Nanoparticle Interaction with Extracellular Matrix Stabilizes Cartilage Tissue Integrity

<b>Statement of Purpose</b>: Arthritic disorders are a leading cause of disability worldwide¹. Current treatments result in unsatisfactory outcomes due to the lack of specificity, fast clearance, and inability to penetrate biological barriers. Advancements in nanotherapeutics have shown that electrostatic interactions and nano-sized particles are able to target and penetrate dense, negative tissues such as the joint cartilage². Previously, we have shown that nanoparticles (NPs) interact with proteins in the joint space, which dictate the NPs’ interactions with cells and cartilage tissue³. As it is known that NPs must interact with the cartilage extracellular matrix (ECM) proteins prior to reaching their cellular target, this study aimed to demonstrate that the NPs’ interactions with ECM can be used as a means to stabilize the tissue and reduce degradation.<br/><b>Methods</b>: A cationic NP panel that differed in size and charge was synthesized based on either poly(amidoamine) (PAMAM) dendrimers (DendritechR, Midland, MI, USA) or poly(lactic-co-glycolide) (PLGA; ResomerR, Essen, Germany) polymer particles (formulated by nanoprecipitation) decorated with either PEG or polyethylenimine (PEI)-PEG modifications. All particles were fluorescently labeled with FITC. NP size and charge were characterized by dynamic light scattering (DLS) as well as electron microscopy. To mimic the thickness of human cartilage, porcine articular cartilage explants were used in this study, and subjected to arthritis-relevant enzymes such as ADAMTS5 for 1 or 2 hours, followed by NP treatment of equivalent duration. Released ECM components including sulfated glycosaminoglycans (GAGs) were assessed by colorimetric assays and polyacrylamide gel electrophoresis (PAGE). NP tissue uptake was evaluated by confocal microscopy, while aggregate formation and tissue topography were determined by atomic force microscopy (AFM). The interactions between the NPs and ECM proteins were evaluated by isothermal titration calorimetry (ITC) and quantitative proteomics.<br/><b>Results</b>: Physicochemical characterization of PAMAM particles revealed sizes of 7.2 nm and 11.0 nm and zeta potentials of +17.0 mV and +4.0 mV for non-PEGylated and PEGylated NPs respectively. Control PLGA-based NP size varied between 270 nm and 264 nm with their respective zeta potentials exhibiting +22.2 mV and +3.78 mV. By treating porcine explants with the different particles after enzymatic stimulation, we measured a reduction of GAG concentration in the supernatants after 2- and 4-hour treatments compared to the control. To investigate the level of engagement between the particles and the tissue, we tracked the fluorescently labeled NPs using confocal fluorescent microscopy. To further analyze the specific interactions with the ECM proteins and individual NPs, proteomic analysis revealed several highly abundant integrity-associated proteins that could account for the NP-matrix stabilization interactions including proteoglycans such as biglycan and fibromodulin, as well as collagen-binding proteins such as matrilins that are essential for tissue integrity.<br/><b>Significance</b>: Soluble GAG concentration was inversely correlated to particle cationic charge, suggesting an effect based on electrostatic interactions with the anionic ECM components. NPs are usually studied as carriers for drug delivery applications, yet their physicochemical effects on the ECM are rarely reported. This study aims to be the first to demonstrate that interactions between cationic NPs and ECM components such as anionic GAGs can result in cartilage tissue stabilization in the catalytic proinflammatory arthritic environment.<br/><br/>References:<br/>1. Allen K. D. N. C. Med. J. 2017, 78 (5), 306–309.<br/>2. He T. J. Control. Release 2020, 318, 109–123.<br/>3. von Mentzer U. Osteoarthr. Cartil. 2022, 30, 88-89.