Developing and testing novel glutamate receptor antagonist delivery systems for the treatment of post traumatic osteoarthritis

Egan, Benjamin 2020. Developing and testing novel glutamate receptor antagonist delivery systems for the treatment of post traumatic osteoarthritis. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Joint injury is a major risk factor towards developing osteoarthritis (OA) later in life. Post traumatic OA (PTOA) accounts for at least 12% of all OA cases, developing in 50–70% of patients with ACL ruptures or meniscal damage, 10–15 years after the injury. Current therapies do not prevent or cure OA and are limited to symptom management and end stage joint replacement. Synovial fluid (SF) glutamate concentrations increase in arthritis. NBQX and DNQX are AMPA/kainate glutamate receptor (GluR) antagonists developed as antiepileptic drugs. Previous work revealed intra-articular NBQX reduced knee swelling, cartilage destruction, synovial inflammation, gait abnormalities and bone erosions in rodent antigen induced arthritis and PTOA. This project aims to determine whether GluR antagonists can be incorporated into sustained release poly(lactic-co-glycolic) acid (PLGA) nanoparticles and thermoresponsive hydrogels and investigates GluR antagonists’ protective effects on inflammatory or mechanically driven pathways in bone in vitro. PLGA nanoparticles were developed within the nanoscale range and demonstrated sustained delivery of both NBQX and DNQX for up to 5 weeks in phosphate buffered saline (PBS). A freeze-drying methodology was developed that did not alter particle physical properties (size, poly-dispersal index) facilitating long term nanoparticle storage. PLGA nanoparticles did not elicit a cytotoxic response from primary human osteoblasts. Sustained GluR antagonist delivery was not detected in bovine SF and nanoparticle physical properties were altered when suspended in bovine SF with a decrease in particle size (5.1-fold, p<0.0001) and zeta potential (5700-fold, p<0.0001) detected vs PBS suspended nanoparticles. These findings indicated a potentially detrimental impact to particle stability in suspension media reflecting in vivo conditions. GluR antagonists were incorporated into thermoresponsive hydrogels for sustained subcutaneous delivery. Pluronic F-127 and Carbopol 934 based hydrogels were rheologically tested across a range of Pluronic F-127 concentrations (19-28% w/v). 22% and 25% w/v Pluronic F-127 hydrogels had appropriate gelation temperatures and viscosity characteristics for gelation upon subcutaneous injection. DNQX delivery was sustained over 27hrs through a semi permeable membrane into PBS and over 24hrs through extracted bovine synovial membranes from 25% w/v hydrogels. Hydrogels reduced primary human osteoblast viability by 8-fold vs controls at 1:1 volumetric ratio hydrogel:media but not at 1:8 hydrogel:media (7 days exposure). The effect of AMPA/KA GluR antagonist delivery was investigated in osteoblasts. Both NBQX and DNQX reduced osteoblast mineralisation 1.8-fold and 1.2-fold (p=0.004) at 200μM concentrations in 2D human primary osteoblast and human Y201 MSCs differentiated to osteoblasts. Human Y201s, were embedded in type I collagen and osteogenically differentiated into dendritic, osteocyte-like cells (expressing bone markers OPG, OCN, ALP and COL-1A1) and either stimulated with IL-6 and sIL-6r, or mechanically loaded, to mimic inflammatory and loading effects on osteocytes relevant to joint degeneration. The effects of either unencapsulated NBQX or unencapsulated NBQX combined with sustained release NBQX loaded PLGA nanoparticles was determined. IL-6/sIL-6r treatment increased nitrite levels indicating oxidative stress. Multivariate analysis identified a decrease following IL-6/sIL-6r treatment on bone turnover (opg 1.9-fold, alp 2.0-fold), inflammatory markers (il-6 2.5-fold) and glutamate signalling machinery (gria-1 3.1-fold) vs controls. Short term NBQX exposure decreased glutamate release vs controls (1.2-fold, p=0.030). alp expression was significantly reduced at 24hrs following IL-6 exposure and corrected by short term NBQX treatment. Antiinflammatory cytokines (IL-4, IL-10, IL-13) release was upregulated by short term NBQX treatment at 24hrs. Nanoparticle treated cells did not differ to IL-6/sIL-6r treated cells except that they increased glutamate and IL- 6 release following extended exposure (72hrs). These observations indicate limited therapeutic benefit of sustained release NBQX encapsulated in nanoparticles which were associated with detrimental effects of raised lactate dehydrogenase and nitrite. These findings supported cell viability assays (MTS) indicating empty nanoparticles at high concentrations (100mg/ml) increased MTS release vs controls across all timepoints analysed (1, 3 and 7 days) by 2.6- (p=0.012), 3.9- (p<0.0001) and 2.9-fold (p=0.048) respectively. NBQX treatment after pathophysiological loading (5000μstrain) modulates the mRNA expression of bone turnover (opg 1.43-fold decrease; alp, 3.1-fold decrease (p<0.0001)), inflammation (il-6, 1.4-fold decrease, p=0.033) and glutamate signalling markers (gria-1, 3-fold decrease, p=0.015). NBQX treatment elicited a repressive effect on all inflammatory biomarkers analysed, as well as on glutamate and OPG release across all time points tested. Two GluR antagonist sustained release delivery vehicles were developed with differing release properties. PLGA nanoparticles sustained release of low (49μg/ml from 20mM NBQX loaded freeze dried nanoparticles) GluR antagonist loads over 3-5 weeks whereas Pluronic-F-127/Carbopol-934 hydrogels sustained release over 27hrs of high (0.74mg/ml from 2.5mM DNQX loaded hydrogels) concentrations. This study also provides evidence of the capacity of NBQX to protect against changes to bone turnover and induce the release of anti-inflammatory cytokines following a pathological inflammatory stimulus and to decrease the expression of bone turnover markers and inflammation following pathological loading.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General) R Medicine > R Medicine (General)
Date of First Compliant Deposit:	4 October 2021
Last Modified:	03 Aug 2022 01:40
URI:	https://orca.cardiff.ac.uk/id/eprint/144634

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