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.

Figure 1. SapC-DOPS-CVM localizes to arthritic joints and is visible by live fluorometric imaging.

Male C57Bl/6 mice three months of age were given 150 µl of sera i.p. as indicated. Seven days after sera injection, mice were imaged by IVIS® (A) five hours following SapC-DOPS-CVM i.v. injection as indicated (- is PBS); (B) two hours following i.v. injection of DOPS-CVM, DOPS-CVM plus uncoupled SapC, or SapC-DOPS-CVM as indicated. Arrows indicate macroscopically swollen paws.

Figure 2. SapC-DOPS-CVM localizes to arthritic joints in CIA.

(A) Light images of DBA/1 mice treated with CII (left, CIA) and non-CII treated (right, Control). (B) IVIS® images of mice in A, 48 hours following SapC-DOPS-CVM i.v injection. Light images of CIA hind paw (C) and forepaw (E) with corresponding IVIS® images in (D) and (F). Solid black arrows indicate macroscopically swollen paws and white arrows indicate macroscopically swollen joints. (G) and (I) IVIS® images of CII-treated mice 48 hours following SapC-DOPS-CVM i.v. injection, with arrows indicating corresponding histological knee sections (H) and (J) stained with hematoxylin and eosin. CVM intensity as measured by live fluorometric imaging is represented by red, yellow, green, blue colorimetric scale with red representing highest and blue representing lowest intensity.

Figure 3. CVM signal intensity increases during the course of K/BxN arthritis and correlates with arthritic parameters.

C57Bl/6 mice were given 150 µl of K/BxN sera i.p. and 84 µg of SapC-DOPS-CVM as indicated. (A) Means of back paw intensity values are graphed as columns with error bars representing standard error of the mean. * p<0.05, Students t-test, n≥4 mice per group. For mice treated with K/BxN sera (n = 7) and given SapC-DOPS-CVM the (B) changes in paw thickness and (C) changes in ankle circumference were rounded to the nearest millimeter and the means (+/− SEM) of IVIS® signal intensity for the corresponding paws are graphed as columns. Lines represent linear regression of values in B and C with correlation coefficients indicated. Correlation of arthritis parameters and IVIS® intensity signals in B and C is significant p<0.005, as determined by Pearson Product Moment Correlation and Spearman Rank Order Correlation.

Figure 4. CVM signal intensity from CII-challenged mice increases over disease course and correlates with arthritic severity.

(A) Mice were injected i.p. with SapC-DOPS-CVM starting on day 23 following primary CII immunization and then every other day. Mice were imaged daily by IVIS®. Columns indicate the mean IVIS® intensity values (average radiance) for paws in each group +/− the SEM. Mice were scored macroscopically for arthritis daily. N of paws imaged in groups were as follows: No SapC-DOPS-CVM, CII+: 16; SapC-DOPS-CVM+, no CII:16; SapC-DOPS-CVM+,CII+macroscopic arthritis: 8; SapC-DOPS-CVM+CII+ no macroscopic arthritis: 24. * = p<0.05 as compared to SapC-DOPS-CVM+, no CII, Mann Whitney (B) Box plots indicate the median values and range for IVIS® intensity of paws receiving the specified macroscopic arthritic score during the time course of disease. The line represents linear regression of mean values with the correlation coefficients indicated. Correlation of arthritis parameter values and IVIS® intensity signal values is significant, p<0.005, Pearson Product Moment Correlation and Spearman Rank Order Correlation.

Figure 5. SapC -DOPS-CVM targets primarily CD11b+Gr-1+ joint cells in the K/BxN model of arthritis.

Joint cells from hind paws were stained with antibodies to cell surface markers as indicated. FACS plots are representative from one animal, with bar graphs indicating the means +/− the SEM of groups. (A) Dot plots indicate cell populations that are CVM+ and positive for other cell surface markers as indicated. (B) CVM+ cells. Columns represent mean of total cells that are CVM+ +/− SEM with treatment of animals as indicated. * = p<0.05. (C) Bar graph indicates means +/− the SEM of the percentage of total cells that are CVM+ and positive for the indicated cell surface marker. * = p<0.05, Students t-test. (D) Dot plots show forward (FSC) and side scatter (SSC) for joint populations with gating for CVM+ cell populations versus side scatter (SSC). Gated CVM+ cells are further analyzed for specific cell-surface markers CD11b, CD11c and Gr-1 with percentages indicated. (E) Columns represent the mean +/− the SEM of the percentage of total cells that are positive for CVM, CD11b, and also positive for either CD11c or Gr-1 * = p<0.05. N = 4 No Sera, SapC-DOPS-CVM; N = 9 Sera, SapC-DOPS-CVM.

Figure 6. SapC -DOPS-CVM targets primarily CD11b+Gr-1+ joint cells in the CIA model of arthritis.

Cells were stained with antibodies to cell surface markers as indicated. FACS plots are representative from one animal, with bar graphs indicating the means +/− the SEM of groups. (A) Dot plots indicate cell populations from mice treated as indicated that are CVM+ and positive for other cell surface markers as indicated on left. (B) CVM+ cells. Columns represent mean of total cells that are CVM+ +/− SEM (error bars) with treatment of animals as indicated. * p<0.0001 Mann Whitney. (C) Columns indicate means +/− the SEM (error bars) of the percentage of total cells that are CVM + and positive for the indicated cell surface marker. * = p<0.05 Mann Whitney. (D) Dot plots show forward (FSC) and side scatter (SSC) for joint populations with gating for CVM +cell populations versus side scatter (SSC). Gated CVM + cells are further analyzed for combinations of the specific cell-surface markers CD11b, CD11c and Gr-1 as indicated. (E) Columns represent the mean +/− the SEM (error bars) of the percentage of total cells that are positive for CVM, CD11b, and also positive for either CD11c or Gr-1. N = 4, No CII, SapC-DOPS-CVM; 8, CII+, SapC-DOPS-CVM+ * p<0.05, Mann Whitney.