparticle having microwave heat sensitization property is spherical or spheroidal, and the average particle diameter is 120nm to 150 nm.

In some embodiments of the invention, the encapsulated amount of the chemotherapeutic agent is 0.5mg to 1 mg.

Furthermore, the wrapping amount of the chemotherapeutic drug is 0.5 mg-0.7 mg.

In some embodiments of the invention, the immunosuppressant loading is 0.4mg to 1 mg.

Furthermore, the loading amount of the immunosuppressant is 0.4 mg-0.6 mg.

According to a second aspect of the present invention, a method for preparing a composite nanoparticle with microwave thermal sensitization characteristics is provided, comprising the following steps:

stirring and reacting zinc nitrate hexahydrate, 2-methylimidazole, chemotherapeutic drugs and immunosuppressant to obtain the composite nano particle with the microwave sensitization characteristic.

In some embodiments of the invention, the ratio of the zinc nitrate hexahydrate to the 2-methylimidazole is 1:1 to 5.

In some embodiments of the invention, the stirring reaction time is 2h to 3 h.

In some preferred embodiments of the invention, the rotation speed of the stirring is 1000r/min to 2000 r/min.

In some preferred embodiments of the invention the chemotherapeutic agent is selected from at least one of doxorubicin, cisplatin, cyclophosphamide.

In some preferred embodiments of the invention, the immunosuppressive agent is at least one of an IDO inhibitor, a PD-1 inhibitor, a CTLA-4 inhibitor.

According to a third aspect of the present invention, a composite porous scaffold is provided, which comprises a three-dimensional porous metal scaffold and the composite nanoparticles with microwave thermal sensitization characteristics of the first aspect, wherein the three-dimensional porous metal scaffold is coupled with the composite nanoparticles with microwave thermal sensitization characteristics through a chemical bond.

In the invention, the three-dimensional porous metal scaffold and the composite nano particles with triple effects of microwave thermotherapy, chemotherapy and immunotherapy are coupled and combined through chemical bonds to construct a composite porous scaffold material, so that residual focuses after osteosarcoma operation are eliminated and bone repair is promoted. After the composite nano particles enter tumor cells, the microwave response releases chemotherapeutic drugs and immunosuppressive agents, the microwave heat sensitization characteristic of ZIF-8 and the chemotherapeutic drugs are utilized to kill osteosarcoma cells and release immunogenic substances, the immunosuppressive agents remove the immunosuppressive effect, the aims of activating the anti-tumor immunity of an organism to kill local residual tumor cells and potential micro metastasis of the whole body and inhibiting tumor recurrence through the immune memory effect are achieved, and meanwhile, the mechanical adaptation microenvironment of the support and the ZIF-8 degradation are utilized to release zinc ions to promote osseointegration.

In some embodiments of the present invention, the material of the three-dimensional porous metal scaffold is selected from at least one of titanium alloy, pure titanium, cobalt alloy, stainless steel.

According to a fourth aspect of the present invention, there is provided a method for preparing a composite porous scaffold, comprising the steps of:

activating groups on the surface of the three-dimensional porous metal support by using an activating agent, adding a coupling agent for reaction, adding the composite nano particles in the first aspect for bonding reaction, and washing to obtain the composite porous support.

In some preferred embodiments of the present invention, the activator is selected from at least one of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

In some preferred embodiments of the present invention, the coupling agent is selected from at least one of N-hydroxysuccinimide, N-hydroxysuccinimide.

According to a fifth aspect of the invention, the application of the composite nano particles or the composite porous scaffold in preparing bone defect repair materials and/or anti-osteosarcoma implant materials is provided.

In some preferred embodiments of the present invention, the composite nanoparticle is the composite nanoparticle having microwave thermal sensitization characteristics according to the first aspect.

In some preferred embodiments of the present invention, the composite porous scaffold is the composite porous scaffold of the third aspect or the composite porous scaffold prepared according to the preparation method of the fourth aspect.

The invention has the beneficial effects that:

1. the composite nano particle has the triple effects of microwave thermotherapy, chemotherapy and immunotherapy, and can release zinc ions to promote osseointegration.

2. The composite porous scaffold provided by the invention utilizes the microwave heat sensitization characteristic of the coupled composite nanoparticles and the effects of killing osteosarcoma cells, releasing immunogenic substances and relieving immunosuppressive effect to achieve the purposes of activating the anti-tumor immunity of an organism, killing local residual tumor cells and potential micrometastasis focus on the whole body and inhibiting tumor recurrence through the immune memory effect, and simultaneously utilizes the mechanical adaptation microenvironment of the scaffold and ZIF-8 degradation to release zinc ions to promote osseointegration.

3. The preparation method of the composite porous scaffold is simple and easy to produce.

4. The composite porous scaffold is applied to preparing bone defect repair materials and/or anti-osteosarcoma implant materials, can solve the problems of photothermal penetrating power and systemic treatment by combining immune check point treatment, can induce immunogenic death of tumor cells and amplify anti-tumor immune reaction of organisms, and can promote osteogenesis to improve osseointegration by active ions generated by degradation of nano materials.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a graph of the killing effect of ZIF-8, ZIF-8@ DOX prepared in comparative examples 1-2 and ZIF-8@ DOX-IDO prepared in example 1 on osteosarcoma cells K7M2 in the absence of and in response to microwaves, where P represents < 0.0001;

FIG. 2 is a flow chart and an analysis chart of ZIF-8 and ZIF-8@ DOX prepared in comparative examples 1-2 and ZIF-8@ DOX-IDO prepared in example 1 for inducing apoptosis of osteosarcoma cells K7M2 without microwave and in response to microwave; wherein A is an apoptosis flow chart, and B is an apoptosis quantitative analysis chart;

FIG. 3 is a graph showing the release of microwave-free and microwave-responsive osteosarcoma cell immunogenic substances prepared in comparative examples 1 to 2 and ZIF-8@ DOX-IDO prepared in example 1 of the present invention; wherein A is an expression quantitative analysis chart of high mobility protein HMGB-1, and B is an expression quantitative analysis chart of calreticulin CRT;

FIG. 4 is a flow chart and an analysis chart of ZIF-8 and ZIF-8@ DOX prepared in comparative examples 1-2 and ZIF-8@ DOX-IDO prepared in example 1 for activating dendritic cell BMDC without microwave and in response to microwave-induced osteosarcoma cells; histogram is a quantitative analysis of the proportion of activated dendritic cells;

FIG. 5 shows that the porous multi-titanium alloy scaffold is respectively coupled with ZIF-8 and ZIF-8@ DOX prepared in comparative examples 1-2 and ZIF-8@ DOX-IDO prepared in example 1 to form a composite porous scaffold which is free of microwaves and responds to microwaves for treating dendritic cells DC and CD8 of tumor tissues of mouse osteosarcoma ⁺ T lymphocytes, CD4 ⁺ Flow charts of T lymphocyte ratio changes; wherein A is flowing type of mouse osteosarcoma tissue DCIn the figure, B is CD8 ⁺ Flow charts of T lymphocytes; c is CD4 ⁺ Flow diagram of T lymphocytes, D from left to right being DC, CD8 ⁺ T lymphocytes, CD4 ⁺ Quantitative analysis chart of T lymphocyte proportion change;

fig. 6a is a graph of the Micro-CT reconstruction scanning effect of the porous titanium alloy scaffold prepared in example 1 of the present invention and the composite porous scaffold TZDI coupled with ZDI composite nanoparticles implanted, grown and taken out, and B is a graph of the bone volume/tissue volume BV/TV, the trabecular bone thickness tb.th, the trabecular bone separation degree tb.sp, the trabecular bone number tb.n and the bone mineral density for quantitative analysis for promoting new bone generation, where P represents less than 0.1; c is a tissue HE staining diagram for promoting new bone formation, the left four diagrams represent results of the titanium alloy porous scaffold, and the right four diagrams represent results of the composite porous scaffold, and the results correspond to the scales of the titanium alloy porous scaffold result diagrams one by one.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Example 1

The embodiment prepares the composite nano particles and the composite porous scaffold, and the specific process comprises the following steps:

weighing 0.3g of zinc nitrate hexahydrate and 1.0g of 2-methylimidazole, dissolving in 4mL of deionized water, stirring at room temperature with the stirring speed of 1200rpm/min for 2h to obtain a ZIF-8 nanoparticle suspension solution, centrifuging to obtain a white precipitate, washing with water for 3 times to remove excessive reactants, drying in vacuum to obtain ZIF-8, weighing 1.0g of DOX and 1.0g of IDO inhibitor, dissolving in 2mL of deionized water to obtain a drug dissolved solution, adding the ZIF-8 nanoparticle suspension solution, stirring at room temperature of 1200rpm/min for 10min, and centrifuging to obtain DOX and IDO loaded composite nanoparticles, namely ZDI composite nanoparticles.

Preparing a 3D printing titanium alloy bracket: porous titanium stent materials with different pore diameters and porosities are designed through the Mimics 17.0 software and the UGS NX 9.0 software, the finite element analysis software ABAQUS 7.0 is used for carrying out structural analysis on the stent, and the pore structure is optimized to determine the optimal parameters. The porous titanium alloy support is prepared by using titanium alloy (Ti6Al4V) powder as a raw material and using a selective laser sintering forming metal 3D printing technology.

Coupling a nano material on the surface of a stent through 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), dispersing a 3D printing titanium stent in a normal saline solution, firstly adding EDC to activate groups on the surface of the stent, reacting for 10min, then adding NHS, continuing reacting for 15min, then adding ZDI composite nano particle to perform bonding reaction for 4h, centrifugally washing, and washing with the normal saline solution for three times, wherein a precipitate is the composite porous stent material of the coupled ZDI composite nano particles, namely TZDI, Ti @ ZDI for short.

Comparative example 1

The composite nano particles and the composite porous scaffold prepared in the comparative example are different from those prepared in the example 1 in that the ZIF-8 does not load chemotherapeutic drugs and immunosuppressants, and the specific process is as follows:

weighing 0.3g of zinc nitrate hexahydrate and 1.0g of 2-methylimidazole, dissolving in 4mL of deionized water, stirring at the stirring speed of 1200rpm/min for 2 hours at room temperature to obtain a ZIF-8 nanoparticle suspension solution, centrifuging to obtain a white precipitate, washing with water for 3 times to remove excessive reactants, and drying in vacuum to obtain the ZIF-8.

Coupling a nano material on the surface of a stent through 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), dispersing a 3D printing titanium stent in a normal saline solution, firstly adding EDC to activate groups on the surface of the stent, reacting for 10min, then adding NHS, continuing to react for 15min, then adding a ZIF-8 nano material to perform bonding reaction for 4h, centrifugally washing, washing for three times with the normal saline solution, and obtaining a precipitate, namely the composite porous stent material coupled with ZIF-8.

Comparative example 2

The composite nano particles and the composite porous scaffold are prepared in the comparative example, and the difference from the example 1 is that the ZIF-8 does not load an immunosuppressant, and the specific process is as follows:

weighing 0.3g of zinc nitrate hexahydrate and 1.0g of 2-methylimidazole, dissolving the zinc nitrate hexahydrate and the 2-methylimidazole in 4mL of deionized water, stirring at the room temperature of 1200rpm/min for 2 hours to obtain a ZIF-8 nanoparticle suspension solution, centrifuging to obtain a white precipitate, washing with water for 3 times to remove excessive reactants, drying in vacuum to obtain a ZIF-8, weighing 1.0g of DOX, dissolving the DOX in 2mL of deionized water to obtain a drug dissolved solution, adding the ZIF-8 nanoparticle suspension solution, stirring at the room temperature of 1200rpm/min for 10 minutes, and centrifuging to obtain the DOX-loaded composite nanoparticle.

1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) are coupled with nano materials on the surface of a stent, a 3D printing titanium stent is dispersed in normal saline solution, EDC is firstly added to activate groups on the surface of the stent, the groups react for 10min, NHS is then added, the reaction is continued for 15min, then ZIF-8 nano materials loaded with DOX are added for bonding reaction for 4h, centrifugal washing is carried out, the normal saline solution is washed for three times, and precipitates are the composite porous stent material coupled with ZIF-8 loaded with DOX.

Test example 1

Mouse osteosarcoma cell line K7M2 in logarithmic growth phase was digested with 0.25% trypsin containing EDTA, collected in 15mL centrifuge tube, centrifuged at 1000r/min for 5min, the supernatant was discarded, 1mL of 10% FBS high-glucose DMEM complete medium was added, the cells were counted in 8 groups of 3 time points of 10000 cells/100 μ L/well for 1 day, 8000 cells/100 μ L/well for 3 days, and 5000 cells/100 μ L/well for 7 days, and each group was set as NC (blank control group), Z (ZIF-8 nanoparticles, comparative example 1), ZD (ZIF-8 nanoparticles carrying DOX chemotherapeutic agent, comparative example 2), ZDI (ZIF-8 nanoparticles carrying DOX chemotherapeutic agent and IDO immunosuppressant, example 1), NC M (porous titanium alloy scaffold control + microwave), Z M (ZIF-8 nanoparticles + microwave, i.e., comparative example 1+ microwave), ZD M (the ZIF-8 nanoparticles load DOX chemotherapeutic agent + microwave, i.e., comparative example 2+ microwave), ZDI M (the ZIF-8 nanoparticles load DOX chemotherapeutic agent and IDO immunosuppressant + microwave, i.e., example 1+ microwave), each group was provided with 5 replicate wells, the required cell amount was calculated respectively,planting in 96-well plate, placing at 37 deg.C and 5% CO ₂ Culturing in a cell culture box.

After culturing for 4h, the cells adhere to the wall, the corresponding nano material is added into each hole according to the concentration of 100 mu g/mL, and the mixture is placed at 37 ℃ and 5% CO ₂ The cells were incubated for 8h in a cell incubator (during which time the cells endocytosed the nanomaterial), the medium was changed, digested with 0.25% pancreatin containing EDTA, collected in 1.5mL EP tubes, centrifuged at 1000r/min for 5min, the supernatant discarded, and 1mL of 10% FBS high-sugar DMEM complete medium was added. Wherein 4 groups of microwave ablation instruments needing microwave ablation are arranged, the power is 1W, the time is 5min, and the microwave ablation antenna is inserted into the bottom of a 1.5mL EP tube to carry out microwave ablation. After microwave ablation, 1.5mL of the cell suspension in EP was transferred to a 96-well plate and continued at 37 ℃ with 5% CO ₂ The cell culture box is used for culturing for 24 hours.

The working solution in each hole is as follows: 10 mu L of CCK-8 mother liquor and 100 mu L of serum-free culture medium are firstly prepared into CCK-8 working solution. Removing culture medium from 96-well plate, washing with PBS for 2 times, adding CCK-8 working solution 110 μ L/well, placing at 37 deg.C and 5% CO ₂ Incubate in cell incubator for 1 h.

And (3) CCK-8 detection: after incubation for 1h, the absorbance was measured with a microplate reader at a wavelength of 450nm (full-wavelength microplate reader, Mutiska Go), and the results are shown in FIG. 1. FIG. 1 is a graph of the viability of the 8 groups of cells examined above. (x, P < 0.0001)

And (4) analyzing results: as can be seen from FIG. 1, at the same time point, the K7M2 activity was significantly reduced in the microwave ablation group compared to the non-microwave ablation group, with statistical differences. In the microwave ablation group, the K7M2 activity for ZDM and the ZDI M group were much lower than the NC M and ZM groups. As the incubation time was extended, the activity of K7M2 was further reduced. The results show that the combined nano-drug of the chemo-thermal treatment can reduce the activity of the K7M2 cells of the osteosarcoma cells.

Test example 2

Digesting mouse osteosarcoma cell line K7M2 in logarithmic growth phase with 0.25% pancreatin containing EDTA, collecting in 15mL centrifuge tube, centrifuging at 1000r/min for 5min, discarding supernatant, adding 1mL 10% FBS high-sugar DMEM complete culture medium, counting cells, dividing into 8 groups with 100000 cells/1.5 mL/well, respectively setting as NC (blank control group), Z (ZIF-8 nanoparticle, namely, ZIF-8 nanoparticle)Comparative example 1), ZD (ZIF-8 nanoparticles loaded with DOX chemotherapeutic agent, i.e., comparative example 2), ZDI (ZIF-8 nanoparticles loaded with DOX chemotherapeutic agent and IDO immunosuppressant, i.e., example 1), NC M (porous titanium alloy scaffold control + microwave), Z M (ZIF-8 nanoparticles + microwave, i.e., comparative example 1+ microwave), ZD M (ZIF-8 nanoparticles loaded with DOX chemotherapeutic agent + microwave, i.e., comparative example 2+ microwave), ZDI M (ZIF-8 nanoparticles loaded with DOX chemotherapeutic agent and IDO immunosuppressant + microwave, i.e., example 1+ microwave), 3 duplicate wells were set for each group, the required cell amount was calculated, seeded in 6-well plates, placed at 37 ℃, 5% CO in 37 ℃, each group ₂ Culturing in a cell culture box.

After culturing for 4h, the cells adhere to the wall, corresponding nano-materials are added into each hole according to the concentration of 100 mu g/mL, and the mixture is placed at 37 ℃ and 5% CO ₂ Culturing in cell culture box for 8h (during which the cells endocytose the nanomaterial), changing the solution, digesting with 0.25% pancreatin containing EDTA, collecting in 1.5mL EP tube, centrifuging at 1000r/min for 5min, discarding the supernatant, and adding 1mL 10% FBS high-sugar DMEM complete medium. And 4 groups of the devices needing microwave ablation are provided with a microwave ablation instrument with the power of 1W and the time of 5min, and the microwave ablation antenna is inserted into the bottom of the 1.5mL EP tube to perform microwave ablation. After microwave ablation, 1.5mL of cell suspension in EP was transferred to a 6-well plate and continued at 37 ℃ with 5% CO ₂ The cell culture box is used for culturing for 24 hours.

Flow-type apoptosis detection: the medium in 6-well plates was collected in 15mL centrifuge tubes, washed 2 times with PBS and collected in 15mL centrifuge tubes, digested with Accutase (Thermal) and placed at 37 ℃ in 5% CO ₂ Digesting in a cell culture box for 1-2 min, collecting cells in a 15mL centrifuge tube, centrifuging at 1000r/min for 5min, and discarding the supernatant. Add 1mL Annexin V Binding Buffer to each tube, resuspend cells, centrifuge at 1000r/min for 5min, discard supernatant. Add 200. mu.L Annexin V Binding Buffer per tube, resuspend cells, set one Annexin V and PI single staining tube (Annexin V stands for early apoptotic cells, PI stands for late apoptotic and necrotic cells).

5 mu L of Annexin V dye is added into the Annexin V single dyeing tube and the experiment group 8 respectively, and the mixture is incubated for 15min at room temperature in a dark place. 2mL of Annexin V Binding Buffer is added into each tube to stop staining, the mixture is centrifuged at 1000r/min for 5min, and the supernatant is discarded. Add 200 u Lannexin V Binding Buffer to each tube, resuspend cells, add 5 u L PI dyes to PI single staining tube and experiment group 8 group separately, incubate 5min at room temperature in dark place. 2mL of Annexin V Binding Buffer is added into each tube to stop staining, the mixture is centrifuged at 1000r/min for 5min, and the supernatant is discarded. Add 200. mu.L Annexin V Binding Buffer per tube and resuspend the cells.

The apoptosis (Beckman Cytoflex) was detected by flow-based detection, and the results are shown in FIG. 2. FIG. 2A is a flow chart showing the apoptosis and necrosis ratios of osteosarcoma cells in each group; FIG. 2B is a diagram of apoptosis quantification. (. about, P < 0.0001)

And (4) analyzing results: as can be seen from fig. 2, the apoptosis rate of the microwave ablation group K7M2 was significantly higher than that of the non-microwave ablation group. The apoptosis rate of the ZDM group and the ZDM group K7M2 is much higher than that of other groups, and the statistical difference shows that the microwave ablation-chemotherapy combination treatment can induce K7M2 cell apoptosis. These results indicate that the microwave ablation-chemotherapy combination therapy has an excellent tumor treatment effect and is expected to be used for in vivo antitumor therapy.

Test example 3

Digesting a mouse osteosarcoma cell line K7M2 in logarithmic growth phase with 0.25% EDTA-containing pancreatin, collecting the cells in a 15mL centrifuge tube, centrifuging the cells for 5min at 1000r/min, discarding the supernatant, adding 1mL of 10% FBS high-sugar DMEM complete medium, counting the cells, and setting as NC (blank control group), Z (ZIF-8 nanoparticle, comparative example 1), ZD (ZIF-8 nanoparticle-loaded DOX chemotherapeutic, comparative example 2), ZDI (ZIF-8 nanoparticle-loaded DOX chemotherapeutic and IDO immunosuppressive, example 1), NC M (porous titanium alloy scaffold control + microwave), Z M (ZIF-8 nanoparticle + microwave, comparative example 1+ microwave), ZD M (ZIF-8 nanoparticle-loaded DOX chemotherapeutic + microwave, comparative example 2+ microwave), ZDI M (ZIF-8 nanoparticles loaded with DOX chemotherapeutic and IDO immunosuppressant + microwave, i.e., example 1+ microwave), each group had 3 duplicate wells, the required cell mass was calculated separately, seeded in 12-well plates, placed at 37 ℃ in 5% CO ₂ Culturing in a cell culture box.

After culturing for 4h, the cells adhere to the wall, corresponding nano material is added into each hole according to the concentration of 100 mu g/ml, and the mixture is placed at 37 ℃ and 5% CO ₂ Culturing for 8h in a cell incubator (during which the cell endocytosis nanometer material), changing the liquid,digested with 0.25% EDTA-containing trypsin, collected in 1.5mL EP tubes, centrifuged at 1000 rpm for 5min, the supernatant discarded, and 1mL of 10% FBS high-glucose DMEM complete medium was added. The power of 4 groups of microwave ablation instruments needing microwave ablation is 1W, the time is 5min, and a microwave ablation antenna is inserted into the bottom of a 1.5mL EP tube to perform microwave ablation.

After microwave ablation, 1.5mL of cell suspension in EP was transferred to 12-well plates and continued at 37 ℃ with 5% CO ₂ The cell culture box is used for culturing for 24 hours.

CRT, HMGB-1ELISA detection in supernatant of osteosarcoma cell line K7M 2: collecting cell supernatant in a sterile 15mL centrifuge tube, and centrifuging for 20min at 2000 r/min-3000 r/min.

Sample adding of the standard: and arranging a standard product hole and a sample hole, wherein 50 mu L of standard products with different concentrations are added into the standard product hole respectively. Blank holes (the blank reference holes are not added with the sample and the enzyme labeling reagent, and the rest steps are operated in the same way) and sample holes to be detected are respectively arranged. 40 mu L of sample diluent is added into sample holes to be detected on the enzyme-labeled coated plate, and then 10 mu L of sample to be detected is added (the final dilution of the sample is 5 times). Adding sample to the bottom of the plate hole of the enzyme label, keeping the sample from touching the hole wall as much as possible, and gently shaking and mixing the sample and the hole wall. Adding an enzyme: add enzyme labeling reagent 100. mu.L to each well except for blank wells. Incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 60 min. Preparing liquid: diluting the 20 times of concentrated washing solution with 20 times of distilled water for later use. Washing: carefully uncovering the sealing plate membrane, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30s, discarding, repeating the steps for 5 times, and patting dry. Color development: adding 50 μ L color-developing agent A into each well, adding 50 μ L color-developing agent B, shaking gently, mixing, and developing at 37 deg.C in dark for 15 min. And (4) terminating: the reaction was stopped by adding 50. mu.L of stop solution to each well (blue color turned to yellow color). And (3) determination: the blank wells were set to zero, and the absorbance (OD value) of each well was measured in order at a wavelength of 450nm, and the measurement was carried out within 15 minutes after the addition of the stop solution. The results are shown in FIG. 3. FIG. 3 is the release pattern of the immunogenic substances from each group of osteosarcoma cells, wherein A is the expression quantitative analysis pattern of the high mobility protein HMGB-1, and B is the expression quantitative analysis pattern of calreticulin CRT. (. about, P < 0.0001)

And (4) analyzing results: from FIG. 3 it can be seen that the molecules CRT and HMGB-1 representing immunogenicity were released more in the ZDM and ZDIM groups with statistical differences. The increase in CRT and HMGB-1 in the ZD and ZDI groups may be associated with partial release of encapsulated DOX acting on K7M 2. The composite nanoparticles are shown to cause immunogenic death of K7M2 cells by being combined with microwave ablation thermal chemotherapy, and the release of CRT and HMGB-1 is increased.

Test example 4

1. Digesting a mouse osteosarcoma cell line K7M2 in a logarithmic growth phase by using 0.25% EDTA-containing pancreatin, collecting the digested cell line in a 15mL centrifuge tube, centrifuging the cell line for 5min at 1000r/min, discarding supernatant, adding 1mL of 10% FBS high-glucose DMEM complete medium, counting cells, and setting the cells as NC (blank control group), Z (ZIF-8 nanoparticle, namely comparative example 1), ZD (ZIF-8 nanoparticle-loaded DOX chemotherapeutic, namely comparative example 2), ZDI (ZIF-8 nanoparticle-loaded DOX chemotherapeutic and IDO immunosuppressant, namely example 1), NC M (porous titanium alloy scaffold control + microwave), Z M (ZIF-8 nanoparticle + microwave, namely comparative example 1+ microwave), ZD M (ZIF-8 nanoparticle-loaded DOX chemotherapeutic + microwave, namely comparative example 2+ microwave), ZDIM (ZIF-8 nanoparticles loaded with DOX chemotherapeutic and IDO immunosuppressant + microwave, i.e., example 1+ microwave), each group was set with 3 duplicate wells, the required cell mass was calculated separately, seeded in 12-well plates, placed at 37 ℃ in 5% CO ₂ Culturing in a cell culture box.

2. After culturing for 4h, the cells adhere to the wall, and according to the concentration of 100 mug/mL, corresponding nano materials are added into each hole of each group, and the mixture is placed at 37 ℃ and 5% CO ₂ Culturing in cell culture box for 8h (during which the cells endocytose the nanomaterial), changing the solution, digesting with 0.25% pancreatin containing EDTA, collecting in 1.5mL EP tube, centrifuging at 1000r/min for 5min, discarding the supernatant, and adding 1mL 10% FBS high-sugar DMEM complete medium. The power of 4 groups of microwave ablation instruments needing microwave ablation is 1W, the time is 5min, and a microwave ablation antenna is inserted into the bottom of a 1.5mL EP tube to perform microwave ablation.

3. After microwave ablation, 1.5mL of cell suspension in EP was transferred to 12-well plates and continued at 37 ℃ with 5% CO ₂ The cell culture box is used for culturing for 24 hours.

Separating and extracting BMDC: a BALB/c mouse (6-10 weeks old) is killed by cervical dislocation, all thighbones and shinbones are taken out by operation, muscle tissues around the bones are removed as clean as possible by scissors and tweezers, the bones are moved to a super clean bench and are soaked for 2-5 min in a sterile culture dish filled with 70% alcohol for disinfection and sterilization, and then the bones are washed for 2 times by sterile PBS; moving the bone into another new culture dish containing PBS, shearing off two ends of the bone by using scissors, extracting the PBS by using an injector, respectively inserting the needle heads into marrow cavities from two ends of the bone, and repeatedly washing out the bone marrow to the culture dish until the bone is completely whitened; collecting bone marrow suspension, and filtering with 200 mesh nylon net to remove small pieces and muscle tissue; centrifuging the filtrate at 1200r/min for 5min, and discarding the supernatant; adding 2mL of ammonium chloride erythrocyte lysate (1x), resuspending the cells, and incubating at room temperature for 3-5 min. The action of the lysate was neutralized by adding 10mL of PBS, followed by centrifugation at 1200r/min for 5min, discarding the supernatant, washing 1 time with PBS, and resuspending the cells in RPMI 1640 medium containing 10% FBS, to obtain.

Induction of BMDCs: cell concentration was adjusted to 2 x 10 using 10% FBS in RPMI 1640 complete medium after bone marrow cell counting in mice ⁵ Per mL; spread to 100mm petri dishes with 10mL cells per dish, and recombinant mouse GM-CSF and IL-4(PeproTech, 20ng/mL) were added at 37 deg.C with 5% CO ₂ Culturing in an incubator. On day 3, 10mL of complete medium containing 20ng/mL recombinant mouse GM-CSF and IL-4 was added to the dishes. Half-volume changing the culture solution respectively at 6 th day and 8 th day, namely collecting the old culture solution, suspending the cell sediment again by using complete culture solution containing 20ng/mL recombinant mouse GM-CSF and IL-4 after centrifugation, and then putting the cell suspension back to the original dish; cells were collected on day 10.

6. The cells and supernatant from step 3 were collected in a sterile 15mL centrifuge tube, centrifuged for 20 minutes (2000-.

7. BMDC cells were counted at 1x 10 ⁶ Each well was laid in a 6-well plate, and the groups were set to NC (blank control group), Z (ZIF-8 nanoparticles, comparative example 1), ZD (ZIF-8 nanoparticles loaded with DOX chemotherapeutic, comparative example 2), ZDI (ZIF-8 nanoparticles loaded with DOX chemotherapeutic and IDO immunosuppressive, example 1), NC M (porous titanium alloy scaffold control + microwave), Z M (ZIF-8 nanoparticles + microwave, comparative example 1+ microwave), ZD M (ZIF-8 nanoparticles loaded with DOX chemotherapeutic + microwave, comparative example 2+ microirnmicrowaveWave), ZDI M (ZIF-8 nanoparticles loaded with DOX chemotherapeutic and IDO immunosuppressive agent + microwave, i.e., example 1+ microwave), each group had 3 multiple wells, 4h later the supernatant from step 6 was added to each well, 37 ℃, 5% CO ₂ The cell incubator was incubated for 24 h.

8. Flow cytometry detection of BMDC activation: scraping cells on a pore plate by using cells or digesting the cells by an Accucate non-destructive enzyme, completely transferring the cells into a 15mL centrifuge tube, centrifuging for 5min at 300g, discarding supernatant, re-suspending cell precipitates by using DPBS (double stranded phosphate buffer solution), centrifuging for 5min at 300g, and washing off redundant culture medium; using 100. mu.L of stabilizing Buffer (corresponding to 10 cells) ⁶ Individually) resuspended centrifuged cell pellets, transferred to a 1.5mL centrifuge tube, 2. mu.L of CD16/32 blocked cell surface Fc receptor was added to each sample, incubated at 4 ℃ for 15min, and blocked by addition of 1mL stabilizing Buffer. Centrifuging at 4 deg.C for 5min at 300g, discarding supernatant, adding 100 μ L of stabilizing Buffer into each tube to resuspend cell suspension, and repeating the washing for 2 times; antibody staining: the antibody was centrifuged before use, 100. mu.L of stabilizing Buffer resuspended cells were added to each tube, 2. mu.L of the corresponding single-stained antibody was added to each tube, and 8. mu.L of mixed antibody (PE-Cy7-CD11c, FITC-MHC-II, APC-CD80, PE-CD86) was added to each tube for the experimental group. The incubation conditions were: keeping out of the sun at 4 ℃ for 30 min; and keeping away from light for 15min at room temperature. After the incubation was completed, 1mL of stabilizing Buffer was added to terminate the reaction. Centrifuging for 5min at 4 ℃ at 300g, discarding the supernatant, adding 100 mu L of stabilizing Buffer to resuspend the cell suspension, repeating the washing for 2 times, finally adding 100 mu L of stabilizing Buffer to resuspend the cell precipitate, and directly detecting on a machine by a flow cytometer: beckman CytoFLEX, the results are shown in FIG. 4. Fig. 4 is a graph showing the effect of each group of osteosarcoma cells on the activation of bone marrow-derived dendritic cell BMDC, and a histogram is a quantitative analysis of the proportion of activated dendritic cells. (. about, P < 0.0001)

And (4) analyzing results: it can be seen from fig. 4 that the ZDM and ZDI M groups of BMDCs are significantly more activated than the NC M, ZM and non-microwave ablation groups. Among them, the ZDI M group has the highest BMDC activation degree, which indicates that the combination of heat chemotherapy and immunotherapy promotes the immunogenic death of osteosarcoma cells, increases the expression of BMDC co-stimulatory molecules CD80 and CD86, and has stronger BMDC activation capability.

Test example 5

1. Constructing a femur in-situ osteosarcoma mouse model, and implanting a stent into the tumor and assisting microwave ablation when the tumor grows to be about 5mm in diameter. The method is divided into groups, namely NC (blank control group, non-implanted), TZ (porous titanium alloy stent loaded with ZIF-8 nano particles), TZD (porous titanium alloy stent loaded with ZIF-8 nano particles containing DOX chemotherapeutic drugs), TZDI (porous titanium alloy stent loaded with ZIF-8 nano particles containing DOX chemotherapeutic drugs and IDO immunosuppressive agents), NC M (porous titanium alloy stent control + microwave), TZ M (porous titanium alloy stent loaded with ZIF-8 nano particles + microwave), TZDM (porous titanium alloy stent loaded with ZIF-8 nano particles containing DOX chemotherapeutic drugs + microwave), TZDI M (porous titanium alloy stent loaded with ZIF-8 nano particles + microwave containing DOX chemotherapeutic drugs and IDO immunosuppressive agents), and 3 repeats are set in each group.

2. The time points for material selection were 3 days, 7 days and 14 days after surgery. Each group of mouse osteosarcoma tissues was isolated and cut into small pieces, and centrifuged at 300g for 5 min.

3. After digestion with collagenase IV and DNase enzymes, the cell suspension was filtered through a 200 mesh filter and centrifuged at 300g for 5 min.

4. The cell suspension was blocked with Fc blocker (TruStain FcX), incubated at room temperature for 15min, blocked by addition of 5mL of Staining Buffer, and centrifuged at 300g for 5 min.

5. The supernatants were discarded and flow antibodies (CD45-BV711 for leukocytes, CD3-FITC, CD4-percp, CD8-BV510 for T cell subclone recognition, CD11c PE/Dazle 594 for dendritic cells) were added, respectively, incubated at room temperature for 15min, blocked by addition of 5mL of Staining Buffer, and centrifuged at 300g for 5 min.

6. The analysis was performed using a flow cytometer and the results are shown in FIG. 5. FIG. 5 shows the immune effect activated by groups of dendritic cells DC, CD8 after microwave ablation in a mouse femoral in situ osteosarcoma model ⁺ T lymphocytes, CD4 ⁺ Flow chart of T lymphocyte proportion change, A is scatter diagram of mouse osteosarcoma tissue DC, B is CD8 ⁺ A scatter plot of T lymphocytes; c is CD4 ⁺ Scattergram of T lymphocytes, D from left to right successively DC, CD8 ⁺ T lymphocytes, CD4 ⁺ Quantitative analysis chart of T lymphocyte proportion change. (. about, P < 0.0001)

And (4) analyzing results: FIG. 5 shows the CD3 of osteosarcoma local infiltration after microwave ablation ^- CD11c ⁺ 、CD3 ⁺ CD8 ⁺ And CD3 ⁺ CD4 ⁺ The proportion of cells is the highest in the ZDI M group, which indicates that the combination of the heat chemotherapy and the immunotherapy can chemotaxis immune cells to tumor parts, increase infiltration and enhance the killing effect on tumors.

Test example 6

Establishing a femoral bone defect model of an SD rat: SD rats (150g, male) were anesthetized with 40 μ L intraperitoneal injection of 3% sodium pentobarbital, shaved around the knee joint, sterilized with iodophor before surgery, incised through an arc incision, and muscle and soft tissue was bluntly separated to fully expose the distal lateral condyle of the femur. The hand drill drills a hole on the lateral condyle at the far end of the femur, the diameter of the hand drill is about 4mm, the hand drill is drilled to touch the opposite cortical bone, the cortical bone is not punched, and chippings and blood are cleaned. Respectively implanting a titanium alloy porous scaffold and a composite porous scaffold TZDI (same as Ti @ ZDI) into the holes, suturing layer by layer, and disinfecting the skin by iodophor. Three days before surgery, continuous intramuscular injection of antibiotics prevents infection.

2. The time points of material drawing are 4 weeks and 12 weeks, after material drawing, Micro-CT scanning and reconstruction analysis (see the result in fig. 6A) and hard tissue slice HE staining and analysis (see the result in fig. 6C) are performed, and fig. 6B is a quantitative analysis chart of new bone formation promoted by the porous titanium alloy scaffold (Ti) and the composite porous scaffold. Figure 6 is a graph of the effect of bone repair in a scaffold in a rat bone defect model. (. P < 0.1)

And (4) analyzing results: as can be seen from the CT reconstruction scan effect graph in fig. 6a and the quantitative analysis of bone volume/tissue volume BV/TV, trabecular bone thickness tb.th, trabecular bone resolution tb.sp, trabecular bone number tb.n and bone mineral density in B, the number of new bones in the composite porous scaffold TZDI group (Ti @ ZDI group) of the present application is greater than that in the porous titanium alloy scaffold group (Ti group), indicating that the composite porous scaffold of the present application has better ability to induce new bones. The HE staining results at 4 and 12 weeks in panel C show that the amount of new bone in the red zone around the TZDI scaffold was higher than in the porous titanium alloy scaffold group, and the composite porous scaffold of the present application accelerated the maturation of new bone.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the embodiments, and various changes can be made without departing from the gist of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Claims

1. The composite nanoparticle with the microwave heat sensitization characteristic is characterized in that the composite nanoparticle has a core-shell structure and comprises a ZIF-8 nanoparticle, a chemotherapeutic agent and an immunosuppressant; the ZIF-8 nano particles wrap the chemotherapeutic drugs, and the immunosuppressants are loaded on the surfaces of the ZIF-8 nano particles.

2. The composite nanoparticle with microwave thermal sensitization property according to claim 1, wherein the composite nanoparticle with microwave thermal sensitization property is spherical or spheroidal, and the average particle diameter is 120 nm-150 nm.

3. The composite nanoparticle with microwave thermal sensitization property according to claim 1, wherein the wrapping amount of the chemotherapeutic agent is 0.5mg to 1 mg.

4. The composite nanoparticle with microwave thermal sensitization property according to claim 1, wherein the loading amount of the immunosuppressant is 0.4 mg-1 mg.

5. A preparation method of composite nanoparticles with microwave sensitization characteristics comprises the following steps:

stirring and reacting zinc nitrate hexahydrate, 2-methylimidazole, a chemotherapeutic agent and an immunosuppressant to obtain the composite nanoparticle with the microwave sensitization property according to any one of claims 1-4.

6. The preparation method of the composite nanoparticle with the microwave sensitization characteristic according to claim 5, wherein the dosage ratio of the zinc nitrate hexahydrate to the 2-methylimidazole is 1: 1-5.

7. A composite porous scaffold, which is characterized by comprising a three-dimensional porous metal scaffold and the composite nanoparticles with microwave thermal sensitization characteristics described in any one of claims 1-4, wherein the three-dimensional porous metal scaffold and the composite nanoparticles with microwave thermal sensitization characteristics are coupled through chemical bonds.

8. The preparation method of the composite porous scaffold is characterized by comprising the following steps of:

activating groups on the surface of the three-dimensional porous metal scaffold by using an activating agent, adding a coupling agent for reaction, adding the composite nano particle with the microwave sensitization characteristic of any one of claims 1-4 for bonding reaction, and washing to obtain the composite porous scaffold of claim 7.

9. The method for preparing a composite porous scaffold according to claim 8, wherein the activator is at least one selected from the group consisting of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide; the coupling agent is at least one selected from N-hydroxysuccinimide and N-hydroxysuccinimide.

10. The application of composite nanoparticles or composite porous scaffolds in preparing bone defect repair materials and/or anti-osteosarcoma implant materials is characterized in that the composite nanoparticles are the composite nanoparticles with the microwave heat sensitization property according to any one of claims 1-4; the composite porous scaffold of claim 5.