Transport of proteins, biopharmaceuticals and small pharmaceutical compounds into normal and injured cartilage by Sangwon Byun.
Massachusetts Institute of Technology. Dept. of Electrical Engineering and Computer Science.
Alan J. Grodzinsky and Eliot H. Frank.
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Traumatic joint injury can induce acute damage to cartilage and surrounding joint tissues accompanied by an inflammatory response, which can significantly increase the risk of developing Qsteoarthritis. The mechanism by which joint injury results in disease development is not fully understood. However, chondrocyte metabolism is greatly affected by the transport properties of cartilage extracellular matrix, which determine the accessibility and the concentrations of various proteins and therapeutic agents to cells and cell receptors. Using in vitro models of mechanical injury to cartilage, we have characterized the uptake and binding of proteins and biopharmaceuticals in normal articular cartilage and have compared the results to those in cartilage subjected to mechanical injury and pro-inflammatory cytokines. We studied equilibrium partitioning and non-equilibrium transport into cartilage of Pfpep, a 760 Da positively charged peptide inhibitor of the pro protein convertase PACE4. Competitive binding measurements revealed negligible binding to sites in the matrix. The uptake of Pf-pep depended on GAG charge density, consistent with predictions of Donnan equilibrium. The diffusivity of Pf-pep was measured to be ~1 x 10-6 cm2/s, close to other similarly-sized non-binding solutes. These results suggest that small positively charged therapeutics will have a higher concentration within cartilage than in the surrounding synovial fluid, a desired property for local delivery; however, such therapeutics may rapidly diffuse out of cartilage unless there is additional specific binding to intratissue substrates that can maintain enhanced intratissue concentration. We have also examined the effect of mechanical injury and inflammatory cytokines, TNFa, on the uptake of anti-IL-6 antibody Fab fragment (48 kDa). Anti-IL-6 Fab was able to penetrate into cartilage, though final equilibrium uptake would likely occur only after 6-10 days within 1 mm thick explant disks. Uptake of anti-IL-6 Fab was significantly increased following mechanical injury of the cartilage in vitro. A further increase in uptake was caused by TNFa treatment combined with mechanical injury. The increase in uptake was accompanied by GAG loss from the tissue, suggesting that there can be greater accessibility of large solutes into cartilage after direct mechanical injury or inflammatory cytokine treatment to the tissue, where the increase in uptake was related with the severity of matrix damage and loss. We also studied the binding and uptake of TNFa in articular cartilage and observed significant binding of TNFa to matrix sites. Binding was stronger for the monomeric form of TNFa compared to trimeric form. Binding of TNFa was not disrupted by pre-treatment of the tissue with trypsin, indicating that the intra-tissue binding sites were not removed by trypsininduced proteolysis of the matirx. These results suggest that matrix binding as well as monomer-trimer conversion of TNFa both play crucial roles in regulating the accessibility of TNFa to cell receptors. The results of this thesis are significant in that they suggest that injurious mechanical loading and inflammatory cytokine applied to cartilage can affect transport processes within the tissue. The resulting altered transport, in turn, can influence the accessibility of proinflammatory cytokines and anti-catabolic drugs which are designed to treat pathogenesis of OA.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2010."June 2010." Cataloged from PDF version of thesis.Includes bibliographical references.
DepartmentMassachusetts Institute of Technology. Dept. of Electrical Engineering and Computer Science.
Massachusetts Institute of Technology
Electrical Engineering and Computer Science.