Carrier-free Bone-targeted Nanomedicine Simultaneously Inhibits Malignant Bone Tumor And Tumor-associated Osteolysis

Objectives:Malignant bone tumors can be divided into primary and secondary types.Primary malignant bone tumor has a high incidence,rapid progression and poor prognosis in adolescents,while Secondary malignant bone tumor is common in the terminal cancer,which greatly shortens the lifetime and reduces the living quality.Surgery and radiotherapy can restrain local tumor growth,such as those with acute symptoms,but it is difficult to control multiple,metastatic malignant bone tumors.Systemic chemotherapy is essential in the treatment of malignant bone tumor,but the side effects as well as the drug resistance make it cease to advance.One reason for the poor therapeutic effect of chemotherapeutic drugs is the lack of drug targeting.Nanotechnology and bone-targeting compounds can provide targeting ability,but the utilization of nanocarriers will increases the difficulty of synthesis and potential bio-security problems.The other reason is the "vicious cycle" between malignant bone tumor and the bone microenvironment.The malignant bone tumor cells and osteoclasts in the microenvironment promote each other through the cytokines they secrete.Therefore,cutting off the relationship between malignant bone tumor cell proliferation and osteoclastogenesis is of great significance to enhance the efficiency for treating malignant bone tumors.Upon the basis,we proposed to design a kind of nanodrug: CPPA,which can inhibit malignant bone tumor and osteoclastogenesis.The drug is made of natural compounds phytic acid(PA)and chemotherapy drugs cisplatinum(CDDP)through a simple "onestep" synthetic method.CPPA nanodrug can address the two causes for bad performance of chemotherapy,which offers a new strategy for the treatment of malignant bone tumors.Methods:1.The "one-step method" was used to connect the chemotherapeutic drug CDDP with small molecule compound PA,and the two were assembled into nanoparticles by adjusting the concentration of reactants;The physical and chemical properties of CPPA were characterized by transmission electron microscope,dynamic light scatterer and X-ray photoelectron spectroscopy.Laser confocal microscopy was used to detect the 3D tumor ball penetration of CPPA nanodrug.The ability of CPPA to promote hemolysis in vitro and the release of lactate dehydrogenase in mouse fibroblasts were detected by spectrophotometer.Blood routine and liver function tests were used to evaluate the hematotoxicity and hepatotoxicity of CPPA nanodrug.2.Inductively coupled plasma mass spectrometer was used to detect the binding ability of CPPA nanodrug to hydroxyapatite bone tablets;The binding ability of CPPA nanodrug to mouse osteoblastic matrix was detected by laser confocal microscopy.MTT assay was used to detect the ability of CPPA nanodrugs to inhibit breast cancer MDA-MB-231 cells and osteosarcoma U2 OS cells in neutral and acidic environments.The AO/EB double staining test was used to evaluate the ability of CPPA nanodrug to promote the apoptosis of MDA-MB-231 cells and U2 OS cells.The capacity of CPPA nanodrug to inhibit the differentiation of bone marrow macrophages into osteoclasts was evaluated by in vitro osteoclastogenesis assay.WB and q PCR assay were used to explore the molecular mechanism of CPPA inhibiting osteoclastogenesis.3.Luciferase were transfected into MDA-MB-231 cells and U2 OS cells by virus vectors;The two kinds of cells were used to construct the mouse tibial tumor-bearing model.These two models were used to detect the bone-targeting ability and metabolic distribution of CPPA nanodrug in vivo.The inhibitory effect of CPPA nanomedicine on the proliferation of malignant bone tumors in vivo was detected by In vivo imaging system.The inhibitory effect of CPPA nanodrug on bone destruction in vivo was detected by Micro-CT.TUNEL and TRAP staining were used to verify the ability of CPPA nanodrug to promote apoptosis and inhibit osteoclastogenesis at the tissue level.H&E stain of visceral organs was used to detect the bio-safety of CPPA nanodrug.Results:1.Through the "one-step method",we synthesized CPPA nanodrug with uniform nano particle size;CPPA nanodrug can keep stable in neutral environment for a long time,while dissociate rapidly in acidic environment.CPPA nanodrug can effectively penetrate3 D tumor cell spheres constructed by MDA-MB-231 cells and U2 OS cells.CPPA nanodrug was nontoxic to mouse fibroblasts.CPPA nanodrug has very low hemolysis tendency.It did not cause damage to blood cells and liver function.2.CPPA nanodrug can effectively target hydroxyapatite bone tablets and mouse osteoblastic matrix;CPPA nanodrug has ability to kill MDA-MB-231 cells and U2 OS cells in response to acid microenvironment.CPPA can promote apoptosis of MDA-MB-231 cells and U2 OS cells.CPPA nanodrug can effectively inhibit the differentiation of mouse bone marrow macrophages into osteoclasts.CPPA nanodrug can inhibit the osteoclasts activation molecular signaling pathway by increasing the expression of tumor necrosis factor receptor-associated factor 3(TRAF3)and decreasing the expression of activated T nuclear factor 1(NFATC1).3.CPPA nanodrug can effectively target the osteoclastic interface of malignant bone tumors in vivo;CPPA nanodrug can effectively inhibit the proliferation of mouse tibial tumor-bearing models in vivo and promote the apoptosis of the malignant bone tumor cells.CPPA nanodrug can effectively inhibit the destruction of adjacent bone tissues in mouse tibial tumor-bearing models of breast cancer and osteosarcoma in vivo and inhibit the activation of osteoclasts differentiation in this region.CPPA nanodrug did not cause significant weight loss and visceral damage in mice.Conclusions:By the dynamic connection and self-assembly of CDDP and PA,we can of can synthesize nanodrug CPPA in "one-step".the reaction is simple,efficient,without carrier introduction.CPPA has nanosize,the capability of long-term circulation,and reliable ability for tumor penetration;At the same time,CPPA nanodrug has low toxicity and high biocompatibility under neutral environment and can effectively kill tumor cells and promote apoptosis in response to acid stimulation.In addition,CPPA nanopdrug can also target the bone resorption interface and inhibit the activation of osteoclastogenesis in tumor regions by the functions of PA.The combination of diverse functions enables CPPA to nanodrug show excellent efficacy in the treatment of malignant bone tumor models.