CN113101409A

CN113101409A - Multifunctional compound bracket and preparation method and application thereof

Info

Publication number: CN113101409A
Application number: CN202110384198.8A
Authority: CN
Inventors: 李明强; 陶玉; 雎恩国; 党文涛; 金圆圆; 易可
Original assignee: Third Affiliated Hospital Sun Yat Sen University
Current assignee: Third Affiliated Hospital Sun Yat Sen University
Priority date: 2021-04-09
Filing date: 2021-04-09
Publication date: 2021-07-13

Abstract

The application belongs to the technical field of biomedical materials, and particularly relates to a multifunctional compound support and a preparation method and application thereof. The multifunctional composite scaffold of the present application comprises a bone tissue scaffold and a functional layer; the functional layer is arranged on the surface of the bone tissue scaffold; the functional layer comprises a photothermal layer and a medicine carrying layer; or the functional layer is a mixed layer which comprises a photothermal reagent and a chemotherapeutic drug; the preparation method of the bone tissue scaffold comprises the following steps: obtaining bone medical image data of a part to be implanted, converting the bone medical image data into three-dimensional model data of bones, and preparing a bone tissue scaffold by 3D printing processing according to the three-dimensional model data. The application provides a multifunctional compound bracket and a preparation method and application thereof, which can efficiently kill residual tumor cells and fill the bone defect position after operation, and can effectively solve the technical problems of single function, low tumor treatment effective rate and difficult removal of residual tumor cells of the traditional bracket for treating bone cancer.

Description

Multifunctional compound bracket and preparation method and application thereof

Technical Field

The application belongs to the technical field of biomedical materials, and particularly relates to a multifunctional compound support and a preparation method and application thereof.

Background

The incidence of bone metastasis of bone tumors and metastatic tumors increases year by year, and great pain is brought to patients. Currently, the main method for clinically treating bone tumor is surgical resection with chemotherapy; however, the operation causes massive bone defects to affect the motor function of the body, and it is often difficult to completely remove tumor cells, and the remaining cancer cells are a major cause of tumor recurrence. Systemic chemotherapy can also damage normal tissue cells while killing tumor cells, causing systemic toxic and side effects to patients. In addition, long-term chemotherapy can cause bone cancer cells to develop drug resistance and reduce the immunity of the organism. Therefore, the development of the biomaterial which can kill residual tumor cells for a long time and fill bone defects has wide clinical requirements and great economic value.

Therefore, the development of a product capable of killing residual tumor cells with high efficiency and filling bone defects is a technical problem to be solved by those skilled in the art.

Disclosure of Invention

In view of the above, the application provides a multifunctional composite scaffold, a preparation method and an application thereof, which can efficiently kill residual tumor cells and fill bone defects, and can effectively solve the technical problems of single function, low tumor treatment efficiency and difficulty in thoroughly removing residual tumor cells of the existing scaffold for treating bone cancer.

The present application provides in a first aspect a multifunctional composite stent comprising:

bone tissue scaffolds and functional layers; the functional layer is arranged on the surface of the bone tissue scaffold;

the functional layer comprises a light-heat layer and a medicine-carrying layer;

or the functional layer is a mixed layer, and the mixed layer comprises a photothermal agent and a chemotherapeutic drug;

the preparation method of the bone tissue scaffold comprises the following steps: obtaining bone medical image data of a part to be implanted, converting the bone medical image data into three-dimensional model data of a bone, and preparing a bone tissue scaffold through 3D printing and processing according to the three-dimensional model data.

In another embodiment, the bone tissue scaffold is made of one or more materials selected from hydroxyapatite, tricalcium phosphate, calcium silicate, bioactive glass ceramic, and degradable polymers.

Specifically, the degradable polymer is selected from one or more of polylactic acid, poly (lactide-co-glycolide) and polycaprolactone.

In particular, the hydroxyapatite, the tricalcium phosphate, the bioactive glass ceramic and the degradable polymer are common materials for bone tissue engineering.

Preferably, the bone tissue scaffold is made of bioactive glass ceramic, and the bioactive glass ceramic is one of ideal materials for bone tissue engineering due to excellent biocompatibility, good bone conductivity and bone inductivity. The bone tissue scaffold is created by a method of printing bone tissue materials layer by layer, so that individualized manufacturing of filling of the bone defect part of a patient is realized.

In another embodiment, the photothermal agent of the photothermal layer is selected from hemoglobin, black phosphorus, graphene, molybdenum disulfide (MoS)₂) Titanium nitride (TiN), titanium dioxide (TiO)₂) Gold nanorods, metal organic framework compound, polydopamine, and ferroferric oxide (Fe)₃O₄) Copper sulfide (CuS) and selenium copper iron ore (CuFeSe)₂) One or more of;

the photothermal reagent is selected from one or more of heme, black phosphorus, graphene, molybdenum disulfide, titanium nitride, titanium dioxide, a nano gold rod, a metal organic framework compound, polydopamine, ferroferric oxide, copper sulfide and selenium copper iron ore.

Specifically, the application discovers that under the action of near infrared light, heme loaded on the surface of the bone tissue scaffold has a thermotherapy effect, and the bone tissue scaffold loaded with the heme can kill solid tumor cells and inhibit the growth of solid tumors by irradiating the near infrared light with tissue penetrability.

In another embodiment, the chemotherapeutic agent of the drug-loaded layer is selected from one or more of doxorubicin, fluorouracil, cyclophosphamide, vincristine, vindesine, daunorubicin, irinotecan, mitoxantrone, methotrexate, paclitaxel, docetaxel, camptothecin, cisplatin, carboplatin, and oxaliplatin;

the chemotherapy drug is one or more selected from adriamycin, fluorouracil, cyclophosphamide, vincristine, vindesine, daunorubicin, irinotecan, mitoxantrone, methotrexate, paclitaxel, docetaxel, camptothecin, cisplatin, carboplatin and oxaliplatin.

In another embodiment, the number of the photo-thermal layers is 1, 2, 3, 4, 5 or 6; the number of the medicine-carrying layers is 1, 2, 3, 4, 5 or 6; the number of the mixed layers is 1, 2, 3, 4, 5 or 6.

In another embodiment, the photothermal layer and the drug-loaded layer are arranged on the surface of the bone tissue scaffold at intervals; the photothermal layer and the drug-loaded layer are sequentially arranged on the surface of the bone tissue bracket; the surface of the bone tissue bracket is sequentially provided with the medicine-carrying layer and the photothermal layer.

Specifically, when the number of the photo-thermal layer and the drug-loaded layer is 1, the preparation method comprises the following steps: soaking the bone tissue scaffold in the photothermal layer solution, taking out and drying, arranging the photothermal layer on the surface of the bone tissue scaffold, soaking the bone tissue scaffold attached with the photothermal layer in the drug-loaded layer solution, taking out and drying to obtain the bone tissue scaffold attached with the functional layer.

Specifically, when the number of the photo-thermal layer and the drug-loaded layer is greater than 1, the preparation method comprises the following steps: soaking the bone tissue scaffold in the photothermal layer solution, taking out and drying; soaking the bone tissue scaffold attached with the 1-layer photothermal layer in the photothermal layer solution, taking out and drying, and repeating the operation to obtain the bone tissue scaffold attached with the multi-layer photothermal layer; soaking the bone tissue scaffold attached with the multiple photothermal layers in the drug-loaded layer solution, taking out and drying, soaking the bone tissue scaffold attached with the 1-layer drug-loaded layer and the multiple photothermal layers in the drug-loaded layer solution, taking out and drying, and repeating the operation to obtain the bone tissue scaffold attached with the multiple photothermal layers and the multiple drug-loaded layers in sequence.

Specifically, when the number of the drug-loaded layer and the number of the photothermal layer are 1, the preparation method comprises the following steps: soaking the bone tissue scaffold in the drug-loaded layer solution, taking out and drying, arranging the drug-loaded layer on the surface of the bone tissue scaffold, then soaking the bone tissue scaffold attached with the drug-loaded layer in the photo-thermal layer solution, taking out and drying to obtain the bone tissue scaffold attached with the functional layer.

Specifically, when the number of the drug-loaded layer and the photothermal layer is greater than 1, the preparation method comprises the following steps: soaking the bone tissue scaffold in the drug-loaded layer solution, taking out and drying; soaking the bone tissue scaffold attached with 1 drug-loaded layer in the drug-loaded layer solution, taking out and drying, and repeating the above operation to obtain the bone tissue scaffold attached with a plurality of drug-loaded layers; soaking the bone tissue scaffold attached with the multiple drug-loaded layers in the photo-thermal layer solution, taking out and drying, soaking the bone tissue scaffold attached with the 1-layer photo-thermal layer and the multiple drug-loaded layers in the photo-thermal layer solution, taking out and drying, and repeating the operation to obtain the bone tissue scaffold attached with the multiple drug-loaded layers and the multiple photo-thermal layers in sequence.

In a second aspect, the present application provides a method for preparing a multifunctional composite scaffold, comprising:

step 1, obtaining bone medical image data of a part to be implanted, and converting the bone medical image data into 3D model data of bones;

taking a bone tissue material as a raw material, and preparing a bone tissue scaffold by 3D printing processing according to the 3D model data;

step 2, soaking the bone tissue scaffold in a functional layer solution, taking out and drying to obtain a multifunctional compound scaffold;

the functional layer solution comprises a photo-thermal layer solution and a drug-loaded layer solution;

or the functional layer solution is a mixed layer solution, and the mixed layer solution comprises a photo-thermal reagent and a chemotherapeutic drug.

In another embodiment, in step 2, the bone tissue scaffold is soaked in the functional layer solution, taken out and dried, and then the dried scaffold is repeatedly soaked in the functional layer solution, taken out and dried, and repeated for a plurality of times. Specifically, the number of repetitions is 1, 2, 3, 4, 5 or 6.

Specifically, the functional layer solution comprises a photothermal layer solution and a drug-loaded layer solution;

Specifically, the photothermal layer solution comprises a photothermal agent and a solvent; the drug-loaded layer solution comprises a chemotherapeutic drug and a solvent.

Specifically, the mixed layer solution comprises a photothermal agent, a chemotherapeutic drug and a solvent.

In another embodiment, the bone tissue material is selected from one or more of hydroxyapatite, tricalcium phosphate, calcium silicate, bioactive glass-ceramic, and degradable polymers;

the photothermal reagent of the photothermal layer solution is selected from one or more of heme, black phosphorus, graphene, molybdenum disulfide, titanium nitride, titanium dioxide, a nano gold rod, a metal organic framework compound, polydopamine, ferroferric oxide, copper sulfide and selenium copper iron ore;

the photothermal reagent is selected from one or more of heme, black phosphorus, graphene, molybdenum disulfide, titanium nitride, titanium dioxide, a nano gold rod, a metal organic framework compound, polydopamine, ferroferric oxide, copper sulfide and selenium copper iron ore;

the chemotherapy drug of the drug-carrying layer solution is selected from one or more of adriamycin, fluorouracil, cyclophosphamide, vincristine, vindesine, daunorubicin, irinotecan, mitoxantrone, methotrexate, paclitaxel, docetaxel, camptothecin, cisplatin, carboplatin and oxaliplatin;

In another embodiment, the solvent is selected from one or more of a dichloromethane solution of polylactic acid, a chloroform solution of polylactic acid, or a hexafluoroisopropanol solution of polylactic acid.

Specifically, the concentration of the polylactic acid in the dichloromethane solution of the polylactic acid is 0.01-0.3 g/mL.

In another embodiment, the photothermal agent of the photothermal layer solution is selected from hemoglobin.

In another embodiment, the chemotherapeutic agent is selected from doxorubicin.

In another embodiment, the concentration of hemoglobin in the photothermal layer solution is 0.001-0.1 g/mL.

In another embodiment, the concentration of the chemotherapeutic agent in the drug-loaded layer solution is 0.01-5 mg/mL.

In another embodiment, the concentration of the heme in the mixed layer solution is 0.001-0.1 g/mL; the concentration of the chemotherapeutic drug in the mixed layer solution is 0.01-5 mg/mL.

In another embodiment, the bone tissue material is a bioactive glass-ceramic.

In another embodiment, a method of making a multifunctional composite scaffold of the present application comprises:

step 1), obtaining bone medical image data of a part to be implanted, and converting the bone medical image data into 3D model data of bones;

step 2), soaking the bone tissue scaffold in a photo-thermal reagent solution, taking out and drying to obtain a bone tissue scaffold doped with a photo-thermal reagent;

and 3) soaking the bone tissue scaffold doped with the photo-thermal reagent in a chemotherapeutic drug solution, taking out and drying to obtain the multifunctional composite scaffold.

In another embodiment, in step 2), the bone tissue scaffold is soaked in the photothermal agent solution, taken out and dried, and then the dried scaffold is repeatedly soaked in the photothermal agent solution, taken out and dried, and repeated for a plurality of times. Specifically, the number of repetitions is 1, 2, 3, 4, 5 or 6.

In another embodiment, in step 3), the bone tissue scaffold doped with the photo-thermal agent is soaked in the chemotherapeutic drug solution, taken out and dried, and then the dried scaffold is repeatedly soaked in the chemotherapeutic drug solution, taken out and dried for several times. Specifically, the number of repetitions is 1, 2, 3, 4, 5 or 6.

step 1)), obtaining bone medical image data of a part to be implanted, and converting the bone medical image data into 3D model data of bones;

step 2)), soaking the bone tissue scaffold in a chemotherapeutic drug solution, taking out and drying to obtain the bone tissue scaffold doped with the chemotherapeutic drug;

and 3), soaking the bone tissue scaffold doped with the chemotherapeutic drugs in a photo-thermal reagent solution, taking out and drying to obtain the multifunctional composite scaffold.

In another embodiment, step 2), the bone tissue scaffold is soaked in the chemotherapeutic drug solution, taken out and dried, and then the dried scaffold is repeatedly soaked in the chemotherapeutic drug solution, taken out and dried, and repeated for a plurality of times. Specifically, the number of repetitions is 1, 2, 3, 4, 5 or 6.

In another embodiment, the bone tissue scaffold doped with the chemotherapeutic drug is soaked in the photothermal agent solution, taken out and dried in step 3)), and then the dried scaffold is repeatedly soaked in the photothermal agent solution, taken out and dried for several times. Specifically, the number of repetitions is 1, 2, 3, 4, 5 or 6.

In another embodiment, the bone tissue scaffold can be soaked in a dichloromethane solution of polylactic acid containing heme and adriamycin simultaneously, so that the heme and the adriamycin are adhered to the surface of the scaffold, and the multifunctional composite scaffold doped with the heme and the adriamycin is prepared.

In another embodiment, the bone tissue scaffold can be soaked in dichloromethane solution of polylactic acid containing adriamycin, then taken out, dried and soaked in dichloromethane solution of polylactic acid containing heme to ensure that adriamycin and heme are sequentially adhered to the surface of the scaffold, and the prepared bone tissue scaffold doped with heme and adriamycin can delay release of adriamycin due to the heme adhered to the outer layer.

In another embodiment, the number of times the bone tissue scaffold is soaked in a dichloromethane solution of polylactic acid containing hemoglobin can be increased by 2, 3, 4, 5 or 6 times, and as the number of soaking times is increased, more hemoglobin adheres to the surface of the scaffold.

In particular, the multifunctional composite scaffold provided by the application can be used for treating bone tumors, or other solid tumors metastasized to bone, such as one or more of metastatic breast tumors, metastatic brain tumors, metastatic liver tumors, metastatic prostate tumors, metastatic head and neck tumors, and metastatic oral tumors.

In a third aspect, the application discloses the multifunctional compound bracket and the application of the multifunctional compound bracket prepared by the preparation method in preparing a medicament for treating bone tumor or solid tumor transferred to bone.

The fourth aspect of the application discloses the multifunctional compound bracket and the application of the multifunctional compound bracket and the near infrared device prepared by the preparation method in the preparation of a solid tumor system for treating bone tumor or transferring to bone.

Specifically, in the application, near infrared light is adopted to irradiate the multifunctional composite scaffold, so that the multifunctional composite scaffold generates photothermal curative effect in bone tumor cells or solid tumor cells transferred to bones.

Specifically, the multifunctional composite scaffold is irradiated by near infrared light, and the multifunctional composite scaffold kills bone tumor cells and inhibits bone tumor, or growth of solid tumor transferred to bone.

Specifically, the solid tumor is selected from one or more of metastatic breast tumor, metastatic skin tumor, metastatic brain tumor, metastatic liver tumor, metastatic prostate tumor, metastatic head and neck tumor and metastatic oral tumor.

In another embodiment, the power density of the near infrared light is 0.3-1.5W/cm²(ii) a The irradiation time of the near infrared light is 0-30 min.

In summary, the present application prepares the bone tissue scaffold applicable to human body by 3D printing technology according to the actual bone tissue model, the photothermal layer and the drug-loaded layer or/and the mixed layer are modified on the surface thereof, and under the action of near infrared light, the photothermal layer and the drug-loaded layer or/and the mixed layer attached to the surface of the scaffold have the functions of thermotherapy and chemotherapy, enhance the anti-tumor effect, and can be used for combined anti-tumor of chemotherapy and thermotherapy.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is an external view and a scanning electron microscope of a BGC-HM stent doped with hemoglobin with different proportions according to an embodiment of the present invention;

FIG. 2 is a BGC-3HM scaffold element scan, XPS spectroscopy and XRD pattern analysis provided in an embodiment of the present application;

FIG. 3 is an appearance, SEM, red fluorescence and DOX release analysis of BGC-1DOX, BGC-2DOX, BGC-3DOX, BGC-4DOX scaffolds provided in the examples herein;

FIG. 4 is a photothermal property analysis of various BGC scaffolds provided in the examples herein;

FIG. 5 is an analysis of the effect of different BGC scaffolds provided in the examples of the present application on killing cancer cells in vitro;

FIG. 6 shows the apoptosis analysis of K7M2wt cells in the presence and absence of Near Infrared (NIR) light for different scaffolds provided in the examples of the present application;

FIG. 7 shows in vivo anti-tumor effect analysis of different BGC scaffolds provided in the examples of the present application;

FIG. 8 is a picture of tumor tissue and H & E (hematoxylin and eosin) staining after 18 days of treatment of mice with BGC, BGC-3HM, BGC-4DOX, BGC-3HM + NIR, BGC-3HM-4DOX + NIR scaffolds provided in the examples of the present application;

FIG. 9 is a graph showing the H & E (hematoxylin and eosin) staining of the major organs (including heart, liver, spleen, lung and kidney) of tumor-bearing mice after BGC, BGC-3HM, BGC-4DOX, BGC-3HM-4DOX, NIR, BGC-3HM-4DOX + NIR treatment provided in the examples of the present application.

Detailed Description

The application provides a multifunctional compound scaffold and a preparation method and application thereof, which are used for solving the technical defects that in the prior art, a bone tissue scaffold for treating solid tumors, particularly after bone tumor and metastatic tumor bone metastasis surgical treatment has single function, low tumor treatment effective rate and difficulty in removing residual tumor cells.

The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The raw materials and reagents used in the following examples are commercially available or self-made.

The polylactic acid of the present application is poly (D, L-lactic acid).

Example 1

The embodiment of the application provides BGC powder, a BGC stent and a preparation method of the BGC stent doped with Heme (HM) and adriamycin (DOX), and the preparation method specifically comprises the following steps:

1. preparation of BGC powder (bioactive glass ceramic powder): preparing BGC powder by a sol-gel method. The process is described as follows: 156.81mL TEOS (Ethyl orthosilicate) was added to 200mL deionized water, followed by 25.00mL HNO₃Solution (2moL/L) was added to the above solution and stirred for 30 min. Next, 59.04g Ca (NO) was added₃)₂·4H₂O and 8.53ml of LTEP (triethyl phosphate) were added to the above solution and stirred for 6 h. The solution was then placed in an air oven at 65 ℃ for 24h, and the product was then dried in an air oven at 120 ℃ for 48 h. The dried product was transferred to a planetary high energy ball mill and milled for 4h at 600 rpm. Then will produceThe material was calcined in a high temperature sintering furnace with a maximum temperature of 800 ℃ for 3 h. After cooling to room temperature, the sintered powder was collected and filtered through a 300 mesh screen to obtain BGC powder.

2. Preparation of BGC scaffolds (bioactive glass ceramic scaffolds): the BGC powder (6g) was poured into a 50mL beaker. 5.5mL of an aqueous F-127 solution (15 wt%) was then added to the beaker and stirred for 10min to obtain a homogeneous BGC ink. Next, the BGC ink was transferred into a printing tube, and then printed using a 3D printer (produced by bubon obi biotechnology limited). The printing speed is 5mm/s and the pressure is 0.5-2.0 Bar. After the printed stent was dried at room temperature for 24h, it was transferred to a high temperature sintering furnace and sintered at a temperature of up to 1200 ℃ for 3h to obtain a BGC stent.

3. Preparation of a BGC scaffold incorporating heme and doxorubicin: the heme powder was added to deionized water and milled by a planetary high energy ball mill at 500rpm for 4 h. After milling, the heme powder was dried and collected. 0.1g of polylactic acid was added to 5mL of methylene chloride and stirred to be completely dissolved. Then, the heme powder was put into a dichloromethane solution of the above polylactic acid. Stirring for 10min, ultrasonically dispersing for 30min to obtain mixed solution of heme and polylactic acid, completely soaking BGC stent in the mixed solution of heme and polylactic acid for 4s, and taking out to obtain BGC-HM stent. 0.1g of polylactic acid was added to 5mL of methylene chloride and stirred to be completely dissolved. Then, doxorubicin was put into the above-mentioned methylene chloride solution of polylactic acid. Stirring for 10min and ultrasonically dispersing for 30min to obtain a mixed solution of adriamycin and polylactic acid, and completely immersing the BGC-HM stent into the mixed solution of adriamycin and polylactic acid. And (3) immersing the BGC-HM stent into the adriamycin-containing mixed solution for 4s, and taking out the BGC-HM stent to obtain the BGC-HM-DOX stent.

Example 2

The embodiment of the application provides a preparation method of a BGC-HM stent doped with different proportions of heme HM, which comprises the following steps:

1. the heme powder was added to deionized water and milled by a planetary high energy ball mill at 500rpm for 4 h. After milling, the heme powder was dried and collected. 0.1g of polylactic acid was added to 5mL of methylene chloride and stirred to be completely dissolved. Then, three different weights of heme powder (0.02g, 0.06g, and 0.12g) were placed in the above-mentioned dichloromethane solution of polylactic acid, respectively. Stirring for 10min and ultrasonically dispersing for 30min to obtain mixed solution of heme and polylactic acid, which is labeled as 1HM mixed solution, 2HM mixed solution and 3HM mixed solution.

2. The BGC stents prepared in example 1 were immersed in the 1HM mixed solution, the 2HM mixed solution, and the 3HM mixed solution, respectively. And completely immersing the BGC bracket into three heme mixed solutions with different concentrations for 4s, and taking out the BGC bracket to prepare the BGC bracket doped with heme correspondingly, which is named as BGC-1HM, BGC-2HM and BGC-3HM brackets respectively.

3. The results of optical micrographs and scanning electron micrographs of the BGC stent prepared in example 1 and the BGC-HM stents prepared above and doped with different proportions of heme (BGC-1HM, BGC-2HM and BGC-3HM) are shown in FIG. 1, and FIG. 1 provides an appearance view and a scanning electron micrograph of the BGC-HM stent doped with different proportions of heme in the example of the present application.

In FIG. 1, A is an external view, A is 2mm, B-D are SEM images with different magnifications, B is 1mm, C is 40 μm, D is 10 μm, 1 is a BGC stent not doped with heme, 2 is BGC-1HM in which the BGC stent is immersed in a 1HM mixed solution, 3 is BGC-2HM in which the BGC stent is immersed in a 2HM mixed solution, 4 is BGC-3HM in which the BGC stent is immersed in A3 HM mixed solution, and the BGC-HM stent doped with heme in different proportions has different colors as can be seen from A1-A4, and the color of the stent changes from white A1 to black A4 as the heme on the surface of the BGC increases; 2-4 stents are SEM images of BGC stents from low to high magnification, as can be seen from B1-B4, C1-C4, and D1-D4, the stents are formed by stacking, and some micropores are observed on the stents (in the images B1-B4., in addition, the surfaces of the stents are distributed, and some micropores are formed in the gaps. for BGC-1HM, BGC-2HM and BGC-3HM stents, the gaps and micropores of the stents are covered by the mixed substance of heme and polylactic acid. from the SEM images of high magnification, a large number of heme particles and polymers are observed on the BGC-1HM, BGC-2HM and BGC-3HM stents.

4. The BGC-3HM stent prepared in example 1 was immersed in a 3HM mixed solution to prepare a BGC-3HM stent. Elemental scanning, XPS spectroscopy and XRD pattern analysis of BGC-3HM scaffolds were performed and the results are shown in FIG. 2.

FIG. 2A is a scanned elemental view of a BGC-3HM stent provided in an embodiment of the present application; FIG. 2B is an XPS spectrum of a BGC-3HM stent provided in an embodiment of the present application; fig. 2C is an XRD pattern of the BGC scaffold provided in the examples of the present application. As can be seen from fig. 2A-C, the corresponding elemental map distribution on the BGC-3HM stent indicates the presence of Si, O, P, C, Ca and Fe, which confirms successful encapsulation of heme on the BGC stent, with a scale of 10 μm in a; XPS analysis was used to further confirm the presence of heme on the BGC scaffold; in addition, XRD patterns show that the BGC scaffold is made of SiO₂、CaSiO₃And Ca₃(PO₄)₃Phase composition.

Example 3

The embodiment of the application provides a preparation method of a BGC-HM stent doped with adriamycin with different proportions, which comprises the following steps:

1. 2mg, 5mg, 10mg and 15mg of doxorubicin were weighed, and each of doxorubicin was added to 5mL of a methylene chloride solution of polylactic acid having a polylactic acid concentration of 0.02 g/mL. After stirring and ultrasonic dispersion, the BGC stent was immersed in the above mixed solution for 4s, and then taken out.

2. The stents are respectively immersed in polylactic acid dichloromethane solution containing 2mg, 5mg, 10mg and 15mg of adriamycin, and the taken out stents are respectively named as BGC-1DOX, BGC-2DOX, BGC-3DOX and BGC-4DOX stents.

3. The BGC-1DOX, BGC-2DOX, BGC-3DOX and BGC-4DOX scaffolds were examined for appearance, SEM, red fluorescence and doxorubicin release, with the results shown in FIG. 3. FIG. 3A is an external view of a BGC-1DOX, BGC-2DOX, BGC-3DOX, and BGC-4DOX stent provided in accordance with an embodiment of the present application; FIGS. 3B and 3C are SEM images of BGC-2DOX and BGC-4DOX stents, respectively; FIG. 3D is a red fluorescence image of a BGC-4DOX stent provided in an embodiment of the present application; FIG. 4E is a graph showing the doxorubicin release profiles for BGC-1DOX, BGC-2DOX, BGC-3DOX, and BGC-4DOX scaffolds provided in the examples herein.

As can be seen from FIGS. 3A-3E, after doxorubicin had been loaded onto the BGC stent, the stent color was observed to become darker as the doxorubicin concentration increased, indicating that doxorubicin was successfully loaded onto the BGC stent (FIGS. 3A 1-A4), with the scale in FIG. A representing 2 mm. SEM images of the BGC-2DOX and BGC-4DOX scaffolds of FIGS. 3B and 3C show that there was little change in morphology after loading of the BGC scaffold with doxorubicin. In addition, the red fluorescence of the BGC-4DOX scaffold further confirmed the successful loading of doxorubicin onto the BGC scaffold (FIG. 3D). The release profile of doxorubicin showed that more and more doxorubicin could be released from the scaffold with increasing time (fig. 3E). The released adriamycin amount is increased along with the increase of the concentration of the adriamycin coated on the stent, so that the dosage of the drug can be flexibly adjusted to reduce the side effect of the drug. The scale of FIGS. 3B and 3C is 20 μm, and the scale of FIG. 3D is 500 μm.

Example 4

The embodiment of the application provides a test of photo-thermal properties of different BGC (BGC) brackets, and the method comprises the following steps:

1. BGC stents were prepared according to the method described in example 1, BGC-1HM, BGC-2HM and BGC-3HM stents were prepared according to the method described in example 2, and BGC-1DOX, BGC-2DOX, BGC-3DOX and BGC-4DOX stents were prepared according to the method described in example 3.

The BGC-3HM stents prepared in the above example 2 were immersed in a solution of polylactic acid dichloromethane containing 2mg, 5mg, 10mg and 15mg of adriamycin, respectively, and taken out therefrom, and named BGC-3HM-1DOX, BGC-3HM-2DOX, BGC-3HM-3DOX and BGC-3HM-4DOX stents.

2. The prepared BGC, BGC-1HM, BGC-2HM, BGC-3HM, BGC-1DOX, BGC-2DOX, BGC-3DOX, BGC-4DOX, BGC-3HM-1DOX, BGC-3HM-2DOX, BGC-3HM-3DOX and BGC-3HM-4DOX stents were subjected to photothermal performance analysis, and the results are shown in FIG. 4.

Placing the above-prepared dry BGC, BGC-1HM, BGC-2HM and BGC-3HM scaffolds into the wells of a 48-well culture plate, and then placing the power density at 0.9W/cm²The NIR laser beam of (a) is irradiated onto these supports. The irradiation time was 5min, and a thermal imager was used to detect temperature changes and capture photothermal images at different time points. At the same time, recordThe temperature at different time points was measured. Adding 250 μ LPBS into 48-well culture plate containing BGC, BGC-1HM, BGC-2HM and BGC-3HM scaffolds to obtain wet scaffold with power density of 0.9W/cm²The NIR laser beam irradiates the support for 10min, and the temperature at different time points is recorded. The results are shown in FIGS. 4A, 4B and 4C.

Placing the dry BGC-3HM scaffold into 48-well culture plate, and irradiating with NIR laser at power density of 0.6W/cm²、0.7W/cm²、0.8W/cm²、0.9W/cm²、1.0W/cm²The irradiation time was 5min and the temperature was recorded. The results are shown in FIG. 4D. The BGC-3HM scaffolds were irradiated with NIR light in the dry state and then subjected to thermographic analysis, the results of which are shown in FIG. 4G.

Placing the above prepared dry BGC, BGC-1DOX, BGC-2DOX, BGC-3DOX and BGC-4DOX scaffolds in the wells of a 48-well culture plate, and then placing the power density at 0.9W/cm²The NIR laser beam of (a) is irradiated onto these supports. Temperature changes were detected using a thermal imager. 250 mu LPBS was added to 48-well culture plates containing BGC, BGC-1DOX, BGC-2DOX, BGC-3DOX and BGC-4DOX scaffolds to obtain wet scaffolds. The power density is 0.9W/cm²The NIR laser beam of (a) was irradiated on these dry and wet scaffolds for 5min for dry irradiation and 10min for wet irradiation, and the resulting temperature was recorded. The results are shown in FIG. 4E.

Placing the prepared dry BGC-3HM, BGC-3HM-1DOX, BGC-3HM-2DOX, BGC-3HM-3DOX and BGC-3HM-4DOX bracket into the holes of 48-hole culture plate, and then placing the power density at 0.9W/cm²The NIR laser beam was irradiated onto these supports for 5min, and the temperature change was detected using a thermal imager. 250 mu L of PBS is added into a 48-hole culture plate containing BGC-3HM, BGC-3HM-1DOX, BGC-3HM-2DOX, BGC-3HM-3DOX and BGC-3HM-4DOX stents to obtain wet stents. The power density is 0.9W/cm²The NIR laser beam of (a) was irradiated to these holders for 10min and the resulting temperature was recorded. The results are shown in FIG. 4F.

FIGS. 4A and 4B are temperature profiles of BGC, BGC-1HM, BGC-2HM and BGC-3HM stents provided by embodiments of the present application after being irradiated by NIR light for different times in the dry state and in the wet state; FIG. 4C is the equilibrium temperature of BGC, BGC-1HM, BGC-2HM and BGC-3HM stents provided by embodiments of the present application after irradiation by the same NIR optical power density in the dry and wet states; FIG. 4D is an equilibrium temperature of a BGC-3HM stent provided by an embodiment of the present application exposed to NIR light of different power densities in the dry and wet states; FIG. 4E is the equilibrium temperature of BGC, BGC-1DOX, BGC-2DOX, BGC-3DOX, and BGC-4DOX stents provided in accordance with embodiments of the present application after exposure to NIR light in the dry and wet states; FIG. 4F is the equilibrium temperature of the BGC-3HM, BGC-3HM-1DOX, BGC-3HM-2DOX, BGC-3HM-3DOX and BGC-3HM-4DOX stents provided in accordance with embodiments of the present application after irradiation by NIR light in the dry and wet states; fig. 4G is a thermal imaging diagram of a BGC-3HM stent provided in an embodiment of the present application after irradiation with NIR light in a dry state.

As can be seen from fig. 4, the BGC scaffold without heme loading did not have photothermal conversion capability. While the equilibrium temperatures after 5min were raised to 68.10 + -1.37 deg.C, 98.60 + -1.73 deg.C and 114.38 + -1.41 deg.C, respectively, when BGC-1HM, BGC-2HM and BGC-3HM stents were exposed to the same NIR laser power density (FIG. 4A). This phenomenon mainly indicates that the heme-loaded BGC scaffold has photothermal conversion capability and increases with increasing heme content on the surface of the scaffold. Meanwhile, the photothermal properties of the BGC-HM scaffold are strongly related to the dry and wet conditions (FIGS. 4B and 4C). In addition, the equilibrium temperature of the stent after NIR illumination increased with increasing optical power density (fig. 4D). In contrast, neither the BGC-DOX nor BGC scaffolds changed significantly in temperature after NIR illumination, either in dry or wet state (fig. 4E). Moreover, the additional loading of doxorubicin coatings on the BGC-1HM, BGC-2HM, and BGC-3HM stent surfaces did not affect the temperature of the stent after NIR illumination (FIG. 4F). Thermography of BGC-3HM stent after NIR illumination further confirmed that heme confers photothermal properties on BGC stents (fig. 4G).

Example 5

The embodiment of the application provides an in vitro cancer cell killing effect test of different BGC (BGC fusion proteins) stents, which comprises the following steps:

The BGC-3HM stents prepared in the above examples were immersed in a solution of polylactic acid in dichloromethane containing 2mg, 5mg, 10mg and 15mg of adriamycin, respectively, and taken out therefrom, and named BGC-3HM-1DOX, BGC-3HM-2DOX, BGC-3HM-3DOX and BGC-3HM-4DOX stents.

2. Cells (4X 10) containing K7M2wt⁴Cells/well) were seeded into 48-well plates (500 μ L of medium). After 24h, the medium was removed and fresh medium (500. mu.L) was added. Then, BGC-1HM, BGC-2HM, and BGC-3HM scaffolds were placed in each well, and cells without scaffold were set as a control group (control), cultured for 24 hours, 250 μ L of the medium was aspirated, and after 10min, these scaffolds were gently removed, and then subjected to MTT test for cell viability. Meanwhile, in order to examine the effect of the NIR light irradiation treatment on the cell activity, after culturing for 24 hours, 250. mu.L of each of the media containing the four scaffolds was removed, and then the NIR light irradiation treatment was performed with a laser power density of 0.9W/cm²Irradiation time 10 min. MTT assay specifically, MTT (40 μ L, 5mg/mL) was added to each well and incubated for 4h to generate formazan. After that, the solution in each well was gently aspirated, and DMSO (300 μ L) was added to each well to dissolve the formazan. Finally, the absorbance at 490nm was measured with a microplate reader (Synergy H1, BioTek). The results are shown in fig. 5A, fig. 5A shows the cell survival rate of the bone cancer cells K7M2wt co-incubated with different scaffolds in the presence and absence of NIR light irradiation, w/o laser shows the cell survival rate of the bone cancer cells K7M2wt co-incubated with different scaffolds in the absence of NIR light irradiation, and withlaser shows the cell survival rate of the bone cancer cells K7M2wt co-incubated with different scaffolds in the presence of NIR light irradiation.

3. Cells (4X 10) containing K7M2wt⁴Cells/well) were seeded into 48-well plates (500 μ L of medium). After 24h of seeding the cells, the medium was removed and fresh medium (500 μ L) was added. Then, the BGC-3HM scaffold was placed in each well, cultured for 24 hours, 250. mu.L of the medium was removed, and the power density (0.6W/cm) was adjusted²、0.7W/cm²、0.8W/cm²、0.9W/cm²) Respectively irradiating the BGC-3HM bracket by the NIR laser for 10min, and then performing MTT measurement. Cells (4X 10) containing K7M2wt⁴Cells/well) was seeded into 48-well plates (500 μ L). After 24h of seeding the cells, the medium was removed and fresh medium (500 μ L) was added. Then, the BGC-3HM scaffold was placed in each well, cultured for 24h, 250. mu.L of the medium was removed, and the power density was 0.7W/cm²The BGC-3HM bracket is irradiated by the NIR laser for 0, 5, 10, 15 and 20min respectively, and then MTT is carried out to evaluate the effect of killing cancer cells. Results as shown in fig. 5B and 5C, fig. 5B shows the survival rate of K7M2wt cells co-incubated with BGC-3HM stent for NIR laser NIR at different power densities according to the examples of the present application, and fig. 5C shows the survival rate of K7M2wt cells after BGC-3HM stent treatment for NIR light at different times according to the examples of the present application.

4. To investigate the effect of the doxorubicin-loaded scaffold on cell viability, cells containing K7M2wt (4X 10)⁴Cells/well) was seeded into 48-well plates (500 μ L). After 24h, the medium was removed and fresh medium (500. mu.L) was added. Then, ten sets of samples (BGC, BGC-1DOX, BGC-2DOX, BGC-3DOX, BGC-4DOX, BGC-3HM-1DOX, BGC-3HM-2DOX, BGC-3HM-3DOX, and BGC-3HM-4DOX scaffolds) were added to the well plates, respectively, and then incubated for 24h, with cells without scaffolds added set as control groups. Finally, MTT assays were performed to assess the survival of K7M2wt cells. The results are shown in fig. 5D, fig. 5D is the viability of K7M2wt cells after 24h incubation with different scaffolds as provided in the examples herein.

5.5 mg of doxorubicin was weighed and added to 5mL of a polylactic acid-dichloromethane solution at a concentration of 0.001 g/mL. After stirring and ultrasonic dispersion, the BGC stent prepared in example 1 was immersed in the above mixed solution for 4 seconds, and then taken out. The removed stents were designated BGC-2DOX stents, respectively. Then, the BGC-2DOX stent was dried and then re-immersed in the above-mentioned doxorubicin-containing polylactic acid dichloromethane solution for 4s, and the taken-out stents were designated as BGC-2DOX-1 stents, respectively. Then, the BGC-2DOX-1 stent was dried, and then re-immersed in the above-mentioned polylactic acid-dichloromethane solution containing doxorubicin for 4s, and the taken-out stents were named BGC-2DOX-2 stents, respectively. BGC stents coated with one, two and three doxorubicin were obtained by the above method.

To study the effect of the number of entrapments on the survival of K7M2wt cells, media (500 μ L) containing K7M2wt cells was seeded into 48-well plates, 24h later, the media was replaced with fresh media (500 μ L), BGC scaffolds coated with different numbers of times were placed into each well and cultured for 24h, and then the MTT assay was performed. Results as shown in fig. 5E, fig. 5E is a graph showing the effect of BGC scaffolds with different doxorubicin entrapment times on the survival rate of K7M2wt cells according to the examples provided in this application.

6. To investigate the synergistic effects of photothermal therapy and chemotherapy, K7M2wt cells (4X 10)⁴Cells/well) were seeded in 48-well plates. After 24h, the medium was replaced with fresh medium (500. mu.L). And 7 groups are set, the first group is a contrast (namely, a bracket and a control are not added), the second group is added with a BGC bracket, the third group is added with a BGC-3HM bracket, the fourth group is added with the BGC-3HM bracket and carries out NIR light irradiation, the fifth group is added with a BGC-2DOX bracket, the sixth group is added with the BGC-3HM-2DOX bracket, and the seventh group is added with the BGC-3HM-2DOX bracket and carries out NIR light irradiation. After 24h incubation, 250. mu.L of medium was aspirated, and the NIR-irradiated group was subjected to NIR irradiation at a NIR power density of 0.7W/cm²Time 10min, and finally MTT assay and apoptosis test. Results as shown in fig. 5F and fig. 6, fig. 5F is the effect of different scaffolds provided in the examples on the survival rate of K7M2wt cells in the presence and absence of NIR light, and fig. 6 is the K7M2wt apoptosis test in the presence and absence of NIR light.

As can be seen from FIGS. 5 and 6, when the bone cancer cell K7M2wt was incubated with the BGC, BGC-1HM, BGC-2HM and BGC-3HM scaffolds, no significant cytotoxicity was observed, indicating that these scaffolds are highly biocompatible (FIG. 5A). However, significant cytotoxicity was observed with the BGC-2HM and BGC-3HM groups when subjected to NIR irradiation. In addition, tumor cell killing ability had optical power density and illumination time dependence (fig. 5B and 5C). As the level of the adriamycin drug in the BGC-DOX or BGC-3HM-DOX scaffolds was increased, more cancer cells could be killed (FIG. 5D). The number of doxorubicin loads on the BGC scaffold had a significant effect on the killing ability of the BGC-DOX scaffold (fig. 5E). This is because as the number of drug loading increases, more doxorubicin is loaded onto the BGC scaffold and, therefore, more doxorubicin is released, resulting in greater cytotoxicity to K7M2wt cells. In addition, the BGC-3HM-2DOX + NIR group was able to minimize the cell survival rate compared to the control, BGC-3HM + NIR, BGC-2DOX, BGC-3HM-2DOX groups, indicating that the synergy of photothermal therapy and chemotherapy was able to kill tumor cells efficiently (FIG. 5F). The apoptosis test of FIG. 6 shows that the BGC group shows negligible apoptosis, less than 20% of the apoptotic cells of the BGC-3HM group, and 55.0% and 39.4% of the apoptotic cells of the BGC-3HM + NIR and BGC-2DOX groups, respectively, and up to 88.8% of the apoptotic cells of the BGC-3HM-2DOX + NIR groups, which indicates that photothermal and chemotherapy lead to the eventual death of cancer cells as apoptosis.

Example 6

The embodiment of the application provides an in vivo antitumor effect test of different BGC (BGC) stents, which comprises the following steps:

1. BGC stents were prepared according to the method described in example 1, BGC-1HM and BGC-3HM stents were prepared according to the method described in example 2, and BGC-4DOX stents were prepared according to the method described in example 3.

The BGC-3HM stent prepared in the above example was immersed in a solution of polylactic acid in dichloromethane containing 15mg of doxorubicin, and the stent was removed therefrom and named BGC-3HM-4DOX stent.

2. Female nude mice of 4-6 weeks of age were selected to construct subcutaneous osteosarcoma models to study the therapeutic efficacy of the scaffolds. K7M2wt cells (8X 10)⁶) Suspended in 200. mu.L sterile PBS solution and injected into the back of the mice. When the tumor volume is about 200mm³When, mice were divided into seven groups. Different stents were then surgically implanted at the tumor site (n-4 per group) and NIR light irradiation was performed on mice with BGC-3HM and BGC-3HM-4DOX stents placed in them. The mice were divided into seven groups, the first group was control, the second group was BGC group, the third group was BGC-3HM group, the fourth group was BGC-4DOX group, the fifth group was BGC-3HM-4DOX group, and the sixth group was contra-placement groupThe BGC-3HM stent is arranged to be irradiated by NIR light (namely a BGC-3HM + NIR group), and the seventh group is arranged to be irradiated by the NIR light (namely the BGC-3HM-4DOX + NIR group) on the BGC-3HM-4DOX stent. The date on which the stent was implanted was marked as day 0. On day 0, the BGC-3HM + NIR group and the BGC-3HM-4DOX + NIR group were exposed to NIR laser light for 10min and the temperature was controlled at 48 ℃. Next, NIR irradiation was performed on the NIR-irradiated group mice for 4 consecutive days, each irradiation time was 10min, and the length and width of the tumor and the body weight of the nude mice were measured every two days for 18 days. The formula for tumor volume is: tumor volume (V) ═ tumor length (x) (tumor width)²2-scaffold volume, scaffold volume 20mm³. Tumor tissues and major organs, such as heart, liver, spleen, lung and kidney, were collected on day 18, washed with PBS, and immediately placed in 4% paraformaldehyde overnight and embedded in paraffin. Paraffin-embedded tissue was cut to a thickness of 5mm and treated with hematoxylin and eosin (H)&E) After staining, histological examination was performed by microscopy. Tumor volume, tumor weight, change in mouse body weight, tumor appearance, H in tumor tissue in mice&E staining, and H of major organs of mice including heart, liver, spleen, lung and kidney&The results of the E-staining analysis are shown in FIGS. 7 to 9. FIG. 7A is the growth curves of the BGC, BGC-3HM, BGC-4DOX, BGC-3HM + NIR, BGC-3HM-4DOX + NIR scaffolds provided in the examples of the present application for treating the tumors of mice respectively; FIG. 7B shows the tumor weights of mice treated with BGC, BGC-3HM, BGC-4DOX, BGC-3HM + NIR, BGC-3HM-4DOX + NIR scaffolds provided in the examples of the present application after 18 days; FIG. 7C shows the body weight changes of mice treated with BGC, BGC-3HM, BGC-4DOX, BGC-3HM + NIR, BGC-3HM-4DOX + NIR scaffolds provided in the examples of the present application after 18 days; FIG. 8A is the appearance of tumors removed after 18 days of treatment of mice with BGC, BGC-3HM, BGC-4DOX, BGC-3HM + NIR, BGC-3HM-4DOX + NIR scaffolds provided in the examples of the present application, and FIG. 8D is a scale of 5 mm; FIG. 8B shows the tumor tissue H treated by BGC, BGC-3HM, BGC-4DOX, BGC-3HM + NIR, BGC-3HM-4DOX + NIR scaffolds provided in the examples of the present application after 18 days of treatment of mice&E staining pattern, scale of FIG. 8E is 50 μm. FIG. 9 is a drawing of this applicationPlease refer to the examples, which show that after BGC, BGC-3HM, BGC-4DOX, BGC-3HM + NIR, BGC-3HM-4DOX + NIR treatment, H in the major organs (including heart, liver, spleen, lung and kidney) of tumor-bearing mice&E stain image, scale bar 50 μm in FIG. 9.

The antitumor effect of the scaffold in vivo was evaluated by constructing an osteosarcoma model by injecting K7M2wt cells subcutaneously in nude mice. Tumors were greatly suppressed in BGC-3HM-4DOX + NIR treated mice (FIGS. 7A and 7B). In contrast, the tumors of BGC and BGC-3HM treated mice grew rapidly. While a certain degree of tumor suppression effect was observed in the BGC-4DOX, BGC-3HM-4DOX and BGC-3HM + NIR groups. The photographs and weights of the tumors taken at day 18 also demonstrate that the synergistic effect of photothermal therapy and chemotherapy is highly effective in inhibiting tumor growth. Furthermore, all mice showed no significant fluctuations in body weight, confirming that NIR laser, DOX drug and heme, as well as BGC scaffolds had negligible effects on the health of nude mice (fig. 7C). The H & E staining results of the tumors collected at 18 days show that compared with the BGC and BGC-3HM groups, the BGC-4DOX, BGC-3HM + NIR and BGC-3HM-4DOX + NIR groups have loose tumor tissue structures, and the disappearance of tumor cell nuclei is obviously observed, so that the anti-tumor effect is further verified.

Fig. 9 is a graph of H & E staining of the heart, liver, spleen, lung, and kidney of treated mice, showing that these major organs are structurally similar to untreated controls after different treatments, indicating that heme, NIR laser irradiation, and treatment of osteosarcoma with an adriamycin drug did not cause significant damage to the organs.

Example 7

The embodiment of the application provides another multifunctional compound bracket, which is a tricalcium phosphate bracket doped with heme and adriamycin, and the preparation method comprises the following steps:

1. preparing a tricalcium phosphate scaffold: 5g of tricalcium phosphate powder (available from Kunshan Huaqiao scientific materials, Inc.) was added to a 50mL beaker. Then 4.5mL of an aqueous solution of F-127 (15 wt%) was added to the beaker and stirred for 20min to obtain a homogeneous tricalcium phosphate ink. The tricalcium phosphate ink was then printed using a 3D printer (produced by bubon, obit biotechnology limited). The printing speed is 5mm/s and the pressure is 0.5-1.5 Bar. And drying the printed support for 24 hours at room temperature, sintering the printed support by using a high-temperature sintering furnace, keeping the temperature for 3 hours at the highest sintering temperature of 1100 ℃, and finally obtaining the tricalcium phosphate support.

2. The heme powder was added to deionized water and milled by a planetary high energy ball mill at 500rpm for 4 h. After milling, the heme powder was dried and collected. 0.1g of polylactic acid was added to 5mL of methylene chloride and stirred to be completely dissolved. Then, 0.06g of heme powder was put into the above-mentioned dichloromethane solution of polylactic acid. Stirring for 10min, and ultrasonically dispersing for 30min to obtain mixed solution of heme and polylactic acid, which is labeled HM mixed solution.

3. And (3) immersing the tricalcium phosphate stent prepared in the step (1) into the HM mixed solution, and taking out the tricalcium phosphate stent after 4s to prepare the tricalcium phosphate stent doped with the heme.

4. 10mg of doxorubicin was weighed and added to 5mL of a methylene chloride solution of polylactic acid having a polylactic acid concentration of 0.1 g/mL. After stirring and ultrasonic dispersion, the tricalcium phosphate scaffold doped with the heme is immersed in the mixed solution for 4s, then taken out and dried to obtain the tricalcium phosphate scaffold doped with the heme and the adriamycin.

The multifunctional composite scaffold prepared by the embodiment has the function of killing cancer cells after in vitro photo-thermal treatment.

Example 8

Another multifunctional composite scaffold is provided in the examples herein, which is a hydroxyapatite scaffold incorporating heme and doxorubicin, and is prepared in a manner similar to that of example 7, except that the hydroxyapatite scaffold is substituted for the tricalcium phosphate scaffold, and the remaining steps are identical to those of example 7. The preparation method of the hydroxyapatite scaffold comprises the following steps: 5g of hydroxyapatite powder (available from Kunshan Huaqiao scientific materials, Ltd.) was poured into a 50mL beaker. Then 4.5mL of an aqueous F-127 solution (15 wt%) was added to the beaker and stirred for 15min to obtain a uniform calcium silicate ink. The hydroxyapatite graphite water was then printed using a 3D printer (produced by yokogaku biotechnology limited). The printing speed is 5mm/s and the pressure is 0.5-2.0 Bar. After the printed scaffolds were dried at room temperature for 24h, they were transferred to a high temperature sintering furnace and sintered at a temperature of up to 700 ℃ for 3h to obtain hydroxyapatite scaffolds.

Example 9

Another multi-functional composite scaffold is provided in the examples herein, which is a calcium silicate scaffold incorporating heme and doxorubicin, and is prepared in a manner similar to that of example 7, except that the calcium silicate scaffold is substituted for the tricalcium phosphate scaffold, and the remaining steps are identical to those of example 7. The preparation method of the calcium silicate scaffold comprises the following steps: 5g of calcium silicate powder (available from Kunshan Huaqiao scientific and technological New materials, Inc.) was poured into a 50mL beaker. Then 4.5mL of an aqueous F-127 solution (15 wt%) was added to the beaker and stirred for 10min to obtain a uniform calcium silicate ink. The calcium silicate ink was then printed using a 3D printer (manufactured by bubon biotechnology limited). The printing speed is 5mm/s and the pressure is 0.5-2.5 Bar. After the printed scaffolds were dried at room temperature for 24h, they were transferred to a high temperature sintering furnace and sintered at a temperature of up to 1250 ℃ for 3h to obtain calcium silicate scaffolds.

Example 10

The embodiment of the application provides another multifunctional compound stent, which is a BGC stent doped with heme and fluorouracil, and the method comprises the following steps:

1. the heme powder was added to deionized water and milled by a planetary high energy ball mill at 500rpm for 4 h. After milling, the heme powder was dried and collected. 0.1g of polylactic acid was added to 5mL of methylene chloride and stirred to be completely dissolved. Then, 0.06g of heme powder was put into the above-mentioned dichloromethane solution of polylactic acid. Stirring for 10min, and ultrasonically dispersing for 30min to obtain mixed solution of heme and polylactic acid, which is labeled HM mixed solution.

2. The BGC stent prepared in example 1 was immersed in the HM mixed solution for 4 seconds, and then removed to prepare a BGC stent doped with heme.

3. 10mg of fluorouracil was weighed and added to 5mL of a dichloromethane solution of polylactic acid having a polylactic acid concentration of 0.02 g/mL. After stirring and ultrasonic dispersion, the BGC stent doped with the heme is immersed in the mixed solution for 4s, then taken out and dried to obtain the BGC stent doped with the heme and the fluorouracil.

Example 11

Another multifunctional complex stent is provided in the embodiments of the present application, which is a BGC stent incorporating heme and paclitaxel, and the method includes: 10mg of paclitaxel was weighed and added to 5mL of a dichloromethane solution of polylactic acid having a polylactic acid concentration of 0.02 g/mL. After stirring and ultrasonic dispersion, the BGC stent doped with heme was immersed in the above mixed solution for 4s, then taken out, and dried to obtain a BGC stent doped with heme and paclitaxel.

Example 12

Another multifunctional composite stent is provided in the embodiments of the present application, which is a BGC stent doped with black phosphorus and docetaxel, and the method includes:

1. 0.1g of polylactic acid was added to 5mL of methylene chloride and stirred to be completely dissolved. Then, 0.1g of black phosphorus nanosheet (purchased from Jiangsu Xiancheng nanomaterial science and technology Co., Ltd.) was placed in the above-mentioned dichloromethane solution of polylactic acid. Stirring for 10min and ultrasonically dispersing for 30min to obtain mixed solution of black phosphorus and polylactic acid, which is labeled as BP mixed solution correspondingly.

2. The BGC stent prepared in example 1 was immersed in the BP mixed solution for 4 seconds, and then taken out to prepare a black phosphorus-doped BGC stent.

3. 10mg of docetaxel was weighed and docetaxel was added to 5mL of a dichloromethane solution of polylactic acid having a polylactic acid concentration of 0.02 g/mL. After stirring and ultrasonic dispersion, the BGC stent doped with the black phosphorus is immersed in the mixed solution for 4s, then taken out and dried to obtain the BGC stent doped with the black phosphorus and the docetaxel.

Example 13

The embodiment of the application provides another multifunctional composite stent, which is a BGC stent doped with titanium nitride and oxaliplatin, and the method comprises the following steps:

1. 0.1g of polylactic acid was added to 5mL of methylene chloride and stirred to be completely dissolved. Then, 0.1g of titanium nitride (purchased from Shanghai Neihou nanotechnology Co., Ltd.) was put into the above-mentioned solution of polylactic acid in methylene chloride. Stirring for 10min and carrying out ultrasonic dispersion for 30min to prepare a titanium nitride and polylactic acid mixed solution, wherein the mixed solution is correspondingly marked as TN mixed solution.

2. The BGC stent prepared in example 1 was immersed in the TN mixed solution for 4 seconds, and then removed to prepare a BGC stent doped with titanium nitride.

3. Oxaliplatin in an amount of 10mg was weighed out and added to 5mL of a dichloromethane solution of polylactic acid having a polylactic acid concentration of 0.02 g/mL. After stirring and ultrasonic dispersion, the BGC stent doped with the titanium nitride is immersed in the mixed solution for 4s, then taken out and dried to obtain the BGC stent doped with the titanium nitride and the oxaliplatin.

Therefore, the BGC stent can be soaked in a dichloromethane solution of polylactic acid containing heme and adriamycin simultaneously, so that the heme and the adriamycin are adhered to the surface of the stent, and the prepared BGC stent doped with the heme and the adriamycin has the performance of BGC-HM-DOX in the embodiment.

Therefore, the BGC stent can be soaked in a polylactic acid dichloromethane solution containing adriamycin, then taken out and dried, and then soaked in a polylactic acid dichloromethane solution containing heme, so that the adriamycin and the heme are sequentially adhered to the surface of the stent, the prepared BGC stent doped with the heme and the adriamycin has the performance of BGC-HM-DOX in the embodiment, and the loaded adriamycin achieves a slow release effect due to the fact that the heme is adhered to the outer layer.

It can be seen that the number of times that the BGC stent is soaked in the methylene chloride solution of polylactic acid containing heme can be increased by 2 times, 3 times, 4 times, 5 times, or 6 times, and as the number of soaking times is increased, more heme adheres to the surface of the BGC stent.

It can be seen that the BGC scaffold in the above embodiments can be replaced with an existing conventional bone tissue scaffold, such as a tricalcium phosphate scaffold, a hydroxyapatite scaffold, or a calcium silicate scaffold; the doxorubicin in the above examples can be replaced with existing conventional chemotherapeutic drugs such as fluorouracil, cyclophosphamide, vincristine, vindesine, daunorubicin, irinotecan, mitoxantrone, methotrexate, paclitaxel, docetaxel, camptothecin, cisplatin, carboplatin, or oxaliplatin; the heme in the above examples can be replaced by conventional photothermal agents, such as black phosphorus, graphene, molybdenum disulfide, titanium nitride, titanium dioxide, gold nanorods, metal organic framework compounds, polydopamine, ferroferric oxide, copper sulfide, and iron selenium copper ore.

The foregoing is only a preferred embodiment of the present application and it should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.

Claims

1. A multi-functional composite stent, comprising:

2. The multifunctional composite scaffold according to claim 1, wherein the bone tissue scaffold is made of one or more materials selected from hydroxyapatite, tricalcium phosphate, calcium silicate, bioactive glass-ceramic, and degradable polymers.

3. The multifunctional composite stent as claimed in claim 1, wherein the photothermal agent is selected from one or more of heme, black phosphorus, graphene, molybdenum disulfide, titanium nitride, titanium dioxide, gold nanorods, metal organic framework compounds, polydopamine, ferroferric oxide, copper sulfide, and selenocopicrite.

4. The multi-functional composite stent of claim 1, wherein the chemotherapeutic agent is selected from one or more of doxorubicin, fluorouracil, cyclophosphamide, vincristine, vindesine, daunorubicin, irinotecan, mitoxantrone, methotrexate, paclitaxel, docetaxel, camptothecin, cisplatin, carboplatin, and oxaliplatin.

5. The multifunctional composite support according to any one of claims 1 to 4, wherein the number of layers of said photothermal layer is 1, 2, 3, 4, 5 or 6; the number of the medicine-carrying layers is 1, 2, 3, 4, 5 or 6; the number of the mixed layers is 1, 2, 3, 4, 5 or 6.

6. The multifunctional composite stent as claimed in claim 5, wherein the photothermal layer and the drug-loaded layer are disposed at intervals on the surface of the bone tissue stent; the photothermal layer and the drug-loaded layer are sequentially arranged on the surface of the bone tissue bracket; the surface of the bone tissue bracket is sequentially provided with the medicine-carrying layer and the photothermal layer.

7. A method of making a multifunctional composite scaffold, comprising:

taking a bone tissue scaffold material as a raw material, and preparing a bone tissue scaffold by 3D printing processing according to the 3D model data;

8. The method of claim 7, wherein the bone tissue material is selected from one or more of hydroxyapatite, tricalcium phosphate, calcium silicate, bioactive glass-ceramic, and degradable polymers;

9. Use of the multifunctional composite scaffold of any one of claims 1 to 6 and the multifunctional composite scaffold prepared by the preparation method of claim 7 or 8 for preparing a medicament for treating bone tumor, or solid tumor transferred to bone.

10. Use of the multifunctional composite scaffold of any one of claims 1 to 6 and the multifunctional composite scaffold and near infrared light device prepared by the preparation method of claim 7 or 8 for preparing a system for treating bone tumor or solid tumor transferred to bone.