- Email: [email protected]
Molecular Imaging Cellular SPIO Uptake with Nonlinear Optical Microscopy
Journal of Medical Imaging and Radiation Sciences
Journal of Medical Imaging and Radiation Sciences 41 (2010) 159-164
Journal de l’imagerie médicale et des sciences de la radiation
www.elsevier.com/locate/jmir
Molecular Imaging Cellular SPIO Uptake with Nonlinear Optical Microscopy Lina Machtoub, PhDa*, Rudolf Pfeiffer, PhDb, Aleksandar Backovic, PhDc, Stefan Frischauf, MDa, and Marius C. Wick, MDa a
b
Universitaetsklinik Fuer Radiodiagnostik, Innsbruck Medical University, Innsbruck, Austria Faculty of Physics, University of Vienna, Vienna, Austria, and Department of Electrical Engineering, California Institute of Technology, Pasadena c CSO-Research and Development at IDC Immumetrics, Vienna, Austria
ABSTRACT We report a novel application to demonstrate and visualize the selective binding of lipids in cells of the reticuloendothelial system to super paramagnetic iron oxide (SPIO) nanoparticles. Ten New Zealand White rabbits that were experimentally injected intravenously with SPIO and five controls were investigated with vibrational microspectroscopy based on surface-enhanced coherent anti-Stokes Raman scattering (SECARS) microscopy. Marked cellular intensity enhancements in hepatic Kupffer cells and melanomacrophages of spleen have been observed in the range of 2850–2875 cm1 in SPIO-injected animals but not in controls. The enhancements are related to the selective association of lipid molecules in cells of the reticuloendothelial system to uptaken SPIO, which can uniquely be visualized with SECARS microscopy.
RE´SUME´ Nous annonc¸ons une nouvelle application qui sert a` de´montrer et a` visualiser la liaison se´lective de lipides dans les cellules du syste`me re´ticulo-endothe´lial avec des nanoparticules d’oxyde de fer superparamagne´tique (OFS). Dix lapins ne´o-ze´landais blancs ayant rec¸u une injection intraveineuse expe´rimentale d’OFS et 5 te´moins ont e´te´ e´tudie´s par microspectroscopie vibratoire fonde´e sur la microscopie SECARS (Surface-Enhanced Coherent anti-Stokes Raman Scattering). Des ame´liorations marque´es de l’intensite´ cellulaire dans les cellules de Kupffer du foie et les me´lanomacrophages de la rate ont e´te´ observe´es dans la plage de 2850 a` 2875 cm1 chez les animaux ayant rec¸u l’injection d’OFS, mais non chez les sujets te´moins. Ces ame´liorations sont lie´es a` l’association se´lective des mole´cules de lipides des cellules du syste`me re´ticulo-endothe´lial avec l’OFS capte´, association visualisable exclusivement par la microscopie SECARS.
Introduction
from two to three orders of magnitude [4]. In these studies, the observed surface enhancement has been partially attributed to the quantum size effect of the iron particles. Novel new applications are recently underway to use magnetic nanoparticles as targeting markers for cancer drug delivery. It has been shown that arginine-glycine-aspartic (RGD) peptide-coated iron oxide can be bonded with an integrin-rich tumor cell, and potentially can be used as targeting marker. In addition, the synthesis of dumbbell-shaped magnetic nanoparticles (Au-Fe3O4) has made such species especially attractive as multifunctional probes for diagnostic and therapeutic applications [5]. Superparamagnetic iron oxide (SPIO) nanoparticles, a potent new class of in vivo magnetic resonance imaging (MRI) contrast agents, have been widely recognized as tissue-specific MRI contrast agents for in vivo detection of malignant tumors in the liver and spleen of patients. The magnetic properties of these materials were first referred to as superparamagnetic in 1955 [6]. The term refers to the large
Surface-enhanced Raman scattering (SERS) nanoparticles have drawn a large degree of interest as targeted imaging probes and therapeutic agents [1, 2]. Recently, SERS microscopy has been exploited to observe trace amounts of biologically relevant molecules that allow for the selective and highly sensitive localization of target molecules. Based on their surface plasmon polaritons, novel metal nanoclusters (eg, gold or silver nanoparticles) provide the key effect for observing enhanced Raman signals for molecules attached to them [3]. It has been reported that iron nanoparticles can have remarkable SERS activity in a range This project was supported by a grant from the Medizinische Forschungsfo¨rderung Innsbruck MFI (MCW, Project 9443). * Corresponding author: Dr. Lina Machtoub, PhD, Universitaetsklinik fuer Radiodiagnostik, Innsbruck Medical University, Anichstrasse 35, A-602Innsbruck, Austria. E-mail address: [email protected] (L. Machtoub). 1939-8654/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi: 10.1016/j.jmir.2010.08.001
magnetic moments they acquire when placed in external magnetic fields. To visualize SPIO nanoparticles in biological systems, a molecular imaging modality has to meet the needs of high sensitivity combined with high spatial and temporal resolution and high multiplexing capacity. For ex vivo tissue analyses from biopsy samples conventional histological techniques are considered gold standard; however, they are not feasible for live tissue imaging. Recently, nonlinear optical microscopy, particularly coherent anti-Stokes Raman scattering (CARS) has taken a large step toward becoming a medical diagnostic tool for both ex vivo and for some cases even in vivo [7]. Nonlinear optical microscopy and microspectroscopy allow for differentiating the spectra fingerprint of many molecules with submicron resolution, resulting in very high multiplexing capabilities and narrow spectral features that can be easily separated from the broadband autofluorescence. Compared with other clinical imaging modalities, CARS microscopy can be tuned to provide a variety of possible tissue contrasts, with an advantage of subcellular spatial resolution and near real-time temporal resolution compared with MRI [8]. The ability to resolve individual nanoparticles is a challenging task and CARS has the ability to chemically selectively detect aggregations or accumulations of nanoparticles in cells or thin tissue slices. It has already been reported that even sub-wavelength sites can be seen if the nonlinearity of the material is sufficiently strong [9]. Apart from its chemical selectivity, the main strength of the method lies in the fact that the nanoparticles do not have to be treated (for example, labeled with a fluorescent marker), but can be imaged in their native state. This is important, because labeling might change the behavior of the (for example, metallic/ organic, soluble/insoluble, functionalized) nanoparticles in cells, and thus influence the outcome of investigations on the fate of the particles. Previous findings reported intense CARS signals observed from the experiments performed on a fish gill with embedded metal oxides nanoparticles (for example, ZnO, TiO2, Fe2O3) [10, 11]. The nanoparticles were visible with a high contrast because of their high thirdorder nonlinear optical susceptibilities. The main goal of this study was to explore the potential of surface-enhanced coherent anti-Stokes Raman scattering (SECARS)-based nonlinear optical microscopy in studying the cellular uptake or clearance of SPIO nanoparticles in tissue samples from an established experimental rabbit model. The animals and tissue samples for our experiments were also used in a study investigating the effect of stress on arterial endothelial cells and in vivo molecular MRI of endothelial expression of stress proteins [12]. For this purpose, a clinically approved established SPIO MRI contrast agent was used that is intravenously (i.v.) injected for the detection of malignant focal hepatocellular lesions in patients (Ferucarbutran, Resovist, Schering, Austria). This SPIO contrast agent is a nonstoichiometric polycrystalline mixture of magnetite (Fe3O4) and maghemite (g-Fe2O3) coated with carboxydextran (total core size of 62 nm). After injection, SPIO cores are phagocytosed by cells of the reticuloendothelial system (RES) and 160
accumulated in the lysosomal compartment. The liver (80%) and the spleen (5–10%) are the known primary organs of SPIO uptake and lysosomal aggregation. Figure 1 shows a representative in vivo MRI of a New Zealand White (NZW) rabbit before (A) and 70 min. after (B) i.v. injection of SPIO demonstrating the uptake of SPIO by the liver. Materials and Methods The animal experiments were surveyed by the Committee for Animal Experimentation and approved by the Austrian Ministry of Sciences (reference number BMWF-66.011/0017-II/10b/ 2008). In the course of an in vivo molecular MRI experiment for the detection of an arterial endothelial expression of stress proteins, 15 normocholesterolemic female NZW rabbits have been injected i.v. Lipopolysaccharide (LPS; a standard endothelial stressor; in a pilot study found to be optimal at 10 mg/kg body weight [12]) and sacrificed 8–10 h after LPS injection. Four hours after stress induction with i.v. LPS, 10 rabbits were experimentally i.v. injected with 0.5–0.7 mL SPIO (¼5 mg Fe/kg KG) to partially saturate iron uptake in the RES. Five rabbits were used as untreated controls. In vivo MRI was performed under general anesthesia in all SPIO-given animals before and at 20-min intervals until 2–4 h after i.v. SPIO according to a standardized protocol on a 1.5T MRI (Siemens, Magnetom Avanto, Erlangen, Germany). In controls, MRI was performed 6 h after LPS. After MRI, euthanasia of all animals was performed under anesthesia with xylanest and ketamine via in vivo perfusion with phosphate-buffered saline (PBS, pH 7.2) followed by perfusion-fixation with 4% PBS-buffered paraformaldehyde. Thereafter, tissue samples were taken from the liver, spleen
Figure 1. In vivo magnetic resonance imaging (ie, a T2-weighted R2-map magnetic resonance sequence) of a New Zealand White rabbit before (A) and 70 min after (B) intravenous injection of 0.7 mL super paramagnetic iron oxide (SPIO) (¼5 mg Fe/kg kg) demonstrating the uptake of SPIO by the liver (circumscribed and arrows).
L. Machtoub et al./Journal of Medical Imaging and Radiation Sciences 41 (2010) 159-164
and fat and stored in 4% PBS-buffered paraformaldehyde until further preparation for CARS, Raman mapping and conventional light microscopy. Histopathologic Preparation The perfusion-fixated tissues were embedded in paraffin blocks and were subsequently cut into slices of 10-mm thickness for histological staining. Turnbull-blue reaction was used to detect the presence and cellular localizations of SPIO at light microscopy with magnifications from 200 to 630. A standard protocol of iron staining was performed, starting with 1 h treatment of the tissues in a freshly prepared 10% ammonium sulfide solution (in aqua dest.). Then, the sections were treated with 1% HCl solution (in aqua dest.) and 20% potassium ferricyanide (III in aqua dest.). This causes ferrous iron to react to form an insoluble bright blue pigment (ferrous ferricyanide). The nuclei were counterstained with nuclear fast red (Carl Roth, Karlsruhe, Germany) and mounted with Kaiser Glycerol gelatin (Merck, Darmstadt, Germany). Light microscopic images of the stained tissues were taken by Nikon eclipse E800 digital camera. Control Experiments Hepatic tissue samples of wild-type mice (on a Western diet) were disintegrated in a porcelain mortar together with a 2:1 mixture of chloroform and methanol. The solution was incubated for 2 h at 4 C before aqua dest. was added to aid in phase separation during a subsequent centrifugation step at 10,000g for 10 min. The chloroform-lipid mixture sat overnight at room temperature to evaporate the chloroform. Subsequently, the lipids were redissolved in ethanol. Murine adipocytes were grown on gelatin-coated glass coverslips and exposed to 5 ng/ mL of SPIO nanoparticles (in total: 15 ng in 3 mL fresh medium). After incubation for 24 h, adipocytes were fixated at room temperature with 4% paraformaldehyde for 30 min. and stored in PBS until further preparation for microscopic measurements. Further control experiments were performed on hepatic and spleen tissues, extracted from the untreated control (NZW) rabbits, without administration of SPIO nanoparticles, subject to similar experimental conditions. Experimental Measurements and ex vivo Imaging Fourier transform Raman spectra (FT-Raman) were measured using a Bruker RFS 100/S spectrometer (Bruker Optics Inc, Billerica, MA). Spectra of all samples were measured at room temperature in a 180 backscattering geometry in a microscope. The absorption measurements were performed using a Hitachi U-3410 Spectrophotometer. Microscopic Raman mapping images were recorded using an alpha 300R instrument (WITec, Ulm, Germany) equipped with a CCD detector. The integration time per pixel was 0.1 s. The samples were irradiated by a He Ne laser at 632.8 nm and focused by a 40 0.65 NA microscope objective lens. CARS measurements were performed using two ps light sources of near-infrared excitation; Ti: sapphire laser (Coherent Mira HP, 3.5W 3 ps, 700–1000 nm), and 1064-nm mode-locked Nd::YVO4. The fundamental lines (830 nm,
1064 nm) are used for the Stokes beam, whereas the signals from the optical parametric oscillators were used as the pump and probe. The corresponding pump beams were tunable in the range of 510–930 nm by using two optical parametric oscillators (APE, Germany). Both oscillators are pumped by part of the fundamental output lines and can be continuously tuned over a wavelength range that can cover the entire chemical essential vibration frequency range of 200–3600 cm1. The beams are scanned over the sample and focused by water immersion objective lens with 1.2 numerical apertures. Both beams have a power of several tens of milliwatts for the sample. Images taken at different wavelengths are collected consecutively by tuning the oscillators without the need for optics realignment. The residual pump and Stokes radiation are separated from the CARS signal by using dielectric filters. The collected signal is filtered by bandpass filters and detected by a photomultiplier tube (Hamamatsu R6357). The images were recorded with a resolution of 5 s total acquisition time for one frame of 512 512 pixels. The microscope is designed for the signal to be detected in both forward and epi-direction. The samples used in the experiment were tissue slices, cell lines and some compounds made in solutions. For most of the samples, the CARS signal was collected in the forward direction; however, for some thick specimens (tissue sections) we used epi-detection because of the back scattering of the strong forward signal from several layers in the tissue. Results Ex vivo CARS measurements on unstrained hepatic tissue samples from NZW rabbits that have been i.v. injected with SPIO showed markedly increased intracellular signal enhancements in regions at the perisinusoidal location of Kupffer cells (Figure 2A). These enhancements have only been observed in the wavelengths range of 2850–2875 cm1, which relates to the symmetric CH2 stretch vibration of lipids (Figure 2B). Conventional light microscopy using Turnbull-blue staining correlated the observed SECARS signals to an intracellular accumulation of SPIO in Kupffer cells (Figure 2C). In contrast, measurements on hepatic specimen from control NZW rabbits showed negative staining for SPIO and no CARS signal enhancements at any vibrational resonance wavelengths. (See Supplemental Figure 1 in supplemental online material.) Similarly, CARS measurements of tissue samples from spleen with SPIO-injected rabbits but not from controls showed strong signals enhancements at 2850–2875 cm1 (Figure 3A). The enhancements correlated to SPIO depositions at perisinusoidal melanomacrophage centres. Histological analysis confirmed the intracellular presence of iron particles in macrophages (Figure 3B). To investigate a selective intracellular SPIO lipid-binding activity, we performed several control CARS measurements on SPIO-treated adipocytes and extracted lipids from hepatic tissue, incubated in vitro with or without SPIO. An intensive intracellular signal enhancement could be found in
L. Machtoub et al./Journal of Medical Imaging and Radiation Sciences 41 (2010) 159-164
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Figure 2. (A) Coherent anti-Stokes Raman scattering (CARS) image of an unstained hepatic tissue specimen from a NZW rabbit that was intravenously injected with super paramagnetic iron oxide (SPIO) nanoparticles, taken at 2850 cm1 (B) The corresponding cross section profile shows intensity enhancement along the indicated line shown in the inset of the CARS image. (C) Light microscopic image of Turnbull blue stained hepatic tissue (arrows indicate SPIO nanoparticles [blue] in Kupffer cells).
the vibrational resonance wavelengths range w2850 cm1 from SPIO-incubated lipids and particular regions highlighting accumulation of SPIO particles in adipocytes. (See Supplemental Figures 2 and 4A in supplemental online material.) The intracellular enhancement has been further investigated on all tissues by Raman imaging chemical mapping measurements (Figure 4B). In contrast to the CARS spectrum, which has resonant and nonresonant contributions, Raman imaging does not suffer from the coherent addition of nonresonant background signal. Clear contrast enhancement is observed in iron nanoparticle–treated tissues and is indicated in the bright regions highlighting high accumulation of iron oxide nanoparticles. Similar measurements on untreated tissues have shown no indication of any
enhancements. (See Supplemental Figure 3 in supplemental online material.) To support the SERS activity of iron oxide nanoparticles, additional controlled microscopic Raman measurements were performed on iron oxide nanoparticles dissolved in a specific heterocyclic organic compound (C5H5N; Pyridine). Clear intensity enhancement could be detected from SPIO nanoparticles in Pyridine compared with the pure solution. (See Supplemental Figure 4 in supplemental online material.) Raman intensity from these solutions has shown considerable dependence on particles concentration. On the other hand, the FT-Raman measurements performed on iron oxide nanoparticles have shown no substantial resonance Raman activity observed in the region of interest. (See Supplemental Figures 5 and 6 in supplemental online material.)
Figure 3. (A) Coherent anti-Stokes Raman scattering image (2850 cm1) of an unstained spleen tissue sample of a New Zealand White rabbit after intravenous administration of super paramagnetic iron oxide (SPIO) nanoparticles. The inset shows SPIO nanoparticles in macrophages. (B) Corresponding light microscopic image of spleen tissue stained with Turnbull blue, SPIO (blue) are indicated by arrow.
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L. Machtoub et al./Journal of Medical Imaging and Radiation Sciences 41 (2010) 159-164
Figure 4. (A) Coherent anti-Stokes Raman scattering image of adipocytes intravenously given super paramagnetic iron oxide (SPIO) nanoparticles, taken at 2850 cm1. The corresponding section profile shows intensity enhancement highlighting the distribution of SPIO nanoparticles. (B) Raman mapping image of SPIO treated hepatic tissue specimen, taken in the range of 2840–2880cm1, shows clear contrast enhancement. The corresponding cross-section profiles of the spectra indicate an enhancement from the bright (1) and dark (2) regions.
Discussion The observed surface-enhanced Raman signal in the vibrational resonance wavelengths range of lipids in tissues from SPIO-treated animals and the results from our control experiments with SPIO-exposed adipocytes and extracted lipids suggest a selective binding of peri- or intracellular lipid molecules to SPIO accumulated in or adjacent to the lysosomal compartment of cells of the RES. The possibility of a binding between lipids and iron has already previously been demonstrated with phosphatidic acid, phosphatidylserine, oleic and stearic acids. These pure lipids have shown an apparent iron binding in filtration assays, the highest affinity at 5- to 10-fold on a molar basis with oleic acid [13]. Similar to the function of Kupffer cells in hepatic sinusoids, macrophages surrounding the spleen sinusoids play an important role in iron uptake from the circulation. Histological analyses from tissue samples of NZW rabbits that have been i.v. injected with SPIO have shown homogenously distributed iron depositions in the liver. However, in the spleen, the iron depositions were scattered and mainly detectable in the melanomacrophage centres. The lipid-binding selectivity of iron has been carefully investigated. Previous findings reported a phenomenon of iron-induced lipid peroxidation, for iron more often than any other transition metal. An in vivo study on rats with experimental chronic iron overload has shown direct evidence of hepatic lipid peroxidation in both mitochondrial
and microsomal membrane lipids [14]. The binding of Fe3þ to erythrocyte cell membranes has been investigated in a system in which lipid peroxidation was proportional to Fe3þ concentration. Such binding of Fe3þ was evaluated by labeling with 59 FeCl3 and measurement of nuclear magnetic resonance water-proton relaxation times. It has been shown that 95% of the Fe3þ was membrane-bound at 100 mM FeCl3 in a 1.5 mg protein/ml membrane suspension. Both spin-lattice (T1) and spin-spin (T2) relaxation times decreased with increasing Fe3þ concentration. Charge transfer to Fe3þ may occur at the membrane binding site with resultant decrease in the Fe3þ effect on water-proton relaxation times. These studies support the hypothesis that Fe3þ binds to the membrane and generates free radicals at the binding site [15]. However, in our experiment, the possibility of lipid peroxidation is less probable because the iron concentration in our experimental animals did not reach highly pathological values and histopathologic evidence of possible cell degeneration was absent on tissue samples. Another possible mechanism of an increased lipid-iron binding activity might be originated from observations that showed lipids representing a substantial proportion of Salmonella typhimurium LPS, which is expected to have high iron-binding abilities. Furthermore, both LPS and an extract of two major outer membrane proteins caused large increases in 55Fe uptake over control (phospholipids only) vesicles. One study has shown that Desulfovibrio vulgaris lipopolysaccharide has a specific iron-binding site within its polysaccharide side chain [16, 17].
L. Machtoub et al./Journal of Medical Imaging and Radiation Sciences 41 (2010) 159-164
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Whether LPS injections, which are used in our animal experiments as an endothelial stressor, per se cause an altered cellular SPIO-lipid binding activity needs further investigations. In summary, using CARS and Raman imaging microscopy, surface-enhanced signals have been observed in hepatic and spleen tissue samples from experimental animals that have been injected with SPIO nanoparticles. The intensity enhancement was markedly higher than in control animals. The observed SECARS signal has been near the Raman resonance of 2,850 cm1 and 2,875 cm1, which matches the C-H stretch vibrations in lipids in biological tissues. Our results indicate a selective iron-binding enhancement from surrounding lipid molecules adsorbed to the surface of iron oxide nanoparticles. With this study, we demonstrate the potential contribution of SECARS microscopy for visualization of SPIO uptake in phagocyticactive macrophages and their metabolic effects in biological systems. The SERS-based optical properties of iron oxide nanoparticles are shown to be promising magnetic optical probes for real-time cell imaging. Acknowledgment The authors thank Prof. S. Schluecker, Department of Physics, University of Osnabruek, for providing facilities for Raman microscopic measurements. Part of this work was performed at the Jena Institute for Photonic Technologies, IPHT-Germany. The authors gratefully acknowledge the facilities provided by Prof. J. Popp and Dr. B. Dietzek and the technical assistance by Dr. D. Akimov and G. Bergner. Special thanks to Prof. Sunney Xie, Harvard University, for providing the opportunity to use Harvard CARS facilities for some material test measurements during the CRS. L.M. thanks Dr. S. Kreutmayer for her great efforts in sample material preparation. The technical assistance in preparation of the animal experiments by C. Kresmer and C. Grudtmann is acknowledged. This project was supported by a grant from the Medizinische Forschungsfo¨rderung Innsbruck MFI (MCW, project 9443). References [1] Gessner, R., Roesch, P., & Petry, R., et al. (2004). The application of a SERS fiber probe for the investigation of sensitive biological samples. Analyst 129, 1193–1199.
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[2] Qian, X., Peng, X., & Ansari, D., et al. (2007). In vivo tumor targeting and spectroscopic detection with surface enhanced Raman nanoparticles tags. Nature Biotechnol 10, 1038–1377. [3] Gellner, M., Koempe, K., & Schluecker, S. (2009). Multiplexing with SERS labels using mixed SAMs of Raman reporter molecules. Anal Bioanal Chem 394, 1839–1844. [4] Guo, L., Huang, Q., Li, X. Y., & Yang, S. (2001). Iron nanoparticles: synthesis and applications in surface enhanced Raman scattering and electrocatalysis. Phys Chem Chem Phys 3, 1661–1665. [5] Xie, J., Chen, K., & Lee, H. Y., et al. (2008). Ultra-small c(RGDyK)coated Fe3O4 nanoparticles and their specific targeting to integrin avß3-rich tumor cells. J Am Chem Soc 130, 7542–7543. [6] Neel, L. (1955). Some theoretical aspects of rock-magnetism. Adv Phys 191–243. [7] Evans, C. L., Potma, E. O., Puoris0 haag, M., Coˆte´, D., Lin, C. P., & Xie, X. S. (2005). Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy. PNAS 102(46), 16807–16812. [8] Evans, C. L., Xu, X., Kesari, S., Xie, X. S., Wong, S., & Young, G. S. (2007). Chemically-selective 15 imaging of brain structures with CARS microscopy. Optics Express 15(19), 2076–12087. [9] Hashimoto, T., Yamada, T., & Yoko, T. (1996). Third-order nonlinear optical properties of sol-gel derived a-Fe2O3, g-Fe2O3 and Fe3O4 thin films. J Appl Phys 80, 3184–3190. [10] Zheng, Y., Holtom, G. R., & Colson, S. (2004). Multichannel multiphoton imaging of metal oxides nanoparticles in biological system. Proc SPIE 5323, 390–399. [11] Moger, J., Johnston, B. D., & Tayler, C. (2008). Imaging metal oxide nanoparticles in biological structures with CARS microscopy. Optics Express 16, 3408–3419. [12] Wick, M. C., Mayerl, C., & Backovic, A., et al. (2008). In vivo imaging of the effect of LPS on arterial endothelial cells: molecular imaging of heat shock protein 60 expression. Cell Stress Chaperones 13, 275–285. [13] Roth, B., Fkelund, M., Fan, B., Haegerstrand, I., & Nilsson-Ehle, P. (1996). Lipid deposition in Kupffer cells after parenteral fat nutrition in rats: a biochemical and ultrastructural study. Intensive Care Med 22, 1224–1231. [14] Morrill, G. A., Kostellow, A., Resnick, L. M., & Gupta, R. K. (2004). Interaction between ferric ions, phospholipid hydroperoxides, and the lipid phosphate moiety at physiological pH. Lipids 39, 881–889. [15] Gangloff, S. C., Ladam, G., & Dupray, V., et al. (2006). Biologically active lipid A antagonist embedded in a multilayered polyelectrolyte architecture. Biomaterials 27, 1771–1777. [16] Bacon, J., Dover, L. G., & Hatch, K., et al. (2007). Lipid composition and transcriptional response of Mycobacterium tuberculosis grown under iron limitation in continuous culture: identificaton of a novel wax ester. Microbiology 153, 1435–1444. [17] van Oosten, M., van Bilt, E., van de Berkel, T., & Kuiper, J. (1998). New scavenger receptor-like receptors for the binding of lipopolysaccharide to liver endothelial and Kupffer cells. Infect Immun 66, 5107–5112.
L. Machtoub et al./Journal of Medical Imaging and Radiation Sciences 41 (2010) 159-164
Journal of Medical Imaging and Radiation Sciences 41 (2010) 159-164
Journal de l’imagerie médicale et des sciences de la radiation
www.elsevier.com/locate/jmir
Molecular Imaging Cellular SPIO Uptake with Nonlinear Optical Microscopy Lina Machtoub, PhDa*, Rudolf Pfeiffer, PhDb, Aleksandar Backovic, PhDc, Stefan Frischauf, MDa, and Marius C. Wick, MDa a
b
Universitaetsklinik Fuer Radiodiagnostik, Innsbruck Medical University, Innsbruck, Austria Faculty of Physics, University of Vienna, Vienna, Austria, and Department of Electrical Engineering, California Institute of Technology, Pasadena c CSO-Research and Development at IDC Immumetrics, Vienna, Austria
ABSTRACT We report a novel application to demonstrate and visualize the selective binding of lipids in cells of the reticuloendothelial system to super paramagnetic iron oxide (SPIO) nanoparticles. Ten New Zealand White rabbits that were experimentally injected intravenously with SPIO and five controls were investigated with vibrational microspectroscopy based on surface-enhanced coherent anti-Stokes Raman scattering (SECARS) microscopy. Marked cellular intensity enhancements in hepatic Kupffer cells and melanomacrophages of spleen have been observed in the range of 2850–2875 cm1 in SPIO-injected animals but not in controls. The enhancements are related to the selective association of lipid molecules in cells of the reticuloendothelial system to uptaken SPIO, which can uniquely be visualized with SECARS microscopy.
RE´SUME´ Nous annonc¸ons une nouvelle application qui sert a` de´montrer et a` visualiser la liaison se´lective de lipides dans les cellules du syste`me re´ticulo-endothe´lial avec des nanoparticules d’oxyde de fer superparamagne´tique (OFS). Dix lapins ne´o-ze´landais blancs ayant rec¸u une injection intraveineuse expe´rimentale d’OFS et 5 te´moins ont e´te´ e´tudie´s par microspectroscopie vibratoire fonde´e sur la microscopie SECARS (Surface-Enhanced Coherent anti-Stokes Raman Scattering). Des ame´liorations marque´es de l’intensite´ cellulaire dans les cellules de Kupffer du foie et les me´lanomacrophages de la rate ont e´te´ observe´es dans la plage de 2850 a` 2875 cm1 chez les animaux ayant rec¸u l’injection d’OFS, mais non chez les sujets te´moins. Ces ame´liorations sont lie´es a` l’association se´lective des mole´cules de lipides des cellules du syste`me re´ticulo-endothe´lial avec l’OFS capte´, association visualisable exclusivement par la microscopie SECARS.
Introduction
from two to three orders of magnitude [4]. In these studies, the observed surface enhancement has been partially attributed to the quantum size effect of the iron particles. Novel new applications are recently underway to use magnetic nanoparticles as targeting markers for cancer drug delivery. It has been shown that arginine-glycine-aspartic (RGD) peptide-coated iron oxide can be bonded with an integrin-rich tumor cell, and potentially can be used as targeting marker. In addition, the synthesis of dumbbell-shaped magnetic nanoparticles (Au-Fe3O4) has made such species especially attractive as multifunctional probes for diagnostic and therapeutic applications [5]. Superparamagnetic iron oxide (SPIO) nanoparticles, a potent new class of in vivo magnetic resonance imaging (MRI) contrast agents, have been widely recognized as tissue-specific MRI contrast agents for in vivo detection of malignant tumors in the liver and spleen of patients. The magnetic properties of these materials were first referred to as superparamagnetic in 1955 [6]. The term refers to the large
Surface-enhanced Raman scattering (SERS) nanoparticles have drawn a large degree of interest as targeted imaging probes and therapeutic agents [1, 2]. Recently, SERS microscopy has been exploited to observe trace amounts of biologically relevant molecules that allow for the selective and highly sensitive localization of target molecules. Based on their surface plasmon polaritons, novel metal nanoclusters (eg, gold or silver nanoparticles) provide the key effect for observing enhanced Raman signals for molecules attached to them [3]. It has been reported that iron nanoparticles can have remarkable SERS activity in a range This project was supported by a grant from the Medizinische Forschungsfo¨rderung Innsbruck MFI (MCW, Project 9443). * Corresponding author: Dr. Lina Machtoub, PhD, Universitaetsklinik fuer Radiodiagnostik, Innsbruck Medical University, Anichstrasse 35, A-602Innsbruck, Austria. E-mail address: [email protected] (L. Machtoub). 1939-8654/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi: 10.1016/j.jmir.2010.08.001
magnetic moments they acquire when placed in external magnetic fields. To visualize SPIO nanoparticles in biological systems, a molecular imaging modality has to meet the needs of high sensitivity combined with high spatial and temporal resolution and high multiplexing capacity. For ex vivo tissue analyses from biopsy samples conventional histological techniques are considered gold standard; however, they are not feasible for live tissue imaging. Recently, nonlinear optical microscopy, particularly coherent anti-Stokes Raman scattering (CARS) has taken a large step toward becoming a medical diagnostic tool for both ex vivo and for some cases even in vivo [7]. Nonlinear optical microscopy and microspectroscopy allow for differentiating the spectra fingerprint of many molecules with submicron resolution, resulting in very high multiplexing capabilities and narrow spectral features that can be easily separated from the broadband autofluorescence. Compared with other clinical imaging modalities, CARS microscopy can be tuned to provide a variety of possible tissue contrasts, with an advantage of subcellular spatial resolution and near real-time temporal resolution compared with MRI [8]. The ability to resolve individual nanoparticles is a challenging task and CARS has the ability to chemically selectively detect aggregations or accumulations of nanoparticles in cells or thin tissue slices. It has already been reported that even sub-wavelength sites can be seen if the nonlinearity of the material is sufficiently strong [9]. Apart from its chemical selectivity, the main strength of the method lies in the fact that the nanoparticles do not have to be treated (for example, labeled with a fluorescent marker), but can be imaged in their native state. This is important, because labeling might change the behavior of the (for example, metallic/ organic, soluble/insoluble, functionalized) nanoparticles in cells, and thus influence the outcome of investigations on the fate of the particles. Previous findings reported intense CARS signals observed from the experiments performed on a fish gill with embedded metal oxides nanoparticles (for example, ZnO, TiO2, Fe2O3) [10, 11]. The nanoparticles were visible with a high contrast because of their high thirdorder nonlinear optical susceptibilities. The main goal of this study was to explore the potential of surface-enhanced coherent anti-Stokes Raman scattering (SECARS)-based nonlinear optical microscopy in studying the cellular uptake or clearance of SPIO nanoparticles in tissue samples from an established experimental rabbit model. The animals and tissue samples for our experiments were also used in a study investigating the effect of stress on arterial endothelial cells and in vivo molecular MRI of endothelial expression of stress proteins [12]. For this purpose, a clinically approved established SPIO MRI contrast agent was used that is intravenously (i.v.) injected for the detection of malignant focal hepatocellular lesions in patients (Ferucarbutran, Resovist, Schering, Austria). This SPIO contrast agent is a nonstoichiometric polycrystalline mixture of magnetite (Fe3O4) and maghemite (g-Fe2O3) coated with carboxydextran (total core size of 62 nm). After injection, SPIO cores are phagocytosed by cells of the reticuloendothelial system (RES) and 160
accumulated in the lysosomal compartment. The liver (80%) and the spleen (5–10%) are the known primary organs of SPIO uptake and lysosomal aggregation. Figure 1 shows a representative in vivo MRI of a New Zealand White (NZW) rabbit before (A) and 70 min. after (B) i.v. injection of SPIO demonstrating the uptake of SPIO by the liver. Materials and Methods The animal experiments were surveyed by the Committee for Animal Experimentation and approved by the Austrian Ministry of Sciences (reference number BMWF-66.011/0017-II/10b/ 2008). In the course of an in vivo molecular MRI experiment for the detection of an arterial endothelial expression of stress proteins, 15 normocholesterolemic female NZW rabbits have been injected i.v. Lipopolysaccharide (LPS; a standard endothelial stressor; in a pilot study found to be optimal at 10 mg/kg body weight [12]) and sacrificed 8–10 h after LPS injection. Four hours after stress induction with i.v. LPS, 10 rabbits were experimentally i.v. injected with 0.5–0.7 mL SPIO (¼5 mg Fe/kg KG) to partially saturate iron uptake in the RES. Five rabbits were used as untreated controls. In vivo MRI was performed under general anesthesia in all SPIO-given animals before and at 20-min intervals until 2–4 h after i.v. SPIO according to a standardized protocol on a 1.5T MRI (Siemens, Magnetom Avanto, Erlangen, Germany). In controls, MRI was performed 6 h after LPS. After MRI, euthanasia of all animals was performed under anesthesia with xylanest and ketamine via in vivo perfusion with phosphate-buffered saline (PBS, pH 7.2) followed by perfusion-fixation with 4% PBS-buffered paraformaldehyde. Thereafter, tissue samples were taken from the liver, spleen
Figure 1. In vivo magnetic resonance imaging (ie, a T2-weighted R2-map magnetic resonance sequence) of a New Zealand White rabbit before (A) and 70 min after (B) intravenous injection of 0.7 mL super paramagnetic iron oxide (SPIO) (¼5 mg Fe/kg kg) demonstrating the uptake of SPIO by the liver (circumscribed and arrows).
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and fat and stored in 4% PBS-buffered paraformaldehyde until further preparation for CARS, Raman mapping and conventional light microscopy. Histopathologic Preparation The perfusion-fixated tissues were embedded in paraffin blocks and were subsequently cut into slices of 10-mm thickness for histological staining. Turnbull-blue reaction was used to detect the presence and cellular localizations of SPIO at light microscopy with magnifications from 200 to 630. A standard protocol of iron staining was performed, starting with 1 h treatment of the tissues in a freshly prepared 10% ammonium sulfide solution (in aqua dest.). Then, the sections were treated with 1% HCl solution (in aqua dest.) and 20% potassium ferricyanide (III in aqua dest.). This causes ferrous iron to react to form an insoluble bright blue pigment (ferrous ferricyanide). The nuclei were counterstained with nuclear fast red (Carl Roth, Karlsruhe, Germany) and mounted with Kaiser Glycerol gelatin (Merck, Darmstadt, Germany). Light microscopic images of the stained tissues were taken by Nikon eclipse E800 digital camera. Control Experiments Hepatic tissue samples of wild-type mice (on a Western diet) were disintegrated in a porcelain mortar together with a 2:1 mixture of chloroform and methanol. The solution was incubated for 2 h at 4 C before aqua dest. was added to aid in phase separation during a subsequent centrifugation step at 10,000g for 10 min. The chloroform-lipid mixture sat overnight at room temperature to evaporate the chloroform. Subsequently, the lipids were redissolved in ethanol. Murine adipocytes were grown on gelatin-coated glass coverslips and exposed to 5 ng/ mL of SPIO nanoparticles (in total: 15 ng in 3 mL fresh medium). After incubation for 24 h, adipocytes were fixated at room temperature with 4% paraformaldehyde for 30 min. and stored in PBS until further preparation for microscopic measurements. Further control experiments were performed on hepatic and spleen tissues, extracted from the untreated control (NZW) rabbits, without administration of SPIO nanoparticles, subject to similar experimental conditions. Experimental Measurements and ex vivo Imaging Fourier transform Raman spectra (FT-Raman) were measured using a Bruker RFS 100/S spectrometer (Bruker Optics Inc, Billerica, MA). Spectra of all samples were measured at room temperature in a 180 backscattering geometry in a microscope. The absorption measurements were performed using a Hitachi U-3410 Spectrophotometer. Microscopic Raman mapping images were recorded using an alpha 300R instrument (WITec, Ulm, Germany) equipped with a CCD detector. The integration time per pixel was 0.1 s. The samples were irradiated by a He Ne laser at 632.8 nm and focused by a 40 0.65 NA microscope objective lens. CARS measurements were performed using two ps light sources of near-infrared excitation; Ti: sapphire laser (Coherent Mira HP, 3.5W 3 ps, 700–1000 nm), and 1064-nm mode-locked Nd::YVO4. The fundamental lines (830 nm,
1064 nm) are used for the Stokes beam, whereas the signals from the optical parametric oscillators were used as the pump and probe. The corresponding pump beams were tunable in the range of 510–930 nm by using two optical parametric oscillators (APE, Germany). Both oscillators are pumped by part of the fundamental output lines and can be continuously tuned over a wavelength range that can cover the entire chemical essential vibration frequency range of 200–3600 cm1. The beams are scanned over the sample and focused by water immersion objective lens with 1.2 numerical apertures. Both beams have a power of several tens of milliwatts for the sample. Images taken at different wavelengths are collected consecutively by tuning the oscillators without the need for optics realignment. The residual pump and Stokes radiation are separated from the CARS signal by using dielectric filters. The collected signal is filtered by bandpass filters and detected by a photomultiplier tube (Hamamatsu R6357). The images were recorded with a resolution of 5 s total acquisition time for one frame of 512 512 pixels. The microscope is designed for the signal to be detected in both forward and epi-direction. The samples used in the experiment were tissue slices, cell lines and some compounds made in solutions. For most of the samples, the CARS signal was collected in the forward direction; however, for some thick specimens (tissue sections) we used epi-detection because of the back scattering of the strong forward signal from several layers in the tissue. Results Ex vivo CARS measurements on unstrained hepatic tissue samples from NZW rabbits that have been i.v. injected with SPIO showed markedly increased intracellular signal enhancements in regions at the perisinusoidal location of Kupffer cells (Figure 2A). These enhancements have only been observed in the wavelengths range of 2850–2875 cm1, which relates to the symmetric CH2 stretch vibration of lipids (Figure 2B). Conventional light microscopy using Turnbull-blue staining correlated the observed SECARS signals to an intracellular accumulation of SPIO in Kupffer cells (Figure 2C). In contrast, measurements on hepatic specimen from control NZW rabbits showed negative staining for SPIO and no CARS signal enhancements at any vibrational resonance wavelengths. (See Supplemental Figure 1 in supplemental online material.) Similarly, CARS measurements of tissue samples from spleen with SPIO-injected rabbits but not from controls showed strong signals enhancements at 2850–2875 cm1 (Figure 3A). The enhancements correlated to SPIO depositions at perisinusoidal melanomacrophage centres. Histological analysis confirmed the intracellular presence of iron particles in macrophages (Figure 3B). To investigate a selective intracellular SPIO lipid-binding activity, we performed several control CARS measurements on SPIO-treated adipocytes and extracted lipids from hepatic tissue, incubated in vitro with or without SPIO. An intensive intracellular signal enhancement could be found in
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Figure 2. (A) Coherent anti-Stokes Raman scattering (CARS) image of an unstained hepatic tissue specimen from a NZW rabbit that was intravenously injected with super paramagnetic iron oxide (SPIO) nanoparticles, taken at 2850 cm1 (B) The corresponding cross section profile shows intensity enhancement along the indicated line shown in the inset of the CARS image. (C) Light microscopic image of Turnbull blue stained hepatic tissue (arrows indicate SPIO nanoparticles [blue] in Kupffer cells).
the vibrational resonance wavelengths range w2850 cm1 from SPIO-incubated lipids and particular regions highlighting accumulation of SPIO particles in adipocytes. (See Supplemental Figures 2 and 4A in supplemental online material.) The intracellular enhancement has been further investigated on all tissues by Raman imaging chemical mapping measurements (Figure 4B). In contrast to the CARS spectrum, which has resonant and nonresonant contributions, Raman imaging does not suffer from the coherent addition of nonresonant background signal. Clear contrast enhancement is observed in iron nanoparticle–treated tissues and is indicated in the bright regions highlighting high accumulation of iron oxide nanoparticles. Similar measurements on untreated tissues have shown no indication of any
enhancements. (See Supplemental Figure 3 in supplemental online material.) To support the SERS activity of iron oxide nanoparticles, additional controlled microscopic Raman measurements were performed on iron oxide nanoparticles dissolved in a specific heterocyclic organic compound (C5H5N; Pyridine). Clear intensity enhancement could be detected from SPIO nanoparticles in Pyridine compared with the pure solution. (See Supplemental Figure 4 in supplemental online material.) Raman intensity from these solutions has shown considerable dependence on particles concentration. On the other hand, the FT-Raman measurements performed on iron oxide nanoparticles have shown no substantial resonance Raman activity observed in the region of interest. (See Supplemental Figures 5 and 6 in supplemental online material.)
Figure 3. (A) Coherent anti-Stokes Raman scattering image (2850 cm1) of an unstained spleen tissue sample of a New Zealand White rabbit after intravenous administration of super paramagnetic iron oxide (SPIO) nanoparticles. The inset shows SPIO nanoparticles in macrophages. (B) Corresponding light microscopic image of spleen tissue stained with Turnbull blue, SPIO (blue) are indicated by arrow.
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Figure 4. (A) Coherent anti-Stokes Raman scattering image of adipocytes intravenously given super paramagnetic iron oxide (SPIO) nanoparticles, taken at 2850 cm1. The corresponding section profile shows intensity enhancement highlighting the distribution of SPIO nanoparticles. (B) Raman mapping image of SPIO treated hepatic tissue specimen, taken in the range of 2840–2880cm1, shows clear contrast enhancement. The corresponding cross-section profiles of the spectra indicate an enhancement from the bright (1) and dark (2) regions.
Discussion The observed surface-enhanced Raman signal in the vibrational resonance wavelengths range of lipids in tissues from SPIO-treated animals and the results from our control experiments with SPIO-exposed adipocytes and extracted lipids suggest a selective binding of peri- or intracellular lipid molecules to SPIO accumulated in or adjacent to the lysosomal compartment of cells of the RES. The possibility of a binding between lipids and iron has already previously been demonstrated with phosphatidic acid, phosphatidylserine, oleic and stearic acids. These pure lipids have shown an apparent iron binding in filtration assays, the highest affinity at 5- to 10-fold on a molar basis with oleic acid [13]. Similar to the function of Kupffer cells in hepatic sinusoids, macrophages surrounding the spleen sinusoids play an important role in iron uptake from the circulation. Histological analyses from tissue samples of NZW rabbits that have been i.v. injected with SPIO have shown homogenously distributed iron depositions in the liver. However, in the spleen, the iron depositions were scattered and mainly detectable in the melanomacrophage centres. The lipid-binding selectivity of iron has been carefully investigated. Previous findings reported a phenomenon of iron-induced lipid peroxidation, for iron more often than any other transition metal. An in vivo study on rats with experimental chronic iron overload has shown direct evidence of hepatic lipid peroxidation in both mitochondrial
and microsomal membrane lipids [14]. The binding of Fe3þ to erythrocyte cell membranes has been investigated in a system in which lipid peroxidation was proportional to Fe3þ concentration. Such binding of Fe3þ was evaluated by labeling with 59 FeCl3 and measurement of nuclear magnetic resonance water-proton relaxation times. It has been shown that 95% of the Fe3þ was membrane-bound at 100 mM FeCl3 in a 1.5 mg protein/ml membrane suspension. Both spin-lattice (T1) and spin-spin (T2) relaxation times decreased with increasing Fe3þ concentration. Charge transfer to Fe3þ may occur at the membrane binding site with resultant decrease in the Fe3þ effect on water-proton relaxation times. These studies support the hypothesis that Fe3þ binds to the membrane and generates free radicals at the binding site [15]. However, in our experiment, the possibility of lipid peroxidation is less probable because the iron concentration in our experimental animals did not reach highly pathological values and histopathologic evidence of possible cell degeneration was absent on tissue samples. Another possible mechanism of an increased lipid-iron binding activity might be originated from observations that showed lipids representing a substantial proportion of Salmonella typhimurium LPS, which is expected to have high iron-binding abilities. Furthermore, both LPS and an extract of two major outer membrane proteins caused large increases in 55Fe uptake over control (phospholipids only) vesicles. One study has shown that Desulfovibrio vulgaris lipopolysaccharide has a specific iron-binding site within its polysaccharide side chain [16, 17].
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Whether LPS injections, which are used in our animal experiments as an endothelial stressor, per se cause an altered cellular SPIO-lipid binding activity needs further investigations. In summary, using CARS and Raman imaging microscopy, surface-enhanced signals have been observed in hepatic and spleen tissue samples from experimental animals that have been injected with SPIO nanoparticles. The intensity enhancement was markedly higher than in control animals. The observed SECARS signal has been near the Raman resonance of 2,850 cm1 and 2,875 cm1, which matches the C-H stretch vibrations in lipids in biological tissues. Our results indicate a selective iron-binding enhancement from surrounding lipid molecules adsorbed to the surface of iron oxide nanoparticles. With this study, we demonstrate the potential contribution of SECARS microscopy for visualization of SPIO uptake in phagocyticactive macrophages and their metabolic effects in biological systems. The SERS-based optical properties of iron oxide nanoparticles are shown to be promising magnetic optical probes for real-time cell imaging. Acknowledgment The authors thank Prof. S. Schluecker, Department of Physics, University of Osnabruek, for providing facilities for Raman microscopic measurements. Part of this work was performed at the Jena Institute for Photonic Technologies, IPHT-Germany. The authors gratefully acknowledge the facilities provided by Prof. J. Popp and Dr. B. Dietzek and the technical assistance by Dr. D. Akimov and G. Bergner. Special thanks to Prof. Sunney Xie, Harvard University, for providing the opportunity to use Harvard CARS facilities for some material test measurements during the CRS. L.M. thanks Dr. S. Kreutmayer for her great efforts in sample material preparation. The technical assistance in preparation of the animal experiments by C. Kresmer and C. Grudtmann is acknowledged. This project was supported by a grant from the Medizinische Forschungsfo¨rderung Innsbruck MFI (MCW, project 9443). References [1] Gessner, R., Roesch, P., & Petry, R., et al. (2004). The application of a SERS fiber probe for the investigation of sensitive biological samples. Analyst 129, 1193–1199.
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