A delivery system targeting bone formation surfaces to facilitate RNAi-based anabolic therapy


Metabolic skeletal disorders associated with impaired bone formation are a major clinical challenge. One approach to treat these defects is to silence bone-formation–inhibitory genes by small interference RNAs (siRNAs) in osteogenic-lineage cells that occupy the niche surrounding the bone-formation surfaces. We developed a targeting system involving dioleoyl trimethylammonium propane (DOTAP)-based cationic liposomes attached to six repetitive sequences of aspartate, serine, serine ((AspSerSer)6) for delivering siRNAs specifically to bone-formation surfaces. Using this system, we encapsulated an osteogenic siRNA that targets casein kinase-2 interacting protein-1 (encoded by Plekho1, also known as Plekho1). In vivo systemic delivery of Plekho1 siRNA in rats using our system resulted in the selective enrichment of the siRNAs in osteogenic cells and the subsequent depletion of Plekho1. A bioimaging analysis further showed that this approach markedly promoted bone formation, enhanced the bone micro-architecture and increased the bone mass in both healthy and osteoporotic rats. These results indicate (AspSerSer)6-liposome as a promising targeted delivery system for RNA interference–based bone anabolic therapy.

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Figure 1: Differential occupancy characteristics of (AspSerSer)6 compared to Asp8 at bone-formation or bone-resorption surfaces in nondecalcified bone sections using a confocal laser scanning microscope.
Figure 2: Organ-selective delivery and gene knockdown in vivo.
Figure 3: Cell-selective delivery and knockdown efficiency in vivo.
Figure 4: In vivo microCT examinations of the three-dimensional trabecular architecture and an ex vivo bone formation evaluation in nondecalcified bone sections in healthy rats.
Figure 5: In vivo microCT examination of the three-dimensional trabecular architecture in OVX-treated rats.


  1. 1

    Black, D.M. et al. The effects of parathyroid hormone and alendronate alone or in combination in postmenopausal osteoporosis. N. Engl. J. Med. 349, 1207–1215 (2003).

    CAS  Article  Google Scholar 

  2. 2

    Hodsman, A.B. et al. Parathyroid hormone and teriparatide for the treatment of osteoporosis: a review of the evidence and suggested guidelines for its use. Endocr. Rev. 26, 688–703 (2005).

    CAS  Article  Google Scholar 

  3. 3

    Cosman, F. et al. Daily and cyclic parathyroid hormone in women receiving alendronate. N. Engl. J. Med. 353, 566–575 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Lindsay, R. et al. Randomised controlled study of effect of parathyroid hormone on vertebral-bone mass and fracture incidence among postmenopausal women on oestrogen with osteoporosis. Lancet 350, 550–555 (1997).

    CAS  Article  Google Scholar 

  5. 5

    Neer, R.M. et al. Effect of parathyroid hormone (1–34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N. Engl. J. Med. 344, 1434–1441 (2001).

    CAS  Article  Google Scholar 

  6. 6

    López-Fraga, M., Martinez, T. & Jimenez, A. RNA interference technologies and therapeutics: from basic research to products. BioDrugs 23, 305–332 (2009).

    Article  Google Scholar 

  7. 7

    Novina, C.D. & Sharp, P.A. The RNAi revolution. Nature 430, 161–164 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Itaka, K. et al. Bone regeneration by regulated in vivo gene transfer using biocompatible polyplex nanomicelles. Mol. Ther. 15, 1655–1662 (2007).

    CAS  Article  Google Scholar 

  9. 9

    Wang, D., Miller, S.C., Kopeckova, P. & Kopecek, J. Bone-targeting macromolecular therapeutics. Adv. Drug Deliv. Rev. 57, 1049–1076 (2005).

    CAS  Article  Google Scholar 

  10. 10

    Wang, D. et al. Osteotropic peptide that differentiates functional domains of the skeleton. Bioconjug. Chem. 18, 1375–1378 (2007).

    CAS  Article  Google Scholar 

  11. 11

    Yarbrough, D.K. et al. Specific binding and mineralization of calcified surfaces by small peptides. Calcif. Tissue Int. 86, 58–66 (2010).

    CAS  Article  Google Scholar 

  12. 12

    Lu, K. et al. Targeting WW domains linker of HECT-type ubiquitin ligase Smurf1 for activation by Ckip-1. Nat. Cell Biol. 10, 994–1002 (2008).

    CAS  Article  Google Scholar 

  13. 13

    Zhang, L. et al. The PH domain containing protein Ckip-1 binds to IFP35 and Nmi and is involved in cytokine signaling. Cell. Signal. 19, 932–944 (2007).

    CAS  Article  Google Scholar 

  14. 14

    Stuart, A.J. & Smith, D.A. Use of the fluorochromes xylenol orange, calcein green, and tetracycline to document bone deposition and remodeling in healing fractures in chickens. Avian Dis. 36, 447–449 (1992).

    CAS  Article  Google Scholar 

  15. 15

    Aubin, J. & JNM., H. Bone cell biology: osteoblast, osteocyte and osteoclasts. in Pediatric Bone 43–47 (Academic Press, San Diego, California, USA, 2002).

  16. 16

    Gronthos, S. et al. Differential cell surface expression of the STRO-1 and alkaline phosphatase antigens on discrete developmental stages in primary cultures of human bone cells. J. Bone Miner. Res. 14, 47–56 (1999).

    CAS  Article  Google Scholar 

  17. 17

    Ishikawa, S. et al. Involvement of FcRγ in signal transduction of osteoclast-associated receptor (OSCAR). Int. Immunol. 16, 1019–1025 (2004).

    CAS  Article  Google Scholar 

  18. 18

    Kim, N., Takami, M., Rho, J., Josien, R. & Choi, Y. A novel member of the leukocyte receptor complex regulates osteoclast differentiation. J. Exp. Med. 195, 201–209 (2002).

    CAS  Article  Google Scholar 

  19. 19

    Posner, A.S. & Betts, F. Synthetic amorphous calcium-phosphate and its relation to bone-mineral structure. Acc. Chem. Res. 8, 273–281 (1975).

    CAS  Article  Google Scholar 

  20. 20

    Hoang, Q.Q., Sicheri, F., Howard, A.J. & Yang, D.S. Bone recognition mechanism of porcine osteocalcin from crystal structure. Nature 425, 977–980 (2003).

    CAS  Article  Google Scholar 

  21. 21

    Midura, R.J. et al. Bone acidic glycoprotein-75 delineates the extracellular sites of future bone sialoprotein accumulation and apatite nucleation in osteoblastic cultures. J. Biol. Chem. 279, 25464–25473 (2004).

    CAS  Article  Google Scholar 

  22. 22

    Steitz, S.A. et al. Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification. Am. J. Pathol. 161, 2035–2046 (2002).

    CAS  Article  Google Scholar 

  23. 23

    Takahashi-Nishioka, T. et al. Targeted drug delivery to bone: pharmacokinetic and pharmacological properties of acidic oligopeptide-tagged drugs. Curr. Drug Discov. Technol. 5, 39–48 (2008).

    CAS  Article  Google Scholar 

  24. 24

    Wang, D., Miller, S., Sima, M., Kopeckova, P. & Kopecek, J. Synthesis and evaluation of water-soluble polymeric bone-targeted drug delivery systems. Bioconjug. Chem. 14, 853–859 (2003).

    CAS  Article  Google Scholar 

  25. 25

    Federman, N. & Denny, C.T. Targeting liposomes toward novel pediatric anticancer therapeutics. Pediatr. Res. 67, 514–519 (2010).

    CAS  Article  Google Scholar 

  26. 26

    Wang, G., Kucharski, C., Lin, X. & Uludag, H. Bisphosphonate-coated BSA nanoparticles lack bone targeting after systemic administration. J. Drug Target. 18, 611–626 (2010).

    CAS  Article  Google Scholar 

  27. 27

    Cullis, P.R., Mayer, L.D., Bally, M.B., Madden, T.D. & Hope, M.J. Generating and loading of liposomal systems for drug-delivery applications. Adv. Drug Deliv. Rev. 3, 267–282 (1989).

    CAS  Article  Google Scholar 

  28. 28

    Vegni, F.E., Corradini, C. & Privitera, G. Effects of parathyroid hormone and alendronate alone or in combination in osteoporosis. N. Engl. J. Med. 350, 189–192, author reply 189–192 (2004).

    CAS  Article  Google Scholar 

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We thank X.-H. Yang for technical support with the confocal imaging, H. Yang for technical support with the flow cytometry and F.C. Chun-Wan for assistance with the bone histomorphometry. This study was supported by the Chinese National Basic Research Programs (2011CB910602), the Hong Kong Competitive Earmarked Research Grant (CUHK479111 and 473011), the Direct Grant of Faculty of Medicine of the Chinese University of Hong Kong (2041478 and 2041525), the Faculty Research Grant of Hong Kong Baptist University (30-08-089), the Chinese National Natural Science Foundation Project (30830029) and the National Key Technologies Research and Development Program for New Drugs (2009ZX09503-002).

Author information




All the authors were involved in conducting, drafting or revising the manuscript. All the authors approved the final version of the manuscript for submission. L.Q. had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and supervision: G.Z., L.Q., L. Zhang, F.H. Design and preparation of delivery system: G.Z., H.W., Z.Y., H.C., Y.L., K.T. Design, synthesis and screening of siRNA sequences: T.T., G.Z., L. Zheng, Z.H., N.D. Analysis and interpretation of data from cell biology and molecular biology: B.G., T.T., B.-T.Z., G.Z., D.L., X.W., L.Q. Analysis and interpretation of data from immunohistochemistry: B.-T.Z., B.G., G.Z., K. Lee, L.Q. Analysis and interpretation of data from biophotonic imaging: B.G., G.Z., G.L., L.Q. Analysis and interpretation of data from microCT and bone histomorphometry: B.G., G.Z., Y.H., Y.W., L.Q. Analysis and interpretation of data for clinical relevance: G.Z., L.Q., X.P., L.H., K. Leung.

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Correspondence to Ge Zhang or Lingqiang Zhang or Ling Qin.

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The authors declare no competing financial interests.

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Supplementary Figures 1–4, Supplementary Tables 1–4, Supplementary Methods, Supplementary Discussion and Supplementary Results (PDF 3361 kb)

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Zhang, G., Guo, B., Wu, H. et al. A delivery system targeting bone formation surfaces to facilitate RNAi-based anabolic therapy. Nat Med 18, 307–314 (2012). https://doi.org/10.1038/nm.2617

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