Poster AA3 - Multiscale In Situ Imaging Of Collagen-Targeting Biomimetic Nanovesicles For Drug Delivery To Post-Traumatic Osteoarthritic Joints

Introduction: Post-traumatic osteoarthritis (PTOA) is characterized by the progressive degeneration of articular cartilage 1. Current clinical treatment of PTOA relies on analgesics and NSAIDs to provide symptomatic relief, not structural or functional disease modification 2. However, any systemic PTOA treatment carries significant concerns over drug side effects and off-target interactions. To enhance the therapeutic efficacy of disease-modifying PTOA drugs, and reduce systemic drug exposure and unwanted side effects, strategies based on intra-articular (i.a.) injection are utilized. However, these also have challenges, including low joint retention time and poor targeting to cartilage’s dense ECM, typically necessitating repeated high-concentration i.a. injections 3. A need exists for drug delivery platforms that localize and retain disease-modifying drugs within the joint space. Recent work from our team has shown that short synthetic peptides, based on elastin- and collagen-like sequences (ELPs & CLPs, respectively), can be conjugated to enable the formation of novel nanovesicles having a high binding affinity for tissues exhibiting collagen damage—thus actively targeting diseased tissues; and sequence-specific behaviors—that allow tuning of drug loading efficiency and passive drug release, as well as thermoresponsive disassembly due to mild cooling (Fig 1A) 4,5. However, the retention, distribution, and targeting of these elastin-collagen nanovesicles (ECnVs) (and their cargo) in situ has yet to be established. In this study, we leveraged multiscale bioimaging approaches (e.g., in vivo, epi-fluorescent, confocal, and super-resolution imaging) to characterize the retention and localization of fluorescently-labeled ECnV (far-red) and their fluorescent cargo (near-IR), within intra- & peri-articular joint tissues following i.a. injection.

Materials and

Methods: Our team synthesized ELP (W6) and CLP (G8) monomers, based upon the sequences (VPGWG)6G and (GPO)8GC, respectively, using standard solid-phase peptide synthesis. 30% of the G8 CLPs were modified to allow fluorescent labeling via an EDC/sulfo-NHS reaction. W6 was conjugated to G8 using copper-catalyzed click chemistry to yield ~1mg/ml W6-G8 conjugates, which self-assemble into (empty-) ECnVs. These ECnVs were then surface-labelled with AZ647-NHS, a far-red fluorophore, to allow their visualization in tissues and organs in vivo/in situ. Particle size (~200nm diameter) and concentration (~1011 per ml) were determined via nanoparticle tracking analysis (NTA). Confocal and super-resolution imaging of ECnVs and their localization and interactions with target tissues necessitates their survival through optical tissue clearing and processing, which involves tissue fixation, clearing, and paraffin embedding. Initially, ECnVs were introduced into intact rat muscle tissue (ex vivo), fixed, and optically cleared (THF-DBE, TDE)6 for ex vivo multiscale imaging. After confirming ECnV stability to tissue processing, AZ647-labeled (empty-) ECnVs were i.a. injected (6uL) into murine joints for macro-scale in vivo imaging (UVP iBox Scientia) and subsequent multiscale 3D confocal imaging of intact lower limbs following THF-DBE or PEGASOS7 clearing (Andor Dragonfly and Zeiss CD7). Intact joints can then be de-cleared and processed for decalcified paraffin embedding for histology and super-resolution imaging.

Results, Conclusions, and Discussions: AZ647-labeled (empty-) ECnVs were successfully synthesized and shown to be robust to temperature-controlled handling, fixation, clearing, and tissue processing conditions. Ex vivo ECnV injection into rat muscle tissue (vastus lateralis) substantiated their in situ stability to tissue clearing, permitting the facile far-red fluorescent confocal visualization of ECnVs in situ at tissue depths exceeding 500µm (to the utilized objective limit; Fig 1F). Following i.a. injection into murine knees, robust ECnV fluorescence was observed—at the macroscale—via in vivo imaging (Fig 1B) and at the micro/nano-scale via confocal microscopy after fixation and clearing (Fig 1C-D). In intact joints, deep-tissue imaging in THF-DBE cleared joints (R.I. 1.56) (Fig 1E) was superior to that of PEGASOS (R.I. 1.54). ECnV localization, clearance, drug delivery, and biosafety will next be assessed after i.a. injection of (empty-)ECnVs vs. (loaded-)ECnVs (e.g. Cy7.5- or Dex-CF) to mouse knees. Overall, these results confirm our ability to directly characterize ECnV presence in situ across several translationally relevant length scales in murine musculoskeletal tissues.
In this study, multiscale bioimaging was used to characterize fluorescently-labeled AZ647-ECnVs within murine joint and muscle tissues across size scales from millimeter to nanometer. Our data demonstrates that our novel ECnV particles survive the handling & imaging necessary for such interrogations. Such multiscale analyses greatly improve our ability to study clinically relevant drug delivery characteristics using smaller sample sizes. Future work will involve i.a. administration of empty- and cargo-laden- ECnVs to both healthy and injured (early PTOA) murine joints, followed by multiscale bioimaging, to comprehensively analyze nanovesicle retention and distribution in and around the joint, as well as to establish the biosafety and efficacy of ECnVs as a targeted and controllable delivery platform for PTOA therapeutics.

Acknowledgements (Optional): 1) Little, Nature Reviews Rheumatol. (2013) 2. Dingle, Z Rheumatol (1999) 3. Karsdal, Osteoarthritis Cartilage (2016) 4. Luo, Biomacromolecules (2017) 5. Dunshee, J Drug Deliv Sci Technol (2022) 6. Berke, PLoS One (2016) 7. Jing, Cell Res (2018)