Melanin-manganese nanoparticles with ultrahigh efficient clearance in vivo for tumor-targeting T₁ magnetic resonance imaging contrast agent

Abstract

Endogenous biomaterials in organisms, with native biocompatibility and biodegradability, appear more advantageous in the development of nanoscale diagnostic and therapeutic systems for future clinical translation. Herein, a novel tumor-targeting Magnetic Resonance Imaging (MRI) contrast agent was developed based on Mn²⁺-chelating ultrasmall water-soluble melanin nanoparticles (MNP-PEG-Mn). The nanoparticles, with a size of about 5.6 nm, presented high chelation stability and showed negligible cytotoxicity as estimated by MTT assay. Moreover, the r₁ longitudinal relaxivity (20.56 mM⁻¹ s⁻¹) of MNP-PEG-Mn was much higher than that of Gadodiamide (6.00 mM⁻¹ s⁻¹), which is a clinically approved MRI contrast agent. In vivo MRI experiments revealed excellent tumor-targeting specificity after tumor-bearing mice were intravenously injected with MNP-PEG-Mn. Additionally, MNP-PEG-Mn could be excreted via renal and hepatobiliary pathways with negligible toxicity to body tissues. These preliminary results indicated the clinically translatable potential of MNP-PEG-Mn as a T₁ MRI contrast agent for tumor-targeted imaging.

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1. Introduction

Magnetic resonance imaging (MRI), as one of the most prominent clinical imaging techniques, providing noninvasive anatomical and physiological information with high spatial and temporal resolution, has shown great potential in the early detection and accurate localization of tumors for decreasing tumor mortality.^1–3 In particular, the rapid advancement of MRI has been accelerated by the development of exogenous contrast agents that can effectively improve diagnostic sensitivity and accuracy by shortening the relaxation times of the protons in water.^4,5 Among MRI contrast agents, gadolinium chelates have been widely used and studied in the clinic due to their predominant positive signal enhancement.^6–8 However, some studies have demonstrated that Gd-based contrast agents increased the risk of nephrogenic systemic fibrosis in patients, which is a rare, idiopathic systemic fibrosing disorder that can be critical to patients with acute or chronic kidney disease, or severely impaired renal function.^9,10 Furthermore, it is also reported that the use of Gd-based contrast agents could lead to brain abnormalities in healthy patients.¹¹ Manganese (Mn)-based contrast agents, as alternatives to Gd-based contrast agents, have been receiving more attention lately for use in MRI as they have better biosafety.^12–14 Manganese is not only a natural cellular constituent but also plays a critical role in cell and mitochondrial function.¹⁵

In recent years, numerous Mn-based nanoscale MRI contrast agents have been developed. Some manganese complexes based on small molecules have attracted considerable interest,^16,17 however low sensitivity, non-specificity and fast elimination from the circulation system still remain challenging problems. Besides, various morphologies of manganese oxide, such as nanoplates,^18,19 mesoporous silica-coated hollow nanoparticles²⁰ and PLGA encapsulated nanocrystals,²¹ have also been reported for MRI due to their potential physiological function and high resolution.^22,23 These nanoscale systems showed enhanced relaxivity and high accumulation in tumors via enhanced permeability and retention (EPR) effects, promoting their potential as MRI contrast agents for cancer diagnosis. However, the non-biodegradability and potential long-term toxicity of these inorganic nanosystems have remarkably impeded their progress for future clinical translation. Endogenous biomaterials in organisms, with native biocompatibility and biodegradability, appear more advantageous and may address the biosafety issues in vivo. Nevertheless, most organic structures, such as cells²⁴ and ferritines,²⁵ need complicated and time-consuming synthetic procedures to develop them into MRI contrast agents, due to their lack of contrast properties. Thus, exploring endogenous materials with high contrast properties is more promising for in vivo MRI and clinical translation.

Melanin, an irregular biopolymer and a ubiquitous natural pigment, presents in many organisms. It has attracted much attention because of its good biocompatibility and biodegradability, contrast properties and metal ion chelation, which are appealing for the fabrication of MRI contrast agents. For example, Ju and co-workers prepared melanin-like nanoparticles and chelated Fe³⁺ for an MRI contrast agent.²⁶ Nevertheless, a good MRI contrast agent not only needs high relaxivity, excellent tumor-targeting ability and good biosafety, but rapid clearance properties also remain an important goal. The large size and poor solubility of the above-mentioned melanin-like nanoparticles apparently increased the difficulty of metabolising the nanoparticles and led to their accumulation in vivo. Recently, we prepared ultrasmall water-soluble melanin nanoparticles (MNPs) that preserve many of the properties of melanin, and can be used as a good endogenous nanoplatform for tumor multimodality imaging and imaging-guided therapy.^27–29 The ultrasmall scale nanostructures have strong surface effects, which allow for enhanced exposure of metal ions and further complexation with more hydrophilic ligands. The high surface area and increased number of hydrophilic ligands enhance the relaxivity of the MRI contrast agent.³⁰ Meanwhile it has been reported that nanoparticles smaller than 6 nm in diameter can undergo renal clearance.^31,32 Therefore, it is highly desirable to explore an ultrasmall water-soluble melanin-Mn based MRI contrast agent with highly efficient clearance for tumor-targeting imaging, while the correlative research has not yet been reported to the best of our knowledge.

Herein, a novel MRI contrast agent has been constructed based on ultrasmall PEG-modified MNP and chelated Mn²⁺ (MNP-PEG-Mn). A schematic illustration of the preparation process is shown in Fig. 1. The physicochemical properties and in vitro behaviors of MNP-PEG-Mn were thoroughly examined with transmission electron microscopy (TEM), dynamic light scattering (DLS), relaxivity measurements, MTT assays, and confocal microscopy. Subsequently, the in vivo MRI performance was studied on tumor-bearing mice. Interestingly, the nanoparticles show significant signal enhancement in tumors and could be entirely cleared by the liver and kidneys. All of these properties make MNP-PEG-Mn highly promising as an MRI contrast agent for potential clinical translation.

Fig. 1 Schematic illustration of the construction of tumor-targeting MRI contrast agent with ultrahigh efficient clearance in vivo, based on Mn²⁺-chelating melanin nanoparticles.

2. Experimental

2.1. Materials

Melanin was acquired from Sigma-Aldrich. Sodium hydroxide (NaOH), hydrochloric acid (37 wt% HCl) and MnCl₂·4H₂O were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. Amine-PEG₅₀₀₀-amine (NH₂-PEG₅₀₀₀-NH₂, 5 kDa) was obtained from Shanghai Zzbio Co., Ltd. All of the reagents were purchased and used without further purification. Phosphate buffered saline (PBS) and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Boster Biological Technology Co., Ltd.

2.2. Preparation of MNP-PEG-Mn

MNPs were prepared according to a previously reported method with slight modifications.²⁷ Briefly, 10 mg Melanin was dissolved in 3 mL of 0.1 M NaOH aqueous solution under vigorous stirring. After dissolving, HCl aqueous solution (0.1 M) was swiftly dropped into the basic melanin solution to adjust the pH to 7.5 under sonication for 2.5 min. A dark black melanin aqueous solution was obtained. The neutralized solution was further centrifuged using 30 kDa molecular weight cut off (MWCO) filters and washed with deionized water, this was repeated several times to remove the NaCl and other small melanin fragments that were produced. The aqueous solvent was removed by freeze-drying to obtain MNPs.

For good biocompatibility, the commercially available NH₂-PEG₅₀₀₀-NH₂ was used to modify the surface of the MNPs. 0.1 M NaOH solution was added to the MNP aqueous solution (1 mg mL⁻¹ of water) to adjust the pH of the solution to 9.5. 20 mg NH₂-PEG₅₀₀₀-NH₂ aqueous solution with pH = 9.5 was added dropwise to the MNP aqueous solution. After vigorous stirring for 12 h, the PEG-modified MNPs were centrifuged and washed several times to remove the free NH₂-PEG₅₀₀₀-NH₂. The resulting products, labeled MNP-PEG, were obtained by freeze-drying for further application.

1 mL of 2 mg mL⁻¹ fresh MnCl₂ solution was added to the MNP-PEG (2 mg mL⁻¹) aqueous solution to chelate Mn²⁺. After incubation for 1 h at room temperature, the complexes were washed with water several times and then purified using a PD-10 column. The products, marked as MNP-PEG-Mn, were washed out by PBS solution and passed through a 0.22 μm Millipore filter into a sterile tube for in vitro and animal experiments.

The stability of manganese ion-chelated MNP-PEG was studied by incubating MNP-PEG-Mn in a PBS solution (pH = 7.4) and acetic acid-sodium acetate buffer solution (pH = 5.5) at 37 °C. MNP-PEG-Mn was placed in a dialysis tube (MWCO 3500D) with magnetic stirring. At a certain time, the dialysate was removed and replaced with fresh PBS solution or acetic acid-sodium acetate buffer solution. The Mn²⁺ concentration in the dialysate was measured by inductively coupled plasma mass spectrometry (ICP-MS, Thermal Elemental X7, Thermo Electron Co., USA) analysis. To evaluate the physiological stability, MNP-PEG-Mn (1 mg) was dispersed into a solution with 50% fatal bovine serum (FBS, 5 mL) and 50% PBS solution (5 mL), and then incubated at 37 °C for 24 h. Its hydrodynamic size was determined at 0 h, 2 h, 4 h, and 24 h by DLS measurements.

2.3. Characterization of MNP-PEG-Mn

TEM images were recorded on a JEM-2100F microscope with an accelerating voltage of 200 kV. The hydrodynamic size and zeta potential were investigated using a Nano-Zetasizer system (Malvern Instruments Ltd). The chemical properties of MNP-PEG-Mn were measured by an iS50 Fourier transform-infrared (FT-IR) spectrometer using the KBr method in transmission mode in the range 500–4000 cm⁻¹.

2.4. Cell culture and animal tumor model

The human laryngeal cancer Hep-2 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The Hep-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplemented with 10% (v/v) fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and 100 μg mL⁻¹ penicillin/streptomycin (HyClone, Logan, UT, USA) in a humidified incubator at 37 °C under 5% CO₂. The cell morphology was monitored daily and cell passaging was done every 3 days at 50% confluence. For cell passaging and cell experiments, cells were detached by incubating with trypsin and collected via centrifugation (5 min, 1000 RPM, 4 °C).

Female nude mice (5–7 weeks, 18 ± 2 g) were purchased from Hunan slack King experimental animal Co. and housed in an isolated animal room with water and rodent food supplements to acclimatize for 2 weeks before use. All animal experiments were conducted in accordance with the relevant laws and institutional guidelines following the approval of the Ethics Committee of the Chinese Academy of Sciences. To induce a tumor, 1 × 10⁶ KB Hep-2 cells were suspended in 100 μL of PBS solution and injected subcutaneously into the flank region of the nude mice. The tumor size was measured periodically using a slide caliper, the animals were observed daily for any behavioral abnormalities and weighed weekly. When the volume of the tumors approached 150 mm³, the mice were used for the MRI studies.

2.5. In vitro cytotoxicity assay

The in vitro cytotoxicity of MNP-PEG-Mn was measured in Hep-2 cells and NIH 3T3 cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were seeded in 96-well plates at a density of 8000 cells per well and incubated for 24 h at 37 °C under 5% CO₂ to allow the cells to attach before the experiment. Then, the cells were washed with PBS solution and replaced with the complete medium with a series of concentrations of MNP-PEG and MNP-PEG-Mn (1600, 800, 400, 200, 100, 50, 25, and 0 μg mL⁻¹) for another 24 h. The culture medium was discarded and the cells were subsequently washed several times with PBS solution. Fresh culture medium, containing 20 μL of MTT solution (5 mg mL⁻¹), was added to each well. After incubation for 4 h in the CO₂ incubator, the MTT solution was carefully removed and replaced with 100 μL of dimethyl sulfoxide. Finally, the absorbance of each well was measured with a microplate reader, using a wavelength of 550 nm as the test wavelength and 650 nm as the reference wavelength (Infinite M1000; TECAN, Morrisville, NC, USA). The absorbance of the untreated cells was used as a control and its absorbance was used as the reference value for calculating 100% cellular viability. All of the experiments were independently performed three times.

2.6. Cellular uptake studies

Confocal fluorescence imaging in vitro. 30 μL Rhodamine aqueous solution (5 mg mL⁻¹) was added to 1 mL of 5 mg mL⁻¹ MNP-PEG-Mn aqueous solution and the mixture was stirred for 1 h at 40 °C to obtain the MNP-PEG-Mn-rhodamine chelator. Then, the chelator was centrifuged using a 30 kDa MWCO filter and washed several times with ultrapure water to remove unchelated rhodamine. The Hep-2 cells were incubated with MNP-PEG-Mn-rhodamine (200 μg mL⁻¹) at 37 °C in a 5% CO₂ incubator for 4 h. After fixing with 4% paraformaldehyde in PBS solution for 20 min at room temperature, the cells were washed with PBS solution and stained with DAPI (4′,6-diamidino-2-phenylindole) for visualization of the nuclei. Finally, fluorescence microscopy images were acquired using a confocal laser scanning microscope (CLSM, FV1200, Olympus, Japan). Rhodamine was excited with light at 517 nm and DAPI was excited at 358 nm.

TEM in vitro. To further study the cellular uptake and confirm the cellular localization, thin slides of cells treated with MNP-PEG-Mn were visualized using TEM. Hep-2 cells were incubated with MNP-PEG-Mn (200 μg mL⁻¹) for 24 h. Then, cells were washed with PBS solution and centrifuged at 1500 rpm for 5 min. The resulting cell pellets were fixed with 2% glutaraldehyde for 2 h and washed with PBS solution several times. After post-fixation with 1% osmium tetroxide for 1 h, they were successively transferred to a series of graded acetone solutions for dehydration. The final pellets were cured in a vacuum oven using Epon 618 at 60 °C and sectioned using a Leica EM UC6 Ultramicrotome and imaged using a JEM-1011 TEM at 80 kV.

2.7. In vitro MRI study

In vitro MRI experiments were performed at 25 °C in a 3.0 T MR clinical scanner (Magnetom Trivo with Tim, Siemens, Germany) with a wrist surface coil (diameter: 11 cm). For the measurement of T₁, relaxivity (r₁) and T₁-weighted MR images, 200 μL of MNP-PEG-Mn and Gadodiamide aqueous solutions with different concentrations (0.025, 0.05, 0.1 and 0.2 mM) were transferred to tubes, and the data were acquired with spin-echo acquisition on a NMR-analyzer (GY-PNMR-10). The imaging parameters were as follows: field of view (FOV), 90 × 60 mm²; slice thickness, 1.5 mm; spacing, 0.5 mm; base matrix resolution, 256 × 256; repetition time (TR), 500 ms; and effective echo time (TE), 20 ms. The r₁ values were calculated from the slope of the curve-fitting of 1/T₁ (s⁻¹) versus the metal ion concentration (mM).

2.8. In vivo MRI study

The animal study was approved by the Institutional Animal Use and Care Committee of Shan Xi Medical University (approval no. 2016LL141). The tumor-bearing mice were anesthetized using an intraperitoneal injection of 1% pentobarbital sodium (40 mg kg⁻¹), then the axial and coronal MR images of the tumor-bearing mice were collected as the data of pre-injection. Afterward, the mice (n = 3) were treated with 200 μL of the sample in PBS solution (8 mg mL⁻¹ based on the MNP concentration) through tail intravenous injection. The axial images of the tumor location were collected at 0.5 h, 24 h and 48 h, and meanwhile, the coronal images were also obtained at particular time intervals (0.5 h, 1 h, 24 h, 48 h, 72 h and 120 h) using 3.0 T clinical MRI equipment (Trivo, Siemens) with a wrist surface coil (diameter: 11 cm). The imaging sequence parameters were as follows: FOV, 90 × 60 mm²; slice thickness, 5 mm; spacing, 0.5 mm; base resolution matrix, 256 × 256; TR, 500 ms; and TE, 20 ms. Imaging analysis was performed using Image J software.

2.9. In vivo toxicity assessment

Distribution of the MRI contrast agent in tissues. To study the biodistribution of our contrast agent in mice, the major organs and tissues (heart, liver, spleen, lung, kidney and tumor) from mice (n = 3) were collected at 24 h, 5 days, and 30 days. After injection into the tail vein, the mice were sacrificed at the indicated time point and the organs were dissected, weighed and stored at −20 °C before analysis. For ICP-MS experiments, the collected organs were wet-weighted and solubilized in chloroazotic acid under heating at the boiling point until digestion was complete. After cooling to room temperature, the solution in each flask was diluted to 10 g with 2% HNO₃. A series of Mn standard solutions (0, 0.005, 0.01, 0.02, 0.1, 0.5, 1, 5 and 10 ppm) were prepared with the above solution. Both the test and standard solutions were measured by ICP-MS. The blood samples were collected at 24 h and 5 days after injection. The blood samples from the healthy mice without injection were used for comparison. The blood urea nitrogen (BUN) and serum creatinine (SCr) in the blood were measured.

Histopathological analysis. For the histology analysis, three mice from 5 days post-injection of MNP-PEG-Mn, and three age-matched healthy mice used as the control, were sacrificed. The harvested organs were fixed in 4% formalin solution at room temperature for 48 h, embedded in paraffin blocks, and sectioned at a thickness of 8 μm. Slices of the organs, including the heart, liver, spleen, lung and kidney, were stained with hematoxylin and eosin (H&E) and observed by an optical microscope (CX-31, Olympus, Japan).

2.10. Statistical analysis

The variance and t tests were used to analyze the data. The data are presented with their mean and standard deviation (SD), and p < 0.05 was considered as statistically significant. All statistical analyses were performed using SPSS 22.0 for Windows (Chicago, IL, USA).

3. Results and discussion

3.1. Preparation and characterization of MNP-PEG-Mn

The ultrasmall water-soluble MNPs were prepared according to a previous method,²⁷ which preserved good biocompatibility and biodegradability, high contrast properties and excellent metal ion chelation. To retain the water-solubility, prolong the circulation time in the blood and further improve the in vivo behavior, polyethylene glycol (PEG) was introduced into the ultrasmall MNPs, which was a simple, time-saving, and low energy-consuming modification process.³³ The successful PEGylation can be verified by the FT-IR spectra (Fig. S1†); the characteristic absorption peaks of MNP-PEG at 2880 cm⁻¹ belong to the alkyl C–H stretching and the peak at 1110 cm⁻¹ is attributed to the C–O–C stretching in the PEG molecules. Fig. S2†shows a representative TEM image and size distribution of MNP-PEG, which suggest that MNP-PEG was well dispersed with uniform size and shape, and the mean hydrodynamic diameter was about 3.6 nm. The zeta potential of MNP-PEG was measured to be −25.4 ± 2.3 mV. The MNPs can bind with metal ions owing to the existence of catechol groups.^27,28,34 After chelation of Mn²⁺, the results of TEM and DLS measurements are shown in Fig. 2a–c. The MNP-PEG-Mn was still monodispersed, while the average diameter became larger and increased to approximately 5.6 nm (Fig. S3†). Moreover, the surface potential decreased to −13.5 ± 0.6 mV because of the introduction of positive Mn²⁺. The number of Mn²⁺ ions chelated to the MNP-PEG was calculated to be about 55 per MNP.

Fig. 2 Characterization of physical properties. (a and b) TEM images and (c) DLS distribution of MNP-PEG-Mn. (d) Mn²⁺ stability study of MNP-PEG-Mn in PBS solution (pH = 7.4) and acidic buffer solution (pH = 5.5).

To check the leakage of Mn²⁺ from MNP-PEG-Mn, the dialysis experiments of MNP-PEG-Mn in PBS solution (pH = 7.4) and acidic buffer solution (pH = 5.5) were performed. The results indicated that only about 1% of the Mn²⁺ ions were released from MNP-PEG-Mn in the first 2 h, and there was no further release at longer incubation time periods, even after 48 h (Fig. 2d), indicating the strong chelating stability of MNP-PEG-Mn. The metal ions released in the first 2 h may derive from those that were absorbed on MNP-PEG-Mn through weak electrostatic interactions. The physiological stability of MNP-PEG-Mn was investigated by measuring the change in the hydrodynamic size distribution of the nanoparticles in serum and the results are shown in Fig. S4.†The hydrodynamic size of MNP-PEG-Mn showed little significant change after incubation for 24 h in serum, indicating good physiological stability.

3.2. In vitro cytotoxicity assay

Before in vivo application of MNP-PEG-Mn, Hep-2 cells were employed to evaluate the cytotoxicity of MNP-PEG and MNP-PEG-Mn with various concentrations over 24 h using a standard MTT assay. The standard non-cancerous cells (NIH 3T3 fibroblasts) were used as a normal contrast group and the results are shown in Fig. 3 and Fig. S5.†The samples presented negligible cytotoxicity before and after Mn²⁺ chelation, whether using Hep-2 cells or NIH 3T3 cells, even at manganese concentrations up to 1600 μg mL⁻¹. This favorable result may be attributed to the low cytotoxicity and the good native biocompatibility of melanin. Thus, the in vitro cell viability results showed that MNP-PEG-Mn can be used as an MRI contrast agent for in vivo tumor diagnosis.

Fig. 3 In vitro experiments. Relative viabilities of Hep-2 cells incubated with MNP-PEG and MNP-PEG-Mn nanoparticles at various concentrations for 24 h using a MTT assay.

3.3. Cellular uptake studies

The cellular uptake behaviors and subsequent localization of MNP-PEG-Mn have been estimated by CLSM and bio-TEM after Hep-2 cells were incubated with 200 μg mL⁻¹ MNP-PEG-Mn for 24 h. The CLSM images of MNP-PEG-Mn chelated with rhodamine are shown in Fig. 4a–c. MNP-PEG-Mn can be clearly seen in the cytoplasm after incubation for 24 h, as shown by the red fluorescence from rhodamine lighting up the nuclei, suggesting the existence of Hep-2 cells which uptake the chelated nanoparticles.

Fig. 4 (a–c) CLSM images of MNP-PEG-Mn-rhodamine in vitro, (a) rhodamine chelated MNP-PEG-Mn (red) in the cytoplasm, (b) cell nuclei stained by DAPI (blue), and (c) merged image. (d and e) TEM images of Hep-2 cells incubated with 200 μg mL⁻¹ MNP-PEG-Mn for 24 h, (d) scale bar = 1 μm and (e) magnified image in scale of 200 nm. The nanoparticles can be detected in the organelles (pink arrows).

To further understand the detailed localization of MNP-PEG-Mn after cellular uptake, Hep-2 cells treated with the same concentration of MNP-PEG-Mn for a period of 24 h were fixed, and infiltrated with resin. Then, thin sections of the fixed cells were imaged using TEM, as shown in Fig. 4d and e. The darker contrast in various regions indicates a high level of aggregation of nanoparticles within the cells. As the magnification was increased, MNP-PEG-Mn could be clearly seen within the organelles, which was likely taken up via passive endocytosis where the nanoparticles were engulfed into the cell and located within endosomes or lysosomes.

3.4. MRI in vitro

To explore the potential of MNP-PEG-Mn as a T₁ MRI contrast agent, the longitudinal relaxivity (r₁), which reflects the ability to shorten the T₁ longitudinal relaxation time of water protons, was measured on a 3 T MRI scanner at 35 °C. At the same time, Gadodiamide, a commercial MRI contrast agent used in clinics, was selected as a control. The final values were calculated from the slope of the curves in Fig. 5a. It can be seen that the MR signal significantly enhanced with increasing concentrations of MNP-PEG-Mn, suggesting the nanoparticles generated a high magnetic field gradient on their surface. The r₁ value of MNP-PEG-Mn is 20.57 mM⁻¹ s⁻¹, which is about 3.4 times higher than commercial Gadodiamide (6.00 mM⁻¹ s⁻¹). The high relaxivity of MNP-PEG-Mn could be attributed to the strong surface effects of the ultrasmall scale nanostructures, which exposed more metal ions and chelated with hydrophilic ligands. Further research on the T₁-weighted images (Fig. 5b) revealed that MNP-PEG-Mn presented significantly brighter images than Gadodiamide at the same metal ion concentration. These results suggested that MNP-PEG-Mn had better MRI enhancement than Gadodiamide.

Fig. 5 In vitro MRI studies of MNP-PEG-Mn and Gadodiamide. T₁ relaxation rate measurements (a) and T₁-weighted imaging (b) at various concentrations of metal ions (0.2 mM, 0.1 mM, 0.05 mM, and 0.025 mM). The measurements were performed on a 3 T MRI scanner at 35 °C.

3.5. MRI in vivo

It has been reported that ultrasmall-sized nanoparticles can cross the blood-brain barrier and accumulate in the tumor region by the EPR effect.⁶ The in vivo performance of MNP-PEG-Mn, used as a tumor-targeting MRI contrast agent, was evaluated with 3.0 T clinical MRI equipment using tumor-bearing mice. Samples were intravenously injected into the mice at a melanin concentration of 1.6 mg per mouse, and the axial T₁-weighted MR images were acquired pre-injection and at appropriate time intervals (0 h, 0.5 h, 24 h and 48 h) after injection (as shown in Fig. 6). It can be seen that obvious enhanced signal and prominent positive contrast effects were achieved in the tumor region at 0.5 h after the injection of the samples, suggesting a great number of nanoparticles have accumulated according to the EPR effect. The MR signals of the tumor tissue became slightly weaker at the 24 h time interval, but could still be clearly detected. Finally, the signal showed a sharp decrease at 48 h, due to the metabolism of MNP-PEG-Mn.

Fig. 6 T ₁-Weighted axial MR images of tumor-bearing mice at different time intervals, (a) 0 h, (b) 0.5 h, (c) 24 h and (d) 48 h, after the intravenous injection of MNP-PEG-Mn using 3.0 T clinical MRI equipment. The white circles highlight the tumor sites.

To better understand the change of the MR signal in the location of the tumor, the signal intensities of the tumor and the muscle, estimated by Image J Software, were quantitatively analyzed. On the whole, the tumor without injection showed a lower signal intensity of 458 ± 2% and the muscle showed a signal intensity of 355 ± 3% as the control. The intensity value at the tumor site increased to 567 ± 7% in 0.5 h after injection, while the intensity in the muscle only showed a small increase of 361 ± 2%, indicating high uptake in the tumor. After 24 h, the tumor site had a reduced signal of about 530 ± 4%, while the signal value of the muscle did not significantly change. Finally, the values of the tumor and the muscle decreased at 48 h to 455 ± 2% and 358 ± 4% respectively, similar to the values pre-injection. According to the published results, the high intensity value of 567 ± 7% for the tumor is sufficient to detect the location of tumor tissues.^8,35 Therefore, MNP-PEG-Mn exhibited excellent contrast effects and high tumor-targeting ability.

To study the metabolic behavior of the nanoparticles in vivo, coronal images were obtained at the same time points, as shown in Fig. 7a–g. Impressively, a high T₁-weighted contrast of the livers and kidneys can be observed, in which the contrast of the renal pelvis is visible after 0.5 h and lights up 1 h post-injection. Moreover, the kidney showed higher brightness than the liver, suggesting that most of the ultrasmall nanoparticles are metabolized by the kidney, which is in agreement with previous reports.^31,32,36 As the kidneys filter metabolites from the bloodstream through the basal lamina, which has pores of approximately 10 nm, some small nanoparticles show enhanced renal accumulation and long retention time (∼24 h), which are also beneficial for kidney imaging. Thus MNP-PEG-Mn can not only accumulate in tumors and allow for tumor imaging due to its EPR effect, but also can serve as a kidney-targeting MRI contrast agent. After 48 h, the MR signals of the liver and kidneys remarkably decreased, and the MR images at the 72 h time point showed little difference compared with those pre-injection, indicating MNP-PEG-Mn could be completely excreted via renal and hepatobiliary pathways.

Fig. 7 T ₁-Weighted MR coronal images of tumor-bearing mice at different times (a–g) after the intravenous injection of MNP-PEG-Mn using 3.0 T clinical MRI equipment. The livers and kidneys are highlighted by yellow arrows and circles, respectively. (h) Quantification analysis of the T₁-weighted MR signals in livers and kidneys.

For quantitative analysis, the signal intensity of the region of interest on the liver and kidney of each T₁-weighted MR image was measured, as shown in Fig. 7h. The average intensity of the liver increased immediately from 452 ± 23% at 0 h to 778 ± 34%, and the intensity of the kidneys enhanced from 512 ± 40% at 0 h to 982 ± 30% at 0.5 h after the injection of MNP-PEG-Mn. The signal intensity of the kidneys continually increased to 1080 ± 45%, while that of liver decreased to 685 ± 26% at 1 h, further confirming that the nanoparticles can be eliminated by the liver and kidneys, especially by the kidneys. After 48 h, the signal contrast was not obvious compared with the image taken pre-injection, indicating that most nanoparticles had been cleared from the body. Therefore, MNP-PEG-Mn not only displayed excellent contrast properties, but also could be very efficiently excreted via the liver and kidney pathways after the MR imaging in vivo, this reduces the toxic effects caused by the long-term accumulation of conventional nanoparticles in various organs, and enhances the biosafety.

The biodistribution of the tumor-bearing mice (n = 3) after tail vein injection with the same concentration of the MRI contrast agent MNP-PEG-Mn after 1, 5 and 30 days is shown in Fig. 8. The mice were sacrificed and then the Mn²⁺ levels were measured in various organs using ICP-MS. The results revealed that the nanoparticles were mainly cleared by the kidneys and liver, which is consistent with the MRI results in vivo. The accumulation in various organs after 1 day is obviously higher than that after 5 days and 30 days. With increasing time, the nanoparticles were gradually cleared. At 5 days, only the kidneys had a small accumulation of 1.26 ± 0.68 %ID g⁻¹, and the nanoparticles in the various organs were undetectable at 30 days. The results suggested that most of the MNP-PEG-Mn could be excreted mainly through the renal and hepatobiliary systems after 5 days.

Fig. 8 The biodistribution of MNP-PEG-Mn in tumor-bearing mice, represented by the Mn percentage in each tissue. Groups of mice (n = 3) were sacrificed at 1, 5 and 30 days after tail vein injection and the Mn²⁺ levels in various organs were measured using ICP-MS.

3.6. Toxicity studies

The in vivo toxicity of MNP-PEG-Mn was also investigated in detail. By intravenous injection of MNP-PEG-Mn, at a dose of 3.2 mg per mouse, which is 2 times that of the in vivo imaging dose, no noticeable abnormal behavior and weight change (as shown in Fig. 9a) were observed among those mice receiving a high dose of MNP-PEG-Mn. Three mice were sacrificed at 24 h post-injection of MNP-PEG-Mn. The photos of the harvested organs including the heart, liver, spleen, lung and kidneys are shown in Fig. 9b. No remarkable differences were observed except in the kidneys. The kidneys became black because most of the melanin nanoparticles are cleared by the kidney (Fig. 7 and 8). The blood chemistry examination serum creatinine (SCr) and urea nitrogen (BUN), the specific serological markers for kidney function, were measured for the blood samples of the above mice. The detection indexes of SCr and BUN remained within the normal ranges (Fig. 9c and d), suggesting that intravenous injection of MNP-PEG-Mn had no major renal toxicity. To further confirm the toxicity of MNP-PEG-Mn, the main organs were analyzed by H&E staining, as shown in Fig. 9e. The results indicated that MNP-PEG-Mn has no appreciable adverse effect on these main organ tissues. In short, these in vivo results validated that MNP-PEG-Mn showed excellent biocompatibility and therefore could be used as a promising contrast agent for MRI.

Fig. 9 (a) The weight change of mice (n = 3) over 30 days after the tail vein was injected with MNP-PEG-Mn and PBS. (b) Photos of major organs from healthy control mice and MNP-PEG-Mn-injected mice after 24 h. Blood chemistry indexes of the tumor-bearing mice before and after being treated with MNP-PEG-Mn. Time course changes of (c) serum creatinine (SCr) and (d) blood urea nitrogen (BUN). (e) H&E-stained images of major organs from healthy control mice and MNP-PEG-Mn-injected mice after 24 h. The scale bars of the H&E-stained images are 200 μm.

4. Conclusions

In summary, a natural biopolymer system based on Mn²⁺-chelating ultrasmall water-soluble melanin nanoparticles were developed as a T₁ MRI contrast agent for tumor-targeted imaging. The melanin nanoparticles exhibited excellent biosafety due to being abundantly present in many organisms. Furthermore, MNP-PEG-Mn had a much higher relaxivity than that of commercial Gadodiamide. More attractively, the results of in vivo MRI displayed good tumor-targeting specificity, and the nanoparticles could be very efficiently eliminated by both the renal and hepatobiliary pathways, avoiding long-term accumulation. Therefore MNP-PEG-Mn showed great translation potential as an MRI contrast agent for tumor diagnosis.

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

This work has been financially supported by the National Natural Science Foundation of China (Grant No. 51372260, 51132009, 51402338, 81471730 and 81571747), the Shanghai Excellent Academic Leaders Program (Grant No. 16XD1404000), Overseas students science and technology Projects, Shanxi Scholarship Council of China (No. 2015057) and the Key research and development project of Shanxi Province (No. 2016021).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: FT-IR spectra of MNP, MNP-PEG and NH₂-PEG₅₀₀₀-NH₂ (Fig. S1). TEM image and hydrodynamic size distribution graph of MNP-PEG (Fig. S2). The hydrodynamic size distribution of MNP-PEG-Mn in intensity and volume modes (Fig. S3). The hydrodynamic size distribution of MNP-PEG-Mn during different incubation periods with serum (Fig. S4). Cell viabilities of the NIH 3T3 cells after 24 h incubation with MNP-PEG-Mn at various concentrations determined by MTT assay (Fig. S5). See DOI: 10.1039/c7bm00635g

‡ These authors contributed equally to this work.

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