Neutralization of Pro-inflammatory Cytokines by Intra-articular Injection of Biofunctionalized Nanoparticles as an Advanced Treatment for Osteoarthritis

Statement of Purpose: Osteoarthritis (OA), a progressive multifactorial degenerative joint disease, affects millions of people worldwide. [1] Pro-inflammatory cytokines, especially tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), play a crucial role in inflammation and cartilage destruction. Since human monoclonal antibodies (Abs) prevent cytokines role in inflammation, biological therapies are promising treatments. [2] However, the therapeutic efficacy of the Abs is limited due to their short half-life, and present serious side effects when injected systemically. A solution to overcome this problem is using nanoparticles (NPs) as a substrate to deliver, protect and prolong the half-life of the Abs after the local delivery in the affected joint. Therefore, in this work, anti-TNF-α and anti-IL-6 Abs were immobilized at the surface of biodegradable polymeric NPs to selectively capture and neutralize those cytokines. Our system aims to extend and increase the Abs therapeutic efficacy, owing to the protection from degradation that the NPs provide, and to reduce the systemic side effects using intra-articular (IA) delivery.

Methods: Chitosan-hyaluronic acid NPs were prepared as previously described. [3] Different concentrations of anti-TNF-α and anti-IL-6 Abs (from 0 to 20 μg/mL) were assessed to determine the maximum Abs immobilization at the NPs surface. The in vitro co-culture system was established using human articular chondrocytes (hACs) and activated M1 macrophages. After 1, 3, 7 and 14 days of treatment (Abs or NPs+Abs), cell viability was assessed by Alamar blue and cell morphology by Scanning Electron Microscopy (SEM). Experimental monoarthritis was induced through an injection of carrageenan into the right knee joint of Wistar rats. The therapeutic effect was assessed by measuring clinical parameters (joint perimeter – edema), nociceptive behavior (mechanical allodynia - flexion/extension test, and mechanical hyperalgesia - pressure application measurement (PAM)) and histological analysis.

Results: The maximum Ab immobilization ability was 10 μg/mL for anti-TNF-α Ab and 15 μg/mL for anti-IL-6 Ab. After hACs co-culture with M1 macrophages there was a significant decrease in cell viability comparatively to the control. The biofunctionalized NPs with both Abs exhibit a prolonged action and stronger efficacy than free Abs, since there was a significant improvement in cell viability. Moreover, biofunctionalized NPs were able to preserve hACs morphology. The results from the in vivo experiment confirmed the safety profile of the NPs and biofunctionalized NPs after IA injection (Figure 1). Furthermore, inflammation of the synovial membrane decreased in NPs+Abs group. We also observed a higher efficacy of the biofunctionalized NPs in comparison with free Abs, since NPs+Abs group had less vocalizations in the flexion/extension test and higher values of limb withdrawal threshold in PAM (Figure 2). Therefore, biofunctionalized NPs significantly decreased mechanical allodynia and hyperalgesia, successfully reversing nociceptive impairments of arthritis.

Conclusions:  Biofunctionalized NPs after local delivery exhibited a safe profile, prolonged action and stronger efficacy than free antibodies, and consequently this approach can lead to a revolution in the standard of care of this debilitating disease.

References: [1] Roy K. Int J Nanomedicine. 2015;10:5407-20. [2] Chevalier X. Nat Rev Rheumatol. 2013;9:400-10. [3] Lima AC. ACS Appl Mater Interfaces. 2018;10:13839-13850.