A hyaluronic acid binding peptide-polymer system for treating osteoarthritis: an in vivo study

Abstract

Purpose: Osteoarthritis (OA) is a degenerative joint disease in which synergistic interactions between synovial fluid lubricants and the cartilage surface are lost. We designed a strategy to bind HA to the cartilage surface via a polymer-peptide binding system, mimicking the function of lubricin on the healthy cartilage surface. This system contains an HA binding peptide and a collagen binding peptide linked by poly(ethylene glycol) (PEG) to target and concentrate HA at the cartilage surface. This technology enhanced HA retention in vivo and cartilage lubrication in vitro. In the current study, the polymer-peptide technology was applied to a small animal model of post traumatic OA. A synthetic peptide (HABP1), RHAMM mimetic peptide (HABP2), and another synthetic peptide (HABP3), each conjugated to 1kDa peg-COLBP and also to an 8-arm peg-colbp, were studied to determine which one had the greatest efficacy reducing OA progression in vivo and in binding HA in vitro. Methods: The anterior cruciate ligament (ACL) of each mouse was transected and OA was allowed to develop over two weeks at which time saline controls or polymer-peptides were injected intra-articularly. Cartilage integrity was assessed using OARSI scoring of histology at 4 weeks post ACLT. Additionally, joints were harvested for quantitative PCR (qPCR) of structural cartilage genes and inflammatory markers at 4 weeks post ACLT. Pain and function was monitored at 2 and 4 weeks post ACLT by hot plate and incapacitance testing, which are functional assessments of sensitivity and pain. In vitro, quartz crystal microbalance with dissipation monitoring (QCM-D) was used to compare binding of different HABP formulations to HA. Results: The average OARSI score was reduced to varying degrees in each treatment group. Among peptides, the highest concentration of each peptide possible was tested. This resulted in a reduction from the average saline control score of 3.28–1.33 for HABP1-peg-colbp, 1.33 for HABP2-peg-colbp, 1.66 for HABP3-peg-colbp, 1.83 for HABP1-8 arm peg-colbp, and 0.833 for HABP2-8 arm peg-colbp (n = 3, HABP2-8 arm peg-colbp was significant at p < 0.05). These preliminary results showed that all peptide treated groups had more intense proteoglycan staining and intact articular cartilage than saline controls; more animals will be added to select treatments based on these preliminary OARSI scores and on in vitro binding outcomes. These OARSI results tended to correlate with the functional pain tests, in which the HABP1-peg-colbp, HABP2- peg-colbp, and HABP2-8 arm peg-colbp injected mice displayed a significant reduction of pain on the hot plate test (p < 0.05). HABP2- peg-colbp and HABP2-8 arm peg-colbp also reduced pain in the incapacitance test (p < 0.05). qPCR analysis was performed 4 weeks post ACLT and fold changes calculated with respect to un-operated, age matched controls. This revealed that, compared to saline controls, HABP1-peg-colbp injected animals expressed significantly lower levels of the matrix metalloproteinase MMP13 (on average 1.16 fold compared to 5.00 fold, n = 4) and inflammatory cytokine IL-6 (1.23 fold compared to 3.10 fold, n = 4). Preliminary qPCR on the other peptide groups showed similar levels of MMP13 and IL-6 compared to HABP1-peg-colbp; however, additional samples need to be analyzed to reach significance. In vitro, QCM-D revealed that all peptides bind to immobilized HA, with HABP2-8 arm peg-colbp exhibiting the most robust signal. Conclusions: The data thus far suggests that HABP2-8 arm peg-colbp is a good candidate for further in vivo studies. This formulation prevented cartilage destruction in the post traumatic OA mouse model and also reduced pain in both functional tests. Additionally, HABP2-8 arm peg-colbp showed the best binding to HA in QCM-D studies. More animal tests will be carried out to confirm the preliminary qPCR results. Overall, this therapy is a promising disease modifying approach for osteoarthritis treatment.