PEG-B-PLA-adenosine prevents osteoarthritis progression

Abstract

Purpose: In a previous study we demonstrated that adenosine plays an important role in maintaining homeostasis in the joint. Adenosine has a short half-life (1-4 seconds in whole blood) limiting its therapeutic potential. Previously we showed that intrarticular injection of adenosine in a liposomal formulation prevents and reverses osteoarthritis (OA) progression in a post-traumatic OA (PTOA) animal model. Here we adopt a different strategy in prolonging the therapeutic half-life of adenosine by conjugating adenosine to the biodegradable carrier poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles. Methods: Six targeted polymeric (PEG-b-PLA) nanoparticles were synthetized by binding PLA-b-PEG400-N3 and PLA-b-PEG2000 block copolymers to adenosine at the 3’,4’-OH, 5’-OH, and 6-NH2 positions with an acetylene group. The efficacy of the new molecules to stimulate cAMP production was tested in RAW264.7 murine macrophage cells. PTOA was induced in Sprague Dawley rats (n=3 for each group) following non-surgical rupture of anterior cruciate ligament (ACL). Rats were treated with intra-articular injections of 100 ul of a suspension containing PLA-PEG2000-3,4-OH-adenosine (Nano-Ade; 1 mg/kg), or PLA-PEG2000 nanoparticles (Nano) or with saline for 6 weeks. The animals received the first injection 7 days after ACL rupture and injections were performed every 10 days. Knee swelling in the rats was measured before every injection. At the end of the experiment rats were sacrificed and both legs were collected for immunohistochemistry and microcomputed tomography (μCT) analysis. The capacity of the various nanoparticle preparations to bind to and activate adenosine receptors was studied as the capacity to stimulate cAMP accumulation and inhibit IL-1-stimulated IL-6, MMP13, col10a1 and NFkB translocation to the nucleus in primary murine chondrocytes. Results: Only adenosine-conjugated nanoparticles in which PEG2000 was bound to adenosine on the 3’,4’ hydroxyl groups stimulated cAMP increase (163% vs control; p<0.05) and this increase were blocked by selective antagonists of both adenosine A2A and A2B receptors. Intra-articular injection of the Nano-Ade in PTOA rats diminished swelling in affected knees (p<0.001 vs Nano). We observed that Nano-Ade diminished the IL-1β-stimulated expression of IL-6 mRNA expression (from 214±107 to 79±89 vs Control group), an effect that was partially reversed by A2AR (ZM241385 and SCH58261, 1μM each) and A2BR (PSB1115) antagonists. In contrast, the Nano-Ade diminished IL-1β-stimulated MMP13 and Collagen-10 mRNA and protein expression via stimulation of A2BR since the effect was reversed by the selective A2B antagonist but not by the A2AR-selective antagonist. Nano-Ade also diminished IL-1β stimulated activation of NF-kB, a central intracellular signal for inflammation (67% decrease vs IL-1β treated cells; p<0.001). In rats treated with Nano-Ade there was markedly reduced fibrillation of the cartilage surface (H&E staining) and less proteoglycan loss (Safranin-O staining) resulting in a significantly decreased OARSI score (Nano=2.99±1.20, Nano-Ade=0.88±0.69; p<0.03 vs Nano). Analysis of μCT scanned images showed an increase in cartilage volume in Nano-Ade treated rats (143% vs Nano; p=0.023). We also found that Nano-Ade reduced RNA expression for MMP-13 (83% reduction compare to IL-1β stimulated cells; p<0.001) and Collagen-10 (81% reduction compare to IL-1β stimulated cells; p<0.01) in primary murine chondrocytes stimulated with Il-1. Conclusions: Taken together these results demonstrate that the adenosine-functionalized particles bind to and activate adenosine A2A and A2B receptors on murine cells. Activation of adenosine receptors by PLA-PEG2000-3,4-OH-adenosine prevents OA progression and reduce pro-inflammatory mediators in murine chondrocytes.