2.1.

Preparation of the IR-676-Doped Micelles

The moderately hydrophobic pentamethine cyanine fluorescent dye IR-676 (1,1′,3,3,3′,3′-hexamethyl-4,5,4′,5′-dibenzoindodicarbocyanine)-iodide [Fig. 1(a)] was selected as a promising candidate, as (1) it has low water solubility (but is still not insoluble), and is moderately soluble in other polar solvents (e.g., lower alcohols) (2) its excitation/emission wavelength maxima differ only marginally from some other widely used “gold standard” NIR fluorophores (Cy5.5, Alexa Fluor 680, etc.), thereby ensuring compatibility with the existing imaging technology and wavelength filters. It has also been previously demonstrated that this dye is an excellent calibration standard for fluorescence tomography systems.²³ IR-676 was obtained in powder form (MW: $610.58\mathrm{g}/\mathrm{mol}$, excitation maximum: 676 nm, emission maximum: 700 nm, Spectrum-Info Ltd., Kyiv, Ukraine). The dye can be easily dissolved in dichloromethane or alcohols.^23,24 Taking into consideration the incompatibility of dichloromethane with most plastic materials, the dye was initially dissolved in methanol at room temperature (RT), and was ultrasonicated then stirred until it formed a clear, deep blue solution with a concentration of $2\times {10}^{-4}\mathrm{M}$. This stable stock solution was either immediately used, or stored at $-20°\mathrm{C}$ protected from light. $100\mu \mathrm{l}$ batches were pipetted into microtubes which were covered with a parafilm membrane pierced by a 27G needle three to four times to allow evaporation of the solvent at a moderate rate. The vials were then centrifuged for at least 60 min in a Speed Vac centrifuge (Savant RVT4104 refrigerated vapor trap, Savant Instruments Inc, Farmingdale, New York), leaving behind a thin film of dried dye at the bottom of the vial, which was stored light protected at 4°C. For the reconstitution, a 5 w/v% solution of the nonionic surfactant Kolliphor HS 15 (Polyethylene glycol-15-hydroxystearate, Sigma-Aldrich) was prepared in sterile deionised water. Kolliphor HS 15 was selected based on its outstanding in vivo safety and previously demonstrated effectiveness for imaging contrast agent formulas such as ICG.^22,25–26 $500\mu \mathrm{l}$ batches of this medium were directly pipetted into the microtubes containing the dye at RT. Complete dissolution of IR-676 could be achieved within 10 to 20 min by vortexing and ultrasonication. The resulting stable solution ($4\times {10}^{-5}\mathrm{M}$) was either immediately used, or stored at 4°C for a maximum of 48 h.

Fig. 1

The structure of IR-676 and the binding ability of the formula to serum proteins. (a) IR-676 displays a pentamethine cyanine backbone structure similarly to other, more common fluorophores (Cy5.5 etc.). As the side chains are single methyl groups, the water-solubility and reactivity of the dye is low. (b) IR-676 fluorescence measurement following native polyacrylamide gel electrophoresis of bovine serum albumin (BSA), fetal bovine serum (FBS), and horse serum (HS) samples incubated with ethanol (20 v/v%) or Kolliphor HS 15 (5 w/v%)-based solutions of IR-676. (c) Fluorescence image of the same gel following protein-staining with Coomassie brilliant blue.

2.2.

Animals

For the experiments, 10 to 12-week-old C57Bl/6 mice (weighing 20 to 25 g) and 5-week-old Wistar rats (weighing 125–150 g) were used, for which the original breeding pairs were obtained from Charles-River Hungary Ltd. (Budapest, Hungary). The animals were bred in the Laboratory Animal House of the Department of Pharmacology and Pharmacotherapy at the University of Pécs, fed with standard rodent chow and water ad libitum, kept at 24°C to 25°C ambient temperature and 12-h light-dark cycles. The studies were approved by the Ethics Committee on Animal Research of the University of Pécs according to the Ethical Codex of Animal Experiments (license No: BA 02/2000-2/2012 and BA02/2000-5/2011).

2.3.

Imaging Protocol

The IR-676-based contrast agent was injected iv. in a dosage of $2\times {10}^{-7}\mathrm{mol}/\mathrm{kg}$ body weight, corresponding to a convenient injectate volume of 100 to $150\mu \mathrm{l}$ for mice or 0.6 to 0.8 ml for rats. The method of delivery was either lateral tail vein (oxazolone- and carrageenan-model) or retro-orbital injection (in the other models). Imaging was performed at several selected time-points with the in vivo imaging system (IVIS) Lumina II (PerkinElmer Ltd., Waltham, Massachusetts) with a 640-nm excitation (535, 570, and 605 for spectral unmixing) and $\sim 700\text{-}\mathrm{nm}$ emission filters, auto exposure time, and a binning factor of 2. Fluorescence was expressed as the total radiant efficiency [$(\text{photons}/\mathrm{s}/{\mathrm{cm}}^2/\text{steradian})/(\mu \mathrm{W}/{\mathrm{cm}}^2)$]. The fluorescence molecular tomography (FMT) 2000 system (PerkinElmer Ltd.) was calibrated using the instrument’s 680-nm laser channel, and a sample solution of $1\times {10}^{-6}\mathrm{M}$ following the instructions of the instrument’s manufacturer. Imaging with the FMT system was performed uniformly 3 h postinjection due to the lower throughput of the three-dimensional (3-D) imaging system using high-resolution acquisition parameters. Fluorescence was expressed as either the calculated amount of fluorophore (pmol) or as surface fluorescence (counts/energy). In the airway inflammation model, the extent of the inflamed area was also expressed in ${\mu \mathrm{m}}^3$. Animals were anesthetized for the injection and imaging with ketamine-xylazine ip. (dosage: $100:5\mathrm{mg}/\mathrm{kg}$). During anesthesia, body temperature was maintained by a heating lamp and vital signs were monitored by observation until recovery. Home cage activity after recovery was also observed.

2.4.

Interaction with Serum Proteins

To determine whether IR-676 binds to serum proteins in its freely dissolved or encapsulated state, ethanol-based (20 v/v%) or micellar (5 w/v%) solutions ($10\mu \mathrm{l}$, $4\times {10}^{-5}\mathrm{M}$) of the dye were incubated with bovine serum albumin (BSA) ($0.5\mathrm{mg}/\mathrm{ml}$), fetal bovine serum (FBS), and horse serum (HS) ($10x$ dilution) samples ($100\mu \mathrm{l}$) at RT. Additionally, the encapsulated dye was also incubated in an equal amount of deionised water. $10\mu \mathrm{l}$ batches of the samples were mixed with an equal amount of loading dye (bromophenol-blue solution) and $10\mu \mathrm{l}$ of the mixtures were pipetted into the gel. To preserve the physiologic protein structure and potential nonspecific binding, samples were separated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (100 V, 1.5 h, Tris/glycine buffer).²⁷ Gels were scanned using the IVIS Lumina II, then stained with Coomassie brilliant blue and imaged again using the same settings (Note: the fluorescence spectra of the two dyes overlap, and the methanol-based Coomassie-staining procedure removes IR-676 from the gel).

2.5.

In Vitro Toxicity Screening

The biological safety of the formula was evaluated on the Chinese Hamster Ovary (CHO-K1) cell line. Cells were trypsinized, washed, and resuspended in PBS at a density of $2\times {10}^5/\mathrm{ml}$. 50 or $100\mu \mathrm{l}$ batches of the imaging formula or the vehicle alone were added to 1 ml amounts of cells; thereafter, the cells were incubated for 5 min at 37°C, pelleted by centrifugation at 1000 rpm for 5 min, after which they were resuspended in 1 ml PBS. Cell viability was evaluated by the fluorescein diacetate-propidium iodide (FDA/PI) assay,²⁸ as $500\mu \mathrm{l}$ batches of each sample were stained with either FDA ($1\mu \mathrm{l}$ of an 500-mM stock solution in dry DMSO) or PI ($5\mu \mathrm{l}$ of 0.75-mM stock solution) and incubated for 10 min. Viable cells fluoresce in green as they enyzmatically convert FDA to fluorescin, whereas dead cells fluoresce in red as PI stains the nuclei after passing through the damaged cell membranes. $2\times {10}^4\text{cells}/\text{cohort}$ were evaluated by a CyFlow flow cytometer (Partec Ltd., Münster, Germany). Untreated and 6% formalin fixed cells were used to set the gates for living and dead cells, respectively. The percentages of viable and nonviable cells were calculated by identical gating for each sample.

2.6.

Preparation of the Frozen Ear Sections for Microscopy

The samples were embedded in a frozen section compound (FSC 22, Surgipath Ltd., Richmond, Illinois), immediately frozen in liquid nitrogen, and stored at $-80°\mathrm{C}$. The samples were cryosectioned ($8\mu \mathrm{m}$), and imaged within 24 h by an Olympus FluoView 1000 confocal laser scanning fluorescence microscope (Olympus, Center Valley, Pennsylvania). Single optical sections were taken from areas of interest using a $40\times$ objective.

2.7.

Oxazolone-Induced Contact Dermatitis Mouse Model

The ear model of oxazolone-induced allergic contact dermatitis was previously validated as a robust, reliable disease model, leading to localized edema and sensitization, as well as inflammatory cell activation primarily mediated by Th1 cell response.²⁹ Moreover, it has been recently demonstrated as a good proof-of-concept model to test potential NIR tracers of vascular leakage.³⁰ Anesthetized mice were sensitized on two consecutive days by applying $50\mu \mathrm{l}$ 2% oxazolone (Sigma-Aldrich) onto the shaved abdomen. Six days later, $15-15\mu \mathrm{l}$ of 2% oxazolone was applied on both the inner and outer surfaces of the right ear, whereas the left ear was treated with the vehicle only (96% ethanol) to serve as control. The oxazolone treatment induces a marked hyperemia and edema within hours, accompanied by leukocyte influx and T cell infiltration. Subjects were injected with the contrast agent 24 h later, and were repeatedly imaged between 5 to 180 min postinjection with the IVIS Lumina II. Thereafter, the mice were sacrificed, and ears were collected for cryosection histology.

2.8.

Lipopolysaccharide-Induced Acute Airway Inflammation Mouse Model

The LPS model is a reliable tool to investigate acute inflammatory changes of the airways and lungs, and has been validated and optimized by our workgroup before.³¹ In the present series of experiments, a modified, direct intratracheal delivery was used, since our preliminary experiments demonstrated that the conventionally used intranasal route produces far too high intra-group variations for in vivo imaging, Mice were anesthetized, and a PE-10 cannula was carefully introduced into the trachea via the mouth by a novel technique allowing simple visualization of the vocal cords.³² The airway inflammation was induced by intratracheal instillation of $60\mu \mathrm{l}$ PBS in which $100\mu \mathrm{g}$ dry, purified Escherichia coli (serotype: 083) endotoxin was dissolved while controls received PBS. 21 h after LPS treatment, the mice were injected with the contrast agent, and they were imaged 3 h later by the FMT 2000 imaging system.

2.9.

K/BxN Serum-Transfer Arthritis Mouse Model

The K/BxN serum-transfer model is a reliable immune-mediated model of rheumatoid arthritis, which is based on the transfer of autoantibody-rich sera from the spontaneously arthritic K/BxN mice into healthy recipients, thus eliciting a transient polyarthritis. Sera were obtained from the K/BxN mice, pooled, and stored at $-80°\mathrm{C}$ as described earlier.³³ To induce a systemic polyarthritis, mice were injected ip. with $300\mu \mathrm{l}$ serum diluted to 1 ml with sterile PBS. Subjects were injected 7 days after arthritis induction, and were imaged 3 h postinjection using the FMT 2000 system.

2.10.

Carrageenan-Induced Acute Paw Inflammation Model of the Rat

This model was selected to evaluate the feasibility of in vivo imaging of larger species with this contrast agent. Carrageenan is a complex polysaccharide which elicits a massive edema formation, local hyperemia, and consequent leukocyte accumulation, thus serving as a reliable model of acute inflammation.³⁴ Wistar rats under anesthesia received an intraplantar injection of $100\mu \mathrm{l}$ 1% carrageenan (Sigma-Aldrich) in the left hindpaw, while the right paw was injected with an equal volume of saline. Two hours later, the animals were injected with the contrast agent, and were imaged 60 and 120 min postinjection with the IVIS Lumina II.

2.11.

Statistical Analysis

Results are expressed as mean±standard error of the mean. Statistical evaluation was performed by Graphpad Prism®. The imaging results in the oxazolone model were evaluated by two-way ANOVA with Sidak’s multiple comparisons test, while other imaging results were achieved by Student’s two-tailed $t$-test. At least $p<0.05$ was considered significant.

3.1.

The IR-676-Based Formula Demonstrates Negligible Binding to Serum Proteins

The nondenaturing-PAGE demonstrated that IR-676 binds weakly if at all to serum proteins regardless of being present as freely dissolved (ethanol) or encapsulated (Kolliphor HS 15). As strong fluorescence was exclusively observed in the wells where micellar IR-676-containing samples were pipetted, which did not propagate in the gel, we can conclude that the formula did not disintegrate even when exposed to strong electric current, and its size prevented it from entering into the gel [Figs. 1(b) and 1(c)].

3.2.

The Dye Formula Has No Impact on Cell Viability in the Fluorescein Diacetate-Propidium Iodide Acute Toxicity Assay

Exposing CHO-K1-cells to two concentrations of the formula or the vehicle alone exerted no apparent acute toxic effects. The percentage of viable and dead cells showed minimal fluctuations between study groups (Table 1). As the higher concentration employed ($100\mu \mathrm{l}$ $\text{formula}/1\mathrm{ml}$ cell suspension) exceeded even the expected peak plasma concentration upon intravenous injection, we assume that the present formula poses minimal if any risks of toxicity within its suggested operational range.

Table 1

Fluorescein diacetate/propidium iodide (FDA/PI) toxicity assay results. Chinese Hamster Ovary (CHO-K1) cells (1 ml of a 2×105  cells/ml suspension) were incubated in the presence of the dye formula or the vehicle alone, and were subsequently stained with FDA (stains viable cells) or PI (stains dead cells) to assess their viability. 2×104  cells were evaluated by flow cytometry to represent each experimental condition.

Although no formal in vivo toxicity tests were performed, we carefully observed IR-676-treated animals to assess any major untoward effect. No lethality occurred throughout the study and no observable pathological sign was ever found upon close inspection of the animals during and after anesthesia. Heart rate, as well as respiratory frequency and depth remained regular, and no changes in skin color and visible mucous membranes were seen. Dye-injected animals regained consciousness normally and afterward showed normal home cage activity (locomotion, eating/drinking, climbing, and grooming). Based on these observations and the in vitro cytotoxicity data, we can conclude that the potential in vivo acute toxicity of the dye is of minor significance.

3.3.

IR-676-Doped Micelles Selectively Accumulate in Areas Affected by Chemically Induced Contact Dermatitis in Mouse Ear

Immediately 10 min postinjection, the contrast agent yielded a more than 260% greater signal in the inflamed ears than on the contralateral noninflamed side. Moreover, the signal strength showed minimal variations in the vehicle-treated ears, demonstrating the importance of body weight-based dosage. In the following 3 h, the signal strength slowly decreased in the control ears indicating the gradual clearance of the dye-loaded micelles from the circulation. At 3 h, the fluorescence intensity in the affected ears was more than 650% higher than in the controls, pointing out the prolonged presence of the extravasated dye molecules in diseased tissues [Figs. 2(a) and (b)]. The confocal fluorescence microscopic examination supported these findings, showing a patchy accumulation of the fluorophore in the perivascular regions within oxazolone-treated ears [Fig. 2(c)].

Fig. 2

Oxazolone-induced acute dermatitis on the mouse ear. (a) Representative in vivo epifluorescence images taken 10 and 180 min following intravenous injection of the formula. (b) The changes of fluorescence intensity in the 96% ethanol-treated control and oxazolone-treated inflamed ears throughout the measurement ($n=7$, ****$p<0.0001$). (c) Representative confocal fluorescence microscopy images of the control and inflamed ears harvested 3 h postinjection clearly demonstrating the tissue localization of the dye (Note: the autofluorescence of the ear cartilage is shown to provide visual aid).

3.4.

Imaging of Deep-Tissue Inflammation of the Mouse Airways

The 3-D tomographic imaging of the LPS-treated mice 3 h postinjection demonstrated considerably greater signal strength in the areas corresponding to the airways. Whereas in the thoracic area of PBS-treated control mice, the calculated amount of IR-676 was only 15 to 20 pmol, LPS-treated mice displayed a 120 to 130 pmol signal strength in the respective regions [Figs. 3(a) and 3(c)]. Additionally, the area of increased plasma extravasation was significantly greater in LPS-treated mice than in the controls (on average 700 versus $200\mu {\mathrm{m}}^3$) demonstrating that a diffuse region was affected by the LPS-induced pulmonary inflammation [Figs. 3(b) and 4]. IR-676 showed a strong accumulation in the liver, similarly to other cyanine dyes. This, however, could be easily demarcated from the specific signal (despite being at least a magnitude greater) in the 3-D tomographic scans. In contrast, it was not possible to separate the specific signal from the liver signal in the 2-D epifluorescence scans [Fig. 3(d)] due to the scattering of the latter.

Fig. 3

Endotoxin-induced acute airway inflammation of the mouse. (a) The measured total amount of fluorophore (pmol) in the thoracic region 24 h following LPS- or PBS-challenge, 3 h after the injection of the probe. (b) The tissue volume (${\mu \mathrm{m}}^3$) affected by increased dye-accumulation. (c) Representative three-dimensional (3-D) fluorescence tomographic reconstructions. (d) Conventional epifluorescence images of the same mice for comparison. ($n=6/\text{group}$, **$p<0.01$, ***$p<0.001$).

Fig. 4

Microcomputer tomography-fluorescence tomography co-registration of an LPS-treated mouse. The animal was immediately transferred upon completion of the FMT-scan into the micro-CT (Skyscan 1176, Bruker, Kontich, Belgium) remaining within the imaging cassette, where a CT-imaging of the respective region was performed. The scans were individually reconstructed, and were merged together using the AMIDE software based on three-dimensional fiducial markers present on the imaging cassette. (Video 1, MPEG, 0.92 MB) [URL: http://dx.doi.org/10.1117/1.JBO.20.1.016022.1].

3.5.

Visualization of Immune-Mediated Polyarthritis of the Mouse Ankle Joints

In the control mice, the fluorescence in the hindlimbs was negligible 3 h after the injection (less than 1 pmol or not quantifiable) and was not localized in the ankle joints. In comparison, the K/BxN serum-treated mice displayed a circumscribed, increased signal accumulation ($\sim 12\mathrm{pmol}$ on average) corresponding to the region of the tibiotarsal joints (affected principally by the disease), in sharp contrast to their controls [Figs. 5(a) to 5(c) and 6]. Conventional 2-D-scans of the same set of mice yielded similar results [Figs. 5(b) to 5(d)].

Fig. 5

K/BxN serum-induced chronic polyarthritis of the mouse. (a) The total amount of fluorophore in the ankle joints of intact and K/BxN serum-treated mice, 7 days after disease induction and 3 h following the probe injection. (b) Fluorescence intensity calculated on the basis of the conventional epifluorescence scans in the same joints. (c) Representative three-dimensional representations and (d) epifluorescence images of the same mice (Note: the numbering of the tail by a permanent marker created artifacts on the 2-D-scans). ($n=6-7/\text{group}$, ****$p<0.0001.$).

Fig. 6

Micro-CT-fluorescence tomography coregistration of an arthritic mouse. (Video 2, MPEG, 0.92 MB) [URL: http://dx.doi.org/10.1117/1.JBO.20.1.016022.2].

3.6.

Imaging Acute Carrageenan-Induced Paw Edema in the Rat

The accumulation of signal in the treated hindpaws of the rats was clearly visible both 1 and 2 h postinjection, the signal strength being 50% and 80% greater than in the sham-treated control paws. The observed lower contrast is potentially caused by the circumscribed, local inflammation around the site of carrageenan-injection which leaves most of the paw unaffected. Thus, only a small part of the paw becomes actually inflamed, causing a comparatively modest signal surplus over the nonspecific ambient background [Figs. 7(a) and 7(b)].

Fig. 7

Carrageenan-induced acute paw inflammation in the rat. (a) Fluorescence intensity in the saline and carrageenan-treated hindpaw (IV injection performed 2 h after disease induction), measured by epifluorescence imaging 60 and 120 min postinjection. (b) Representative images of the vehicle (right) and carrageenan-treated (left) hindlimbs (Note: the tail was covered to prevent possible interference caused by the concentrated dye present on the site of the IV injection). ($n=4$, **$p<0.01$)