Onâ Nanopomegranate for In Vivo

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A Self-Assembled “Off/On” Nanopomegranate for In Vivo Photoacoustic and Fluorescence Imaging: Strategic Arrangement of Kupffer Cells in Mouse Hepatic Lobules Qiaoya Lin, Deqiang Deng, Xianlin Song, Bolei Dai, Xiaoquan Yang, Qingming Luo, and Zhihong Zhang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b07283 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 4, 2019

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A Self-Assembled “Off/On” Nanopomegranate for In Vivo Photoacoustic and Fluorescence Imaging: Strategic Arrangement of Kupffer Cells in Mouse Hepatic Lobules Qiaoya Lin† § ‡, Deqiang Deng† § ‡, Xianlin Song† §, Bolei Dai† §, Xiaoquan Yang† §, Qingming Luo† §* & Zhihong Zhang† §* †Britton

Chance Center for Biomedical Photonics, Wuhan National Laboratory for

Optoelectronics-Huazhong University of Science and Technology, Wuhan, Hubei, 430074, China §MoE

Key Laboratory for Biomedical Photonics, Collaborative Innovation Center for Biomedical

Engineering, School of Engineering Sciences, Huazhong University of Science and Technology, Wuhan, Hubei, 430074, China E-mail: [email protected], [email protected] KEYWORDS: macrophages, nanoparticles, dual-modality imaging, distribution, phagocytic and degradative function

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ABSTRACT:

Kupffer cells (KCs), potent scavenger cells located in hepatic sinusoids, constantly phagocytize and degrade foreign materials to maintain metabolism and clearance. Understanding the strategic KC arrangement which link to their spatial location and function in hepatic lobules, the basic functional unit in the liver, is highly valuable for characterizing liver function. However, selectively labeling KCs and characterizing their function in vivo remains challenging. Herein, a fast self-assembled pomegranate structure-like nanoparticle with “nanopomegranate seeds” of dye aggregate has been developed, which has dual-modality “off/on” capability. This nanopomegranate shows good photostability, a high extinction coefficient, a high KC labeling efficiency (98.8%) and better visualization of KC morphology than commercial FluoSpheres. In vivo photoacoustic (PA) and fluorescence imaging consistently visualize that KCs are strategically distributed along the central vein (CV)-portal triad (PT) axis in each liver lobule: more and larger KCs exist in areas closer to the PTs. The high-resolution PA quantitative data further revealed that the density of KCs was linearly dependent on the rn/rmax ratio (their relative location along CV-PT axis) (R2=0.7513), and the KC density at the outermost layer is almost 246-fold that at the innermost layer (Each layer: 8 μm). Notably, the phagocytic ability of KCs located in layers with rn/rmax ratios of 0.167-0.3 varies in a zig-zag pattern, as evidenced by their different PA intensities. Additionally, the fluorescence imaging quantitation suggests similar fluorescence activation of nanopomegranate in KCs. Nanopomegranates combined with dual-modality imaging reveal the strategic arrangement of KCs in vivo, greatly extending our understanding of liver physiology.

The liver is a heterogeneous tissue made of functional units called hepatic lobules. The lobules (diameter: sub-millimeter in mice) consist of hepatocyte plates, portal triads (PTs) at the periphery


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and a central vein (CV) in the center, and the blood in liver sinusoids (diameter: 7-14 m in mice) moves along the PT-CV axis.1-3 Kupffer cells (KCs), which constitute the largest population of tissue-resident macrophages in the entire body and are located in liver sinusoids, are potent scavenger cells that constantly phagocytize and degrade cellular debris or foreign materials, thus performing a vital function in maintaining metabolism and clearance.4 A detailed understanding of the strategic arrangement of KCs (e.g., cell density, spatial distribution, phagocytic function as well as their relationship) in hepatic lobules will provide great value for characterizing liver function and evaluating its physiological and pathological status. Ultrasound imaging has been used to evaluate KC phagocytic function with Sonazoid (microbubble) contrast agent, but the resolution (0.5~1 mm) is too low to differentiate cells.5, 6 High resolution fluorescence imaging via confocal or two-photon laser scanning microscopy with dye-conjugated anti-F4/80 antibody7, 8 or 0.5-μm fluorescent latex (Lx) particles9 has also been utilized to visualize KCs in vivo. However, the imaging field of view and depth limit the ability of this technique to visualize the complex structure of hepatic lobules.7,

8

Optical-resolution

photoacoustic microscopy (OR-PAM) has attracted much attention since OR-PAM provides optical absorption contrast with high spatial resolution, a large imaging field of view and relatively deep tissue penetration. OR-PAM has been successfully used for in vivo anatomical imaging of single capillaries due to the strong absorption of endogenous hemoglobin and for functional imaging with exogenous absorption contrasts.10-13 Therefore, OR-PAM will be a powerful tool for the fine structural imaging of hepatic sinusoids, and holds great promise for visualization of KCs in the hepatic lobule with suitable absorption contrast agents. However, to indicate both phagocytic and degradative function of KCs, the single photoacoustic imaging based on photoacoustic contrast agent is far from enough.


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Some “off/on” activation nanoparticles,14-16 such as cyanine dye-based nanoparticles,17-20 porphyrin-based nanoparticles,21-25 squaraine dye-based nanoparticles26,

27

and semiconducting

polymer nanoparticles,28-30 have been used for dual photoacoustic (PA) and fluorescence imaging.31 However, these reported “dual-modality off/on” agents could not selectively label KCs and characterize their function. Thus, to investigate the strategic arrangement of KCs in vivo, the ideal probe should not only have high selectivity for KCs but also possess excellent optical properties and a “dual-modality off/on” switching capability to reflect the phagocytic and degradative ability of KCs. Specifically, it can provide imaging feedback of particles uptake by PA imaging, and particles disassociation by the fluorescence activation over time. As is well known, KCs can phagocytose and then degrade materials with a size of several hundreds of nanometers.32, 33 In this work, to visualize the detailed distribution and function of KCs in terms of hepatic lobule structure in vivo, we developed a fast (one minute) self-assembled nanoparticle with a pomegranate-like structure, termed a nanopomegranate. In this nanostructure, almost hundreds of thousands of 4~5 nm “nanopomegranate seeds” loaded with DiR-BOA, a bisoleate-modified near-infrared (NIR) fluorophore, formed ~400 nm spherical nanoparticles. The nanopomegranate has excellent optical properties with a high extinction coefficient, good photostability and biocompatibility, and a high hepatic signal-to-noise ratio and possesses a “dualmodality off/on” switching capability, which is suitable for in vivo dual-modality photoacoustic and fluorescence imaging. In intact nanopomegranates, DiR-BOA is tightly packed in the “nanopomegranate seeds”, and its fluorescence is completely self-quenched, a state denoted as a “dark” nanopomegranate with an “off” fluorescence signal and an “on” photoacoustic signal. Upon disassociation, the nanopomegranate becomes a “bright” nanopomegranate, as DiR-BOA is released to switch the fluorescence signal “on” and gradually “turn down” the photoacoustic signal


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(Figure 1a). Most importantly, intravenously administered nanopomegranates are exclusively selective for KCs, thus enabling specific dual-modality imaging of KCs in vivo. Therefore, nanopomegranates with structure-modulated dual-modality (fluorescence and photoacoustic imaging) switching “off/on” capability, enable not only for visualizing the KC distribution but also for characterizing their phagocytic function with fine spatial information in vivo. Based on the KC characterization, we illustrated the strategic arrangement of KCs in hepatic lobules, which has great implications for our basic understanding of liver physiology. RESULTS AND DISCUSSION


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Figure 1. Characterization of the nanopomegranate. a) Schematic representation of the nanopomegranate suitable for dual-modality PA and fluorescence imaging. b) Electron micrographs of stained nanopomegranates. c) Photographic images and DLS size profiles of the nanopomegranate. d) Wide-field fluorescence imaging of the nanopomegranate in the intact (left, “dark” nanopomegranate) versus disrupted (right, “bright” nanopomegranate) state (with detergent). e) Fluorescence emission of the nanopomegranate in the intact (red) versus disrupted


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(blue) state. f) Absorbance of the nanopomegranate in the intact (red) versus disrupted (blue) state. g) Normalized photoacoustic amplitudes of the nanopomegranate excited at 744 nm as a function of mass concentration. h) Photostability of the indicated groups versus laser scanning time.

Without any template, nanopomegranates formed spontaneously and rapidly (one minute) via gently dissolving the near-infrared (NIR) hydrophobic dye DiR-BOA34 in a DMSO/PBS solution (v:v=1:80). A transmission electron microscope (TEM) image showed that DiR-BOA (250 μM) self-assembled in the solution, resulting in particles with a size of hundreds of nanometers: nearly hundreds of thousands of “nanopomegranate seeds” (4.35±0.16 nm) formed spherical aggregates (diameter of 399.1±38.7 nm) with one layer of empty nanovesicles (diameter of 13.87±0.62 nm) covering the outer sphere (Figure 1b). The intact nanopomegranate size of approximately 400 nm was also confirmed by the dynamic light scattering (DLS) size profile (Figure 1c). As shown in Figure 1d, e, the results of fluorescence intensity and spectrum measurements indicated that the nanoscale intact pomegranate-like structure enabled a “dark” nanopomegranate with extremely strong DiR-BOA fluorescence quenching (at 99.74%). Fluorescence with a peak at 796.5 nm was successfully liberated when the packed structure of the nanopomegranate was disrupted in a 0.5% solution of Triton X-100. The quantum yield of nanopomegranate after breakdown by using Triton-X100 was 0.49, which is higher than many NIR cyanine dyes, such as indocyanine green (ICG) and IR-783.35, 36 As shown in Figure S1a in the nanopomegranate formulation (supporting information), when the DiR-BOA amount in the DMSO/PBS mixed solution (200 μl) was increased from 0.2 μg to 100 μg, the nanopomegranate size increased from 137.05±9.32 nm to 533.93±28.6 nm, and all formulations remained in the “dark” state, as evidenced by negligible fluorescence signal of DiR-


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BOA (Figure S1b, supporting information). For very low DiR-BOA amount of 0.002~0.02 μg, the formulation was not detectable with DLS. Considering the size dependence of phagocytosis by macrophages, we chose the formulation with 50 μg DiR-BOA and a size of ~400 nm for all further experiments. Once the nanopomegranate was formed, the size of nanoparticles (~400 nm) won’t change by continual dilution. To check the robustness of this simple, fast self-assembly method, we applied other hydrophobic dyes, such as Fluo-BOA37 and DiR to make nanopomegranates. A similar pomegranate-like structure was formed by using these hydrophobic dyes. Fluo-BOA and DiR also possessed a high fluorescence quenching efficiency (90.72% and 99.93%, respectively) but a smaller size (~300 nm and ~270 nm, respectively) than DiR-BOA at the same concentration of 250 μM (Figure S2a-d, supporting information). Hydrophilic dyes such as ICG and IR-783 did not form any nanostructures by the same procedure. Thus, the similar structure between the nanopomegranates formed with hydrophobic dyes (e.g., DiR-BOA, Fluo-BOA, and DiR) suggests that the aggregation of DiR-BOA in the DMSO/PBS mixed solution may occur through hydrophobic interactions15 and that this simple, fast, template-free self-assembly method may have high broad applications in the construction of organic dye aggregate-based supramolecular nanostructures. To check the photoacoustic property of the nanopomegranate, the OR-PAM technique with high resolution (1-2 m) and a millimeter-sized scan range established in our lab previously13, 38, 39

was utilized in this study. As shown in Figure 1f, the “dark” nanopomegranate had strong

absorbance in the NIR region from 655-900 nm and weak absorbance from 474-531 nm, which is suitable for parallelly acquisition of PA signals from both nanopomegranates (excited with a 744nm pulsed laser) and hemoglobin (excited with a 523-nm pulsed laser) in the liver with good signal-to-noise and contrast. The PA signals of “dark” nanopomegranates in the PBS solution were


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linearly dependent on the concentration (R2=0.999) from 0-500 μg mL−1 (Figure 1g). Importantly, the estimated extinction coefficient of the intact nanoparticles was extremely high (ε764nm = 2.13×1011 M-1cm-1) and was comparable to that of most gold nanostructures. To test the photostability of nanopomegranate, we use a 744-nm pulsed laser to irradiate samples multiple times (0-40). The nanopomegranate treated with detergent was set as a negative control without a PA signal. As shown in Figure 1h, no significant PA signal attenuation was observed in the nanopomegranate group, while the PA signal of ICG, an FDA-approved NIR dye also used as a PA contrast agent, gradually decreased with scanning time. Thus, the excellent optical properties of “dark” nanopomegranates make it highly promising for in vivo hepatic photoacoustic imaging.


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Figure 2. Evaluation of the fluorescence activation ability of nanopomegranates in vitro. a) Confocal imaging showing time-dependent fluorescence activation of nanopomegranates (5 μM DiR-BOA concentration) in Raw 264.7 cells 2 h after incubation. b) Representative flow cytometry histograms of nanopomegranate fluorescence activation at different time points in Raw 264.7 cells (top panel), which were gated from living cells as shown in the flow cytometry plots (bottom panel). c) Quantitative results of the normalized photoacoustic amplitudes (left Y axis) and percentage of fluorescent cells (right Y axis) among total Raw 264.7 cells detected at different time points 2 h after incubation with nanopomegranates. d) Photoacoustic imaging and fluorescence imaging of nanopomegranates incubated with 10% serum in a gel phantom for the different indicated time points; the control is the nanopomegranates alone. After characterizing the physicochemical properties of nanopomegranates, we used the Raw 264.7 mouse macrophage cell line to evaluate the intracellular fluorescence activation of nanopomegranates. Raw 264.7 cells were incubated with nanopomegranates for 2 h and then rinsed with PBS to remove excess nanopomegranates. Confocal images taken at different time points are shown in Figure 2a. The nanopomegranate fluorescence was minimally activated in Raw 264.7 cells before 12 h; starting from 18 h, the nanopomegranate fluorescence gradually increased. To further quantify the dynamic dual-modality imaging with “off/on” switching in Raw 264.7 cells, a flow cytometry assay (Figure 2b) and the OR-PAM system were utilized to measure the timedependent fluorescence activation and PA signal attenuation, respectively, under the same regimen as described above for confocal imaging. As shown with the quantitative data in Figure 2c, the percentage of Raw 264.7 cells with fluorescence activation gradually increased from 11.06% at 2 h to 28.57% at 12 h while the PA signal was relatively stable. From 18 h to 48 h, the fluorescence activation percentage increased from 39.23% to 51.73% while the PA signal dramatically


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decreased from 88.65% to 23.73%. We also noticed that there was no further fluorescence increase between 24 h and 48 h while the PA signal sharply decreased. This phenomenon might be induced by a complex process of nanopomegranate digestion in macrophages. During nanopomegranate degradation, free NIR dyes were released from the large spherical nanoparticles. On the other hand, the released NIR dyes might be digested and cleared from cells over time. Moreover, we verified the biocompatibility of nanopomegranates by the MTT assay and its stability in serum by dualmodality imaging. The data showed no obvious toxicity in Raw 264.7 cells incubated with nanopomegranates for DiR-BOA concentrations of 3.125 μM to 25 μM (Figure S3a, supporting information). And the cytotoxicity of the free DiR-BOA solution to Raw 264.7 cells in vitro showed a slight increase compared to the nanopomegranate solution (Figure S3b, supporting information), the data suggested that the nanopomegranate shielded DiR-BOA, which reduced the toxicity. The nanopomegranates were relatively stable in 10% serum at 37 °C for 4 h, as evidenced by the maintenance of a high 89.5% photoacoustic signal and a weak fluorescent signal (Figure 2d), indicating that most of nanopomegranates maintained their integrity before being taken up by macrophages in vivo. We also confirmed that the nanopomegranates were quite stable in 10% FBS, PBS, or water; the intact structure persisted in the fluorescence “off” state even at 24 h of incubation, as evidenced by its undetectable fluorescence (Figure S4, supporting information). Thus, these nanopomegranates with excellent biocompatibility and moderate stability are a sensitive indicator that can reflect the phagocytic and degradative function of macrophages in vivo.


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Figure 3. In vivo evaluation of the specific uptake ability of nanopomegranate by KCs. a) In vivo spinning-disk confocal imaging of a mouse liver with intravenous administration of nanopomegranates (200 μl, DiR-BOA concentration of 250 μM) at 2 h post injection. Anti-BV421F4/80 antibodies were injected 10 min before imaging. b) Flow cytometry analysis including the


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gating strategy (top panel) and the mean fluorescence intensity (MFI) of nanopomegranate uptake in KCs (F4/80highCD11bint), LSECs (CD45-CD146+), and monocyte-derived macrophages (CD11bhighly6Chigh and CD11bhighly6Clow). c) Representative flow cytometry histograms of PBS (blue) versus the nanopomegranate (2 h after injection, red) showing the ratio of nanopomegranatepositive KCs (top panel) and a confocal fluorescence image of nanopomegranate-labeled KCs (white arrow) located at hepatic sinusoids (yellow arrow) with a 60× objective. Next, we investigated the labeling efficiency of nanopomegranates and their specificity for KCs in vivo using spinning-disk confocal microscopy and the nanopomegranate fluorescence. As shown in Figure 3a, 2 h after the intravenous injection of nanopomegranates, cells in the hepatic sinusoids (dark area showing contrast with the rest of the image in the green (autofluorescence) channel) became “bright” with the strong and uniform red fluorescence from the DiR-BOA in the nanopomegranates. These cells were also validated to be F4/80+ KCs,7, 8, 40, 41 as evidenced by their blue fluorescence from anti-BV421-F4/80 antibodies. Further, quantitative flow cytometry data (Figure 3b) showed that the fluorescent signal of nanopomegranates was mostly found in KCs (F4/80highCD11bint

cells)

but

was

rarely

found

in

monocyte-derived

macrophages

(CD11bhighly6Chigh and CD11bhighly6Clow subsets) and liver sinusoidal endothelial cells (LSECs, CD45-CD146+ cells). Additionally, nearly all of KCs were efficiently labeled by nanopomegranates, as indicated by the presence of a DiR-BOA fluorescent signal in 98.8% of KCs (Figure 3c, top panel). Notably, compared to another KC-labeling agent, FluoSpheres (diameter: 0.5 μm), the nanopomegranates visualized morphology better. Morphologically, the nanopomegranate-labeled KCs presented an irregular shape with filopodia or lamellipodia (Figure 3c, bottom panel), while FluoSpheres-labeled KCs showed only some spot-like signals without a visible cellular outline.9 Additionally, the fluorescence activation time of nanopomegranates in


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KCs (2 h) in vivo was obviously shorter than that in Raw 264.7 cells (18~48 h) in vitro. As KCs are well known as one of most potent scavenger cells in organism, this difference was mainly due to their different phagocytic and degradation capacities, especially in the normal physiological environment. In addition, fluorescence molecular tomographic (FMT) imaging was also performed to evaluate the distribution of nanopomegranates at the whole-body level. The data in Figure S5 (supporting information) showed that the nanopomegranates mainly accumulated in the liver at different time points ranging from 30 min to 48 h post injection. And the biodistribution data also confirmed that liver, followed by spleen and lung, has a high uptake of nanopomegranate at 2 h post injection. Those are all resident macrophage-enriched organs (Figure S6, supporting information). Thus, the fluorescence imaging results demonstrated that nanopomegranates had high selectivity for KCs in liver, indicating that nanopomegranates constitute an ideal dualmodality probe for characterizing the distribution and function of KCs in vivo.


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Figure 4. In vivo photoacoustic and fluorescence imaging of KCs in mouse hepatic lobules. a) In vivo dual-wavelength photoacoustic mapping of hepatic sinusoids (523 nm, red) and KCs (744 nm, green) obtained using the OR-PAM system b) In vivo spinning-disk confocal imaging of KCs (Ex: 640 nm, red) and the autofluorescence of liver lobules (Ex: 488 nm, green) 2 h after the intravenous injection of nanopomegranates. c) Schematic representation of fine layers of KCs in the hepatic lobule (rn-rn-1=8 μm). d) KC density per layer against the rn/rmax ratio. The quantified data of e) the average area of the PA signal in individual KCs and f) the mean PA intensity of individual KCs per layer against the rn/rmax ratio. To accurately characterize the distribution and function of KCs in the liver, PA images of both KCs and hemoglobin were simultaneously acquired by using our homemade OR-PAM system 2 h after intravenous injection. As shown in Figure 4a and supporting movie 1, the 3D structural information of hepatic sinusoids and crisscrossing arrayed hepatic lobules were clearly reconstructed according to the PA signal of hemoglobin excited with a 523-nm pulsed laser. The average diameter of hepatic lobules, the CV and hepatic sinusoids was 481.2±19.82 μm, 35.68±7.78 μm and 11.04±1.61 μm, respectively. Dual-wavelength PA imaging showed that the PA signal of nanopomegranates (green color in the image, excited at a wavelength of 744 nm) was located in the red area corresponding to the PA signal of hemoglobin. The data indicated the nanopomegranate-labeled KCs were exactly located in hepatic sinusoids of each hepatic lobule (Figure 4a). Interestingly, both dual-color photoacoustic maximum amplitude projection (MAP) (image field of view: 2 mm×2 mm; image depth: 300 μm) and 3D images visually displayed that the KCs were inclined to distribute at the PT area rather than at the CV area of hepatic lobules. This combination of spatial location and this distribution pattern of KCs was also confirmed by intravital fluorescence imaging using spinning-disk confocal microscopy with 55 stitched images


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(image field of view: 1658.8 μm×1658.8 μm; image depth: 10 μm, Figure 4b). The accuracy of the KC distribution determined in vivo by using PA imaging and confocal imaging was further validated under the KC-depleted condition with the use of clodronate liposomes (Figure S7, supporting information), which resulted in nearly no visible cells in both images. To quantify the distribution trend along the CV-PT axis and clarify the relationship between the density of KCs and their position relative to CV, we performed linear regression analysis. According to the average width of KCs in hepatic sinusoids (8.561.55 μm), we divided the hepatic lobules into n layers with 8 μm intervals from the CV to PTs (Figure 4c). The relative location of KCs along the CV-PT axis was represented as the rn/rmax ratio, in which rn and rmax are the radius of Circlen and hepatic lobules, respectively. The KC density per layer was calculated with the formula Drn/rmax = (Nn-Nn-1)/[π (rn2-rn-12)], where Nn represents the number of KCs in the circle with radius rn. As shown in Figure 4d and supporting table 1, the density of KCs was linearly dependent on the rn/rmax ratio (R2=0.7513) according to the equation Y = 21.90X-2.103, and the density of KCs in the outermost layer was almost 246-fold that in the innermost layer. Thus, dualcolor PA images provided intuitive evidence that the KC distribution in each liver lobule followed a linear pattern: more KCs were located in regions closer to the PT area. In addition, Figure 4e shows the positive correlation (Pearson coefficient R2=0.7209) between the area of the PA signal in individual KCs per layer and the rn/rmax ratio, with increases in the area as the rn/rmax ratio increased along the CV-PT axis. Additionally, to investigate the relationship between KC function and their spatial location, the mean PA intensity of individual KCs per layer was quantified and shown in a histogram of the rn/rmax ratio. Notably, the phagocytic ability of KCs varied along different layers of the CV-PT axis in the hepatic lobules, as evidenced by their different PA intensities (Figure 4f, supporting table


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2). The layers with rn/rmax ratios between 0.167 and 0.3 (close to the CV) had a zig-zag pattern, with minimum and maximum values of 34.59±3.80 (71.1% of the average) and 52.26±4.42 (116.5% of the average) at the rn/rmax ratios of 0.2 and 0.233, respectively. Given the report that blood flow influences KC uptake in vitro,42 the fluctuating phagocytic abilities in the layers from 0.167-0.3 may result from sharp differences in flow velocity at this location; further study with accurate flow rate monitoring in vivo is required to clarify this issue in the future. Besides, in vivo dual-wavelength PA imaging of liver at 24 h, 48 h, 72 h and 7 d post-injection of nanopomegranate was also performed in our study. PA imaging of mouse liver showed that the PA intensity of individual KCs slightly decreased to 83.37% at 24 h post-injection, dramatically decreased to 62.68% at 48 h and 40.16% at 72 h, and was very weak (16.53%) at 7 d post-injection (Figure S8b, supporting information). Similar attenuation was observed in the numbers of KCs with detectable PA signals (Figure S8c, supporting information). We also quantified the fluorescence signal of each KC at 2 h post injection (Figure S9, supporting information) and found no significant difference between cells, suggesting similar fluorescence activation of nanopomegranate in KCs. We noted the variation of PA and fluorescent signals in KCs at different positions. There were two major reasons for this phenomenon. One was the heterogeneity of KCs in phagocytic and degradative function, which might be related to their spatial location and microenvironment (e.g., blood velocity, oxyhemoglobin saturation, and cytokines)42-44. The other was the different image depths of PA imaging and fluorescence imaging: PA imaging obtained a signal with 300 μm depth of field, and confocal fluorescence imaging only showed a signal with dozens of microns of depth because of abundant hemoglobin in liver. Thus, PA imaging combined with nanopomegranate gives the advantage of quantitative investigation of


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the function and distribution of KCs in vivo, due to a millimeter-sized scan range, 300 μm image depth, high resolution (1-2 μm) and dual-wavelength image capability of our OR-PAM system. To further verify the capability of nanopomegranates in evaluating the phagocytic and degradative function of KCs, we prepared a model with impaired KC function, in which C57BL/6 mice were intravenously administered with a single low dose of clodronate liposome. As shown in Figure S10 (a), supporting information, both the number of KCs and their area decreased after 12 h clodronate liposome-treatment compared to normal conditions, as seen by in vivo photoacoustic imaging. In addition, minimal fluorescence activation was observed on spinningdisk confocal imaging (Figure S10 (b), supporting information). The quantification data also confirmed that the KC density was dramatically decreased to 25.3% of that of the normal group (Figure S10 (d), supporting information). And the KC distribution pattern altered, no longer fitting a linear pattern. No relationship was found between the area of KCs and their spatial location (Figure S10 (e), supporting information). As shown in Figure S10 (f), the average PA intensity of individual KCs was 49.84±2.26, which was comparable to the normal condition but with no significant difference between different layers. Combining the PA and fluorescence imaging, the data indicate that with a single low dose of clodronate liposome-treatment, the KCs nearly lost their degradative capacity while keeping their phagocytic ability and lost their strategic arrangement in hepatic lobules. Thus, our self-assembled “off/on” nanopomegranate for in vivo photoacoustic and fluorescence imaging could be used to evaluate the function of phagocytes under disease states.


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Figure 5. No adverse effects of nanopomegranates on healthy C57BL/6 mice. Healthy C57BL/6 mice (female, 8-10 weeks old) were intravenously administered nanopomegranates (200 μl, DiRBOA concentration of 250 μM). Liver function (ALB, ALP and ALT) and renal function (BUN, CRE and GLOB) were assessed 24 h after injection, and all the organs were excised 7 days after injection. Error bars represent the SEM. Student’s t-test (two-tailed) was used to determine significance, and p < 0.05 were considered significant. ALB: Albumin; ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; BUN: Blood urea nitrogen; CRE: Creatinine; GLOB: Globulin.


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Moreover, to evaluate whether nano-pomegranates could induce any adverse effect, healthy C57BL/6 mice were subjected to the same concentration of nano-pomegranates with the imaging dose and their biochemical effects were then assessed. Mice injected with 200 μl of PBS were used as controls. When compared with PBS controls, the nano-pomegranate treatment group did not show any measurable adverse effect on liver function (ALB: 26.33 ± 0.88 g/l; ALP:237.30 ± 6.23 IU/l; and ALT: 18.33 ± 2.60 IU/l) as well as renal function (BUN: 8.37±0.52 mmol/l; CRE: 77.00 ± 5.13 μmol/l; and GLOB: 26.67 ± 2.03 g/l) (Figure 5a). In addition, there was no significant difference in histology between nano-pomegranates groups and PBS controls, and all organs including hearts, livers, spleens, lungs and kidneys did not show any significant pathologic abnormality (Figure 5b). Besides, the oxygen saturation imaging of liver also was taken by using OR-PAM system. No significant difference of oxygen saturation in liver was found between PBS and nano-pomegranate groups (Figure S11, supporting information). These data indicate that there was no measurable adverse effect of nano-pomegranates. CONCULSIONS In summary, with the aid of our self-assembled pomegranate structure-like nanoparticles, we illustrate the strategic arrangement of KCs in vivo, including their fine spatial location and distribution and their phagocytic and degradative function in hepatic lobules in the normal physiological environment. Dual-wavelength PA imaging with high spatial resolution (1~2 μm) and a large field of view (2 mm×2 mm) visualized the 3D structure of hepatic sinusoids and crisscrossing arrayed hepatic lobules and the spatial location of nanopomegranate-labeled KCs. Dual-modality PA and fluorescence imaging revealed that KCs were strategically distributed along the PT-CV axis in each liver lobule following a linear pattern, with more and larger KCs with similar degradation capacity in regions closer to the PT area in vivo. Notably, the phagocytic


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abilities of KCs located between the regions with rn/rmax ratios of 0.167-0.3 fluctuated in a zig-zag pattern. The strategic arrangement of KCs which link to their spatial location and function not only has great implications for our basic understanding of liver physiology but also might provide therapeutic approaches involving KCs in the future. METHODS Materials.

The

hydrophobic

NIR

dye

DiR-BOA

(1,1’-dioctadecyl-3,3,3’,3’-

tetramethylindotricarbocyanine iodide bis-oleate) and Fluo-BOA ((Z)-octadec-9-enyl 2-(3-((Z)octadec-9-enyloxy)-6-oxo-6H-xanthen-9-yl) benzoate or dioleyl fluorescein) were synthesized as previously reported.34, 37 DMSO, Triton X-100, ICG, Hoechst 33248, propidium iodide (PI), low temperature agarose, DNA enzyme, collagenase IV, and xylazine were all purchased from SigmaAldrich (Saint Louis, MO, USA). Fluorescent latex (Lx) particles were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Clodronate liposomes were purchased from Liposomal BV Company (Amsterdam, Netherlands). Nanoparticle preparation. Nanoparticles were formed spontaneously and rapidly as illustrated below. First, 0.05 mg of DiR-BOA was dissolved in 2.5 μl of DMSO (Sigma-Aldrich cell) under sterile condition at room temperature according to standard pharmaceutical guidelines. Then, the DiR-BOA-DMSO solution was added to 200 μl of sterile prechilled PBS buffer (4 °C, pH=6.87.0) and gently mixed until the solution became clear (usually, 20 s). Nanoparticle characterization. The particle size distributions of the nanoparticles were measured using DLS (photon correlation spectroscopy) with a Zetasizer Nano-ZS90 system (Malvern Instruments, Worcestershire, UK). TEM imaging was performed using an HT7700-type TEM (Hitachi High Technology Company) with an accelerating voltage of 100 kV. The fluorescence


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emission spectrum was obtained by a Luminescence spectrometer LS55 (PerkinElmer Instrument Co., Ltd.), and the absorbance spectrum was measured by using a UV2550 UV-VIS spectrophotometer (Shimadzu Enterprise Management (China) Co., Ltd). The quantum yield of nanopomegranate was measured by using a 25/35 Lambda UV-Vis Spectrophotometer UV (PerkinElmer, USA) and FP-6500 fluorescence spectrometer (Japan Spectrum Co., Ltd.). The comparative method was used to calculate the quantum yield according to the vendor’s procedure (FL6500 Fluorescence Spectrometer ((PerkinElmer, USA)) with the formula: 𝑄𝑠 = 𝑄𝑟 2

(𝐴𝑟𝐴𝑠)(𝐸𝑟𝐸𝑠)(𝑛𝑟𝑛𝑠)

(Q = fluorescence quantum yield; n = refractive index of the solvent; A = absorbance

of the solution; E = integrated fluorescence intensity of the emitted light; subscripts ‘r’ and ‘s’ refer to the reference and unknown), which compared the fluorescence intensity of the sample to the reference dye DiR, a commercial dye with a known quantum yield in methanol (ΦF = 0.28) and optical properties closely matching DiR-BOA. A custom-made wide-field fluorescence imaging system was used to detect the fluorescence quenching of DiR-BOA with a near-infrared filter set (excitation=716/40 nm; emission=775/46 nm). Photoacoustic characterization was conducted with a homemade optical-resolution photoacoustic microscopy (OR-PAM) system with a high resolution (micrometers) as previously described.13 For photostability testing, we scanned the samples continuously at the same position 40 times with 2000 steps and a step size of 4 μm, the 744-nm laser energy per pulse was measured to be approximately 200 nJ, and each scan took 2 s (1 ms per step) by our OR-PAM system. Cell culture. Raw 264.7 cell were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). The Raw 264.7 cells were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 100 U ml-1 penicillin, and 0.1 mg ml-1 streptomycin. All cells were cultured under 5% CO2 at 37 °C in an incubator (Thermo).


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In vitro confocal fluorescence imaging. The Raw 264.7 cells (1×105 per well) were seeded into a confocal cell culture dish (NEXS, USA) and incubated for 24 h at 37 °C in a humidified incubator with 5% CO2. Then, the nanopomegranate solution with 5 μM DiR-BOA was added. After 2 h of incubation, the Raw 264.7 cells were rinsed three times with PBS to remove excess nanopomegranate and reincubated in fresh cell culture medium. Hoechst 33258 (0.5 μg ml-1) was added 15 min before imaging for nuclear staining. Images were taken at 2, 4, 6, 12, 18, 24 and 48 h under the same conditions. The confocal image of cells was acquired using a spinning-disk confocal microscope (PerkinElmer Instrument Co., Ltd., Germany) with an excitation wavelength of 405 nm for Hoechst 33258 and 640 nm for DiR-BOA. In vitro flow cytometry analysis. The Raw 264.7 cells were given same regimen as above confocal study. PI (4 μg ml-1) was added for staining dead cells. Then, the 5.4×104 Raw 264.7 cells for each time point were detected by flow cytometry (Guava microcapillary cell flow analysis platform) with a red channel (690/50 nm) for PI and NIR-II channel (785/70 nm) for DiR-BOA. In vitro photoacoustic imaging. The Raw 264.7 cells were subjected to the same regimen as described above for the confocal study. Then, for each time point, 5×104 Raw 264.7 cells in 200 μl of sterile PBS were injected into the capillary tube to measure the photoacoustic signal by the homemade OR-PAM system (744-nm laser, 200 nJ). Stability evaluation of nanoparticles. To check the nanoparticle stability, the nanopomegranate solution was incubated with 10% mouse serum at 37 °C for 1, 2, 4, and 6 h and then loaded into in a low temperature agarose gel phantom (0.8%). The samples were then imaged with the homemade wide-field fluorescence imaging system. The DiR-BOA fluorescence was measured with a near-infrared filter set (excitation=716/40 nm; emission=775/46 nm) and an exposure time


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of 10 s; the bright field images were acquired with an exposure time of 0.02 s; and all images were processed by using MATLAB software. Mice. Female C57BL/6 mice (8-12 weeks old) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, Hunan, China). All of the mice were maintained in a specific pathogen-free (SPF) barrier facility at the Animal Center of Wuhan National Laboratory for Optoelectronics. All animal studies were conducted in compliance with protocols that had been approved by the Hubei Provincial Animal Care and Use Committee and were in compliance with the experimental guidelines of the Animal Experimentation Ethics Committee of Huazhong University of Science and Technology. Animal regimen. Before confocal imaging and photoacoustic imaging, 10 C57BL/6 mice (female 8-10 weeks old) were subjected to the following procedures. The preoperative analgesic was injected subcutaneously into animals 30 min before the surgery. Then, the mice were injected intraperitoneally with ketamine at dose of 120 mg kg-1 and xylazine at dose of 18 mg kg-1 for anesthesia. The intravital imaging liver model was utilized in our study in a manner similar to a previous report.45-47 In vivo confocal fluorescence imaging. The liver of the C57BL/6 mice in the different groups was imaged by spinning-disk confocal microscopy (PerkinElmer Instrument Co., Ltd., Germany) in vivo 2 h after the intravenous injection of nanopomegranate (200 μl, DiR-BOA concentration of 250 μM). To evaluate whether nanopomegranate uptake was specific to KCs, anti-BV421-F4/80 antibody (6 μg) was injected 10 min before imaging. The liver images were taken with an excitation wavelength of 488 nm for autofluorescence (90% laser power, exposure time of 200 ms) and 640 nm for DiR-BOA (30% laser power, exposure time of 50 ms).


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Flow cytometry analysis of liver tissue. Flow cytometry analysis of hepatic cells was performed (Beckman CytoFLEX, Indianapolis, USA) as described previously.9,

48

In brief, livers were

digested with collagenase type-IV and DNase I at 37 °C after a low-pressure intraportal perfusion with collagenase type-IV solution. Extracts were filtered using 70 μm cell strainers (BD), and parenchymal cells were removed by centrifugation for 1 min at 100 g. Staining of nonparenchymal cells was performed using combinations of the following monoclonal antibodies: anti-mouse CD45, anti-mouse F4/80, anti-mouse Ly6C, anti-mouse CD146, and anti-mouse CD16/32 (BioLegend); and anti-mouse Ly-6G and anti-mouse CD11b (BD-Bioscience; Franklin Lake, New Jersey, USA). Data were analyzed with FlowJo (TreeStar). In vivo photoacoustic imaging. Dual-wavelength photoacoustic MAP images were taken in vivo with the homemade OR-OAM system with 523-nm and 744-nm pulsed lasers at 200 nJ at 2 h after the intravenous injection of nanopomegranates (200 μl, DiR-BOA concentration of 250 μM) in the C57BL/6 mice in different groups. The images were acquired by performing the B-scan process, each B-scan consisting of 1000 steps with a step size of 2 microns. The cross-sectional B-scan image data indicated that the image depth of OR-PAM for the hepatic sinusoid in mouse liver was 300 μm (Figure S12, supporting information). The oxygen saturation images were acquired as previously reported with 576-nm, 580-nm and 584-nm pulsed lasers.49 Blood hemanalysis and biochemical analyses. To assess the adverse effects of nanopomegranates, N=4 healthy C57BL/6 mice (female, 8-10 weeks old) were intravenously administered 200 μl of nanopomegranates (DiR-BOA concentration of 250 μM), and the other 4 mice were intravenously administered 200 μl of PBS as controls. Blood samples were collected from the mice 24 h after the intravenous injection of nanoparticles or PBS control. The blood cell and biochemical analyses were performed using a hematology analyzer (BC-3200, Mindray,


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Shenzhen, China) and an automatic biochemical analyzer (Spotchem EZ SP-4430, Arkray Inc., Kyoto, Japan), respectively. Histopathological analyses. Livers, spleens, kidneys, and lungs were extracted from experimental group and control group mice and fixed in a 4% paraformaldehyde solution for 10 h. The organs were embedded in paraffin, sectioned, and processed for hematoxylin and eosin (HE) staining. The HE sections were imaged on a Nikon Ni-E microscope (Nikon, Minato, Tokyo, Japan). All images were acquired with the software NIS-Elements and further analyzed with Image J software. Quantification of KCs per layer. The KC density per layer was calculated with the formula Drn/rmax = (Nn-Nn-1)/[π(rn2-rn-12)] via MATLAB software, where Nn represents the number of KCs in a circle with radius rn; (rn-rn-1=8 μm); and rmax represents the radius of liver lobule. The average area of the PA signal in individual KCs and the mean PA intensity of individual KCs per layer was calculated by Fiji and MATLAB software (n=120 for cell density measurements, n=615 for the mean PA intensity quantification and n=396 for cell area calculations). Statistical analysis Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, CA). Error bars represent the SEM. For comparisons of two groups, the two-tailed unpaired t-test was used. Significant differences between or among the groups are indicated as follows: ns for no significant difference, * for P < 0.05, ** for P < 0.01, and *** for P < 0.001. ASSOCIATED CONTENT Supporting Information.


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The following files are available free of charge via the Internet at http://pubs.acs.org. Supporting Information Figure S1. The size of nanopomegranates examined by DLS increased as the DiR-BOA amount in the DMSO/PBS mixed solution increased. In addition, all formulations remained in the “dark” state, as evidenced by the negligible fluorescence signal observed. Supporting information Table S1. The quantum yield of DiR and DiR-BOA in different states. Supporting Information Figure S2. A similar nanopomegranate structure with the use of hydrophobic dye Fluo-BOA or DiR. Supporting information Figure S3. Evaluation of cell toxicity of the DiR-BOA-loaded nanopomegranate solution and free DiR-BOA solution by MTT assay. Supporting Information Figure S4. Stability test of nanopomegranate solution in vitro. Supporting Information Figure S5. FMT imaging at the whole-body level revealed that the nanopomegranates were targeted to organs enriched with tissue-resident macrophages, mainly the liver. Supporting Information Figure S6. The biodistribution of nanopomegranates in normal C57BL/6 mice at 2 h post-injection. Supporting Information Figure S7. In vivo photoacoustic and fluorescence imaging of nanopomegranates under the KC-depleted condition. Supporting Information Figure S8. In vivo dual-wavelength PA imaging of liver at 24 h, 48 h, 72 h and 7 d post-injection of nanopomegranate. Supporting Information table S2. The quantification data of different layers. Supporting Information Figure S9. No significance difference was found in the MFI between each individual KC along the CV-PT axis in liver lobules.


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Supporting Information Figure S10. In vivo photoacoustic and fluorescence imaging of KCs in mouse hepatic lobules under clodronate liposome (Liposomal BV) treatment. Supporting Information Figure S11. No significant difference in oxygen saturation in the liver was found between the PBS and nanopomegranate groups. Supporting Information Figure S12. Evaluation of the image depth of OR-PAM using the crosssectional B scan image method. (file type, PDF) Supporting information movie S1. The 3D structure of hepatic sinusoids and crisscrossing arrayed hepatic lobules and the spatial location of nanopomegranate-labeled KCs (file type, mp4)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected]. Author Contributions ‡These authors contributed equally. ACKNOWLEDGMENT We thank the Optical Bioimaging Core Facility of WNLO-HUST for the support in data acquisition. We thank Professor Gang Zheng and Dr. Juan Chen (University of Toronto, Toronto, ON, Canada) for manuscript discussion. This work was supported by National Science Fund for Distinguished Young Scholars (81625012), the Major Research plan of the National Natural Science Foundation of China (Grant No. 91442201), Science Fund for Creative Research Groups of the National Natural Science Foundation of China (Grant No. 61421064), National Natural


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