Photothermal conversion material, photothermal conversion biological material, preparation method and application

Abstract

The invention relates to a photothermal conversion material, a photothermal conversion biological material, a preparation method and application. And the photothermal conversion material comprises cerium-substituted molybdenum-based polyoxometallate. The photo-thermal conversion biological material comprises a photo-thermal conversion material and a biological active material, and the biological active material is loaded with the photo-thermal conversion material. The preparation method has the advantages that the preparation method of the thermal conversion material is simple, the whole reaction process is non-toxic, and the biological safety is ensured; the photothermal conversion material shows an outstanding photothermal effect in a near-infrared window, so that effective PTT-mediated tumor ablation can be caused; the photothermal conversion material can be used as an ROS scavenger to quickly eliminate active oxygen rising after PTT, so that the generation of inflammatory reaction is inhibited; the photothermal conversion biological material has high osteogenic induction activity and promotes bone repair.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a photothermal conversion material, a photothermal conversion biomaterial, a preparation method and an application thereof. Background Art

[0002] Osteosarcoma, also known as osteogenic sarcoma, is a common malignant bone tumor in adolescents. Conventional treatments include chemotherapy or radiotherapy, limb preservation therapy, immunotherapy, etc. The latest treatment method is photothermal therapy. Photothermal therapy uses a stent loaded with a material with photothermal conversion ability to be implanted in the affected area to perform photothermal treatment on osteosarcoma and repair bone defects.

[0003] However, existing photothermal therapy has some drawbacks, as follows:

[0004] 1) Existing stents loaded with materials capable of photothermal conversion only consider the removal of tumors, while ignoring the protection of surrounding tissues. The large amount of heat generated by photothermal therapy not only ablates tumors, but also burns normal tissues, leading to inflammatory reactions, causing further damage to surrounding tissues, and hindering the treatment of osteosarcoma;

[0005] 2) Ordinary 3D printed bioactive glass scaffolds have good biocompatibility, but their osteogenic properties are limited and are still insufficient for large bone defects caused by osteosarcoma.

[0006] Currently, no effective solution has been proposed for the problems existing in related technologies, such as photothermal conversion materials burning normal tissues, causing inflammatory reactions, and poor osteogenesis. Summary of the invention

[0007] The purpose of the present invention is to provide a photothermal conversion material, a photothermal conversion biomaterial, a preparation method and an application in view of the deficiencies in the prior art, so as to solve the problems existing in the related art such as photothermal conversion materials burning normal tissues, causing inflammatory reactions, and poor osteogenesis.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] In a first aspect, a photothermal conversion material is provided, comprising a cerium-substituted molybdenum-based polyoxometalate.

[0010] In a second aspect, a method for preparing a photothermal conversion material is provided, which is used to prepare the photothermal conversion biomaterial as described in the first aspect, comprising:

[0011] mixing an inorganic cerium salt and a molybdate under acidic conditions to obtain a mixed solution;

[0012] The mixed solution is dried to obtain cerium-substituted molybdenum-based polyoxometalate.

[0013] In some embodiments, the inorganic cerium salt includes cerium chloride, cerium nitrate, and cerium acetate.

[0014] In some embodiments, the molybdate includes sodium molybdate, potassium molybdate, and ammonium molybdate.

[0015] In some embodiments, the molar ratio of the inorganic cerium salt to the molybdate is 1:4 to 1:10.

[0016] In some of the embodiments, including:

[0017] Dissolving cerium chloride and hydrazine hydrochloride in deionized water to obtain a first solution;

[0018] adding hydrochloric acid to the first solution to obtain a second solution;

[0019] adding sodium molybdate to the second solution to obtain a third solution;

[0020] The third solution is sequentially subjected to heating drying and freeze drying to obtain cerium-substituted molybdenum-based polyoxometalate.

[0021] In some of the embodiments, including:

[0022] Dissolve 20 mg of CeCl3·7H2O and 3 mg of hydrazine hydrochloride in 5 mL of deionized water to obtain a first solution;

[0023] Add 0.4 mL of 1 M hydrochloric acid to the first solution to obtain a second solution;

[0024] adding 50 mg of Na2MoO4·2H2O to the second solution to obtain a third solution;

[0025] The third solution is sequentially subjected to heating drying and freeze drying to obtain cerium-substituted molybdenum-based polyoxometalate.

[0026] In a third aspect, a photothermal conversion biomaterial is provided, comprising:

[0027] The photothermal conversion material as described in the first aspect, or the photothermal conversion material prepared by the preparation method as described in the second aspect;

[0028] A bioactive material, wherein the bioactive material carries the photothermal conversion material.

[0029] In some embodiments, the bioactive material includes bioactive glass, polylactic acid, polycaprolactone, and hydroxyapatite.

[0030] In some embodiments, the mass ratio of the photothermal conversion material to the bioactive material is 1:200 to 1:4000.

[0031] In a fourth aspect, a method for preparing a photothermal conversion biomaterial is provided, which is used to prepare the photothermal conversion biomaterial as described in the third aspect, comprising:

[0032] The photothermal conversion material is loaded on a bioactive material to obtain a photothermal conversion biomaterial.

[0033] In some embodiments, the method for preparing the bioactive material comprises:

[0034] mixing the bioactive material with the polyvinyl alcohol solution to obtain a slurry;

[0035] The slurry is printed using a 3D printing method to obtain a bioactive material scaffold.

[0036] In some embodiments, loading the photothermal conversion material on a bioactive material to obtain a photothermal conversion biomaterial comprises:

[0037] adding a cerium-substituted molybdenum-based polyoxometalate solution dropwise to a bioactive material scaffold to obtain an initial material;

[0038] The initial material is subjected to a heat treatment to obtain a bioactive material scaffold loaded with cerium-substituted molybdenum-based polyoxometalate.

[0039] In some embodiments, the mass ratio of the bioactive material to the polyvinyl alcohol solution is 1.2:1 to 1.8:1.

[0040] In some embodiments, the mass percentage of the polyvinyl alcohol solution is 8% to 12%.

[0041] In some embodiments, the concentration of the cerium-substituted molybdenum-based polyoxometalate solution is 0.1 mg / mL to 2.5 mg / mL.

[0042] In some of the embodiments, including:

[0043] (I) Preparation of bioactive materials

[0044] The 80S bioactive glass and 11 wt % polyvinyl alcohol were mixed in a mass ratio of 1.5:1 to obtain a slurry;

[0045] The slurry is 3D printed to obtain an initial scaffold, wherein the 3D printing parameters are: the nozzle inner diameter is 400 μm, the extrusion pressure is 2.8 bar to 4.4 bar, the printing speed is 8 mm / s to 18 mm / s, and the single layer thickness is 0.32 mm;

[0046] The initial scaffold is sequentially dried and sintered to obtain a bioglass active scaffold;

[0047] (II) Preparation of photothermal conversion biomaterials

[0048] Adding 20 μL of 1.2 mg / mL cerium-substituted molybdenum-based polyoxometalate solution dropwise to the bioglass active scaffold to obtain an initial material;

[0049] The initial material is dried at room temperature for 30 to 60 minutes to obtain a bioactive glass scaffold loaded with cerium-substituted molybdenum-based polyoxometalate.

[0050] In the fifth aspect, there is provided an application of the photothermal conversion material as described in the first aspect, or the photothermal conversion material prepared by the preparation method as described in the second aspect, or the photothermal conversion material as described in the third aspect, or the photothermal conversion biomaterial prepared by the preparation method as described in the fourth aspect in the preparation of materials for treating bone tumors.

[0051] In a sixth aspect, there is provided a use of the photothermal conversion biomaterial as described in the third aspect or the photothermal conversion biomaterial prepared by the preparation method as described in the fourth aspect in the preparation of bone defect repair materials.

[0052] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0053] The present invention discloses a photothermal conversion material, a photothermal conversion biomaterial, a preparation method and an application. The preparation method of the photothermal conversion material is simple, the overall reaction process is non-toxic, and biosafety is guaranteed; the photothermal conversion material exhibits an outstanding photothermal effect in the near-infrared window, which can lead to effective photothermal therapy (PTT)-mediated tumor ablation; the photothermal conversion material can be used as a ROS scavenger to quickly eliminate the reactive oxygen species that rise after PTT, thereby inhibiting the generation of inflammatory reactions; the photothermal conversion biomaterial has high osteogenic induction activity and promotes bone repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1A This is a SEM image of a CMB crystal, and the inset is the element distribution map of Mo, Ce, and O;

[0055] Figure 1B The CMB crystal structure model constructed based on the single crystal XRD results of CMB;

[0056] Figure 2A is the morphology of pure BG and CMB@BG scaffolds with different loading amounts;

[0057] Figure 2B is the XRD characterization diagram of pure BG and CMB@BG scaffold;

[0058] Figure 2Cis the SEM-element distribution image of the CMB@BG scaffold, and the small picture is the element distribution map of Mo, Ce, Ca, Si, O, and P;

[0059] Figure 3 The photothermal performance test diagram of CMB@BG bracket, where a is the CMB@BG under 1064nm laser (power density 0.25-1.25W / cm 2 ) irradiated, b is the heating curve of CMB@BG under 1064nm laser (power density 1W / cm 2 ) irradiated, c is the heating curve of CMB@BG under 1064nm laser (power density 1W / cm 2 ) irradiated, d is the temperature rise-cooling curve of CMB@BG under 1064nm laser (power density 1W / cm 2 ) on tumor cells in vitro. e is the killing effect of CMB@BG on tumor-bearing nude mice irradiated with 1064 nm laser (power density 1.5 W / cm 2 ) irradiated by 1064 nm laser (power density 1.5 W / cm 2 ) in vitro tumor tissue after 2 weeks of irradiation treatment. g is the nude mice bearing tumors irradiated with 1064nm laser (power density 1.5W / cm 2 ) Tumor clearance rate (TGI) 2 weeks after irradiation therapy;

[0060] Figure 4 The ROS scavenging performance test diagram of CMB@BG scaffold, where a is the DPPH experiment, b is the ABTS experiment, c is the rapid scavenging experiment of hydroxyl radical (·OH) (Start is the start of the reaction, Mid is 5 minutes after the reaction, and End is 10 minutes after the reaction), d is the superoxide anion (O2 ·- ) (In the figure, Start is the start of the reaction, Mid is 5 minutes after the reaction, and End is 10 minutes after the reaction), e is the intracellular ROS level of BMSCs under hydrogen peroxide stimulation, f is the intracellular ROS level of RAW264.7 cells under lipopolysaccharide (LPS) stimulation, g is the level of TNF-α under LPS stimulation, h is the level of IL-6 under LPS stimulation, and i is the level of IL-1β under LPS stimulation;

[0061] Figure 5: is a test diagram of the ROS scavenging performance of the CMB@BG scaffold, wherein a is HE staining to observe tissue inflammation and ROS levels in mice implanted with BG or CMB@BG scaffolds and local injection of LPS, b is DHE fluorescence staining of reactive oxygen probe to observe tissue inflammation and ROS levels in mice implanted with BG or CMB@BG scaffolds and local injection of LPS, c is ELISA to detect the level of IL-6 in serum inflammatory factors in mice implanted with BG or CMB@BG scaffolds and local injection of LPS, d is ELISA to detect the level of IL-1β in serum inflammatory factors in mice implanted with BG or CMB@BG scaffolds and local injection of LPS, and e is ELISA to detect the level of IL-10 in serum inflammatory factors in mice implanted with BG or CMB@BG scaffolds and local injection of LPS;

[0062] Figure 6 Figure 1 is a test diagram of the osteogenic performance of the CMB@BG scaffold, where a is the expression of osteogenic-related proteins (RUNX2 and OPN) of BMSCs inoculated on CMB@BG or simple BG scaffolds, b is the fluorescence intensity of osteogenic-related proteins (RUNX2 and OPN) of BMSCs inoculated on CMB@BG or simple BG scaffolds, c is the mRNA level of BMSCs osteogenic-related genes (RUNX2, COL1 and OPN) detected by qPCR, d is micro-CT showing the bone tissue repair in the rat skull defect model implanted with CMB@BG and simple BG at different time points, e is the trabecular bone density (Tb.BMD), f is the total porosity, and g is the trabecular bone volume fraction (BV / TV) value. DETAILED DESCRIPTION

[0063] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0064] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0065] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0066] Example 1

[0067] This embodiment relates to the photothermal conversion material, preparation method and application of the present invention.

[0068] An exemplary embodiment of the present invention provides a method for preparing a photothermal conversion material, comprising:

[0069] Step S102, mixing the inorganic cerium salt and the molybdate under acidic conditions to obtain a mixed solution;

[0070] Step S104: drying the mixed solution to obtain cerium-substituted molybdenum-based polyoxometalate (CMB).

[0071] In step S102, the inorganic cerium salt includes but is not limited to cerium chloride, cerium nitrate, and cerium acetate.

[0072] In step S102, the molybdate includes but is not limited to sodium molybdate, potassium molybdate, and ammonium molybdate.

[0073] In step S102, the molar ratio of the inorganic cerium salt to the molybdate is 1:4 to 1:10.

[0074] In step S102, the acidic condition is a pH of 1 to 2.5.

[0075] In some of the embodiments, in step S102, it further includes:

[0076] The inorganic cerium salt, the reducing agent and the molybdate are mixed under acidic conditions to obtain a mixed solution.

[0077] In some of these embodiments, the reducing agent includes but is not limited to hydrazine hydrochloride.

[0078] In some embodiments, step S102 specifically includes:

[0079] Step S1021, dissolving cerium chloride and hydrazine hydrochloride in deionized water to obtain a first solution;

[0080] Step S1022, adding hydrochloric acid to the first solution to obtain a second solution;

[0081] Step S1023, adding sodium molybdate to the second solution to obtain a third solution.

[0082] In step S1021, the molar ratio of cerium chloride to hydrazine hydrochloride is 5:1 to 1:4.

[0083] In step S1021, in the first solution, the concentration of cerium chloride is 4 mg / mL to 40 mg / mL.

[0084] In step S1021, in the first solution, the concentration of hydrazine hydrochloride is 0.6 mg / mL to 12 mg / mL.

[0085] In step S1022, the pH of the second solution is 1-2.5.

[0086] In step S1022, the concentration of hydrochloric acid is 0.5M to 5M.

[0087] In step S1022, the molar ratio of hydrochloric acid to cerium chloride is 4:1 to 10:1.

[0088] Furthermore, in step S1021 to step S1023, the first solution, the second solution, and the third solution need to be mixed respectively.

[0089] The mixing methods include but are not limited to stirring, vortexing, ultrasound, and pipetting.

[0090] In step S104, drying includes but is not limited to heating drying and freeze drying.

[0091] In some embodiments, step S104 specifically includes:

[0092] The third solution is sequentially subjected to heating drying and freeze drying to obtain cerium-substituted molybdenum-based polyoxometalate.

[0093] In some embodiments, the heating and drying temperature is 30° C. to 100° C., and the heating and drying time is 2 h to 5 h.

[0094] In some embodiments, heating and drying the third solution comprises:

[0095] transferring the third solution to a sealed device;

[0096] The sealed device is placed in a drying device for heating and drying.

[0097] The sealing device comprises a sealing bottle and a sealing cover, wherein the sealing cover is a through structure, and the sealing cover is detachably connected to the sealing bottle (such as a threaded connection).

[0098] In some embodiments, the sealing cover includes a cover member and a through-hole member, wherein the cover member is detachably connected to the sealing bottle; and the through-hole member is arranged through the cover member to allow the gas generated by the evaporation of the liquid in the sealing bottle to flow outward.

[0099] In some embodiments, the sealing cover comprises a cover member, a groove member, an elastic member and a through-hole member. The cover member is detachably connected to the sealed bottle; the groove member is arranged through the cover member; the elastic member is arranged inside the groove member to seal the groove member; and the through-hole member is arranged through the elastic member to allow the gas generated by the evaporation of the liquid in the sealed bottle to flow outward.

[0100] There may be one through-hole member or several through-hole members. In the case where there are several through-hole members, the several through-hole members are evenly distributed.

[0101] In some of the embodiments, the freeze-drying temperature is -80°C to -10°C, the freeze-drying vacuum degree is 0.001 kPa to 0.04 kPa, and the freeze-drying time is 10 h to 72 h.

[0102] Furthermore, the preparation method of the photothermal conversion material includes:

[0103] Dissolve 20 mg of CeCl3·7H2O and 3 mg of hydrazine hydrochloride in 5 mL of deionized water to obtain a first solution;

[0104] Add 0.4 mL of 1 M hydrochloric acid to the first solution to obtain a second solution;

[0105] Add 50 mg of Na2MoO4·2H2O to the second solution to obtain a third solution;

[0106] The third solution is sequentially subjected to heating drying and freeze drying to obtain cerium-substituted molybdenum-based polyoxometalate.

[0107] The photothermal conversion material (CMB) prepared by the present invention can be used to prepare materials for treating bone tumors.

[0108] For the photothermal conversion material (CMB) prepared by the present invention, Figure 1A to Figure 1B As shown in Figure 2, the CMB crystals obtained by SEM observation have a typical rhombus morphology, which is consistent with the typical morphology of Mo-based POM crystals. Element Mapping characterizes its elemental composition, and it can be seen that Mo, Ce, and O elements are evenly distributed on the CMB crystals ( Figure 1A ). The CMB single crystal obtained by single crystal XRD characterization obtained the crystal structure of CMB. It can be seen that CMB retains the classic ring structure of molybdenum blue, which is composed of Mo8, Ce Ⅲ , Mo1, three basic structures constitute the ring structure of CMB ( Figure 1B ).

[0109] The advantages of the present invention are as follows:

[0110] 1) The preparation method of photothermal conversion materials is simple, the overall reaction process is non-toxic, and biosafety is guaranteed;

[0111] 2) Photothermal conversion materials exhibit outstanding photothermal effects in the near-infrared window, which can lead to effective photothermal therapy (PTT)-mediated tumor ablation;

[0112] 3) Photothermal conversion materials can act as ROS scavengers to quickly eliminate the reactive oxygen species that rise after PTT, thereby inhibiting the occurrence of inflammatory responses.

[0113] Example 2

[0114] This embodiment relates to the photothermal conversion biomaterial, preparation method and application of the present invention.

[0115] An illustrative embodiment of the present invention is a method for preparing a photothermal conversion biomaterial, comprising:

[0116] Step S202, preparing bioactive materials;

[0117] Step S204: Load the photothermal conversion material described in Example 1 onto a bioactive material to obtain a photothermal conversion biomaterial.

[0118] In step S202, the bioactive material includes but is not limited to bioactive glass, polylactic acid, polycaprolactone, and hydroxyapatite.

[0119] In step S202, the preparation method of the bioactive material includes but is not limited to 3D printing.

[0120] In some embodiments, step S202 specifically includes:

[0121] Step S2021, mixing the bioactive material with the polyvinyl alcohol solution to obtain a slurry;

[0122] Step S2022: Print the slurry using a 3D printing method to obtain a bioactive material scaffold.

[0123] In step S2021, the concentration of the bioactive material in the slurry is 54% to 65%.

[0124] In step S2021, the mass ratio of the bioactive material to the polyvinyl alcohol solution is 1.2:1 to 1.8:1.

[0125] In step S2021, the mass percentage of the polyvinyl alcohol solution is 8% to 12%.

[0126] In step S2022, the 3D printing device is an air pump type 3D printer.

[0127] In step S2022, the 3D printing parameters are: nozzle inner diameter is 400 μm, extrusion pressure is 2.8 bar to 4.4 bar, printing speed is 8 mm / s to 18 mm / s, and single layer thickness is 0.32 mm.

[0128] In step S204, the mass ratio of the photothermal conversion material to the bioactive material is 1:200 to 1:4000.

[0129] In some embodiments, step S204 specifically includes:

[0130] Step S2041, dripping a cerium-substituted molybdenum-based polyoxometalate solution onto a bioactive material scaffold to obtain an initial material;

[0131] Step S2042: heat the initial material to obtain a bioactive material scaffold loaded with cerium-substituted molybdenum-based polyoxometalate.

[0132] In step S2041, the concentration of the cerium-substituted molybdenum-based polyoxometalate solution is 0.1 mg / mL to 2.5 mg / mL.

[0133] In step S2042, the heating process includes but is not limited to drying and sintering.

[0134] In some embodiments, step S2042 includes:

[0135] The initial material is dried and sintered in sequence to obtain a bioactive material scaffold loaded with cerium-substituted molybdenum-based polyoxometalate.

[0136] In some embodiments, the drying temperature is 20° C. to 60° C., and the drying time is 2 h to 24 h.

[0137] In some embodiments, the sintering temperature is 800° C. to 1100° C., and the sintering time is 1 h to 5 h.

[0138] In some embodiments, step S204 includes:

[0139] Repeat steps S2041 to S2042 several times to obtain bioactive material scaffolds loaded with cerium-substituted molybdenum-based polyoxometalates with different loading amounts.

[0140] Furthermore, the method for preparing the photothermal conversion biomaterial comprises:

[0141] (I) Preparation of bioactive materials

[0142] The 80S bioactive glass and 11 wt % polyvinyl alcohol were mixed in a mass ratio of 1.5:1 to obtain a slurry;

[0143] The slurry was 3D printed to obtain an initial scaffold, wherein the 3D printing parameters were as follows: the inner diameter of the nozzle was 400 μm, the extrusion pressure was 2.8 bar to 4.4 bar, the printing speed was 8 mm / s to 18 mm / s, and the single layer thickness was 0.32 mm;

[0144] The initial scaffold is sequentially dried and sintered to obtain a bioglass active scaffold (BG);

[0145] (II) Preparation of photothermal conversion biomaterials

[0146] 20 μL of 1.2 mg / mL cerium-substituted molybdenum-based polyoxometalate solution was added dropwise to the bioglass active scaffold to obtain the initial material;

[0147] The initial material was dried at room temperature for 30 to 60 minutes to obtain a bioactive glass scaffold loaded with cerium-substituted molybdenum-based polyoxometalate.

[0148] The photothermal conversion biomaterial (CMB@BG) prepared by the present invention can be used to prepare materials for treating bone tumors.

[0149] The photothermal conversion biomaterial (CMB@BG) prepared by the present invention can be used to prepare bone defect repair materials.

[0150] For the photothermal conversion biomaterial (CMB@BG) prepared by the present invention, Figure 2A to Figure 2C As shown in the figure, the prepared CMB@BG retains the fine 3D structure of the 3D printed BG scaffold: the scaffold is composed of regularly arranged trabeculae stacked layer by layer, the three-dimensional morphology of the scaffold can be adjusted according to the actual shape of the bone defect, and the trabeculae width and spacing can also be changed arbitrarily according to requirements. The printed circular scaffold shown in the figure has a diameter of 7.5mm and a height of 1.6mm; the trabeculae are 400μm wide, the spacing between adjacent trabeculae is 400μm, and the height of each layer is 320μm. With the increase of CMB loading, CMB@BG shows a deeper blue color ( Figure 2A ). XRD characterization of blank BG scaffold and CMB@BG scaffold shows that their composition is mainly silicon dioxide and basic calcium phosphate, which is consistent with the composition of BG ( Figure 2B ). Element mapping characterizes its elemental composition, and it can be seen that Mo, Ce, and O elements are evenly distributed on the bracket, indicating the successful loading of CMB ( Figure 2C ).

[0151] The advantages of the present invention are as follows:

[0152] 1) The preparation method of photothermal conversion biomaterials is simple, and the overall reaction process is non-toxic, ensuring biosafety;

[0153] 2) Photothermal conversion biomaterials exhibit outstanding photothermal effects in the near-infrared window, which can lead to effective photothermal therapy (PTT)-mediated tumor ablation;

[0154] 3) Photothermal conversion biomaterials can act as ROS scavengers to quickly eliminate the reactive oxygen species that rise after PTT, thereby inhibiting the occurrence of inflammatory responses;

[0155] 4) At the bone defect site, the proliferation and differentiation promoting abilities of photothermal conversion biomaterials can be continuously exerted both in vitro and in vivo and effectively accelerate bone regeneration.

[0156] Example 3

[0157] This embodiment is a specific preparation embodiment of the light-to-heat conversion material of the present invention.

[0158] A method for preparing a photothermal conversion material, comprising:

[0159] Dissolve 20 mg CeCl3·7H2O and 3 mg hydrazine hydrochloride in 5 ml water;

[0160] Add 0.4 mL 1 M HCl with stirring;

[0161] While maintaining stirring, add 50 mg of Na2MoO4·2H2O to the mixture;

[0162] Continue stirring for 10-20 minutes to obtain a clear dark blue solution;

[0163] Transfer it to a 20 ml vial sealed with a metal cap with a rubber septum; pierce the rubber septum with a hollow needle with an inner diameter of 1.2 mm to place the sample under controlled evaporation;

[0164] After heating at 100 °C in an oven for 3 days, a dark blue solution containing CMB was obtained;

[0165] It was freeze-dried and stored for later use.

[0166] Example 4

[0167] A method for preparing a photothermal conversion material, comprising:

[0168] Weigh 20 mg of CeCl3·7H2O, 3 mg of hydrazine hydrochloride, and 50 mg of Na2MoO4·2H2O, and dissolve them in 1 ml of water to obtain 1 ml of each of the three solutions;

[0169] Take 2 mL of deionized water, add 1 mL of the aforementioned CeCl3 solution and 1 mL of the aforementioned hydrochloric acid hydrazine solution under stirring;

[0170] While stirring, add 0.4 mL 1 M HCl;

[0171] While maintaining stirring, add 1 mL of the aqueous solution containing Na2MoO4 described above dropwise to the mixture;

[0172] Continue stirring for 10-20 minutes to obtain a clear dark blue solution;

[0173] This was transferred to a 20-mL vial sealed with a metal cap with a rubber septum;

[0174] The rubber septum was pierced with a hollow needle with an inner diameter of 1.2 mm to put the sample in a state of controlled evaporation;

[0175] After heating at 100 °C in an oven for 3 days, a dark blue solution containing CMB was obtained;

[0176] It was freeze-dried and stored for later use.

[0177] Example 5

[0178] This embodiment is a specific preparation embodiment of the light-to-heat conversion material of the present invention.

[0179] A method for preparing a photothermal conversion biomaterial, comprising:

[0180] (I) Preparation of bioactive materials

[0181] 80S bioactive glass and 11wt% polyvinyl alcohol solution were mixed at a mass ratio of 1.5:1 and stirred vigorously to obtain a uniform white slurry;

[0182] The slurry is then transferred to an air pump 3D printer for 3D printing. The printing shape can be designed according to the shape of the bone defect. The slurry is extruded from a nozzle with an inner diameter of 400 microns, the extrusion pressure is 2.8-4.4 bar, the printing speed is 8-18 mm / s, and the single layer thickness is about 0.32 mm to obtain a scaffold;

[0183] The printed scaffold was dried (37°C, overnight) and sintered (1060°C, 3 hours) to obtain the BG scaffold.

[0184] (II) Preparation of photothermal conversion biomaterials

[0185] 20 μL of 1.2 mg / mL CMB solution was dropped onto the BG scaffold and dried at room temperature for 30–60 min;

[0186] By repeating the process of CMB dropwise addition-drying, CMB@BG scaffolds with different CMB loading amounts were obtained.

[0187] In this embodiment, the CMB@BG scaffold with repeated dripping for 3 times was used for the treatment of osteosarcoma.

[0188] Example 6

[0189] This embodiment is a specific preparation embodiment of the light-to-heat conversion material of the present invention.

[0190] A method for preparing a photothermal conversion biomaterial, comprising:

[0191] (I) Preparation of bioactive scaffold materials

[0192] 45S bioactive glass and 11wt% polyvinyl alcohol solution were mixed at a mass ratio of 1.8:1 and stirred vigorously to obtain a uniform white slurry;

[0193] The slurry is then transferred to an air pump 3D printer for 3D printing. The printing shape can be designed according to the shape of the bone defect. The slurry is extruded from a nozzle with an inner diameter of 400 microns, the extrusion pressure is 2.8-4.4 bar, the printing speed is 8-18 mm / s, and the single layer thickness is about 0.32 mm to obtain a scaffold;

[0194] The printed scaffolds were dried (37°C, overnight) and sintered (900°C, 3 hours) to obtain blank BG scaffolds.

[0195] (II) Preparation of photothermal conversion biomaterials

[0196] 20 μL of 1.2 mg / mL CMB solution was dropped onto the BG scaffold and dried at room temperature for 30–60 min;

[0197] By repeating the process of CMB dropwise addition-drying, CMB@BG scaffolds with different CMB loading amounts were obtained.

[0198] In this embodiment, the CMB@BG scaffold with repeated dripping for 3 times was used for the treatment of osteosarcoma.

[0199] Example 7

[0200] This embodiment involves the verification of various performance indicators of the photothermal conversion biomaterial of Example 5.

[0201] 1. Photothermal performance test

[0202] 1. Evaluate the photothermal performance of CMB@BG.

[0203] The CMB@BG stent was irradiated with a 1064nm laser, and the temperature change of the stent was observed by a photothermal camera to further evaluate the photothermal performance of the CMB@BG stent. First, the CMB@BG stent was irradiated with 1064nm lasers of different power densities. It was observed that under a mild power density (0.5W / cm2) of 1064nm laser, the equilibrium temperature of CMB@BG easily reached above 50°C, which was sufficient to induce tumor cell ablation, and under a power density of 1.25W / cm2, the temperature could reach above 80°C, indicating that the CMB@BG stent has a fairly strong photothermal effect ( Figure 3 ab). Subsequently, the laser was repeatedly turned on and off to illuminate the CMB@BG scaffold, and it can be seen that in the 6 heating-cooling cycles, the CMB@BG scaffold observed a highly repetitive time-temperature curve, indicating that the CMB@BG scaffold has good photothermal stability ( Figure 3 c).

[0204] 2. Detect the tumor cell killing effect induced by CMB@BG photothermal treatment.

[0205] To detect photothermal-induced tumor cell ablation, the cell viability of MG63 osteosarcoma cells treated differently on BG or CMB@BG scaffolds was detected using the CCK-8 method. There was no significant change in cell viability of MG63 cells seeded on culture dishes (control group) or ordinary BG scaffolds, regardless of whether they were irradiated with 1064nm laser. However, 1064nm laser irradiation on the CMB@BG group can lead to tumor cell ablation due to the high photothermal conversion efficiency of CMB@BG ( Figure 3 d).

[0206] 3. The anti-tumor effect of CMB@BG stent combined with photothermal therapy in vivo was then further evaluated.

[0207] Methods: First, a subcutaneous tumor-bearing model of nude mice was established. Female Balb / c nude mice aged 4 weeks were selected. The concentration of MG63 cell suspension was adjusted to 3×107cellsmL-1, and 0.1mL of MG63 cell suspension was subcutaneously injected into the inguinal region of each mouse to establish a subcutaneous osteosarcoma animal model. The tumor-bearing mice were divided into 4 groups and treated differently: BG group, BG+NIR-II group, CMB@BG group, and CMB@BG+NIR-II group. Under isoflurane anesthesia, an arc incision was made along the edge of the tumor, and the space under the subcutaneous tumor was carefully exposed by blunt dissection. Then, BG or CMB@BG scaffolds (Φ5mm×1.2mm) were implanted at the bottom of the tumor. After implantation, the subcutaneous tissue and skin were sutured with absorbable sutures. The tumor site was then irradiated with a 1064nm laser for PTT.

[0208] Results: Osteosarcoma-bearing mice were randomly divided into 4 groups and given different treatments: BG group, BG+NIR group, CMB@BG group, and CMB@BG+NIR group. CMB@BG or ordinary BG stents were surgically implanted into the gap between the subcutaneous tumor and the underlying healthy tissue. After the stent was placed, the skin incision was sutured, and then the tumor area was irradiated with a 1064nm laser (1.5W / cm2, 10min), and photothermal therapy was performed on the BG+NIR group and CMB@BG+NIR group. Infrared thermography detected that the CMB@BG stent had a strong thermal effect under near-infrared-Ⅱ irradiation, and the temperature of the tumor site increased sharply from 32.8℃ to 52.0℃ within 10 minutes, while no obvious temperature increase was observed in the ordinary BG group, indicating that it has a good therapeutic effect on tumor ablation ( Figure 3 e). CMB@BG combined with NIR-II laser irradiation can significantly inhibit tumor growth and eradicate the tumor within 2 weeks, while the tumor volume of other groups increases rapidly ( Figure 3 fg).

[0209] 2. ROS removal performance test (I)

[0210] 1. The ROS scavenging activity of CMB was evaluated by DPPH and ABTS free radical scavenging assays using UV-visible spectroscopy.

[0211] Methods: In the DPPH test, 0.04 mg mL-1 DPPH ethanol solution was prepared in advance, and then 2 mL of CMB solution with different concentrations was mixed with 2 mL of DPPH solution for 30 min, and then the absorbance at 519 nm was measured by UV-visible spectroscopy.

[0212] In the ABTS assay, 0.2 mL of ABTS solution (7.4 mmol L-1) and 0.2 mL of potassium persulfate solution (2.6 mmol L-1) were mixed and reacted in the dark for 24 h to generate ABTS. ·+ Then dilute 50 times with PBS buffer and react with CMB. 0.6 mL of CMB solution with different concentrations was mixed with 2.4 mL of ABTS ·+ After the solution was mixed and reacted for 10 min, the absorbance was measured at a wavelength of 734 nm.

[0213] Results: In both DPPH and ABTS tests, the absorbance at the characteristic peaks of free radicals (DPPH at 519 nm; ABTS at 724 nm) decreased, indicating that CMB has good free radical scavenging ability ( Figure 4 ab).

[0214] 2. Subsequently, the electron spin resonance (ESR) spectroscopy was used to determine the scavenging ability of CMBs on representative intracellular ROS, including superoxide anion radicals (O2 ·- ), and hydroxyl radical (·OH)( Figure 4 cd).

[0215] Methods: The CMB was detected by ESR spectroscopy for ·OH and O2 ·- ·OH is produced by the Fenton reaction between FeSO4 and H2O2. · - Produced by xanthine / xanthine oxidase reaction. TEMP and DMPO were selected as ·OH and O2, respectively · -spin trapping agent.

[0216] Results: O2 was detected at 0, 5 and 10 minutes after the start of the reaction. ·- After incubation with 100 μg / mL CMB for 10 min (reaction end point), the O2 ·- The characteristic peaks of ·OH and ·OH were sharply weakened to near the baseline level, indicating that CMB has a strong ROS elimination efficiency ( Figure 4 cd).

[0217] 3. CMB@BG reduces intracellular ROS levels and inhibits the release of inflammatory factors.

[0218] Methods: The inhibitory effect of CMB@BG on intracellular ROS levels and inflammation was verified in BMSCs and RAW264.7 macrophage cell lines. BG or CMB@BG was co-incubated with BMSCs cells and then treated with 200 μM H2O2 for 24 h to establish a BMSCs oxidative stress model. The DCFH-DA fluorescent probe was then used to detect the intracellular ROS level.

[0219] Similarly, RAW264.7 macrophage cell lines were plated and co-incubated with BG or CMB@BG scaffolds, and stimulated with 10 ng / mL LPS to construct an M1 polarized macrophage model. The DCFH-DA fluorescent probe was then used to detect the intracellular ROS level; representative proinflammatory cytokines such as TNF-α, IL-6, and IL-1β were detected using ELISA.

[0220] Results: ROS fluorescent probe DCFH-DA was used to detect the intracellular ROS levels of BMSCs treated with different methods. Under the conditions of H2O2-induced oxidative stress, CMB@BG scaffolds could significantly inhibit the increase of ROS levels in BMSCs ( Figure 4 e).

[0221] Considering that the body's inflammatory response is often associated with the M1 polarization of macrophages, the potential antioxidant-induced anti-inflammatory effect of CMB@BG on M1 polarized macrophages was further investigated. The CMB@BG scaffolds were incubated with lipopolysaccharide (LPS)-stimulated RAW264.7 cells (M1 macrophages), and then the antioxidant effect of CMB@BG was determined by the DCFH-DA method ( Figure 4 f). The DCFH-DA method detected an increase in the intracellular ROS level in M1 polarized macrophages, while the application of CMB@BG scaffolds significantly reduced the ROS signal of M1 macrophages. To evaluate the anti-inflammatory effect of CMB@BG, representative pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β were detected using ELISA ( Figure 4 gi). In LPS-treated RAW264.7 cells, the expression levels of pro-inflammatory cytokines were increased, indicating the pro-inflammatory transformation of M1 macrophages; while the co-incubation of CMB@BG could effectively reduce the levels of inflammatory factors.

[0222] 3. ROS removal performance test (II)

[0223] In vivo ROS scavenging and anti-inflammatory effects evaluation of CMB@BG.

[0224] Methods: To evaluate the level of inflammation in vivo, 4-week-old Balb / c mice were selected and divided into 6 groups: control group, BG group, CMB@BG group, BG+LPS group, and CMB@BG+LPS group. Ordinary BG or CMB@BG scaffolds were implanted into the subcutaneous tissue anterior to the quadriceps femoris of the right lower limb. Two weeks after surgery, LPS (0.4 μg per mouse) was subcutaneously injected at the scaffold implantation site. The animals were euthanized the next day after LPS injection, and the soft tissues near the injection site were collected for histological analysis, including H&E staining and DHE staining. Peripheral blood serum was also collected to detect the levels of inflammatory factors IL-1β, IL-6, and IL-10.

[0225] Results: H&E staining of subcutaneous tissue at the stent implantation site showed that the BG+LPS group had typical inflammatory reactions, such as tissue congestion, edema, and inflammatory cell aggregation, while no obvious inflammatory pathological changes were observed in the control group and CMB@BG+LPS group. At the same time, the fluorescent probe dihydroethidium (DHE) was used to detect the increased level of ROS in the subcutaneous tissue of the BG+LPS group, indicating that inflammation and ROS levels were increased. CMB@BG implantation effectively reduced the level of ROS in the tissue ( Figure 5 ab). ELISA was used to detect the levels of serum inflammatory factors in different groups. The levels of IL-1β, IL-6, and IL-10 in the serum of the BG and CMB@BG control groups injected with PBS remained stable, indicating that the scaffold had reliable biocompatibility. CMB@BG had an ideal anti-inflammatory effect, which was manifested by the downregulation of IL-1β, IL-6, and IL-10 levels ( Figure 5 ce).

[0226] 4. Osteogenesis Performance Test

[0227] 1. To further evaluate the osteogenic ability of CMB@BG scaffolds, BMSCs were seeded on blank BG scaffolds or CMB@BG scaffolds. After 2 weeks of osteogenic differentiation, immunofluorescence staining of osteogenic genes RUNX2 and OPN was performed ( Figure 6 ab); RT-PCR was used to further detect the expression of osteogenic genes (OPN, RUNX2 and COL1) in BMSCs ( Figure 6 c).

[0228] Methods: Sodium β-glycerophosphate (10 mM), L-ascorbic acid (50 μg / mL), and dexamethasone (10 nM) were added to DMEM medium. BMSCs were cultured in the obtained osteogenic differentiation medium for 14 days. Immunofluorescence staining of osteogenic genes RUNX2 and OPN was performed. RT-PCR was used to further detect the expression of osteogenic genes (OPN, RUNX2, and COL1) in BMSCs.

[0229] Results: Compared with BMSCs on ordinary BG scaffolds, BMSCs on CMB@BG scaffolds showed significantly higher expression levels of RUNX2 and OPN, which indicated that CMB@BG scaffolds induced more effective osteoblast differentiation ( Figure 6 ab). RT-PCR was used to detect the expression levels of representative osteogenic genes such as RUNX2, OPN and collagen I (COL1). Figure 6 c). CMB@BG has a higher ability to induce osteogenic formation compared with conventional BG: the relative mRNA expression levels of RUNX2, COL1, and OPN were significantly increased in BMSCs seeded on CMB@BG scaffolds.

[0230] 2. The bone repair effect of CMB@BG scaffold was verified using the SD rat skull defect model.

[0231] Methods: Circular bone defects with a diameter of 5 mm were established in the bilateral parietal bones of SD rats, and ordinary BG or CMB@BG scaffolds (Φ5 mm×1.2 mm) were implanted into the defect sites. Rat skulls were collected at different time points for further evaluation. The dissected rat skulls were fixed in 4% paraformaldehyde fixative for 3 days. The skull samples were then scanned using a micro-CT device (NemoNMC-100, PING SENG Healthcare Inc.), and the scan results were analyzed using Avatar software 1.6.6 to obtain three-dimensional reconstructed images and trabecular bone volume / tissue volume (Tb.BV / TV), trabecular bone mineral density (Tb.BMD), and total porosity results.

[0232] Results: Three-dimensional reconstruction images and corresponding micro-computed tomography (micro-CT) analysis showed that more newly formed bone tissue appeared in the defect site where CMB@BG was implanted ( Figure 6 d). It is noteworthy that the parietal bone defect was almost completely healed 12 weeks after surgical intervention. Micro-CT quantitative analysis of the defect site, including trabecular bone volume / tissue volume (Tb.BV / TV), trabecular bone mineral density (Tb.BMD), and total porosity, further revealed the changes in trabecular bone parameters after CMB@BG implantation ( Figure 6 Compared with the defects implanted with ordinary BG scaffolds, the BV / TV value of the defects treated with CMB@BG increased, the total porosity decreased, and the BMD increased, indicating that the volume of new bone tissue in the defect area (TB.BV / TV) increased relatively, and the average bone density (Tb.BMD and total porosity) increased.

[0233] From the above tests, it can be seen that the CMB@BG bracket of the present invention has the following technical effects:

[0234] 1) The prepared CMB@BG scaffold can be used to show outstanding photothermal effect in the near-infrared-Ⅱ (NIR-Ⅱ) window, leading to effective photothermal therapy (PTT)-mediated tumor ablation;

[0235] 2) CMB, as a powerful ROS scavenger, rapidly eliminated the reactive oxygen species (ROS) that rose after PTT, thereby inhibiting the resulting inflammatory response;

[0236] 3) At the bone defect site, the proliferation and differentiation-promoting abilities of CMB@BG scaffolds can be continuously exerted both in vitro and in vivo and effectively accelerate bone regeneration;

[0237] 4) The CMB@BG scaffold combines the powerful NIR-Ⅱ-activated PTT effect, antioxidant-induced anti-inflammatory ability, and enhanced osteogenesis, making it easy to become a practical treatment for osteosarcoma, providing groundbreaking insights into the comprehensive treatment strategy for osteosarcoma.

[0238] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A photothermal conversion material, It is characterized in that Includes cerium substituted molybdenum based polyoxometalates.

2. A method for preparing a photothermal conversion material, for preparing the photothermal conversion biomaterial as claimed in claim 1, It is characterized in that include: mixing an inorganic cerium salt and a molybdate under acidic conditions to obtain a mixed solution; The mixed solution is dried to obtain cerium-substituted molybdenum-based polyoxometalate.

3. The preparation method according to claim 2, It is characterized in that The inorganic cerium salt includes cerium chloride, cerium nitrate, cerium acetate; and / or The molybdate includes sodium molybdate, potassium molybdate, ammonium molybdate; and / or The molar ratio of the inorganic cerium salt to the molybdate is 1:4 to 1:

10.

4. The preparation method according to claim 2 or 3, It is characterized in that include: Dissolving cerium chloride and hydrazine hydrochloride in deionized water to obtain a first solution; adding hydrochloric acid to the first solution to obtain a second solution; adding sodium molybdate to the second solution to obtain a third solution; The third solution is sequentially subjected to heating drying and freeze drying to obtain cerium-substituted molybdenum-based polyoxometalate.

5. The preparation method according to claim 4, It is characterized in that include: 20 mg CeCl 3 7H 2 O and 3 mg of hydrazine hydrochloride are dissolved in 5 mL of deionized water to obtain a first solution; Add 0.4 mL of 1 M hydrochloric acid to the first solution to obtain a second solution; Add 50 mg Na2MoO 4 ·2H 2 O to the second solution to obtain a third solution; The third solution is sequentially subjected to heating drying and freeze drying to obtain cerium-substituted molybdenum-based polyoxometalate.

6. A photothermal conversion biomaterial, It is characterized in that include: The photothermal conversion material according to claim 1, or the photothermal conversion material prepared by the preparation method according to any one of claims 2 to 5; A bioactive material, wherein the bioactive material carries the photothermal conversion material.

7. The photothermal conversion biomaterial according to claim 6, It is characterized in that The bioactive material includes bioactive glass, polylactic acid, polycaprolactone, hydroxyapatite; and / or The mass ratio of the photothermal conversion material to the bioactive material is 1:200 to 1:4000.

8. A method for preparing a photothermal conversion biomaterial, for preparing the photothermal conversion biomaterial as claimed in any one of claims 6 to 7, It is characterized in that include: The photothermal conversion material is loaded on a bioactive material to obtain a photothermal conversion biomaterial.

9. The preparation method according to claim 8, It is characterized in that The preparation method of the bioactive material comprises: mixing the bioactive material with the polyvinyl alcohol solution to obtain a slurry; Printing the slurry using a 3D printing method to obtain a bioactive material scaffold; and / or Loading the photothermal conversion material on a bioactive material to obtain a photothermal conversion biomaterial comprises: adding a cerium-substituted molybdenum-based polyoxometalate solution dropwise to a bioactive material scaffold to obtain an initial material; The initial material is subjected to a heat treatment to obtain a bioactive material scaffold loaded with cerium-substituted molybdenum-based polyoxometalate.

10. The preparation method according to claim 8, It is characterized in that The mass ratio of the bioactive material to the polyvinyl alcohol solution is 1.2:1 to 1.8:1; and / or The mass percentage of the polyvinyl alcohol solution is 8% to 12%; and / or The concentration of the cerium-substituted molybdenum-based polyoxometalate solution is 0.1 mg / mL to 2.5 mg / mL.

11. The preparation method according to claims 8 to 10, It is characterized in that include: (I) Preparation of bioactive materials The 80S bioactive glass and 11 wt % polyvinyl alcohol were mixed in a mass ratio of 1.5:1 to obtain a slurry; The slurry is 3D printed to obtain an initial scaffold, wherein the 3D printing parameters are: the nozzle inner diameter is 400 μm, the extrusion pressure is 2.8 bar to 4.4 bar, the printing speed is 8 mm / s to 18 mm / s, and the single layer thickness is 0.32 mm; The initial scaffold is sequentially dried and sintered to obtain a bioglass active scaffold; (II) Preparation of photothermal conversion biomaterials Adding 20 μL of 1.2 mg / mL cerium-substituted molybdenum-based polyoxometalate solution dropwise to the bioglass active scaffold to obtain an initial material; The initial material is dried at room temperature for 30 to 60 minutes to obtain a bioactive glass scaffold loaded with cerium-substituted molybdenum-based polyoxometalate.

12. Use of the photothermal conversion material according to claim 1, or the photothermal conversion material prepared by the preparation method according to any one of claims 2 to 5, or the photothermal conversion material according to any one of claims 6 to 7, or the photothermal conversion biomaterial prepared by the preparation method according to any one of claims 8 to 11 in the preparation of materials for treating bone tumors.

13. Use of the photothermal conversion biomaterial as claimed in any one of claims 6 to 7 or the photothermal conversion biomaterial prepared by the preparation method as claimed in any one of claims 8 to 11 in preparing bone defect repair materials.

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