In Vivo Long-Term Magnetic Resonance Imaging Activity of Ferritin

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In vivo long-term MRI activity of ferritin-based magnetic nanoparticles versus a standard contrast agent Elsa Valero, Silvia Fiorini, Stefano Tambalo, Heriberto Busquier, Jose CallejasFernandez, Pasquina Marzola, Natividad Gálvez, and José M. Dominguez-Vera J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5004446 • Publication Date (Web): 05 Jun 2014 Downloaded from http://pubs.acs.org on June 12, 2014

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Journal of Medicinal Chemistry

In vivo long-term MRI activity of ferritin-based magnetic nanoparticles versus a standard contrast agent Elsa Valero,† Silvia Fiorini,‡ Stefano Tambalo,‡ Heriberto Busquier,§ José Callejas-Fernández, ∥ Pasquina Marzola,*, ‡ Natividad Gálvez,*,† and José M. Domínguez-Vera*,† †

Departamento de Química Inorgánica, Instituto de Biotecnología, Facultad de Ciencias,

Universidad de Granada, Granada, 18071, Spain ‡

Dipartimento di Informatica, Università degli Studi di Verona, Verona, I-37134, Italy

§

Hospital Virgen de las Nieves, Sección de Neurorradiología, Granada, 18014, Spain

∥Departamento

de Física Aplicada, Facultad de Ciencias, Universidad de Granada, Granada,

18071, Spain KEYWORDS. Magnetic nanoparticles, MRI, Ferritin, Contrast agent, Biodegradation

ABSTRACT. New long-circulating maghemite nanoparticles of 4 and 6 nm coated with the apoferritin protein capsid exhibit useful properties to act as MRI contrast agents. A full in vivo study of the so-called Apomaghemites, reveals that their long-term MRI properties are better than those of a standard SPIO widely used in biomedical applications. The biodistribution of Apomaghemites and the standard SPIO was investigated by MRI in mice at two different

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concentrations, 6 and 2.5 mg Fekg-1 over 60 days. Significant differences are found at low dose (2.5 mg Fekg-1). Thus, whereas Apomaghemites are active for MR bioimaging of liver for 45 days, the standard SPIO is not effective beyond 7 days. Based on our data, we may concluded that Apomaghemites can act as new long-term MRI liver contrast agents allowing first, the diagnosis of a liver pathology and then, monitoring after treatment without the need of a second injection.

INTRODUCTION During last decades, there has been a growing interest to develop contrast agents for their use in biomedical imaging modalities. Magnetic resonance imaging (MRI) ranks among the best noninvasive methodologies today available in clinical medicine thanks to its superb soft tissue contrast and high spatial resolution. It provides information to the clinician about anatomy and function of tissues. MRI provides good contrast between the different soft tissues of the body. However, sometimes intravenous injection of contrast agents is required to enhance the appearance of blood vessels, tumors, inflamed tissues or other pathologies. Thus, there is a need for the development of better diagnostic probes in order to enhance the in vivo MRI performance.1-7 Superparamagnetic iron oxide nanoparticles (SPIO) are used in medicine because of their magnetic properties and biocompatibility.7-16 They are specifically uptake by the monocytemacrophage system and therefore, they are good candidates to be MRI probes for tissues with high macrophage phagocytic activity, as liver or spleen.2-4 In fact, common clinical targets of SPIOs have been liver diseases.13-15


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On the other hand, Kupffer cells and macrophages at the liver, spleen, lymph and bone marrow sequester a great amount of SPIOs, which are phagocytosed producing a drop in T2 relaxation times. Primary liver tumor or liver metastasis lack Kupffer cells and therefore do not retain SPIOs and do not exhibit change in MRI signal. This difference produces a strong contrast between tumor and normal tissues, thereby enabling a more precise MRI detection. Nevertheless, SPIOs are rapidly excreted by the liver to the blood plasma, leading to a limitation in SPIO’s ability for assessing anatomical and biological functions by MRI.2 In this sense, design and preparation of MRI diagnostic agents capable of exhibiting long-term activity is a very important step in the expansion of their applications in medicine. These longterm agents could overcome two crucial issues: the difficulty of conducting time-dependent imaging studies, due to the rapid clearance from intravascular and interstitial space,17-19 and the administration of high and repeated doses. The development of long-term MRI probes could allow first, the diagnosis of a pathology and then, the monitoring of the therapy efficiency without an additional injection. After experimental evidences reported in last years, ferritin, the iron storage protein, is increasingly being recognized as the final iron form after biodegradation of the SPIOs core at macrophages.20-23 Once the SPIO coating is chemically degraded the iron core is metabolized and it is progressively incorporated into the iron-storage protein ferritin. This metabolic mechanism evidences that ferritin is more stable to degradation than SPIOs. Therefore, we have coated magnetic nanoparticles with apoferritin (the ferritin shell) for creating new SPIOs with improving stability, thus remaining non-degraded and active in MRI for a longer period of time. In this context, we have recently reported on the successfully preparation of 4 and 6 nm-based maghemite nanoparticles encapsulated into the apoferritin cavity (Apomag-4 and Apomag-6).24



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In this article we study the pharmacokinetics: biodistribution, long-term activity and toxicity of these ferritin-based nanoparticles and we compare their in vivo features with a SPIO widely used in medicine.25-30

RESULTS AND DISCUSSION In vivo MRI properties The MR imaging capability of Apomaghemites and the standard SPIO during their in vivo degradation was parallel investigated. First, the two Apomaghemite samples and the standard were individually administered through the tail vein into separate, normal Balb-c mice at a high dose of 6 mg Fe kg-1. We collected representative pre-contrast and post-contrast T2*-weighted images of the regions of interest (ROI) in the liver, kidneys, fat and muscle. The post-contrast T2*-weighted images, acquired 2h after injection, showed that the Apomaghemites preferentially accumulate at the liver (Figure 1) and to a lesser extent at the spleen. Quantitative comparison between the efficiency of Apomaghemites and the standard SPIO was obtained by measuring the time dependence of the MRI signal intensity (S.I.) drop (Figure 1a). Normalized S.I. was calculated as (Liver S.I.)/(Muscle S.I.). We used S.I. from the muscle, which can be considered as not influenced by the contrast agent, to obtain relative enhancement data from liver. Results show that Apomaghemites induce signal drops similar to the standard SPIO. R2 values24 and DLS measurements (Supporting Information Figure S1), points out the existence of some aggregation in Apomaghemite samples, probably through the protein capsids, as it occurs in native ferritin. The MRI liver signal drop did not practically change after 30 min. Therefore, in


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the first two hours, both Apomag-4 and Apomag-6 showed a similar behavior to the standard SPIO.

Figure 1. a) Biodistribution of the Apomaghemite nanoparticles at the liver over 2h (green line: standard SPIO, black line: Apomag-4 and red line: Apomag-6). b) Whole body images of mice: A before (pre-contrast) and C 120 min after (post-contrast) administration of Apomag-4; B before (pre-contrast) and D 120 min after (post-contrast) administration of Apomag-6; E before (pre-contrast) and F 120 min after (post-contrast) administration of the standard SPIO. The arrows highlight the liver. Long-term MRI study of liver The in vivo long-term efficiency of Apomaghemites was investigated at liver over a 30-60 days period using a BioSpec 4.7T. Both Apomaghemites and the standard SPIO were individually administered through the tail vein into separate, normal Balb-c mice at doses of 6 and 2.5 mg Fe kg-1 and the animals underwent T2*-weighted MRI at multiple time points post



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injection to evaluate the hepatic uptake and clearance. The T2*-weighted MRI was performed before injection and after 10 min, 30 min, 1h, 2h, 24 h, 7 days, 14 days, 30 days, 45 days and 60 days post-injection at a field of 4.7 T. Figure 2a shows MRI signal intensities of Apomag-4, Apomag-6 and standard SPIO at a dose of 6 mg Fe kg-1 over time. The three nanoparticles accumulate quickly (within 30 min) in the liver and gradually degrade in this organ during the time of the experiment, allowing MRI acquisitions until 30 days for Apomag-4 and 60 days for Apomag-6. We found that, in broad terms, the biodegradation profile of all samples is slow. However, it should be highlighted that after 15 days the MRI signal induced by both Apomaghemites becomes different from each other. Thus, whereas MRI signal for Apomag-4 remains stable for the first 15 days and then, has a gradual restoration to normal level after 30 days, that of Apmag-6 has a long period of plateau up to 30 days, followed by a gradual restoration to nearly the normal level not before than 60 days. The signal drop for the standard SPIO, at a dose of 6 mg Fe kg-1, is more pronounced than for the two Apomaghemites and its biodegradation slower. However, the T2* images obtained were saturated, which makes difficult to infer any conclusion. Although the explanation of the different biodegradation behavior between 4 and 6 nm nanoparticles requires a further study, one possible interpretation could be that there is a higher degree of protein self-assembling around the 6 nm nanoparticles than around the 4 nm ones, probably because 6 nm size is closer to the 8 nm cavity size of native protein. Namely, protein assembling is less effective around the 4 nm nanoparticle template thus producing a more rapid biodegradation.


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Figure 2. a) Representative biodistribution curves of Apomaghemites and the standard SPIO at a 6 mg Fe kg-1 dose (green line: standard SPIO, black line: Apomag-4 and red line: Apomag-6). b) Whole body images of a mouse before (pre-contrast) and after (post-contrast) administration of Apomag-6 nanoparticles over a period of 60 days. The arrows highlight the liver. As it can be observed for whole body acquisitions (Figure 2b), the T2* signal obtained at a dose of 6 mg Fe kg-1 is dark and intense, which makes difficult the examination of images. Therefore, we decided to use a lower Fe dose to acquire a more adequate T2* signal. For this purpose, the Apomag-6 (the best long-term MRI Apomaghemite) and the standard SPIO were injected at a dose of 2.5 mg Fe kg-1. T2*-weighted pre-contrast images were acquired before the injection and after 10 min, 1h, 2 h, 24 h, 7 days, 14 days, 30 days and 45 days post-injection from the 4.7 T MRI system.



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Figure 3a shows as Apomag-6 nanoparticles are quickly accumulated at the liver and lead to an adequate drop of the MRI signal. Likewise, it can be observed that the liver clearance of Apomag-6 at this dose is extremely slow since the restoration of the MRI signal takes place after 45 days. In contrast, the standard SPIO exhibited a different pattern. Thus, the signal drop of the standard SPIO is less pronounced and permanently remains above that of Apomag-6 (Figure 3a). On the other hand, the standard SPIO undergoes a gradual restoration to the normal level of MRI signal after 7 days, whereas Apomag-6 is active until 45 days. Moreover, whole body images (Figure 3b) show that the T2* signal obtained at a dose of 2.5 mg Fe kg-1 with Apomag-6 is not saturated and it is optimal to acquire MRI images. Briefly, two iron dosages were tested, 6 and 2.5 mg Fe kg-1, to collect representative precontrast and post-contrast T2*-weighted images. We found that at a dose of 6 mg Fe kg-1 Apomaghemtites, as well as standard SPIO, induced a very intense T2* signal making it difficult the clinical exploration of images. Nevertheless, the T2* signal obtained from the 2.5 mg Fe kg-1 dose permitted us to monitor MRI liver signal even 45 days after injection compared to 7 days for standard SPIO. These results are consistent with those obtained by Kalber et al.31 They obtained a recovered of r2 values to control levels for the standard SPIO after 10 days of administration for a 2.5 mg Fe kg-1 dose. This is precisely the human clinical dose used when working with USPIOs.4 This means that for our system, it is not necessary to inject a large dose of contrast agent to achieve high stability over time and that the 2.5 mg Fe kg-1 is the ideal dose to be used as long-term MRI liver contrast agent. The 6 mg Fe kg-1 dose is not useful because of the saturation in contrast of MR images. Finally, the presence of the apoferritin shell around the maghemite nanoparticles makes them stable over a long period of time. The r2 values,24,32 as well as the in vivo pre-contrast and post-


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contrast T2*-weighted images, suggest that Apomaghemites can act as new contrast agents for MRI. The long-time stability of Apomaghemites at the liver can be useful in the clinical field to monitor the progress of a liver pathology. Therefore our system can be used in long-term MR imaging to conduct many time-dependent imaging studies at the liver.

Figure 3. a) Representative biodistribution curves of Apomag-6 (red line) and the standard SPIO (green line) at a 2.5 mg Fe kg-1 dose. b) Whole body images of a mouse before (pre-contrast) and after (post-contrast) administration of Apomag-6 over a period of 45 days. Liver is highlighted by an arrow. Figure 3a shows that the liver clearance of Apomag-6 at this dose is extremely slow. It is interesting to note that the increase of MRI signal to the pre-contrast baseline after 45 days for Apomag-6 indicates the dissolution of nanoparticles at liver. Interestingly, Apomag-6 and the standard SPIO exhibited a drastically different signal pattern. Thus, the signal drop of the standard SPIO is less pronounced and permanently remains above the Apomag-6 signal. The



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standard SPIO undergoes a gradual restoration to the normal level after 7 days whereas Apomag6 is active until 45 days. Moreover, whole body images (Figure 3b) show that the T2* signal obtained at a dose of 2.5 mg Fe kg-1 with Apomag-6 is not saturated and it is optimal to acquire MRI images (Fig. 4b). Histopatological Evaluation Tissue staining was performed on various tissues (liver and kidneys) to evaluate toxicity and iron distribution (Figure 4) on mice administered with Apomag-6, Apomag-4 and the standard SPIO at the highest dose of 6 mg Fe kg-1. Sections of liver and kidney tissues were harvested from mice 2h and 45 days after receiving nanoparticles injection, fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin, eoxin (H&E) and/or with Prussian Blue. After 2h of administration iron oxide nanoparticles (PB staining) are visible in the case of Apomaghemites at the liver and kidneys. There is no PB staining on kidneys for standard SPIO after 2h, probably because of the larger size of standard SPIO nanoparticles (80-150 nm). It is known that, usually, nanoparticles smaller than 10 nm are cleared out of organism by the kidneys and the largest ones by the liver. After 45 days of administration, PB staining on the liver for the three samples detected a few residual iron particles. No iron particles were detected on the kidneys for all samples. H&E staining showed no evidence of toxicity in any stained tissues. In general 60 days was time enough for liver and kidney regions to be cleared out of iron nanoparticles.


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Figure 4. Tissue staining images: a) and b) Prussian blue stained tissue sections of liver and kidney for Apomaghemites and standard SPIO after 2h of administration.; c) and d) Hematoxylin (blue nuclei) & Eosin (pink cytoplasm) and PB (iron particles) stained tissue sections of liver and kidney for Apomaghemites and standard SPIO after 45 days of administration. CONCLUSIONS In summary, our results indicate that Apomaghemites accumulates preferentially at the liver and remain stable (and functional) in this organ for long time. In particular, Apomag-6 at 2.5 mgFekg-1 dose succeeds to be MRI active for 45 days after injection, significantly higher than the standard SPIO. This capability confirms our hypothesis that the apoferritin shell is an adequate capsid since its chemical robustness delays the biodegradation of the magnetic core.



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The lack of toxicity evaluated by histopathological assays was negative. Likewise, mice were still alive after 2 months with no obvious evidence of toxicity. Negative health effects were not observed. Apomaghemites therefore represent a new generation of long-term MRI contrast agents capable of giving us, not only a first diagnosis but also information regarding the progress of a medical treatment over time, without the need of a second injection of contrast. This aspect would be very helpful, for example, in measuring the effectiveness of an anticancer therapy or inflammatory process because it would allow anatomical and morphological information of liver for a long period of time.

EXPERIMENTAL SECTION Preparation of Apomaghemite nanoparticles The synthesis of Apomaghemite nanoparticles is reported elsewhere.24 Briefly, magnetite nanoparticles were prepared by coprecipitation of Fe2+ [(NH4)2Fe(SO4)2 amonium iron(II) sulfate hexahydrate] and Fe3+ [Fe(NO3)3] salts in stoichiometry of 0.5 (based on Massart’s method).33 By adjusting both pH (12.0 and 11.0 for 4 and 6 nm, respectively) and ionic strength (2 and 1 M NaNO3 for 4 and 6 nm, respectively), the size of the resulting magnetite nanoparticles can be controlled.34,35 All solutions were carefully deaerated with argon. After oxidation with HClO4, a colloid of maghemite nanoparticles stable at pH 2.0 was obtained. Then, the maghemite nanoparticles were entrapping inside the protein cavity by adjusting the pH. The pH of a 4 mL solution of Apoferritin (5 mg mL-1) was slowly lowered to pH 2.0 with 0.1 M HCl. After being stirred for 15 min, the solution was mixed with 1 mL of a diluted nanoparticles solution at pH 2.0 (200 µL to 1 mL) and stirred for 30 min. The pH of the resulting solution was then adjusted to 7.0 with 0.1 M NaOH to allow the proper subunit assembly into the 24-mer protein and the


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simultaneous encapsulation of nanoparticles. Finally, the resulting clear reddish dark solution was purified to remove unfolded subunits or large nanoparticles aggregates by centrifugation (10,000 rpm, 1 h). Next, the solution was chromatographed on a Sephadex G-25 column to isolate the protein-containing fractions which are concentrated with Amicon® Ultra Centrifugal Filters 10,000 MWCO (8,500 rpm, 20 min) to obtain the desired iron concentration. Dynamic Light Scattering Values of average hydrodynamic diameter in dilute dispersions were obtained by dynamic light scattering measurements using a 4700C System from Malvern Instruments (UK) and a 3DDLS from LS Instruments (Switzerland), respectively. In both cases, a Helium-Neon laser operating at 632.8 nm wavelength and about 20 mW was used. The experiments were performed at 25 ºC and scattering angle of 90º (q = 0.018 nm-1). The CONTIN analysis of the intensity autocorrelation function reveals the existence of two population sizes, the first one centered on ~ 30 nm and the second one at ~ 200 nm (Figure S1a). The Figure S1b shows the intensity autocorrelation function of the scattered light, G2(q,τ). MRI measurements. In vivo experiments. MRI measurements were performed with a 4.7T Biospec Tomograph System (Bruker, Karlsruhe, Germany) operating at 200 MHz and equipped with a 33 cm bore magnet (Oxford Ltd., UK). In the in vivo experiments, a total of 30 normal Balb-c mice weighing about 20 g were used (thirteen animals for Apomag- 6, eight for Apomg-4 and nine animals for Endorem®). Animals were anesthetized with gas anaesthesia (a mixture of O2 and air containing 1-1.5% of isofluorane), placed in a heated animal bed and inserted in a 3.5 cm internal diameter bird-cage coil. All procedures were performed with the approval of the Ethical Committee for Animal Experimentation of the University of Verona. T2-weighted images of the mice body were



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acquired at 4.7 T using a T2-weighted sequence with the following parameters: FOV= 6x3 cm2, MTX = 256x128 pixels, Slice thickness= 0.2 cm, TE= 5.9 ms TR= 2000 ms NEX=2, Echos=16. T2* weighted images were acquired using a Gradient-Echo sequence with the following parameters: TE= 4.4 ms, TR=1,000 ms, FOV 6X3 cm2, NEX= 2, MTX= 256X128, Slice Thickness=0.2 cm, flip angle 20°. Contrast agents were administered through the tail vein at doses of 6 mg Fe kg-1 and 2.5 mg Fe kg-1. The images were acquired before, and at different time points after contrast agent administration. After the MRI investigation, the mice were sacrificed by anesthesia overdose. Hematoxylin and eoxin staining. After 2h and 45 days of nanoparticles administration, liver and kidney were extracted from the sacrificed mice. The extracted organs were then fixed in 10% formalin and embedded in paraffin blocks by standard procedure. All paraffin-blocked samples were cut in sections of 4 µm thick using a microtome and adhered on slide glasses. Sections were then deparaffinised in xylene for an hour and rehydrated in ethanol (100, 95, 90, 80, and 75%) and distilled water for 10 min at each step. Nuclei were stained with hematoxylin for 2 min and rinsed with top water and then with distilled water many times. Cytoplasm of the tissues was stained by eosin for 30 s. The eosin-stained sections were rinsed with distilled water and dehydrated in ethanol (75, 80, 90, 95, 100%) for 5 min at each step. The stained tissues were dipped into xylene for 10 min and mounted in DPX. Finally, all slides were observed using a Zeiss Primo Star optical microscope equipped with a CCD camera. Prussian Blue staining. The sections were incubated with Prussian Blue solution (5% hydrochloric acid mixed with 5% potassium ferrocyanide) for 30 min. After washing, the sections were dehydrated, cover


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slipped with Entellan, and observed using a Zeiss Primo Star optical microscope equipped with a CCD camera.33

ASSOCIATED CONTENT Supporting Information Available: DLS measurements of Apomag-6 sample. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * e-mail: [email protected], phone: +34958249386, e-mail: [email protected], phone: +34958248097, Departamento de Química Inorgánica. Facultad de Ciencias. Universidad de Granada, Granada, 18071, Spain. * e-mail: [email protected], phone: +39458027614, Dipartimento di Informatica, Università degli Studi di Verona, Verona, I-37134, Italy.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This work was supported by MINECO: projects CTQ2012-32236 and IT2009-0060, Junta de Andalucía project: P11-FQM-8136, by Fondazione Cariverona (Verona, Italy): project “Verona Nanomedicine Initiative” and by MIUR through FIRB project RBAP114AMK - RI.NA.ME. “Rete Integrata per la Nanomedicina”.



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ABBREVIATIONS MRI, magnetic resonance imaging; SPIO, superparamagnetic iron oxide; ROI, regions of interest; SI, signal intensity; USPIO, ultrasmall superparamagnetic iron oxide; H&E, hematoxylin eoxin; PB, Prussian Blue; DLS, Dynamic Light Scattering.

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TABLE OF CONTENTS GRAPHIC


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In Vivo Long-Term Magnetic Resonance Imaging Activity of Ferritin

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