2.1.

Chemicals

BSA and GmSA, which contained 23 galactosamine molecules on a single albumin molecule, were purchased from Sigma Chemical (St. Louis, Missouri). Amine-reactive biotin, succinimidyl-6-(biotin-amido)hexanoate (NHS-LC-Biotin), and avidin were purchased from Pierce (Rockford, Illinois). Amine-reactive Cy5.5 (peak emission wavelength at $691\mathrm{nm}$ ) and Cy7 (peak emission wavelength at $776\mathrm{nm}$ ) were purchased from GE Healthcare Limited (Piscataway, NJ).

2.2.

Synthesis of BSA- and GmSA-Conjugated Near-Infrared Contrast Agents

At room temperature, $400\mu \mathrm{g}$ $\left(5.7\mathrm{nmol}\right)$ of GmSA or $400\mu \mathrm{g}$ $\left(5.9\mathrm{nmol}\right)$ of BSA in $193\mu \mathrm{L}$ of ${\mathrm{Na}}_2\mathrm{H}\mathrm{P}{\mathrm{O}}_4$ was incubated with $70\mathrm{nmol}$ $(7\mu \mathrm{L}∕10\mathrm{mM})$ of Cy5.5 or Cy7 in dimethyl sulfoxide (DMSO) at pH 8.5 for $15\min$ . The mixture was purified with Sephadex G50 (PD-10; GE Healthcare, Milwaukee, Wisconsin). All conjugated samples were concentrated with a Centricon 30 (Millipore Co., Billerica, Massachusetts), and concentration was adjusted to $2\mathrm{mg}∕\mathrm{mL}$ in phosphate buffered saline (PBS) at pH 7.4. GmSA-conjugated Cy5.5 and Cy7 (GmSA-Cy5.5 and GmSA-Cy7, respectively) and BSA-conjugated Cy5.5 and Cy7 (BSA-Cy5.5 and BSA-Cy7, respectively) samples were refrigerated at $4°\mathrm{C}$ . The protein concentration of GmSA-Cy5.5, GmSA-Cy7, BSA-Cy5.5, and BSA-Cy7 samples was determined with a Coomassie Plus protein assay kit (Pierce Chem Co., Rockford, Illinois) by measuring the absorption at $595\mathrm{nm}$ with a UV-Vis system (8453 Value UV-Bis system, Agilent Technologies, Palo Alto, California) using standard solutions of known concentrations of GmSA or BSA (100, 200, and $400\mu \mathrm{g}∕\mathrm{mL}$ ). The concentration of Cy5.5 and Cy7 was then measured by the absorption at 679 and $756\mathrm{nm}$ , respectively, with a UV-Vis system (8453 Value UV-Bis system, Agilent Technologies) to confirm the number of fluorophore molecules conjugated with each GmSA or BSA molecule. The number of fluorophore molecules per GmSA or BSA was 2.0 for Cy5.5 and 2.5 for Cy7, respectively.

2.3.

Synthesis of Biotinylated BSA-Conjugated Near-Infrared Contrast Agents

At room temperature, $120\mathrm{nmol}$ $(12\mu \mathrm{L}∕10\mathrm{mM})$ NHS-LC-Biotin dissolved in DMSO was added to $400\mu \mathrm{g}$ $\left(5.9\mathrm{nmol}\right)$ of BSA in $281\mu \mathrm{L}$ of ${\mathrm{Na}}_2\mathrm{H}\mathrm{P}{\mathrm{O}}_4$ and incubated for $30\min$ . Then $70\mathrm{nmol}$ $(7\mu \mathrm{L}∕10\mathrm{mM})$ of Cy5.5 or Cy7 in DMSO at pH 8.5 was added and incubated for $15\min$ . Unreacted biotin and Cy5.5 or Cy7 were separated from the protein by gel filtration using a Sephadex G50 (PD-10; GE Healthcare). All conjugated samples were concentrated with a Centricon 30 (Millipore Co.) and adjusted to a concentration of $2\mathrm{mg}∕\mathrm{mL}$ in PBS pH 7.4. Biotinylated BSA conjugated to Cy5.5 and Cy7 (b-BSA-Cy5.5 and b-BSA-Cy7, respectively) samples were refrigerated at $4°\mathrm{C}$ .

The protein concentration of b-BSA-Cy5.5 and b-BSA-Cy7 samples was determined with a Coomassie Plus protein assay kit (Pierce Chem Co.) by measuring the absorption at $595\mathrm{nm}$ with a UV-Vis system (8453 Value UV-Bis system, Agilent Technologies) using standard solutions of known concentrations of BSA (100, 200, and $400\mu \mathrm{g}∕\mathrm{mL}$ ). The concentration of Cy5.5 and Cy7 was then measured by the absorption at 679 and $756\mathrm{nm}$ , respectively with a UV-Vis system (8453 Value UV-Bis system, Agilent Technologies) to confirm the number of fluorophore molecules conjugated with each BSA molecule. The number of fluorophore molecules per BSA was 1.8 for Cy5.5 and 2.2 for Cy7, respectively.

The biotin labeling ratio was determined by the HABA assay (Pierce Chem. Co.), and $1\mathrm{mg}$ of avidin and $60\mu \mathrm{L}$ of $10\text{-}\mathrm{mM}$ HABA were added to $1.94\mathrm{mL}$ of PBS (HABA/Avidin solution). The HABA/Avidin solution $\left(900\mu \mathrm{L}\right)$ was put in a $1\text{-}\mathrm{mL}$ cuvette, and the absorbance of this solution was measured at $500\mathrm{nm}$ and recorded as the ${\mathrm{A}}_{500}$ HABA/Avidin. Then, $100\mu \mathrm{L}$ of b-BSA-Cy5.5 or b-BSA-Cy7 was added to the HABA/Avidin solution, and the absorbance at $500\mathrm{nm}$ was measured and recorded as the ${\mathrm{A}}_{500}$ HABA/Avidin/Biotin. The number of biotin conjugated per BSA was calculated based on the Beer Lambert law:

2.4.

Two-Color Wavelength-Resolved Contrast-Enhanced Dynamic Optical Imaging

All in vivo procedures were carried out in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), National Research Council, and approved by the Animal Care and Use Committee. Twelve-week-old normal athymic mice were anesthetized with intraperitoneal injection of $1.15\text{-}\mathrm{mg}$ sodium pentobarbital (Dainabot, Osaka, Japan). The tail vein was then cannulated with a 30-gauge needle, and in vivo contrast-enhanced dynamic imaging study was performed. This in vivo dynamic contrast-enhanced study consists of two protocols:

For the one-shot injection study, $75\text{-}\mu \mathrm{L}$ mixed solution of $0.7\text{-}\mathrm{nmol}$ BSA-Cy5.5 and $1.5\text{-}\mathrm{nmol}$ GmSA-Cy7 or $75\text{-}\mu \mathrm{L}$ mixed solution of $0.7\text{-}\mathrm{nmol}$ GmSA-Cy5.5 and $1.5\text{-}\mathrm{nmol}$ BSA-Cy7 was injected as a mixture. Mice were placed in the supine position, and wavelength-resolved spectral fluorescence imaging was carried out (Maestro In-Vivo Imaging System, CRi Inc., Woburn, Massachusetts). For $1\min$ before and $10\min$ after injection, the contrast agent mixture (either BSA-Cy5.5 and GmSA-Cy7 or GmSA-Cy5.5 and BSA-Cy7), spectral fluorescence imaging was performed every $20\mathrm{s}$ . At $10\min$ after injection, images were obtained every $1\min$ for another $10\min$ . For the multi-injection study, three sequential injections of $50\text{-}\mu \mathrm{L}$ b-BSA-Cy7 $\left(1.5\mathrm{nmol}\right)$ , $25\text{-}\mu \mathrm{L}$ BSA-Cy5.5 $\left(0.7\mathrm{nmol}\right)$ , and $1.5\text{-}\mathrm{nmol}$ avidin in $50\text{-}\mu \mathrm{L}$ PBS or $25\text{-}\mu \mathrm{L}$ b-BSA-Cy5.5 $\left(0.7\mathrm{nmol}\right)$ , $50\text{-}\mu \mathrm{L}$ BSA-Cy7 $\left(1.5\mathrm{pmol}\right)$ , and $0.7\text{-}\mathrm{nmol}$ avidin in $25\text{-}\mu \mathrm{L}$ PBS were administered at $200\text{-}\mathrm{s}$ intervals. Spectral fluorescence imaging was performed every $20\mathrm{s}$ starting $1\min$ before and $13\min$ after the initial injection (b-BSA-Cy7 or b-BSA-Cy5.5).

The excitation bandpass filter of $575\text{to}605\mathrm{nm}$ was used. The tunable filter within the camera was automatically stepped in $10\text{-}\mathrm{nm}$ increments from $650\text{to}950\mathrm{nm}$ maintaining the same exposure time for images captured at each wavelength. Collected images were analyzed by the Maestro software, which uses spectral unmixing algorithms to separate the fluorescence from each of the NIR contrast agents and from background autofluorescence. Then composite images consisting of two spectrally unmixed images representing the distribution of the two optically labeled probes were made. All experiments were performed in triplicate.

2.5.

Image Analysis

For the one-shot injection study, regions of interest (ROIs) were drawn over the liver and a superficial vessel. ROIs were selected based on regions with the least movement and the lowest background signal during the course of the dynamic series of scans, and then the liver-to-vessel signal intensity ratio (SIR) was calculated as the signal intensity of the liver divided by the signal intensity of the vessel using ImageJ software (http://rsb.info.nih.gov/ij/plugins/mri-analysis.html). The dynamics of SIR on the spectrally unmixed images of the two different contrast agents were compared by plotting the SIR values as a function of the time.

For the avidin chase study, ROIs were drawn over the liver and a superficial vessel, and the liver-to-vessel SIR was calculated as the signal intensity of the liver divided by the signal intensity of the vessel in spectrally unmixed images corresponding to the two contrast agents. The SIR values were plotted from $60\mathrm{s}$ before avidin injection to $380\mathrm{s}$ after avidin injection. A comparison of SIR kinetics after avidin injection was made between the GmSA and BSA labeled probes.

3.1.

Spectrally Resolved Two-Color Dynamic Contrast-Enhanced Imaging Simultaneously Visualizes Two Different Protein Conjugates

Spectrally resolved two-color dynamic contrast-enhanced optical imaging was performed after intravenous injection of a mixed solution consisting of BSA-Cy5.5 and GmSA-Cy7. At $40\mathrm{s}$ after injection, the spectrally unmixed BSA-Cy5.5 image and GmSA-Cy7 image demonstrated enhancement of the skin and the superficial vessels [Fig. 1a ]. The BSA-Cy5.5 unmixed images demonstrated enhancement of the skin and the vessels for up to $1000\mathrm{s}$ after injection, but the liver was not enhanced [Fig. 1a]. The spectrally unmixed images after GmSA-Cy7 injection likewise demonstrated strong enhancement of the skin and the superficial vessels $40\mathrm{s}$ after injection, but the enhancement of the skin and the vessels substantially decreased over time. Meanwhile, the liver had enhanced significantly by $1000\mathrm{s}$ .

Fig. 1

Biodistribution of optically labeled BSA and GmSA following intravenous injection. (a) Sequential spectral two-color dynamic contrast-enhanced optical images after BSA-Cy5.5 and GmSA-Cy7. At $40\mathrm{s}$ after injection, the superficial vessels were (arrowheads) enhanced on the spectrally unmixed BSA-Cy5.5 and GmSA-Cy7 images. The liver signal (arrows) gradually increases while the enhancement of the vessels decreases on the unmixedGmSA-Cy7 images. Spectrally unmixed BSA-Cy5.5 images showed minimal enhancement of the liver, while the enhancement of the skin and the vessels persisted for at least $1000\mathrm{s}$ after injection. (b) Sequential optical images after coinjection of GmSA-Cy5.5 and BSA-Cy7. At $40\mathrm{s}$ after injection, the superficial vessels (arrowheads) were enhanced on both spectrally unmixed GmSA-Cy5.5 and BSA-Cy7 images. The liver (arrows) was slightly enhanced at $40\mathrm{s}$ in the BSA-Cy7 image, but the enhancement of the liver decreased at $1000\mathrm{s}$ . Conversely, the GmSA-Cy5.5 images demonstrated gradual enhancement of the liver while the vascular enhancement substantially decreased by $1000\mathrm{s}$ .

The optical labels were then switched (BSA-Cy7 and GmSA-Cy5.5). Using these agents, the unmixed images at $40\mathrm{s}$ after coinjection demonstrated the enhancement of the skin and the superficial vessels [Fig. 1b]. The unmixed images of BSA-Cy7 demonstrated enhancement of the skin and the vessels for up to $1000\mathrm{s}$ after injection, but the liver demonstrated only slight enhancement. By $1000\mathrm{s}$ , the skin and the vascular enhancement remained high. However, on unmixed images of GmSA-Cy5.5, the skin and the vascular enhancement substantially decreased over time, while liver uptake increased substantially [Fig. 1b]. Thus, the biodistribution of the optical probes was dictated by the protein and not by the NIR fluorophore.

To semiquantitatively assess the biodistribution differences between BSA and GmSA labeled with NIR fluorophores, ROIs were drawn on the liver and a superficial vessel, and liver-to-vessel SIR was calculated by ImageJ software (http://rsb.info.nih.gov/ij/plugins/mri-analysis.html) as the signal intensity of the liver divided by the signal intensity of a superficial vessel in spectrally unmixed images (BSA-Cy5.5 and GmSA-Cy7 or BSA-Cy7 and GmSA-Cy5.5). Immediately after coinjection of BSA-Cy5.5 and GmSA-Cy7, the SIR of BSA-Cy5.5 slightly increased but remained practically stable for up to $1000\mathrm{s}$ after injection, while the SIR of GmSA-Cy7 gradually increased, doubling by $1000\mathrm{s}$ (Fig. 2 ). Similar findings were observed when the optical NIR fluorophores were switched and the injected solution contained GmSA-Cy5.5 and BSA-Cy7. Immediately after coinjection of BSA-Cy7 and GmSA-Cy5.5, the SIR of BSA-Cy7 slightly increased but then decreased during the observation period, while the SIR of GmSA-Cy5.5 consistently increased, ultimately increasing tenfold by $1000\mathrm{s}$ after injection (Fig. 2). The liver signal intensity of BSA-Cy7 was higher than BSA-Cy5.5 probably because of better tissue penetration of Cy7 leading to consistently higher liver-to-blood SIRs for BSA-Cy7 compared to BSA-Cy5.5. These findings were consistent among all three mice in each group, in which the same combination of agents was injected.

Fig. 2

Semiquantitative assessment of the biodistribution of BSA and GmSA shows unique behavior. Regions of interest were drawn over the liver and a superficial vessel (left panels; images taken $1000\mathrm{s}$ after coinjection of agents), and the liver-to-vessel signal intensity ratio (SIR) was calculated as the signal intensity of the liver divided by the signal intensity of the vessel on each of the images obtained after injection of a mixture of either BSA-Cy5.5 and GmSA-Cy7 or GmSA-Cy5.5 and BSA-Cy7. The SIRs of BSA-Cy5.5 and BSA-Cy7 demonstrated a slight increase immediately after injection that gradually decreased over $1000\mathrm{s}$ . Conversely, the SIRs of GmSA-Cy7 and GmSA-Cy5.5 demonstrated a gradual increase over $1000\mathrm{s}$ , indicating abundant liver uptake of GmSA-Cy7 and GmSA-Cy5.5.

3.2.

Spectrally Resolved Two-Color Dynamic Optical Imaging Tracks the Fate of Two Optical Probes After Avidin Chase

At $200\mathrm{s}$ after the injection of biotinylated-BSA-Cy7 (b-BSA-Cy7) and nonbiotinylated BSA-Cy5.5, an injection of avidin was administered to clear the b-BSA-Cy7 from the circulation and deposit it in the liver. At $40\mathrm{s}$ after b-BSA-Cy7 and BSA-Cy5.5 injection (prior to avidin), the skin and the superficial vessels were enhanced on the spectrally unmixed images [Fig. 3a ]. At $200\mathrm{s}$ after injection, avidin was administered to selectively remove b-BSA-Cy7 from the circulation and have it bind to the liver (also known as an avidin “chase”). At $40\mathrm{s}$ after avidin injection, the signal intensity of the superficial vessels decreased on the b-BSA-Cy7 image but not the BSA-Cy5 image, while the signal intensity of the liver slightly increased on the b-BSA-Cy7 image. At $400\mathrm{s}$ after avidin injection, the superficial vessels were almost undetectable, while the liver and the spleen were strongly enhanced on the b-BSA-Cy7 images. The signal intensity changes on the BSA-Cy5.5 images were not changed by the avidin chase [Fig. 3a].

Fig. 3

The biodistribution of b-BSA and BSA was imaged before and after an avidin chase. Sequential injections of biotinylated BSA (b-BSA) and nonbiotinylated BSA-conjugated with either Cy5.5 or Cy 7 (BSA-Cy5.5 or BSA-Cy7, respectively) were performed, followed by an avidin injection to “chase” the b-BSA complex. (a) Sequential injections of b-BSA-Cy7, BSA-Cy5.5, and avidin were performed at intervals of $200\mathrm{s}$ . At $40\mathrm{s}$ after injection of b-BSA-Cy7, superficial vessels (arrowhead) and the liver (arrow) were visualized only on the b-BSA-Cy7 image. At $40\mathrm{s}$ after the subsequent injection of BSA-Cy5.5, the superficial vessels were visualized on both the b-BSA-Cy7 image and the BSA-Cy5.5 image. The liver was almost undetectable on both b-BSA-Cy7 images and BSA-Cy5.5 images. At $40\mathrm{s}$ after the injection of avidin, the liver was slightly enhanced while the enhancement of the superficial vessels slightly decreased on the b-BSA-Cy7 image. However, by $400\mathrm{s}$ after injection with avidin, the liver demonstrated marked enhancement, while the superficial vessels were almost undetectable on the b-BSA-Cy7 image. The liver was never visualized on the BSA-Cy5.5 image. The persistent enhancement (yellow arrows) noted on both the BSA-Cy5.5 image and the b-BSA-Cy7 image were due to extravasation of the contrast agents. (b) Sequential injection of b-BSA-Cy5.5, BSA-Cy7, and avidin was performed at intervals of $200\mathrm{s}$ . At $40\mathrm{s}$ after injection of b-BSA-Cy5.5, several superficial vessels (arrowheads) and the liver (arrows) were visualized only on the b-BSA-Cy5.5 image. At $40\mathrm{s}$ after injection of BSA-Cy7, the superficial vessels were visualized on both the b-BSA-Cy5.5 image and the BSA-Cy7 image. The liver was barely detectable on the b-BSA-Cy7 image and the BSA-Cy5.5 image. However, at $40\mathrm{s}$ after injection of avidin, the liver became increasingly higher in signal on the b-BSA-Cy7 image, while the enhancement of the superficial vessels diminished. At $400\mathrm{s}$ after avidin injection, the liver was strongly enhanced, while the superficial vessels were almost undetectable on the b-BSA-Cy7 image. The enhancement of the liver and the skin were minimally changed on the BSA-Cy7 image.

To confirm the independence of the NIR fluorophores labeled to the proteins, Cy5.5-labeled b-BSA (b-BSA-Cy5.5) and Cy7-labeled BSA (BSA-Cy7) were synthesized and the experiment was repeated. At $40\mathrm{s}$ after b-BSA-Cy5.5 injection, the skin and superficial vessel enhancement was noted [Fig. 3b]. At $40\mathrm{s}$ after the additional injection of BSA-Cy7, the enhancement of the skin and the superficial vessels was noted on both spectrally unmixed b-BSA-Cy5.5 and BSA-Cy7 images [Fig. 3b]. Avidin was injected $200\mathrm{s}$ after the BSA-Cy7 injection to clear the b-BSA-Cy5.5 from the circulation. At $40\mathrm{s}$ after avidin injection, the signal intensity of the superficial vessels decreased on the b-BSA-Cy5.5 image and the signal intensity of the liver slightly increased. By $400\mathrm{s}$ , the superficial vessels were undetectable but the signal intensity of the liver markedly increased on the unmixed b-BSA-Cy5.5 images. The signal intensity changes on the BSA-Cy7 images were minimal during the same time period [Fig. 3b]. Thus, the avidin chase removed b-BSA from the circulation and led to its deposition in the liver, a process that could be monitored by in vivo spectral imaging. These findings were consistent within each group ( $n=3$ animals/group), in which the same combination of agents was injected.

To semiquantitatively assess the differences between b-BSA and BSA biodistribution during avidin chase, ROIs were drawn over the liver and a superficial vessel, and the liver-to-vessel SIR was calculated as the signal intensity of the liver divided by the signal intensity of the vessel on spectrally unmixed images using ImageJ software (http://rsb.info.nih.gov/ij/plugins/mri-analysis.html). After avidin injection, the SIR on the unmixed b-BSA-Cy7 image gradually increased, while the SIR on the unmixed BSA-Cy5.5 slightly decreased over the same $400\mathrm{s}$ (Fig. 4 ). Similar results were observed when the fluorophores were switched to b-BSA-Cy5.5 and BSA-Cy7: the SIR on the unmixed b-BSA-Cy5.5 image increased, while the SIR on the unmixed BSA-Cy7 image slightly decreased over the $400\mathrm{s}$ after avidin injection (Fig. 4).

Fig. 4

Semiquantitative assessment of the SIR demonstrates that b-BSA and BSA exhibit different biodistributions after an avidin chase. Sequential injections of b-BSA-Cy7 and BSA-Cy5.5 or b-BSA-Cy5.5 and BSA-Cy7 were performed in two animals followed by an avidin chase. Regions of interest were drawn over the liver and a superficial vessel (left panels; images obtained $400\mathrm{s}$ after avidin injection), and then the liver-to-vessel SIR was calculated. The SIRs of BSA-Cy5.5 and BSA-Cy7 demonstrated a gradual decrease after avidin injection, while the SIRs of b-BSA-Cy7 and b-BSA-Cy5.5 demonstrated a gradual increase over $400\mathrm{s}$ after avidin injection, indicating that b-BSA-Cy7 and b-BSA-Cy5.5 were accumulating in the liver after the avidin chase.