Oligopeptide/Aptamer-conjugated Dendrimer-based Nanocarrier For Bone Dual-targeting Delivery

Objective:Bone targeting is one of the most potentially valuable therapeutic methods for medically treating bone diseases,such as osteoarthritis,osteoporosis,nonunion bone defects,bone cancer,and myeloma-related bone disease,but its efficacy remains a challenge due to the unfavorable bone biodistribution,off-target effects,and lack of cell specificity.To address these problems,we synthesized a new dual-targeting nanocarrier for efficient bone targeting delivery by covalently modifying the G4.0 PAMAM dendrimer with the C11 peptide and CH6 aptamer(CH6-PAMAM-C11).Methods:~1H-NMR and FT-IR were used to detect the molecular structure of the nanocarriers.Dynamic light scattering particle size analyzer was used to detect the particle size and Zeta potential.TEM was used to observe the morphology and size of the nanocarriers.PAMAM,CH6aptamer and C11 peptide were modified with 3 kinds of fluorescence,respectively.After cellular uptake,the co-localization of the 3 kinds of fluorescence in the cells was observed by a laser confocal scanning microscope,thus test whether the materials were successfully synthesized,and the targeting properties of nanocarriers to osteoblasts and hydroxyapatite in vitro.The VitD3 was taken as a small molecule model drug,the microplate reader was used to detect the OD value of VitD3 to analyze the drug-loading capacity of nanocarriers.The CCK-8 assay was used to research the long-term effects of nanocarriers on cell viability.After the administration of the fluorescent-labeled nanocarriers by tail vein,in vivo imaging system was used to observe the distribution of nanoparticles in rats,the confocal laser scanning microscope was used to observe the distribution of nanocarriers in bone tissue and detect the targeting ability to osteoblast.Results:The results of ~1H-NMR and FT-IR showed CH6 aptamer and C11 peptide were successfully conjugated to PAMAM via PEG.CLSM showed that the three kinds of fluorescence co-localized well in the cell,which confirmed the successful synthesis of the nanocarriers.In addition,the accumulation in the targeted cells,mineralized areas and tissues was successfully observed.DLS and TEM demonstrated that CH6-PAMAM-C11 was approximately 40～50 nm in diameter.In vitro targeting experiments confirmed that the C11 ligand had a high affinity for HAP,while the CH6 aptamer had a high affinity for osteoblasts.In vivo biodistribution analysis showed that CH6-PAMAM-C11 could rapidly accumulate in bone within 4 h and 12 h and then deliver to sites of osteoblast activity.The components of CH6-PAMAM-C11 were well excreted via the kidneys.The accumulation of many more CH6-PAMAM-C11 dual-targeting nanocarriers than single-targeting nanocarriers was observed in the periosteal layer of the rat skull,along with aggregation at sites of osteoblast activity.Conclusion:All of results indicate that CH6-PAMAM-C11 may be a promising nanocarrier for the delivery of drugs to bone,particularly for the treatment of osteoporosis,and our research strategy may serve as a reference for research in targeted drug,small molecule drug and nucleic acid delivery.