Long-Circulating Iodinated AlbuminâGadolinium Nanoparticles as

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Long-circulating Iodinated Albumin-Gadolinium Nanoparticles as Enhanced Magnetic Resonance and Computed Tomography Imaging Probes for Osteosarcoma Visualization Qianliang Wang, Ling Lv, Zhuoyan Ling, Yangyun Wang, Yujing Liu, Guodong Liu, Liubing Li, Liqin Shen, Jun Yan, and Yong Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504752a • Publication Date (Web): 27 Mar 2015 Downloaded from http://pubs.acs.org on April 2, 2015

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Analytical Chemistry

Long-Circulating Iodinated Albumin-Gadolinium Nanoparticles as Enhanced Magnetic Resonance and Computed Tomography Imaging Probes for Osteosarcoma Visualization Qianliang Wang†,#, Ling Lv†,#, Zhuoyan Ling†,#, Yangyun Wang‡,ǁ, Yujing Liu†, Liubing Li†, Guodong Liu†, Liqin Shen†, Jun Yan†,*, Yong Wang‡,ǁ,* † The Second Affiliated Hospital of Soochow University, 1055 Sanxiang Road, Suzhou, 215004, China ‡ Center for Molecular Imaging and Nuclear Medicine, School for Radiological and Interdisciplinary Sciences (RAD-X), Soochow University, 199 Renai Road, Suzhou Industrial Park, 215123, China ǁ Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, 199 Renai Road, Suzhou Industrial Park, 215123, China KEYWORDS magnetic resonance, computed tomography, osteosarcoma

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ABSTRACT Multimodal imaging probes represent an extraordinary tool for accurate diagnosis of diseases due to the complementary advantages of multiple imaging modalities. The purpose of the work was to fabricate a simple dual-modality MR/CT probe for osteosarcoma visualization in vivo. Protein-directed synthesis methods offer a suitable alternative to MR/CT probe produced by synthetic chemistry. Bovine serum albumin (BSA) bound to gadolinium nanoparticles (GdNPs) was first prepared via a biomimetic synthesis method, and subsequently iodinated by chloramine-T method. The final iodinated BSA-GdNPs (I-BSA-GdNPs) showed excellent chemical stability and biocompatibility, intense X-ray attenuation coefficient, and good MR imaging ability. However, an iodinated protein nanoparticles synthesis for MR/CT imaging, as well as its useful application, has not been reported yet. Intravenous injection of I-BSA-GdNPs into orthotopic osteosarcoma-bearing rats led to its accumulation and retention by the tumor, allowing for a noninvasive tumor dual-modality imaging through the intact thigh. The longcirculating dual-model I-BSA-GdNPs probes possess potential application for image-guided drug delivery and image-guided surgery. Our study is therefore highlighting the properties of albumin in this field combined with its useful use in dual-model MR/CT osteosarcoma visualization, underlining its potential use as a drug carrier for a future therapy on cancer.


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Osteosarcoma is a malignant tumor of the bone that primarily affects adolescents between 10 and 25 years old.[1] Despite the effective surgical resection of the primary tumor and consequent chemotherapy, the rate of long-term survival is only 15% to 20% because of pulmonary metastases, unresponsiveness to therapy, or disease relapse.[2] Therefore, early detection and differentiation of osteosarcoma from osteoid osteoma, aneurysmal bone cyst, infectious or inflammatory processes, would be advantageous for a better and appropriated management of this malignant disease.[3] Imaging techniques play an important role on improving human health through an early detection of several diseases including cancer, with their ability to locate tumors, assess the tumor activity, as well as providing an useful guide to perform the best surgery or radiotherapy.[4] However, among all the single modality imaging techniques available, none of them is suitable and appropriate to gain all the required information to adequately face the disease.[5] Magnetic resonance (MR) is a powerful tool for the soft tissues diffusion-weighted imaging, but unenhanced MR contrast imaging could be hard to discriminate tumor and the surrounding healthy tissue due to the low contrast sensitivity.[6] Computed tomography (CT) can form 3D visual reconstruction of the tissues of interest as a result of the high resolution, but its intrinsic low sensitivity causes poor contrast among soft tissues.[7] The combination of enhanced MR and CT imaging might therefore offer synergistic advantages over any other single modality for soft tissues imaging.[8] However, combining two different types of imaging probes is not enough to enhance the contrast imaging, unless these probes possess identical pharmacological properties.[9] Therefore, the same multimodal probe should be able to overcome this obstacle when used for different imaging techniques.


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Nanoprobes are a class of representative nanoparticles extensively applied in medical and biological diagnostics that are emerging as potential contrast agents.[10] Up to now, MR/CT multimodal nanoprobes have been synthesized and utilized for imaging in vivo.[11-16] For example, gadolinium-chelated gold nanoparticles pioneered the development of MR/CT multimodal nanoprobes.[11] Extraordinarily, Hyeon’s group reported a Fe3O4/TaOx core/shell nanoparticles as MR and CT probes can provide complementary information for tumorassociated blood vessels and tumor microenvironment.[13] Recently, various commercial iodine compound coated gadolinium oxide nanoparticles were developed and used as MR/CT imaging probes in vivo for the first time.[15] These designs inspired the design of MR/CT imaging probes for the application in vivo. However, the previous design strategies are still relatively complicated, and require the rigorous synthesis condition. Therefore, a simple method to synthetize nanoparticle should be thus developed to obtain suitable MR/CT multimodal probes with high biocompatibility, optimal functionality and with an easy-modifiable structure. Protein-directed synthesis methods offer a suitable alternative to inorganic nanoprobes.[17-19] Moreover, proteins containing tyrosine residues can be easily iodinated to obtain a CT probe.[20,21] However, an iodinated protein synthesis for CT imaging, as well as its useful application, has not been reported yet. Furthermore, nanoparticles can passively target and accumulate within the tumor matrix because of the enhanced permeability and retention (EPR) effect of the tumor mass deriving from the leaky tumor vasculature combined with a poor lymphatic drainage.[22] The most important factor influencing the EPR effect of the particles is their molecular size that need to be larger than 40 kDa.[23] The molecular size of bovine serum albumin (BSA) is 67 kDa, which make it extremely suitable for passive targeted nanoprobe


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synthesis. The best of our knowledge, the development of a protein-templated MR/CT multimodal nanoprobe has not been reported yet. Here, we report the synthesis of multifunctional iodinated albumin-gadolinium nanoparticles and their use in dual-model MR/CT osteosarcoma visualization (Scheme 1). BSA was firstly served as a stabilizer for the biomineralization synthesis of gadolinium nanoparticles (GdNPs). Moreover, BSA molecule possesses several chemically active groups including 21 potential tyrosine residues, which can be completely iodinated to form an iodinated BSA-GdNPs (I-BSAGdNPs) complex. The newborn I-BSA-GdNPs complex shows excellent chemical stability and biocompatibility, intense X-ray attenuation coefficient, and good MR imaging ability. Animal studies suggest that the I-BSA-GdNPs may accumulate for a long time within the tumor matrix via EPR effect. As far as we know, this is the first study reporting iodinated BSA-nanoparticles probes for MR/CT multimodal imaging in vivo. Scheme 1. Schematic Diagram for the Fabrication of I-BSA-GdNPs as a MR/CT Dual Modality Nanoprobe for Osteosarcoma Visualization.

EXPERIMENTAL SECTION


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Materials and Chemicals. All chemicals of analytical grade were used without further purification. Ultrapure water (18 MΩ*cm) was used throughout this work. Gd(NO3)3·6H2O was purchased from Alfa Aesa (Shanghai, China). BSA, NaOH, KI, chloramine-T and phosphate buffer solution (PBS) were purchased from Sinopharm (Shanghai, China). Instrumentation and Characterization. High-resolution transmission electron microscopy (HRTEM) measurements were carried out with Tecnai G2 F20 S-TWIN TMP microscopes (FEI, USA) operating at 200 kV. Zetasizer Nano ZS90 DLS system equipped with a red (633 nm) laser and an avalanche photodiode detector (APD) (quantum efficiency > 50% at 633 nm) (Malvern, England) was used for the measurement of the hydrodynamic diameters. X-ray photoelectron spectroscopy (XPS) was performed with an Axis Ultra DLD spectrometer fitted with a monochromatic Al Kα X-ray source (hν = 1486.6 eV), hybrid (magnetic/electrostatic) optics, a multichannel plate, and a delay line detector (Kratos Analytical, Manchester, UK). The concentration of Gd and I elements in the I-BSA-GdNPs were determined by using an inductively coupled plasma mass spectrometer (ICPMS) (Thermo Elemental X series, UK). Synthesis of I-BSA-Gd NPs. The BSA-GdNPs were synthesized according to the literature.[17] Generally, Gd(NO3)3 (1 mL, 50 mM) was gradually added to 9 mL of ultrapure water containing 25 mg mL-1 BSA solution under vigorous stirring. After 5 min, 1 mL of 2 M NaOH was added the mixture and allowed reaction for 12 h at 37 °C. The synthetized BSA-GdNPs was purified via dialysis for 24 h to remove the excess of unbound reagents. The chloramines-T method was served for the synthesis of I-BSA-GdNPs. Four mL of 5 M KI was added to the purified BSAGdNPs solution, and then 1 mL chloramines-T (5 mg mL-1) was introduced to the previous solution under vigorous stirring for 10 min at 37 °C. The iodinated mixture was dialyzed to remove excess of iodine. Finally, the solution was concentrated to 4 mL via centrifugal filtration


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at 6000 g for 30 min with an ultrafiltration centrifuge tube (Millipore Amicon Ultra-15, MA). The concentration of Gd and I in the concentrated solution was measured by ICPMS. Stability Test. The hydrodynamic size of I-BSA-GdNPs (25 mg mL-1) in PBS buffer (pH 7.4) was measured at constant time point. For the stability of I attached BSA-GdNPs, the I-BSAGdNPs solution was centrifugally filtered with 6000 g in constant time. The concentration of I in the concentrated solution and in the filtrate was measured by ICPMS. Cell Culture. The murine K7M2Wt osteosarcoma cells were cultured in DMEM contained 10% FBS. The cells were cultured in a humidified atmosphere of 5% CO2 at 37 °C and the medium was updated every other day. Trypsin was used to detach the confluent monolayers and to dissociate the cell clumps into a single cell suspension for further cell culture. Animal Models. Animal experiments were approved by the Institutional Animal Care and Use Committee of Soochow University (Suzhou, China). Ten female SD rats three weeks old of approximately 110 g were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China) at least one week before the beginning of the experiment. The SD rats were anesthetized with 200 µL pentobarbital sodium solution via intraperitoneal injection. The knee joint of the left tibia was made a 2-mm midline skin incision. A 10 µL cell suspension (5×107 cells/mL) was injected into the intramedullary cavity from the skin incision with a 31 G latex-free insulin syringe (MSW, China). A 6-0 suture was used for closing the skin. The tumor growth status was monitored by X-ray imaging in 2-week intervals. Biodistribution and Toxicity Analysis. Six female BALB/c mice 6-8 weeks old were divided into three mice per group. A group was administrated I-BSA-GdNPs (580 mg I/kg, 50 mg Gd/kg) by intravenous injection in the tail vein. For the control group, another group was intravenously


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injected with 0.2 mL PBS buffer. The mice were euthanized after 24 h using carbon dioxide (CO2) and the major organs were captured, such as heart, liver, spleen, lung, and kidney. Organs were fixed in PBS contained 10% formalin. All organs were routinely made to pathological section, and then the slides were stained with Hematoxylin and Eosin (H&E) for light microscopy observation. At the same time point, five additional mice administrated I-BSAGdNPs were euthanized using CO2 and the same organs were collected and weighed for ICPMS analysis MR and CT imaging in vitro. I-BSA-GdNPs and the clinical contrast agent (Gd-DTPA and Ioversol) at different concentrations were used to fill 96-well plates, not containing any cells, and placed in the scanning holder. The MR scans and relaxivity were performed on 3.0-T clinical MRI instrument with T1-weighted FSE-XL/90 sequence (GE, Signa 3.0T). MR imaging parameters were as follows: FOV, 80×80; TR/TE, 375/24 ms; matrices, 216×218; slices, 3; slices thickness, 1 mm; averages, 5. CT scans and X-ray absorption ability were carried out with 16detector CT (GE, Light Speed VCT). CT imaging parameters were as follows: voltage, 100 kV; current, 300 µA; field of view, 512 × 512; slice thickness, 0.625 mm; table speed, 40 mm/rotation; gantry rotation time, 0.5 s; pitch, 0.984:1. MR and CT imaging in vivo. Orthotopic osteosarcoma rats (n=6) were anesthetized by intraperitoneal injection of pentobarbital sodium solution (0.5 mL, 1 wt%), then 1 mL I-BSAGdNPs (580 mg I/kg, 50 mg Gd/kg, same as the exposure dose we used in mice) were intravenously injected into four osteosarcoma rats with a 1.0 mL insulin syringe. MR imaging of two osteosarcoma rats were performed on the same as the in vitro process. CT imaging of two osteosarcoma rats was obtained on a 16-detector CT scanner. As the CT imaging control group, the Ioversol (580 mg I/kg) was intravenously injected into the rest of the two osteosarcoma rats.


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Over a period of 24 h post injection, the series of images were captured by MRI and CT instrument. RESULTS AND DISCUSSION

Figure 1. (A) TEM image of I-BSA-GdNPs. (B) HRTEM image of I-BSA-GdNPs. (C) The detached rate of I in the I-BSA-GdNPs solutions in 8 days (D) XPS spectra of Gd 4d for I-BSAGdNPs. (E) XPS spectra of O 1s for I-BSA-GdNPs. (F) XPS spectra of I 3d for I-BSA-GdNPs. The black lines represent the experimentally measured data points, pink lines represent the background, and the other peaks of different colors correspond to the I-BSA-GdNPs analysed by CasaXPS software.


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Synthesis and Characterization of I-BSA-GdNPs. To obtain stable I-BSA-GdNPs with intense CT and MR imaging efficiency, BSA-GdNPs was first prepared via a one-step biomineralization approach. Then, the 21 tyrosine groups of the BSA can be iodinated for the fabrication of I-BSA-GdNPs by the chloramines-T method. The iodinated mixture was purified by dialysis for characterization. The concentration of I and Gd in the concentrated solution were 127.6 mg mL-1 and 11 mg mL-1, respectively, measured by ICPMS measured, respectively. The size, morphology and composition of the as-prepared I-BSA-GdNPs were characterized with HRTEM and XPS. The as-prepared I-BSA-GdNPs showed monodisperse spheres with a average diameter of 2.5 ± 0.3 nm (Figure 1A). The lattice fringes (d222 = 0.312 ± 0.3 nm) of a single IBSA-GdNPs were clearly visible in the HRTEM image (Figure 1B), which was consistent with that of Gd2O3.[24] The stability was monitored against time by hydrodynamic size and the content of I in the prepared I-BSA-GdNPs. The hydrodynamic size of the prepared I-BSA-GdNPs (25 mg mL-1) was about 25 nm (Figure S1A in the supporting information), and no aggregation was observed over 1 week. The concentration dependency of the hydrodynamic sizes was evaluated, which indicated that high concentrations may cause aggregation of the I-BSA-GdNPs (Figure S1B in the supporting information). The iodinated stability of the prepared I-BSA-GdNPs in PBS showed that the release of I was less than 10% from the labeled materials within 8 days (Figure 1C). These results indicated that the prepared I-BSA-GdNPs exhibited excellent stability in aqueous media. The XPS peaks at 142.5 eV corresponded to Gd2O3 (red line), the peak of 148.8 eV (green line) belonged to the remaining traces of Gd(OH)3 (Figure 1D). The O 1s spectrum showed two peaks (Figure 1E), the peaks at 531.5 eV corresponded to the oxygen in Gd2O3 (green line), the peak at 532.6 eV derived from the oxygen in the carbonyl and carboxyl groups in the BSA molecule (red line). The I 3d spectrum showed two peaks of 618.5 eV and 630.2 eV,


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corresponding to the I bound to tyrosine groups in I-BSA-GdNPs (red line), while the peaks at 620.5 eV and 631.8 eV (green line) corresponded to the remaining traces of iodine (Figure 1F). The above results confirmed the successful synthesis of a stable I-BSA-GdNPs.

Figure 2. (A) r1 relaxivity curves of I-BSA-GdNPs and Gd-DTPA. (B) T1-weighted MR phantom images of I-BSA-GdNPs and Gd-DTPA at different concentrations. (C) CT values (HU) of IBSA-GdNPs and Ioversol. (D) CT phantom images at different concentrations of I-BSA-GdNPs and Ioversol. MR and CT Imaging in Vitro. To evaluate the potential of the above probe for MR/CT multimodal imaging, the I-BSA-GdNPs was compared to the clinically available MR and CT imaging contrast agent. The longitudinal proton relaxation time (T1) was measured in I-BSAGdNPs and Gd-DTPA solutions at different Gd3+ concentrations. The inverse relaxation times 1/T1 (r1) was then plotted as a function of Gd3+ concentration. The measured r1 value was 12.03 mM−1 s−1, almost four times more than the value of Gd-DTPA (3.19 mM−1 s−1) (Figure 2A). Furthermore, I-BSA-GdNPs showed stronger T1 signals than Gd-DTPA with the same Gd


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concentration (Figure 2B). The high relaxivity coefficients of the I-BSA-GdNPs were attributed to the efficient longitudinal relaxation of the water protons on the surface of gadolinium oxide nanoparticles where a large amount of Gd3+ retaining high magnetic moments.[25] To further investigate the CT contrast efficacy, the X-ray absorption of I-BSA-GdNPs was compared to the absorption of Ioversol in vitro. The results demonstrated that I-BSA-GdNPs produced a contrast similar to the contrast produced by Ioversol. The HU values and signal increased linearly with concentration for both I-BSA-GdNPs and Ioversol (Figure 2C and D). The above results indicated that I-BSA-GdNPs exhibited good MR and CT imaging ability in vitro. These results are of extreme importance for in vivo application. Biodistribution and Toxicity Analysis. To evaluate the biodistribution and toxicity of the IBSA-GdNPs, mouse organs were collected at 24 h after its injection. The biodistribution of I and Gd in the organs was consistent (Figure 3 A and B), but the concentration of I in the homologous organ was much lower than that of injection. So we deduced that the partial I-BSA-GdNPs might be hydrolyzed by deiodinase.[26] The I-BSA-GdNPs could be accumulated in the reticuloendothelial systems as spleen and liver, similar to the in vivo behaviors of previous gadolinium oxide nanoparticles.[27] Moreover, the high accumulation of I-BSA-GdNPs in the kidney proved that the BSA-nanoparticles could cross the glomerulus and could be excreted via the urine. These encouraging data showed the metabolism of the I-BSA-GdNPs in the body, suggesting that I-BSA-GdNPs might be promising for in vivo imaging. To analyze whether IBSA-GdNPs caused a harmful effect to other susceptible organs, the evaluation of histological changes in these organs was performed. No tissue damage was found associated with the administration of I-BSA-GdNPs (Figure 3C). Based on the above results, I-BSA-GdNPs showed


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a good biocompatibility and exhibited more promising properties for its potential use on MR/CT multimodal imaging.

Figure 3. Biodistribution and toxicity analysis of I-BSA-GdNPs at 24 h in BALB/c mice. The concentration of I (A) and Gd (B) in the organs was evaluated by ICPMS (n=5). (C) Histological changes in the organs after injection of I-BSA-GdNPs. MR and CT Imaging in Vivo. The orthotopic osteosarcoma animal model was performed for in vivo imaging (Figure 4A), and the tumor growth was followed by X-ray imaging until reaching a visible size (Figure S3 in the supporting information). After I-BSA-GdNPs was injected in the osteosarcoma models, MR contrast enhancement of osteosarcoma could be observed at 30 min (Figure 4B). A distinct enhanced signal for the osteosarcoma could be detected within 24 h. The results indicated that I-BSA-GdNPs possess a long-circulating and enhanced retention property in vivo. The long circulation and retention in vessels is very important

for

many

biomedical

applications,[28,29]

including

visualization

of

tumor

neovascularization, as well as monitoring of the tumor status. Enhanced CT signals from osteosarcoma also arose from the 3D-renderings of CT images within the constant period of time


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(Figure 4C). The exact profile of the tumor were indicated in comparison with other tissues at 30 min. I-BSA-GdNPs can be eliminated through renal excretion and the signal was increased rapidly in the bladder, which is consistent with the literature.[30] After 24 h, the CT signal of tumor were higher than the previous image at 30 min, indicating further applications in the passive tumor targeting imaging (Table S1 in the Supporting Information). As a negative control, clinically available Ioversol was intravenously injected into different osteosarcoma models for CT imaging. Ioversol was mainly accumulated in the kidney and the bladder at 30 min due to its short time in the circulation (Figure 4C), which limited the application of Ioversol for tumor targeting imaging and angiography. The imaging results clearly illustrated that the I-BSA-GdNPs could be used as enhanced MR/CT multimodal imaging probe for osteosarcoma visualization.

Figure 4. (A) Orthotopic osteosarcoma animal models. (B) In vivo T1-weighted MRI images of orthotopic osteosarcoma rats before and at 30 min, 2 h, and 24 h after I-BSA-GdNPs injection. (C) CT 3D images of orthotopic osteosarcoma rats after Ioversol and I-BSA-GdNPs injection. CONCLUSIONS In conclusions, we reported the synthesis of multifunctional iodinated albumin-gadolinium nanoparticles for dual-model MR/CT osteosarcoma visualization. BSA was used as a stabilizer in the biomineralization synthesis of BSA-GdNPs. Moreover, BSA molecule possesses several


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active chemical groups including 21 potential tyrosine residues, which could be completely iodinated for the synthesis of I-BSA-GdNPs. The synthetized I-BSA-GdNPs showed excellent chemical stability and biocompatibility, intense X-ray attenuation coefficient, and good MR imaging ability. Animal studies suggest that the long-circulating I-BSA-GdNPs could accumulate in the tumor matrix via EPR effect. The long-circulating MR/CT multimodal imaging probe possesses potential application for image-guided drug delivery and image-guided surgery. ASSOCIATED CONTENT Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Tel/Fax: (86) 512-65880052. E-mail addresses: [email protected] (J. Yan), [email protected] (Y. Wang). Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Q. Wang provided mouse models, performed MRI, CT experiments, and analyzed data. L. Lv synthesized and characterized I-BSA-GdNPs, performed MRI, CT and histology experiments. Z. Ling provided mouse models, performed MRI, CT experiments. Y.Y. Wang and Y. Liu designed and performed the biodistribution experiments. L. Li helped create the research environment, performed histology experiments. L. Li, L. Shen and


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G. Liu provided mouse models. J. Yan co-initiated the project, designed the experiments. Y. Wang initiated the project, designed and performed the experiments, analyzed the data and wrote the manuscript, supervised and coordinated all investigators for the project. #These authors contributed equally. ACKNOWLEDGMENT This work was supported by A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection, Jiangsu Planned Projects for Postdoctoral Research Funds (1401062C, 1402109C), Project Funded by China Postdoctoral Science Foundation (2014M560442), the Jiangsu Provincial Natural Science Foundation (BK20140298), and the National Natural Science Foundation of China (81401500). REFERENCES [1] Sun, X.; Niu, G.; Yan, Y.; Yang, M.; Chen, K.; Ma, Y.; Chan, N.; Shen, B.; Chen, X. Clin. Cancer Res. 2010, 16, 4268. [2] Mutsaers, A. J.; Walkley, C. R. Bone 2014, 62, 56. [3] Janeway, K. A.; Walkley, C. R. Bone 2010, 47, 859. [4] Weissleder, R. Science 2006, 312, 1168. [5] Weissleder, R.; Pittet, M. J. Nature 2008, 452, 580. [6] Gallo, J.; Long, N. J.; Aboagye, E. O. Chem. Soc. Rev. 2013, 42, 7816. [7] Lusic, H.; Grinstaff, M. W. Chem. Rev. 2012, 113, 1641. [8] Arifin, D. R.; Long, C. M.; Gilad, A. A.; Alric, C. E.; Roux, S.; Tillement, O.; Link, T. W.; Arepally, A.; Bulte, J. W. M. Radiology 2011, 260, 790. [9] Louie, A. Chem. Rev. 2010, 110, 3146.


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