Rhein Modified Spherical Hydroxyapatite Nanocarriers For Targeted Therapy Of Bone Tumors

One potential way to enhance the clinical efficacy of bone tumor is the bone-targeting treatments,which can minimize skeletal morbidity and preserve the patients’quality of life and physical function.Rhein,an anti-inflammatory agent isolated from Rheum palmatum L（Chinese rhubarb）,and it has a similar chemical structure to tetracycline.Although the biocompatibility and bone affinity of Rhein have been confirmed in the previous reports,and few studies focus on the bone-targeting treatments of bone tumor using Rhein.Based on the affinity of Rhein for bone,we developed a novel Rhein-polyethylene glycol（PEG）-nanohydroxyapatite（n HA）carrier to deliver doxorubicin（DOX）and phosphorus-32(³²P)simultaneously for enhanced the effect of chemo-radiotherapy on bone tumors.The spherical n HA was firstly synthesized successfully by biomimetic method using Ca（NO₃）₂·4H₂O and（NH₄）₂HPO₄ in the presence of PEG.Then,the spherical n HA was modified with PEG,and the NHS-ester of Rhein was condensed with PEG to prepare Rhein-PEG-n HA nanoparticles.The structure of Rhein-PEG-n HA was confirmed by Scanning electron microscopy（SEM）,Fourier transform infrared spectrometer（FT-IR）and Thermogravimetric analyzer（TGA）.The synthetic Rhein-PEG-n HA conjugates were sphere in shape with an average diameter of～120 nm.Furthermore,the in vitro studies reveal that Rhein-PEG-n HA had a significant affinity to bone fragments compared with the PEG-n HA.The DOX@Rhein-PEG-n HA and ³²P@Rhein-PEG-n HA were prepared using the physical absorption method.In the present study,the loading capacity of the DOX was19.68%,and the radiolabeling yield of ³²P was 45%.In vitro release pattern indicated that the release profiles of DOX depend on p H condition,and a faster DOX release can be found at lower p H value.And DOX@Rhein-PEG-n HA show a sustained release behavior favorable for long-term therapy.At the same time,³²P from the³²P@Rhein-PEG-n HA can be observed under different p H conditions,and ³²P exhibited good stability.In the animal level experiment,the animal model of bone tumor was successfully established.In order to track the in vivo distribution of the Rhein-PEG-n HA,^99mTc was used to label the Rhein-PEG-n HA nanoparticles through the physical absorption method.A time-dependent increase of SPECT signals was observed in tumor,which demonstrated that the efficient tumor targeting of ^99mTc@Rhein-PEG-n HA.After 24 h of the injection,the uptake rate of tumor tissue in 9.57%ID/g.In terms of curative effect,by comparing the relative tumor volume,tumor weight,tumor burden and tumor inhibition rate（TIR）,it was found that Rhein-PEG-n HA and DOX@PEG-n HA did not exhibit better effect on tumor inhibition compared with Saline group.By contrast,DOX@Rhein-PEG-n HA and ³²P@Rhein-PEG-n HA showed a significant tumor inhibition due to their good bone tumor targeting capacity.Furthermore,DOX/³²P@Rhein-PEG-n HA exhibited highest tumor growth inhibition effects among the different groups,and the TIR was 77.38%.Pathological sections of H&E and TUNEL staining show greater tumor cell degeneration and cell apoptosis,and an same tendency of curative effect can be found.The skeletal morphology of tumor-bearing tibias after different treatments were evaluated using CT.A more complete structure of tumor-bearing tibias of mice in DOX/³²P@Rhein-PEG-n HA group were observed,which suggested that DOX/³²P@Rhein-PEG-n HA group could efficiently protect the skeletal structure.Additionally,blood chemistry tests,whole blood cell analysis and H&E staining confirms that Rhein-PEG-n HA nanoparticles have better safety and non-toxic.Overall,the tumor growth can be significantly inhabited after the DOX or ³²P loaded bone targeted n HA treatments,and the inhibition effect can be further enhanced using the chemo-radiation combined therapy,indicating that Rhein-PEG-n HA nanocarrier provides an efficient strategy for the bone targeting treatment of bone metastases.