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Organic−Inorganic Hybrid Nanoparticles for Tracking the Same Cells Seamlessly at the Cellular, Tissue, and Whole Body Levels Koichiro Hayashi,*,† Yusuke Sato,† Hiroki Maruoka,‡ Wataru Sakamoto,† and Toshinobu Yogo† †

Division of Materials Research, Institute of Materials and Systems for Sustainability, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan ‡ Technical Research Laboratory Applied Development Group, Kurabo Industries Ltd., 14-30, Shimokido-Cho, Neyagawa, Osaka 572-0823, Japan S Supporting Information *

ABSTRACT: Techniques to elucidate the kinetics and distribution of the same cells in the whole body and in tissues are necessary for further studies of cancer, immunity, and regenerative medicine. Fluorescent imaging is a powerful technique for visualization of cells. However, current fluorescent probes are applicable in either the ultraviolet (UV)−visible (Vis) region (300−650 nm) or the biological transparency window (BTW, 650−900 nm), but not both. Thus, they cannot serve as fluorescent probes for both in vivo and in vitro imaging, and it is difficult to achieve imaging of the same cells seamlessly from the cellular level to the whole body and tissue levels using currently available fluorescent probes. Accordingly, in this paper, we describe organic−inorganic hybrid nanoparticles (HNPs) that could be used to achieve seamless tracking of the same cells. Within the HNPs, a porphyrin molecule, Vis-fluorophore, was surrounded by a siloxane chain, preventing the aggregation of porphyrin molecules. As a result, the porphyrin fluorescence was not quenched. Furthermore, indocyanine green (ICG), a BTW fluorophore, was localized on the HNP surface, leading to fluorescence resonance energy transfer (FRET) from porphyrin to ICG only near the HNP surface. Through the above structural design, the HNPs acquired both excitation (λex) and emission (λem) wavelengths in the visible region and BTW, respectively, as well as large Stokes shifts. The HNP-labeled immune cells successfully and the labeled cells were separated easily from unlabeled cells by fluorescenceactivated cell sorting. The kinetics of the labeled cells in the whole body were revealed by fluorescence imaging within BTW. Furthermore, the distributions of the same labeled cells were elucidated by histological analysis within the UV−vis region. Thus, the HNPs served as fluorescent probes for seamless tracking of the same cells. KEYWORDS: organic−inorganic hybrid, nanoparticles, fluorescence imaging, nanomedicine

1. INTRODUCTION Fluorescence imaging is a necessary technique for studies in the biomedical field.1 In particular, fluorescence imaging has been shown to be a powerful tool for investigation of the behaviors of cells within the whole body, organs, and tissues in various research fields, such as cancer,2 immunity,3 and regenerative medicine.4 In general, fluorescence imaging of cells and tissues (i.e., in vitro imaging and histological analysis) is conducted in the ultraviolet (UV)-visible (Vis) region (wavelength [λ] = 300− 650 nm).5 However, UV−vis light has poor permeability into the body and strong autofluorescence occurs in the UV−vis region, which decreases the signal-to-noise ratio (SNR).6 In contrast, light in the biological transparency window (BTW; λ = 650−900 nm) penetrates deeply into the body compared with UV−vis light, and autofluorescence is weak in the BTW.6 Therefore, fluorescence imaging of the whole body (in vivo imaging) is conducted in the BTW. Accordingly, fluorescence probes for in vivo imaging are distinguished from those for in vitro imaging or histological analysis.7 Thus, cells visualized by in vivo imaging are different from those visualized by in vitro © XXXX American Chemical Society

imaging or histological analysis. However, to investigate the mechanisms of cancer metastasis and the therapeutic effects of regenerative medicine, cells visualized by in vivo imaging should be the same as those visualized by in vitro imaging or histological analysis. In vivo imaging, histological analysis, and in vitro imaging should be achieved seamlessly using a single type of fluorescent probe in order to observe the same cells at the whole body, organ, tissue, and cellular levels. This type of imaging is called seamless imaging. If seamless imaging is achieved, researchers may realize cell labeling with fluorescent probes, collection of labeled cells by flow cytometry, tracking of labeled cells within the body by in vivo imaging, and elucidation of the distribution of the labeled cells in organs and tissues by histological analysis. Fluorescent probes with both excitation (λex) and emission wavelengths (λ em ) in the UV−vis region and BTW, respectively, are required to achieve seamless imaging. Received: March 23, 2017 Accepted: April 13, 2017 Published: April 13, 2017 A

DOI: 10.1021/acsbiomaterials.7b00181 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 1. (A) Synthesis scheme of ICG-HNPs. (B) Conceptual diagram of seamless imaging of the same cells using ICG-HNPs.

Furthermore, these fluorescent probes should have large Stokes shifts to obtain images at good SNRs. Quantum dots (QDs) are typical fluorescent probes that have λex and λem in either UV− vis or BTW, but not both.8 In addition, most QDs are toxic.9−12 In contrast, silica incorporated with fluorophores/pure silica core/shell nanoparticles, called Cornell dots, function as bright, stable, and biocompatible fluorescent probes.13,14 However, indiscrete incorporation of fluorophores into silica nanoparticles causes aggregation of fluorophores in the nanoparticles, resulting in proximity quenching owing to excited state energy transfer from one fluorophore to another.15 Fluorescence resonance energy transfer (FRET) is an effective method for increasing the Stokes shift of a fluorescent probe.16 FRET occurs when the λem of the UV−vis fluorophore is nearly equal to the λex of the BTW fluorophore and the distance between the UV−vis and BTW fluorophores is short enough to allow for energy transfer from the former to the latter. However, FRET from UV−vis to BTW fluorophores occurs almost completely when both fluorophores are incorporated into the inner part of silica nanoparticles. As a result, visible fluorescence is not obtained or is very weak. Hence, proper design of nanoparticle structure and selection of fluorophores are necessary to prevent proximity quenching, increase the Stokes shift, and provide both λex and λem in UV−vis and BTW, respectively. In this study, we synthesized fluorophores-siloxane hybrid nanoparticles (HNPs) with both λex and λem in UV−vis and BTW, respectively, by incorporating one fluorophore, porphyrin, into the HNP framework via covalent bonds and localizing the other fluorophore, indocyanine green (ICG), on the HNP surface (Figure 1A). The main points of the HNP structure design are as follows: (1) the siloxane chains may act as separators for isolating porphyrin molecules, resulting in prevention of proximity quenching caused by aggregation of porphyrins; and (2) the FRET from porphyrins to ICG may occur only near the HNP surface, which may provide large Stokes-shifted visible and BTW fluorescences. Furthermore, we demonstrated that the HNPs enabled the seamless tracking of

the same immune cells at the cellular, whole body, and tissue levels (Figure 1B).

2. EXPERIMENTAL SECTION 2.1. Materials. Tetrakis(4-carboxyphenyl)porphyrin (TCPP), 3aminopropyltriethoxysilane (APTES), (3-mercaptopropyl)trimethoxysilane (MPTMS), 1-(3-dimethyl aminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC), N-hydroxysuccinimide (NHS), and N,N-dimethylformamide (DMF) were purchased from Tokyo Chemical Industry (Tokyo, Japan). Maleimide-binding ICG (ICGmal) was purchased from Goryo Chemical (Sapporo, Japan). Ammonia−water (28%) was purchased from Kishida Chemical (Osaka, Japan). 2.2. Preparation of the Precursor. Alkoxysilane having porphyrin within a molecule, 4,4′,4″,4‴-(porphyrin-5,10,15,20tetrayl)tetrakis(N-(3-(triethoxysilyl)propyl) benzamide) (por-silane), was prepared as a precursor by the method described in our previous paper.17,18 In brief, APTES (15 mM) and TCPP (3.75 mM) were dissolved in DMF. EDAC (15 mM) and NHS (15 mM) were added to this solution. The mixture was stirred for 24 h at room temperature to form por-silane via the amidation reaction between the amino group of APTES and the carboxylic acid groups of TCPP. 2.3. Synthesis of Porphyrin-Siloxane HNPs. Porphyrin-siloxane HNPs were synthesized by hydrolysis and condensation of por-silane and MPTMS. Here, MPTMS was used for introducing thiol groups on the HNP surface in one step. The HNPs were synthesized as follows. The por-silane (0.75 mM) and MPTMS (27 mM) were dissolved in DMF. Ammonia−water (4.4 M) was added to the above solution. The solution was stirred for 24 h at 80 °C. The product was collected by centrifugation at 36 000 × g for 20 min and was then redispersed in distilled water (DW). This processing was repeated at least three times to wash the products. 2.4. Modification of HNPs with ICG. The HNPs were modified with ICG via the Michael addition reaction between the thiol groups on the HNP surface and maleimide group of ICG-mal, as follows. The PS HNPs were dispersed in DW at 1.0 mg/mL. ICG-mal (42 μM) was then added to the dispersion. The dispersion was stirred at 30 °C for 3 h. The product was collected by centrifugation at 22 140 × g for 20 min and was then redispersed in DW. This processing was repeated at least three times to wash the products. 2.5. Characterization. The sizes and shape of products were observed using a transmission electron microscope (TEM; H-800; B

DOI: 10.1021/acsbiomaterials.7b00181 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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cells labeled with ICG-HNPs and Hoechst (1 × 107 cells/mouse) using a multispectral fluorescence imager (KURAVIC-vivo; Kurabo, Osaka, Japan) at λex of 655 nm and λem of 700, 720, 740, 760, 780, 800, 820, and 840 nm preinjection and at 15 min, 4 h, and 24 h postinjection (n = 5). The fluorescent intensity per square meter of tissue was measured by ex vivo fluorescence imaging of organs excised from mice at 4 h postinjection. 2.13. Histological Analyses. Livers were excised from mice 4 h after intravenous injection of cells labeled with ICG-HNPs and Hoechst and then fixed for 48 h using 4% paraformaldehyde solution. The fixed livers were embedded in paraffin and 3-μm-thick paraffin sections were prepared using a microtome. The sections were stained with hematoxylin and eosin. Unstained sections were also prepared to determine the distributions of cells labeled with ICG-HNPs and Hoechst in the liver tissues.

Hitachi, Tokyo, Japan). Hydrodynamic diameter was measured using a dynamic light scattering (DLS) instrument (DelsaMax PRO equipped with DelsaMax ASSIST; Beckman Coulter, CA, USA). Solid state 29Si nuclear magnetic resonance (NMR) spectra of the HNPs modified with ICG (ICG-HNPs) were recorded with an Avance 300 spectrometer (Bruker, MA, USA). The Fourier transform infrared (FTIR) spectra were recorded with an FTIR spectrometer (Nicolet iS10; Thermo Fisher Scientific, MA, USA). The thermal behavior of the ICG-HNPs was analyzed by thermogravimetric (TG)/differential thermal analysis (DTA; TG 8120; Rigaku, Tokyo, Japan). Hydrodynamic diameters and zeta potentials of the HNPs and ICG-HNPs were measured using a light scattering analyzer (DelsaMax PRO equipped with DelsaMax ASSIST; Beckman Coulter, CA, USA). Absorption and fluorescence properties were investigated using a UV− vis spectrophotometer (V-570; JASCO, Tokyo, Japan) and fluorescence spectrophotometer (FP-8600; JASCO). 2.6. Quantification of Thiols on the HNP Surface. Quantification of thiols on the HNP surface was performed using the thiol-specific reagent 4-dithiodipyridine (4-DPS), which reacts with thiol to form a stoichiometric amount of the chromogenic compound 4-thiopyridone (4-TP) with absorption at 324 nm.19 4DPS (5 mM) was dissolved in 0.1 M phosphate buffer. The HNPs (0.17 mg/mL) were dispersed in 0.1 M phosphate buffer. The 4-DPS solution (20 μL) was added to the solution containing the HNPs (200 μL). After incubation at 30 °C for 10 min, the supernatant was collected by centrifugation at 22,140 × g for 20 min. The absorption spectrum of the supernatant was measured using a UV−vis spectrophotometer. The amount of thiol on the HNP surface was estimated using the following equation

cSH = A324 /(ε324lm)

3. RESULTS AND DISCUSSION TEM images of the HNPs revealed that the HNPs had a primary particle diameter of 38.5 nm and that the particles formed aggregates (Figure 2A). The hydrodynamic diameter of

(1)

,where cSH is the amount of thiol per gram of HNPs (mol/g), A324 is the absorbance of the supernatant at 324 nm, ε324 is the molar absorbance coefficient of 4-TP at 324 nm (19,800 M−1 cm−1),20 l is the light path (1 cm), and m is the concentration of HNPs. 2.7. Quantification of ICG Molecules on the HNP Surface. The amount of ICG on the HNPs was estimated from differences in the thiol amount before and after surface modification of the HNPs with ICG. 2.8. Cell Lines and Animals. RAW264.7 macrophages were obtained from RIKEN Cell Bank (Tsukuba, Japan). Cells were maintained in Dulbecco’s modified Eagle’s medium containing fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37 °C. BALB/c-nu/nu mice (female; 6 weeks of age) were purchased from Japan SLC (Shizuoka, Japan) and maintained in a specific pathogen-free facility in the Center for Animal Research and Education of Nagoya University. All animal experiments were conducted with the approval of the Animal Care Committee of Nagoya University in accordance with the Fundamental Academic Research Institution of Japan. 2.9. Cytotoxicity Assay. ICG-HNPs (50−400 μg/mL) were added to culture medium in the presence of RAW264.7 cells (5 × 105 cells/mL). The cells were incubated in a humidified atmosphere containing 5% CO2 at 37 °C for 24 h. Cell survival rate was estimated by the water-soluble tetrazolium (WST)-1 assay. 2.10. Cell Labeling. ICG-HNPs (50 μg/mL) were added to culture medium in the presence of RAW264.7 cells (5 × 105 cells/ mL). The cells were incubated in a humidified atmosphere containing 5% CO2 at 37 °C for 24 h. Labeling of cells with ICG-HNPs was confirmed by fluorescence imaging using a fluorescence microscope (EVOS FL Cell Imaging System; Thermo Fisher Scientific, MA, USA) at an excitation wavelength (λex) of 425 nm and emission wavelength (λem) of 655 nm. The nuclei of live cells were labeled with Hoechst and the fluorescence was observed at λex of 357 nm and λem of 447 nm. 2.11. Cell Sorting. ICG-HNP-labeled cells were collected by fluorescence-activated cell sorting (FACS) using a flow cytometer (LSRFortessa X-20; BD Biosciences, CA, USA; λex = 405 nm and λem = 670 nm; λex = 640 nm and λem = 780 nm). 2.12. In Vivo Cell Tracking. In vivo cell tracking was conducted by fluorescence imaging of mice subjected to intravenous injection of

Figure 2. (A) TEM image of ICG-HNPs. (B) Particle size distribution of ICG-HNPs in PBS. (C) Solid-state 29Si NMR spectrum of ICGHNPs. (D) FTIR spectrum of HNPs and TCPP.

the HNPs in phosphate buffered saline (PBS), measured by DLS, was 41 nm (Figure 2B). Thus, the primary diameter estimated from the TEM images was nearly equal to the hydrodynamic diameter, indicating the HNPs were dispersed uniformly in PBS and did not aggregate. The HNP aggregates observed in the TEM image were considered to probably have formed by solvent evaporation from the TEM grids in the process of preparation of samples for TEM. The two resonance peaks at −58.8 and −68.8 ppm in the solid-state 29Si NMR spectrum of the HNPs (Figure 2C) could be ascribed to the Si environments of T2 and T3, respectively.21 These results established that two or three of the alkoxy groups of por-silane and MPTMS underwent hydrolysis and condensation to form a siloxane network. The FTIR spectra of TCPP and the HNPs showed that the absorptions attributed to the pyrrole rings in the porphyrin appeared at 3309 and 3313 cm−1, respectively (Figure 2D).22 The absorption bands owing to the amine of porphyrin were observed at 3309 and 3313 cm−1 in the FTIR spectra of TCPP and the HNPs, respectively.22 The absorption attributed to carboxylic acid appeared at 1708 cm−1 in the C

DOI: 10.1021/acsbiomaterials.7b00181 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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surface was 7 μmol/g HNPs. Thus, the thiol groups on the HNP surface decreased by 36 μmol after modification of HNPs with ICG. This result indicated that 36 μmol ICG/g HNPs bound to the HNP surface. Because 42 μmol ICG-mal/g HNPs was used for surface modification of the HNPs, the surface modification efficiency was 86%. Absorptions due to porphyrin and ICG were observed at 415 and 810 nm, respectively, in the spectrum of ICG-HNPs (Figure 3A). In contrast, absorption owing to ICG was not observed in the spectrum of unmodified HNPs. Furthermore, absorption owing to ICG in the spectrum of ICG-HNPs redshifted by 30 nm compared with that in the spectrum of ICG alone. ICG is known to form J-aggregates at high concentrations, which leads to a red shift in the absorption.24 These findings demonstrated that ICG bound to the HNP surface densely, consistent with the results showing that there were abundant ICG molecules present on the HNP surface, as estimated from changes in the amount of thiols before and after surface modification of HNPs with ICG. ICG-HNPs had λex and λem values in both the visible region and BTW; i.e., λex = 425 nm and λem = 650 nm in the visible region (Figure 3C) and λex = 650 nm and λem = 885 nm in the BTW (Figure 3D). Thus, Stokes shifts were 225 and 235 nm in the visible region and BTW, respectively, which were large enough to eliminate the harmful influence of autofluorescence and obtain high SNR images. The main λexs of ICG were 610 and 760 nm, and ICG had no λex at 650 nm (Figure S2A). ICG emitted fluorescence at 820 nm in the case of λex = 760 nm (Figure S2B), indicating that the Stokes shift was 60 nm, which was significantly smaller than Stokes shift of ICG-HNPs. These findings demonstrated that FRET occurred from HNPs to ICG on the HNP surface and that FRET provided a significantly large Stokes shift in the BTW. The cytotoxicity of ICG-HNPs was estimated using WST-1 assays. The majority of cells survived at concentrations of ICGHNPs below 200 μg/mL and the cell survival rate was 54% when the concentration of ICG-HNPs was 400 μg/mL. Thus, ICG-HNPs exhibited little toxicity, at least below 200 μg/mL. We confirmed that ICG-HNPs labeled immune cells in RAW 264.7 macrophages by in vitro fluorescence imaging within the visible region (Figure 4). The nuclei of live cells were stained with Hoechst. The fluorescence derived from ICG-HNPs was observed in the cellular cytoplasm, indicating that the ICGHNPs were localized in the cytoplasm. The nuclei of cells internalizing the ICG-HNPs were stained with Hoechst, indicating that ICG-HNPs labeled the cytoplasm without causing cell death. These results demonstrated that the ICGHNPs served as nontoxic fluorescent probes for visualizing the cytoplasm by in vitro imaging. The separation of ICG-HNP-labeled cells and unlabeled cells is necessary to obtain accurate data on the biodistributions of

spectrum of TCPP.22 However, this band disappeared, and the absorptions attributed to the amide appeared at 1658 cm−1 in the spectrum of the HNPs.22 These findings revealed that the porphyrin molecules were incorporated in the siloxane network of the HNPs via amide linkages. In the TG curve of the HNPs (Figure S1), a weight loss of 53% was observed at 800 °C and the residue was 47%. Thus, the HNPs were composed of 53% organics and 47% siloxane. The HNPs showed absorption due to TCPP at 425 nm (Figure 3A). In the spectrum of the ICG-HNPs, absorption due

Figure 3. (A) Absorption spectra of ICG-HNPs, HNPs, ICG, and TCPP. (B) Photographs of HNPs and ICG-HNPs. (C) Excitation (blue) and fluorescence (red) spectra of ICG-HNPs; the excitation spectrum was measured at λem = 650 nm, and the fluorescence spectrum was measured at λex = 425 nm. (D) Excitation (blue) and fluorescence (red) spectra of ICG-HNPs; the excitation spectrum was measured at λem = 885 nm, and the fluorescence spectrum was measured at λex = 650 nm.

to ICG and TCPP was observed. Furthermore, the ICG-HNPs were green in color to reflect the ICG color, whereas the HNPs were pink in color (Figure 3B). These results demonstrated that the HNPs were successfully modified with ICG. The amount of ICG on the ICG-HNP surface was estimated from changes in the amount of thiol on the HNP surface, as measured by Ellman’s method,23 before and after modification of HNPs with ICG. The unmodified HNPs had 42 μmol thiol groups/g HNPs. The amount of thiol groups on the ICG-HNP

Figure 4. In vitro fluorescence images of cells incubated with ICG-HNPs and Hoechst. Hoechst: λex = 357 nm and λem = 447 nm. ICG-HNPs: λex = 425 nm and λem = 655 nm. D

DOI: 10.1021/acsbiomaterials.7b00181 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 5. In vivo fluorescence images of mice subjected to intraperitoneal injection of cells labeled with (A) ICG-HNPs and (B) CellVue Maroon: pink indicates fluorescence derived from ICG-HNPs and yellow indicates mouse autofluorescence and feed fluorescence. (C) In vivo fluorescence image of a mouse subcutaneously injected with ICG-HNP-labeled cells. The injected cell numbers were 2 × 105, 1 × 105, 0.5 × 105, and 0. (D) Average fluorescence intensity of the areas injected with the ICG-HNP-labeled cells per square millimeter, estimated from Figure 5C. (E) In vivo fluorescence images of mice injected with cells labeled with ICG-HNPs and Hoechst. (F) Ex vivo fluorescence images of uninjected mice and mice at 24 h postinjection of cells labeled with ICG-HNPs and Hoechst. (G) Average fluorescence intensity of the liver per square millimeter, estimated from image E. (H) Bright-field and fluorescence images of organs excised from an uninjected mouse and a mouse at 2 h postinjection of cells labeled with ICG-HNPs and Hoechst (H, heart; Lu, lungs; Li, liver; Sp, spleen; St, stomach; I, intestine; K, kidney; SG, seminal gland; B, bladder; and C, cecum). (J) Average fluorescence intensity of organs at 2 h postinjection per square millimeter, estimated from image I. All fluorescence images were acquired at λex = 655 nm and λem = 700, 720, 740, 760, 780, 800, 820, and 840 nm.

injected ICG-HNP-labeled cells decreased in the range from 2 × 105 to 0.5 × 105 cells (Figure 5D). The ICG-HNP-labeled cells were detectable even at the cell number of 0.5 × 105 in vivo, which was significantly lower than the detectable number of cells labeled with common fluorescent dyes. For example, green fluorescent protein could not detect even 1 × 108 cells and tdTomato and DsRed2-N1 required 1 × 106 and 2 × 107 cells to detect, respectively. Thus, the ICG-HNPs enabled extremely sensitive detection of cells as compared with common fluorescent dyes. To establish that ICG-HNPs were useful for in vivo cell tracking and the investigation of cell distribution within tissues, cells labeled with ICG-HNPs and Hoechst were injected intravenously into mice. In vivo fluorescent images showed that the fluorescence of the labeled cells was detectable separately from mouse autofluorescence and feed fluorescence even in the case of intravenous injection owing to the large Stokes shift of ICG-HNPs (Figure 5D). In vivo images showed that the labeled cells accumulated in the liver and spleen at 2 h postinjection (Figure 5D). The fluorescent images of the organs excised from mice at 2 h postinjection also showed that the fluorescence of the labeled cells was observed in the liver and spleen, whereas little fluorescence was observed in the other organs (Figure 5H, I). Thus, the fluorescent images of excised organs confirmed the validity of the results of in vivo imaging. Following 2 h postinjection, the fluorescence of the labeled cells in the liver gradually decreased with time (Figure 5E, G). However, the labeled cells did not disappear completely and remained in the liver even at 24 h postinjection as observed

the cells. We attempted to separate the ICG-HNP-labeled and unlabeled cells by FACS at λex = 405 nm and λem = 670 nm. The fluorescence intensity of ICG-HNP-labeled cells was 100− 10 000 times higher than that of unlabeled cells (Figure S3). Hence, ICG-HNP-labeled cells were easily separated from unlabeled cells. These results demonstrated that ICG-HNPs were usable as fluorescent probes for flow cytometry. To confirm that the fluorescence from ICG-HNPs-labeled cells was detectable in distinction from the autofluorescence of mice and fluorescence of feed, we injected ICG-HNP-labeled cells intraperitoneally into mice. For comparison, we also intraperitoneally injected cells labeled with a commercial fluorescent cell labeling dye (CellVue Maroon, λex = 647 nm and λem = 667 nm), which had an excitation wavelength nearly equal to ICG-HNPs. In vivo fluorescence images showed that fluorescence from ICG-HNP-labeled cells was detectable in distinction from mouse autofluorescence and feed fluorescence (Figure 5A). In contrast, fluorescence from cells labeled with the commercial dye could not be distinguished because the λex and λem of the dye overlapped mouse autofluorescence and feed fluorescence (Figure 5B). These results demonstrated that the significantly large Stokes shift of ICG-HNPs overcame the harmful influence of mouse autofluorescence and feed fluorescence and enabled in vivo tracking of the labeled cells. To investigate the relationship between cell number and fluorescence intensity, we subcutaneously administered the ICG-HNP-labeled cells to the mouse back at cell numbers of 2 × 105, 1 × 105, 0.5 × 105, and 0 cells (Figure 5C). The fluorescence strength decreased linearly as the number of E

DOI: 10.1021/acsbiomaterials.7b00181 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 6. (A) HE-stained tissue section of liver samples excised from mice at 4 h after intravenous injection of cells labeled with ICG-HNPs and Hoechst. (B) Unstained tissue sections of the region enclosed by the square in Figure 6A. Hoechst: λex = 357 nm and λem = 447 nm. ICG-HNPs: λex = 425 nm and λem = 655 nm.

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in in vivo fluorescent images (Figure 5E). The in vivo fluorescence image at 24 h postinjection was coincident with the ex vivo fluorescence image at 24 h postinjection (Figure 5F), indicating that the ICG-HNPs enabled in vivo cell tacking over a period of 24 h. The above results demonstrated that ICG-HNPs could serve as fluorescent probes for investigation of the biodistribution of cells within the whole body. We prepared tissue sections from liver samples after ex vivo imaging to reveal the localization of the labeled cells within the liver. The unstained tissue sections showed that fluorescence derived from ICG-HNPs was observed at the same position as fluorescence derived from Hoechst (Figure 6). Thus, fluorescence derived from ICG-HNPs indicated the position of cells injected into mice. These results demonstrated that the ICG-HNPs revealed the disposition of labeled cells not only within the whole body but also within the tissues.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00181. TG-DTA curves of HNPs; excitation and fluorescence spectra of ICG; and histograms of cells labeled with ICGHNPs and unlabeled cells (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Koichiro Hayashi: 0000-0002-3147-5784 Notes

The authors declare no competing financial interest.

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4. CONCLUSION

ACKNOWLEDGMENTS We are grateful to the Center for Animal Research and Education (CARE) and the Technical Center at Nagoya University. This work was supported by a Grant-in-Aid for Young Scientists (A) (26709050) and a Grant-in-Aid for Exploratory Research (15K14146) from the Japan Society for the Promotion of Science (JSPS). Additionally, the work was partly supported by a Health Labor Sciences Research Grant from the Ministry of Health Labor and Welfare and a Longrange Research Initiative (LRI) grant from the Japan Chemical Industry Association (JCIA).

HNPs with large Stokes shifts in both the visible region and BTW were successfully obtained by isolation of porphyrin with siloxane chains within HNPs and localization of ICG on the HNP surface. The large shift in BTW was provided by FRET from porphyrin to ICG near the HNP surface. The ICG-HNPs labeled immune cells. The labeled cells were separated easily from unlabeled cells by FACS. The kinetics and distribution of the labeled cells in the whole body were revealed by in vivo fluorescence imaging. Furthermore, the distribution of the labeled cells in tissues was revealed by histological analyses. Thus, the ICG-HNPs enabled tracking of the same cells seamlessly at the cellular, tissue, and whole body levels with facility. However, it is difficult to quantitate the number of cells accurately by fluorescence imaging using ICG-HNPs. These are inherent limitations of fluorescence imaging. Positron emission tomography (PET) imaging potentially overcomes these limitations, although it requires special and expensive installation. Furthermore, it is difficult to track the same cells for a long period of time by PET because of the short half-life period of radioactive tracers, as the half-life periods of common tracers are 2 to 110 min.25 Thus, fluorescence imaging using ICG-HNPs in this study has the potential to become a powerful technique to trace the same cells seamlessly at the cellular, tissue, and whole body levels for a long period of time with facility at low cost, although it is difficult to quantitate the number of cells accurately. This seamless imaging technique using ICG-HNPs may contribute to advancements in the study of cancer, immunity, and regenerative medicine.

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REFERENCES

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DOI: 10.1021/acsbiomaterials.7b00181 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsbiomaterials.7b00181 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX