Chemical Homing to Localize Anabolic Extracellular Matrix Cues to Accelerate Bone Fracture Repair
Small molecule drug conjugates to deliver therapeutics to bone are one of the most promising strategies to increase the efficacy and safety of drugs used to treat bone fractures and bone-related diseases1,2. Traditionally, administration of bone anabolic agents (such as extracellular matrix (ECM) fragments) for fracture repair, has relied entirely on local surgical applications. Yet, because the surgical applications are invasive, use and development of bone anabolic agents has been limited3. Therefore, we have aimed to develop a drug delivery system that could safely and effectively be used to deliver therapeutics directly to the site of a bone fracture, without surgical implantation. By conjugating bone anabolic agents to bone-homing molecules (molecules that localize very selectively to bone), fracture treatment can be performed through minimally invasive subcutaneous administration. ECM cues were found to localize specifically to the site of bone damage following subcutaneous injection. The noninvasive administration route allows for more of the drug to be delivered as needed making it much easier to maintain therapeutic levels throughout the entirety of the fracture repair process. This noninvasive method reduces the systemic side effects due to implanted compound leakage associated with ECM-derived anabolics, increases their efficacy relative to nontargeted ECM fragments, and opens the possibility of developing these compounds as pharmaceuticals for noninvasive treatment throughout fracture repair.
Bone targeting relies on the exposure of raw hydroxyapatite that occurs with a bone fracture which allows these high-affinity targeting ligands to chelate the calcium component of hydroxyapatite and localize primarily to the fracture site3,4. While several bone-homing molecules (such as bisphosphonates and tetracycline targeting) have been developed to treat osteoporosis5,6, these molecules all have some toxicity associated with them7and have never been shown to target bone fractures. We have found that mimicking bone homing of bone sialoprotein by using short acidic oligopeptides is sufficient to localize to bone fractures with high selectivity and have very low toxicity compared to bisphosphonates and tetracyclines. The adjacent carboxylic acid side chains in the polymers of acidic oligopeptides allow for the chelation of multiple calcium ions in the hydroxyapatite and provide high affinity for the bone. We have demonstrated the ability of these acidic oligos to target bone with numerous payloads of small molecules linked via labile linkers. We have also demonstrated that these can be used to target peptides including hydrophobic, neutral, cationic, anionic amino acids as well as peptides of varying length. This is particularly useful because many bone anabolics are peptide in nature8. Additionally, we have found that acidic oligos have better persistence at the site of the fracture than bisphosphonate-targeted therapeutics. We have also seen that increasing the length of the acidic oligo targeting ligand has increased the selectivity for the fracture site over that of the healthy bone. Taken together, this method allows for a systemic administration of bone anabolics to treat bone fractures, which it achieves by accumulating the bone anabolic at the fracture site.
One example of these anabolic agents that are limited to surgical applications is that of ECM fragments. Tissue engineering has long understood the importance of the extracellular matrix in promoting wound repair, often relying on decellularized ECMs to act as scaffolds or isolating anabolic fragments from the ECM to promote wound repair. Current investigative strategies utilizing ECMs for bone fracture repair have relied on the local application of ECM peptide-coated implants, -impregnated cements, or demineralized bone to stimulate and accelerate the repair. Some of the cements and coatings have relied on using fragments of collagen9–13, fibronectin14–17, laminin18,19,20, osteonectin21,22, bone sialoprotein23,24, and osteopontin25–27. These cements and implants have proved effective, but they are incredibly invasive to implant and are limited to a single application. Furthermore, their use is limited to open bone fracture repair. We, however can utilize these ECM cues as therapeutics by using our fracture-targeted drug delivery system to deliver them to the site of a bone fracture. We can constantly amplify the cues from the ECM to promote repair in just one region from a systemic administration, thus making the surgical application and coating of implants unnecessary. This bone fracture targeted platform allows for greater specificity of the drugs accumulation leading to lower required doses and lower side effects. We achieved this by synthesizing targeted fragments of collagen, fibronectin, osteopontin, bone sialoprotein, and osteonectin. The conjugates were tested in a mid-shaft stabilized femur fracture model in 12-week-old Swiss Webster mice. The mice were dosed subcutaneously daily for 3 weeks (n=10). Healing was assessed both structurally (Micro CT) and mechanically (4-point bend). The best performing ECM cue was ITGA5, which is a synthetic integrin alpha 5 ligand that binds to the integrin alpha 5 integrin on the surface of mesenchymal stem cells and promotes their differentiation into osteoblasts via the integrin-mediated cell signals FAK and ERK1/2-MAPKs16,17,28. We found that, by targeting this compound, we were able to achieve an 89% increase in BV/TV of the fracture callus relative to saline control. We were also able to achieve a significant 216% increase relative to a saline control in work-to-fracture. Additionally, targeted ITGA demonstrated a significant 98% increase in stiffness and a 75% increase in maximum force relative to the saline control. In the targeted drugs, no side effects were observed even at very high doses. A histological evaluation of the liver and kidneys also demonstrated no significant difference between saline and the treated groups. No off-target skeletal effects were observed. The treated contralateral femurs in every ECM drug group showed no significant difference when compared to the saline control contralateral femur. We found that by stabilizing the disulfide cyclization in ITGA the activity of the drug could be increased by improving the stability. The targeted ECM fragments were able to improve fracture healing and stimulate a more rapid bone formation at the site of injury. The ITGA molecule was able to develop a hard callus fast enough for the fractured femur to reach the average pre-fracture strength in three weeks compared to eight weeks for the saline-treated mice—a dramatic reduction in healing time that could reduce the time patients spend suffering from immobility. Additionally, the cardiovascular comorbidities associated with patients’ immobility could decrease.
This new drug administration for these anabolic ECM fragments has the potential to provide a new non-invasive treatment course in which a more sustained therapeutic effect can be elicited with less drug. It has helped create a safe alternative to surgically implanting these compounds, allowing them to have a more widespread use. This technology has the potential to be applied to different classes of anabolic drugs that improve healing via different mechanisms and can also deliver drugs where damaged hydroxyapatite is exposed as a result of varying diseases. The impact of this platform development could extend far beyond bone fractures and ECM proteins to encompass the many other musculoskeletal diseases. Furthermore, it opens the door for a new way of treating the prevalent afflictions of broken bones and associated deaths of severe fractures.
1. Srinivasarao, M. & Low, P. S. Ligand-Targeted Drug Delivery. Chem. Rev 117, 12133–12164 (2017).
2. Low, S. A. et al. Biodistribution of fracture-targeted GSK3β inhibitor-loaded micelles for improved fracture healing. Biomacromolecules 16, 3145–3153 (2015).
3. Stapleton, M. et al. Development of Bone Targeting Drugs. Int. J. Mol. Sci. 18, 1–15 (2017).
4. Rawat, P. et al. Revisiting bone targeting potential of novel hydroxyapatite based surface modi fi ed PLGA nanoparticles of risedronate : Pharmacokinetic and biochemical assessment. Int. J. Pharm. 506, 253–261 (2016).
5. Cole, L. E., Vargo-gogola, T. & Roeder, R. K. Targeted delivery to bone and mineral deposits using bisphosphonate ligands. Adv. Drug Deliv. Rev. 99, 12–27 (2016).
6. Chai, G. & Hu, F. Tetracycline-grafted PLGA nanoparticles as bone-targeting drug delivery system. Int. J. Nanomedicine 10, 5671–5685 (2015).
7. Newman, M. R. Local and targeted drug delivery for bone regeneration. 125–132 (2017). doi:10.1016/j.copbio.2016.02.029.Local
8. Amso, Z., Cornish, J. & Brimble, M. A. Short Anabolic Peptides for Bone Growth. Med. Res. Rev. 36, 579–640 (2016).
9. Pedersen, R. H., Rasmussen, M., Overgaard, S. & Ding, M. Effects of P-15 Peptide Coated Hydroxyapatite on Tibial Defect Repair In Vivo in Normal and Osteoporotic Rats. Biomed Res. Int. 2015, 1–14 (2015).
10. Gomar, F., Orozco, R. & Luis, J. P-15 small peptide bone graft substitute in the treatment of non-unions and delayed union . A pilot clinical trial. Int. Orthop. 31, 93–99 (2007).
11. Bhatnagar, R. S. et al. Design of Biomimetic Habitats for Tissue Engineering with P-15, a Synthetic Peptide Analogue of Collagen. Tissue Enginering 5, 53–65 (1999).
12. Agrawal, V. et al. Recruitment of Progenitor Cells by an Extracellular Matrix Cryptic Peptide in a Mouse Model of Digit Amputation. TISSUE Eng. Part A 17, 2435–2444 (2011).
13. Agrawal, V. et al. An Isolated Cryptic Peptide Influences Osteogenesis and Bone Remodeling in an Adult Mammalian Model of Digit Amputation. TISSUE Eng. Part A 17, 3033–3044 (2011).
14. Brun, J. et al. Peptide-Based Activation of Alpha5 Integrin for Promoting Osteogenesis Olivia. J. Cell. Biochem. 3038, 3029–3038 (2012).
15. Feng, Y. & Mrksich, M. The synergy peptide PHSRN and the adhesion peptide RGD mediate cell adhesion through a common mechanism. Biochemistry 43, 15811–21 (2004).
16. Ringe, J. et al. Priming integrin ␣ 5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis. PNAS 106, 1–5 (2009).
17. Gandavarapua, N. R., Algeb, D. L. & Anseth, K. S. Osteogenic differentiation of human mesenchymal stem cells on α5 integrin binding peptide hydrogels is dependent on substrate elasticity. Biomater Sci. 2, 352–361 (2015).
18. Lin, X., Takahashi, K., Liu, Y. & Zamora, P. O. Enhancement of cell attachment and tissue integration by a IKVAV containing multi-domain peptide. Biochim. Biophys. Acta - Gen. Subj. 1760, 1403–1410 (2006).
19. Yeo, I. S. et al. Adhesion and spreading of osteoblast-like cells on surfaces coated with laminin-derived bioactive core peptides. Data Br. 5, 411–415 (2015).
20. Kang, H. K. et al. The effect of the DLTIDDSYWYRI motif of the human laminin α2 chain on implant osseointegration. Biomaterials 34, 4027–4037 (2013).
21. Hove, A. H. Van, Burke, K., Antonienko, E., Brown, E. & Benoit, D. S. W. Enzymatically-responsive pro-angiogenic peptide-releasing poly(ethylene glycol) hydrogels promote vascularization in vivo. J Control Release 118, 6072–6078 (2016).
22. Pickart, L. The human tri-peptide GHK and tissue remodeling. J. Biomater. Sci. Polym. Ed. 19, 969–988 (2008).
23. Rapuanoa, B. E. & MacDonald, D. E. Structure-activity relationship of human bone sialoprotein peptides. Eur J Oral Sci 185, 974–981 (2013).
24. Choi, Y. J., Lee, J. Y., Chung, C. & Park, Y. J. Enhanced osteogenesis by collagen-binding peptide from bone sialoprotein in vitro and in vivo. 547–554 (2012). doi:10.1002/jbm.a.34356
25. Lee, J. et al. Assembly of collagen-binding peptide with collagen as a bioactive scaffold for osteogenesis in vitro and in vivo. Biomaterials 28, 4257–4267 (2007).
26. Lee, J. et al. Injectable gel with synthetic collagen-binding peptide for enhanced osteogenesis in vitro and in vivo. Biochem. Biophys. Res. Commun. 357 357, 68–74 (2007).
27. Kyoung, M. et al. A novel collagen-binding peptide promotes osteogenic differentiation via Ca 2 + / calmodulin-dependent protein kinase II / ERK / AP-1 signaling pathway in human bone marrow-derived mesenchymal stem cells. Cell. Signal. 20, 613–624 (2008).
28. Saidak, Z. et al. Wnt / β -Catenin Signaling Mediates Osteoblast Differentiation Triggered by Peptide-induced α 5 β 1 Integrin Priming in Mesenchymal Skeletal Cells *. J. Biol. Chem. 290, 6903–6912 (2015).