The fate of ultrasmall fluorescent silica nanoparticles as drug delivery vehicles in cartilage explants: differential release kinetics between matrix and chondrocytes

Introduction: The effectiveness of intra-articular (IA) drug delivery approaches for osteoarthritis (OA) is hindered by the dense, avascular extra-cellular matrix (ECM) of cartilage and the rapid synovial fluid clearance time on the order of hours [1,2]. Nano- and micro-particle systems have been engineered to deliver therapeutic payloads, yielding modest improvements in cartilage penetration and joint residence time compared to free therapeutics [3,4,5]. However, little is known regarding the fate of these vehicles within joint tissues.
Ultrasmall (d¬h ~ 6 nm) fluorescent poly(ethylene glycol) coated core-shell silica nanoparticles (C’ Dots) have been used in multiple investigational new drug (IND) FDA-approved diagnostic and therapeutic human clinical trials in oncology [6,7]. Our work aims to further engineer this nanotechnology for applications in cartilage and OA. We have shown previously that C’ Dots penetrate the articular surface of healthy cartilage within 2 hours (Deff = 2 µm2/s) to reach tissue-resident chondrocytes in vitro [8]. Separately, we characterized the clearance of IA-injected C’ Dots in rat knees and observed joint retention for 3+ months after a single injection [9].
Few studies have reported on the role of tissue-resident chondrocytes in nanoparticle retention and therapeutic delivery. Understanding and leveraging these biological and transport phenomena may inform the design of OA therapeutic delivery systems. In this study, we utilized C’ Dots as an ultrabright and photostable fluorescent tracker to probe release kinetics in whole cartilage explants over 6 days. We hypothesized that C’ Dots would exhibit differential partitioning and release kinetics in chondrocytes and ECM in whole explants.

Materials and

Methods: C’ Dot Synthesis: Cyanine 5 fluorophore-encapsulating C’ Dots were synthesized following previously established methods [10,11]. Cellular Internalization: Cylindrical cartilage plugs (d = 6 mm, h = 2 mm, n = 3 per group) were cut from the patellofemoral groove of 1-3 day old bovine stifle joints and randomly assigned to C’ Dot (EXP) or Control (CTRL) groups. C’ dots in media (2 µM) passively diffused through the articular surface and radial edges of submerged cartilage plugs [12]. After 24 hours, plugs were moved to fresh media and washed once per day until imaging. Imaging: Cartilage plugs were bisected with a lubricated blade for staining (30 minutes Calcein, AM & Sytox Blue nucleic acid stain, 30 minutes lysosome stain, or 24 hours endosome transfection). Tissue penetration and cellular internalization of C’ Dots were assessed at the bisected plane using a Zeiss 710 inverted confocal microscope and the following excitation wavelengths: Sytox Blue (405 nm), Calcein, AM (461 nm), Endosome-GFP (488 nm), LysoView 540 (540 nm), tissue reflectance (561 nm), and Cyanine-5 (633 nm). Image Analysis: A custom MATLAB code created logical masks to compartmentalize each image into regions corresponding to cells and ECM. Sample viability was calculated from Calcein and Sytox Blue cell counts (samples with < 1000 total cells were excluded from further analysis). Statistical Analysis: Viabilities and Mean Pixel Intensities were compared using a linear mixed-effects model with Kenward-Roger approximation (α = 0.05).

Results, Conclusions, and Discussions: Fluorescent C’ Dot puncta were observed within live cell cytoplasm after 24 hours of incubation and remained detectable after 5 days of washing (Fig. 1A). C’ Dot signal was co-localized with endosomal and lysosomal fluorescent markers (arrows), but unlabeled C’ Dot-containing vesicular structures were also present (diameter ~ 0.5 – 4 µm) (Fig. 1B).
Area-normalized C’ Dot signal in the cell and ECM compartments trended towards decreasing with longer washing, with ~50% reduction observed from 0 d to 5 d (Fig. 1C (i), p = 0.79, p = 0.97, respectively). C’ Dot signal was highly heterogeneous within cells; this organization into subcellular vesicular structures contributed to the observed variation in cell pixel intensity. Vesicular C’ Dot signal decreased from 1d to 5d (p = 0.036) and was distinct from total cell and ECM compartments at 5 d (Fig. 1C (ii), p = 0.037, p = 0.014, respectively). Viabilities were not different between groups (Fig. 1C (i), p > 0.05).

C’ Dots were internalized within 24 hours by chondrocytes in 3D cartilage explants and remained detectable in cell, ECM, and vesicle compartments over 5 days of washing. While we expect that C’ Dots will be released from chondrocytes at later timepoints, these unexpected release kinetics offer insight into the role of chondrocyte dynamics in drug delivery. To our knowledge, this study is the first to quantify compartmental retention of nanoparticles for longer than 48 hours, and the first to use whole tissue explants to investigate chondrocyte processing dynamics with high spatial resolution.
The role of chondrocytes in processing injected particles may offer a previously unexplored mechanism for increasing tissue retention of OA therapeutics and site-specific delivery within cartilage, including in chondrocytes. Further investigation of complex tissue transport kinetics on the timescale of days to weeks is necessary to bridge the gap in timescale between in vitro studies (e.g. hours-days) and preclinical models (e.g. months-years). This study highlights the active role of chondrocytes in mediating nanoparticle presence in whole cartilage tissue and yields important insights into chondrocyte-mediated mechanisms to extend particle residence and specific targeting in joints.

Acknowledgements (Optional): This work was funded by the Cornell IGNITE program and the NSF GRFP and made use of the Cornell Biotechnology Resource Center which is supported through the NYSTEM CO29155 and NIH S10OD018516.

References
[1] DiDomenico+ Nat Rev Rheum 2018; [2] Doan+ Acta 2019; [3] He+ J Contr Rel 2020; [4] Pontes+ Front Bioeng Biotech 2023; [5] Aldrich+ ORS 2023; [6] Phillips+ Sci Transl Med 2014; [7] Zanoni JAMA 2021; [8] Fortin+ ORS 2023; [9] Fortin+ ORS 2024; [10] Ma+ Chem Mat 2015; [11] Ma+ Chem Mat 2017; [12] DiDomenico+ J Biomech 2017.