Study On Bisphosphonate-Based Drug Delivery Strategy And Antitumor Therapy

Osteosarcoma is the most common primary malignant tumor of bone,which is common in children and adolescents.The malignant degree of osteosarcoma is extremely high.Although the 5-year survival rate of osteosarcoma is effectively increased from 10%～20%to 65%～70%by new chemotherapy,some patients still die quickly after metastasis or tumor recurrence.The most common metastatic site of osteosarcoma is lung,followed by bone.With the application of targeted drugs and the development of imaging technology,the survival time of osteosarcoma patients in advanced stage is obviously prolonged,but the incidence of brain metastasis in later stage is increasing.Tumor metastasis is the final product of a multi-stage cell biological process,which involves the diffusion of the anatomically distant organs of cancer cells and adapts to the new tissue microenvironment slowly.Include genetic changes of tumor cell gene and enrichment of non-tumor interstitial cells.The synergistic stimulation of these aspects provides the required characteristics for the initial metastatic cells.Bisphosphonates,BPs)have therapeutic effects on various bone diseases and calcium metabolic diseases.It can specifically target hydroxyapatite(HAP)to bone tissue in vivo,and has been widely used in osteoporosis,tumor bone metastasis and so on.The essential condition for bisphosphonate to produce activity is the basic structure of P-C-P.Because of its unique physical and chemical properties,its quantitative methods are digestion method,pre-column or post-column chromatographic derivatization detection.Although the identification method of this compound is mature,its quantitative method still has some problems,such as complicated detection steps and expensive instruments and equipment.In view of this,this paper focuses on the characteristics of bisphosphonates,improves the detection method of bisphosphonates,and uses its biological function to design a series of nano-drugs to inhibit tumor metastasis and treat tumor.(1)Improved detection method of bisphosphonateFrom the point of view of drug analysis,bisphosphonates have high polarity,and most bisphosphonates have no chromophore and are not volatile,so they cannot be directly detected by liquid/gas chromatography.In this chapter,based on the traditional phosphorus determination method,the original phosphorus determination reagent was improved by alcohols,and the improved phosphorus determination reagent directly reacted with bisphosphonate.The method has mild conditions,simple reaction and easy operation,and the color reaction can take place in a short time without extreme conditions.Then,the effects of reaction time,reaction temperature,different alcohols and alcohol content on color reaction were investigated.Finally,the linear relationship between absorbance and bisphosphonate concentration was calculated at 660 nm.The modified phosphorus determination reagent has a good linear relationship with the color development of bisphosphonates,and can be used for the content detection of bisphosphonates.(2)Preparation of hydroxyapatite and its application in anti-tumor with RISScreening needle-like hydroxyapatite with high biocompatibility and Si-doped flower-like hydroxyapatite by biomimetic synthesis.FTIR,XRD,XPS,TEM and SEM were used to characterize the synthesized nano HAP.The HAP with good biocompatibility was used to adsorb RIS,and the anti-angiogenesis and anti-tumor metastasis effects of HAP loaded with RIS in vitro were studied.(3)Bone targeted alendronate-low molecular weight heparin conjugate delivery system The bone-targeted alendronate sodium is combined with low molecular weight heparin,LMWH is the ligand of CD44v3,which is overexpressed in many types of tumor cells.The preliminary results showed that ALN-LMWH(AH)in vivo has a significant therapeutic effect on the growth of bone metastasis tumors.AH not only reduced osteolysis caused by tumor,but also significantly reduced the tumor size of proximal tibia in mice.AH retains the most important ability of its component ALN to resist resorption in vivo and inhibit osteoclast formation in vitro.On the other hand,although AH contains heparin,AH does not prolong APTT and improve its bioavailability.(4)Preparation of RIS modified Fe3O4 nanoparticles and its application in anti-tumor therapy The risedronate sodium modified ferroferric oxide superparamagnetic nanoparticles(RIS-FeNPs)have uniform particle size and good stability in solvent.The nanoparticles were characterized by IR,XRD and TEM vibrating sample magnetometer.In vitro and in vivo results show that the magnetic nanoparticles have only a low inhibitory effect on the growth of tumor cells,but can inhibit the migration function of tumor cells and inhibit angiogenesis.In vivo targeting results show that the magnetic nanoparticles can carry RIS well and actively target to the tumor site through an external magnetic field.After targeting the tumor site,the temperature measuring probe can detect the obvious temperature rise of the tumor site during the magnetocaloric process.In vivo experimental results of RIS-FeNPs also show that the superparamagnetic nanoparticles can inhibit tumor migration and metastasis.The RIS-FeNPs can improve the tumor microenvironment and down-regulate the proportion of M2 type macrophages in the tumor microenvironment through magnetocaloric effect.(5)Study on the treatment of osteosarcoma with magnetically targeted liposomes prepared by the risedronate modified Fe3O4 nanoparticlesThe prepared RIS-FeNPs were coated with liposomes and loaded with desalinated adriamycin.The results of physical and chemical characterization show that the RFe/DOX Lipo prepared by this method has uniform particle size,and the liposome has a particle size of about 200 nm,with good dispersibility.The liposome can wrap RIS-FeNPs well.In the unstained electron microscope,RIS-FeNPs has a structure similar to grape clusters.In addition,the liposome has good stability in the environment of 10%fetal bovine serum,and the drug is released slowly at 37℃.RFe/DOX Lipo has good tumor targeting under external fixed magnetic field,which proves the feasibility of magnetic targeting drug delivery strategy.In vivo efficacy experiment,tumor growth of mice in RFe/DOX Lipo+M group was effectively inhibited,and in vivo treatment results showed the feasibility of RIS-FeNPs combined with DOX treatment strategy.In the investigation of therapeutic mechanism in vivo,the expression of related proteins in tumor and immunohistochemical results of immune factors show that the combination of them can not only inhibit tumor angiogenesis,but also repolarize M2 macrophages in tumor.In RFe/DOX Lipo+M group,the expression of CD206,a marker of M2 macrophages(M),is hardly detected,and the proportion of M1 macrophages(iNOS)is increased.The result of Tunel showed that tumor apoptosis was obvious in RFe/DOX Lipo+M group,which indicated that RFe/DOX Lipo could induce apoptosis of tumor cells and inhibit the growth of tumor cells.Immunohistochemical results prove that RFe/DOX Lipo combined with magnetic targeting therapy can reshape the immune microenvironment of K7 tumor,repolarize or remove M2 macrophages,and inhibit the metastasis of lung and liver of tumor.The RFe/DOX Lipo drug delivery system constructed in this study is safe and effective with low side effects.Combined magnetic targeted therapy strategy has a good effect in tumor treatment and tumor metastasis inhibition,and has a potential application prospect.(6)Study on overcoming osteosarcoma by magnetocaloric therapy of magnetic targeted liposome prepared by the risedronate modified Fe3O4 nanoparticlesTreatment of K7 osteosarcoma in situ by RFe/DOX Lipo liposome combined with magnetocaloric therapy.The liposome can be heated to 45℃ in alternating magnetic field.In vivo,the results showed that RFe/DOX Lipo combined with magnetocaloric therapy could completely inhibit the growth of tumor after only three courses of treatment.The flow results showed that RFe/DOX Lipo combined with magnetocaloric therapy can reduce the proportion of M2 type m,reduce the proportion of Treg cells and increase the proportion of CD8+TNF-α+T and CD8+Ki67+T in tumor microenvironment(TME),thus improving tumor microenvironment.Immunohistochemical results showed that combined treatment inhibited tumor angiogenesis.HE staining showed that RFe/DOX Lipo combined with magnetocaloric therapy could inhibit tumor metastasis.CT results showed that RFe/DOX Lipo combined with magnetocaloric therapy could significantly inhibit bone resorption.RFe/DOX Lipo combined with magnetocaloric therapy has a good effect in tumor treatment and tumor metastasis inhibition,and has a potential application prospect.