Facile preparation of hyaluronic acid-modified Fe₃O₄@Mn₃O₄ nanocomposites for targeted T₁/T₂ dual-mode MR imaging of cancer cells

Abstract

We report a facile approach to synthesizing hyaluronic acid (HA)-modified Fe₃O₄@Mn₃O₄ nanocomposites (NCs) for targeted T₁/T₂ dual-mode magnetic resonance (MR) imaging of cancer cells. In this work, branched polyethyleneimine (PEI)-coated Fe₃O₄@Mn₃O₄ NCs (Fe₃O₄@Mn₃O₄-PEI NCs) were first synthesized via a one-pot hydrothermal route, followed by modification of HA on the particle surface via PEI amines. The formed Fe₃O₄@Mn₃O₄-PEI-HA NCs were well characterized via different techniques. Our results manifest that the formed Fe₃O₄@Mn₃O₄-PEI-HA NCs possess good water dispersibility, colloidal stability, cytocompatibility in the studied concentration range, and targeting specificity to CD44 receptor-overexpressing cancer cells. Due to the coexistence of Fe₃O₄ and Mn₃O₄ in the particles, the Fe₃O₄@Mn₃O₄-PEI-HA NCs display relatively high r₂ (143.26 mM⁻¹ s⁻¹) and r₁ (2.15 mM⁻¹ s⁻¹) relaxivities, and can be used as an efficient nanoprobe for targeted T₁/T₂ dual-mode MR imaging of cancer cells in vitro. The developed Fe₃O₄@Mn₃O₄-PEI-HA NCs may hold great promise to be used as a nanoplatform for theranostics of different biological systems.

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Introduction

Early-stage diagnosis of cancer is critical to decrease the death rate.¹ The presently used diagnostic techniques include magnetic resonance (MR) imaging,^2–4 computed tomography (CT) imaging,^5–7 optical imaging,^8,9 ultrasonic (US) imaging,^10,11 and position emission tomography (PET) imaging,^12,13 etc. Although each imaging modality has its own advantages, single mode imaging usually fails to provide complete and accurate information of the diseases.^14,15 Therefore, it is strongly recommended to develop dual mode or multi-mode imaging techniques for disease diagnosis with improved accuracy, such as T₁/T₂-weighted MR imaging,^16–19 MR/CT imaging,^20–22 MR/optical imaging,^23,24 MR/US imaging,^25–27 and MR/PET imaging.^28,29

For high quality molecular imaging applications, contrast agents are generally required. For instance, gadolinium (Gd)- or manganese (Mn)-based nanodevices have been widely studied for T₁-weighted MR imaging;^30–32 superparamagnetic iron oxide (Fe₃O₄) nanoparticles (NPs) are used as ideal probes for T₂-weighted MR imaging;^33,34 gold (Au) NPs have received much attention as contrast agents for CT imaging;^35,36 and quantum dots have been designed for optical imaging.^37,38 Currently, multifunctional nanocomposites (NCs) consisting of different imaging components have been proposed as molecular probes for dual- or multi-mode imaging applications. As an example, Yang et al.³⁹ developed a multifunctional Gd-labeled Fe₃O₄ NPs for targeted T₁- and T₂-weighted dual-mode MR imaging of tumors. An et al.⁴⁰ synthesized hollow silica nanospheres with a size of 400 nm and conjugated them with Gd(III) complexes and arginine–glycine–aspartic acid peptide for targeted MR/US dual-modal imaging of tumors. In our previous work,^41,42 we fabricated Fe₃O₄@Au NC particles via the combination of layer-by-layer self-assembly process and dendrimer chemistry for dual-mode MR/CT imaging applications. These prior successes witnessed the rapid development of imaging probes for biomedical applications. However, preparation of multifunctional NCs for dual mode accurate disease diagnosis still remains an open area and offer great challenges.

Recently, we developed a facile one-pot hydrothermal route to synthesize Fe₃O₄ NPs,⁴³ 3-aminopropyltriethoxysilane (APTS)-coated Fe₃O₄ NPs,⁴⁴ and polyethyleneimine (PEI)-coated Fe₃O₄ NPs (Fe₃O₄-PEI NPs).⁴⁵ Especially, the formed Fe₃O₄-PEI NPs can be further modified with targeting ligands folic acid (FA) or hyaluronic acid (HA) for targeted MR imaging of tumors overexpressing FA receptors (FAR) or CD44 receptors, respectively.^46–48 Further, the hydrothermal route can be extended to synthesize Fe₃O₄@Au composite NPs or gadolinium hydroxide (Gd(OH)₃)-doped Fe₃O₄ NPs for highly efficient in vivo dual-mode MR/CT and T₁/T₂-weighted MR imaging, respectively.^49,50 Recently, manganese oxide (Mn₃O₄) NPs were well developed as contrast agents for T₁-weighted MR imaging applications because of their high relaxivities and good biocompatibility.^3,51 In addition, it has been reported that Mn₃O₄ NPs can be prepared by a facile hydrothermal route.^52,53 It is reasonable to speculate that Fe₃O₄@Mn₃O₄ NCs may also be prepared by a hydrothermal route for T₁/T₂-weighted MR imaging applications.

In this present work, we present a facile hydrothermal method to synthesize PEI-coated Fe₃O₄@Mn₃O₄ NCs. The PEI-coated Fe₃O₄@Mn₃O₄ NCs were then conjugated with targeting ligand HA for targeted T₁- and T₂-weighted dual-mode MR imaging applications (Scheme 1). The formed Fe₃O₄@Mn₃O₄-PEI-HA NCs were characterized via different techniques. The cytotoxicity of the NCs, the binding specificity of the Fe₃O₄@Mn₃O₄-PEI-HA NCs to CD44 receptor-overexpressing cancer cells, and the potential to use the NCs for T₁- and T₂-weighted MR imaging of cancer cells in vitro were investigated in detail. To our knowledge, this is the first report related to the development of Fe₃O₄@Mn₃O₄-PEI-HA NCs via a hydrothermal method for dual mode T₁- and T₂-weighted MR imaging of cancer cells.

Scheme 1 Schematic representation of the synthesis of the Fe₃O₄@Mn₃O₄-PEI-HA NCs.

Experimental

Materials

Branched PEI (M_w = 25 000) was supplied by Sigma-Aldrich (St. Louis, MO). HA (M_w = 31 200) was obtained from Zhenjiang Dong Yuan Biotechnology Corporation (Zhenjiang, China). 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from J&K Chemical Ltd. (Shanghai, China). HeLa cells (a human cervical carcinoma cell line) were from Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, China). Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Jinuo Biomedical Technology (Hangzhou, China). Ferrous chloride tetrahydrate (FeCl₂·4H₂O), manganese acetate (MnAc·4H₂O), dimethyl sulfoxide (DMSO) and all other chemicals and solvents were from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and used as received. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was acquired from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Water in all experiments was purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA) with a resistivity higher than 18.2 MΩ cm.

Synthesis of Fe₃O₄@Mn₃O₄-PEI NCs

The Fe₃O₄@Mn₃O₄-PEI NCs were prepared through a previously reported hydrothermal route with some modifications.^45,50 Briefly, FeCl₂·4H₂O (0.10 g, 0.50 mmol) and MnAc·4H₂O (1.55 g, 6.33 mmol) were first co-dissolved in water (7.75 mL). Then, ammonium hydroxide (6.25 mL) was added into the above mixture under vigorous magnetic stirring and the suspension was left stirring in the air atmosphere for 40 min. After that, the reaction mixture together with an aqueous solution of PEI (0.50 g dissolved in 5 mL water) was transferred into a 50 mL autoclave (KH-50 autoclave, Shanghai Yuying Instrument Co., Ltd., Shanghai, China). The reaction mixture was blended thoroughly and then sealed. After reaction at 160 °C for 16 h, the autoclave was cooled down to room temperature. The product was collected by magnetic separation, washed with water for several times, and then dispersed in DMSO or water (9 mg mL⁻¹) for further use.

Synthesis of Fe₃O₄@Mn₃O₄-PEI-HA NCs

The conjugation of HA onto the surface of Fe₃O₄@Mn₃O₄-PEI NCs was performed according to our previous work.⁴⁷ Briefly, 5 mL of DMSO solution containing EDC (4.80 mg) and NHS (2.90 mg) was added dropwise into an aqueous solution of HA (78.12 mg, 5 mL) under vigorous magnetic stirring. After 3 h, the activated HA was added dropwise into a DMSO solution of Fe₃O₄@Mn₃O₄-PEI NCs (45 mg, 5 mL). The reaction mixture was continuously shaken for 3 days, then the product was collected by magnetic separation and sequentially washed with DMSO and water for several times. Finally, the formed Fe₃O₄@Mn₃O₄-PEI-HA NCs were redispersed in 15 mL water and stored at 4 °C for further use.

Characterization techniques

X-ray diffraction (XRD) was performed using a Rigaku D/max 2550 PC X-ray diffractometer (Tokyo, Japan) equipped with a Cu Kα radiation (λ = 0.154056 nm, 40 kV, 200 mA) in the scan range (2θ) of 10° to 90°. A Malvern Zetasizer Nano ZS model ZEN3600 (Worcestershire, UK) equipped with a standard 633 nm laser was used to measure the surface potential and hydrodynamic size of the particles. Thermal gravimetric analysis (TGA) was carried out on a TG 209 F1 (NETZSCH Instruments Co., Ltd., Bavaria, Germany) thermogravimetric analyzer under nitrogen from 30 °C to 800 °C at a rate of 20 °C min⁻¹. TEM images were taken using a JEOL 2010F transmission electron microscopy (JEOL, Tokyo, Japan) operating at 200 kV and the samples were prepared by dropping 7 μL of the water suspension onto a carbon-coated copper grid and left until dried. A Leeman Prodigy inductively coupled plasma-atomic emission spectroscopy (ICP-AES) system (Hudson, NH) was used to analyze the element concentration of the solution. The T₁/T₂ relaxation times of the particle suspension with different Fe concentrations (0.005–0.08 mM) or Mn concentrations (0.0125–0.2 mM) in water were measured by a 0.5 T NMI20-Analyst NMR Analyzing and Imaging system (Shanghai Niumag Corporation, Shanghai, China). The parameters were set as follows: CPMG sequence, point resolution = 156 mm × 156 mm, section thickness = 0.6 mm, TR = 600 ms, TE = 160 ms, number of excitation = 1 for T₁-weighted MR imaging, and CPMG sequence, point resolution = 156 mm × 156 mm, section thickness = 0.6 mm, TR = 4000 ms, TE = 60 ms, number of excitation = 1 for T₂-weighted MR imaging. The relaxivity (r₁ and r₂) was calculated by a linear fitting of the inverse relaxation time as a function of the Fe or Mn concentration.

Cell culture

HeLa cells overexpressing CD44 receptors were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U mL⁻¹), and streptomycin (100 μg mL⁻¹) at 37 °C and 5% CO₂. HeLa cells grown in normal HA-free DMEM overexpressed high-level CD44 receptors (denoted as HeLa-HCD44 cells), while HeLa cells pre-treated with 2.0 mM free HA for 2 h or longer overexpressed low-level CD44 receptors (denoted as HeLa-LCD44 cells). Unless otherwise stated, the term of HeLa cells always represents the HeLa-HCD44 cells.

Cytotoxicity assay

MTT assay was carried out to determine the cytotoxicity of the Fe₃O₄@Mn₃O₄-PEI-HA NCs. HeLa cells were seeded into 96-well plates with 200 μL fresh medium at a density of 1 × 10⁴ cells per well and cultured at 37 °C and 5% CO₂ overnight. The next day, the medium was discarded and cells were incubated with fresh medium containing PBS (control) or Fe₃O₄@Mn₃O₄-PEI-HA NCs at different Mn concentrations (0.2–2.0 mM) or Fe concentrations (0.03–0.32 mM) for 24 h. Then, MTT (5 mg mL⁻¹, in 20 μL phosphate buffered saline (PBS)) was added to each well, and the cells were further incubated at 37 °C for 4 h. Then the medium was discarded carefully and 200 μL of DMSO was added into each well to dissolve the formazan crystals. The absorbance of each well was measured at 570 nm using a Thermo Scientific Multiskan MK3 ELISA reader (Thermo scientific, Waltham, MA). The cells cultured in normal medium without supplement of MTT were used as the sample blank to eliminate the effect of particles on the absorbance. The relative cell viability was calculated according to the equation of “Cell viability = (OD_sample − OD_{sample blank})/(OD_control − OD_{control blank}) × 100%”. Five parallel wells for each sample were measured to report the mean and standard deviation. Likewise, the morphology of cells after treated with the Fe₃O₄@Mn₃O₄-PEI-HA NCs at different Mn concentrations (0–2.0 mM) for 24 h was also observed by phase contrast microscopy (Leica DM IL LED inverted phase contrast microscope) with a magnification of 200 × for each sample.

In vitro cellular uptake assay

To qualitatively confirm the uptake of the Fe₃O₄@Mn₃O₄-PEI-HA NCs by HeLa cells, Prussian blue staining of cells was performed according to our previous work.⁴⁵ Briefly, HeLa-HCD44 or HeLa-LCD44 cells were seeded into 24-well plates at a density of 2 × 10⁵ cells per well, and cultured for 12 h to bring the cells to confluence. Thereafter, the medium in each well was replaced with fresh medium containing the Fe₃O₄@Mn₃O₄-PEI-HA NCs at different Mn concentrations (0, 0.6, or 1.0 mM). After being continuously incubated for 6 h, the cells were washed 3 times with PBS, fixed with formaldehyde solution (2.5%) at 4 °C for 15 min, and stained with Prussian blue reagent at 37 °C for 30 min. The images of the stained cells were taken by phase contrast microscopy with a magnification of 200× for each sample.

ICP-AES was also used to quantitatively assay the in vitro cellular uptake of the Fe₃O₄@Mn₃O₄-PEI-HA NCs by HeLa-HCD44 or HeLa-LCD44 cells. Both kinds of cells were seeded into 12-well plates at a density of 2 × 10⁵ cells per well and cultured overnight to lead the cells to adherence. Then, the medium was replaced with fresh medium containing the Fe₃O₄@Mn₃O₄-PEI-HA NCs at different Mn concentrations (0–0.6 mM) and the cells were further incubated for 6 h. Afterwards, the medium was removed carefully and the cells were washed 3 times with PBS, treated with trypsin, suspended in fresh medium, and counted by hemocytometry. The remaining cells were collected by centrifugation at 1000 rpm for 5 min and lysed with 1.0 mL aqua regia solution for 24 h. The Mn content was determined by ICP-AES after the samples were diluted 2 times with PBS.

In vitro targeted T₁/T₂ MR imaging of cancer cells

HeLa-HCD44 or HeLa-LCD44 cells were seeded into 6-well plates at a density of 2 × 10⁶ cells per well and then cultured at 37 °C and 5% CO₂ for 12 h. After discarding the medium, the cells were incubated with 2 mL fresh DMEM containing the Fe₃O₄@Mn₃O₄-PEI-HA NCs at the Mn concentration of 0, 0.1, 0.2, 0.4, 0.8 mM or Fe concentration of 0, 0.0175, 0.035, 0.07, 0.14 mM for 6 h. Next, the cells were washed with PBS for 3 times, treated with trypsin, collected by centrifugation, and resuspended in 1 mL PBS (containing 0.5% agarose) before MR imaging. Both T₁- and T₂-weighted MR images were acquired using a 1.5 T Signa HDxt superconductor clinical MR system (GE Medical Systems, Milwaukee, WI). The parameters were set as follows: point resolution = 156 mm × 156 mm, section thickness = 0.6 mm, TR = 500 ms, TE = 8.6 ms, and number of excitation = 1 for T₁-weighted MR imaging. To perform T₂-weighted MR imaging, the parameters were set as follows: point resolution = 156 mm × 156 mm, section thickness = 0.6 mm, TR = 3000 ms, TE = 90 ms, and number of excitation = 1.

Statistical analysis

One-way ANOVA analysis was carried out to evaluate the significance of the experimental data. A p value of 0.05 was chosen as the significance level, and the data were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively.

Results and discussion

Synthesis and characterization of Fe₃O₄@Mn₃O₄-PEI-HA NCs

Hydrothermal method has been conveniently used to prepare Fe₃O₄-PEI NPs or Fe₃O₄@Au composite NPs for MR imaging of different biological systems.⁴⁸ Herein, via the same hydrothermal approach, Fe₃O₄@Mn₃O₄-PEI NCs were formed, followed by modification of HA via EDC chemistry (Scheme 1). XRD was firstly carried out to confirm the crystalline structure of the formed Fe₃O₄@Mn₃O₄-PEI NCs (Fig. 1a). The diffraction peaks at 18.33, 29.60, 34.72, 42.62, 52.90 and 61.67° well match the (111), (220), (311), (400), (422) and (440) planes of Fe₃O₄ crystals (JCPDS card no. 19-0629). In addition, the peaks at 18.01, 31.20, 32.34, 35.91, 44.50, 50.10, 54.00, 56.03, 58.50, 59.64, 64.20, 65.70, 67.62, 69.81, 73.10, 74.12, 77.60, 80.51, 84.96, 86.40 and 88.11° represent the (101), (200), (103), (211), (220), (105), (312), (303), (321), (224), (400), (323), (411), (305), (420), (413), (404), (316), (431), (415) and (512) planes of the Mn₃O₄ crystals (JCPDS card no. 24-0734). The results of XRD pattern confirmed the formation of Fe₃O₄@Mn₃O₄ NCs.

Fig. 1 (a) XRD pattern of the Fe₃O₄@Mn₃O₄-PEI NCs; (b) TGA curves of the Fe₃O₄@Mn₃O₄-PEI and Fe₃O₄@Mn₃O₄-PEI-HA NCs.

HA is known to be an efficient target for CD44 receptors overexpressed on the surface of cancer cells,⁵⁴ and HA with a larger M_w has a better targeting ability than the one with a lower M_w.⁴⁷ Herein, HA with an M_w of 31 200 was chosen to be modified onto the surface of the Fe₃O₄@Mn₃O₄-PEI NCs. TGA was used to quantitatively characterize the weight loss of the NCs before and after the modification (Fig. 1b). It is clear that the Fe₃O₄@Mn₃O₄-PEI NCs have a weight loss of 6.70% due to the PEI coating. After further HA modification, the weight loss of the Fe₃O₄@Mn₃O₄-PEI-HA NCs increases to 8.79%. Hence, the HA modification can be deduced to be 2.09%. The surface modification of HA was also confirmed by zeta potential and dynamic light scattering (DLS) measurements (Table 1). The positive surface potential of the Fe₃O₄@Mn₃O₄-PEI NCs (30.93 ± 0.21 mV) switches to be negative after conjugation of HA to form the Fe₃O₄@Mn₃O₄-PEI-HA NCs (−20.37 ± 0.25 mV).⁴⁷ In addition, the hydrodynamic size of the Fe₃O₄@Mn₃O₄-PEI-HA NCs was determined to be 292.47 ± 10.68 nm, which is much larger than that of the Fe₃O₄@Mn₃O₄-PEI NCs (213.86 ± 9.77 nm) before HA modification.

Table 1 The surface potential, hydrodynamic size, and polydispersity index (PDI) of the Fe₃O₄@Mn₃O₄-PEI and Fe₃O₄@Mn₃O₄-PEI-HA NCs. Data are provided as mean ± S.D. (n = 3)

Sample	Surface potential (mV)	Hydrodynamic size (nm)	PDI
Fe3O4@Mn3O4-PEI NCs	30.93 ± 0.21	213.86 ± 9.77	0.28 ± 0.03
Fe3O4@Mn3O4-PEI-HA NCs	−20.37 ± 0.25	292.47 ± 10.68	0.37 ± 0.02

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TEM was used to characterize the morphology of the Fe₃O₄@Mn₃O₄-PEI-HA NCs (Fig. 2a and b). Clearly, the NCs possess a spherical or quasi-spherical shape with a mean size of 23.6 ± 6.5 nm. Some aggregated or interconnected particles can be observed in the TEM image. This is likely due to the TEM sample preparation process that includes an air-drying step of the aqueous suspension of the samples. The air drying process often leads to the aggregation or interconnection of the particles, especially for Fe₃O₄ NPs or Fe₃O₄-based composite NPs synthesized in aqueous phase.⁴⁹ In addition, the size measured by TEM is much smaller than the hydrodynamic size measured by DLS in water. This is because TEM measures a single core particle in a dry state, while DLS measures the sizes of large clusters of particles in aqueous solution that may be composed of many single particles.^47,50 High-resolution TEM image clearly reveals the crystal lattices of metallic oxide and transparent polymer shell (Fig. 2c). What's more, energy-dispersive spectroscopy (EDS) demonstrates the existence of Fe, Mn and O elements in the products (Fig. 2d). The elemental composition of the Fe₃O₄@Mn₃O₄-PEI-HA NCs was further analyzed by ICP-AES, where the Mn/Fe molar ratio was estimated to be 5.73 : 1. Combining the results of TGA, we can calculate that the relative amount of Fe₃O₄, Mn₃O₄, PEI and HA is 13.70%, 77.51%, 6.70% and 2.09% in the NCs, respectively. It should be pointed out that the Fe₃O₄@Mn₃O₄-PEI-HA NCs still display a strong magnetic property even if the content of Fe₃O₄ is very low, since they can be easily collected by a magnet in several minutes (Fig. S1†).

Fig. 2 TEM image (a), size distribution histogram (b), high-resolution TEM image (c), and EDS spectrum (d) of the Fe₃O₄@Mn₃O₄-PEI-HA NCs, respectively.

Prior to biological imaging applications, we checked the colloidal stability of the Fe₃O₄@Mn₃O₄-PEI-HA NCs by DLS (Fig. S2†). The hydrodynamic size of the NCs in water does not have obvious change over a period of 15 days. Besides, after exposing the Fe₃O₄@Mn₃O₄-PEI-HA NCs to water, PBS, or cell culture medium for 15 days, they are still colloidally stable and no precipitate occurs (Fig. S3†). All of these results suggest that the prepared Fe₃O₄@Mn₃O₄-PEI-HA NCs possess an excellent colloidal stability, which is amenable for MR imaging applications.

MR relaxivity measurements

Because of the favorable biocompatibility, high detection accuracy and high T₂ relaxivity, Fe₃O₄ NPs have been used as contrast agents for T₂-weighted MR imaging.⁴⁸ On the other hand, Mn₃O₄ NPs have been reported to be a desired positive contrast agent for T₁-weighted MR imaging.³ As expected, the prepared Fe₃O₄@Mn₃O₄-PEI-HA NCs may be used as a dual mode contrast agent for both T₁- and T₂-weighted MR imaging applications.

To explore the T₁-weighted imaging performance, MR phantom studies of the Fe₃O₄@Mn₃O₄-PEI-HA NCs dispersed in water at different Mn concentrations were investigated (Fig. 3). As shown in Fig. 3a, the MR signal intensity increases as a function of the Mn concentration (0.0125–0.2 mM). The r₁ relaxivity of the Fe₃O₄@Mn₃O₄-PEI-HA NCs was calculated to be 2.15 mM⁻¹ s⁻¹ after linear fitting of the inverse T₁ relaxation time (1/T₁) versus Mn concentration (Fig. 3b). It should be noted that the r₁ relaxivity of the NCs is much higher than that of the previously reported Mn₃O₄ NPs,^3,55,56 likely due to the synergistic effect of Fe₃O₄ and Mn₃O₄ on the r₁ relaxivity in this situation.

Fig. 3 Color T₁-weighted images (a) and linear fitting of 1/T₁ (b) of the Fe₃O₄@Mn₃O₄-PEI-HA NCs with different Mn concentrations. The color bar from blue to red indicates the gradual increase of MR signal intensity.

To demonstrate the potential to use the NCs for T₂-weighted MR imaging, MR phantom studies of the NCs as a function of Fe concentration was also carried out (Fig. 4). T₂-Weighted MR images (Fig. 4a) reveal that the brightness of the MR image becomes darker with the increase of the Fe concentration. Meanwhile, the T₂ relaxation rate (1/T₂) increases linearly with the Fe concentration, and the slope (r₂ relaxivity) is calculated to be 143.26 mM⁻¹ s⁻¹ (Fig. 4b). Considering the high r₁ and r₂ relaxivities, the developed Fe₃O₄@Mn₃O₄-PEI-HA NCs may be used as a contrast agent for dual mode T₁/T₂ MR imaging. It is interesting to note that the Mn/Fe molar ratio was optimized by varying the weight of precursors to have a better T₁- and T₂-weighted MR imaging performance. If the Mn/Fe molar ratio was higher than 5.73, the NCs displayed a reduced r₂ relaxivity. Similarly, lowering the Mn/Fe molar ratio than 5.73 resulted in a reduced r₁ relaxivity.

Fig. 4 Color T₂-weighted images (a) and linear fitting of 1/T₂ (b) of the Fe₃O₄@Mn₃O₄-PEI-HA NCs with different Fe concentrations. The color bar from red to blue indicates the gradual decrease of MR signal intensity.

Cytotoxicity assay

We next evaluated the cytotoxicity of the developed Fe₃O₄@Mn₃O₄-PEI-HA NCs via MTT viability assay of HeLa cells (Fig. 5). The cell viability displays a concentration-dependent effect and the viability of HeLa cells still remains above 80% even at the Mn concentration up to 2.0 mM. Our data clearly indicates that the Fe₃O₄@Mn₃O₄-PEI-HA NCs are non-cytotoxic in the given concentration range.

Fig. 5 MTT assay of HeLa cell viability after treatment with PBS (control) and the Fe₃O₄@Mn₃O₄-PEI-HA NCs at different Mn concentrations (a) and different Fe concentrations (b) for 24 h.

In addition, the morphology of HeLa cells treated with the Fe₃O₄@Mn₃O₄-PEI-HA NCs at different Mn concentrations for 24 h was observed by phase contrast microscopy to further assess the cytotoxicity of the NCs (Fig. S4†). Clearly, the cells display a healthy morphology after treatment with the Fe₃O₄@Mn₃O₄-PEI-HA NCs at the given concentrations, similar to the control cells treated with PBS. Taken together with the results of MTT assay, we can safely conclude that the Fe₃O₄@Mn₃O₄-PEI-HA NCs have an excellent cytocompatibility in the given concentration range.

Cellular uptake assay

The modification of HA onto the NCs is expected to render the NCs with targeting specificity to CD44 receptor-expressing cancer cells. The specific cellular uptake of the Fe₃O₄@Mn₃O₄-PEI-HA NCs by HeLa-HCD44 cells was confirmed by Prussian blue staining (Fig. 6). In contrast to control cells treated with PBS that do not display blue staining (Fig. 6a and d), cells treated with the Fe₃O₄@Mn₃O₄-PEI-HA NCs display the blue staining, and the staining is more obvious with the increase of Mn concentration for both HeLa-HCD44 and HeLa-LCD44 cells (Fig. 6b, c, e and f). More importantly, HeLa-HCD44 cells display much more obvious blue staining than HeLa-LCD44 cells under the same treatment, especially at the high Mn concentration of 1.0 mM (Fig. 6c and f).

Fig. 6 Phase contrast microscopic images of the HeLa-HCD44 cells (a–c) and HeLa-LCD44 cells (d–f) after Prussian blue staining. (a)–(c) Panels show the HeLa-HCD44 cells treated with PBS (a), and the Fe₃O₄@Mn₃O₄-PEI-HA NCs at the Mn concentration of 0.6 mM (b) or 1.0 mM (c). (d)–(f) Panels show the HeLa-LCD44 cells treated with PBS (d) and the Fe₃O₄@Mn₃O₄-PEI-HA NCs at the Mn concentration of 0.6 mM (e) or 1.0 mM (f) for 6 h. The scale bar in each panel represents 100 μm.

The targeted cellular uptake of the Fe₃O₄@Mn₃O₄-PEI-HA NCs by HeLa-HCD44 cells was further quantitatively confirmed by ICP-AES (Fig. 7). The cellular Mn uptake displays a concentration-dependent manner for both cells, and higher Mn concentration of the NCs leads to an increased Mn uptake in the cells. However, the Mn uptake in the HeLa-HCD44 cells is much higher than that in the HeLa-LCD44 cells at the same Mn concentrations. The enhanced cellular uptake of the NCs should be associated with the grafted HA ligands that can direct the specific targeting of the NCs to HeLa-HCD44 cells via ligand–receptor interaction, in agreement with our previous work.^47,54

Fig. 7 Mn uptake by HeLa-HCD44 and HeLa-LCD44 cells after treated with PBS or Fe₃O₄@Mn₃O₄-PEI-HA NCs at the Mn concentration of 0.2, 0.4, or 0.6 mM for 6 h.

Targeted MR imaging of cancer cells

We next explored the feasibility to use the NCs as a probe for targeted dual mode T₁/T₂ MR imaging of cancer cells. We firstly applied them for targeted T₁-weighted MR imaging of cancer cells in vitro (Fig. 8). As shown in Fig. 8a, the MR signal intensity increases with the Mn concentration for both cells, however the increasing trend of HeLa-HCD44 cells is much more significant than that of HeLa-LCD44 cells at the same Mn concentrations. This can be further verified by quantitative analysis of the MR signal intensity (Fig. 8b).

Fig. 8 T₁-Weighted MR images (a) and MR signal intensity analysis (b) of HeLa-HCD44 and HeLa-LCD44 cells after treated with the Fe₃O₄@Mn₃O₄-PEI-HA NCs at different Mn concentrations for 6 h.

Different from T₁-weighted MR imaging, the same probe is also able to decrease the MR signal intensity of cancer cells with the Fe concentration in the T₂-weighted MR images (Fig. 9a). Thanks to the HA-mediated targeting, HeLa-HCD44 cells display a more significant MR signal intensity dropdown than HeLa-LCD44 cells at the same Fe concentrations. By plotting the MR signal intensity of the cells as function of Fe concentration (Fig. 9b), we can see that the MR signal intensity of both treated cells is much lower than that of the control cells treated with PBS, and the MR signal intensity decreases with the Fe concentration. Compared with HeLa-LCD44 cells, the HeLa-HCD44 cells show a much lower MR signal intensity at the same Fe concentrations. Our results suggest that with the HA-mediated targeting, HeLa-HCD44 cells display enhanced uptake of the NCs, thus allowing for their effective targeted T₁/T₂ MR imaging.

Fig. 9 T₂-Weighted MR images (a) and MR signal intensity analysis (b) of HeLa-HCD44 and HeLa-LCD44 cells after treated with the Fe₃O₄@Mn₃O₄-PEI-HA NCs at different Fe concentrations for 6 h.

Conclusion

In summary, we successfully synthesized the Fe₃O₄@Mn₃O₄-PEI-HA NCs for targeted T₁- and T₂-weighted MR imaging of cancer cells. Under a hydrothermal condition, Fe₃O₄@Mn₃O₄-PEI NCs can be synthesized using mixed Fe and Mn precursors. Further conjugation of HA via EDC chemistry leads to the formation of the Fe₃O₄@Mn₃O₄-PEI-HA NCs with good water dispersibility, colloidal stability, and cytocompatibility in the given concentration range. Because of the co-existence of both Fe₃O₄ and Mn₃O₄ in the NCs, the developed probe possess a high r₁ (2.15 mM⁻¹ s⁻¹) and r₂ (143.26 mM⁻¹ s⁻¹) relaxivities. The developed NCs are able to be used as an effective probe for targeted dual mode T₁/T₂ MR imaging of cancer cells overexpressing CD44 receptors thanks to the attached HA ligand. The developed Fe₃O₄@Mn₃O₄-PEI-HA NCs may hold a great promise to be used for targeted dual mode T₁/T₂ MR imaging of other CD44 receptor-expressing cancer cells in vitro and in vivo.

Acknowledgements

This research is financially supported by the Science and Technology Commission of Shanghai Municipality (15520711400 for M. Shen), the Sino-German Center for Research Promotion (GZ899, for X. Shi), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of High Learning (for X. Shi), and the Fundamental Research Funds for the Central Universities (for M. Shen).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra05648b

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