Highly enhanced optical properties of indocyanine green/perfluorocarbon nanoemulsions for efficient lymph node mapping using near-infrared and magnetic resonance imaging
© Bae et al.; licensee Springer 2014
Received: 8 November 2013
Accepted: 20 December 2013
Published: 14 March 2014
The near-infrared (NIR) fluorescence probe has better tissue penetration and lower autofluorescence. Indocyanine green (ICG) is an NIR organic dye for extensive biological application, and it has been clinically approved for human medical imaging and diagnosis. However, application of this dye is limited by its numerous disadvantageous properties in aqueous solution, including its concentration-dependent aggregation, poor aqueous stability in vitro, and low quantum yield. Its use in molecular imaging probes is limited because it loses fluorescence after binding to nonspecific plasma proteins, leading to rapid elimination from the body with a half-life of 2 – 4 min. In this study, the multifunctional perfluorocarbon (PFC)/ICG nanoemulsions were investigated with the aim of overcoming these limitations. The PFC/ICG nanoemulsions as a new type of delivery vehicle for contrast agents have both NIR optical imaging and 19 F-MR imaging moieties. These nanoemulsions exhibited less aggregation, increased fluorescence intensity, long-term stability, and physicochemical stability against external light and temperature compared to free aqueous ICG. Also, the PFC/ICG bimodal nanoemulsions allow excellent detection of lymph nodes in vivo through NIR optical imaging and 19 F-MR imaging. This result showed the suitability of the proposed nanoemulsions for non-invasive lymph node mapping as they enable long-time detection of lymph nodes.
Medical imaging has experienced explosive growth over the past few decades and now plays a central role in noninvasive imaging technologies for biomedical research. Multimodal imaging will allow clinicians not only to see where a tumor is located in the body but also to visualize the expression and activity of specific molecules and biological processes that influence tumor behaviour and/or its response to therapy. The multimodal imaging probes with optical imaging dyes and magnetic resonance (MR) imaging contrast agents have been exploited for targeted molecular imaging, disease diagnosis, and in vivo animal studies [1, 2]. Targeted magnetic resonance imaging (MRI) has emerged as a promising diagnostic approach that offers a high-resolution depiction of pathological anatomy and the ability to detect associated disease biomarkers [3, 4]. Fluorophores have long been used as luminescence probes in various biological and biomedical applications [5–7].
Perfluorocarbons (PFCs) have been widely employed as 19 F-MR agents for imaging modalities due to their biological and chemical inertness. PFCs have been used clinically as blood substitutes because of their high gas dissolving capacity for oxygen and carbon-dioxide as well as their chemical and metabolic stability . The 19 F in the PFC nanoparticles have low background biological abundance and provide excellent signal sensitivity compared with 1H [9–11]. The encapsulation or conjugation of a wide variety of contrast agents onto PFCs for multimodal imaging and therapeutics, in combination with antibodies or other targeting ligands, causes them to accumulate in specific sites, holding great potential for medical applications. The PFC nanoemulsions have been widely explored for various applications, such as ultrasound imaging as a vehicle for targeted delivery of contranst agents and drug delivery , partial liquid lung ventilation , and blood substitutes [8, 14].
Indocyanine green (ICG) is a water-soluble tricarbocyanine dye with substantial absorption and fluorescence in the near-infrared region (NIR) [15, 16]. The United States Food and Drug Administration (FDA)  has approved ICG for use in diagnostic applications in clinical settings including its use as an optical contrast agent in the imaging of cardiac and hepatic vascular systems [18, 19], the retinal and choroidal vasculature , and lymphatic systems [21–23]. ICG has also been investigated in laser-mediated therapeutic applications. This dye can transform from the absorbed NIR light energy into free-oxygen species and heat, which further expands its therapeutic application to photothermal and photodynamic therapies [24–26]. Moreover, ICG, a NIR fluorescence probe, has several advantages over visible optical probes for in vivo imaging applications, including improved deep-tissue penetration, lower absorption and scattering by blood and tissue components, and minimal autofluorescence [18, 27]. However, ICG has several intrinsic limitations in optical imaging applications: 1) ICG is rapidly cleared from the circulatory system (half-life of 2-4 min) [28, 29]; 2) ICG forms aggregates depending on its concentration and interacts non-covalently with various proteins such as lipoproteins, plasma proteins, and human serum albumin via physical mechanisms due to its amphiphilic properties [30, 31]; 3) in aqueous solutions, ICG is unstable as the compound undergoes physicochemical transformations such as thermal degradation and photodegradation [28, 32]. Such changes result in discoloration, decreased light absorption, decreased fluorescence, and a shift in the wavelength of maximum absorption.
Recently, researchers have proposed nanomaterial-based ICG probes to overcome the high degradation rate and the short plasma half-life of ICG [33–35]. Polymeric nanoparticles and inorganic nanoparticles containing ICG could increase the stability of ICG and improve its physicochemical stability [34, 35]. Also, nanoparticles with a polymer core and lipid shell provided great targeting capability . Liposomes have been widely used as fluorescence probes for in vivo applications. In this study, we synthesized multifunctional PFC/ICG nanoemulsions as a new type of delivery vehicles for ICG to overcome the aforementioned limitations. The PFC/ICG nanoemulsions have both NIR optical imaging and 19 F-MR imaging moieties. We demonstrated the stability and fluorescence intensity of PFC/ICG nanoemulsions in vivo and in vitro and their physicochemical stability against exterior light and temperature. Also, we used multi-modal PFC/ICG nanoemulsions to indentify sentinel lymph nodes.
Perfluoro-15-crown ether (PFCE) was obtained from SynQuest Laboratories Inc. (Alachua, FL). L-a-phosphatidylcholine (Egg PC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] (DSPE-mPEG2000) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Indocyanine green, and cholesterol were obtained from Sigma-Aldrich Co. (St. Louis, MO).
Preparation of PFC/ICG nanoemulsions
To synthesize the PFC/ICG nanoemulsions, PFCE liquids were emulsified in an aqueous solution using a lipid mixture. The lipid compositions of the PFC/ICG nanoemulsions were PC/cholesterol/DSPE-mPEG2000 in a molar ratio of 70:20:10, respectively. The lipid mixture was reacted for 1 h at room temperature by the addition of 2 mg ICG, evaporated with a rotary evaporator to ensure the production of a thin lipid film, and dried in a vacuum oven (25°C) for 24 h. The lipid film was rehydrated with phosphate-buffered saline (PBS), and the resulting solution was sonicated in a bath sonicator followed by five cycles of freezing and thawing. The rehydrated lipid mixture (2% w/v) and PFCE solution (20% v/v) were mixed for 4 min using a homogenizer, followed by microfluidisation . A M-110S microfluidiser (Microfluidics Inc., Newton, MA) operating at a liquid pressure of approximately 20,000 psi was used for nanoemulsion preparations. The PFC/ICG nanoemulsions were stored at 4°C.
Characterization of PFC/ICG nanoemulsions
To evaluate the characteristics of the PFC/ICG nanoemulsions, a JEOL FE-TEM (transmission electron microscope) was utilized, and the TEM images were captured at 200 kV using a device from Tecnai. The PFC/ICG nanoemulsions were drop-cast onto carbon-coated TEM grids preliminarily stained with 2% uranyl acetate, and the solution was dried in a vacuum oven.
The emission and absorption spectra were obtained on a Perkin-Elmer LS-55 and a Beckman Coulter UV–VIS spectrophotometer (DU 800). The size of the PFC/ICG nanoemulsions was analyzed via dynamic light scattering using an electrophoretic light scattering photometer (ELS-Z, Otsuka Electronics, Osaka, Japan). The NIR fluorescence images of the PFC/ICG nanoemulsions were obtained using the IVIS Lumina imaging system (Caliper Life Science, MA) with an ICG filter set.
ICG and PFC loading efficiency
The ICG loading efficiency was analyzed using a previously reported method . The quantity of ICG loaded into the PFC/ICG nanoemulsions was determined from the free ICG that was not incorporated into the PFC/ICG nanoemulsions. A 1-mL sample of the PFC/ICG nanoemulsion was centrifuged, and the supernatant was removed and stored in a centrifuge tube; the PFC/ICG nanoemulsion was dispersed in a PBS solution. The centrifugation was repeated, and the collected supernatants were combined. The ICG concentration was quantified via UV–Vis spectroscopy. The quantity of ICG inside the PFC/ICG nanoemulsions was also measured to verify the accuracy of the method. Selected PFC/ICG nanoemulsion samples were treated with an HNO3 solution to induce capsule disassembly and to release the ICG into the solution. For all tested samples, the quantity of ICG released and the unencapsulated ICG equaled the quantity of the ICG precursor, indicating that the mass balance was conserved. The loading efficiency was calculated as the mass of ICG incorporated by the PFC/ICG nanoemulsions divided by the total ICG mass added to the nanoemulsion aggregate suspension.
To evaluate the PFC loading efficiency in PFC/ICG nanoemulsions, 19 F-MR imaging was performed on serial dilutions (0 – 0.4 ml) of PFCE liquids using a 4.7 T Bruker scanner (Biospec, Rheinstetten, Germany). The 19 F MR signal intensity was determined from the PFC signals originating from the PFCE liquids within a region of interest (ROI). We generated a calibration curve from the serial dilutions of the PFCE liquids and calculated the PFC loading efficiency in PFC/ICG nanoemulssions. The loading efficiency of PFC onto the PFC/ICG nanoemulsions was approximately 75.5 ± 3.2%.
Physicochemical stability of PFC/ICG nanoemulsions
Y denotes the y values of input curve, and Y’ is the normalized curve.
The HeLa (human cervical cancer cells) and Raw264.7 (Murine macrophage cells) cell lines were obtained from the American Type Culture Collection (Rockville, MD). These cell lines were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 IU/ml penicillin, and 50 μg/ml streptomycin. The cultures were maintained at 37°C/5% CO2 in tissue culture plates. The DC2.4 cells, previously characterized as an immature murine dendritic cell line, were obtained from Dr. Kenneth L. Rock (Dana-Farber Cancer Institute, Boston, MA) . This cell line was grown and maintained in DMEM supplemented with 10% heat-inactivated FBS, 50 IU/ml penicillin, and 50 μg/ml streptomycin.
Cell fluorescence imaging
To determine the intracellular delivery capacity of PFC/ICG nanoemulsions, the HeLa, Raw264.7, and DC2.4 cells were incubated with 10 μl/ml PFC/ICG nanoemulsions in μ-slide 8-well microscopy chamber at a density of 1 × 104 cells per well for 6 h at 37°C. The culture medium was then carefully aspirated, and the cells were washed three times. The labeled cells were fixed with 4% paraformaldehyde and stained with DAPI. The NIR fluorescence images were obtained on a Deltavision RT deconvolution microscope (Applied Precision Technologies, Issaquah, WA) using a filter set (excitation: 775/50, emission: 845/55; Omega Optical, Brattleboro, VT).
In vitro 19 F-MR and NIR fluorescence imaging
HeLa, Raw, or DC2.4 cells (1 × 106) were seeded on each well of a 6-well plate and grown for 24 h. The cells were then incubated with a medium containing 10 μl/ml PFC/ICG nanoemulsions. After 6 h, the medium was removed, and the cells were washed three times with PBS. The cell pellets were suspended with a 2% solution of low-melting agarose. The cells were collected in 0.2-mL tubes, and the MR and NIR fluorescence signals were measured. All 19 F-MR imaging of the PFC/ICG nanoemulsions was performed with a 4.7 T Bruker scanner using a double-tuned 1H/19F quadrature birdcage RF resonator. The 19 F-MR image was captured with a FLASH sequence (128 × 128 matrix; 30 × 30 mm2 FOV; 50 ms TR; 2.6 ms TE; 10 mm slice thickness; 256 NEX). The NIR fluorescence images were obtained using the IVIS Lumina imaging system (Caliper Life Science, MA) with an ICG filter set.
Cell cytotoxicity assays
The cell cytotoxicity was assessed using a modified 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Raw, HeLa, or DC2.4 cells were seeded in a 96-well plate (Corning Costar, Cambridge, MA) at 1 × 104 cells/well. After incubation for 24 or 48 h, several different concentrations of the prepared PFC/ICG nanoemulsions (0.38 ug/ul of ICG, 0.3 ul/ul of PFC) were poured into the wells. After incubation for a predetermined time, the residual nanoemulsions were removed, and a 2.5 mg/ml MTT solution was added to each well. The wells were then incubated in a humidified CO2 incubator at 37°C for 2 h. An acidified isopropanol/10% Triton X-100 solution (100 μl) was then added, and the plates were shaken to dissolve the formazan products. The absorbance was measured using a microplate reader at 570 nm. The cell survival rate in the control wells without the PFC/ICG nanoemulsions was considered 100% cell survival. The cytotoxic concentration (CC80) was defined as the concentration of the compound that reduced the absorbance of the control samples by 80%.
In vivo tracking of PFC/ICG nanoemulsions using NIR fluorescence and 19 F-MR imaging
Female hairless mice, 5–6 weeks of age, were purchased from SLC, Inc. (Japan). The mice were maintained at the KRIBB animal facility under pathogen-free conditions. All animal care and experimental procedures were approved by the Animal Care Committees of the KRIBB.
For the in vivo NIR fluorescence and 19 F-MR imaging of the sentinel lymph nodes, hairless mice were injected with 20 μl (25 μM of ICG, 15 ul/ml of PFC) of the PFC/ICG nanoemulsions (n = 5) or free ICG solutions (n = 5) in the footpad of the foreleg. Prior to the fluorescence imaging experiments, the mice were anesthetized with 200 μl of a 2.5% avertin solution (2, 2, 2-tribromoethanol-tert amyl alcohol, Sigma) throughout the experiments. After a predetermined time, the fluorescence intensity was quantitatively analyzed using the IVIS Lumina imaging system. Thereafter, the 19 F-MR images of the mice were obtained with a 4.7 T Bruker scanner using a double-tuned 1H/19F Birdcage coil design (inner diameter: 35 mm; length: 78 mm). After acquiring the morphological 1H images, the resonator was tuned to 19 F. For the 19 F-MR image, the mouse was imaged with a gradient echo sequence (128 × 128 matrix; 3 cm FOV; 56.0 ms TR; 2.6 ms TE; 20 mm slice thickness; 60° flip angle; 256 NEX; 30 min total scan time).
The statistical evaluations of the experiments were performed by ANOVA analysis followed by a Newman-Keuls multiple comparison test.
Results and discussion
Characteristics of PFC/ICG nanoemulsions
Summary of the physicochemical properties and cytotoxicity activity of the PFC/ICG nanoemulsions
Emission peak (nm)
Loading efficiency (%)
119.1 ± 25.1
95.1 ± 2.2
63.4 ± 15.6
20 ± 3.5
Physicochemical stability of PFC/ICG nanoemulsions
In vitro fluorescence and 19 F-MR imaging
Lymph node mapping
In summary, we encapsulated ICG molecules inside the PFC/ICG nanoemulsions as a novel bimodal imaging probe to allow for simultaneous 19 F-MR imaging and NIR optical imaging. The conjugation chemistry of ICG molecules is difficult due to their amphiphilicity and few functional groups. However, ICG molecules were encapsulated through a simple method to improve their properties. The ICG molecules protected the PFC/ICG nanoemulsions from aggregation and thus decreased the fluorescence self-quench. Therefore, these nanoemulsions could significantly increase the in vivo and in vitro stability and fluorescence intensity of ICG and improve its physicochemical stability against external light and temperature. Also, we used multi-modal PFC/ICG nanoemulsions to indentify the sentinel lymph nodes. Lymph nodes were detected by NIR optical imaging and 19 F-MR imaging. For sentinel lymph node biopsy, the incision procedure of sentinel nodes should be performed within short time (~30 min) because of the easy diffusion of free ICG and the decrease of fluorescence signal. The accuracy of sentinel lymph node biopsy depends upon the detection of sentinel nodes with high sensitivity and long-lasting vital dye. This result showed the suitability of the proposed nanoemulsions for noninvasive lymph node mapping as they enable long-time detection of lymph nodes. In the future, molecular probes in combination with various imaging modalities will provide more effective image-guided therapeutic tools for diagnostics, prognostics, and the treatment of diseases in diverse clinical settings.
Dr. Pan Kee Bae is currently a postdoctoral research associate at BioNanotechnology Research Center in KRIBB. He obtained his B.S. from ChungNam University in 2000 and Ph.D. from department of Biology, Yonsei University in 2006. His recent research is focused on the design of nanoparticles for bio-imaging against cancers and infectious disease.
Dr. Juyeon Jung is a senior research scientist in the BioNanotechnology Research Center in KRIBB. She obtained her B.S. degrees in Biochemistry from Michigan State University in 1998 and her Ph.D. from State University of New York at Stony Brook in 2004. After a postdoctoral study at Weill Medical College of Cornell University, she joined Elusys Therapeutics, Inc. in 2007 and then moved to KRIBB in 2009. Her current research focuses on the development of diagnostic/therapeutic antibodies and biointerfacing technologies for efficient biomolecules immobilization on solid surfaces.
Prof. Dr. Bong Hyun Chung received his PhD degree at Korea Advanced Institute of Science and Technology (KAIST) in 1987. He did his postdoctoral work at Caltech from 1990 to 1991. He is currently a director of the BioNanotechnology Research Center in KRIBB. He is also a professor of Nanobio Major at the University of Science and Technology (UST). His main research interests include (a) bio-nano interfacing science, (b) biochip/biosensor, (c) nanomedicine.
This research was supported by the Pioneer Research Center Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (Grant No.2011-0001952). This work was also supported by the KRIBB Initiative Research Program, Republic of Korea.
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