Thiol-Organosilica Particles Internally Functionalized with Propidium

Sep 18, 2012 - In the ventral view, fluorescence was detected on the upper abdomen until 1 h but was detected in the middle and lower abdomen after 1...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/cm

Thiol-Organosilica Particles Internally Functionalized with Propidium Iodide as a Multicolor Fluorescence and X‑ray Computed Tomography Probe and Application for Non-Invasive Functional Gastrointestinal Tract Imaging Michihiro Nakamura,*,† Aziz Awaad,† Koichiro Hayashi,† Kazuhiko Ochiai,‡ and Kazunori Ishimura† †

Department of Anatomy and Cell Biology, University of Tokushima Graduate School of Medical Sciences, 3-18-15 Kuramoto, Tokushima 770-8503, Japan ‡ Department of Basic Science, School of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1-7-1 Kyonan, Musashino, Tokyo 180-8602, Japan S Supporting Information *

ABSTRACT: Propidium iodide (PI) is a fluorescent nucleic acid dye that contains iodide molecules. Thiol-organosilica particles that were internally functionalized with PI (PI particles) were prepared for X-ray computed tomography (CT) and multicolor fluorescence imaging. PI particles of various sizes were synthesized in a one-pot process. The particles showed unique fluorescent signals and X-ray absorption, with enhancement of the fluorescence intensity of PI located inside organosilica particles. PI particles had multicolor fluorescence, including original fluorescence and near-infrared (NIR). Orally administered PI particles were observed in the gastrointestinal tract (GIT) using fluorescent imaging devices and X-ray CT. An in vivo fluorescence imaging system could detect the NIR fluorescence of PI particles in the GIT specifically. Multipurpose zoom fluorescence microscopy was used to noninvasively visualize the real-time passage of particles and the movement of the GIT. The passage and distribution of particles over time in the GIT were demonstrated using X-ray CT. A correlation analysis between the fluorescent and X-ray CT data demonstrated the characteristics, limitations, and novel potential for noninvasive functional GIT dual modal imaging. KEYWORDS: organosilica particles, propidium iodide, multicolor fluorescence imaging, near-infrared, X-ray computed tomography, gastrointestinal tract

■

INTRODUCTION Organosilica particles are novel materials that are prepared from a single organosilicate coupling agent such as 3mercaptopropyltrimethoxysilane (MPMS).1−6 Organosilica particles are both structurally and functionally different from typical silica particles prepared from tetraethoxyorthosilicate because these organosilica particles contain both interior and exterior functionalities. Typical silica particles can be internally functionalized with fluorescent dyes, and are applied to biomedical fields as fluorescent nanoparticles.7−10 Recently core−shell fluorescent silica nanoparticles (C dots), received FDA approval for a first-in-human clinical trial as a cancerspecific probe.10 The organosilica particles can also be internally functionalized with various fluorescent dyes and fluorescent nanomaterials using a one-pot synthesis.1−6 Organosilica particles that are internally functionalized with fluorescent materials were useful for various fluorescence imaging (FI) techniques such as in vivo fluorescent imaging,6 cell labeling,3−6 single cell functional analysis,11 and molecular tracing.6 © 2012 American Chemical Society

Propidium iodide (PI) is an intercalating agent and a fluorescent dye and is commonly used as a DNA staining reagent.12−15 Once PI binds to nucleic acids, its fluorescence intensity is enhanced, and its fluorescent properties are changed. PI is useful for DNA staining or viability assays using fluorescence microscopy or flow cytometry, and it contains iodide molecules and has X-ray absorption. Because of these properties, organosilica particles that are internally functionalized with PI can be used as dual mode imaging probes for X-ray computed tomography (CT) and FI. We prepared thiol-organosilica particles that were internally functionalized with PI (PI particles). We found unique changes in the PI fluorescence properties when it was embedded in thiol-organosilica particles; therefore, we used PI particles for multicolor FI and X-ray CT of the mouse gastrointestinal tract (GIT). Received: July 26, 2012 Revised: September 18, 2012 Published: September 18, 2012 3772

dx.doi.org/10.1021/cm3023677 | Chem. Mater. 2012, 24, 3772−3779

Chemistry of Materials

■

■

EXPERIMENTAL SECTION

Article

RESULTS AND DISCUSSION Preparation of PI Particles. PI particles were prepared by a one-step, one-pot synthesis using PI and MPMS (Figure 1A).

Materials. MPMS and rhodamine B were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO). PI was obtained from Calbiochem (San Diego, CA). Ethanol and 28% NH4OH were purchased from Katayama Chemical Industries (Osaka, Japan). Disposable animal feeding needles were purchased from Fuchigami (Kyoto, Japan). Preparation of PI Particles. For the preparation of particles, 6.25, 12.5, 25, 50, 100, or 200 mM MPMS was mixed with 40.6, 81.3, 162.5, 325, 650, or 1,300 μM PI or rhodamine B. The mixtures were mixed with 27% NH4OH and incubated at 100 °C for 24 h. To evaluate the effects of PI on the particle size, 325, 163, 82, 41, 20, 10, or 5 μM PI was mixed with 100 mM MPMS and 27% NH4OH and incubated at 100 °C for 24 h. After incubation, the reaction mixtures were subjected to centrifugation to remove any remaining reagents, and the pellet was sonicated. The particles were washed extensively with 70% ethanol and water. Electron Microscopy. The particles were fixed on a 400-mesh copper grid coated with nitrocellulose, and transmission electron microscopy (TEM) images were obtained with a Hitachi H7650 electron microscope (Hitachi, Tokyo, Japan). The sizes and standard deviations of approximately 200 particles were analyzed using Imagepro plus software (MediaCybernetics, Inc., Georgia, U.S.A.). Fluorescence Studies. The fluorescent intensities of a 6.6 μM PI solution and a 1:100 diluted PI particle solution (synthesized with 100 mM MPMS, 660 μM PI, and 27% NH4OH) were obtained with an F2500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Fluorescent intensities were normalized to those in water. Flow Cytometry. Flow cytometry analysis of the PI particles was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) with a 488 nm excitation laser. Fluorescence was detected on the FL1 (530/30 nm bandpass filter), the FL2 (585/30 nm bandpass filter), and the FL3 (670 nm long-pass filter) channels. Oral Administration of PI Particles. All experiments involving mice were performed with consent from the Animal Experimentation Committee of the University of Tokushima. The mice (age 6−8 weeks) were fasted overnight with free access to water before oral administration of 2 mL of a PEG solution (Niflec, Ajinomoto Pharmaceutical Co, Tokyo, Japan) over 2 h (0.5 mL/15 min) using gavage needles. The mice were then fasted with free access to water for 6 h before oral administration of a solution containing PI particles. We used PI particles prepared from 100 mM MPMS and 650 μM PI. We orally administered 4 mg of PI particles suspended in 400 μL of distilled water and 650 μM PI dissolved in 400 μL of phosphatebuffered saline with a disposable animal feeding needle. In Vivo Fluorescence Imaging. Whole-body fluorescence was observed using an IVIS Spectrum (Caliper Life Sciences, Hopkinton, MA) at 24 and 48 h after oral administration. Excitation and emission wavelengths were chosen as 605 nm (passband 590−620 nm) and 660 nm (passband 650−670 nm), respectively. The total photon counts per second per cm2 were measured using the Living Image software (Caliper Life Sciences, Hopkinton, MA). In Vivo Functional Fluorescence Imaging. Fluorescence in the GIT was observed with a multipurpose zoom microscope (AZ100M, Nikon, Kanagawa, Japan) equipped with a 100-W mercury lamp as a light source and a CCD camera (Digital sight DS-Ri1, Nikon, Kanagawa, Japan). Excitation and emission wavelengths were chosen as 540 and 605 nm, respectively. X-ray Computed Tomography. Mice were anesthetized with pentobarbital and placed in a CT scanner chamber for small animals (Latheta LCT-200, Aloka, Tokyo, Japan) at 1 h, 3 h, 6 h, and 9 h after oral administration. The CT scanner was calibrated according to protocols provided by the manufacturer. CT scanning was performed at 192 μm intervals, and 3-dimensional CT pictures were reconstructed using OsiriX software (OsiriX Foundation, Geneva, Switzerland).

Figure 1. Synthesis of PI particles. (A) Schematic illustration of the one-pot synthesis of PI particles. (B) Transmission electron micrographs of PI particles as a function of MPMS and PI concentration. Scale bars: 6.25, 12.5, and 25 mM, 400 nm; 50, 100, and 200 mM, 2,000 nm. (C) Mean particle diameter as a function of MPMS concentration: PI particles (circles), rhodamine B-doped particles (triangles), and plain organosilica particles (square). (D) Mean particle diameter as a function of PI concentration for PI particles.

The monodisperse particles were easily dispersed in water after centrifugation. As indicated by TEM (Figure 1B and Table 1), various sizes of PI-particles with a narrow distribution were prepared according to MPMS concentrations. The sizes of the PI particles were larger than those of plain and thiolorganosilica particles, which contained rhodamine B prepared from the same concentrations of MPMS (Figure 1C and Table 1). Next, we prepared PI particles with 100 mM MPMS and various concentrations of PI. As shown in Figure 1D, the particle sizes were dependent on the PI concentration. These results indicated that the introduction of PI to organosilica particles affected the particle size. MPMS and PI particles contain mercaptopropyl residues. As PI is a less hydrophobic, cationic, and divalent intercalating agent, it is possible that PI was integrated into the thiol-organosilica network via electrostatic interactions with the thiol and silanol residues. This 3773

dx.doi.org/10.1021/cm3023677 | Chem. Mater. 2012, 24, 3772−3779

Chemistry of Materials

Article

above 630 nm (Figure 2D). These results suggested that the fluorescent property of PI was changed in organosilica particles. Because PI particles showed NIR fluorescence, we evaluated the possibility of in vivo imaging of PI particles using an in vivo FI system. As shown in Figure 2E, both the fluorescence intensities at excitation/emission of 605/660 nm and at 500/ 600 nm were detected in a concentration-dependent manner with high sensitivity. These results demonstrated that PI particles had multicolor properties with visible and NIR fluorescence. To distinguish whether the multicolor property of the PI particles was derived from multicolor in single particles or from a mixture of various color particles, we evaluated the fluorescence of single PI particles using flow cytometry. As shown in Figure 2F, the fluorescence of the PI particles was detected at various emission wavelengths (530 nm, 585 nm, and 670 nm) and an excitation wavelength of 488 nm to show the multicolor property. Each fluorescence intensity, FL2 and FL3, from a single particle had a linear relationship. These results indicated that the ratio of each fluorescent intensity of a single particle was almost the same. Therefore, each single particle had similar multicolor properties. After PI binds to nucleic acids, the fluorescent intensity of PI is enhanced 20 to 30 times, the excitation maximum is blueshifted by approximately 30−40 nm, and the emission maximum is blue-shifted by approximately 15 nm. The PI organosilica particles demonstrated similar changes to that of PI bound to nucleic acids. This report is the first describing a change of the fluorescent property of PI in organosilica particles. The internal structure of organosilica particles and its interaction with PI might affect the fluorescent properties of PI. The introduction of PI affects the size of the thiol-organosilica particles, which also indicates that there are interactions between PI and organosilicate. In addition, the PI particles had multicolor properties such as the extension of the wavelength of excitation and emission for fluorescence. In a previous paper,6 we reported unique changes to the fluorescence property of quantum dots (QDs) upon encapsulation within the thiol-organosilica layer. The fluorescence profile of the thiol-organosilica encapsulated QDs had an extended range of excitation wavelengths toward longer wavelengths, which is similar to the effects on the PI particles. Periodic mesoporous organosilica (PMO) can transfer energy and electrons between the pore walls and the mesochannels. Fluorescent organic dyes can be located in spatially separated regions in PMO. PMO doped with fluorescent organic dyes have unique fluorescence properties, such as light-harvesting antenna properties,16,17 color-tunable fluorescence emission,18−20 and highly efficient fluorescence resonance energy transfer.16,21 The particles have an organosilica network connected by a mercaptopropyl chain and are organic/inorganic hybrid structures. It is possible that there is an energy and electron transfer between PI and the organosilica structure. The mechanisms by which encapsulation of PI in organosilica alters the fluorescence properties are under investigation. In Vivo Fluorescence Imaging of Orally Administered PI-Particles. In vivo FI was performed at excitation/emission wavelengths of 605/660 nm on mice that were orally administered PI particles. As shown in Figure 3A, the fluorescence from particles was successfully detected from the ventral and the right and/or left lateral views of the abdomen. The location, shape, and intensity of the fluorescence changed

Table 1. Size Evaluation of Dye-Doped Thiol-Organosilica Particles MPMS conc. (mM)

dye propidium iodide propidium iodide propidium iodide propidium iodide propidium iodide propidium iodide rhodamine rhodamine rhodamine rhodamine rhodamine rhodamine none none none none none none propidium iodide propidium iodide propidium iodide propidium iodide propidium iodide propidium iodide propidium iodide

dye conc. (μM)

diameter (nm)

CV (%)

200

1,300

2650.5

26.0

100

650

1487.5

15.4

50

325

1052.2

8.3

25

163

600.3

20.0

81

203.6

15.0

6.25

41

127.5

9.2

200 100 50 25 12.5 6.25 200 100 50 25 12.5 6.25 100

1,300 650 325 163 81 41 0 0 0 0 0 0 325

1663.5 785.6 446.5 325.4 240.8 120.4 1864.2 720.9 423.1 297.0 192.1 115.2 1388.9

41.8 8.1 15.7 10.9 11.3 19.4 14.3 10.6 5.8 8.1 6.4 8.5 9.2

100

163

1022.1

6.1

100

82

977.0

5.9

100

41

873.6

6.5

100

20

868.6

7.4

100

10

856.3

5.3

100

5

798.0

6.0

12.5

B B B B B B

report is the first describing the effect of size modification of an internal fluorescent dye on organosilica particles. Multicolor Properties of PI Particles. We performed comprehensive 3-dimensional analysis of the excitation, emission, and fluorescence intensities of PI and PI particles (Figure 2A and 2B). These samples represented the pre- and postsynthesis reaction mixtures and were diluted 1:100 with water. A fluorescence profile of the PI particles was different from that of only PI. The solution of PI had excitation/ emission maxima of 500/605 nm (7.90 A.U.). The excitation/ emission maxima of the PI particles were 520/575 nm (24.6 A.U.) and were 6.5 times higher than those of PI. As shown in Figure 2C, PI particles had a higher fluorescent intensity than only PI at the emission wavelength of 500 nm. In addition, extended ranges of the excitation and emission wavelengths were observed in the fluorescence profile of the PI particles. At excitation wavelengths of 400 to 600 nm, the fluorescent intensities of the PI particles at emission wavelengths of 550 to 605 nm were higher than the maximum intensity observed for PI. However, PI showed almost no fluorescence at emission wavelengths above 565 nm, and PI particles showed high fluorescence with emission wavelengths 3774

dx.doi.org/10.1021/cm3023677 | Chem. Mater. 2012, 24, 3772−3779

Chemistry of Materials

Article

Figure 2. Fluorescence properties of PI particles. A comprehensive 3-dimensional analysis of the excitation, emission, and fluorescence intensities for PI (A) and PI particles (B). Fluorescence profiles of PI (black lines) and PI particles (red lines) at emission wavelengths of 500 nm (C) and 600 nm (D). (E) Fluorescence intensities of PI particles under excitation/emission wavelengths of 500/600 nm (black line) and 605/660 nm (red line). (F) Flow cytometry analysis of PI particles on FL1 (530 nm) (green line), FL2 (585 nm) (red line), and FL3 (670 nm) (brown line) channels.

administered GIT imaging. PI particles were not absorbed from the GIT and demonstrated GIT specific imaging, revealing an advantage of using PI particles for GIT imaging. The PI is known to intercalate into DNA, which is generally carcinogenic. Because organosilica particles can contain a fluorescent dye for a long period3 and are not absorbed from the GIT very well,31 the carcinogenic effect of the PI particle is lower than that of PI. However, further evaluations of and investigations for safety are required to use PI particles for humans. The mice were also observed with a multipurpose zoom microscope. The excitation and emission wavelengths were 540/605 nm. As shown in Figure 3C, fluorescence from particles in the GIT was detected from outside the body. After an abdominal section, fluorescence from each part of the GIT containing PI particles was observed clearly (Figure 3Cb−c). Other organs, such as the liver and lung, did not show any fluorescence from the PI particles. The PI particles were exclusively localized within the GIT.

according to the amount of time, indicating the movement of the particles in the GIT. In the ventral view, fluorescence was detected on the upper abdomen until 1 h but was detected in the middle and lower abdomen after 1.5 h. For the ventral view at 0.25, 1, and 1.5 h, fluorescence was detected in the right abdomen, but not the left abdomen. However, fluorescence was observed in the left lateral view. There was some inconsistency between the findings of the ventral view and the left lateral view. As a control experiment, imaging was performed for mice that were orally administered PI at excitation/emission wavelengths of 500/600 nm. As shown in Figure 3B, the fluorescence from PI was different from that of PI particles. After 1.5 h, fluorescence was detected in part of the abdomen and chest compared with the findings of preinjection. After 5 h, systemic fluorescence was detected, including in the lower leg, with a reduced area detected at 24 h. These findings indicated that PI diffused across the GIT and circulated systemically in the blood. Therefore, small molecules are not suitable for orally 3775

dx.doi.org/10.1021/cm3023677 | Chem. Mater. 2012, 24, 3772−3779

Chemistry of Materials

Article

Figure 3. In vivo fluorescence imaging of orally administered PI particles. (A) In vivo fluorescence imaging was performed for mice that had orally administered PI particles using an imaging system at excitation/emission wavelengths of 605/660 nm. Fluorescence from the particles was detected on the abdomen from the ventral (V), right (R) and/or left lateral view (L). (B) Imaging was performed for mice that were orally administered PI molecules at an excitation/emission of 500/600 nm as a control experiment. (C) The mouse was observed using a multipurpose zoom microscope at an excitation/emission of 540/605 nm. Fluorescence from particles in the GIT was detected before (a) and after the abdominal section (b−d). Bright field images (B) and fluorescence images (F) were merged (M).

Figure 4. Functional fluorescence imaging of orally administered PI particles. A mouse that was administered PI particles was observed with a multipurpose zoom microscope to evaluate the real time movement of the gastrointestinal tract at an excitation/emission of 540/605 nm. Fluorescence images (F) were merged with bright field images (M).

imaging. Indocyanine green (ICG), an NIR excitable dye molecule, was applied to dynamic NIR FI of the GIT.23,26,28 Intravenous or intradermal administered ICG enabled in vivo dynamic NIR FI for the peristaltic and segmental motions of the intestine. However, there was background fluorescence from the bile or liver. This image was influenced by liver and bile function because ICG was metabolized in the liver, secreted into the bile, and then secreted from bile into the duodenum. In addition, ICG and other common fluorescent organic dyes are affected by various factors, such as quenching because of high concentration, environmental effects including low pH, and resonance energy transfer.29,30 Therefore, ICG and organic dyes require additional evaluation because the GIT has various pHs depending on the location and can change content concentrations by absorption and secretion. Normal mouse chow was applied as a contrast agent for its NIR autofluorescent property.28 Because mouse chow has suitable NIR properties and is inexpensive, this approach is expected to reveal functional GIT FI in a truly physiological setting. As another approach, we would like to propose the application of fluorescent particles for functional GIT FI. PI particles were prepared from PI and MPMS with a narrow size distribution and a uniform surface structure. Fluorescent organosilica particles can retain fluorescence intensity at low pH6,11 and can even pass through the stomach (pH < 2) of a mouse gastrointestinal tract.31,32 In addition, it is reported that thiolorganosilica particles exhibited very good ability to adhere to mucosal surfaces recently.33 The disulfide bond formation between thiol groups on the surface of the thiol-organosilica particles and cystein-rich domains in mucins on mucosal

In addition, we performed noninvasive functional imaging of the mouse GIT using a multipurpose zoom microscope. As shown in Figure 4 and Supporting Information, Movie S1, fluorescence was detected in the GIT, and its movements were observed noninvasively. After oral administration, fluorescence was detected, and the various contractility and movement of the fluorescence in the small intestine was observed. Figure 4 represents selected frames taken after administration of PI particles, showing the intestinal transit of the particles at different times. Orally administered PI particles could be detected in the mouse GIT using two types of FI devices under different conditions. The excitation/emission of in vivo FI and multipurpose zoom microscope were 605/660 nm and 540/ 605 nm, respectively. Because PI particles had a multicolor property, we used two conditions to observe the PI particles in the GIT. The NIR wavelength conditions were used to detect the specific signal of PI particles in deeper sites with reduced autofluorescence in vivo. The visible wavelength conditions were used to strengthen the emission light of the PI particles in a focused area and enabled movies to be shot (600 ms/frame) of the of local abdomen. The multicolor property of PI particles was useful for two types of FI devices. FI has been applied to functional GIT imaging.22−28 Because FI has advantages such as high temporal resolution and high sensitivity, FI is a very attractive approach for functional GIT 3776

dx.doi.org/10.1021/cm3023677 | Chem. Mater. 2012, 24, 3772−3779

Chemistry of Materials

Article

attenuation coefficient. PI particles at 100 mg/mL (50.5 nM) had a CT value of 206 Hounsfield units (H.U). The PI particles attenuated X-rays because the organosilica particles contain iodide in their structures. The in vivo contrast properties of the PI particles were investigated by oral administration of PI particles to mice (Figure 5B). The CT images at 1 h show an enhancement of the stomach and the duodenum. The transverse and coronal views show the enhancement of the stomach (black arrowheads) and duodenum (white arrowheads). The 3-dimensional views show the location of the stomach (black arrowheads) as well as duodenum (white arrowheads) in the abdomen. The CT images at 3 h and at 6 h show an enhancement of the small intestine. The transverse and coronal views at 3 h show PI particles located in various parts of small intestine. The 3dimensional views show the enhancement of small intestine moved from the right side at 3 h to the left lower part at 6 h. The images at 9 h show an enhancement of the small intestine (white arrowheads) and the cecum (white arrow). The 3dimensional views show that PI particles located in the right lower part of abdomen mainly. The enhancements of the PI particles in the small intestine and the cecum at 9 h were higher than those in the stomach at 1 h and in the duodenum at 3 h. These results demonstrated that PI particles could be detected clearly in the GIT and that the concentration of the PI particles could be evaluated. Correlation Analysis between Fluorescent Imaging and X-ray CT Findings. We evaluated the correlation between in vivo FI (Figure 6A) and X-ray CT imaging (Figure 6B and C) of a mouse that was administered PI particles. The CT images contain 3-dimensional (Figures 6B), transverse, coronal, and sagittal views (Figures 6C) of the same mouse at the same times after oral administration of the PI particles. However, the left lateral view of the FI had weak fluorescent intensity (maximum 46,910 photons) in the stomach (Figure 6AL, red circles), and the CT images showed a high CT signal intensity (maximum 1,241 H.U.) (Figure 6BL and 6Ca, red circles). In addition, the CT images had high CT signal intensity in the left lower abdomen (maximum 3,610 H.U.) (Figure 6BV and 5Cd, green circles), and all views of FI showed no fluorescence (Figure 6A, green circles). On the other hand, the ventral view of the FI showed strong fluorescent intensity (maximum 76,150 photons) in the right abdomen (Figure 6 AV, yellow circles), but the CT images had a low CT signal intensity (maximum 843 H.U.) (Figure 6 BL and 6Cb-c, yellow circles). As shown in Figure 6C, surface plots of the CT signal demonstrated the particle high distribution on the mucosal surfaces of the stomach and intestine. These findings indicated that thiol-organosilica particles had very good ability to adhere to mucosal surfaces as reported previously.33 As mice were examined in a supine position, there were dominant, high intensity areas on posterior walls of the stomach and intestine. We measured the depth of the high intensity areas from the surface of the body using Figure 6Ca for the stomach and Figures 6Cb and 6Cd for the intestine. The minimum distances of the high intensity areas of the stomach from the ventral surface and the left lateral surface were 5.7 mm and 2.3 mm, respectively. In the transverse view in Figure 6Cb, the distance from the high intensity area of the posterior wall of the intestine from the ventral surface was 3.6 mm. Between the anterior and posterior walls, the lumenal distance was 2.6 mm distance. In a

surfaces was proposed as a possible mechanism. These properties are suitable to normalize a functional evaluation of the GIT. Previously, we investigated the biodistribution and immunoresponse of the mouse GIT to various sizes of organosilica particles and discovered size-dependent differences.31,32 The composition and size heterogeneities of digested mouse chow may affect GIT function. Homogeneous contrast reagents with stable fluorescence intensities would be better for functional FI and evaluations of the GIT. As another advantage, FI can be performed with various fluorescent probes for multichannel imaging. Various types of NIR particles have been prepared,34,35 and some of them could be useful for GIT FI. An application of multichannel GIT FI would be the attachment of various therapeutic agents to various fluorescence particles. Fluorescent particles have a high potential for novel in vivo functional GIT FI. In Vivo X-ray Imaging of Orally Administered PI Particles. The X-ray attenuation of PI particles was evaluated as a function of concentration (Figure 5A). A linear relationship existed between the PI particle concentration and the X-ray

Figure 5. X-ray CT images of orally administered PI particles. (A) CT values as a function of PI particle concentration. (B) X-ray CT images of transverse, coronal, and 3-dimensional views of the mouse after oral administration of PI particles. The CT images show enhancements of the stomach (black arrowheads), the small intestine (white arrowheads), and cecum (white arrow). 3777

dx.doi.org/10.1021/cm3023677 | Chem. Mater. 2012, 24, 3772−3779

Chemistry of Materials

Article

the functional analysis of in vivo GIT FI would be improved. Using X-ray CT, the concentration of PI particles, the location of air and liquid in the abdominal cavity, and the condition of the abdominal wall can be evaluated. At the stomach, a ratio between maximum X-ray CT signal intensity (Figure 6BL and 6Ca, red circles) and maximum fluorescent intensity of FI (Figure 6AL, red circles) (max. X-ray CT: max. FI) was 1: 37.8. On the other hand, max. X-ray CT: max. FI at the right abdomen (Figure 6AV, Figure 6BL, and 6Cb−c, yellow circles) was 1:90.3. There was predominant difference between the ratios of stomach and intestine. These results demonstrated that the maximum fluorescent signal from the intestine near the abdominal wall is above 2 fold higher than that from the stomach. In this case maximum fluorescent intensity in the stomach could be corrected as 112,064 photons, and was higher than that of the intestine. On the basis of X-ray CT images, the fluorescence intensities of FI could be corrected. According to tissue, and part, heterogeneity, and variability of the abdomen, corrections of fluorescence intensities of FI would be possible. Dual modal PI organosilica particles would be useful for X-ray CT and FI and functional GIT FI.

■

CONCLUSION Thiol-organosilica particles that were internally functionalized with PI demonstrated enhanced fluorescence intensity, multicolor fluorescence, and X-ray absorption. The interactions between PI and the organosilica particles changed the fluorescence property of PI and the size of the particle. These results indicate that internal functionalization of organosilica particles with functional molecules creates new functions and morphology. These properties could be useful for the development of novel imaging systems, photocatalytic systems, and solar energy applications. PI particles are prepared with just one additional functional molecule, but are applicable to multicolor fluorescence imaging, X-ray CT, and correlation analysis. The application of PI particles is a novel approach for functional GIT FI of animals. The combination of fluorescence imaging and X-ray CT enables the development of quantitative functional GIT imaging.

Figure 6. Correlation analysis between in vivo fluorescence imaging and X-ray CT images. Fluorescence images (A) were compared with X-ray CT images of 3-dimensions (B) and plain images (C) of oral administration of the PI particles. A left lateral view (L) of fluorescence imaging showed weak fluorescent intensity in the stomach ((A)L, red circles), but the CT images showed high CT signal intensity ((B)L and (C)a, red circles). However, the X-ray CT images showed high CT signal intensity in left lower abdomen ((B)V and (C)d, green circles). All views of fluorescence imaging showed no fluorescence (6(B), green circles). The right lateral view (R) and ventral view (V) of fluorescence imaging showed strong fluorescent intensity in the right abdomen ((A)V and (A)R, yellow circles), but the CT images showed low CT signal intensity (maximum 843) ((B)L and 5(C)b, yellow circles).

■

ASSOCIATED CONTENT

S Supporting Information *

Further details are given in the Movie S1. This material is available free of charge via the Internet at http://pubs.acs.org.

transverse view of Figures 6Cd, the distance of a high intensity of the intestine from ventral surface was 2.9 mm. There were various patterns of inconsistencies between findings of the FI and CT images. It is known that in vivo FI are not quantitative, and the image information is surface-weighted. In the abdomen, there were various factors that influenced the FI, including the skin, muscles, and peritoneum in the abdominal wall, and some part contains fasciae and bones. The abdominal cavity contains the GIT, liver, bile, and so forth. Some parts of the GIT may contain air or liquid. Light from excitation can pass through air, but not bones and fasciae. There is heterogeneity in the abdomen that can influence FI. As the intestines can move, the position of and the contents in the intestine can be different in the same region; this variability can influence FI. It is important to consider this variability of the abdomen for FI. Recently, various images of in vivo functional GIT FI were reported,22−28 but the authors did not take into consideration the variability of the abdomen. The X-ray CT images are relatively quantitative. By evaluating the influence of heterogeneity and variability of the abdomen using X-ray CT,

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +81-88-633-9220. Fax: +81-88-633-9426. E-mail: [email protected] or michi.nakamura@ tokushima-u.ac.jp. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Nakamura, M.; Ishimura, K. J. Phys. Chem. C 2007, 111, 18892− 18898. (2) Nakamura, M.; Ishimura, K. Langmuir 2008, 24, 5099−5108. (3) Nakamura, M.; Ishimura, K. Langmuir 2008, 24, 12228−12234. (4) Nakamura, M. Nanostructured Oxides; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2009; pp 109−161. (5) Nakamura, M.; Ozaki, S.; Abe, M.; Doi, H.; Matsumoto, T.; Ishimura, K. Colloids Surf., B 2010, 79, 19−26. 3778

dx.doi.org/10.1021/cm3023677 | Chem. Mater. 2012, 24, 3772−3779

Chemistry of Materials

Article

(6) Nakamura, M.; Ozaki, S.; Abe, M.; Matsumoto, T.; Ishimura, K. J. Mater. Chem. 2011, 21, 4689−4695. (7) Yao, G.; Wang, L.; Wu, Y.; Smith, J.; Xu, J.; Zhao, W.; Lee, E.; Tan, W. Anal Bioanal Chem. 2006, 385, 518−524. (8) Bae, S. W.; Tan, W.; Hong, J. I. Chem. Commun. 2012, 48, 2270− 2282. (9) Vivero-Escoto, J. L.; Huxford-Phillips, R. C.; Lin, W. Chem. Soc. Rev. 2012, 41, 2673−2685. (10) Benezra, M.; Penate-Medina, O.; Zanzonico, P. B.; Schaer, D.; Ow, H.; Burns, A.; DeStanchina, E.; Longo, V.; Herz, E.; Iyer, S.; Wolchok, J.; Larson, S. M.; Wiesner, U.; Bradbury, M. S. J. Clin. Invest. 2011, 121, 2768−2780. (11) Nakamura, M.; Miyamoto, K.; Hayashi, K.; Awaad, A.; Ochiai, M.; Ishimura, K. Nanomedicine: NBM 2012, in press (doi: 10.1016/ j.nano.2012.05.018). (12) Taylor, I. W.; Milthorpe, B. K. J. Histochem. Cytochem. 1980, 28, 1224−1232. (13) Vuist, W. M. J.; Buitenen, F. V.; De Rie, M. A.; Hekman, A.; Ruemke, P.; Melief, C. J. M. Cancer Res. 1989, 49, 3783−3788. (14) El-Naggar, A. K.; Batsakis, J. G.; Teague, K.; Garnsey, L.; Barlogie, B. Cytometry 1991, 12, 330−335. (15) Souchier, C.; Ffrench, M.; Benchaib, M.; Catallo, R.; Bryon, P. A. Cytometry 1995, 20, 203−309. (16) Inagaki, S.; Ohtani, O.; Goto, Y.; Okamoto, K.; Ikai, M.; Yamanaka, K.; Tani, T.; Okada, T. Angew. Chem., Int. Ed. 2009, 48, 4042−4046. (17) Takeda, H.; Goto, Y.; Maegawa, Y.; Ohsuna, T.; Tani, T.; Matsumoto, K.; Shimada, T.; Inagaki, S. Chem. Commun. 2009, 6032− 6034. (18) Mizoshita, N.; Goto, Y.; Tani, T.; Inagaki, S. Adv. Mater. 2009, 21, 4798−4801. (19) Mizoshita, N.; Goto, Y.; Maegawa, Y.; Tani, T.; Inagaki, S. Chem. Mater. 2010, 22, 2548−2554. (20) Maegawa, Y.; Mizoshita, N.; Tani, T.; Inagaki, S. J. Mater. Chem. 2010, 20, 4399−4403. (21) Mizoshita, N.; Yamanaka, K.; Hiroto, S.; Shinokubo, H.; Tani, T.; Inagaki, S. Langmuir 2012, 28, 3987−3994. (22) Tennvassas, T. L.; Skretting, A. Eur. J. Nucl. Med. 1998, 25, 1531−1536. (23) Behrendt, F. F.; Tolba, R. H.; Overhaus, M.; Hirner, A.; Minor, T.; Kalff, J. C. Eur. Surg. Res. 2004, 36, 210−218. (24) de Backer, O.; Blanckaert, B.; Leybaert, L.; Lefebvre, R. A. Neurogastroenterol. Motil. 2008, 20, 700−707. (25) Fuhrmann, G.; Leroux, J. C. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 9032−9037. (26) Kwon., S.; Sevick-Muraca, E. M. Neurogastroenterol. Motil. 2011, 23, 1−6. (27) Loginova, Y. F.; Dezhurov, S. V.; Zherdeva, V. V.; Kazachkina, N. I.; Wakstein, M. S.; Savitsky, A. P. Biochem. Biophys. Res. Commun. 2012, 419, 54−59. (28) Kwon, S.; Davies-Venn, C.; Sevick-Muraca, E. M. Neurogastroenterol. Motil. 2012, 24, 494−497. (29) Bjornsson, O. G.; Murphy., R.; Chadwick, V. S. Experientia 1982, 38, 1441−1442. (30) Lakowicz, J. R. Anal. Biochem. 2001, 298, 1−24. (31) Awaad, A.; Nakamura, M.; Ishimura, K. Nanomedicine: NBM 2012, 8, 627−636. (32) Awaad, A.; Nakamura, M.; Ishimura, K. Int. J. Nanomedicine 2012, 7, 1423−1439. (33) Irmukhametova, G. S.; Mun, G. A.; Khutoryanskiy, V. V. Langmuir 2011, 27, 9551−9556. (34) He, X.; Wang, K.; Cheng, Z. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2010, 2, 349−366. (35) Melancon, M. P.; Zhou, M.; Li, C. Acc. Chem. Res. 2011, 44, 947−956.

3779

dx.doi.org/10.1021/cm3023677 | Chem. Mater. 2012, 24, 3772−3779