Saposin C Coupled Lipid Nanovesicles Specifically Target Arthritic Mouse Joints for Optical Imaging of Disease Severity

Abstract

Rheumatoid arthritis is a chronic inflammatory disease affecting approximately 1% of the population and is characterized by cartilage and bone destruction ultimately leading to loss of joint function. Early detection and intervention of disease provides the best hope for successful treatment and preservation of joint mobility and function. Reliable and non-invasive techniques that accurately measure arthritic disease onset and progression are lacking. We recently developed a novel agent, SapC-DOPS, which is composed of the membrane-associated lysosomal protein saposin C (SapC) incorporated into 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) lipid nanovesicles. SapC-DOPS has a high fusogenic affinity for phosphatidylserine-enriched microdomains on surfaces of target cell membranes. Incorporation of a far-red fluorophore, CellVue Maroon (CVM), into the nanovesicles allows for in vivo non-invasive visualization of the agent in targeted tissue. Given that phosphatidylserine is present only on the inner leaflet of healthy plasma membranes but is “flipped” to the outer leaflet upon cell damage, we hypothesized that SapC-DOPS would target tissue damage associated with inflammatory arthritis due to local surface-exposure of phosphatidylserine. Optical imaging with SapC-DOPS-CVM in two distinct models of arthritis, serum-transfer arthritis (e.g., K/BxN) and collagen-induced arthritis (CIA) revealed robust SapC-DOPS-CVM specific localization to arthritic paws and joints in live animals. Importantly, intensity of localized fluorescent signal correlated with macroscopic arthritic disease severity and increased with disease progression. Flow cytometry of cells extracted from arthritic joints demonstrated that SapC-DOPS-CVM localized to an average of 7–8% of total joint cells and primarily to CD11b+Gr-1+ cells. Results from the current studies strongly support the application of SapC-DOPS-CVM for advanced clinical and research applications including: detecting early arthritis onset, assessing disease progression real-time in live subjects, and providing novel information regarding cell types that may mediate arthritis progression within joints.

<em>In Vivo</em> Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

“…The determinant of this binding is SapC, a fusogenic lysosomal protein with a strong affinity for anionic phospholipids such as phosphatidylserine 7,10,11 . When conjugated to a fluorescent probe (CVM), systemically injected SapC-DOPS can be traced to tumor and arthritic sites by fluorescence imaging.…”

“…2. Mix SapC protein as previously described 7,10,11 . Mix DOPS (0.18 mg) and CVM (0.03 mg) in a glass tube and use nitrogen gas to evaporate lipid solvents.…”

“…3. Add SapC protein powder (0.32 mg) to the mixture, suspend the dry mixture in 1 ml of PBS buffer and bath sonicate for about 15 min as previously described 7,10,11 . Then pass the suspension through a Sephadex G25 column (PD-10) to remove free CVM dye.…”

Head-to-Head Comparison of Protocol Modifications for the Generation of Collagen-Induced Arthritis in a Specific-Pathogen Free Facility Using DBA/1 Mice

“…Severity of arthritis was scored using an arthritic index scoring system ranging from 0 to 4 per paw (0 = no detectable arthritis, 1 = swelling and/or redness of paw or 1 digit, 2 = two digits involved, 3 = three digits involved, and 4 = severe arthritis of the entire paw and all digits) with a maximum score of 16 per mouse (26). …”

Hydrophobic cyanine dye-doped micelles for optical in vivo imaging of plasma leakage and vascular disruption

“…in vitro, prior to use [15][16][17][18] or (2) synthesizing fluorescently labeled microparticles whose extravasating capabilities are limited by their size. [19][20][21] The latter method could especially provide additional benefits, as the incorporation of the dye into a nanoprobe minimizes the potential cytotoxicity and delays systemic elimination, thereby leading to prolonged plasma half-life. Here, we have employed Kolliphor HS 15 for this purpose, a nonionic solubilizer that forms stable micelles in water and physiologic buffers with an average size of 12 nm, and has a negligible toxicity on its own.…”