Inhibiting C3 Toxin from a Protein–DNA Hybrid
Total Page：16
File Type：pdf, Size：1020Kb
FULL PAPER Bone Defects www.advhealthmat.de Spatiotemporally Controlled Release of Rho-Inhibiting C3 Toxin from a Protein–DNA Hybrid Hydrogel for Targeted Inhibition of Osteoclast Formation and Activity Jasmina Gacˇanin, Anna Kovtun, Stephan Fischer, Victoria Schwager, Johanna Quambusch, Seah Ling Kuan, Weina Liu, Felix Boldt, Chuang Li, Zhongqiang Yang, Dongsheng Liu, Yuzhou Wu,* Tanja Weil, Holger Barth,* and Anita Ignatius* 1. Introduction In osteoporosis, bone structure can be improved by the introduction of thera- Bone turnover is an active process peutic molecules inhibiting bone resorption by osteoclasts. Here, biocompatible resulting from the well-orchestrated hydrogels represent an excellent option for the delivery of pharmacologically interplay between bone-forming cells active molecules to the bone tissue because of their biodegradability, inject- (osteoblasts) and bone-resorbing cells ability, and manifold functionalization capacity. The present study reports the (osteoclasts). Their cross-talk is tightly preparation of a multifunctional hybrid hydrogel from chemically modified regulated by hormones, including para- [1,2] human serum albumin and rationally designed DNA building blocks. The hybrid thormone, cytokines, for example, interleukin 6,[3] and growth factors, hydrogel combines advantageous characteristics, including rapid gelation including bone morphogenetic proteins through DNA hybridization under physiological conditions and a self-healing (BMPs) 2 and 7.[1] Disequilibrium in this and injectable nature with the possibility of specific loading and spatiotempo- complex interplay leads to an imbalance rally controlled release of active proteins, making it an advanced biomaterial for between bone formation and resorption, the local treatment of bone diseases, for example, osteoporosis. The hydrogels which results in severe bone diseases, including osteoporosis, and affects regen- are loaded with a recombinant Rho-inhibiting C3 toxin, C2IN-C3lim-G205C. This eration processes after both injuries and toxin selectively targets osteoclasts and inhibits Rho-signaling and, thereby, surgical intervention.[4,5] Therefore, opti- actin-dependent processes in these cells. Application of C2IN-C3lim-G205C mizing skeletal repair in the context of toxin-loaded hydrogels effectively reduces osteoclast formation and resorption osteoporosis is an important scientific and activity in vitro, as demonstrated by tartrate-resistant acid phosphatase staining medical aim. Bone structure improvement and the pit resorption assay. Simultaneously, osteoblast activity, viability, and can be achieved by enhancing bone forma- tion with therapeutic molecules, including proliferation are unaffected, thus making C2IN-C3lim-G205C toxin-loaded BMPs,[6,7] and repressing resorption hybrid hydrogels an attractive pharmacological system for spatial and selective using, for example, bisphosphonates.[7–9] modulation of osteoclast functions to reduce bone resorption. To achieve local delivery of therapeuti- cally active compounds in bone defects or J. Gacˇanin, J. Quambusch, W. Liu, F. Boldt, Prof. Y. Wu, Prof. T. Weil Dr. C. Li, Prof. Z. Yang, Prof. D. Liu Institute of Organic Chemistry III Key Laboratory of Organic Optoelectronics and Molecular University of Ulm Engineering of the Ministry of Education 89081 Ulm, Germany Department of Chemistry E-mail: [email protected] Tsinghua University Dr. A. Kovtun, Prof. A. Ignatius 100084 Beijing, China Institute of Orthopedic Research and Biomechanics Prof. Y. Wu Trauma Research Center School of Chemistry and Chemical Engineering University of Ulm Huazhong University of Science and Technology 89081 Ulm, Germany 430074 Wuhan, China E-mail: [email protected] S. Fischer, V. Schwager, Prof. H. Barth © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Institute of Pharmacology and Toxicology Weinheim. This is an open access article under the terms of the Creative University of Ulm Commons Attribution-NonCommercial License, which permits use, dis- 89081 Ulm, Germany tribution and reproduction in any medium, provided the original work is E-mail: [email protected] properly cited and is not used for commercial purposes. J. Quambusch, Dr. S. L. Kuan, Prof. Y. Wu, Prof. T. Weil The copyright line was changed 5 September 2017 after initial Max Planck Institute for Polymer Research publication. 55128 Mainz, Germany DOI: 10.1002/adhm.201700392 Adv. Healthcare Mater. 2017, 6, 1700392 1700392 (1 of 12) © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.advhealthmat.de weak bone, for example, immediately surrounding implants, an yet biocompatible possibility for well-directed cargo release. To appropriate carrier is crucial to achieve sustained in situ release address the excessive bone resorption by osteoclasts in osteo- of therapeutic molecules. Here, biocompatible hydrogels could porosis, the targeted pharmacological modulation of osteo- represent one of the best options for achieving an efficient local genesis or osteoclast activity represents a favorable strategy. delivery system. The high water content imparts biocompat- The clostridial Rho-inhibiting C3 toxins could be attractive ibility and a large number of therapeutics could be loaded and candidates for this purpose, because the treatment of cultured delivered via a controllable slow-release mechanism.[10] osteoclast-like cells with these enzymes effectively decreased It has been shown previously that the delivery of thera- both osteoclast formation from monocytic progenitor cells and peutic molecules into bone tissue is possible via absorption their resorption activity in vitro.[25] The C3 toxins (≈25 kDa) of the therapeutic within a synthetic hydrogel,[11] physically mono-ADP-ribosylate Rho in the cytosol inhibits Rho-mediated crosslinked alginate-gel[12] or Matrigel[13] during hydrogel prepa- signal-transduction and results in reorganization of the actin ration. Recently, we developed a biodegradable and biocom- cytoskeleton accompanied by a dramatic change in cell mor- patible protein–DNA hybrid hydrogel, which is crosslinked by phology and inhibition of central actin-associated cellular func- DNA hybridization without the application of reactive organic tions.[26,27] Moreover, C3 toxins, such as C3bot from Clostridium reagents or catalysts. DNA hybridization is based on sequence- (C.) botulinum[28] and C3lim from C. limosum,[29] are efficiently specific, supramolecular interactions, providing the hydrogel taken up into the cytosol of cells of the monocyte/macrophage- with beneficial features, including being injectable and self- line by an endocytic mechanism but not into the cytosol of healing.[14] Importantly, it is this injectable nature, which allows other cell types, including epithelial cells and fibroblasts.[26] The for simple, local administration of the protein–DNA hybrid recombinant fusion toxin C2IN-C3lim contains C3lim and the hydrogel via injection at the diseased site. In contrast to the nontoxic C2IN portion of the C. botulinum C2 toxin,[26] which incorporation of guest molecules via absorption, protein–DNA likely enhances the selective uptake of C2IN-C3lim into the hydrogels offer the opportunity to readily assemble the desired cytosol of monocytes/macrophages and osteoclasts, because bioactive molecules within the hydrogel using the appropriately both cell populations are derived from the same lineage.[25] In functionalized DNA adaptors. Even different kinds of func- these earlier studies, we identified C2IN-C3lim as the most tional proteins could be immobilized successfully following this efficient Rho-inhibitor for RAW 264.7 macrophages among approach.[14] In this fashion, a highly controlled protein loading the clostridial C3 enzymes. Additionally, we demonstrated in combination with local release of the bioactive molecules that this fusion toxin ADP-ribosylates Rho in RAW 264.7 cells could be achieved at the diseased site by nuclease-mediated results in reorganization of F-actin, as demonstrated by DAPI/ cargo release. In contrast to conventional hydrogel scaffolds phalloidin-staining of C2IN-C3lim-treated cells, and finally in based on synthetic polymers or undefined mixtures, including the characteristic changes of cell morphology.[25] We believe Matrigel, with high batch-to-batch variations,[15] the protein– that incorporating C2IN-C3lim enzyme into the biocompatible DNA hydrogels described herein feature a precisely defined protein–DNA hybrid hydrogel opens a new avenue for efficient composition and highly reproducible synthesis. Moreover, pro- and cell type-selective treatment of increased bone resorption, tein–DNA hybrid hydrogels provide several advantageous fea- featuring local application of therapeutics, ensuring a high local tures, including adaptable polypeptide chain lengths, precisely concentration and spatiocontrolled cargo release (Figure 1). defined amino acid sequences, and high biocompatibility.[16] In Herein, we demonstrate the unique potential of this novel addition, further chemical modifications are facilitated by the protein–DNA hybrid hydrogel for local application and con- presence of different functional groups along the main chain trolled release of C2IN-C3lim for the targeted pharmacological of the polypeptide backbone. The same strategy that converts inhibition of osteoclast formation and resorption activity proteins into versatile biomaterials combining biocompatibility, without affecting osteoblasts. biodegradability,