Charge based intra-cartilage delivery of single dose dexamethasone using Avidin nano-carriers suppresses cytokine-induced catabolism long term

Abstract

Objective Avidin exhibits ideal characteristics for targeted intra-cartilage drug delivery: its small size and optimal positive charge enable rapid penetration through full-thickness cartilage and electrostatic binding interactions that give long half-lives in-vivo. Here we conjugated Avidin with dexamethasone (DEX) and tested the hypothesis that single-dose Avidin-delivered DEX can ameliorate catabolic effects in cytokine-challenged cartilage relevant to post-traumatic OA. Methods Avidin was covalently conjugated with DEX using fast (ester) and slow, pH-sensitive release (hydrazone) linkers. DEX release kinetics from these conjugates was characterized using 3H-DEX-Avidin (scintillation counting). Cartilage explants treated with IL-1α were cultured with or without Avidin-DEX conjugates and compared to soluble DEX. Sulfated-glycosaminoglycan (sGAG) loss and biosynthesis rates were measured using DMMB assay and 35S-incorporation, respectively. Chondrocyte viability was measured using fluorescence staining. Results Ester linker released DEX from Avidin significantly faster than hydrazone under physiological buffer conditions. Single dose Avidin-DEX suppressed cytokine-induced sGAG loss over 3-weeks, rescued IL-1α-induced cell death, and restored sGAG synthesis levels without causing cytotoxicity. The two Avidin-DEX conjugates in 1:1 combination (fast:slow) had the most prominent bioactivity compared to single dose soluble-DEX, which had a shorter-lived effect and thus needed continuous replenishment throughout the culture period to ameliorate catabolic effects. Conclusion Intra-cartilage drug delivery remains inadequate as drugs rapidly clear from the joint, requiring multiple injections or sustained release of high doses in synovial fluid. A single dose of Avidin-conjugated drug enables rapid uptake and sustained delivery inside cartilage at low intratissue doses, and potentially can minimize unwanted drug exposure to other joint tissues.

Development of in vitro joint tissue co-culture models for evaluating transport and biological crosstalk

“…Frequent injections are painful, costly and inconvenient to the patients and also cause local joint and systemic toxicity. It is critical to develop therapies that can target the affected tissue inside the knee joint, bind there and provide a sustained drug release over several weeks following their intra-articular administration [37]. In vitro, the difference between administering a single injection of a conjugated and unconjugated drug can be modeled.…”

“…For example, recently it was shown that the effect of, dexamethasone (DEX) was time dependent and short lived in rescuing the IL-1α induced catabolic activity in vitro [37]. By 12 days of culture, the effect of single dose DEX had tapered off in suppressing the loss of sulfated glycosaminoglycans (sGAG) compared to the continuous dose DEX condition [37].…”

“…By 12 days of culture, the effect of single dose DEX had tapered off in suppressing the loss of sulfated glycosaminoglycans (sGAG) compared to the continuous dose DEX condition [37]. The single dose of DEX mimics a single IA injection which is then followed by drug clearance, similar to in vivo.…”

“…The single dose of DEX mimics a single IA injection which is then followed by drug clearance, similar to in vivo. The continuous dose of DEX mimics a longer in vivo retention time and shows that a single IA injection would be ineffective, thus proving the need for the development of a novel drug delivery system designed to increase retention time [37]. The effects of dosing various therapeutics in a single and continuous pattern will be further explored in Chapter 3.…”

“…The single versus continuous dose has previously been modeled using dexamethasone (DEX), a corticosteroid often used in the treatment of arthritic diseases due to its anti-inflammatory and immunosuppressant effects [37,68]. Bovine cartilage explants were given 1 ng/mL IL-1α and dosed with 100 nM DEX in either a single or continuous pattern [37].…”