Targeting damaged collagen for intra-articular delivery of therapeutics using collagen hybridizing peptides

Abstract

The objective of this study was to investigate the potential of collagen hybridizing peptides (CHPs), which bind to denatured collagen, to extend the retention time of near-infrared fluorophores (NIRF) following intra-articular (IA) injection in rat knee joints. CHPs were synthesized with a NIRF conjugated to the N-terminus. Male Sprague-Dawley rats were assigned to one of four experimental groups: healthy, CHP; osteoarthritis (OA), CHP; healthy, scrambled-sequence CHP (sCHP), which has no collagen binding affinity; or OA, sCHP. Animals in the OA groups received an IA injection of monosodium iodoacetate to induce OA. All animals then received the corresponding CHP injection. Animals were imaged repeatedly over 2 weeks using an in vivo fluorescence imaging system. Joint components were isolated and imaged to determine CHP binding distribution. Safranin-O and Fast Green histological staining was performed to confirm the development of OA. CHPs were found to be retained within the joint following IA injection in both healthy and OA animals for the full study period. In contrast, sCHP signal was negligible by 24-48 h. CHP signal was significantly greater (p < 0.05) in OA joints when compared to healthy joints. At the 2-week end point, multiple joint components retained CHPs, including cartilage, meniscus, and synovium. CHPs dramatically extended the retention time of NIRFs following IA injection in healthy and OA knee joints by binding to multiple collagenous tissues in the joint. These results support the pursuit of further research to develop CHP based therapeutics for IA treatment of OA.

Keywords: articular cartilage; collagen; collagen hybridizing peptides; knee; osteoarthritis.

Figure 1.

Time course of retention of NIRF imaging markers following IA injection in MIA-induced OA rat knee joints. Each row shows a single animal in one of the four groups, and each column shows the specific imaging time. In both OA and healthy animals, NIRF-CHPs were retained for the entire imaging period of 336 hrs. In contrast, NIRF-sCHPs in the OA and healthy animals were not visible after 24–48 hours. Representative animal from each group shown ($\mathrm{n}=4$ per group).

Figure 2.

Total radiant efficiency over the time course of IVIS imaging. CHPs were retained to a greater extent in OA joints (solid red line) compared to healthy joints (solid blue line). sCHPs were cleared from the joint quickly and the total radiant efficiencies were not statistically significant between OA and healthy groups. Data presented as mean ± 95% confidence interval. Asterisk indicates statistical significance between OA CHP and healthy CHP at each time point $(\mathrm{p}<0.05)$.

Figure 3.

Dissected stifle joint at 336 hr post-injection of CHPs. Left) Macroscopic images of healthy (top) and OA (bottom) dissected joint components. Healthy joint shows no apparent disruption to cartilage health following CHP injection. OA joint shows visible lesions to the cartilage, as well as discoloration typical of OA joint destruction. Right) IVIS imaging of healthy (top) and OA (bottom) joints showing binding of CHPs to tibial and femoral cartilage surfaces, medial and lateral menisci, patella, and patellar tendon. In both cases, the highest signal intensity is localized to the cartilage surfaces. CHP binding is significantly greater on the synovial membrane in the osteoarthritic joint.

Figure 4.

Histological staining of healthy and osteoarthritic tibiae. Coronal sections of the tibiae were stained with Safranin-O and Fast Green at 336 hr end point. A) CHP injected stifle joint demonstrates healthy cartilage. B) 21-days post-injection of 10 mg/mL MIA. There is an apparent loss of proteoglycans, loss of chondrocytes, and an increase in calcified cartilage at the osteochondral interface. Representative animal selected for each condition ($\mathrm{n}=4$ per group).