Targeted zwitterionic near-infrared fluorophores for improved optical imaging

Abstract

The signal-to-background ratio (SBR) is the key determinant of sensitivity, detectability and linearity in optical imaging. As signal strength is often constrained by fundamental limits, background reduction becomes an important approach for improving the SBR. We recently reported that a zwitterionic near-infrared (NIR) fluorophore, ZW800-1, exhibits low background. Here we show that this fluorophore provides a much-improved SBR when targeted to cancer cells or proteins by conjugation with a cyclic RGD peptide, fibrinogen or antibodies. ZW800-1 outperforms the commercially available NIR fluorophores IRDye800-CW and Cy5.5 in vitro for immunocytometry, histopathology and immunoblotting and in vivo for image-guided surgery. In tumor model systems, a tumor-to-background ratio of 17.2 is achieved at 4 h after injection of ZW800-1 conjugated to cRGD compared to ratios of 5.1 with IRDye800-CW and 2.7 with Cy5.5. Our results suggest that introducing zwitterionic properties into targeted fluorophores may be a general strategy for improving the SBR in diagnostic and therapeutic applications.

Figure 1. Targeted NIR fluorophores and improved SBR during cell-based assays

(a) Chemical structure, molecular weight (MW), logD, net surface charge and 3D modeling of the geometrical position of charge and hydrophobicity of cRGD-ZW800-1, cRGD-CW800 and cRGD-Cy5.5 at pH 7.4. Red = negative charge; Blue = positive charge; Gray = hydrophobicity. (b) Live cell binding assay for cRGD-ZW800-1, cRGD-CW800 and cRGD-Cy5.5 (top 2 rows) or NIR fluorophores alone (bottom row) in M21 (α_vβ₃-positive) and M21-L (α_vβ₃-negative) melanoma cell lines after incubation with 2 μM of each molecule for 30 min at 37 C. Scale bars = 100 μm. All NIR fluorescence images have identical exposure times and are normalized to peak signal. (c) Antibody binding assay for secondary antibodies conjugated with ZW800-1, CW800 and Cy5.5 in MDA-MB-361 (Her2-positive) and MDA-MB-231 (Her2-negative) human breast cancer cell lines in the presence (top 2 rows) or absence (bottom 2 rows) of the anti-Her2 primary antibody. Scale bars = 50 μm. All NIR fluorescence images have identical exposure times and are normalized to peak signal.

Figure 1. Targeted NIR fluorophores and improved SBR during cell-based assays

(a) Chemical structure, molecular weight (MW), logD, net surface charge and 3D modeling of the geometrical position of charge and hydrophobicity of cRGD-ZW800-1, cRGD-CW800 and cRGD-Cy5.5 at pH 7.4. Red = negative charge; Blue = positive charge; Gray = hydrophobicity. (b) Live cell binding assay for cRGD-ZW800-1, cRGD-CW800 and cRGD-Cy5.5 (top 2 rows) or NIR fluorophores alone (bottom row) in M21 (α_vβ₃-positive) and M21-L (α_vβ₃-negative) melanoma cell lines after incubation with 2 μM of each molecule for 30 min at 37 C. Scale bars = 100 μm. All NIR fluorescence images have identical exposure times and are normalized to peak signal. (c) Antibody binding assay for secondary antibodies conjugated with ZW800-1, CW800 and Cy5.5 in MDA-MB-361 (Her2-positive) and MDA-MB-231 (Her2-negative) human breast cancer cell lines in the presence (top 2 rows) or absence (bottom 2 rows) of the anti-Her2 primary antibody. Scale bars = 50 μm. All NIR fluorescence images have identical exposure times and are normalized to peak signal.

Figure 1. Targeted NIR fluorophores and improved SBR during cell-based assays

(a) Chemical structure, molecular weight (MW), logD, net surface charge and 3D modeling of the geometrical position of charge and hydrophobicity of cRGD-ZW800-1, cRGD-CW800 and cRGD-Cy5.5 at pH 7.4. Red = negative charge; Blue = positive charge; Gray = hydrophobicity. (b) Live cell binding assay for cRGD-ZW800-1, cRGD-CW800 and cRGD-Cy5.5 (top 2 rows) or NIR fluorophores alone (bottom row) in M21 (α_vβ₃-positive) and M21-L (α_vβ₃-negative) melanoma cell lines after incubation with 2 μM of each molecule for 30 min at 37 C. Scale bars = 100 μm. All NIR fluorescence images have identical exposure times and are normalized to peak signal. (c) Antibody binding assay for secondary antibodies conjugated with ZW800-1, CW800 and Cy5.5 in MDA-MB-361 (Her2-positive) and MDA-MB-231 (Her2-negative) human breast cancer cell lines in the presence (top 2 rows) or absence (bottom 2 rows) of the anti-Her2 primary antibody. Scale bars = 50 μm. All NIR fluorescence images have identical exposure times and are normalized to peak signal.

Figure 2. NIR fluorophore-conjugated antibodies and improved SBR during histopathological analysis

(a) H&E (left columns) and NIR fluorescence immunostaining (right columns) of prostate tissue using a rabbit anti-human AMACR primary antibody (top row), goat anti-rabbit secondary and NIR-conjugated mouse anti-goat tertiary antibodies alone (middle row) and NIR fluorophores alone (2 μM each; bottom row). All NIR fluorescence images have identical exposure times and are normalized to peak signal. (b) H&E (left columns) and NIR fluorescence immunostaining (right columns) of breast tissue using a rabbit anti-human Her2 primary antibody (2 row), goat anti-rabbit secondary and NIR-conjugated mouse anti-goat tertiary antibodies alone (middle row) and NIR fluorophores alone (2 μM each; bottom row). All NIR fluorescence images have identical exposure times and are normalized to peak signal. Scale bars = 200 μm.

Figure 2. NIR fluorophore-conjugated antibodies and improved SBR during histopathological analysis

(a) H&E (left columns) and NIR fluorescence immunostaining (right columns) of prostate tissue using a rabbit anti-human AMACR primary antibody (top row), goat anti-rabbit secondary and NIR-conjugated mouse anti-goat tertiary antibodies alone (middle row) and NIR fluorophores alone (2 μM each; bottom row). All NIR fluorescence images have identical exposure times and are normalized to peak signal. (b) H&E (left columns) and NIR fluorescence immunostaining (right columns) of breast tissue using a rabbit anti-human Her2 primary antibody (2 row), goat anti-rabbit secondary and NIR-conjugated mouse anti-goat tertiary antibodies alone (middle row) and NIR fluorophores alone (2 μM each; bottom row). All NIR fluorescence images have identical exposure times and are normalized to peak signal. Scale bars = 200 μm.

Figure 3. Improved in vivo optical imaging using zwitterionic, NIR fluorescent, targeted small molecules and proteins

(a) Real-time intraoperative melanoma detection using targeted small molecules. 3 nmol of cRGD-ZW800-1 (left), 3 nmol of cRGD-CW800 (middle), or 10 nmol of cRGD-Cy5.5 (right) was injected intravenously into melanoma tumor mice. Shown are representative (n = 5) images of color and NIR fluorescence immediately pre-injection (0 h) and at 4 h post-injection. T(+) = integrin α_vβ₃-positive tumor; T(−) = integrin α_vβ₃-negative tumor; arrows = kidneys; red dotted circle = ROI used for TBR background measurement. Quantitation of image signal and background is also shown at 4 h post-injection. All NIR fluorescence images have identical exposure times and are normalized to peak signal. (b) Real-time intraoperative liver (left) and lung (right) tumor detection using targeted small molecules. 10 nmol of cRGD-ZW800-1 (top), cRGD-CW800 (middle), or cRGD-Cy5.5 (bottom) was injected intravenously into each tumor mouse 4 h prior to imaging. Shown are representative (n = 5) images of color image, NIR fluorescence and a pseudo-colored merge of the two. AW, abdominal wall; GB, gallbladder; He, heart; In, intestine; Li, liver; Lu, lungs; St, stomach; TW, thoracic wall. Arrows = tumors. All NIR fluorescence images have identical exposure times and are normalized to peak signal. (c) Real-time intraoperative thrombus detection using targeted proteins. 40 pmol/g of FBG-ZW800-1 (left), FBG-CW800 (middle), or FBG-Cy5.5 (right) was injected intravenously into SD rats 1 h after mucosal resection (arrows) in the stomach (shown) or mesenteric vessels (Supplementary Fig. 6). CBR (lower graphs) was calculated from the ratio of the signal at the site of injury site versus nearby normal tissue background. Arrows = injury sites. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison test (*P < 0.05; **P < 0.01). All NIR fluorescence images have identical exposure times and are normalized to peak signal. Scale bars = 1 cm.

Figure 3. Improved in vivo optical imaging using zwitterionic, NIR fluorescent, targeted small molecules and proteins

(a) Real-time intraoperative melanoma detection using targeted small molecules. 3 nmol of cRGD-ZW800-1 (left), 3 nmol of cRGD-CW800 (middle), or 10 nmol of cRGD-Cy5.5 (right) was injected intravenously into melanoma tumor mice. Shown are representative (n = 5) images of color and NIR fluorescence immediately pre-injection (0 h) and at 4 h post-injection. T(+) = integrin α_vβ₃-positive tumor; T(−) = integrin α_vβ₃-negative tumor; arrows = kidneys; red dotted circle = ROI used for TBR background measurement. Quantitation of image signal and background is also shown at 4 h post-injection. All NIR fluorescence images have identical exposure times and are normalized to peak signal. (b) Real-time intraoperative liver (left) and lung (right) tumor detection using targeted small molecules. 10 nmol of cRGD-ZW800-1 (top), cRGD-CW800 (middle), or cRGD-Cy5.5 (bottom) was injected intravenously into each tumor mouse 4 h prior to imaging. Shown are representative (n = 5) images of color image, NIR fluorescence and a pseudo-colored merge of the two. AW, abdominal wall; GB, gallbladder; He, heart; In, intestine; Li, liver; Lu, lungs; St, stomach; TW, thoracic wall. Arrows = tumors. All NIR fluorescence images have identical exposure times and are normalized to peak signal. (c) Real-time intraoperative thrombus detection using targeted proteins. 40 pmol/g of FBG-ZW800-1 (left), FBG-CW800 (middle), or FBG-Cy5.5 (right) was injected intravenously into SD rats 1 h after mucosal resection (arrows) in the stomach (shown) or mesenteric vessels (Supplementary Fig. 6). CBR (lower graphs) was calculated from the ratio of the signal at the site of injury site versus nearby normal tissue background. Arrows = injury sites. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison test (*P < 0.05; **P < 0.01). All NIR fluorescence images have identical exposure times and are normalized to peak signal. Scale bars = 1 cm.

Figure 3. Improved in vivo optical imaging using zwitterionic, NIR fluorescent, targeted small molecules and proteins

(a) Real-time intraoperative melanoma detection using targeted small molecules. 3 nmol of cRGD-ZW800-1 (left), 3 nmol of cRGD-CW800 (middle), or 10 nmol of cRGD-Cy5.5 (right) was injected intravenously into melanoma tumor mice. Shown are representative (n = 5) images of color and NIR fluorescence immediately pre-injection (0 h) and at 4 h post-injection. T(+) = integrin α_vβ₃-positive tumor; T(−) = integrin α_vβ₃-negative tumor; arrows = kidneys; red dotted circle = ROI used for TBR background measurement. Quantitation of image signal and background is also shown at 4 h post-injection. All NIR fluorescence images have identical exposure times and are normalized to peak signal. (b) Real-time intraoperative liver (left) and lung (right) tumor detection using targeted small molecules. 10 nmol of cRGD-ZW800-1 (top), cRGD-CW800 (middle), or cRGD-Cy5.5 (bottom) was injected intravenously into each tumor mouse 4 h prior to imaging. Shown are representative (n = 5) images of color image, NIR fluorescence and a pseudo-colored merge of the two. AW, abdominal wall; GB, gallbladder; He, heart; In, intestine; Li, liver; Lu, lungs; St, stomach; TW, thoracic wall. Arrows = tumors. All NIR fluorescence images have identical exposure times and are normalized to peak signal. (c) Real-time intraoperative thrombus detection using targeted proteins. 40 pmol/g of FBG-ZW800-1 (left), FBG-CW800 (middle), or FBG-Cy5.5 (right) was injected intravenously into SD rats 1 h after mucosal resection (arrows) in the stomach (shown) or mesenteric vessels (Supplementary Fig. 6). CBR (lower graphs) was calculated from the ratio of the signal at the site of injury site versus nearby normal tissue background. Arrows = injury sites. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison test (*P < 0.05; **P < 0.01). All NIR fluorescence images have identical exposure times and are normalized to peak signal. Scale bars = 1 cm.

Figure 4

Modification of a primary amine by commercially available anionic NIR fluorophores (top) and zwitterionic NIR fluorophores (bottom).