Protein corona formation on particles in bovine synovial fluid and in a rat knee model of osteoarthritis

Abstract

Purpose: In osteoarthritis (OA), molecular changes occur throughout the joint, causing changes in the joint milieu. Micro- and nanoparticles can be used to characterize these molecular changes. Specifically, proteins within the synovial fluid non-specifically adsorb onto the surface of particles. As such, changes in the protein corona formation on the particle surface may relate to molecular changes in OA. While protein corona formation is commonly studied in serum and cell culture media, it has not been assessed in OA animal models. In this study, protein corona formation was assessed in bovine synovial fluid and in a rat OA model. Methods: First, commercially available, magnetic microparticles (Dynabeads) were placed in bovine synovial fluid and separated using magnetic separation or centrifugation. The resulting protein corona was characterized using mass spectrometry. Then, 12 male and 12 female Lewis rats (9 weeks old) received a unilateral, simulated medial collateral ligament and meniscal injury. At 2, 5, or 8 weeks post-surgery, operated and contralateral limbs were injected with magnetic particles (n=4 per sex). After a 4-hour incubation, animals were euthanized, particles were recovered from the joint, and the protein corona was characterized using an Orbitrap Fusion Mass Spectrometer. Results: In bovine synovial fluid, a total of 116 proteins were identified, of which 35 were different between magnetic separation and centrifugation. In the rat OA model, 454, 464, and 342 proteins were identified at 2, 5, and 8 weeks post-surgery, respectively. Of the identified proteins on the particle surface, 35, 59, and 13 were different between the operated and contralateral limbs at 2, 5, and 8 weeks, respectively (p < 0.05, see Figure 1). Using principal component analysis, changes in the protein corona were found to relate with OA progression. Conclusions: Data from the bovine synovial fluid demonstrated that the protein corona that forms on the particle surface varies based on the method of particle separation from synovial fluid. Moreover, the in vivo data demonstrates that the protein corona layer on a particle surface changes at various stages of OA progression. Understanding how the particle-protein interface changes as a function of disease progression may be useful for biomarker discovery and informing the design of intra-articular drug delivery vehicles.