Three-Dimensional Highly Sensitive Diffusion Reflection-Based Imaging Method for the in Vivo Localization of Atherosclerosis Plaques Following Gold Nanorods Accumulation.

In this work, we present a novel, simple, and highly accurate three-dimensional (3D) diffusion reflection (DR) imaging system and method for the detection of accumulation sites of gold nanorods (GNRs) within the tissue. GNRs are intensively used for diagnosis purposes of varied diseases, mainly because of their ability to well absorb visible light, which introduces them as terrific contrast agents in various imaging and theranostics methods. Lately, these GNRs unique absorption properties have served in DR intensity-based measurements, suggesting a novel diagnostic tool, DR-GNRs. In this paper, we show a new measurement system and method for DR, based on its radial collection from the tissue. These radial measurements enabled a unique 3D presentation of the DR-GNR, introducing the dimensions ρ for the radius, θ for the angle, and Γ for the reflected intensity. On the basis of the diffusion model, which enables to correlate between the sample's optical properties and its reflectance, a unique, radial map is presented. This map introduces the slopes of the DR curves in each measured angle, which are linearly correlated with the tissue's optical properties and with the GNRs concentrations within the tissue, thus enables the exact radial localization of the GNRs in the sample. We show the detection of macrophage accumulation in tissue-like phantoms, as well as the localization of unstable plaques in hyperlipidemic mice, in vivo. This highly accurate, powerful technology paves the way toward a real-time detection method that can be successfully integrated in the rapid increasing field of personalized medicine.

Introduction

The development of technologies for in vivo therapeutic and diagnostic applications is essential for enhancing the therapeutic chances of some diseases. Nowadays, the most applied imaging methods in clinical routines are the X-ray,[1,2] computed tomography (CT),[3] positron emission tomography (PET),[4,5] and magnetic resonance imaging (MRI).[6,7] These are powerful, highly successive methods for all body imaging. Still, they present lack of advantages because of ionizing radiation[8] and high power laser intensity (which might cause some thermal effects to the surrounding tissue) and are usually expensive and sophisticated to use. Another intensively used imaging method is the ultrasound (US),[9,10] which is safe and easy to use, yet suffers from limited resolution and penetration depth.[11] A real need exists in the development of additional diagnosis technologies.

Diffusion reflection (DR) spectroscopy is a simple, safe, and easy-to-apply diagnostic technique that has the potential to provide important morphological information regarding biological tissues without requiring high radiation intensities or high penetration depth.[12−15] On the basis of this technique, a pair of source and detector fibers placed along the tissue surface, separated by a distance origin from few millimeters to few centimeters, to enable the DR collection.[16] Previous research studies have suggested that DR measurements can serve for functional diagnosis and therapeutic monitoring of cancer diseases.[17−20] This includes, for example, dynamic near-infrared (NIR) technique, denoted diffuse correlation spectroscopy[21,22] and diffuse wave spectroscopy.[23,24] Still, similar to other NIR spectroscopies, these methods use natural endogenous tumor-to-normal contrasts available in tissues, such as oxy-deoxy and total hemoglobin, water, lipids, and blood,[25] and therefore suffer from relatively low signal-to-noise ratio (SNR) because of the high scattering properties of the tissue in the optics region. Our previous works have suggested an improved SNR of the diffusion-based spectroscopy by the insertion of biocompatible contrast agents into the tissue, to increase the optical contrast between the site of interest and its surrounding.[26] Gold nanorods (GNRs) have served as successful candidates for this purpose, which significantly improved the SNR by the increase of the absorption properties of the damaged site. The GNRs exhibit unique absorption properties in the NIR region, where light penetration through the tissues is relatively high (up to few centimeters). Up to now, the GNR-based DR measurements have proved to be a successful tool for the detection of head and neck cancer,[27,28] oral cancer,[29,30] and atherosclerosis unstable plaques.[31,32]

Till far, DR-GNR measurements were performed in a one-dimensional scanning form, resulting with a decreasing DR curve as a function of the radial distance from the light source.[27] We showed that on the basis of a simple form of the optical diffusion equation, we can correlate between the tissue’s optical properties and the DR curves. In this paper, we present a major achievement of the DR-GNR detection method, introducing a spatial DR method with high sensitivity and improved resolution. The radial measurements enabled the three-dimensional (3D) presentation of the irradiated tissue, introduced by ρ, the radial distance between the light source and the detector; θ, the radial angle; and Γ, the diffused reflected intensity. The 3D presentation enabled the easy and fast identification of the GNR sites within the tissue. We show the detection of GNRs accumulation in tissue-like phantoms; moreover, the precise detection of unstable, macrophage-rich plaques in hyperlipidemic mice, which served as an atherosclerosis model.

Results and Discussion

Spatial Diffusion Reflection of Homogeneous Tissue-like Phantoms

Spatial DR measurements of tissue-like phantoms were carried out using the method described in the Methods section below. The phantoms were irradiated with 650 nm illumination, according to the absorption peak of the GNRs (see Methods, Figure ). Figure introduces the spatial diffuse reflected intensity from phantoms with and without GNRs (basic and GNR phantoms, Figure a,b, respectively). The spatial intensities present a clear difference between the reflectance of the basic and GNR phantoms, whereas the intensities from the basic phantom begin from ∼13 and present a moderate decrease toward 0, and the intensities from the GNR phantom begin from ∼8 and decrease rapidly toward zero intensity. The low initial intensity, as well as its rapid decrease, is due to the absorption of light by the GNRs, providing a clear indication for the GNRs presence in the measured phantom. 3D figures are attached in the Supporting Information file, enabling the simple rotation of the figures.

Figure 8

Absorption spectrum of the GNRs. The absorption peak is at 640 nm.

Figure 1

Spatial diffusion reflection of basic (a) and GNR (b) phantoms. The color bar presents the intensity of the reflected diffusion, whereas the red colors are for higher intensity values and the dark blue colors are for lower values of intensities. The numbers around the circle are for the measured angles, whereas the interval between each two measurements was 30°. The basic phantom presents higher intensities and a very moderate decrease compare to the GNR phantom, enabling the easy and fast identification of the GNRs in tissue-like conditions. At least three repetitive measurements were performed for each sample.

Figure presents a polar plot of the DR intensities of Figure , presented in the logarithmic form ln(ρ2Γ(ρ)). In our previous works, we have introduced the mathematical correlation which best fit between the optical properties of the irradiated tissue and its DR profile measured by our optical setup[27]

Figure 2

Logarithmic form of the spatial DR plotted according to eq . The radial DR of the basic (a) and GNR (b) phantoms presented according to eq . One can notice that using this presentation, the GNR phantom is more distinguishable from the basic phantom, compare to the “classical” DR presentation in Figure . This is because this presentation is linearly correlated with the sample’s optical properties, thus the high absorption of the GNR phantom has high values. (c) Radial distribution of the average μ (resulting from an average of at least three different measurements) for both basic (solid outline) and GNR (dashed outline) phantoms. The angles were measured with intervals of Δθ = 30°, as presented around the circles. The vertical axis is for the μ values. This radial presentation enabled clear distinguishing between the basic and GNR phantoms, as the basic phantom presents low, uniform μ values compare to the GNR phantom effective attenuation coefficients. The GNR phantom μ values are not uniformly distributed because of some inhomogeneous mixing of the GNR in the phantom solution; thus, some regions in the phantom present higher GNR concentrations than others.

While μ is the effective attenuation coefficient, given bywhen ρ is the distance between the light source and the detector, Γ is the reflectance intensity, C1 is a constant which depends on the optical setup apertures, and μ is the effective attenuation coefficient which depends on μa and μs′, the absorption and the reduced scattering coefficients of the irradiated sample, respectively. Presenting the DR in this logarithmic form suggests high correlation with the samples’ optical properties, as shown in Figure . Thus, the resulted spatial curves of the basic and GNR phantoms are more distinguishably compared to the regular intensity-based presentation in Figure . Furthermore, using this presentation, the DR slopes of each specific measured angle were calculated according to eq , resulting with μ, the effective attenuation coefficient, and then were plotted in a radial map. This radial presentation of μ is highly sensitive to the optical properties of the tissue, introducing a very simple way to distinguish between the two irradiated samples. The μ presentation suggests not only a major difference between the basic and GNR phantoms but also their radial distribution within the measured site. Thus, for example, angles 0, 30, and 60 present the highest effective attenuation coefficient values for the GNR phantom (0.66, 0.55, 0.67, 0.66, compare to ∼0.47 in average for the other measured angles), suggesting a region with higher GNR concentration (this is due to some inhomogeneity of the GNRs distribution in the solidified phantom, caused by an unperfect component mixing process).

Spatial Diffusion Reflection of Mixed Tissue-like Phantoms

Identifying GNRs Specific Accumulation Sites

The spatial DR measurements were performed on basic phantoms presenting a GNR region within it (illustrated in Figure a). The purpose of these experiments was to use the novel DR technique for the identification of the GNR site within a phantom, mimicking the identification of macrophages, following their GNR uptake, in the case of an unstable atherosclerotic plaque and can also serve, later, for tumor margin identification. Accumulation of GNRs in an atherosclerotic active plaque is expected based on the known finding that phagocyte cells, including macrophages, can uptake metal nanoparticles[33,34] because macrophages are major components of the unstable, inflammatory active atherosclerotic plaque.[35,36] This experiment was performed with two different mixed phantoms: first, a phantom presenting a GNR spot in its center and second, a phantom with solidified macrophages, following their 24 h incubation with the GNRs.

Figure 3

Spatial DR of a phantom with a GNR accumulation site within it. (a) Illustration of the three different measured regions in the phantom; (i) the basic region, (ii) the GNR spot, and (iii) the mixed area, containing GNR and basic regions in the same scan. The arrows in the figure are to show a sample of the different measurements’ radii, indicating that the GNR site was on the left side of the mixed area (iii). (b) Spatial diffused reflection from the mixed phantom. The three different regions, i, ii, and iii, are according to the regions illustrated in (a). It is well-noticed that the DR of the site (iii) is a composition of figures (i) and (ii), with a specific indication for the GNR region at 210°–330°, as in this radial region, the DR intensities intensively decrease, similar to figure (ii). (c) Radial distribution of the logarithmic DR average slopes, according to eq , for the three different measured regions; the solid outline is for the region (i), the dashed outline is for the region (ii), and the dotted outline is for the region (iii). It is well seen that in angles 210–300, the μ values are very similar to the μ values in region (ii), indicating the GNRs presence in this site. Each sample was measured three times, and the presented slopes are average of at least three different experiments.

GNR Spot within a Basic Phantom

Phantoms with a spot of GNRs (its preparation procedure is described in the Methods section below) were measured using the spatial DR method. Figure a presents different regions that were measured in this phantom: (i) a site with no GNRs, (ii) the GNRs accumulation site, and (iii) a mixed area, which enabled to measure both basic and GNR sites in the same scan. A representative spatial reflection is presented in Figure b. Whereas the (i) and the (ii) scans present the expected DR spatial pattern, similar to the spatial diffusion presented in Figure a,b, respectively, the (iii) scan suggests a mixed DR pattern, composed of the two DR patterns (i) and (ii), as indicated by the arrows. In region (iii), the steep decreasing pattern is in the radial region of angles 240–300, suggesting that the GNRs which are concentrated is in this radial area. The DR slopes polar presentation in Figure c clearly shows the GNR site, as the μ values for 240°, 270°, and 300° are almost identical to the μ values of the GNR phantoms, whereas for the other measured angles, the effective attenuation coefficients were lower (∼0.2 compare to an average slope of 0.5 for the GNR and basic regions, respectively). In the Supporting Information file, the 3D figures can be found, enabling to rotate the DR profile, for the facile location of the GNRs accumulation site in the phantom. We found this radial presentation a very simple way to identify the exact location of the GNRs accumulation site in a phantom.

Macrophages within a Phantom

Macrophages were incubated with GNRs (0.2 mg/mL) for 24 h and then were dissociated from the surface with trypsin and solidified within a phantom. The GNR uptake by the macrophages was then verified by flow cytometry measurements (FCM), and results suggested a nice uptake of GNRs after 24 h of incubation (see Supporting Information Figure S2). The spatial DR of the phantoms’ different regions, with and without macrophages, was measured. Representative DR images are shown in Figure . It is well-noted that the spatial DR pattern of the macrophage accumulation region is different from the DR of the basic phantom. More particularly, the DR from the macrophages presents two major regions with different decreasing patterns: the first is for the angles 60–120, with high initial intensity (∼9 au) that decreases to zero intensity very fast. The second region is for the angles 240–300, where the initial intensity is the lowest, then decreases to zero in intensity (similar to the pattern in Figure b, for the GNR phantom). Figure c presents the slopes that were extracted from the decreasing ln(ρ2Γ(ρ)) curves (can be found in the Supporting Information, Figure S1), for the same intensities profile presented in Figure a. This radial presentation of the slopes, resulting with μ, clearly shows two different sites, indicated as i and ii, in which the macrophages where accumulated, presenting the highest μ values for 60–120 and 240–300 in degrees. These results indicate the GNRs uptake by the macrophages but more important, the highly sensitive detection of the macrophages following their GNRs, in tissue-like conditions. It paved the way for in vivo radial DR measurements of an atherosclerotic vascular disease in hyperlipidemic mice.

Figure 4

Representative spatial DR of a phantom with macrophages following their 24 h incubation with GNRs. The DR of a basic phantom (a) and a phantom with macrophages (b). The inhomogeneity of the macrophages phantom is well seen, especially in comparison to the basic phantom. In addition, the macrophages phantom presented two interesting regions: (i) 60°–120°, in which the intensity starts from high values and decreased to very low values and region (ii) the radial angles of 240–300 in which the initial intensity is very low, an indication to the macrophages presence in this radial region of the phantom. (c) Radial distribution of the average μ values for the basic phantom (solid outline) and the phantom with the macrophages following their GNR uptake (dashed outline). The phantom containing the macrophages presented two regions with effective attenuation coefficients higher than the basic phantom, for the radial region of 60–120 (i) and 240–300 (ii) in angles, emphasizing the different macrophage sites, as shown in (b). The μ values are average of at least three repetitive measurements of three different samples.

Spatial Scanning of Hyperlipidemic Mice Carotids

Atherosclerotic vascular disease in mice was induced by a hyperlipidemic diet, as described in the Methods section. The mice carotids were scanned, noninvasively, before and 24 h post the GNR injection. Figure a,b shows a representative spatial DR, for the normal diet mouse’s carotid (a) and for the hyperlipidemic mouse (b). There is a clear difference between the two spatial DR patterns, whereas Figure a presents higher reflectance intensities, moderately decreasing toward zero intensity, the hyperlipidemic mouse presents intensities very low in values decreasing steeply toward zero. Figure c,d presents the logarithmic form of the reflectance (according to eq above), with high sensitivity to the tissue’s optical properties thus to the GNRs presence in the tissue. The hyperlipidemic mouse presents higher μ values compare to that of the normal diet mouse for almost all measured angles. In Figure e, the polar distribution of the average DR slopes (of at least three different measurements) is presented with higher values for the hyperlipidemic mouse, indicating that a plaque exists in its artery, whereas the normal diet mouse presented much lower μ values. The highest μ values appear in the 120, 180, and 240 radial angles, indicating the radial scanning site in which the atherosclerosis is more developed. For additional validation of the unstable plaque in the hyperlipidemic mouse artery, an ex vivo CT scan was further performed.

Figure 5

The spatial DR intensity map for the healthy (a) and sick (b) mice’s carotids. The hyperlipidemic mouse presents a radial intensity distribution similar to the GNR phantom, presented in Figure b above, with very low intensity values. (c,d) Logarithmic presentation of the DR, for the normal diet mouse (c) and for the hyperlipidemic mouse (d). This presentation is highly sensitive to the tissue’s optical properties; thus, the right panel, for the hyperlipidemic mouse, shows high values, resulting from the increase in the tissue’s absorption because of the GNRs accumulation. (e) Radial map of the DR slopes for the normal (solid outline) and hyperlipidemic (dashed outline) mice. The map presents higher μ for the hyperlipidemic mouse, resulting from the high absorption of the GNRs in the plaque.

An ex vivo CT scan of a hyperlipidemic mouse carotid is shown in Figure . The figure shows the GNRs accumulation in different sites in the artery, 24 h post the GNRs injection, indicating that the hyperlipidemic diet has induced atherosclerosis in the mouse.[37] The CT results provide a proof for the DR in vivo results, which has revealed the GNRs presence in the same artery. Figure S3, in the Supporting Information, illustrates the measurements’ radii over the CT image, suggesting that once the radius of measurement has “met” the GNRs, the DR slope is different. Moreover, according to the CT image, the artery width, and as a sequence, the GNRs accumulation site, was less than 500 μm, suggesting a very good resolution for the DR measurement.

Figure 6

Ex vivo high-resolution CT scan of hyperlipidemic mouse’s carotid. The distortion of the artery indicates the atherosclerosis formation because of the hyperlipidemic diet. The GNRs were accumulated in different sites within the artery, most probably because of their accumulation in macrophages or in other mononuclear cells. The image resolution was 500 in micrometer, and some GNR sites were even less in diameter, indicating the radial DR imaging method ability to track the AS sites in a very high resolution.

In this paper, we aimed to introduce a novel measurement procedure based on the DR imaging system and method, with GNRs as contrast agents. The novel procedure enables a 3D presentation of the DR from the tissue surface and a polar plot for the very easy identification of GNRs accumulation sites in tissue conditions. Similar works have been performed using targeted GNRs as markers in surface-enhanced Raman scattering imaging of living cells[38,39] or for naked eye glucose detection in human urine.[40] However, in contrast to the targeted GNRs used in these and similar works, the DR uses bare, untargeted GNRs for the simple detection of AS. By the extraction of the decreasing intensity slopes, we were able to create a radial map that enables to locate the GNR accumulation sites in a very high resolution. This new method was successfully used for the exact localization of atherosclerosis unstable plaques in a hyperlipidemic mice model.

Other diagnostic methods resolving spatial DR measurements were previously developed based on the extraction and differentiation of the tissue’s chromophores of healthy and sick tissues.[41,42] Thus, for example, O’Sullivan et al.[43] have reported the detection of tumor breast using spatially and temporally modulated light. Similarly, diffuse optical tomography[44,45] was developed, to improve the accuracy of the measured optical properties. Tomography is also critical for the identification of localized heterogeneities such as tumors in tissue by the reconstruction of the spatial distribution of optical/physiological properties at each point (or volume element) in the tissue, from measurements of fluence rate on the tissue surface.[46] These methods are successful for these specific diagnosis purposes, but because they are based on the detection of endogenous chromophores, it uses more sophisticated tools in order to best extract the tissue’s optical properties and avoid low SNR. In our DR-GNR method, we use a very simple measurement procedure, as well as a simple algorithm, for the tissue diagnosis, as the GNRs serve as excellent contrast agents in these kinds of measurements.

The 3D DR-GNR method presented in this paper was successfully used in the detection of AS unstable plaques. Current imaging techniques are limited to detect early ASVD. Different noninvasive methods have been developed in order to detect atherosclerotic disease before it becomes symptomatic. These included imaging techniques such as coronary CT angiography,[47] fluorodeoxyglucose-PET, and MRI.[48,49] While comparing the DR method with other 3D methods used for atherosclerosis detection, our method presents important advantages, such as fast data acquisition, high sensitivity, spatial resolution, and low cost (see in the Supporting Information, Table S1). Other techniques, such as angiography intravascular US, are invasive and widely employed to visualize the inside, or lumen, of blood vessels, with particular emphasis on the coronary arteries.[50]

The current presented spatial DR-GNR method can also be a part of the ongoing increasing field of personalized medicine, which is critical to many aspects of human health. Because this multidisciplinary field of personalized medicine aims to tailor therapeutics to individual patients, on the basis of their exact need and predicted response, the development of novel detection methods which aims to track the recovery progression of a patient, as a response to a certain treatment, is essential. In Figure S4, in the Supporting Information section, we show an ongoing research of us, the recovery of an atherosclerosis unstable plaque using GNRs attached to high-density lipoprotein (HDL). The conjugation between the GNRs and HDL enables the simultaneous detection and therapy of these plaques. Thus, this novel spatial DR-GNR method can serve for the specific tracking of the medical influence of HDL on the patients’ health progression.

Thus, this paper introduces a unique, novel measurement method that enables the simple localization of GNRs accumulation by a radial map presentation. This novel method was here utilized for atherosclerosis detection, but further work should be carried out for its additional application for cancer and other diseases in which GNRs can be targeted and accumulated in the desired site. Moreover, by the development of a scanning tube which will enable the “one-shot” radial scan of the desired site, the way toward the clinical use of this imaging method is paved.

Methods

Diffusion Reflection Measurements

Our noninvasive DR optical technique (NEGOH-OP Technologies, Israel)[51,52] was used for the radial DR measurements. The setup included a laser diode with a wavelength of 650 nm as an excitation source. Irradiation was carried out using a 125 μm diameter optic fiber to achieve a pencil beam illumination. We used a portable photodiode as a photodetector. The photodiode was kept in close contact with the tissue surface to prevent ambient light from entering the detection system and to avoid potential light loss through specimen edges. The distance between the light source and the photodiode is ρ, and the initial distance was ∼1 mm. A consecutive reflected light intensity Γ(ρ) measurement was enabled using a micrometer plate which was attached to the optical fiber. The micrometer plate was moved automatically by incremental steps of 250 μm each. As a result, the reflected light intensity was collected from 20 source–detector distances with ρ varying between 1 and 6 mm. The reflected intensity profile, Γ(ρ) (presented as arbitrary units), was collected using a DAQ (National Instruments, Israel), and the data were processed using the LabView program. At least three repetitive measurements were performed for each sample.

The DR measurements were carried out by a radial scanning of the phantoms’ surface, with intervals of Δθ = 30° in angle (apart the in vivo experiments, in which Δθ = 60° due to some technical limitation regarding the mouse anesthetizing time) between each two measurements. The measurements were performed in a radial distance starting from 1 up to 6 mm from the light source. Measurements have started from 1 mm in order to avoid ballistic photons and to collect diffusive photons only, in order to use optical diffusion equation required for slope extraction. The radial scanning is illustrated in Figure .

Figure 7

Illustration of the radial DR measurement. The light source is located on a single point on the sample’s surface, and the detector is moving in two ways: first, from 1 to 6 mm in distance from the light source and then, from 0° to 360°, in a radial direction, with increments of 30° between every two measurements.

GNR Fabrication

The GNRs were synthesized using the seed-mediated growth method.[53] A solution of GNRs suspended in cetyltrimethylammonium bromide (CTAB) (Sigma-Aldrich, USA) was centrifuged at 11 000g for 10 min, decanted, and resuspended in water to remove excess CTAB. To prevent aggregation and to stabilize the particles in physiological solutions, a layer of polyethylene glycol (mPEG-SH, MW 5000 g/mol) (creative PEGWorks, Winston Salem, USA) was adsorbed onto the GNRs. A 200 μL mixture of mPEG-SH (5 mM) (85%) and SH-PEG-COOH (1 mM) (15%) was added to 1 mL of GNR solution. The mixture was stirred for 24 h at room temperature. The GNR extinction coefficient spectrum was determined using a spectrophotometer, and the resultant extinction peak was 640 nm (see Figure ). The absorption spectrum of PEGylated GNR solution presented the same absorption peak at 640 nm (data not shown).

Macrophage Cell Preparation

Human peripheral blood mononuclear cells (PBMCs) were isolated from BUFFY COAT donated from healthy blood donors (from Sheba, Tel Hashomer Hospital Blood Bank, Ramat Gan, Israel) by density gradient centrifugation on Ficoll-Hypaque. Monocytes were purified by adherence to plastic in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics. PBMCs (106 cells mL) were first seeded into 24-well plates (0.5 mL per well); after 2 h, nonadherent cells were removed by several washes with warm PBS. Freshly isolated monocytes differentiated into macrophages in complete RPMI 1640 supplemented with a human recombinant macrophage colony stimulating factor (100 ng/mL) for 6 days.

Tissue-like Phantom Preparation

Solid phantoms were prepared in order to simulate skin tissues with specific optical properties.[54] The phantoms were prepared using India ink 0.1%, as an absorbing component, and intralipid (IL) 20% (Lipofundin MCT/LCT 20%, B. Braun Melsungen AG, Germany), as a scattering component. All phantoms presented the same ink and IL concentrations. Agarose powder 1% (SeaKem LE Agarose, Lonza, USA) was added to convert solution into a gel. The solutions were heated and mixed at a temperature of approximately 90 °C, while the agarose powder was slowly added. The phantoms were cooled under vacuum conditions to avoid bubbles. The phantoms’ solutions were stirred continuously (except for the period in which they were solidified in a vacuum) to obtain high uniformity.

Five types of solid phantoms were prepared for different measurements: basic phantoms, containing ink and IL only, phantoms presenting 0.2 mg/mL GNRs, phantoms with a spot of 0.2 mg/mL GNRs located within basic phantoms, and phantoms with ∼2 × 106 cells/mL macrophage cells post 24 h of incubation with GNRs. The phantoms with the macrophages were prepared in a 500 μL Eppendorf, to maintain a significant concentration of macrophages within the phantom.

Flow Cytometry (FCM) Analysis of GNR Loading

The GNR uptake by macrophages was analyzed by FCM scatter measurements (FACS Aria III cell sorter, BD, USA). FCM data were analyzed with the FACSDiva software (version 4.0, BD Biosciences, San Jose, CA). The side scatter (SSC) and forward scatter (FSC) were determined simultaneously with the 488 nm blue laser. Macrophages within the purified monocytes were identified by direct immunostaining with FITC-conjugated antihuman antibody directed against CD11b/Mac-1 (Biolegend Inc., San Diego, CA, USA). As an isotype control, IgG-FITC (Biolegend Inc., San Diego, CA, USA) was used.

The laser setup of the FCM included filters/mirrors as followed: SSC/FSC measurements were performed using a 488 nm laser with a 488/10 filter and 655, 502 low pass (LP) mirrors. The GNRs were irradiated with a 633 nm laser with a 660/20 filter and 735 LP mirror.

In Vivo Experiments

The in vivo experiments were performed on C57BL mice with an atherogenic diet for 12 weeks. For the induction of atherosclerosis, 8 week old C57BL mice were fed and maintained on an atherogenic diet containing 15% fat, 1.25% cholesterol, and 0.5% sodium cholate (TD 90221, Food-Tek, Inc) for 12 weeks.[34] As a control set, four C57BL mice were maintained with a normal diet for 12 weeks. The mice were intravenously injected with 6 mg of GNR and maintained on respective diet up to experimentation. Before the DR measurements, the mice were anesthetized by intraperitoneal injection of ketamine/xylazine and fur from their throat side was removed to have a clear scan of carotids, without hurting the animal.