Development of a multimodal imaging probe by encapsulating iron oxide nanoparticles with functionalized amphiphiles for lymph node imaging

PET is a nuclear imaging modality that uses positron-emitting radiopharmaceuticals designed specific to target organs. It can demonstrate functional differences of organs at molecular levels and detect the molecular events noninvasively with high sensitivity and specificity. However, it cannot provide accurate anatomical information due to its low spatial resolution. Thus, the combination of computed tomography (CT) with PET to construct a PET/CT dual-modality imaging system could greatly improve the resolution of diagnostic images for obtaining detailed anatomical information.

MRI is a powerful medical imaging technique to provide excellent contrast between soft tissues, and enables us to obtain more detailed anatomical information than CT. Because MRI uses radiofrequencies instead of x-rays for image acquisition, it can also avoid the risks arising from radiation exposure which is unavoidable in CT. A combined system of PET/MRI emerged about a decade ago for providing better diagnostic information, and this technique has been improved with significant technological advancement ever since [1,2]. Recently, the number of studies about the preclinical usage of PET/MRI system has been increased dramatically [3–11]. However, most of the PET/MRI systems relying on radiopharmaceuticals such as 18 fluorodeoxyglucose ([¹⁸F] FDG) are suitable for PET imaging only but not for MRI. Therefore, it is necessary to develop multimodal imaging agents, which are capable of simultaneously providing both PET and MRI information, for expanding the applicability of PET/MRI dual modality imaging system.

Nanoparticles (NPs) have been actively investigated for use in medical realms owing to the numerous advantages. NPs are nanometer sized particles and some of them can be used for radioisotope delivery in living organisms. In particular, iron oxide (IO) NPs have been applied as an MRI contrast agent in clinical trials owing to their desired super paramagnetism and low toxicity [12–15]. The use of IO-based NPs made of biocompatible iron might be safer than the use of other types of NPs, for example, quantum dots made of binary alloys such as cadmium selenide or cadmium sulfide containing toxic heavy metal cadmium. The core of IO NPs is composed of iron and oxygen atoms (mostly magnetite, Fe₃O₄; or magnemite, γ-Fe₂O₃), which can be utilized for hemoglobin synthesis. Both superparamagnetic iron oxide and ultrasmall superparamagnetic iron oxide NPs are suspended colloids of IO NPs, and their application as T2 signal contract agents for MRI have been extensively investigated [16–21].

On the basis of the previous studies, we decided to label the magnetic IO NPs with ⁶⁸Ga (T_1/2 = 68 min), which is one of the most promising positron emitters due to the availability of the convenient ⁶⁸Ge/⁶⁸Ga-generator system [22–24], to develop a novel and potent PET/MRI multimodal imaging probe. To label IO NPs with ⁶⁸Ga, an effective bifunctional chelating agent such as 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) should also be introduced to the IO NPs [25–27].

The aim of the present study is to develop a specific PET/MRI dual-imaging probe for targeting lymph nodes. A facile encapsulation method based on the use of specific amphiphiles was adopted for the modification of the surfaces of IO NPs [28]. Moreover, polysorbate 60 was used to solubilize IO NPs. In addition, S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid conjugated with stearylamine (NOTA-SA) and α-D-mannopyranosylphenyl isothiocyanate conjugated with stearylamine (Man-SA) were used as specific amphiphiles for ⁶⁸Ga labeling and macrophage targeting, respectively (Figure 1).

Materials & methods

General

Magnetic iron oxide nanoparticles (Fe₃O₄) in nonpolar solvent (chloroform, d = 5 nm) were purchased from MKnano (MK Impex Corp., ON, Canada) [22]. All other reagents and solvents were purchased from Sigma-Aldrich (MO, USA). 1H-nuclear magnetic resonance spectroscopy was performed at 600 MHz on a Bruker Avance-600 spectrometer (Bruker Corporation, MA, USA). A Waters 3100 liquid chromatography-mass spectroscope (LC–MS, MA, USA) and a FC203B fraction collector (Gilson, Inc., WI, USA) were used for the purification of as-prepared nanoparticles. The hydrodynamic diameter and size distribution of nanoparticles were analyzed by a dynamic light scattering (DLS) system Zetasizer Nano ZS90 (Malvern Instruments Ltd, Worcestershire, UK) and JEM-1010 transmission electron microscope (TEM; JEOL, Tokyo, Japan). Furthermore, a Varian 820-MS was used for inductively coupled-mass spectrometry (Varian, Inc., CA, USA) analysis. All spectra were recorded with a Sinco S-3100 UV/Vis spectrometer (SCINCO America, WI, USA). The ⁶⁸Ge-⁶⁸Ga generator was purchased from ITG GmbH (Munich, Germany). Instant thin layer chromatography-silica gel (ITLC-SG) plates were obtained from Agilent Technologies, Inc. (CA, USA). In addition, radio-thin layer chromatography (radio TLC) was performed with a Bio-Scan AR-2000 System imaging scanner (Bioscan, WI, USA). Radioactivity was measured by a gamma scintillation counter (Packard Cobra II, GMI, NM, USA). Cell counting kit-8 CK04 was used for cell viability assay (Dojindo Laboratories, Kumamoto, Japan). PET imaging was carried out on a Vista micro PET/CT scanner (eXplore Vista PET/CT, GE Healthcare, CT, USA), and MR imaging was performed for both phantom and animal studies on a 3-T MR scanner (MAGNETOM Trio, Siemens Healthcare, Erlangen, Germany). Both Olympus BX-51 light microscope (Olympus, PA, USA) and Leica DFC280 digital camera (Leica Microsystems CMS GmbH, Wetzlar, Germany) were used for image acquisition.

All animal experiments were performed according to the related guidelines in Seoul National University Hospital, Seoul, South Korea, which was fully accredited by Association for Assessment and Accreditation of Laboratory Animal Care International (2007).

Synthesis of NOTA-stearylamine

Triethylamine (TEA) (0.112 ml, 0.804 mmol, 3.0 eq) was added into the solution of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) (150 mg, 0.268 mmol, 1.0 eq) dissolved in 2 ml of CHCl₃ with stirring agitation at room temperature for 10 min. Afterwards, stearylamine (145 mg, 0.536 mmol, 2.0 eq) was added into the reaction mixture, which was stirred at room temperature overnight and concentrated on a rotary vacuum evaporator at 40°C. The resulting oil was purified by silica gel (∼20 g) column chromatography using a mixture of MeCl₂ and MeOH (90:10; Rf = 0.1) as eluent. Mass spectrum (ESI⁺), (M⁺H⁺): 721.8. HRMS (M⁺H⁺): observed 720.4731, calculated 720.4734.

Synthesis of mannose-stearylamine

TEA (0.133 ml, 0.957 mmol, 3.0 eq) was added into the solution of α-d-mannopyranosylphenyl isothiocyanate (100 mg, 0.319 mmol, 1.0 eq) dissolved in 2 ml of CHCl₃ with stirring agitation until the mixture became clear. Stearylamine (172 mg, 0.638 mmol, 2.0 eq) was then added into the reaction mixture, which was stirred at room temperature overnight and concentrated on the rotary vacuum evaporator at 40°C. The resulting organic layer was purified by silica gel (∼20 g) column chromatography using the mixture of MeCl₂ and MeOH (90:10; Rf = 0.1) as eluent. Mass spectrum (ESI⁺), (M⁺H⁺): 583.4. HRMS (M⁺H⁺): observed 583.3785, calculated 583.3781.

Preparation of nonspecific IO NPs by encapsulation with polysorbate 60

Polysorbate 60 solutions at concentrations of 1–10% (v/v) in distilled water were prepared and sonicated for 30 min. Each 10 μl of 5 nm IO NPs solution (5 mg/ml in CHCl₃) was added into a 4-ml glass vial, which was flushed with nitrogen gas to evaporate CHCl₃. Afterwards, 1 ml of the prepared polysorbate 60 solution was added into each vial containing IO NPs followed by sonication for 3 h. The reaction mixtures were centrifuged at 15,000 rpm at 25°C for 5 min to separate the supernatants and pellets. Both supernatants and pellets were analyzed by inductively coupled plasma-mass spectrometer (ICP–MS) to determine the concentration of ferric ion (Fe³⁺).

Preparation of IO NPs with NOTA & mannose residues

Encapsulation of IO NPs with specific amphiphiles was performed by the literature method with a minor modification [28]. Briefly, 0.94 ml of 1 mg/ml NOTA-SA in CHCl₃ (equivalent to 2 mol% of polysorbate 60) and 1.95 ml of 1 mg/ml Man-SA in the mixture of CHCl₃ and MeOH (1:1; equivalent to 5 mol% of polysorbate 60) were added into a 4-ml glass vial, followed by evaporation of all organic solvents using a rotary vacuum evaporator. One milliliter of 8% (v/v) polysorbate 60 was added into the vial, which was sonicated for 30 min to generate the micelle-based reaction mixture. After that, 200 μl of presonicated 5 mg/ml IO NPs in CHCl₃ was added into the reaction mixture during sonication, which continued for another 30 min with heating at 80°C to evaporate CHCl₃. The residual CHCl₃ was eliminated by using the rotary vacuum evaporator until the reaction solution became clear, and the solution was then ultrasonicated for 3 h (amplitude = 70%; cycle = 177.8 W). The reaction mixture was purified by gel-filtration chromatography on a Sephacryl^® S500-HR column (14.5 × 150 mm; V0 = 2.37 ml) equipped with a fraction collector (0.5 ml × 80) with distilled water used as eluent. The collected fractions containing NOTA-IO-Man were concentrated by using an Amicon Ultra-0.5 ultra-filtration device (100 kDa cutoff) at 5000 × g at 25°C for 5 min.

Size analysis

The hydrodynamic diameter and size distribution of encapsulated IO NPs were measured by a DLS instrument. The concentrated IO NPs solutions were diluted 100 times (2 μM) with distilled water and homogeneously dispersed by sonication for 1 min. The particle size and distribution were measured as number percent (%) values at a scattering angle of 90° at 25°C. The morphology and size of IO NPs were characterized by TEM analysis, where the final product was dropped onto a TEM grid, and the images were captured at an accelerating voltage of 80 keV.

⁶⁸Ga labeling

50 μl of encapsulated IO NPs solution (0.200 mM) was added into 100 μl of 1 M sodium acetate buffer (pH 5.6), followed by the addition of 500 μl of ⁶⁸GaCl3 in 0.05 M HCl. The mixture was vortexed at 50°C for 10 min to allow the reaction to proceed. The radiochemical purity was measured with a radio-TLC system using 10 cm ITLC-SG eluted with 0.1 M citric acid. After ⁶⁸Ga labeling, 0.5 ml of reaction mixture was concentrated using ultrafiltration (Amicon Ultra-0.5, 100 kDa, 5000 g, 25°C, 5 min). To the 0.1 ml of concentrated solution, 0.4 ml of distilled water was added, vortexed and concentrated by the same procedure. This procedure was repeated 3 times.

Stability test

Moreover, we examined the stability of IO NPs encapsulated with functionalized amphiphiles in distilled water and in NaCl solutions. Briefly, 100 μl of encapsulated IO NPs were mixed with 1 ml of 0.9, 1.8 and 3.6% NaCl solutions. The mixtures were incubated at room temperature for 1, 3 and 24 h, followed by the measurement of the size distribution of IO NPs using the DLS system. Furthermore, the stability of ⁶⁸Ga-labeled IO NPs in human serum was also investigated. An amount of 100 μl of ⁶⁸Ga-labeled IO NPs solution was added into 1 ml of human serum, followed by incubation in a shaking incubator at 37°C for 1 h. Afterwards, 500 μl of the incubated sample was loaded onto the Sephacryl S-500 HR gel filtration column (14.5 × 200 mm) equipped with a fraction collector and eluted with distilled water. Eighty 0.5-ml fractions were collected, and the radioactivity of each fraction was measured with a gamma scintillation counter. In addition, radio-TLC analysis of the ⁶⁸Ga-labeled IO NPs was performed using 10 cm ITLC-SG eluted with 0.1 M citric acid after incubation in human serum for 0, 10, 30, 60 and 120 min at 37°C.

Phantom imaging study

The encapsulated IO NPs and ferumoxide (Feridex IV; Advanced Magnetics, MA, USA) at the concentration of 0.2 mM were diluted serially in an agarose phantom designed for T2 relaxivity measurement by the 3-T MR scanner. Fast spin-echo MR images of the phantom were acquired by using the following parameters: relaxation time = 5000 ms, echo time = 16, 32, 48, 64, 20, 40, 60, 80, 50 or 100 ms, flip angle = 180°, ETL = 18 fields of view, FOV = 77 × 110 mm², matrix = 256 × 117, slice thickness/gap = 1.4 mm/1.8 mm, and NEX = 1.

Cell viability assay

Cell viability was assayed by a colorimetric procedure with Cell Counting Kit-8 (CCK-8) system. HeLa (a human epithelial carcinoma cell line), MDA-MB-231 (a human breast adenocarcinoma epithelial cell line), RAW 264.7 (a mouse macrophage cell line) were incubated at 37°C in 5% CO₂ humidified incubator. Individual wells of 96-well microculture plate were filled with 800 cells, 200 μl culture media containing 0.1–0.8 mg/ml of NOTA-IO-Man for 1, 24, 48, 72 h. Plates were incubated at 37°C in 5% CO₂ humidified incubator. Subsequently, culture media were carefully removed and 200 μl of DPBS was added for washing. CCK-8 solution (10 μl per 100 μl of culture medium) was added to each well of the plate. The plate was incubated for 2 h in the incubator. The absorbance of each well was measured at 450 nm using a microplate reader.

In vivo imaging

The ⁶⁸Ga-labeled IO NPs were concentrated to 12.02 MBq/10 μl by using the Amicon Ultra-0.5 ultrafiltration device under the conditions described in the Methods section. Eight-week-old male Sprague–Dawley^® rats (average bodyweight: 303.16 g) were anesthetized with isoflurane/O₂ (2:1) before PET/CT analysis. An amount of 10 μl of ⁶⁸Ga-labeled encapsulated IO NPs (12.02 MBq) was subcutaneously injected into the right footpad of the rat using a Hamilton syringe. The dynamic PET/CT images were obtained in list mode format for 60 min. After PET/CT scanning, 100 μl of 30% Zoletil:Rompun solution (4:1) in normal saline, used as anesthetics, was intramuscularly injected into the right thigh of each rat. T2-weighted MR images were then acquired to locate the particle uptake in lymph nodes using the following parameters for a spin-echo multislice sequence: TR = 51 ms, TE = 20 ms and slice thickness = 0.6 mm.

Ex vivo study

Nuclear-fast red solution was prepared by dissolving 0.1 g of nuclear-fast red and 5 g of aluminum sulfate in 100 ml of distilled water. The mixture was slightly heated for complete dye dissolution and filtered through a 0.2-μm syringe filter.

The animals were sacrificed via CO₂ inhalation after in vivo imaging. Both left and right popliteal lymph nodes were isolated, embedded in paraffin and dissected into 4-μm thick sections, which were loaded onto glass coverslips. For Prussian blue staining, the tissue sections were deparaffinized and soaked with a 1:1 mixture of 20% HCl and 10% K₃Fe(CN)6, as a working solution, for 90 min in black plastic cases, and then rinsed with distilled water for 10 min. For Nuclear-fast red staining, the samples were dipped into Nuclear-fast red solution for 15 min and washed with tap water for 10 min. The prepared specimens were dehydrated and mounted onto glass coverslips for further inspections.

Results

Encapsulation of IO NPs with polysorbate 60

IO NPs were encapsulated with polysorbate 60 in aqueous solutions, and the precipitates were removed by centrifugation of the reaction mixtures. The encapsulated IO NPs solutions were obtained as brown transparent liquids. The precipitates were formed at the polysorbate 60 concentration of lower than 8%. Thus, we decided to use 8% polysorbate 60 to prepare the encapsulated IO NPs for all subsequent experiments.

Preparation of NOTA-IO-Man by encapsulation with specific amphiphiles

IO NPs were encapsulated with 8% polysorbate 60, NOTA-SA, and Man-SA by following the protocols described in the Methods section. The hydrodynamic diameter of NOTA-IO-Man increased to 10.12 ± 1.46 nm, as compared with the unencapsulated IO NPs, determined by the DLS data (Figure 2A). The zeta-potential was measured to be -2.87 ± 4.47 mV, and the polydispersity index was 0.611. Furthermore, TEM imaging confirmed the homogeneous dispersion of NOTA-IO-Man with a narrow particle size distribution (Figure 2B).

⁶⁸Ga labeling of NOTA-IO-Man

NOTA-IO-Man was labeled with ⁶⁸Ga in high radiochemical yield (>95%), and separation of ⁶⁸Ga-NOTA-IO-Man from unincorporated ⁶⁸Ga was achieved easily. When ITLC-SG plate was eluted with 0.1 M citric acid, the free ⁶⁸Ga moved along with solvent front (Rf = 1.0), while ⁶⁸Ga-labeled NOTA-IO-Man remained at the origin (Rf = 0).

Stability test of NOTA-IO-Man in NaCl solutions

The stability of NOTA-IO-Man in various concentrations of NaCl solutions at room temperature was determined by measuring the hydrodynamic size distributions after incubation for 0, 1, 3 and 24 h using the DLS system (Table 1). NOTA-IO-Man did not show any particle size change after incubation in NaCl solution at the concentration up to 3.27%, which was >3.6-times higher than that of normal physiological saline, indicating the absence of particle aggregation or decomposition under this condition (p < 0.05) (Figure 3).

Stability test of ⁶⁸Ga-NOTA-IO-Man in human serum

The stability of ⁶⁸Ga-NOTA-IO-Man in human serum was tested by gel filtration at 37°C. The resultant elution profile was similar to that of control, indicating the absence of either particle aggregation or decomposition under this condition (Figure 4A). This result also suggests that ⁶⁸Ga-NOTA-IO-Man is stable in human serum. Furthermore, radio-TLC study also confirmed that ⁶⁸Ga-NOTA-IO-Man was stable in human serum at 37°C for 2 h (Figure 4B).

T2 relaxivity of the particle

Phantom study was performed to determine the T2 relaxivity of the NOTA-IO-Man, which was compared with that of ferumoxide. NOTA-IO-Man showed a lower T2-weighted signal intensity than ferumoxide at the same iron concentration (Figure 5). The T2 relaxivity (r2) of NOTA-IO-Man was measured to be 449.9 mM-1s-1, which was higher than that of ferumoxide (180.38 mM-1s-1).

Cell viability assay

Cell viabilities were tested with NOTA-IO-Man for 1, 24, 58, 72 h using the CCK-8 assay method. NOTA-IO-Man did not cause cellular toxicity in RAW 264.7, HeLa and MDA-MB-231 cell lines (Figure 6).

In vivo study of ⁶⁸Ga-NOTA-IO-Man

The in vivo studies of ⁶⁸Ga-NOTA-IO-Man were performed by conducting PET/CT and MR scans. After the injection of ⁶⁸Ga-NOTA-IO-Man into the footpad of the rat, the popliteal lymph node showed pronounced brightness under PET scan (Figure 7A). This result suggests that ⁶⁸Ga-NOTA-IO-Man could be drawn from the intercellular space toward lymphatic system and could be specifically taken up by macrophages in the lymph node without aggregation in the in vivo system. If ⁶⁸Ga-NOTA-IO-Man was aggregated, it could not move from the intercellular space into lymphatic system [29]. On MR images obtained for the same animal immediately after PET/CT scanning, a dark signal intensity was demonstrated at the same popliteal lymph node (Figure 7B). When compared with MR images before the injection of IO NPs, the decreased signal at the popliteal lymph node after injection represents the successful uptake of ⁶⁸Ga-NOTA-IO-Man at the target.

Ex vivo study of 68Ga-NOTA-IO-Man

Microscopic examination of the histopathology of the popliteal lymph nodes was performed after the staining with Prussian blue and Nuclear-fast red. The tissue section of the left popliteal lymph node showed a distinctive blue pigment, indicating the existence of ferric iron (Fe³⁺) (Figure 8A). Whereas, no staining was observed in the right popliteal lymph node (Figure 8B), suggesting the presence of ⁶⁸Ga-NOTA-IO-Man in the left lymph node only.

Discussion

Medical imaging of sentinel lymph nodes (SLNs) has shown great clinical significance in disease diagnosis. In particular, the detection and mapping of SLNs of melanoma and breast cancer during surgical operation is of critical importance for determining the dissection range [30–32]. A mannose receptor is an immune receptor that participates in the endocytosis of glycoproteins through macrophages. Thus, the presence of mannose receptors has been generally used as an indicator of SLN location, which contributes to the detection of metastatic cancer cells in the SLNs [33–35]. On the basis of these results, the medical imaging of lymph nodes offers an effective way for the accurate diagnosis of cancer at early stages.

NPs, which are present in various shapes such as rods, stars, spheres, cylindroids, sheets and cubes, have demonstrated great potentials for multimodal and multispecific medical imaging. The circulation of NPs in vivo requires adequate time for particles to accumulate in the targeted organ. In addition, the surface modification of NPs with targeting molecules or imaging ligands can result in high-capacity functional NPs, owing to their large surface-to-volume ratio.

However, there are also several problems of NPs in medical applications remaining unresolved. For example, the uncontrollable aggregation of powder-form NPs due to van der Waals’ force attraction is a serious problem for managing the distribution of NPs. In addition, NPs are highly reactive and catalytic, because of their high surface-to-volume ratio, leading to the high potential to aggregate under both in vitro and in vivo conditions. Furthermore, to prevent particle degradation under physiological conditions such as pH variation, body temperature, and enzymatic reaction is also an important aspect of their biological applications. Thus, surface coating of NPs is necessary to improve their stability and solubility under those harsh conditions. Unfunctionalized NPs could be accumulated in tumor tissues in a nonspecific passive way by enhanced permeability and retention effect [36,37]. Hence, to prevent nonspecific uptake of NPs and to achieve specific diagnosis, the development of functionalized NPs is required for effectively targeting the diseased site.

Surface modification is one of the most important steps of NPs functionalization for medical imaging applications. Several different types of ligands should be conjugated to the surface of NPs to induce multimodality and multispecificity, and this process usually requires multiple steps of chemical reactions by using conventional methods. The reactions are normally performed serially. Thus, the overall yield would be largely decreased because of the accumulation of the yield loss incurred in each step. Furthermore, the reaction mixture has to be concentrated at the end of every step to reduce the volume for reaction in the next step. A significant amount of NPs will be therefore aggregated and lost because of the repeated concentration process, resulting in an extremely low production yield.

The aim of this study was to encapsulate IO NPs with specially designed functional amphiphiles to modify the particle surfaces, enhance their solubility and improve the production yield, which cannot be easily achieved by using the conventional method [28]. Polysorbate 60 is a nonionic detergent and a mixture of branched polyethylene glycol (PEG) conjugates of mixed partial stearic (47.0–55.0%) and palmitic acid (35.0–50.0%). It was used as a surfactant and a functional amphiphile in this study due to its effect of ‘stealth’ from immune system in vivo. PEG is a favorable material for the surface modification of NPs to improve the particle solubility and stability [38–41]. Due to its water-soluble property and easy coupling with the hydrophobic surface of NPs to enhance surface hydrophilicity, the US FDA approved the use of polysorbate 60 as a food additive with an acceptable daily intake of 0–25 mg/kg bodyweight per day at the 17th meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1973.

Advanced studies using polysorbate 60 for encapsulating quantum dots have been already reported [28]. In the present study, the polysorbate 60 solution at a slightly higher concentration (8%) was used for encapsulating IO NPs.

NOTA-SA and Man-SA were used as specific amphiphiles for 68Ga-labeling and for targeting lymph nodes, respectively. They are mixed with 8% polysorbate 60 solution to encapsulate IO NPs. The encapsulation mixture was purified twice on a Sephacryl S-500 HR size-exclusion column. The micelles with the IO NPs were completely eliminated by this gel filtration method.

The radiolabeling yield (%) of IO NPs was high (>95%). No significant particle aggregation, degradation or size change was found under various harsh conditions. The radiochemical purity of prepared ⁶⁸Ga-NOTA-IO-Man was very high (>99%) after purification and was stable in human serum for 120 min. The T2 relaxivity (r₂) of ⁶⁸Ga-NOTA-IO-Man (449.9 mM⁻¹s⁻¹) was higher than that of ferumoxide (180.38 mM⁻¹s⁻¹), which can provide a better delineation of lesions at lymph node in MR imaging. The specificity of ⁶⁸Ga-NOTA-IO-Man was confirmed showing both PET/CT and MRI signal at the same site. The tissue section of the left popliteal lymph node of the rat after injection showed a distinctive blue pigment confirming the existence of ferric iron in the lymph node. In the aspect of dual modality imaging, the simultaneous detection of ⁶⁸Ga-NOTA-IO-Man at the same site on both PET/CT and MRI can enhance the specificity of the imaging probes.

Conclusion

In this study, we prepared a multimodal IO NPs containing PEG, NOTA and mannose residues at the surface by one-pot encapsulation method with polysorbate 60 and specific amphiphiles. We confirmed that the prepared NPs were stable and multifunctional under various physiological conditions both in vitro testing and in vivo imaging. This technology has a huge potential, because it can be applied not only for lymph node imaging but also for various cancer targeting.

Future perspective

This encapsulation method can be applied for developing more specific cancer targeting multimodality imaging agents such as angiogenesis, prostate specific membrane antigen, folic acid receptor and CXCR4 receptor. For example, we already reported PET/optical dual modal angiogenesis imaging agent, and a preliminary result of prostate specific membrane antigen targeting PET/MRI dual modal imaging agent developed by this method. Furthermore, development of PET/MRI/optical multimodal imaging agents specific for cancer would also be possible. Radiolabeling with therapeutic radionuclides such as 90Y and 177Lu can be tried for theranostics because the NPs have longer retention in the body which would be helpful to deliver enough radiation dose to the cancer. Thus, numerous clinical application fields would be open if this method is readily applied for developing multimodal agents.