Extended intra-articular presence of fluorescent C’ Dot nanoparticles in naïve and arthritic rat knee joints

Introduction:: Intra-articular (IA) drug delivery approaches for osteoarthritis (OA) are hindered by the dense, avascular cartilage matrix and rapid synovial fluid clearance time on the order of hours. Both challenges prevent therapeutics from reaching the tissue-resident chondrocytes that mediate disease progression. For example, small molecule therapeutics for rheumatoid arthritis, such as anakinra (Kineret) and adalimumab (Humira), are cleared from the joint within 1-3 days and are not effective for OA [1]. Recent studies have extended residence time by 2-4 weeks using nanomaterials; however, standard clinical recommendations limit the frequency of IA injections to 3-6 months [2].

In previous work, we have shown that ultrasmall fluorescent silica poly(ethylene glycol) coated nanoparticles (C’ dots) penetrate the articular surface of healthy cartilage to reach tissue-resident chondrocytes in vitro. C’ dots have versatile surface chemistry for conjugation of multiple different functional groups. Additionally, C’ dots are used in FDA-approved human oncology clinical trials and show favorable biodistribution and pharmacokinetics due to their small size, which enables renal clearance [3]. Given this versatile functionality and safety profile, the C’ dots are a promising platform for IA therapeutic delivery. In this study, we employ a rat surgical model of post-traumatic OA (PTOA) to investigate joint residence times of untargeted C’ dots in naïve and arthritic stifle joints over 8 weeks. Preliminary findings indicate that C’ dots are present in naïve stifles at 14 weeks, suggesting great potential utility as a vehicle for the delivery of OA therapeutics.


Materials and Methods:: Cyanine-5 fluorescent C’ dots were synthesized following previously established methods [4,5]. Adult male Sprague Dawley rats (n=18, 300-325g) were randomly assigned to: unoperated with intra-articular injections of (1) bilateral Cy5 (n=2), (2) unilateral C’ dot with contralateral saline (n=4), (3) bilateral C’ dot (n=4), and (4) animals receiving ipsilateral anterior cruciate ligament transection (ACLT) with contralateral sham surgery and bilateral C’ dot injection one-week postoperatively (n=6). 25 µl of saline, 10 mM Cy5 in saline or 34 µM C’ dots in saline were injected via a parapatellar approach. Fluorescence imaging: Total joint fluorescence was captured longitudinally over 8 weeks using the In Vivo Imaging System (IVIS) Kinetic. IVIS imaging parameters were identical for all images. Total Radiant Efficiency (TRE) was quantified in manually aligned region-of-interest boundaries (ROIs) of identical dimensions. The signals obtained from saline injected knees were averaged and this value was subtracted as background. TRE was then normalized to the peak signal in each stifle for clearance analysis. Analysis: Models of mono-exponential of bi-exponential decay were fit to normalized TRE using one time constant for Cy5 injections or two time constants for C’ dot injections, respectively. Statistics: Time constants for each group were statistically compared using linear mixed-effects model with Kenward-Roger approximation (p < 0.05).


Results, Conclusions, and Discussions:: Cy5 fluorophore and C’ dot fluorescence were detected within the joint ROI and these signals decreased over time (Figure 1A). Interestingly, C’ dot fluorescence was visible in opened joints from animals sacrificed at 14 weeks post-injection (Figure 1B-C). Furthermore, C’ dots appear to have localized to ligaments, synovium, and menisci. This observation suggests the potential of even untargeted C’ dots for extended delivery of therapeutics.

Clearance of C’ dots from the joint and surrounding tissues is much slower than clearance of free Cy5 (half-lives 8 h and 0.4 h, respectively) (Figure 2A). Cy5 behavior was well described with a mono-exponential decay (one time constant), while C’ dot clearance was better represented with a bi-exponential decay (two time constants) (Figure 2A-C). There was no statistical difference between unilateral and bilateral injections of C’ dots (half-lives 8 h and 2 weeks, p > 0.05) (Figure 2 B-C). The shorter time constant describing C’ dot clearance is comparable to half-lives of similarly sized solutes [6]. The longer time constant, however, may be describing rate-limited release of C’ dots that initially diffused into tissues such as articular cartilage, meniscus, and ligaments.

C’ dots injected in PTOA and sham stifles exhibit similar behavior to naïve stifles (Figure 3A). Total joint clearance is also well modeled with a bi-exponential (two time constants), trending towards slower clearance than in naïve stifles. ACLT and sham-operated limbs exhibit no difference in clearance between conditions (p > 0.05) (Figure 3B-C). It is known that clearance of injected therapeutics from the joint is dependent on physicochemical properties and disease state, consistent with our observations [7]. The dramatically slowed clearance of C’ dots from PTOA stifles is promising for therapeutic delivery in joints with mild to moderate OA.

We employed an in vivo surgical model of PTOA to characterize total joint clearance of fluorescent C’ dots from naïve, sham, and PTOA stifles. C’ dot clearance is well modeled by a bi-exponential decay, indicating two distinct time-dependent physiological processes. These results and the versatility and safety of this technology illustrate the potential of C’ dots for extended therapeutic delivery in the knee.


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References (Optional): : [1] Doan+ Acta., 2019; [2] Ayhan+ World J Orthop., 2014; [3] Phillips+ Sci Trans Med., 2014; [4] Ma+ Chem. Mat. 2015; [5] Ma+ Chem Mat. 2017; [6] Brown+ Acta., 2019; [7] Mwangi+ J Contr Rel., 2018