Article pubs.acs.org/cm
Multimodal [GdO]+[ICG]− Nanoparticles for Optical, Photoacoustic, and Magnetic Resonance Imaging Marieke Poß,† Robert J. Tower,‡ Joanna Napp,§,∥ Lia Christina Appold,⊥,@ Twan Lammers,⊥,@ Frauke Alves,§,∥ Claus-Christian Glüer,‡ Susann Boretius,‡,# and Claus Feldmann*,† †
Institute of Inorganic Chemistry, Karlsruhe Institute of Technology (KIT), Engesserstraße 15, 76131 Karlsruhe, Germany Section Biomedical Imaging, Department of Radiology and Neuroradiology, University Hospital Schleswig-Holstein, Campus Kiel, Arnold-Heller-Straße 3, 24105 Kiel, Germany § Department of Molecular Biology of Neuronal Signals, Max-Planck-Institute for Experimental Medicine, Hermann-Rein-Straße 3, 37075 Göttingen, Germany ∥ Department of Diagnostic and Interventional Radiology and Department of Haematology and Medical Oncology, Goettingen University Medical Center Goettingen, Robert Koch Strasse 40, 37075 Goettingen, Germany ⊥ Department of Nanomedicine and Theranostics, Institute for Experimental Molecular Imaging, RWTH Aachen University Clinic, Pauwelsstrasse 30, 52074 Aachen, Germany @ Department of Targeted Therapeutics, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands # German Primate Center, Leibnitz Institute of Primate Research, Kellnerweg 4, 37077 Göttingen, Germany ‡
S Supporting Information *
ABSTRACT: Multimodal contrast agents with high biocompatibility and biodegradability, as well as low material complexity, are in great demand for clinical diagnostics at different scales of resolution and/or for translating preclinical diagnosis into intraoperative imaging. Multimodality, however, often results in multicomponent and multistructured materials with complexity becoming a severe restriction for synthesis, approval, and use in routine clinical practice. Here, we present sulfonate-based saline [GdO]+[ICG]− (ICG, indocyanine green) inorganic-organic hybrid nanoparticles (IOH-NPs with an inorganic [GdO]+ cation and an organic [ICG]− anion) as a novel, multimodality contrast agent for optical, photoacoustic, and magnetic resonance imaging (OI, PAI, MRI). [GdO]+[ICG]− IOH-NPs have a plain composition based on clinically used constituents and are prepared as an insoluble saline compound in water. The high [ICG]− content (81 wt %) ensures intense near-infrared emission (780−840 nm) and a strong photoacoustic signal. First, in vitro studies demonstrate longer detectability and greater emission intensity for [GdO]+[ICG]− IOH-NP suspensions than for ICG solutions, as well as a reduced toxicity compared to that of Gd-DTPA, a standard MRI contrast agent. Conceptual in vivo studies confirm the utility of the [GdO]+[ICG]− IOH-NPs for optical and magnetic resonance imaging with a T1 relaxivity better than that of Gd-DTPA. Taken together, [GdO]+[ICG]− represents a new compound and nanomaterial that can be highly interesting as a multimodal contrast agent.
■
INTRODUCTION
high resolution. In contrast to OI and PAI, however, MRI requires cumbersome instrumentation and time-consuming data acquisition.8,9 Therefore, contrast agents allowing multimodal detection are in great demand for visualization on different scales of resolution, the presentation of different types of tissues (e.g., soft tissue via MRI, blood vessels via PAI, and single cells via OI), the translation from preclinical to clinical
Contrast agents allowing for multimodal imaging are highly relevant to medicine for combining the specific assets of different imaging techniques.1−4 Optical imaging (OI), for instance, can unveil the organ distribution with deep-tissue information (e.g., whole body of a mouse).5,6 OI, however, often suffers from low spatial resolution (e.g., because of optical scattering). Photoacoustic imaging (PAI) allows for higher optical contrast and spatial resolution like those of other optical techniques, but only small areas can be imaged.7 Magnetic resonance imaging (MRI), finally, is widely used in clinical practice and suitable for obtaining deep-tissue information at © 2017 American Chemical Society
Received: December 21, 2016 Revised: March 28, 2017 Published: March 30, 2017 3547
DOI: 10.1021/acs.chemmater.6b05406 Chem. Mater. 2017, 29, 3547−3554
Article
Chemistry of Materials
with DAPI. Images were acquired with an Axiovert 200 M inverted microscope (Carl Zeiss Microscopy GmbH) equipped with a nearinfrared (NIR)-sensitive ORCA-ER digital camera (Hamamatsu) using a 708 ± 37.5 nm excitation filter and a 809 ± 40.5 nm emission filter for ICG and a 365 ± 12.5 nm excitation filter and a 445 ± 25 nm emission filter for DAPI. Image generation and processing were performed with AxioVision release 4.6 and FIJI (National Institutes of Health, Bethesda, MD), respectively.19 For toxicity analysis, cells were plated on 96-well plates (5000 cells per well) and allowed to attach. On the next day, the medium was replaced with cell culture medium supplemented with increasing concentrations (0−0.200 mmol/mL) of either [GdO]+[ICG]− IOHNPs, dextran-coated [GdO]+[ICG]− IOH-NPs, an ICG solution, GdDOTA (Dotarem, Sulzbach, Germany), or Gd-DTPA (Magnograf, Marotrast, Jena, Germany). Cell viability was measured after incubation for 0, 24, 48, and 72 h using a CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). The absorbance at 490 nm was measured using a Wallac Victor2 1420 multiplate reader (PerkinElmer). PAI Experiments. Photoacoustic imaging was performed using the VEVO LAZR photoacoustic imaging system equipped with a LZ250 transducer (VisualSonics, Amsterdam, The Netherlands). Polyethylene tubes with an inner diameter of 0.58 mm and an outer diameter of 0.96 mm immersed in water were used for blood vessel phantom measurements. ICG in solution and [GdO]+[ICG]− IOH-NPs in 30 mM HEPES or 5% dextran 40 were measured at concentrations of 12.9 μM, 1.29 μM, 129 nM, 12.9 nM, and 1.29 nM (correlating with 10, 1, 0.1, 0.01, and 0.001 μg/mL, respectively). PA spectra were recorded from 680 to 968 nm in 2 nm steps with a center frequency of 21 MHz. The focus depth was set at 11 mm. Thirty microliters of ICG in H2O (c = 12.9 μM) and 30 μL of [GdO]+[ICG]− IOH-NPs (c = 12.9 μM) in HEPES were injected at different depths in a chicken breast or subcutaneously into a dead mouse prior to the recording of PA spectra. MRI Experiments. MRI was performed at a field strength of 7 T (ClinScan, 70/30 USR, Bruker Biospin). An inversion recovery spin echo sequence (two-dimensional), an echo time (TE) of 6.3 ms, a repetition time (TR) of 3000 ms, and 16 inversion times (TI = 50− 2000 ms) were used to calculate the T1 relaxation times of different nanoparticle solutions in water [gadolinium concentration (cGd) between 0.001 and 1.055 mmol/L] on a pixel by pixel basis. From that, the specific relaxivity (r1) per gadolinium was calculated by
imaging, and/or the translation of preoperational to intraoperative imaging.1−4 Nanoparticles have turned out to be extremely promising for multimodal imaging. Thus, materials with different functionalities were integrated with high virtuosity into complex nanoarchitectures, including, for instance, superparamagnetic iron oxide nanoparticles (SPIONs) and inorganic fluorescent nanoparticles (e.g., quantum dots and lanthanide-doped oxides) or molecular fluorescent dyes (e.g., coumarins, rhodamines, oxazines, and cyanines) that were encapsulated in or attached to inorganic or organic matrices (e.g., SiO2, calcium phosphate, polymers, liposomes, and dendrimers).1−4 Such matrices, however, reduce the amount of active contrast agent per nanoparticle and, thus, reduce the detection limit. Moreover, the complexity and sheer number of constituents can be restrictions in themselves as all constituents and combinations must be verified individually for clinical approval. Thus, in vivo application in practice becomes more prohibitive the greater the complexity and the more multicomponent the employed materials are.10 To generate multimodal nanoparticles with low material complexity and high biocompatibility, synthesis, composition, and structure should be kept as simple as possible and preferentially involve constituents that are already in clinical use. These requirements were implemented with the sulfonatebased, saline [GdO]+[ICG]− inorganic-organic hybrid nanoparticles (IOH-NPs). Hereof, ICG (indocyanine green) is one of the very few clinically approved dyes.11 Gadolinium is a standard constituent used in MRI (e.g., Gd-DTPA; DTPA, diethylenetriamine pentaacetate).12 Surprisingly, a combination of paramagnetic Gd and fluorescent ICG was yet only realized by encapsulating comparably low concentrations of both in SiO2 matrices13,14 or by elaborate core-shell structures.15−17 The here presented [GdO]+[ICG]− IOH-NPs are a new multimodal contrast agent for use in OI, PAI, and MRI that is distinguished by an uncomplex composition and structure, a water-based synthesis, the exceptional load of [ICG]− (81 wt %) and [GdO]+ (19 wt %), the absence of inert matrices, and the improved performance in comparison to those of conventional ICG and Gd-DTPA in solution.
■
1 1 = + r1cGd T1(cGd) T1(cGd = 0)
EXPERIMENTAL SECTION
Magnograf (Gadopentetic acid) (Gd-DTPA) (MaRotrast) was used as a commercial Gd contrast control. In vivo MRI of mice was performed before and 5 h after intravenous nanoparticle injection (8 mg/kg of mouse) utilizing a radially encoded gradient echo sequence (2D spoiled FLASH; TE and TR values of 1.3 and 3000 ms, respectively; flip angle of 15°; spatial resolution of 300 μm × 300 μm; slice thickness of 700 μm). T1 values were calculated pixel by pixel from a series of images with varying inversion times (TI = 50−2000 ms). Animal Experiments. C57BL/6 albino mice bred in house were used for all animal experiments. All animals were kept in a temperature- and humidity-controlled environment, with a 12 h light/dark cycle, and access to food and water ad libitum. Animal experiments and care were in accordance with the guidelines of institutional authorities and approved by the Ethics Committee for Animal Experiments at the Christian-Albrechts-Universität-zu-Kiel (V 312-72241.121). Mice were anesthetized with intraperitoneal injections of 80 mg of ketamine/kg (Aveco Pharmaceutical) and 10 mg of xylazine/kg (Rugby Laboratories). Mice were intravenously injected via the tail vein with 8 mg of prepared nanoparticle solutions per kilogram. In vivo scans were made using the 800 nm channel of the FMT2500LX instrument from Visen Medical (PerkinElmer). Post mortem and organ fluorescence images were acquired using the
Synthesis. [GdO]+[ICG]− IOH-NPs were synthesized by dissolving 39.5 mg (0.05 mmol) of Na+[ICG]− (ABCR) in 60 mL of demineralized water. Thereafter, 0.5 mL of an aqueous solution containing 18.5 mg (0.05 mmol) of GdCl3·6H2O (Aldrich, 99%) was injected, resulting in an instantaneous nucleation of nanoparticles. After being intensely stirred for 2 min, the [GdO]+[ICG]− IOH-NPs were separated via centrifugation (25000 rpm, 15 min). To remove all remaining salts and starting materials, the dark green [GdO]+[ICG]− IOH-NPs were rinsed three times with H2O. Finally, the as-prepared [GdO]+[ICG]− IOH-NPs were resuspended in H2O, HEPES buffer, or a dextran solution (5% dextran in water). For suspensions that were used in vitro (macrophages) and in vivo (mice model), all solvents were sterilized using 0.2 μm nylon filters prior to usage. In Vitro Studies. Immortalized and adherent mouse alveolar macrophage cell line MH-S (CRL-2019, ATCC) was used.18 Cells were cultivated at 37 °C in a humidified atmosphere of 5% CO2 in complete RPMI medium, supplemented with 10% FCS (fetal calf serum) and 0.05 mM 2-mercaptoethanol. For fluorescence microcopy, cells were grown on 25 mm poly-Llysine-coated coverslips and incubated for 24 h with 10 μg/mL [GdO]+[ICG]− IOH-NPs in cell culture medium at either 37 or 4 °C, washed twice with PBS, fixed for 10 min with ice-cold 4% paraformaldehyde, and mounted in Prolong Gold Antifade Reagent 3548
DOI: 10.1021/acs.chemmater.6b05406 Chem. Mater. 2017, 29, 3547−3554
Article
Chemistry of Materials
Figure 1. Synthesis of [GdO]+[ICG]− IOH-NPs. (a) Structure of the [ICG]− anion. (b) Aqueous synthesis. (c) Scheme of dextran-coated [GdO]+[ICG]− IOH-NPs in water.
Figure 2. Material properties of [GdO]+[ICG]− IOH-NPs. (a) Particle size in water according to DLS. (b) Photograph of the aqueous suspension (0.01 mg/mL). (c) ζ potential of the aqueous suspension. (d) Particle size according to electron microscopy. (e) Excitation and emission spectra (ICG solution as a reference). (f) Storage stability of the [GdO]+[ICG]− suspension in comparison to an ICG solution at an identical ICG concentration (0.01 μmol/mL, 3 °C, darkness). NightOwl planar imaging system and Indigo software (Berthold Technologies, Bad Wildbad, Germany).
and biological buffers (e.g., HEPES and aqueous dextran). It is important to note that the [GdO]+[ICG]− IOH-NPs are readily available as colloidally stable aqueous suspensions immediately following synthesis. As a major advantage in comparison to semiconductor-type quantum dots (e.g., CdSe),1−4,23 advanced multistep synthesis, core-shell structures, or specific demands regarding the crystallinity of materials or surface conditioning for dispersion in water are not required, which facilitates the preparation enormously. On the basis of our synthesis strategy, [GdO]+[ICG]− IOH-NPs are available in large quantities (10 g of [GdO]+[ICG]− nanoparticles made in 1 L of water) and as concentrated suspensions (≤10 mg/ mL). According to dynamic light scattering (DLS), [GdO]+[ICG]− IOH-NPs exhibit a mean hydrodynamic diameter of 50 ± 9 nm with a narrow size distribution for the as-prepared aqueous suspensions (Figure 2a,b). The realization of such small and uniform nanoparticles immediately after synthesis without the need for any specific agent for size control and surface stabilization is notable and can be ascribed to intrinsic charge stabilization. Thus, ζ potential measurements prove negative surface charging (−25 to −35 mV) for the [GdO]+[ICG]− nanoparticles in water at physiologically relevant pH values (6−8) (Figure 2c). Scanning electron microscopy (SEM) confirms the presence of uniform spherical particles with a mean diameter of 49 ± 8 nm [calculated by statistical evaluation of 250 particles (Figure 2d)]. The chemical composition of the [GdO]+[ICG]− IOH-NPs was validated by energy dispersive X-ray analysis (EDX) and Fourier transform infrared spectroscopy (FT-IR), confirming the presence of Gd3+ and [ICG]− qualitatively (Figure S1a).
■
RESULTS AND DISCUSSION Synthesis and Characterization of Materials. The sulfonate-based saline compound [GdO]+[ICG]− is insoluble in water and contains equimolar amounts of paramagnetic Gd3+ as the inorganic cation and the organic fluorescent dye [ICG]− as the anion (Figure 1). Because of this chemical composition, the weight load of the fluorescent dye per nanoparticle is extraordinarily high, reaching 81 wt % [ICG]−. In general, the synthesis strategy follows our recent concept of phosphatebased nanohybrids [ZrO]2+[RfunctionOPO3]2− (Rfunction is a functional organic group) that combine optical imaging and drug release.20,21 This concept, however, is limited by the small number of available functional organic anions containing phosphate functionalities. Moreover, phosphate-functionalized dyes for red and infrared emission, being most relevant for OI, are often very expensive (e.g., Alexa dyes and quantum dots). In contrast, almost all conventional fluorescent dyes are available with sulfonate functionalities that are widely used to make the dyes water-soluble. Hence, sulfonate-based inorganic-organic hybrids can significantly broaden the materials base of IOHNPs. The synthesis of [GdO]+[ICG]− is performed via straightforward forced hydrolysis in water by mixing of aqueous solutions of GdCl3·6H2O and Na+[ICG]− (Figure 1). To obtain nanoparticles and colloidally stable suspensions, general aspects for controlling particle nucleation and particle growth have to be considered following the LaMer model. 2 2 The [GdO]+[ICG]− nanoparticles can be easily suspended in polar solvents (e.g., water, ethanol, and diethylene glycol) 3549
DOI: 10.1021/acs.chemmater.6b05406 Chem. Mater. 2017, 29, 3547−3554
Article
Chemistry of Materials
Figure 3. In vitro uptake and biocompatibility of [GdO]+[ICG]− IOH-NPs. (a) Fluorescence images of [GdO]+[ICG]− (10 μg/mL of medium) after incubation for 24 h with MH-S murine macrophages at 37 °C and (b) at 4 °C (red, [GdO]+[ICG]−; blue, DAPI-stained cell nuclei). (c) Metabolic activity of MH-S murine macrophages after cultivation for 0, 24, 48, and 72 h with [GdO]+[ICG]− and dextran-coated [GdO]+[ICG]− suspensions. ICG, Gd-DOTA, and Gd-DTPA solutions as references (0−0.200 mmol/mL of [GdO]+[ICG]−, ICG, Gd-DOTA, and Gd-DTPA; measured in triplicate; average values shown after subtraction of the absorbance of medium). All statistical analysis was performed using the program Past. A two-tailed unpaired Student’s t test was used to compare two sets of data, and statistical significance was defined as p < 0.005 and indicated with asterisks.32
these limitations, it has been suggested to encapsulate ICG in nanoparticulate polymers, liposomes, micelles, or silica.28,29 All these matrices, however, reduce the total amount of ICG per nanoparticle and are biorelevant by themselves, including toxicity, biodegradability, approval, etc. [GdO]+[ICG]− IOH-NP suspensions, in general, show fluorescence similar to that of ICG solutions. Thus, the highly stable, dark green suspensions in water exhibit intense deep red emission upon visible excitation (Figure 2e and Figure S2a). Although the quantum yield of ICG is limited, the quasi-infinite number of fluorescence centers in [GdO]+[ICG]− guarantees intense emission. Even after certain photobleaching, the reservoir of intact [ICG]− in the nanoparticles maintains considerable emission. Upon comparison of aqueous [GdO]+[ICG]− IOH-NP suspensions and aqueous ICG solutions at an identical ICG concentration (0.01 μmol/mL), the [GdO]+[ICG]− IOH-NPs show storage stability that is better than those of ICG solutions (stored at 3 °C in darkness) (Figure 2f) as well as greater emission intensity (Figure S2b). Whereas ICG solutions degraded continuously, the [GdO]+[ICG]− suspensions remained at ∼60% of the initial emission intensity even after 8 days. Taken together, [GdO]+[ICG]− IOH-NPs outperform ICG solutions in terms of storage stability and emission intensity, both being highly relevant for OI.
For quantification, thermogravimetry (TG) and elemental analysis (EA) were employed and showed a weight loss of 83.3% (TG) due to total combustion of [ICG]− (calcd, 78.2%) and contents (EA) of 55.2 wt % C, 5.4 wt % H, 2.9 wt % N, and 7.1 wt % S [calcd, 55.8 wt % C, 5.2 wt % H, 3.0 wt % N, and 6.9 wt % S (Figure S1b)]. In summary, the chemical composition of [GdO]+[ICG]− is validated by different independent analytical methods. Optical Properties and in Vitro Studies. In view of OI and fluorescence detection, ICG is optimal for medical application. On one hand, its strong visible absorption (700− 820 nm) and its NIR emission (780−840 nm) are ideal for deep-tissue penetration minimizing absorbance by water and hemoglobin (Figure 2e).1−6 Moreover, ICG is well-tolerated (LD50 = 50−80 mg/kg),24 has been approved for clinical use by the Food and Drug Administration,6 and is already widely used in the clinic and for optical imaging.25,26 On the other hand, ICG as a dissolved molecule has significant weaknesses, including (i) rapid binding to human serum albumin and high-density lipoproteins causing agglomeration and rapid clearance via the liver, (ii) a very short circulation time (halflife of only 2−4 min), (iii) a low fluorescence quantum yield (∼5% in water), (iv) low chemical stability under physiological conditions because of fast biodegradation, and (v) rapid photobleaching upon light exposure.11,25−27 To overcome 3550
DOI: 10.1021/acs.chemmater.6b05406 Chem. Mater. 2017, 29, 3547−3554
Article
Chemistry of Materials
Figure 4. Photoacoustic properties of [GdO]+[ICG]− IOH-NPs. (a) Photoacoustic signal of [GdO]+[ICG]− suspensions and ICG solutions at different concentrations in a blood vessel phantom. (b) Photoacoustic spectra of [GdO]+[ICG]− suspensions and ICG solutions. (c) Photoacoustic signal in chicken breast phantoms, exemplifying detection at depths of ≤15 mm. (d) Visualization of a [GdO]+[ICG]− suspension and ICG solutions subcutaneously injected into a dead mouse phantom and tracking of drainage of [GdO]+[ICG]− IOH-NPs from the injection side (arrow).
with [GdO]+[ICG]− IOH-NPs is slightly lower than that of ICG but higher than those of Gd-DOTA and Gd-DTPA solutions [at similar concentrations of ICG and Gd (Figure 3c)]. In particular, Gd-DTPA is outperformed after 72 h by [GdO]+[ICG]−. Taken together, a comparable to partly improved behavior of the novel [GdO]+[ICG]− IOH-NPs in comparison to that of the clinically approved ICG, Gd-DTPA, and Gd-DOTA solutions with the experimental conditions used here is very promising and points to the potential of the IOHNPs. Finally, the fact that only [GdO]+[ICG]− can be used as a multimodal contrast agent whereas standard ICG, Gd-DOTA, and Gd-DTPA solutions are not suitable for MRI or OI/PAI should be taken into account. Next, the photoacoustic signal of [GdO]+[ICG]− IOH-NPs was compared to that of standard ICG solutions that are routinely used as a contrast agent for PAI.33−35 This was done in blood vessel phantoms (Figure 4a,b), chicken breast phantoms (Figure 4c), and dead mouse phantoms (Figure 4d), demonstrating that the PAI signal intensity and depths of detection are identical for [GdO]+[ICG]− IOH-NP suspensions and ICG solutions. We note that the shoulder at 900 nm in the ICG spectrum is caused by aggregates that are formed in the ICG solution. Altogether, [GdO]+[ICG]− IOH-NPs can be used for OI as well as for PAI. In Vivo Imaging and Biodistribution. Besides uncomplicated, water-based synthesis, extraordinarily high [ICG]− loading, and use as an OI and PAI contrast agent, multimodal
With the aim being in vitro and in vivo studies, the [GdO]+[ICG]− IOH-NPs can be coated with dextran.30,31 Upon incubation of MH-S murine macrophages with dextrancoated [GdO]+[ICG]− IOH-NPs,18 the IOH-NPs show excellent uptake and an emission that can be easily detected via NIR fluorescence microscopy (Figure 3a). A clear uptake of the [GdO]+[ICG]− nanoparticles is demonstrated after incubation for 24 h at 37 °C. MH-S cells incubated with the nanoparticles at 4 °C, where the levels of cellular metabolism and therefore internalization are strongly reduced, did not show any comparable fluorescence (Figure 3b), which evidences an active acquisition of nanoparticles by the macrophages. Despite massive internalization, the [GdO]+[ICG]− IOHNPs have a weak effect on the metabolic activity and/or viability of MH-S cells, as measured by the absorbance of the metabolized compound using cell proliferation assays (Figure 3c). Increasing concentrations of IOH-NPs of ≤0.200 mmol/ mL, as expected, resulted in a concentration-dependent decrease in metabolic activity. The same concentrationdependent decrease in metabolic activity was observed upon incubation of the macrophages with ICG, Gd-DOTA, or GdDTPA solutions and can be simply explained by the partial replacement of cell culture medium by the tested solutions, reducing the amount of available nutrients. Thus, we note that at the highest concentration (0.200 mmol/mL) half of the amount of the cell culture medium was replaced by the nanoparticle solution. The viability of the MH-S cells treated 3551
DOI: 10.1021/acs.chemmater.6b05406 Chem. Mater. 2017, 29, 3547−3554
Article
Chemistry of Materials
Figure 5. [GdO]+[ICG]− IOH-NPs as a MR contrast agent in vivo showing reduced T1 relaxation times at 7 T. (a) Maps of T1 relaxation time calculated for varying concentrations of uncoated and dextran-coated nanoparticles. (b) Mice were imaged before and 5 h after injection of [GdO]+[ICG]− IOH-NPs. Images show T1 relaxation time heat maps with a noticeably reduced relaxation in the gall bladder and liver (by 35%).
detection of [GdO]+[ICG]− IOH-NPs is complemented by MRI. Here, Gd-containing contrast agents are standard (e.g., Gd-DTPA).8,9,12 To realize dual-modality MRI and OI, such Gd-based coordination complexes need to be combined with molecular fluorophores (e.g., rhodamine, cyanine, and Alexa dyes)36−39 via coordination of the fluorophore to Gd3+ or attached to fluorescent nanoparticles (e.g., liposomes, dendrimers, silica, and quantum dots).14,36,40−43 Such systems, however, become more and more complex and require advanced synthesis and partly expensive constituents (e.g., Alexa dyes and quantum dots). Because dissolved Gd3+ shows certain toxicity, it is also essential to guarantee a low concentration of freely dissolved Gd3+, either by strong coordination in the coordination complexes mentioned above or by the low solubility of the [GdO]+[ICG]− IOH-NPs (Figure 3c and Figure S3). To determine the utility of the [GdO]+[ICG]− IOH-NPs to serve as a MR contrast agent, we have first characterized the change in T1 relaxation time observed in response to increasing concentrations of IOH-NPs (Figure 5a). As expected, increasing the nanoparticle concentration resulted in a drastic decrease in T1 relaxation. The specific relaxivities (r1) per gadolinium at 7 T were 6.6 ± 0.4 and 8.0 ± 0.4 mM−1 s−1 for uncoated and dextran-coated nanoparticles, respectively, which are higher than that of conventional Gd-based MR contrast agents (3−5 mM−1 s−1). The additional decrease in the T1 relaxation time (increase in 1/T1) by the dextran coating is a commonly observed effect and most likely related to changes in the overall correlation time.44 In vivo, mice were imaged before and 5 h after intravenous injection of [GdO]+[ICG]− IOH-NP suspensions (Figure 5b). T1 relaxation heat maps show a noticeable decrease in relaxation time in the gall bladder and liver, demonstrating that the new [GdO]+[ICG]− IOH-NPs can serve as an in vivo MRI contrast agent with magnetic properties similar to or even better than those of the MRI standard contrast agent Gd-DTPA. In parallel with MRI, mice were imaged in vivo using fluorescence molecular tomography (FMT) and post mortem after the organs had been exposed using fluorescence reflectance imaging (FRI) (Figure 6). As seen for MRI, we observed an accumulation of the [GdO]+[ICG]− IOH-NPs in the liver and, later, excretion through the intestinal tract. To further characterize the biodistribution of the [GdO]+[ICG]− nanoparticles, organs were excised and imaged. The resulting fluorescence intensity and the fluorescence distribution were quantified (Figure 6a). In contrast to ICG solutions, optical
Figure 6. Biodistribution and clearance of [GdO]+[ICG]− IOH-NPs. (a) In vivo and post mortem fluorescence images of mice acquired 0, 5, and 24 h after [GdO]+[ICG]− injection using fluorescence molecular tomography (FMT) and fluorescence reflectance imaging (FRI). Organs were removed, and the fluorescence distribution was quantified for (b) dextran-coated [GdO]+[ICG]−.
imaging based on [GdO]+[ICG]− IOH-NP suspensions is possible over a time range of several hours. Along with the primary clearance through the liver, we also observed some fluorescence clearing through the kidneys (possibly representing the smaller or partially degraded nanoparticle fraction) and also an accumulation within the lungs (possibly representing particle agglomerates). To modulate both the clearance time and the distribution, dextran-coated [GdO]+[ICG]− IOH-NPs were applied and the changing organ intensity and relative organ distribution quantified (Figure 6b and Figure S4). Indeed, dextran-coated [GdO]+[ICG]− IOH-NPs show passaging through the liver and intestine as well as a decreased clearance time, as illustrated by the loss of fluorescence in the intestine from 5 to 24 h. Moreover, no notable accumulation in the lungs was observed (Figure 6b). Combined, these data demonstrate that [GdO]+[ICG]− IOH-NPs can serve as in vivo, multimodal contrast agents. 3552
DOI: 10.1021/acs.chemmater.6b05406 Chem. Mater. 2017, 29, 3547−3554
Article
Chemistry of Materials
■
Ag2Se Quantum Dots for Instant Efficient Labeling and Whole-Body High-Resolution Multimodal Real-Time Tracking of Cell-Derived Microvesicles. J. Am. Chem. Soc. 2016, 138, 1893−1903. (2) Nakamura, T.; Sugihara, F.; Matsushita, H.; Yoshioka, Y.; Mizukami, S.; Kikuchi, K. Mesoporous Silica Nanoparticles for 19F Magnetic Resonance Imaging, Fluorescence Imaging, and Drug Delivery. Chem. Sci. 2015, 6, 1986−1990. (3) Song, G.; Liang, C.; Gong, H.; Li, M.; Zheng, X.; Cheng, L.; Yang, K.; Jiang, X.; Liu, Z. Core-Shell MnSe@Bi2Se3 Fabricated via a Cation Exchange Method as Novel Nanotheranostics for Multimodal Imaging and Synergistic Thermoradiotherapy. Adv. Mater. 2015, 27, 6110−6117. (4) Doolittle, E.; Peiris, P. M.; Doron, G.; Goldberg, A.; Tucci, S.; Rao, S.; Shah, S.; Sylvestre, M.; Govender, P.; Turan, O.; Lee, Z.; Schiemann, W. P.; Karathanasis, E. Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis. ACS Nano 2015, 9, 8012−8021. (5) Nie, L.; Chen, X. Structural and Functional Photoacoustic Molecular Tomography aided by Emerging Contrast Agents. Chem. Soc. Rev. 2014, 43, 7132−7170. (6) Fujimoto, J. G.; Farkas, D. Biomedical Optical Imaging; Oxford University Press: Oxford, U.K., 2009. (7) Kunjachan, S.; Ehling, J.; Storm, G.; Kiessling, F.; Lammers, T. Noninvasive Imaging of Nanomedicines and Nanotheranostics: Principles, Progress, and Prospects. Chem. Rev. 2015, 115, 10907− 10937. (8) Shin, T.-H.; Choi, Y.; Kim, S.; Cheon, J. Recent Advances in Magnetic Nanoparticle-based Multi-Modal Imaging. Chem. Soc. Rev. 2015, 44, 4501−4516. (9) Weissleder, R.; Nahrendorf, M.; Pittet, M. J. Imaging Macrophages with Nanoparticles. Nat. Mater. 2014, 13, 125−138. (10) Cheng, Z.; Al Zaki, A.; Hui, J. Z.; Muzykantov, V. R.; Tsourkas, A. Multifunctional Nanoparticles: Cost versus Benefit of Adding Targeting and Imaging Capabilities. Science 2012, 338, 903−910. (11) Giraudeau, C.; Moussaron, A.; Stallivieri, A.; Mordon, S.; Frochot, C. Indocyanine Green: Photosensitizer or Chromophore? Still a Debate. Curr. Med. Chem. 2014, 21, 1871−1897. (12) Verwilst, P.; Park, S.; Yoon, B.; Kim, J. S. Recent Advances in Gd-Chelate Based Bimodal Optical/MRI Contrast Agents. Chem. Soc. Rev. 2015, 44, 1791−1806. (13) Cao, M.; Wang, P.; Kou, Y.; Wang, J.; Liu, J.; Li, Y.; Li, J.; Wang, L.; Chen, C. Gadolinium(III)-Chelated Silica Nanospheres Integrating Chemotherapy and Photothermal Therapy for Cancer Treatment and Magnetic Resonance Imaging. ACS Appl. Mater. Interfaces 2015, 7, 25014−25023. (14) Sharma, P.; Bengtsson, N. E.; Walter, G. A.; Sohn, H. B.; Zhou, G.; Iwakuma, N.; Zeng, H.; Grobmyer, S. R.; Scott, E. W.; Moudgil, B. M. Gadolinium-Doped Silica Nanoparticles Encapsulating Indocyanine Green for Near Infrared and Magnetic Resonance Imaging. Small 2012, 8, 2856−2868. (15) Liu, Y.; Kang, N.; Lv, J.; Zhou, Z.; Zhao, Q.; Ma, L.; Chen, Z.; Ren, L.; Nie, L. Deep Photoacoustic/Luminescence/Magnetic Resonance Multimodal Imaging in Living Subjects Using HighEfficiency Upconversion Nanocomposites. Adv. Mater. 2016, 28, 6411−6419. (16) Zeng, Y.; Zhang, D.; Wu, M.; Liu, Y.; Zhang, X.; Li, L.; Li, Z.; Han, X.; Wei, X.; Liu, X. Lipid-AuNPs@PDA Nanohybrid for MRI/ CT Imaging and Photothermal Therapy of Hepatocellular Carcinoma. ACS Appl. Mater. Interfaces 2014, 6, 14266−14277. (17) Mathew, S.; Murakami, T.; Nakatsuji, H.; Okamoto, H.; Morone, N.; Heuser, J. E.; Hashida, M.; Imahori, H. Exclusive Photothermal Heat Generation by a Gadolinium Bis(naphthalocyanine) Complex and Inclusion into Modified HighDensity Lipoprotein Nanocarriers for Therapeutic Applications. ACS Nano 2013, 7, 8908−8916. (18) Mbawuike, I. N.; Herscowitz, H. B. MH-S, A Murine Alveolar Macrophage Cell Line: Morphological, Cytochemical, and Functional Characteristics. J. Leukocyte Biol. 1989, 46, 119−127.
CONCLUSION Taken together, the sulfonate-based saline [GdO]+[ICG]− inorganic-organic hybrid nanoparticles (IOH-NPs) are a new multimodal contrast agent for OI, PAI, and MRI. Because of the high load of [ICG]− (81 wt %) and [GdO]+ (19 wt %), the [GdO]+[ICG]− IOH-NPs can be easily visualized in vitro (macrophages), in chicken breast and mouse phantoms, as well as in vivo (mouse model). In contrast to existing multimodal imaging agents, moreover, the [GdO]+[ICG]− IOH-NPs can be obtained via straightforward aqueous synthesis that directly yields colloidally stable suspensions of high concentrations (≤10 mg/mL). [GdO]+[ICG]− IOH-NPs combine clinically used constituents (i.e., gadolinium and indocyanine green) in a new sulfonate-based saline nanomaterial with low material complexity with significantly improved performance and applicability as compared to those of conventional ICG solutions and the standard contrast agent Gd-DTPA. In practice, this can be highly relevant for the visualization at different scales of resolution, the presentation of different types of tissue, and the translation from preclinical to clinical imaging and/or preoperational to intraoperative imaging. Besides tumor imaging, localization of inflammation centers or adenolymphitis may also become highly relevant.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.6b05406. Details regarding analytical tools, synthesis, and material characterization of the as-prepared [GdO]+[ICG]− IOHNPs (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Twan Lammers: 0000-0002-1090-6805 Claus Feldmann: 0000-0003-2426-9461 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors thank the Deutsche Forschungsgemeinschaft (DFG), the state of Schleswig-Holstein, and the European Union ERDF-European Regional Development Fund (MOIN CC, Zukunftsprogramm Wirtschaft) for financial support. M.P. is grateful to the Karlsruhe School of Optics and Photonics (KSOP) for a scholarship. We further thank K. Kötz for her expert technical support with MRI and O. Penate-Medina for helpful discussion. Animal studies and part of the in vitro studies were performed at the Molecular Imaging North Competence Center (MOIN CC) supported by a grant of the European Regional Development Fund (ERDF), the Zukunftsprogramm Wirtschaft of Schleswig-Holstein, and the DFGForschergruppe FOR 1586.
■
REFERENCES
(1) Zhao, J.-Y.; Chen, G.; Gu, Y.-P.; Cui, R.; Zhang, Z.-L.; Yu, Z.-L.; Tang, B.; Zhao, Y.-F.; Pang, D.-W. Ultrasmall Magnetically Engineered 3553
DOI: 10.1021/acs.chemmater.6b05406 Chem. Mater. 2017, 29, 3547−3554
Article
Chemistry of Materials (19) Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; Tinevez, J.-Y.; White, D. J.; Hartenstein, V.; Eliceiri, K.; Tomancak, P.; Cardona, A. Biological Imaging Software Tools. Nat. Methods 2012, 9, 676−710. (20) Heck, J. G.; Napp, J.; Simonato, S.; Möllmer, J.; Lange, M.; Reichardt, H. R.; Staudt, R.; Alves, F.; Feldmann, C. Multifunctional Phosphate-Based Inorganic-Organic Hybrid Nanoparticles. J. Am. Chem. Soc. 2015, 137, 7329−7336. (21) Roming, M.; Feldmann, C. U.S. Patent 12/867,348, 2011. (22) La Mer, V. K.; Dinegar, R. H. Theory, Production, and Mechanism of Formation of Monodispersed Hydrosols. J. Am. Chem. Soc. 1950, 72, 4847−4754. (23) Wegner, K. D.; Hildebrandt, N. Quantum Dots: Bright and Versatile in Vitro and in Vivo Fluorescence Imaging Biosensors. Chem. Soc. Rev. 2015, 44, 4792−4834. (24) Taichman, G. C.; Hendry, P. J.; Keon, W. J. The Use of CardioGreen for Intraoperative Visualization of the Coronary Circulation: Evaluation of Myocardial Toxicity. Texas Heart Institute Journal 1987, 14, 133−138. (25) Hua, R.; Liu, L.; Wang, X.; Chen, L. Imaging Evidence of Diabetic Choroidopathy in Vivo: Angiographic Pathoanatomy and Choroidal-Enhanced Depth Imaging. PLoS One 2013, 8, e83494. (26) Proulx, S. T.; Luciani, P.; Derzsi, S.; Rinderknecht, M.; Mumprecht, V.; Leroux, J.-C.; Detmar, M. Quantitative Imaging of Lymphatic Function with Liposomal Indocyanine Green. Cancer Res. 2010, 70, 7053−7062. (27) Ashokan, A.; Gowd, G. S.; Somasundaram, V. H.; Bhupathi, A.; Peethambaran, R.; Unni, A. K. K.; Palaniswamy, S.; Nair, S. V.; Koyakutty, M. Multifunctional Calcium Phosphate Nano-Contrast Agent for Combined Nuclear, Magnetic and Near-Infrared in Vivo Imaging. Biomaterials 2013, 34, 7143−7157. (28) Yoon, H. K.; Ray, A.; Koo Lee, Y. E.; Kim, G.; Wang, X.; Kopelman, R. Polymer-Protein Hydrogel Nanomatrix for Stabilization of Indocyanine Green towards Targeted Fluorescence and Photoacoustic Bio-imaging. J. Mater. Chem. B 2013, 1, 5611. (29) Morgan, T. T.; Muddana, H. S.; Altinoglu, I.; Rouse, S. M.; Tabakovic, A.; Tabouillot, T.; Russin, T. J.; Shanmugavelandy, S. S.; Butler, P. J.; Eklund, P. C.; Yun, J. K.; Kester, M.; Adair, J. H. Encapsulation of Organic Molecules in Calcium Phosphate Nanocomposite Particles for Intracellular Imaging and Drug Delivery. Nano Lett. 2008, 8, 4108−4115. (30) Chao, Y.; Karmali, P. P.; Mukthavaram, R.; Kesari, S.; Kouznetsova, V. L.; Tsigelny, I. F.; Simberg, D. Direct Recognition of Superparamagnetic Nanocrystals by Macrophage Scavenger Receptor SR-AI. ACS Nano 2013, 7, 4289−4298. (31) Bogart, L. K.; Taylor, A.; Cesbron, Y.; Murray, P.; Levy, R. Photothermal Microscopy of the Core of Dextran-Coated Iron Oxide Nanoparticles During Cell Uptake. ACS Nano 2012, 6, 5961−5971. (32) Hammer, Ø.; Harper, D. A. T. I; Ryan, P. D. PAST: Paleontological Statistics Software Package for Education and Data Analysis. Palaeontologia Electronica 2001, 4, 1−9. (33) Zhang, H. F.; Maslov, K.; Stoica, G.; Wang, L. V. Functional Photoacoustic Microscopy for High-Resolution and Noninvasive in Vivo Imaging. Nat. Biotechnol. 2006, 24, 848−851. (34) Hannah, A.; Luke, G.; Wilson, K.; Homan, K.; Emelianov, S. Indocyanine Green-Loaded Photoacoustic Nanodroplets: Dual Contrast Nanoconstructs for Enhanced Photoacoustic and Ultrasound Imaging. ACS Nano 2014, 8, 250−259. (35) de la Zerda, A.; Liu, Z.; Bodapati, S.; Teed, R.; Vaithilingam, S.; Khuri-Yakub, B. T.; Chen, X.; Dai, H.; Gambhir, S. S. Ultrahigh Sensitivity Carbon Nanotube Agents for Photoacoustic Molecular Imaging in Living Mice. Nano Lett. 2010, 10, 2168−2172. (36) Hüber, M. M.; Staubli, A. B.; Kustedjo, K.; Gray, M. H. B.; Shih, J.; Fraser, S. E.; Jacobs, R. E.; Meade, T. J. Fluorescently Detectable Magnetic Resonance Imaging Agents. Bioconjugate Chem. 1998, 9, 242−249. (37) Jo, M.-H.; Ali, B. A.; Al-Khedhairy, A. A.; Lee, C. H.; Kim, B.; Haam, S.; Huh, Y.-M.; Ko, H. Y.; Kim, S. A Reverse Complementary
Multimodal Imaging System to Visualize MicroRNA9-Involved Neurogenesis Using Peptide Targeting Transferrin Receptor-Conjugated Magnetic Fluorescence Nanoparticles. Biomaterials 2012, 33, 6456−6467. (38) Yan, H.; Wang, L.; Wang, J.; Weng, X.; Lei, H.; Wang, X.; Jiang, L.; Zhu, J.; Lu, W.; Wei, X.; Li, C. Two-Order Targeted Brain Tumor Imaging by Using an Optical/Paramagnetic Nanoprobe across the Blood Brain Barrier. ACS Nano 2012, 6, 410−420. (39) Lee, S. Y.; Jeon, S. I.; Jung, S.; Chung, I. J.; Ahn, C.-H. Targeted Multi-Modal Imaging Modalities. Adv. Drug Delivery Rev. 2014, 76, 60−78. (40) Vuu, K.; Xie, J.; McDonald, M. A.; Bernardo, M.; Hunter, F.; Zhang, Y.; Li, K.; Bednarski, M.; Guccione, S. Gadolinium-Rhodamine Nanoparticles for Cell Labeling and Tracking via Magnetic Resonance and Optical Imaging. Bioconjugate Chem. 2005, 16, 995−999. (41) Boswell, C. A.; Eck, P. K.; Regino, C. A. S.; Bernardo, M.; Wong, K. J.; Milenic, D. E.; Choyke, P. L.; Brechbiel, M. W. Synthesis, Characterization, and Biological Evaluation of Integrin αvβ3-Targeted PAMAM Dendrimers. Mol. Pharmaceutics 2008, 5, 527−539. (42) Liu, Y.; Ai, K.; Yuan, Q.; Lu, L. Fluorescence-Enhanced Gadolinium-Doped Zinc Oxide Quantum Dots for Magnetic Resonance and Fluorescence Imaging. Biomaterials 2011, 32, 1185− 1192. (43) Mulder, W. M.; Castermans, K.; van Beijnum, J.; Oude Egbrink, M. A.; Chin, P. K.; Fayad, Z.; Löwik, C. G. M.; Kaijzel, E.; Que, I.; Storm, G.; Strijkers, G.; Griffioen, A.; Nicolay, K. Molecular Imaging of Tumor Angiogenesis Using αvβ3-Integrin Targeted Multimodal Quantum Dots. Angiogenesis 2009, 12, 17−24. (44) Debroye, E.; Parac-Vogt, T. N. Towards Polymetallic Lanthanide Complexes as Dual Contrast Agents for Magnetic Resonance and Optical Imaging. Chem. Soc. Rev. 2014, 43, 8178−8192.
3554
DOI: 10.1021/acs.chemmater.6b05406 Chem. Mater. 2017, 29, 3547−3554
