Avidin as a Carrier for Drug Delivery into Cartilage: Electrostatic Interactions Enable Rapid Penetration, Enhanced Uptake, and Retention in Rat Knee Joints Ambika Goel Bajpayee 1, Alfredo M. Scheu 2, Ryan M. Porter 2, Alan J. Grodzinsky, PhD 1. 1 MIT, Cambridge, MA, USA, 2 BIDMC, Harvard Medical School, Boston, MA, USA. Disclosures: A.G. Bajpayee: None. A.M. Scheu: None. R.M. Porter: None. A.J. Grodzinsky: None. Introduction: Local drug delivery into cartilage remains a challenge due to its dense extracellular matrix of proteoglycans enmeshed within a collagen fibril network. We previously showed that solutes with hydrodynamic diameter 10 nm can penetrate through the full thickness of cartilage explants while larger sized solutes were trapped in the superficial zone [1]. The high negative fixed charge density of cartilage offers the opportunity to utilize electrostatic interactions to augment transport, binding, and retention of drug carriers. We used a 66 kda (7nm diam), highly positively charged protein, Avidin (Av) as a model for charge driven transport and showed that Av penetrated through the deep zone of bovine cartilage within 24h, it had a significantly higher uptake than its neutral counterpart, NeutrAvidin (Nu), and was retained inside the cartilage for over 2 weeks. With the goal of using Av as a drug delivery carrier, we injected it into healthy rat knee joints to investigate its kinetics, distribution, and retention in vivo over 7 days. Av penetrated throughout the rat cartilage within a short time of 6h, and 10% of the absorbed amount at 24h was retained after 7 days. Methods: In vitro bovine cartilage studies: Cartilage disks (3mm diam, 1mm thick) were harvested from the femoropatellar grooves of 1-2 week old bovine calf knee joints. The disks were incubated for specific times in 300 µl of known concentration (3µM) of FITC-Av and FITC-Nu, supplemented with protease inhibitors at 37 deg C in a 96 well plate format. After removal from the absorption baths, the disks were rinsed, gently wiped and then incubated in 1X or 10X PBS supplemented with protease inhibitors for 24 h or longer as specified. The fluorescence signal in the absorption and desorption baths was quantified using a plate reader; the solute content inside the cartilage disk was determined from the difference between the fluorescence reading of the absorption/desorption baths before and after incubation. The uptake ratio (Fig 1) was calculated as the concentration of the FITC-solute in the cartilage (per intra-tissue water weight) normalized to the concentration of FITC-solute in the equilibration bath. N=6 disks, n=3 animals. In vivo Rat Studies: Knee joints of healthy Fisher rats were injected with 50 μl of 75μM Av conjugated with Texas Red. Following intra-articular (IA) injection, rat knees were flexed and extended to distribute the injected Av throughout the intra-articular space. Contralateral knees were used as controls. Rats were sacrificed at different time points (6h, 1d, 4d, 7d) and 4 tissue types were extracted: Cartilage (from patellar groove, femoral condyle and tibial plateau), Meniscus, Ligament (ACL & PCL) and Patellar Tendon. 6h tissue samples were imaged using a confocal microscope at 10X magnification by taking Z stacks (Fig 2). Samples from all time points were then desorbed in 10X PBS for 48h in 37 deg incubator to disrupt electrostatic interactions and release Av into the bath. Av concentration was determined by using a plate reader and normalized by dry weight of tissue (Fig 3). As a control experiment, free dye (FITC ~400 Da) was injected into rat joints and its uptake in different tissue types was measured after sacrifice at 24h. Data are presented as Mean +/- SD; N=2 joints per condition. Results: Av penetrated throughout the full thickness of bovine cartilage explants within 24h while Nu did not. Fig 1A-B revealed 400 times higher uptake for Av than Nu in normal bovine cartilage. >90% Av retained inside cartilage for at least 15 days. Desorption in 10X PBS reduced Av retention to 50% of the absorbed amount in 24h, confirming electrostatic interactions (Fig 1C). In vivo rat studies suggested that Av penetrated throughout the deep zones of rat cartilage within 6h (RED, Fig 2). Fig 3 shows retention of Av in different tissue types of rat joints over 7 days; Av concentration in cartilage reduced 10 times from 24h to 7 days. Av concentration at 6 h scaled similarly with the sgag content (by DMMB assay) of each tissue type (Fig 4). The contralateral control knee showed no fluorescence in all tissue types. Similarly, cartilage from the rat joint that was injected with free dye showed no fluorescence after 24h. Discussion: The results show that Av penetrated rapidly through the full thickness of rat cartilage within 6h after IA injection. By 24h, Av concentration in rat cartilage (in vivo) was ~5 times lower than that in bovine (in vitro). This may be explained by the lower sgag concentration in rat cartilage (measured to be 3.5 times lower) than that in bovine cartilage. Av was present inside the tissue until at least 7 days, but a significant drop in its retention was observed compared to day 1. We reported >90% retention of Av in bovine cartilage (in vitro) for at least 15 days. A drop in retention is expected because of the absence of convective transport in the in vitro models. However, the 10% retention at 7 day time points in rat studies is most likely due to 10 times lower thickness of rat cartilage as compared to bovine. Since the diffusion-binding time constant scales as square of thickness, the transport rate in rat cartilage is expected to be 100 times faster, explaining the shorter retention duration. Our in vivo data show that the electrostatic interactions between the highly positively charged Av and the negatively charged groups in cartilage augmented its transport rate due to Donnan partitioning, enabling distribution throughout the joint and penetration deep into the tissues before being cleared by the lymphatic system. This suggests that Av or a similar structure, due to its ideal
size and a high positive charge, can provide a system for local delivery of drugs into cartilage. We have previously shown that small doses of Dexamethasone (~100nM) are effective in treating early stage cartilage degradation relevant to PTOA [2]. Due to its low MW (~400Da), DEX is known to be cleared from the joint in ~2 hours [3]. We demonstrated this by injecting a free dye of similar MW into the rat joints. By 24h, no traces of dye were found in the rat tissues. A vehicle like Avidin, however, can transport such drugs into cartilage at a fast rate, bind with the negatively charged groups in the tissue thereby creating a drug depot inside the tissue. Additionally, Avidin s high uptake in ligaments suggests that it can also be used for delivering proanabolic growth factors to injured ligaments. Ongoing in-vivo studies focus on increasing the sample size for the current work and also on testing Av functionalized DEX in a PTOA animal model. Significance: Our in vivo rat data suggests that electrostatic interactions can be utilized for augmenting transport and increasing retention of small MW drugs like DEX inside cartilage; Avidin exemplifies an ideal structure (size and a high positive charge) that can be conjugated with DEX and enable such drug delivery. Acknowledgments: This work is supported by the MIT Deshpande Center and NIH. References: [1] Bajpayee+, ORS 2013; [2] Lu+, Arth Res Ther 13(5):R142, 1-15, 2011, [3] Larsen+, J Pharm Sci 97:4622-54,
ORS 2014 Annual Meeting Poster No: 0310