Development of an Effective Tumor-Targeted Contrast Agent for Magnetic Resonance Imaging Based on Mn/H-Ferritin Nanocomplexes

Magnetic resonance imaging (MRI) is one of the most sophisticated diagnostic tools that is routinely used in clinical practice. Contrast agents (CAs) are commonly exploited to afford much clearer images of detectable organs and to reduce the risk of misdiagnosis caused by limited MRI sensitivity. Currently, only a few gadolinium-based CAs are approved for clinical use. Concerns about their toxicity remain, and their administration is approved only under strict controls. Here, we report the synthesis and validation of a manganese-based CA, namely, Mn@HFn-RT. Manganese is an endogenous paramagnetic metal able to produce a positive contrast like gadolinium, but it is thought to result in less toxicity for the human body. Mn ions were efficiently loaded inside the shell of a recombinant H-ferritin (HFn), which is selectively recognized by the majority of human cancer cells through their transferrin receptor 1. Mn@HFn-RT was characterized, showing excellent colloidal stability, superior relaxivity, and a good safety profile. In vitro experiments confirmed the ability of Mn@HFn-RT to efficiently and selectively target breast cancer cells. In vivo, Mn@HFn-RT allowed the direct detection of tumors by positive contrast enhancement in a breast cancer murine model, using very low metal dosages and exhibiting rapid clearance after diagnosis. Hence, Mn@HFn-RT is proposed as a promising CA candidate to be developed for MRI.

S2 diphenyl-2H-tetrazolium bromide (MTT) stock solution previously diluted in medium without phenol red following the manufacturer's instructions (Promega). After incubation, 0.1 mL of MTT solubilizing solution was added to each well to solubilize the MTT formazan crystals. Absorbance was read using a testing wavelength of 570 nm and a reference wavelength of 630 nm. The results are expressed as mean ± standard deviation (SD) of viability percentage calculated versus the untreated sample (n = 4-6). Cells immunodecorated with the secondary antibody only were used as control.
Cellular uptake by immunodecoration and confocal detection. 3 × 10 5 HCC1954 cells were cultured on cover glass slips precoated with polylysine and, after 24 h, incubated with 0.1 mg/mL of FITC-Mn@HFn-RT suspended in complete medium. Then, cells were washed PBS, fixed with 4% paraformaldehyde (37 °C, 20 min) and then subjected to membrane and nucleus staining by incubation with Wheat Germ Agglutinin (WGA)-Alexa Fluor 555 conjugate (1 μg/mL) and 4',6diamidin-2-phenylindole (DAPI) (1 μg/mL), respectively. After washing with PBS, cover slips were mounted with ProLong Antifade reagent (Thermo Fisher Scientific, MA, USA) and examined by Nikon A1 Confocal Microscope (Nikon Instruments) equipped with laser excitation lines 405, 488 and 555 nm. Images were acquired at 1024 × 1024 pixel resolution and with a 63 × magnification oil-immersion lens. In order to confirm the correct setup of FITC channel, an untreated sample was as well analyzed, and no signal was found.

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Epifluorescence analysis of AF660-HFn performed in vivo and ex vivo. HCC1954 cells were cultured and implanted subcutaneously in four nude mice in a mixture of 1 × 10 7 cells suspended in growth media and Matrigel. Animals were monitored and treated as described in Materials and Method section, until the tumor reached a dimension around 100-200 mm 3 . To observe the biodistribution of HFn in mice body, the protein was labeled with Alexa Fluor™ 660 NHS Ester (AF660-HFn) and injected in the tail vein of mice (5 mg/kg HFn). PBS was used as control. Mn@HFn-HT revealed a light brown color ( Figure S1a). The protein recovery efficiency was 70% (± 4%) for Mn@HFn-RT, while a poor reproducibility along with a lower efficiency (56% (± 12%)) was observed when the reaction temperature was increased to 65 °C (Table 1).
Native gel electrophoresis showed that the protein maintained the original structure both in Mn@HFn-RT and Mn@HFn-HT samples ( Figure S1b), while the hydrodynamic diameter assessed by DLS analysis (12.2 ± 0.5 nm and 13.5 ± 1.1 nm (Figure 1c), respectively) was consistent with the size reported for native ferritin (12 nm). 2 TEM analysis confirmed that the protein preserved its distinctive structure after the reaction: indeed, core-shell architecture was clearly detectable with an S4 inner and outer effective diameter of 7.4 ± 2.2 nm and 12.1 ± 1.4 nm ( Figure 1b). These results suggest that, in both samples, the maintained structural integrity of HFn should be able to promote the effective recognition of TfR1 receptor and the consequent cellular uptake.
A first evidence of Mn encapsulation in HFn was given by TEM analysis conducted in absence of the negative staining ( Figure S1c): thanks to the metal complexation, it was possible to focus the protein cages in Mn@HFn-RT and Mn@HFn-HT samples. After protein disassembly in acidic environment, Mn was quantified by ICP-OES analysis and the Mn/HFn molar ratio was estimated as 218 (± 33) for Mn@HFn-RT and 1087 (± 173) for Mn@HFn-HT (Table 1).
Then, the relaxivity (r) of Mn@HFn-RT and Mn@HFn-HT was calculated after measuring the relaxation time as a function of CAs concentration. Besides the r 1 values already discussed in the main text, this analysis revealed that both Mn@HFn-RT and Mn@HFn-HT are suitable as positive CAs with a r 2 /r 1 rate of 2.3 (± 0.3) and 2.9 (± 0.4), respectively (Table 1). 3 Afterwards, the dependence of the oxidation state of Mn from the reaction conditions was assessed by a colorimetric assay. 1 Figure S2a).
Cell viability assay. As required prior to the in vivo experiments, the cellular toxicity of Mn@HFn nanocomplexes was investigated. For this purpose, a viability assay was conducted incubating TfR1expressing cells HCC1954 (expression confirmed in Figure S3) with Mn@HFn-RT and Mn@HFn-

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HT at the same protein concentration (0.1 mg/mL) and the cellular viability was evaluated at 24, 48 and 72 h by using MTT. In this experiment, unloaded HFn was used as a control to assess any nanocarrier-related effect, whereas MnCl 2 was tested at two different concentrations corresponding to the Mn ions amount contained in Mn@HFn-RT and Mn@HFn-HT samples (80 μM and 327 μM, respectively). The results showed in Figure S4 revealed that cell viability was slightly affected by Mn@HFn-RT (above 85% over time), while in cells incubated with Mn@HFn-HT the cellular toxicity was significantly higher exhibiting a cell viability around 50%. This effect could be attributable to higher metal concentration, which could alter the normal cell cycle. The latter hypothesis was confirmed comparing this data with the controls (HFn and MnCl 2 ): indeed, the protein itself does not impair the cellular growth, while free Mn 2+ ions displayed a concentration-dependent effect on cell viability. These findings were confirmed also conducting the test with a different cell line (HeLa) with a significant overexpression of TfR1 ( Figure S3 and S4).