The present studies investigate the utility and underlying mechanism(s) of SapC-DOPS nanovesicles as an optical imaging agent to detect tissue damage in inflammatory arthritic joints. In two separate animal models of inflammatory arthritis, SapC-DOPS labeled with the fluorophore CVM was visualized locally in arthritic inflammatory joints. Furthermore, throughout the course of both K/BxN and CIA inflammatory arthritis SapC-DOPS intensity increased and correlated with increases in multiple independent parameters of arthritic disease assessment. Joint cells targeted by SapC-DOPS-CVM were primarily positive for both CD11b and Gr-1—cell markers indicative of neutrophils—that have previously been shown to be critical mediators of inflammatory events in arthritis 
. The use of SapC-DOPS to target imaging agents to areas of joint inflammation provides a potentially novel, noninvasive, and robust tool in the assessment of inflammatory arthritis.
Optical imaging has great promise for use in inflammatory arthritis, particularly in RA, in which the peripheral joints are the most highly affected. Assessment of joint damage in RA has been measured primarily through x-ray imaging analysis, which does not readily provide information regarding the earliest local pathogenic events (i.e., inflammation), which occur prior to joint damage. MRI and ultrasound are beginning to be used as modalities to assess soft tissue damage; however, each of these modalities has significant factors hindering their routine use in the clinic. Optical imaging in humans has primarily involved imaging of vasculature and lymphatics using the non-specific, near-infrared dye indocyanine green 
, with more recent advances using fluorescent-labeled folate receptor-alpha targets for tumor-specific, fluorescence-assisted cytoreductive surgery 
. For RA patients, visualization of indocyanine green uptake focusing on the finger and wrist joints has been demonstrated with newly developed optical imaging instrumentation 
. In animal models of arthritis, anti-E-selectin labeled with a near-infrared fluorophore demonstrated specificity for arthritic joints in CIA mice 
. In addition, antibody and folate receptor targets have been used to image activated macrophages in arthritis models and other inflammatory settings 
Using optical imaging our studies indicate that SapC-DOPS-CVM appears to localize to arthritic joints. Our prior optical imaging studies regarding tissue-specific distribution of SapC-DOPS-CVM signal demonstrated fluorescent signal in only liver and spleen of otherwise unchallenged nude mice that diminished over time and was not detectable 48 hours following injection 
. Our present data indicate that in two separate models of arthritis, SapC-DOPS-CVM is detected by optical imaging in arthritic joints. Similar results were obtained in a third model of arthritis in which chronic polyarthritis is driven solely by overexpression of human TNF-alpha via a transgene 
. SapC-DOPS-CVM was clearly localized to arthritic joints of TNF-alpha transgenic mice but not to joints of littermate control transgene negative animals (data not shown). Thus, this novel compound appears to selectively target arthritic tissues, regardless of disease etiology, giving it a monumental advantage over imaging through the use of non-specific fluorophores.
SapC-DOPS-CVM localization as assessed by optical imaging may be useful as a marker for both the detection of early pre-clinical disease and for closely monitoring disease progression. Analysis of SapC-DOPS-CVM accumulation in joints over the course of both K/BxN and CIA arthritis indicated an increase in CVM signal as arthritis progressed. A significant finding of the present study is that clinical parameters, such as paw thickness and ankle circumference in the K/BxN model showed significant correlations with CVM intensity (i.e., increased severity of disease correlated with higher signal intensity). Non-sera treated mice that received SapC-DOPS-CVM every other day, accumulated significant low-level signal that plateaued early in disease. This result indicates that such low level of fluorescent signal may accumulate non-specifically due to the repeated administration of the fluorescent agent but would not hinder detection of early arthritic disease. While our studies focus on proof-of-principle for assessing joint damage in a mouse model over time, in clinical practice low level signal accumulation would not be expected since multiple injections over a short period of time would not be necessary. In parallel CIA analysis, signal intensity of SapC-DOPS-CVM localization to paw joints also increased in CII-challenged mice. In CII-treated mice, macroscopically arthritic paws showed a significant correlation with increased CVM signal intensity. Interestingly and specifically pertinent to early detection of inflammation and disease, in CII challenged mice a limited number of paw joints that were not overtly arthritic showed an increase in CVM signal intensity, which we believe may represent the earliest stages of arthritis. Future studies will histologically assess microscopic arthritis over the time course of disease to directly assess the histological changes that might be occurring sub-clinically in these paws as related to SapC-DOPS-CVM accumulation. Our results did not indicate any affects on arthritic disease progression and severity as both K/BxN and CIA arthritic disease progressed similarly to previous experiments performed in our laboratory; however, the results of the current study do not absolutely exclude the possibility that SapC-DOPS alone alters the kinetics and severity of arthritic disease.
Analysis of SapC-DOPS-CVM targeted cells extracted from arthritic joints indicates that the main cell type targeted is CD11b and Gr-1 positive. Mature granulocytes, particularly neutrophils that reside in tissues, express Gr-1 on their cell surface 
. Neutrophils are short-lived cells that are involved in innate immunity and host defense but have also been shown to be involved in mechanisms that drive RA 
. Deficits in the ability of neutrophils to undergo apoptosis have been associated with autoimmune disorders including RA 
. Disruptions in neutrophil activation mechanisms (i.e., degranulation, oxidative burst) have been shown to limit the local tissue damage commonly associated with inflammatory joint disease 
. Neutrophil activation with production of reactive oxygen species leads to phospholipid oxidation and subsequent externalization of PS 
. Neutrophils are one of the most abundant cell types in synovial fluid from RA patients and products of neutrophil activation are present in RA synovial fluid as well 
. Thus, SapC-DOPS targeting of this cell type is not surprising, as activated neutrophils readily externalize PS. Furthermore, the resistance of neutrophils to apoptosis in RA, potentiates the usefulness of SapC-DOPS binding as a clinical measure. A large portion of targeted cells did not stain positive for CD11b or Gr-1, indicating that other cell types are also targeted by SapC-DOPS-CVM, and future studies with more extensive cell markers will reveal additional targeted cell types. However, other cell types targeted by SapC-DOPS-CVM within our studies include CD19+ cells, which is a B-cell marker; CD31, a marker for endothelial cells; and CD55, a marker that is expressed at high levels on synovial fibroblasts 
. All of these cells are involved in the early inflammatory stages of arthritic disease, providing further support for use of this agent in early detection of inflammatory joint disease 
A significant finding from these studies was that SapC-DOPS is able to target arthritic joints in multiple disease models that exhibit distinct etiologies 
. The targeting of SapC-DOPS correlated directly to the course and pathogenesis of disease in both arthritis models. In K/BxN mice 100% penetrance of hind paws is normally observed by day 8 of disease. Our data indicated a clear targeting of SapC-DOPS-CVM to hind paws in K/BxN arthritis over the time course of disease and a direct correlation with paw thickness and ankle circumference. In addition, joint cells taken at day 8 of disease showed SapC-DOPS targeting of CD11b+Gr-1+ cells, which is consistent with a neutrophil phenotype. Neutrophils have been implicated as a major driver of pathogenesis in the K/BxN arthritis model 
. For CIA, disease incidence is slightly less with ~50–100% of mice developing overt macroscopic disease with both distal (i.e., paw joints) and proximal (i.e., knees) joint involvement that is not uniform, similar to the sporadic joint involvement exhibited by RA patients. In contrast with the K/BxN model in which disease appears quickly, macroscopic signs of arthritis in CIA can become apparent weeks following the secondary collagen immunization 
. In addition, the higher degree of variability observed in the analysis of cell types targeted by SapC-DOPS in the CIA model are likely reflective of both the variability and time course of joint involvement in the CIA model. Given the more complex disease process of CIA 
, SapC-DOPS is likely targeting multiple cell types involved in the pathogenesis of arthritis. Nevertheless, the significant correlation between SapC-DOPS-CVM intensity and arthritic severity observed in the both the K/BxN and CIA models suggests that common pathological mechanisms (i.e., the influx of myeloid derived cells of the innate immune system) exist that are being targeted by SapC-DOPS. In total, our observations strongly suggest that targeting arthritic joints using SapC-DOPS may have far-reaching applicability in a variety of arthritic contexts.
The striking observation on arthritis-selective targeting of the novel compound SapC-DOPS suggests that this molecule may be used to detect inflammation in a variety of settings and contexts. Studies directed at evaluating whether SapC-DOPS can detect subclinical inflammation; other arthritides; and/or other inflammatory disorders remain to be performed; however, the results presented here argue that SapC-DOPS facilitated imaging may be used as a clinical parameter that could be highly valuable to assess inflammatory joint disease. One of the advantages to SapC-DOPS is its flexibility in incorporating other agents within the nanovesicles and targeting inflammatory regions. This technology also has the promising potential for specific delivery of agents to joints, which would be beneficial particularly with respect to therapies that when administered globally have undesirable side effects.