Theranostic immunoliposomes for osteoarthritis

Abstract

Although there have been substantial advancements in the treatment of inflammatory arthritis, treatments for osteoarthritis (OA) have lagged and currently are primarily palliative until joints become totally dysfunctional and prosthetic replacement is needed. One obstacle for developing a preventive therapy for OA is the lack of good tools for efficiently diagnosing the disease and monitoring its progression during the early stages when the effect of therapeutic drugs or biologics is most likely to be effective. We have developed near infrared immunoliposomes conjugated with type II collagen antibody for diagnosis and treatment of early OA. These immunoliposomes bind to damaged but not normal cartilage. Utilizing these reagents, we can quantitate exposure of type II collagen during cartilage degradation in individual joints in vivo in a guinea pig. Immunoliposomes could be used to determine the effectiveness of therapeutic interventions in small animals as well as vehicles for localized drug delivery to OA chondrocytes.

From the clinical editor: This team of authors have developed near infrared immunoliposomes conjugated with type II collagen antibody for diagnosis and treatment of early OA, with promising results demonstrated in a guinea pig model.

Keywords: Extracellular matrix (ECM); Immuno-liposomes (ILs); Near-infrared (NIR) fluorescent; Osteoarthritis (OA); Theranostic (therapeutic and diagnostic); Type II collagen (CII).

Figure 1. Characterization of immuno-liposomes

(A) Kinetics of binding to type II collagen. To determine the binding characteristics of the MAbCII-coupled liposomes, the liposomes were pre-incubated with a known concentration of purified type II collagen overnight, then incubated with a collagen II coated ELISA plate. Liposomes bound to type II collagen were detected with an alkaline phosphatase coupled secondary antibody, using a PNPP substrate. Using a logarithmic regression curve, it was determined that the K_d was 3.0μg/mL, similar to the value observed for the free antibody (1.7μg/mL). (B) Size distribution by DLS. Analysis by DLS showed only one population with a Z-average of 200nm for the immuno-liposomes mixture. (C) Characterization by SEM. The DOPC liposomes are uniform in size with a range of diameter of 150–250 nm. After coupling to antibody, the shape and size of the liposomes remains constant (left side of C). The liposomes were multilamellar structures (right side of C) with 1–4 layers.

Figure 2. Localization of immuno-liposomes on cultured chondrocytes by SEM & fluorescence microscopy

Images of SEM (A, B, C and D); (A and C) Control group: Cultured chondrocytes were treated with control liposomes that were not coupled to antibody, (A is 500x magnitude, C is 12800x magnitude); (B and D) Experimental group: cultured chondrocytes were treated with the immuno-liposomes which were conjugated to CII antibody, (B is 500x magnitude, C is 12800x magnitude); Images of fluorescence microscopy of cultured chondrocytes incubated with control and immuno-liposomes filled with peroxidase and stainined with DAB substrate (E and F); (E) Control group: control liposomes showed no staining with DAB substrate, (F) Experimental group: HRP encapsulated in immuno-liposomes showed localization in proximity to chondrocytes

Figure 3. in vitro model of creating articular cartilage lesions with trypsin digestion and exclusive localization of immuno-liposomes to damaged cartilage explants

Articular cartilage explants were incubated with (C and D) trypsin to mimic a damaged cartilage tissue, or without (A and B) trypsin for intact tissue, then incubated for 1 hr with peroxidase-filled nanosomes coupled to type II collagen antibody (immuno-liposomes, A and C) or peroxidase-filled liposomes alone (control liposomes, B and D). After a secondary punch with a 4mm biopsy punch to remove the sides that were mechanically damaged from the initial harvesting, the cartilage tissues were incubated with the peroxidase substrate DAB. Only the articular cartilage treated with trypsin (C) binds to the immuno-liposomes.

Figure 4. IVIS imaging of DH guinea pigs intravenously injected with MAbCII liposome

Animals were IVIS imaged at 24 hours after injection. (A and B) IVIS scan images of young and older guinea pig (n=12 for each age group). (C) IVIS imaging of cartilages from dissected knee joints; (H) total integrated intensity of fluorescence (ROI) in femoral and tibial condyles was quantitated and normalized using imaging software. (D, E, F and G) Histopathology of Hematoxylin and Eosin (H&E) stained sections of articular knee cartilage from young & old GP showing regional variation in morphology. Tibial condyles were decalcified, embedded and 60 H&E-stained sections from each specimen were prepared with a constant interval of 100 μm to survey the articular cartilage. (I) These sections were then graded by an observer using a modified Mankin scale (normal; grade 0–2, slight; grade 3–5, moderate; grade 6–8 and severe; grade 9–11).