Research On Fabrication, Characterization, Anti-tumor And Bone Repair Mechanism Of ZOL-Loaded Bifunctional Nanocomposite Artificial Bone

Background: The clinical treatment of bone tumors usually adopts systemic treatment based on surgical resection.The problem of repairing huge bone defects caused by surgery and tumor recurrence and metastasis caused by residual tumor cells are major challenges in the treatment of tumor bone defects.The current method of treating bone defects is to fill them with local bone grafts,so the development of artificial bones with anti-tumor and osteogenic properties is of great significance for the treatment of tumor bone defects.Zoledronic acid(ZOL)has a strong ability to inhibit osteoclast,and also has certain antitumor ability.The construction of artificial bone loaded with ZOL is expected to treat tumor bone defects.However,the current preparation strategies for loading ZOL artificial bone include impregnation,direct addition,drug coating,etc.,which have the problems of low drug loading and easy burst release of drugs.Mesoporous silicon is a nanomaterial with large specific surface area,large pore volume,and easy surface modification.It has a wide range of applications in the field of drug delivery.Using mesoporous silicon as the drug carrier and poly-L-lactic acid(PLLA)as the matrix material,the preparation of bifunctional artificial bone with ZOL sustained release ability by SLS technology may be an effective strategy for the treatment of tumor bone defects.Purpose: The development of ZOL-loaded bifunctional nanocomposite artificial bone with anti-tumor and osteogenic properties provides a feasible strategy for the treatment of tumor-induced bone defects.Methods: In this study,functionalized mesoporous silicon SBA15NH2 was used as a carrier to construct loaded ZOL nanoparticles,and the PLLA/SBA15NH2-ZOL artificial bone was prepared by SLS technology.In addition,mesoporous silicon-wrapped graphene oxide(GO/MSN)nanoparticles were also synthesized,and nano drug-loaded particles GO/MSN-ZOL were constructed,and a new type of photothermal therapy and chemotherapy in one nanometer was prepared by using SLS.Composite artificial bone PLLA/GO/MSN-ZOL.(1)Synthesis of drug-loaded nanoparticles SBA15NH2-ZOL.The structure of nanoparticles was characterized by scanning electron microscopy,X-ray diffraction,Fourier transform infrared spectroscopy,Zeta potential test,N2 adsorption and desorption and other experiments.Nanocomposite artificial bone PLLA/SBA15NH2-ZOL was prepared by SLS,and the physical and chemical properties and drug release of the prepared artificial bone were studied.The biocompatibility,anti-tumor performance and mechanism of artificial bone were studied by CCK-8experiment,live-dead cell staining,flow cytometry,and RT-q PCR.The osteoclast inhibition performance of artificial bone was studied by TRAP staining,F-actin experiment,bone resorption pit experiment and RTq PCR.(2)Synthesize mesoporous silicon-wrapped graphene oxide(GO/MSN),and construct a new nano-drug loading platform GO/MSNZOL.A new type of artificial bone PLLA/GO/MSN-ZOL was prepared by SLS,and its physical,chemical and photothermal properties were studied.The anti-tumor properties of artificial bone were detected by CCK-8 assay,live-dead cell staining,and RT-q PCR.The synergistic mechanism between photothermal therapy and chemotherapy was explored by nanoparticle fluorescent tracer.ALP staining,alizarin red staining,TRAP staining,bone resorption pit experiments,etc.were used to study the osteogenic differentiation and osteoclast inhibition of artificial bone.(3)The biological properties of the prepared artificial bone were studied in vivo through the skull defect model and subcutaneous tumor formation in nude mice.The bone repair performance of artificial bone was studied by Micro-CT,HE staining,Masson staining and immunofluorescence.The anti-tumor ability was studied by HE staining and TUNEL staining.Results:(1)The experimental results of SEM,FTIR,XRD,N2 adsorption and desorption,and Zeta potential showed that the drugloaded nanoparticles were successfully constructed,and the ZOL loading rate could reach 23.653%.The PLLA/SBA15NH2-ZOL artificial bone successfully prepared by SLS has good mechanical properties,and its compressive modulus can reach 16.76±0.74 Mpa.The artificial bone is biodegradable and can release ZOL continuously for more than 6 weeks in vitro.Biocompatibility: The results of CCK-8 experiment and livedead cell staining showed that PLLA/SBA15NH2-ZOL had no significant effect on the viability of r BMSCs.Anti-tumor properties: CCK-8 assay and dead cell staining results showed that PLLA/SBA15NH2-ZOL could cause MG-63 cell death and inhibit its proliferation.Flow cytometry results showed that PLLA/SBA15NH2-ZOL could induce tumor cell apoptosis and cell cycle arrest.Promoting bone repair performance:TRAP staining and F-acin test results showed that the number of TRAPpositive osteoclasts in the PLLA/SBA15NH2-ZOL group was significantly less than that in other groups and no F-actin ring formation was observed.In the bone resorption pit experiment,the number and size of the bone resorption pit in the PLLA/SBA15NH2-ZOL group were significantly smaller than those in the control group.(2)Synthesized a new nano-platform GO/MSN and prepared a new type of PLLA/GO/MSN-ZOL artificial bone,which has good physical and chemical properties,photothermal properties,and can rapidly heat up under short-term near-infrared light(NIR)irradiation to around 50°C.CCK-8 experiment,living dead cell staining experiment and expression detection of apoptosis-related genes showed that the anti-tumor performance of artificial bone was significantly improved after combined photothermal therapy.The results of lysosomal fluorescent probe staining experiments showed that more nanoparticles were phagocytosed into tumor cells under the action of light and heat.The results of ALP staining and alizarin red staining experiments showed that alkaline phosphatase and mineralized nodules in the PLLA/GO/MSN-ZOL group were significantly more than those in the control group(3)Micro-CT,HE staining,Masson The results of staining and immunofluorescence showed that PLLA/SBA15NH2-ZOL artificial bone had good bone repair performance,and the bone repair performance of the new PLLA/GO/MSN-ZOL was significantly improved compared with the former.The results of subcutaneous tumor formation in nude mice showed that PLLA/SBA15NH2-ZOL can play an effective anti-tumor effect in vitro,while the anti-tumor performance of PLLA/GO/MSN-ZOL combined with photothermal and chemotherapy was significantly improved compared with the former.Conclusion:(1)The PLLA/SBA15NH2-ZOL artificial bone prepared by SLS in this study has good physical and chemical properties,biocompatibility and excellent ZOL sustained release ability.It can play an anti-tumor effect by inducing tumor cell apoptosis and cell cycle arrest,and promote bone repair by inhibiting the differentiation and maturation of osteoclasts.(2)PLLA/GO/MSN-ZOL artificial bone can enhance the anti-tumor ability of artificial bone through the synergistic effect of photothermal therapy and chemotherapy,and can improve the bone repair ability of artificial bone by inducing osteoblast differentiation through GO,providing a new solution for tumor bone.The study of defect repair materials provides a feasible idea.