Lysosomal pH Decrease in Inflammatory Cells Used To Enable

Jun 2, 2015 - Herein, we reported the observation of lysosome pH which is significantly acidified in inflammatory cells. Taking advantage of lysosome ...
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Lysosomal pH Decrease in Inflammatory Cells Used To Enable Activatable Imaging of Inflammation with a Sialic Acid Conjugated Profluorophore Mingzhu Yu,† Xuanjun Wu,† Bijuan Lin,† Jiahuai Han,‡ Liu Yang,† and Shoufa Han*,† †

Department of Chemical Biology, College of Chemistry and Chemical Engineering, State Key Laboratory for Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, the MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, and Innovation Center for Cell Signaling Network, and ‡State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, 361005, China S Supporting Information *

ABSTRACT: Inflammation causes significant morbidity and mortality, necessitating effective in vivo imaging of inflammation. Prior approaches often rely on combination of optical agents with entities specific for proteinaceous biomarkers overexpressed in inflammatory tissues. We herein report a fundamentally new approach to image inflammation by targeting lysosomes undergoing acidification in inflammatory cells with a sialic acid (Sia) conjugated near-infrared profluorophore (pNIR). Sia−pNIR contains a sialic acid domain for in vivo targeting of inflamed tissues and a pNIR domain which isomerizes into fluorescent and optoacoustic species in acidic lysosomes. Sia−pNIR displays high inflammation-to-healthy tissue signal contrasts in mice treated with Escherichia coli, Staphylococcus aureus, or lipopolysaccharide. In addition, inflammation-associated fluorescence is switched off upon antibiotics treatment in mice. This report shows the potentials of Sia−pNIR for activatable dual-modality inflammation imaging, and particularly the use of lysosomes of inflamed cells as a previously unappreciated biomarker for inflammation imaging.

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Lysosomes are the major intracellular acidic compartments. The acidity of lysosomes (pH 4.0−5.5) is critical for a variety of biological activities ranging from autophagy, energy metabolism, to immunological responses, etc.21 Aberrant lysosomal pH has been documented during cell death, cancer metastasis, and cell maturation.22−26 However, changes of lysosomal pH in cells undergoing inflammation have not been examined. We previously reported the use of acid-activatable profluorophores for high-performance tumor imaging in mice by protontriggered fluorogenic isomerization of the profluorophore within lysosomes of tumor cells.27,28 Albeit widely targeted for cancer imaging, lysosomes of inflammatory cells have not been employed for activatable imaging of inflammation.

nflammation is associated with a myriad of pathological events including bacterial infection, injury, cancer, etc. Substantial morbidity and mortality are caused by both chronic and acute inflammation, necessitating technologies for in vivo inflammation imaging. 1 Prior strategies often rely on integration of dyes or magnetic resonance agents with antibodies or peptides specific for biomarkers abundant in inflammatory tissues, e.g., intercellular adhesion molecule-1,2−5 vascular cell adhesion molecule,6,7 and selectins.8−12 Alternatively, passive targeting of inflammation has also been achieved with dye-encapsulating liposomes via enhanced permeation and retention (EPR) effects of inflamed tissues.13 Agents with “always-on” signals are of intrinsic high backgrounds for in vivo bioimaging. By contrast, probes that could be switched to signal-on states within targets of interest while remaining silent in off-target settings are beneficial to achieve low background signals.14−20 © XXXX American Chemical Society

Received: March 2, 2015 Accepted: June 2, 2015

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Analytical Chemistry Scheme 1. Schematic of Dual-Modality Imaging of Inflammation with Lysosome-Activatable Sia−pNIRa

Nonfluorescent Sia−pNIR undergoes proton-mediated opening of intramolecular lactam to give fluorescent and optoacoustic species within acidic lysosomes. a

Figure 1. Acid-activatable fluorescence of Sia−pNIR (A) and Glu−pNIR (B). A series of phosphate buffer solutions (pH 3.9−9.4) containing Sia− pNIR (200 μM) or Glu−pNIR (200 μM) was analyzed for fluorescence emission using an excitation wavelength of 715 nm.

responses.29 Extensive efforts have been devoted to intelligent systems activatable to acidic microenvironment or acidic lysosomes of tumor cells.14,15,18,30 In contrast, acidic lysosomes of inflammatory cells and the acidified microenvironment of inflammatory sites have been largely unexplored for bioimaging. Sialic acids (Sia), derivatives nine-carbon monosaccharide Nacetyl-neuraminic acid, are typically located at termini of cell surface glycans.31−34 Sialic acid binding lectins modulate immunological activities of lymphocytes by interplay with cognate cell surface sialosides. Selectins, which recognize sialylated glycans, are up-regulated on the surface of endothelial cells during inflammation and mediate leukocyte extravasation from the bloodstream into inflammatory tissues. The sialosidemediated immunological recognition suggests feasibility of sialic acid decorated imaging systems for targeting of inflamed tissues. As such, we investigate the use of Sia−pNIR, recently

Herein, we reported the observation of lysosome pH which is significantly acidified in inflammatory cells. Taking advantage of lysosome acidification in inflamed cells, a sialic acid conjugated with acidic-responsive near-infrared profluorophore (Sia− pNIR) was utilized for imaging of inflammation in mice via proton-triggered “turn-on” fluorescence and optoacoustic contrasts in inflamed cells (Scheme 1). The advantageous biomedical properties of Sia−pNIR show the feasibility of lysosome as the target for in vivo inflammation detection and also the potentials of Sia−pNIR for inflammation imaging and monitoring effects of anti-inflammatory therapies.

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RESULTS AND DISCUSSION Inflammation is hallmarked by influx of circulatory immunological cells into inflammatory sites, whereupon the local pH is often decreased to pH 5.5 or lower owing to immunological B

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Figure 2. Lysosomal acidity-mediated fluorescence-on of Sia−pNIR (A) and Glu−pNIR (B). Raw 264.7 and HeLa cells pretreated with or without BFA (50 nM) were cultured in DMEM supplemented with Sia−pNIR (100 μM) or Glu−pNIR (100 μM) for 2 h, respectively. The cells were washed with PBS and then resuspended in DMEM or acidic buffer (pH 4) and imaged by fluorescence microscopy. Bar: 10 μm.

employed for tumor imaging,35 for inflammation detection via fluorescence switched on by lysosomal acidity in inflammatory cells. pH Responsiveness of Sia−pNIR and Glu−pNIR. To compare the targetability of Sia−pNIR for in vivo imaging, pNIR was conjugated with 2-amino-2-deoxy-D-glucose (Glu) to give Glu−pNIR, which was used as the control. To assess proton-triggered fluorescence of Glu−pNIR, Sia−pNIR and Glu−pNIR were spiked into a series of buffers of pH 3.9−9.4. The buffered samples were analyzed for fluorescence emission and UV−vis−NIR absorbance. Figure 1A shows Sia−pNIR is responsive to acidic pH with optimal turn-on fluorescence within pH 4.0−5.5, which is consistent as previously reported.35 Analogous to Sia−pNIR, Glu−pNIR is nearly nonfluorescent at alkaline conditions and displays enhanced fluorescence emission centered at 740 nm as the buffer pH decreases (Figure 1B), confirming acidic pH-mediated fluorogenic opening of the intramolecular ring of the pNIR domain (Scheme 1). Maximal fluorescence observed in pH 4.0−5.5 overlaps physiological lysosomal acidity window. These results confirm occurring of activatable NIR fluorescence by protontriggered isomerization of Sia−pNIR and Glu−pNIR. Cellular Uptake and Lysosomal Activation of Sia− pNIR and Glu−pNIR. To probe cellular uptake of Sia−pNIR, Raw 264.7 cells and HeLa cells were cultured with Sia−pNIR or Glu−pNIR in Dulbecco’s modified Eagle’s medium (DMEM) and then visualized by fluorescence microscopy. Increased intracellular NIR fluorescence was observed over incubation time (Figure S1, Supporting Information), showing time-dependent cellular uptake of both Sia−pNIR and Glu− pNIR. Lysosomes are the major intracellular acidic vesicles with lumen pH of 4.0−5.5. To determine effects of lysosomal pH on observed intracellular NIR signals, HeLa cells and Raw 264.7 cells were first stained with lysotracker green DND-26 (referred

to as lysotracker green) and further cultured with Sia−pNIR or Glu−pNIR. Confocal microscopic analysis reveals colocalization of NIR fluorescence with lysotracker green (Figure 2). As lysotracker green is a lysosome specific dye, the colocalization shows that pNIR is activated to give fluorescence inside lysosomes. Next, we probed the effects of lysosomal pH on intracellular activation of pNIR. HeLa and Raw 264.7 cells were treated with or without brefeldin (BFA) and then loaded with Sia−pNIR or Glu−pNIR. BFA is a potent inhibitor of VATPase and could effectively alkalinize the acidic pH of lysosomes. As shown in Figure 2, the intracellular fluorescence largely disappeared in cells prestained with BFA, whereas the NIR signal is unaffected in BFA-free cells, suggesting acidic lysosomal pH-mediated fluorogenic isomerization of pNIR moiety outlined in Figure 1. In principle, the vanished fluorescence in BFA-treated cells could originate from either alkalined lysosomal pH-mediated fluorescence-off or compromised cellular uptake of Sia−pNIR by BFA. Hence, HeLa and Raw 264.7 cells dual labeled with BFA and Sia−pNIR or Glu−pNIR were suspended in acidic buffer (pH 4). Microscopic images reveal renaissance of intensive fluorescence in cells dispersed in acidic medium relative to cells maintained in fresh DMEM (pH 7.2), showing effective uptake of Glu−pNIR and Sia−pNIR by BFA-stained cells. These results clearly demonstrate that Glu−pNIR and Sia−pNIR undergo signal activation upon cellular endocytosis into acidic lysosomes. Imaging of Bacteria-Triggered Inflammation in Mice with Sia−pNIR. With lysosome acidity-triggered fluorescence, Sia−pNIR was evaluated for its performance to image inflammation in mice. Escherichia coli (E. coli) was injected into thigh muscle of ICR mice. The mice were maintained for 24 h to allow development of inflammation and then intravenously administered with Sia−pNIR or Glu−pNIR via tail vein. The mice were monitored for whole-body NIR C

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levels of NIR fluorescence were present in kidney, heart, spleen, and lung (Figure 4). NIR signal in liver suggests hepatic uptake

fluorescence over time. No obvious NIR signals could be observed right after probe injection (Figure 3), proving that

Figure 3. Differential illumination of E. coli-induced inflammation by Sia−pNIR (A) and Glu−pNIR (B). ICR mice were injected with E. coli in the muscle of the right thigh and then administered with Sia− pNIR (40 mg kg−1) or Glu−pNIR (40 mg kg−1), respectively, by tail vein. At indicated time points after probe administration, the mice were imaged for whole-body NIR fluorescence.

Figure 4. Biodistribution of Glu−pNIR (A) and Sia−pNIR (B) in E. coli-infected mice. ICR mice loaded with E. coli in the right thigh were intravenously injected with Sia−pNIR (40 mg kg−1) or Glu−pNIR (40 mg kg−1). The mice were sacrificed 24 h after probe injection. The inflamed muscle slice and representative organs were dissected and imaged for ex vivo fluorescence emission (C).

carbohydrate-conjugated pNIR remained silent during circulation in bloodstream (pH 7.4). At 24−96 h postinjection of Sia−pNIR, intense NIR fluorescence was identified in the site injected with E. coli, showing the capability of Sia−pNIR to illuminate inflamed tissues. Fluorescence-off was noticed in mice at 168 h post probe injection, which is largely due to immunological elimination of injected E. coli and thus dampening of inflammation in live mice. In contrast, no detectable NIR fluorescence was observed in E. coli-triggered inflammatory site in mice treated with Glu−pNIR (Figure 3) under identical experimental conditions. The difference clearly demonstrates the critical roles of sialic acid moiety of Sia−pNIR for effective inflammation detection in mice. To probe the effects of natural Sia on cellular uptake of Sia−pNIR by Raw 264.7 cells were incubated with Sia−pNIR in the presence of increasing levels of Sia and then treated with or without E. coli or lipopolysaccharide (LPS). No obvious blockade of cellular internalization and lysosomal activation of Sia−pNIR was identified under these conditions (Figure S5, Supporting Information), suggesting that Sia−pNIR is a superior substrate for cellular uptake relative to Sia. To ascertain in vivo biodistribution of Sia−pNIR, the inflamed muscle and representative organs were dissected from E. coli-bearing mice at 48 h after Sia−pNIR injection and probed by ex vivo fluorescence analysis. Consistent with the aforementioned whole-body imaging results (Figure 3), strong fluorescence was present in inflammatory muscle, whereas low

and clearance of injected Sia−pNIR. Collectively, these results validate preferential in vivo uptake and signal activation of Sia− pNIR over Glu−pNIR by inflammatory tissues. With effective in vivo imaging of inflammation triggered by Gram-negative E. coli, we proceeded to evaluate the efficacy of Sia−pNIR to image inflammation elicited by Gram-positive bacteria. Sia−pNIR was intravenously injected into mice that had been injected with Staphylococcus aureus (S. aureus) in thigh muscle. Strong signal is present in the inflamed site at 48−96 h postinjection of Sia−pNIR over the rest part of the body, whereas no NIR signals could be identified in S. aureus-treated mice with Glu−pNIR (Figure S2, Supporting Information). Taken together, these results established the efficacy of Sia− pNIR to detect inflammation triggered by both Gram-negative and Gram-positive bacteria. Imaging of Lipopolysaccharide-Triggered Inflammation in Mice with Sia−pNIR. Lipopolysaccharide is an endotoxin derived from Gram-negative bacterial cell wall and induces strong inflammatory responses in animals. Shown to detect bacteria-elicited inflammation, Sia−pNIR was then evaluated for its capability to image LPS-induced inflammation D

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Figure 5. Elevation of Sia−pNIR fluorescence in live cells in the presence of bacteria and LPS. Raw 264.7 (A) and HeLa cells (B) prestained with Sia−pNIR (100 μM) were cultured in DMEM containing E. coli, S. aureus, LPS, respectively, or no addition (control). The cells were washed with PBS and then imaged by fluorescence microscopy. Bar: 10 μm.

enhancement in the mean intracellular fluorescence (MF) upon stimulation with E. coli, S. aureus, or LPS (Figure S4, Supporting Information), confirming the boosted NIR signals identified in microscopic images (Figure 5). The dramatic elevated intracellular fluorescence shows that the lysosomal acidity is significantly decreased in the presence of bacteria or LPS. Albeit the cellular mechanisms underlying lysosome acidification in cells stimulated with bacteria or LPS remain to be elucidated, alteration of lysosome acidity demonstrated herein is a previously unappreciated process that could be targeted by acid-activatable imaging systems. Imaging of Antibacteria Effects of Antibiotics in Mice with Sia−pNIR. Antibiotics are widely used to treat bacterial infection. Shown to be responsive to bacterium-triggered inflammation, Sia−pNIR was explored to track the effects of antibiotics on inflammation. A cohort of nude mice injected with E. coli in thigh muscle were then treated with streptomycin or phosphate buffer saline (PBS). Sia−pNIR was injected to E. coli-bearing mice after treatment with streptomycin or PBS. For instance, streptomycin is effective against both Gram-negative bacteria. Time course whole-body imaging revealed quenching of the inflammation-associated fluorescence in muscle at 96 h post-treatment of streptomycin, whereas no loss of NIR signal could be observed in PBS-treated bacteria-bearing mice (Figure 6), showing inflammation is effectively dampened by streptomycin, whereas PBS has no effects on inflammation. These results suggest the feasibility to screen anti-inflammatory compounds in animal models by signal-off of Sia−pNIR. Optoacoustic Imaging of Inflammation with Sia− pNIR. Optoacoustic imaging uses weakly scattered ultrasound produced from inpulsed optical excitation and thus could be used to image objects several centimeters deep in tissues.10,44,45 By contrast, photons suffer from strong diffusion in biological tissues, limiting fluorescence imaging technologies for tissues millimeters in depth. We previously showed that pNIR moiety encapsulated in a polymeric vesicle could be used for turn-on optoacoustic imaging of tumors.46 Given the advantages of optoacoustic imaging for clinical applications, Sia−pNIR was examined for optoacoustic inflammation imaging. To determine pH-correlated photoacoustic property, Sia− pNIR was spiked into buffer of various pH. Optoacoustic

in mice. ICR mice with LPS injected in thigh muscle were then intravenously administered with Sia−pNIR by tail vein. Strong fluorescence is observed in the LPS-treated area with performance similar that of E. coli (Figure S3, Supporting Information), demonstrating the capability of Sia−pNIR for selective detection of LPS-associated inflammation. Historically, a number of molecular systems featuring bacterium-binding entities have been developed for optical detection of invaded bacteria.36−39 Distinct from these systems, we detect bacteria-triggered inflammation by an alternative approach, which is independent of invading pathogens and is instead specific for inflamed host cells. It is anticipated that this approach could be applicable to imaging of inflammation induced by a broad spectrum of bacteria. Alternatively, imaging of inflammation has been reported with chemodosimeters that stoichiometrically react with reactive oxygen species generated in inflammatory tissues to give optical read-out.40,41 Compared with irreversible nature of these chemodosimeters,40,41 Sia− pNIR is of reversible signals allowing differentiation of lysosomes in inflamed tissues over healthy organs, which is beneficial for in vivo dynamic reporting of inflammation status. Decreased Lysosomal pH in Inflammatory Cells Leads to Enhanced Fluorescence of Sia−pNIR. Lysosome mediates a variety of biological events, such as plasma membrane repair, endocytosis, and cellular clearance, etc.21 Lysosomal functions respond to environmental cues, and yet, little is known about how lysosomal parameters varies in different cells, tissues, and pathological conditions.21 It is reported that lysosomal pH is significantly altered during cell death.42,43 Herein, we wish to determine the effects of selected proinflammatory materials on lysosomal pH, which is of significance for activatable inflammation imaging. Raw 264.7 and HeLa cells were stained with Sia−pNIR for 1 h and then rinsed with PBS to remove extracelluar Sia−pNIR. The two cell lines containing identical levels of Sia−pNIR were cultured for 4 h in fresh DMEM supplemented with E. coli, S. aureus, LPS, respectively, or no additions, and then quantitated for intracellular fluorescence intensities. Fluorescence microscopic images show obviously enhanced NIR signals in cells treated with E. coli, S. aureus, or LPS as compared to control cells (Figure 5). Flow cytometric analysis reveals 7−15-fold E

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Figure 6. Antibiotics-mediated fluorescence-off of Sia−pNIR in inflammatory tissues. ICR mice were injected with E. coli in muscle of the right thigh. Sia−pNIR (40 mg kg−1) was intravenously injected into mice, and then the mice were injected with streptomycin (20 mg kg−1) or PBS (100 μL) in the left thigh 2 days after administration of the bacteria. Whole-body fluorescence was visualized at 48 and 96 h postinjection of PBS and streptomycin. Figure 8. Optoacoustic imaging of S. aureus-elicited inflammation in mice with Sia−pNIR. Nude mice were injected with S. aureus in thigh muscle and then intravenously injected with PBS (100 μL) or Sia− pNIR (40 mg kg−1). At 0 and 24 h post probe injection, the inflamed spots were imaged for optoacoustic contrasts.

contrasts of the solutions were recorded over buffer pH. Intense optoacoustic signals are observed in acidic buffers of pH 6.5−4.4, whereupon the intensity increases as pH decreases (Figure 7). Given the weak signals present in buffer of pH 6.4− 8.4, the titration shows acidic pH-mediated turn-on optoacoustic signals of Sia−pNIR, which is consistent with the reported activatable optoacoustic property of pNIR encapsulated in polymeric micelles.46 Next, Sia−pNIR was intravenously administered into S. aureus-bearing mice. The inflammatory site displays intense optoacoustic contrast, whereas control mice treated with PBS displays no optoacoustic signals (Figure 8), clearly proving the use of Sia−pNIR for optoacoustic inflammation imaging.

pH in mammalian cells in the presence of E. coli, S. aureus, and LPS, which gives rise to dramatic elevation of Sia−pNIR fluorescence in these inflamed cells. Sia−pNIR contains a monosaccharide domain of sialic acid for effective targeting of inflamed tissues and a domain of NIR profluorophore which displays turn-on optical and optoacoustic signals under acidic settings. Sia−pNIR exhibits high inflammation-to-background signal contrasts in mice with injected E. coli, S. aureus, or LPS. The bacterium-triggered fluorescence is switched off upon treatment with antibiotics. These results show the use of Sia− pNIR for activatable and targetable inflammation imaging and also the potentials of altered lysosome acidity in host cells undergoing inflammation as a new biomarker for inflammation diagnosis.

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CONCLUSIONS Imaging of inflammation is of biomedical significance. NIR dyes responsive to reactive oxygen species (ROS) have been successfully developed for in vivo imaging of inflammation.47,48 Complementing to the use of ROS-activatable imaging approaches,47,48 we observed significant decrease of lysosomal

Figure 7. Acidic pH-mediated activation of optoacoustic signals of Sia−pNIR. Sia−pNIR was spiked into sodium phosphate buffer (100 mM, pH 4.5−9.5) to a final concentration of 1 mg mL−1. The solutions were recorded for optoacoustic contrasts (A) and visual images (B). The optoacoustic intensity was plotted over buffer pH (C). F

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EXPERIMENTAL PROCEDURE Materials and Methods. Lipopolysaccharide, DMEM, and fetal bovine serum (FBS) were purchased from Sigma. Sia− pNIR and Glu−pNIR were prepared according to published procedures.35 LysoTracker green DND-26 was purchased from Invitrogen. All other chemicals were used as received from Alfa Aesar. HeLa cells, and Raw 264.7 cells were obtained from American Type Culture Collection and grown at 37 °C under 5% CO2 in DMEM. Confocal microscopic images were performed on a Leica SP5 using the following filters: λex at 488 nm and λem at 500−530 nm for Lysotracker green; λex at 633 nm and λem at 700−790 nm for pNIR fluorescence. Fluorescence images of mice and the dissected organs were obtained on a Carestream FX PRO in vivo imaging system using an excitation filter of 690 nm and an emission filter of 750 nm. Fluorescence quantification data were analyzed with Carestream MI SE software. Flow cytometric analysis was performed using λex at 630 nm (λem at APC-Cy7-A channel) and fluorescence emission at 700−790 nm. All animal experiments were performed in accordance with the guidelines of Xiamen University’s Animal Care and Use Committee. Staining of Lysosomes with Sia−pNIR and Glu−pNIR. HeLa cells and Raw 264.7 cells were cultivated for 24 h in DMEM with 10% FBS. The cells were cultured with Sia−pNIR (100 μM) or Glu−pNIR (100 μM), respectively, for 0, 2, or 12 h, and then visualized by confocal microscopy for intracellular NIR signal. Lysosomal Acidity-Mediated Turn-On Fluorescence of Sia−pNIR and Glu−pNIR. HeLa cells and Raw 264.7 cells were first cultured in DMEM spiked without or with BFA (50 nM) for 10 h. The cells were then incubated with Sia−pNIR (100 μM) or Glu−pNIR (100 μM) for 2 h in DMEM supplemented with 10% FBS. The cells were washed with PBS (1 mL) for three times and then stained with Lysotracker green (1 μM) in DMEM for 20 min. The cells were then probed by fluorescence microscopy. In parallel assays, portions of Sia− pNIR- and BFA-treated cells were washed with PBS and then resuspended in sodium phosphate buffer (pH 4, 100 mM) for 10 min. The resultant cells were visualized with a confocal fluorescence microscope. Imaging of Bacteria-Elicited Inflammation in Mice Models. S. aureus (100 μL, 1 × 107−108 CFU), E. coli (100 μL, 1 × 107−108 CFU), or LPS (10 μL, 10 mg mL−1), respectively, was injected into muscle of the right thigh in ICR mice (male, 4 weeks of age). At 1−2 days after agent injection, Sia−pNIR (40 mg kg−1) or Glu−pNIR (40 mg kg−1), respectively, was administered intravenously via tail vein into the bacteria-bearing mice. At 0−168 h following administration of Sia−pNIR or Glu−pNIR, the mice were anesthetized with intraperitoneal injection of 0.5% chloral hydrate (0.01 g mL−1) and then analyzed for whole-body NIR fluorescence. Enhanced Lysosomal Activation of Sia−pNIR in Inflammatory Cells. Raw 264.7 cells and HeLa cells were cultured for 1 h in DMEM spiked with Sia−pNIR (100 μM). The cells were washed with PBS (1 mL) and further incubated with E. coli (1 μL, 1 × 107−108 CFU), LPS (1 μL, 10 mg mL−1), S. aureus (1 μL, 1 × 107−108 CFU) for 4 h in DMEM free of FBS. The resultant cells were analyzed by confocal fluorescence microscopy. In Vivo Biodistribution of Sia−pNIR and Glu−pNIR in Mice. A cohort of ICR mice (female, 4 weeks of age) was injected with E. coli (100 μL, 1 × 107−108 CFU) into muscle of

the right thigh. At 1−2 days after injection, the mice were injected intravenously via the tail vein with Sia−pNIR (40 mg kg−1) or Glu−pNIR (40 mg kg−1). At 0−168 h following probe injection, the mice were anesthetized with intraperitoneal injection of 0.5% chloral hydrate (0.01 g mL−1) and then analyzed for whole-body NIR fluorescence. The anesthetized mice were then sacrificed and the inflamed tissue and selected organs were excised, washed with PBS, and then subjected to ex vivo analysis for the NIR fluorescence. Effects of Antibiotics on Inflammation Imaging. ICR mice (male, 4 weeks of age) were in the muscle of the right thigh by injections of E. coli (100 μL, 1 × 107−108 CFU). At 2 days after bacteria injection, the mice were injected with streptomycin (20 mg kg−1) or PBS (100 μL) three times a day in muscle of the left thigh for 4 days. These mice were administered with Sia−pNIR (40 mg kg−1) via the tail vein into mice roughly the same time as the first streptomycin treatment. At 48 and 96 h following Sia−pNIR injection, the mice were anesthetized with intraperitoneal injection of 0.5% chloral hydrate (0.01 g mL−1) and then analyzed for whole-body NIR fluorescence. Optoacoustic Inflammation Imaging in Mice with Sia−pNIR. Nude mice that had been injected with S. aureus (100 μL, 1 × 10 7−10 8 CFU) in thigh muscle were intravenously injected with Sia−pNIR (40 mg kg−1) or PBS (100 μL). At 0 or 24 h after Sia−pNIR injection, the mice were anesthetized and then analyzed for photoacoustic contrast in the inflamed sites.

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ASSOCIATED CONTENT

S Supporting Information *

pH-mediated fluorescence and optoacoustic contrast of SiapNIA and Glu−pNIR, flow cytometric analysis on bacteria- and LPS-induced fluorescence increase in Sia−pNIR-loaded cells, and effects of Sia on cellular uptake of Sia−pNIR. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b00847.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +865922181728. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the 973 program 2013CB933901, NSF China (21272196, 21305116), Natural Science Foundation of Fujian Province of China (2011J06004), PCSIRT, and an open project grant from State Key Laboratory of Chemo/biosensing and Chemo-metrics (2012002). Dr. J. Han was supported by grants from NSF China (91429301, 31420103910, 31330047, 31221065), the National Scientific and Technological Major Project (2013ZX10002-002), the HiTech Research and Development Program of China (863program; 2012AA02A201), the 111 Project (B12001), the Science and Technology Foundation of Xiamen (3502Z20130027), the National Science Foundation of China for Fostering Talents in Basic Research (J1310027), and The Open Research Fund of State Key Laboratory of Cellular Stress Biology, Xiamen University. G

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DOI: 10.1021/acs.analchem.5b00847 Anal. Chem. XXXX, XXX, XXX−XXX