Multiscale characterization of ultrasmall fluorescent core-shell silica nanoparticles in cartilage and synovial joints reveals rapid cartilage penetration and sustained joint residence

Abstract

Development of non-surgical disease-modifying interventions for knee osteoarthritis (OA) remains a persistent challenge despite decades of efforts. Therapeutic transport to cartilage in synovial joints is hindered by the dense, negatively charged cartilage matrix, and further challenged by rapid synovial fluid clearance within hours to days. In this study, we investigated ultrasmall (d_h ∼ 6 nm) fluorescent core-shell silica nanoparticles (Cornell Prime Dots, or C' Dots), which have received FDA-investigational new drug approval for multiple human clinical trials in oncology, as cartilage-penetrating delivery vehicles for applications in knee OA. Across multiple length and time scales, we examined the relationship between C' Dot tissue and cellular transport kinetics and whole joint clearance. In vitro, C' Dots penetrated cartilage explants within 30 min (D ∼ 2 µm²/s). C' Dots were internalized by chondrocytes within 24 h and were retained in vesicular structures for up to 5 days. In vivo, C' Dot clearance following intra-articular knee injection was well described by two distinct time constants (τ₁ ∼ 18 hours, τ₂ ∼ 3 weeks), consistent with mechanisms of synovial- and tissue-mediated clearance. C' Dot clearance rates were not affected by surgically-induced cruciate ligament transection. Notably, C' Dots remained in the knee longer than 3 months after a single injection and were localized to cartilage, meniscus, ligaments, and synovium. Collectively, these results illustrate the potential of C' Dots for long-term delivery of conjugated therapeutics in the knee. STATEMENT OF SIGNIFICANCE: This research explores a cartilage-penetrating platform nanotechnology for applications in drug delivery for arthritis. The properties inherent to this particle system enabled rapid tissue penetration, chondrocyte internalization and retention, and persistence in rat knees for longer than 3 months after a single injection. The study demonstrates that ultrasmall nanoparticle delivery platforms can use tissue localization to partially avoid clearance by the synovium, while simultaneously enabling chondrocyte targeting. When paired with a therapeutic, C' Dots may be a versatile platform in early-stage OA and PTOA to protect cartilage from further degeneration. These findings inform future design and engineering of biocompatible drug delivery vehicles for other applications where access to dense tissues is needed.

Keywords: Cartilage; Injection; Intra-articular; Osteoarthritis; Pharmacokinetics; Silica nanoparticles.

Fig. 1.. Schematic of C’ Dot molecular structure, in vitro cartilage explant studies, and in vivo whole joint pharmacokinetics study.

(A) Molecular rendering of a C’ Dot [58]. (i) 1–2 Cyanine-5 (Cy5) fluorophores were covalently encapsulated in the silica core, which was (ii) coated with a dense brush layer of covalently attached PEG oligomers. (B) Patellofemoral groove cartilage explants were dissected from neonatal bovine knee joints for use in (i) diffusion experiments and (ii) chondrocyte internalization and retention experiments. (C) Healthy (naïve) and surgically injured (ACLT or sham) rat knees received a single injection of C’ Dots and were imaged longitudinally to characterize whole joint pharmacokinetics of C’ Dots over 8 weeks. Images were partially created with Biorender.

Fig. 2.. C’ Dots rapidly penetrated the articular surface of healthy cartilage explants.

(A) C’ Dot penetration profiles were captured in the center (v) of prepared cylindrical explants. (B) C’ Dots were observed up to 0.5 mm in depth from the articular surface in merged confocal fluorescence and reflectance micrographs. Scale bar = 200 μm. (C) C’ Dot fluorescence profiles acted as a surrogate for C’ Dot concentration in our system. (D) Diffusion coefficients were produced from Fickian 1D transport analysis of the penetration profiles from 100–400 μm in depth. (E) Coefficients, D, were consistent between 1 h and 2 h observations, indicating that our model captured this transient process (n = 5–6). Shared letters denote no significant difference between groups (α = 0.05).

Fig. 3.. C’ Dots were internalized by native chondrocytes in cartilage explants and were retained intracellularly for 5 days.

(A) (left) C’ Dots were robustly internalized by chondrocytes in explants (arrows; green – Calcein, AM, blue – Sytox Blue, red – C’ Dots; z-stack spans 10 μm) and (right) were retained intracellularly for 5 days of washing (n = 3). (B) More C’ Dot fluorescence signal was detected in ECM than in chondrocytes. C’ Dot signal decreased rapidly over the first day of washing. (C) From area-normalized data analysis (e.g. concentration), C’ Dot fluorescence was higher in chondrocytes than in the ECM. (D) C’ Dot signal in chondrocytes was heterogeneous and localized to vesicular structures. (E) C’ Dot fluorescence within vesicular structures (d = 0.6 μm) decreased during washing. (F) C’ Dots localized to fluorescently labeled endosomes and lysosomes (arrows) and were also detected in unlabeled vesicular structures after incubation (0 d) and after washing (5 d). Shared letters denote no significant difference between groups (α = 0.05).

Fig. 4.. C’ Dot fluorescence signal was detectable up to 8 weeks after a single intra-articular injection. C’ Dots cleared from the knee via two time-distinct physiological processes independent of surgical injury.

(A) C’ Dots were detected in intact joints using the IVIS system up to 8 weeks post-injection demonstrating long-term joint retention. (B) Clearance of Cy5 dye was well-described by a single-exponential decay, but two time constants were necessary to describe clearance of C’ Dots (model fits = dashed lines). C’ Dots persisted in the joint up to 8 weeks post-injection. (C) From the amplitudes of the fits, the majority of injected C’ Dots were cleared via a fast process, while ~1/3 cleared via a slow process, and 5–10 % of the initial signal persisted in the joint. (D) Synovial clearance of C’ Dots was significantly slower than clearance of Cy5 dye. Synovium- and tissue-mediated clearance rates of C’ Dots were independent of surgical injury. Shared letters denote no statistical difference between groups (α = 0.05).