| Background:Osteosarcoma（OS）is the most common primary malignant tumor in children and adolescents,it has a high metastasis rate and recurrence rate.At present,the standard chemotherapy regimen resulted in a five-year survival rate of 60%to 70%,and remains as the main means of treating osteosarcoma before and after surgery.However,most chemotherapeutic drugs lack targeting,have greater toxicity to normal human tissues and organs,and produce primary and secondary resistance.Another current main cause of poor prognosis of osteosarcoma is the recurrence and metastasis due to drug resistance.Therefore,an effective treatment for osteosarcoma is urgently needed.In the recent decades the nano drug-loading systems came into being and solved this problem.According to the specific receptors on the surface of tumor cells,the corresponding ligands on the surface of the novel nano drug-loading system were designed,and the nanoscaled drug-loading system was taken up into tumor cells by specific recognition of receptors and ligands.According to the internal environment characteristics of tumor cells,some sensitive materials can be designed to achieve the fixed release of the drug and the like.Therefore,the nanoscaled drug-loading system was expected to solve the problem of poor targeting of the common drug-loading system and large systemic side effects.Aim and Objective:The aim of this study was to develop and investigate a bone-targeting and redox-sensitive liposomal system for the improved treatment of OS.Alendronate（ALN）and hyaluronic acid（HA）were used as bone and CD44（overexpressed in cancer cells）targeting moiety respectively.Hypothesis and rational of study design:This study designed bone-targeting,cell-targeting and reduction-responsive multifunctional liposomes based on the minerals released by osteosarcoma,the surface receptors of osteosarcoma cells and the internal environment of tumor cells,and the effect of this liposome co-administration with a tumour-homing cell penetrating cyclic peptide,iRGD,was further studied.Osteosarcoma causes hydroxyapatite,the main mineral of bone,to be released into the blood.As hydroxyapatite can be specifically recognized by the alendronate,it can be used a bone-targeting material on the surface of multifunctional liposomes.The CD44 receptor is highly expressed on the surface of human osteosarcoma cell line MG-63 and can specifically bind by hyaluronic acid（HA）on the surface of multifunctional liposome.In addition,the concentration of glutathione（GSH）in tumor cells is greater than that of the normal cells,when reduction-sensitive material is used in the liposome such as those contain disulfide bond（-SS-）.The cleavage of-SS-result in the destruction of liposome,allowing drug rapidly released in cells.Therefore,liposomes containing bone and cell targeting ligands,and disulfide bonds（-SS-）can be preferentially recognized by OS site and tumor cells,produce rapid intracellular drug release,thereby improving efficacy and reducing toxic side effects.Methods:First,to synthesize the target amphiphilic polymer ALN-HA-SS-Lipid（ALN-HA-SS-L）.First,alendronate（ALN）and hyaluronic acid（HA）are combined by an amide bond to form a polymerization.ALN-HA,then ALN-HA is covalently bonded withcystaminedihydrochloride（CYS）toformtheintermediatepolymer ALN-HA-SS-NH2,and finally the amine of polymer ALN-HA-SS-NH2 and the carboxyl group of DSPE-PEG2000-COOH（lipid）reacts to form ALN-HA-SS-L.At the same time,HA-SS-L without bone-targeting moiety HA-L without disulfide bond were prepared and to be used in control liposomes.The structures of the products were identified by nuclear magnetic resonance spectroscopy（1H NMR）To obtain functionalised liposomes,first,plain liposomes（non coated）were prepared using thin film dispersion method.Doxorubicin hydrochloride was used as a model drug and then actively loaded with ammonium sulfate gradient.Various polymers is embedded in the liposome using a post-insertion method to obtain ALN-HA-SS-L liposome（ALN-HA-SS-L-L）,and various reference liposomes,HA-SS-L-L liposomes（HA-SS-L-L）,HA-L-L liposomes（HA-L-L）,PEG liposomes（PEG-L）（without bone targeting or CD44 targeting or redox insensitive）.The prepared multifunctional liposome was initially characterized,including:particle size,zeta potential,dispersion,encapsulation efficiency and drug loading.The prepared multifunctional liposome was added to a buffer containing dithiothreitol（DTT）,and the reduction sensitivity was examined by observing the change in particle size and morphology.In vitro release experiments were carried out to investigate the release of doxorubicin-loaded liposomes under different concentrations of GSH,further demonstrating the reduction responsiveness of drug-loaded multifunctional liposomes.Inverted fluorescence microscopy and flow cytometry were used for qualitative and quantitative investigation of tumor cell uptake.The cytotoxicity of drug-loaded liposomes and blank liposomes was examined by MTT assay.In vivo animal experiments,an orthotopic animal model of osteosarcoma was constructed to investigate the tissue distribution,antitumor effect and safety of multi-functional drug-loaded liposomes.42 Tumor-bearing female nude mice showing tumor sizes（100 mm3150 mm3）were randomly divided into seven groups（n=6）.The mice were treated through tail vein with saline,free DOX,PEG-L/DOX,HA-L-L/DOX,HA-SS-L-L/DOX,and ALN-HA-SS-L-L/DOX（with and without co-administration of 4μmol/kg iRGD）respectively at DOX dose of 5 mg/kg at 3 day’s intervals for 3 times.Tumor volumes,animal body weight and the survival of the animals were monitored for 70 days.In addition,to compared the bone/OS targeting property of various liposomes loaded with DiR were injected to mice.Fluorescence signals of DiR were captured under the in vivo imaging system at 1,2,6,12 and 24 h post-administrationResults and Discussion:All the polymers were successfully synthesized and confirmed with proton NMR spectra.After conjugation with the boning targeting moiety ALN,the degree of substitution（DS）of the COOH groups in HA was found to be 17%,leaving the majority of COOH groups in the HA-ALN conjugate free for CD44 binding.Under the transmission electron microscope,the structure of the liposome was observed to be nearly spherical,and the size was uniform.The phospholipid bilayer was observed,which proved that the liposome was successfully prepared.The prepared liposome was placed in an environment of 4°C,and its stability was examined.The change of the particle size of the liposome within 15 days was observed,it was found that the particle size of the liposome did not change significantly and the stability was good.By observing the changes in the particle size and morphology of liposomes induced by DTT,it was shown that ALN-HA-SS-L liposome（ALN-HA-SS-L-L）has reducing sensitivity,and also shows HA-SS-L lipid.Reduction sensitivity of the body（HA-SS-L-L）.In vitro release studies have shown that liposomes have significantly improved drug release performance under high reducing environments（10 mM GSH）in tumor cells.The cytotoxicity of drug-loaded liposomes and blank liposomes was examined by MTT assay.The cell model of this subject is human osteosarcoma cell MG-63,which is highly expressed on the surface of CD44 receptor.The experimental results showed that the cytotoxicity of doxorubicin-loaded liposomes and free doxorubicin was dose-dependent.IC500 value DOX<HA-SS-L-L/DOX<ALN-HA-SS-L-L/DOX<HA-L-L<PEG-L/DOX.Interestingly,when iRGD is present,the IC500 value:ALN-HA-SS-L-L/DOX/iRGD<ALN-HA-SS-L-L/DOX.These results are the effect of the penetration of iRGD,the bone targeting of ALN,the cellular targeting of HA,and the reduction of sensitive bonds（-SS-）.Flow cytometry quantitatively demonstrated the uptake ability of MG-63 cells to doxorubicin-loaded liposomes and free doxorubicin,indicating that the uptake ability of MG-63 cells to HA-SS-L-L/DOX was significantly higher than that of other carriers.Drug liposomes,and when iRGD is present,the uptake capacity is stronger,which is consistent with the results observed by inverted fluorescence microscopy.In situ tibia injection of MG-63 cells was successful to establish a nude mouse model of orthotopic osteosarcoma,confirmed by CT-scanning.The small animal imaging results showed that the fluorescence intensity of ALN-HA-SS-L-L/DiR at the tumor site was significantly higher than that of the other groups,and the fluorescence intensity was stronger when iRGD and ALN-HA-SS-L-L/DiR were injected intravenously.This is consistent with the ex vivo imaging of tumors and normal organs（heart,liver,spleen,lungandkidney）.Intheorthotopicosteosarcomanudemousemodels,ALN-HA-SS-L-L/DOX showedremarkably higher tumor growth suppression accompanied with a prolonged survival rate.This was further improved by the co-administration of iRGD.The antitumor effect of various liposomes were ranked in the same order as the tumor biodistribution by in vivo imaging:ALN-HA-SS-L-L co-administered with iRGD>ALN-HA-SS-L-L>HA-SS-L-L>HA-L-L>DOX.ALN-HA-SS-L-L/DOX（with or without iRGD）also significantly decreased the cardiotoxicity of DOX while prevented lung metastases.Conclusion:Taken together,this study demonstrated that ALN-HA-SS-L-L/DOX,equipped with bone-and CD44-dual targeting abilities and redox-sensitivity,could be a promising osteosarcoma-target therapy.Co-administration of tumour-homing cell penetrating peptide iRGD with the nanoliposomes showed as a promising approach to enhance the OS tumour accumulation and possible the cancer cell uptake and thus enhance the efficacy. |
