Arthritis imaging using a near-infrared fluorescence folate-targeted probe

Abstract

A recently developed near-infrared fluorescence-labeled folate probe (NIR2-folate) was tested for in vivo imaging of arthritis using a lipopolysaccharide intra-articular injection model and a KRN transgenic mice serum induction mouse model. In the lipopolysaccharide injection model, the fluorescence signal intensity of NIR2-folate (n = 12) and of free NIR2 (n = 5) was compared between lipopolysaccharide-treated and control joints. The fluorescence signal intensity of the NIR2-folate probe at the inflammatory joints was found to be significantly higher than the control normal joints (up to 2.3-fold, P < 0.001). The NIR2-free dye injection group showed a persistent lower enhancement ratio than the NIR2-folate probe injection group. Excessive folic acid was also given to demonstrate a competitive effect with the NIR2-folate. In the KRN serum transfer model (n = 4), NIR2-folate was applied at different time points after serum transfer, and the inflamed joints could be detected as early as 30 hours after arthritogenic antibody transfer (1.8-fold increase in signal intensity). Fluorescence microscopy, histology, and immunohistochemistry validated the optical imaging results. We conclude that in vivo arthritis detection was feasible using a folate-targeted near-infrared fluorescence probe. This receptor-targeted imaging method may facilitate improved arthritis diagnosis and early assessment of the disease progress by providing an in vivo characterization of active macrophage status in inflammatory joint diseases.

Figure 1

Immunoperoxidase staining of (a) Mac-3 and (b) folate receptor (FR) at an arthritic ankle 72 hours after lipopolysaccharide induction. The Mac-3-positive and FR-positive cells morphologically correlated well in adjacent tissue sections. Magnification, 400 ×.

Figure 2

In vivo near-infrared fluorescent (NIRF) imaging of inflammatory joints in the lipopolysaccharide (LPS) induction model. The NIR2-folate probe was intravenously injected 2 days after LPS intra-articular injection. (a) White-light images obtained 48 hours after intra-articular LPS injection at the right ankle joint; soft tissue swelling was noted at the affected joint. (b) NIRF images obtained 24 hours after NIR2-folate injection. Note the strong fluorescence signal in the LPS-treated ankle compared with the opposite control side (enhancement ratio = 2.31). (c) A merged NIRF signal with a white-light image showing specific increased fluorescence signal intensity at the affected joint. (d) H&E-stain section of the right ankle joint showing abundant inflammatory cell infiltration at subsynovial tissues. Original magnification, 100 ×. (e) NIRF images of a longitudinal section of the LPS-treated ankles. Pseudo-color coding was used to demonstrate the stronger fluorescence signal surrounding the ankle joint.

Figure 3

Enhancement ratio of lipopolysaccharide (LPS)-treated inflamed ankles in NIR2-folate (n = 12) and NIR2-free dye (n = 5) injection groups at different time points. A significantly higher enhancement ratio was noted in the NIR2-folate injection group at 24-hour, 48-hour and 72-hour time points (P < 0.05).

Figure 4

Colocalization of Mac-3-positive cells and NIR2-folate uptake cells in a lipopolysaccharide-induced arthritic ankle 48 hours after NIR2-folate injection. (a) Immunofluorescence staining for activated macrophage revealed in the FITC channel. (b) NIR2-folate uptake cells are revealed in the near-infrared fluorescent channel. (c) Superimposed image shows Mac-3-positive cells colocalized well with NIR2-folate cellular origins. (d) A negative control without primary antibody. Original magnification, 400 ×.

Figure 5

Establishment of the KRN serum transfer model. (a) Discoloration and swelling (arrow) of the right third proximal interphalangeal joint is noted in a healthy C57BL/6 mouse 4 days after KRN serum transfer. (b) Near-infrared fluorescent imaging of the right paw showed increase fluorescence signal intensity at the inflammatory joint (enhancement ratio = 1.9). (c) Correlated H&E-stain section showed abundant inflammatory cells infiltration with pannus-like formation. Original magnification, 100 ×. (d) Immunoperoxidase staining of Mac-3. Mac-3-positive cell infiltration among polymorphonuclear cells was noted in the pannus. Original magnification, 400 ×.

Figure 6

Early detection (30 hours after KRN serum transfer) of the inflammatory joint by NIR2-folate. (a) White-light image showed no remarkable swelling at bilateral paws. (b) Merged near-infrared fluorescent signal with a white-light image showed increase fluorescence signal intensity at the dorsal aspect of the right wrist, which has a 1.8-fold increase compared with the left wrist. (c) H&E-stain histology of the right wrist showed polymorphonuclear cell infiltration at the dorsal aspect of the right wrist (arrow). Magnification, 20 × (400 ×, insert). (d) Histology of the left wrist showed no remarkable inflammatory cell infiltration. Magnification, 20 × (400 ×, insert). (e) Immunohistochemistry of the right wrist showed Mac-3-positive cell infiltration at subsynovial tissues. Magnification, 400 ×.