T₁–T₂ molecular magnetic resonance imaging of renal carcinoma cells based on nano-contrast agents

Introduction

Among the various biomedical imaging modalities, magnetic resonance imaging (MRI) – which provides high-resolution anatomical images in a noninvasive manner – is one of the most powerful tools.^1,2 In 40%–50% cases of clinical application, magnetic resonance (MR) contrast agents (CAs) are introduced to enhance the contrast, presenting improved sensitivity and quality of the images for more accurate diagnosis.³ MRI CAs, generally in the form of T₁-positive and T₂-negative CAs, greatly improve the sensitivity of MRI by affecting the rate of water proton relaxation. For T₁-weighted CAs, paramagnetic complexes containing gadolinium (Gd³⁺) and manganese (Mn²⁺) ions are commonly used to effectively accelerate the T₁ relaxation process to produce a brighter image.^4–9 In contrast, T₂-weighted CAs, such as the superparamagnetic iron oxide nanoparticles (SPIOs), exhibit strong magnetization when an external magnetic field is applied. This causes microscopic field inhomogeneity and activates the dephasing of protons to produce decreased signal intensity in T₂- and T₂*-weighted MR images.^10,11 However, each modal CA has its own unique advantages and limitations. For example, the commonly used T₁ CAs are relatively small molecules, which are fast excreted by the kidneys, thereby resulting in difficulty with high-resolution image acquisition. Moreover, the functionalization of paramagnetic complexes with targeting molecules is hampered by the arduous and complex modification process.^12–14 On the other hand, SPIOs as T₂ MR CAs are very useful for biomedical applications because they are not subject to strong magnetic interactions in dispersion and are readily stabilized under physiological conditions.¹⁵ However, the biggest issue with using T₂ CAs is that it is difficult to distinguish the signal from an air bubble or image artifact. Due to the negative contrast effect and susceptibility artifacts, it is difficult to distinguish the region of signals induced by SPIOs from low-level background MR signals, such as signals arising from adjacent tissues, bone, or vasculature.¹¹ Furthermore, independent T₁ or T₂ MR imaging suffers from false-positive signals that are ubiquitously observed in tissues. Last but not the least, T₁–T₂ dual imaging displays obvious advantages over single imaging with regard to water and fat molecules in tissue. For example, some tissue components such as the bone and fiber components present low signals on T₁WI similar to water. Moreover, some tissue components such as bleeding and mucus present high signals on the T₁WI similar to fat. It is difficult to distinguish water and fat with single imaging. However, with T₁–T₂ dual imaging, these issues can be addressed. Water presents a low signal on T₁WI and a high signal on T₂WI. Fat presents a high signal on both T₁WI and T₂WI. Thus, it is easy to recognize water and fat with dual imaging. Therefore, the development of T₁–T₂ dual CAs is emerging attractively because they have the ability to validate reconstruction and visualization of the data in a more accurate and reliable manner, and to acquire complementary and self-confirmed information to permit meaningful interpretation.¹⁶ Several kinds of dual CAs have been reported to improve the diagnostic accuracy of diseases.^17–24 Choi et al developed a nanoparticle (NP)-based dual CA – MnFe₂O₄@SiO₂@Gd₂O(CO₃)₂ – which not only possessed superior MR contrast effects, but also had the unique capability of displaying “AND” logic signals in both T₁ and T₂ modes.²² Bae et al synthesized Gd-labeled Fe₃O₄ NPs and demonstrated their capability as dual CAs for T₁- and T₂-weighted MRI.²¹

For the design of T₁–T₂ dual CAs, two main aspects should be concerned. Firstly, to obtain high sensitivity, the T₁ and T₂ CAs themselves should possess good MRI relaxivities. High relaxivity is the primary requisite for the development of molecular imaging MR probes and targeting probes.^25–27 Several strategies for increasing the sensitivity (relaxivity) of MRI CAs have been well reviewed.^28–30 Secondly, the undesirable T₁ signal quenching of the paramagnetic T₁ CA by the magnetic field generated by a superparamagnetic T₂ CA when they are in proximity should be avoided. To address these issues, in this study, we aimed to synthesize the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex (Scheme 1) and explore its potential as a T₁–T₂ dual CA to obtain better MRI contrast enhancement and specific tumor cell targeting. BSA-Gd₂O₃ NPs and Fe₃O₄ NPs were chosen as T₁ and T₂ MRI CAs. High-Gd density paramagnetic ultrasmall gadolinium oxide nanoparticles (Gd₂O₃ NPs) have emerged to present larger T₁ relaxivity, good biocompatibility, and easy conjugation with other biomolecules or imaging agents for molecular MRI (mMRI) and multimodal molecular imaging.^18,31,32 The mesoporous silica nanoshell was introduced to coat Fe₃O₄ NPs and load BSA-Gd₂O₃ NPs as well as to act as a spacer to minimize the magnetic spin interactions between T₁ and T₂ CAs. It has been proved that the magnetic field generated by a superparamagnetic T₂ CA could perturb the electronic spins of paramagnetic T₁ CA when they are in close proximity (<12 nm).²² Furthermore, to realize the specific tumor cell imaging, an AS1411 aptamer, which can selectively bind with nucleolin overexpressed on a variety of cancer cells, was employed as the targeting molecule for the fabrication of the mMRI nanoprobe – Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411.^33–35 Its ability for the specific targeting of renal cell carcinoma (RCC) was confirmed by the T₁–T₂-weighted MRI.

Scheme 1 Schematic illustration of the fabrication process of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3-AS1411 nanoprobes.

Materials and methods

Materials and reagents

Bovine serum albumin (BSA) was obtained from Solarbio (Beijing, People’s Republic of China). Gd(NO₃)₃·6H₂O, hexadecyl trimethyl ammonium bromide (CTAB), and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co. Ltd., Beijing, People’s Republic of China Tetraethylorthosilicate (TEOS), poly(dimethyl diallyl ammonium chloride) (PDDA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). DNA oligos were synthesized and purified by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, People’s Republic of China); 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sunshine Biotech. Co. Ltd. (Nanjing, People’s Republic of China). Cell culture media including HDMEM, RPMI 1640, and fetal calf serum were obtained from Kangmei Biotechnology Co. Ltd (Xuzhou, People’s Republic of China). All other reagents were of analytical grade and used without purification. All aqueous solutions were prepared using ultrapure water from a Milli-Q system (Millipore, Boston, MA, USA). The DNA sequence is listed as follows.

COOH-AS1411:

5′-COOH-GGTGGTGGTGGTTGTGGTGGTGGTGG-3′.

Apparatus and characterization

The size and morphology of NPs were observed by transmission electron microscopy (TEM; TECNAI G2, FEI Company, Hillsboro, OR, USA). Fourier transform infrared absorption (FTIR) spectra were obtained through FTIR spectroscopy (Bruker, Billerica, MA, USA). Zeta potentials were determined by Nano ZS90 (Malvern Panalytical Ltd, Malvern, England). X-ray powder diffraction (XRD) patterns were obtained by using a X-ray powder diffractometer (D8 ADVANCE, Bruker). The determinations of Gd and iron ion concentrations were undertaken with inductively coupled plasma-mass spectrometry (ICP-MS) (Optima 5300DV, PerkinElmer, San Diego, CA, USA). The absorbances for MTT assay were determined by a microplate reader (Multiskan MK3, Thermo Fisher Scientific, Waltham, MA, USA) at 490 nm. MRI scanning was conducted on a Signa HDxt 3.0T, General Electric Company, Boston, Massachusetts, USA. Hematologic and biochemical analysis was carried out with a hematology analyzer (Mindray BC-5390, Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, People’s Republic of China) and cobas connection modules (Roche Diagnostics, Basel, Switzerland), respectively.

Cells and cell culture conditions

The 786-0 renal carcinoma cells and NIH-3T3 mouse fibroblast cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, People’s Republic of China). The 786-0 renal carcinoma cells were cultured in a 10% FBS-containing RPMI 1640 medium (Sijiqing, People’s Republic of China) supplemented with penicillin (100 mg/mL) and streptomycin (100 mg/mL). NIH-3T3 cells were propagated in 10% FBS-containing Dulbecco’s Modified Eagle’s Medium (Gibco, Grand Island, NY, USA) supplemented with penicillin (100 mg/mL) and streptomycin (100 mg/mL). All cells were grown in a humidified incubator (Thermo Fisher Scientific) at 37°C under 5% CO₂ atmosphere.

Preparation of Fe₃O₄ NPs

The Fe₃O₄ NPs were synthesized using a previously reported method.³⁶ In brief, approximately 10 mL water containing 1.0 mol/L Fe³⁺ and 0.5 mol/L Fe²⁺ ions (molar ratio 2:1) was stirred at 400 rpm on a stir plate for 20 min. Then, 30 mL ammonium hydroxide was slowly added to the solution and the speed was increased to 900 rpm in order to uniformly precipitate magnetic NPs. Fe₃O₄ NPs were obtained 30 min later. They were washed with distilled water until the pH value decreased to 7.0. Finally, the obtained Fe₃O₄ NPs were dispersed in distilled water at a concentration of 0.24 mg/mL until further use.

Synthesis of Fe₃O₄@mSiO₂ core-shell NPs

In a typical procedure,³⁷ CTAB (50 mg) was dissolved in 20.8 mL distilled water. In total, 4.2 mL Fe₃O₄ and NaOH solution (2.00 M, 160 μL) were added to the above CTAB solution sequentially. Then, the mixture was heated to 70°C with stirring, and 250 μL TEOS and 500 μL ethylacetate were added to the reaction solution in sequence. The mixture was stirred for 3 h at 70°C. The resultant precipitate was washed 3 times with ethanol to remove the unreacted species and then dispersed in 20 mL ethanol. To extract CTAB, 20 μL HCl was added to the dispersion mixture (pH>1.0) and stirred for 3 h at 60°C. To obtain hollow mesoporous silica, more HCl was used for extraction (pH <1.0). After washing three times with ethanol, the Fe₃O₄@mSiO₂ NPs were dried at 50°C.

Preparation of BSA-Gd₂O₃ NPs

BSA-Gd₂O₃ NPs were synthesized according to the methods specified in the literature with some modifications.³⁸ In total, 0.25 g BSA was dissolved in 9 mL distilled water. Then, 1 mL 50 mM Gd(NO₃)₃ was added to the above solution slowly under vigorous stirring. After the introduction of 1 mL 2 M NaOH 5 min later, the mixture was allowed to react under vigorous stirring at 37°C for 12 h. Finally, the BSA-Gd₂O₃ thus prepared was dialyzed against distilled water (1:1,000, v/v) to remove excess precursors.

Fabrication of Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 mMRI nanoprobe

The Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ was prepared through electrostatic interaction.³⁹ Initially, Fe₃O₄@mSiO₂ (3 mg) and PDDA solution (2 mL, 2 mg/mL, 0.2 M NaCl) were mixed under sonication for 40 min, and the excess polyelectrolyte was removed by washing with water. Then, the positively charged magnetic particles were dispersed in distilled water (500 μL), and BSA-Gd₂O₃ (50 μL, 1.06 μM Gd) was added and sonicated for 40 min. After washing with water, the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ were dispersed in 2 mL water for MRI scanning. Meanwhile, to obtain the best MRI behavior, different concentrations of BSA-Gd₂O₃ NPs (0.212, 0.636, 1.06, 1.484, and 1.908 μM Gd) and Fe₃O₄ (1, 3, and 5 mg) were tested. At the optimized condition, the AS1411 aptamer was functionalized onto the surface of Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ NPs by covalent coupling between the amino group of BSA-Gd₂O₃ and the carboxyl group modified at the 5′ end of AS1411 aptamer with the help of EDC and NHS.⁴⁰ Briefly, 100 μM COOH-AS1411 (50 μL) was mixed with EDC (100 μL, 10 mg mL⁻¹) in 300 μL PBS (10 mM, pH 7.4) and incubated at 37°C for 15 min to form an active carboxyl group. Then, NHS (100 μL, 10 mg mL⁻¹) and Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ (500 μL, 5.0 mg mL⁻¹) were added to the mixture and reacted at 37°C for 2 h. The unreacted biomolecules were removed by two centrifugation/washing cycles. Finally, the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 nanoprobes were dispersed in 1 mL PBS (10 mM) at pH 7.4 for further use.

Relaxivity calculation of Fe₃O₄ NPs, BSA-Gd₂O₃ NPs, Gd-DTPA, and Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex

The MRI behavior test of BSA-Gd₂O₃ and Fe₃O₄ was conducted with a 3.0 T human MR scanner (General Electric Company). Various concentrations of BSA-Gd₂O₃ and Fe₃O₄ solution were prepared before MRI scanning, which varied from 0.212 to 1.908 μM with a volume of 600 μL for BSA-Gd₂O₃ NPs, and 1–5 mg for Fe₃O₄ NPs. The following parameters were adopted in data acquisition. 1) T₁-weighted images: echo time (TE)=16.5 ms, repetition time (TR)=420 ms, field of view (FOV)=14×14 cm, matrix=384×256, slice thickness=2.0 mm, spacing=0.2 mm; and 2) T₂-weighted images: echo time (TE)=90.7 ms, repetition time (TR)=4,500 ms, field of view (FOV)=14×14 cm, matrix=384×256, slice thickness=2.0 mm, spacing=0.2 mm. Quantitative T₁ and T₂ relaxation maps were reconstructed from datasets using function software at a workstation (ADW 4.2). The signal intensities of the samples were measured, and the T₁ and T₂ values were calculated accordingly. MRI scanning of Gd-DTPA and the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex with different concentrations was carried out in the same way.

The T₁ relaxivity values of the BSA-Gd₂O₃, Gd-DTPA, and Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex were determined by measuring longitudinal proton relaxation time (T₁) as a function of Gd concentration. The T₂ relaxivity values of the Fe₃O₄ and Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex were determined by measuring the lateral proton relaxation time (T₂) as a function of Fe concentration.

MTT assay

The 786-0 cells and NIH-3T3 cells were cultured on 96-well plates at a density of 10⁴ cells each well. After 24-h incubation, the medium was substituted with 100 μL fresh medium containing different concentrations of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex (0, 200, 400, 600, 800, 1,000, 1,200, and 1,400 μg mL⁻¹). After 24-h incubation, the medium was removed, and fresh medium (100 μL) containing MTT (20 μL, 5 mg mL⁻¹) was added into each well. Four hours later, the culture medium was carefully removed and washed, and 100 mL DMSO was added to each well to dissolve the formazan crystals for 10 min. The absorbance at 490 nm was measured by a microplate reader (Multiskon MK3, USA).

Stability test

The Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex was dispersed in RPMI 1640 culture medium. At different time points (0, 2, 4, 6, 8, 12, 24, 48, and 72 h), 100 μL samples were collected and centrifuged at 25,000 rpm for 30 min to obtain the supernatant. The concentrations of Fe and Gd ions in the supernatant were determined by ICP-MS. For the pH stability test, the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex was dispersed in solutions of different pHs (pH 4, 5, 6, 7, 8, 9, and 10) and incubated for 24 h. The concentrations determined for Fe and Gd ions were the same as mentioned earlier.

Specific MR imaging of 786-0 cells with Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 mMRI nanoprobe in vitro

The 786-0 cells and NIH-3T3 cells were seeded into six-well plates at a density of 10⁵ cells/well and cultured for 24 h in a humidified incubator at 37°C under 5% CO₂ atmosphere, respectively. Then, the culture media was removed and the cells were washed with PBS twice. In total, 500 μL of different concentrations of Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 nanoprobe (0.5, 1, and 2.5 mg mL⁻¹) and Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex (0.5, 1, and 2.5 mg mL⁻¹) were added into the well and incubated for 1 h at 37°C. After discarding the nanoprobe, the cells were washed and lysed by trypsin. The harvested cells were fixed with 600 μL 1% agarose for MRI scanning. The untreated cells that were incubated with PBS were taken as the control.

Biodistribution of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex in the mouse

The residual nanocomplex in heart, liver, kidneys, and lungs after 1 h and 24 h post-injection of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex was determined by the quantification of the Gd element by ICP-MS. Briefly, six Kunming mice were divided into two groups: 1-h post-injection and 24-h post-injection. Each mouse was injected with the same concentration of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex (0.16 mmol Gd per kg body weight). The mice were sacrificed and the major organs, including heart, liver, kidneys, and lungs, were collected at 1 h and 24 h post-injection. To these organs, 2 mL aqua regia (HCl:HNO₃=3:1) was added and the organs in aqua regia were heated to 200°C for 1 h. After being cooled to room temperature, 2 mL H₂O was added and a 0.22-μm filter was introduced to remove undigested tissues.⁴¹ The Gd concentrations were then analyzed by ICP-MS.

In vivo toxicity evaluation of Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex

Hematologic and biochemical analyses were introduced to evaluate the in vivo toxicity of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex. In total, six Kunming mice were divided into two groups: the normal saline group and the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex group. The mice were injected with normal saline and the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex (0.16 mmol Gd per kg body weight) via tail vein, respectively. After 1 week, blood was harvested by cardiac puncture. Hematologic analysis including platelets (PLT), hematocrit (HCT), hemoglobin (HGB), red blood cells (RBC), white blood cells (WBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) was undertaken on a hematology analyzer. The biochemical effects of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex for alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), creatinine (CREA), total protein (TP), albumin (ALB), and total bilirubin (TBIL) were evaluated by cobas connection modules.

In vivo MRI

Animal experiments were conducted in accordance with the National Institutes of Health guidelines on the use of animals in research, and were approved by the Animal Care Committee of Xuzhou Medical University. Healthy BALB/c mice weighing 20–30 g were chosen for in vivo MRI study. The mice were anesthetized by intraperitoneal injection of 2% mebumalnatrium (4 μL/g bodyweight), followed by tail-vein administration of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex (0.16 mmol Gd per kg body weight). Then, at scheduled intervals, T₁WI images and T₂WI images were acquired by a 3.0 T MR imaging system (GE Discovery 750W, USA) with the animal T/R coil (Magtron Inc., Jiangyin, People’s Republic of China). The sequences for T₁-weighted images were set as follows: TE=12 ms; TR=400 ms; FOV=80×80 mm; slice thickness=2 mm; spacing=0.2 mm; and matrix=256×256. The sequences for T₂-weighted images were set as follows: TE=58 ms; TR=300 ms; FOV=80×48 mm; slice thickness=2 mm; spacing=0.2 mm; and matrix=256×256. To compare the signal intensity at different time points, the same protocols, prone position, and the same scanning landmark of MRI were required. The relative intensity changes of the same region of interest on the same slice in the same window width and window level were recorded by software in the workstation GE AW4.6.

Results

Characterization of Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 mMRI nanoprobe

For the fabrication of the T₁–T₂ dual CA, 10.7-nm-sized-Fe₃O₄ NPs as T₂ CA were first encased with an mSiO₂ nanoshell (Figure S1). An approximately 20-nm nanoshell could reduce the quenching effect of Fe₃O₄ NPs on the T₁ signal to the extent that was possible. Then, BSA-Gd₂O₃ NPs were deposited onto the Fe₃O₄@mSiO₂ surface through layer-by-layer assembly with the help of polyelectrolytes and PDDA. Driven by the electrostatic force, a uniform monolayer of positively charged PDDA and negatively charged BSA-Gd₂O₃ NPs were alternatively adsorbed onto the negatively charged Fe₃O₄@mSiO₂ NPs. This assembly process was monitored by the determination of the changes of zeta potentials. As shown in Figure 1A, the potential value of Fe₃O₄@mSiO₂ NPs was −33.5 mV and changed to 47.8 mV with PDDA assembly. The zeta potential of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex was 26.9 mV, which might derive from the negatively charged BSA. The corresponding hydrodynamic diameters of Fe₃O₄@mSiO₂, Fe₃O₄@mSiO₂/PDDA and Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ were changed from 100.2 to 297.4 and 345.6 nm, accordingly (Figure 1B). From XRD patterns in Figure S2, the typical diffraction peaks of Fe₃O₄ were observed. The amorphous hump of SiO₂ at around 26° and the characteristic peaks of Fe₃O₄ could be observed in Fe₃O₄@mSiO₂ and Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃, indicating the successful fabrication of the nanocomplex.⁴² Then, the further conjugation of the AS1411 aptamer with the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex through a covalent coupling reaction was confirmed by the FTIR absorption spectrum and UV-vis absorption spectrum. The emerging absorption peak at 1,630 and 1,652 cm⁻¹ was ascribed to acylamide vibration in the FTIR absorption spectrum (Figure 1C), and a characteristic UV-vis absorbance peak of the AS1411 aptamer could be observed in the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 nanoprobe (Figure 1D), both indicating the successful preparation of the mMRI nanoprobe.

Figure 1 Characterization of the assembly process of Fe3O4@mSiO2/PDDA/BSA-Gd2O3 with zeta potential (A), hydrodynamic diameter determination (B), and the fabrication of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3-AS1411 nanoprobe with fourier transform infrared absorption (FTIR) spectra (C) and UV-vis spectra (D).

MRI behavior of Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex

To explore its potential as an mMRI nanoprobe, the MRI behavior of our synthesized Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex was first evaluated. With different concentrations of BSA-Gd₂O₃ NPs, the T₁ and T₂ relaxation times of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex varied. As shown in Figures 2 and 3, with the increase of the BSA-Gd₂O₃ NP concentration, the T₁ relaxation time of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex was enhanced and reached a maximum of approximately 320 ms in the presence of 1.06 μM Gd. However, the further increased concentration of BSA-Gd₂O₃ NPs (1.484 and 1.908 μM Gd) influenced the T₁ relaxation time of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex non-obviously. Unlike the T₁ relaxation time, the T₂ relaxation time of Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex changed irregularly with the increase in the concentration of BSA-Gd₂O₃ NP. However, when the BSA-Gd₂O₃ NP concentration was 1.06 μM Gd, the T₂-map color image was darkest. The influence of Fe₃O₄ concentrations on the T₁ and T₂ relaxation times were not as strong as that of BSA-Gd₂O₃ NPs. As shown in Figure S3 and Table S1, with the increase of the Fe₃O₄ concentration, the T₁ relaxation time of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex was weakened; however, its T₂ relaxation time changed little. For overall consideration, 50 μL 1.06 μM BSA-Gd₂O₃ and 1 mg Fe₃O₄ NPs were chosen for the fabrication of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex in the following experiments.

Figure 2 T1-weighted magnetic resonance (MR) images and T1-map images as well as T2-weighted MR images and T2-map images of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex prepared with different concentrations of BSA-Gd2O3 NPs. 3.0 T human magnetic resonance scanner was used.

Figure 3 T1 and T2 relaxation time of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex prepared with different concentrations of BSA-Gd2O3 NPs. BSA-Gd2O3 NPs (0 μM) were treated as the control group, and other groups were all compared with the control group with one-way ANOVA. *Refers to statistically significant differences compared with the control group with one-way ANOVA (p<0.05). #Refers to statistically significant differences compared with control group with one-way ANOVA (p<0.05). A 3.0 T human magnetic resonance scanner was used.

To testify the influence of BSA-Gd₂O₃ NPs of our nanocomplex on T₂WI, we also synthesized the Fe₃O₄@mSiO₂ nanocomplex and the mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex. As shown in Figure S4 and Table S2, the T₂ signals of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex and the Fe₃O₄@mSiO₂ nanocomplex were all less than the T₂ signal of the mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex; however, there was little change between the T₂ signal of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex and the T₂ signal of the Fe₃O₄@mSiO₂ nanocomplex. Thus, in our prepared nanocomplex, the BSA-Gd₂O₃ NPs had no obvious effect on T₂WI, and Fe₃O₄ NPs influenced the T₂ signal obviously. From the above results, we can conclude that, for the synthesis of T₁–T₂ dual MRI CA Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃, T₁ and T₂ signals are ascribed to the BSA-Gd₂O₃ NPs and Fe₃O₄ core, respectively.

Then, the T₁ relaxivity values of Gd-DTPA, BSA-Gd₂O₃ NPs, and the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex were determined and compared by measuring longitudinal proton relaxation time (T₁) as a function of Gd concentration. As shown in Figure 4, the r₁ value of BSA-Gd₂O₃ was 12.2 s⁻¹ mM⁻¹, which was three times that of the commercial MRI CAs, Gd–DTPA (r₁=4.3 s⁻¹ mM⁻¹). The T₁ relaxivity of Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex was 11.5 s⁻¹ mM⁻¹. For the determination of the T₂ relaxivity values of Fe₃O₄ and the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex, lateral proton relaxation times (T₂) as a function of Fe concentration were measured. The T₂ relaxivity of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ (195.1 s⁻¹ mM⁻¹ Fe) was lower than that of Fe₃O₄ (345.9 s⁻¹ mM⁻¹ Fe), but higher than the clinically used Feridex (108 s⁻¹ mM⁻¹ Fe)¹ and the relaxivity reported for Fe₃O₄@mSiO₂ NPs (171.6 s⁻¹ mM⁻¹ Fe; Figure 5).^43,44 Such excellent MR imaging behavior indicated the ability of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex (the molar value of Fe/Gd equals 0.539; r₁/r₂ equals 0.0567) as a promising T₁–T₂ dual MRI CA for the fabrication of an mMRI nanoprobe to specifically image tumor cells.

Figure 4 T1 relaxivity curves (A) and T1-weighted magnetic resonance (MR) images (B) of Gd-DTPA (1), BSA-Gd2O3 NPs (2), and Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex (3) with various Gd concentrations. A 3.0 T human MR scanner was used.

Figure 5 T2 relaxivity curves and T2-weighted magnetic resonance (MR) images of the Fe3O4 (A), and Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex (B) with various Fe3O4 nanoparticle concentrations. A 3.0 T human MR scanner was used.

In vitro cytotoxicity test

Before the use of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex for specific tumor cell imaging, it is vital to assess its safety in vitro. The MTT assay was conducted to evaluate the cell toxicity of the nanocomplex and identify potentially safe concentrations for the following targeting experiments. The Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex with eight different concentrations, ranging from 0 to 1,400 μg/mL, was incubated with 786-0 and NIH-3T3 cells for 24 h, respectively. As shown in Figure 6, the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex displayed good biocompatibility, and no significant cytotoxicity was observed both on 786-0 renal carcinoma or NIH-3T3 cells.

Figure 6 Cell viability of 786-0 renal carcinoma cells and NIH-3T3 mouse fibroblast cells after exposure to various concentrations of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex, determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Stability test

The leaching of metallic ions is a potential harmful effect of their biological application. To test the possibility of Gd and Fe ions leaching from the nanocomplex in biological media, we determined the concentrations of Gd and Fe ions in the supernatant with the passage of time from when the nanocomplex was dispersed in the cell culture medium and in different pH solutions incubated for 24 h. As shown in Figure 7, no obvious Gd and Fe ion leaching could be observed with time in the cell culture medium or different pH solutions, indicating the good stability of our fabricated nanocomplex.

Figure 7 Iron and gadolinium ions released from the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex in cell culture medium over time (A) and in different pH solutions incubated for 24 h (B).

In vitro specific T₁–T₂ dual MR imaging of tumor cells

Finally, the specific cellular targeting of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 mMRI nanoprobe was evaluated by T₁–T₂ MRI. AS1411 is a nucleolus of specific nucleic acid aptamers, which can specifically combine with nucleolin on the surface of the tumor cell. As shown in Figure S5, the AS1411 modification did not produce a significant influence on the MRI behavior of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex, indicating the potential use of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 as an mMRI nanoprobe.

To validate the specific targeting ability of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 mMRI nanoprobe, the 786-0 cells were incubated with the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex and Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 mMRI nanoprobes, respectively. For the achievement of the best signal-to-noise value, 0.5, 1, and 2.5 mg mL⁻¹ nanoprobes were introduced and compared. As shown in Figures 8 and 9, the higher the concentration of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 nanoprobe, the brighter the T₁ signal intensity and the darker the T₂ signal intensity from 786-0 cells. Furthermore, the presence of the AS1411 aptamer could facilitate more of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 nanoprobe to bind with 786-0 cells and present a brighter T₁ signal and a darker T₂ signal at all three concentrations. However, when the concentration of the nanoprobe reached 2.5 mg mL⁻¹, the non-specific adsorption obviously increased. Thus, 1 mg mL⁻¹ of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 nanoprobe was chosen for the specific MRI finally. To further confirm such AS1411 aptamer-based specific targeting, NIH-3T3 cells were introduced as the control and treated with 1 mg of the mL⁻¹ Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 nanoprobe, Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex, respectively. No significant difference existed between these two groups in both T₁ and T₂ signal, indicating the specific MRI ability of our synthesized nanoprobe to ccRCC in vitro (Figure S6, Tables S3 and S4).

Figure 8 T1-weighted magnetic resonance (MR) images and T1-map images (A) as well as T2-weighted MR images and T2-map images (B) of 786-0 renal carcinoma cells treated with different concentrations of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex (−) and Fe3O4@mSiO2/PDDA/BSA-Gd2O3-AS1411 nanoprobe (+) (0, 0.5, 1, 2.5 mg/mL−1). A 3.0 T human MR scanner was used.

Figure 9 T1 (A) and T2 relaxation time (B) of 786-0 renal carcinoma cells treated with different concentrations of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex (−) and Fe3O4@mSiO2/PDDA/BSA-Gd2O3-AS1411 nanoprobe (+). Notes: *Refers to statistically significant differences compared with the control group using one-way ANOVA (p<0.05). #Refers to statistically significant differences compared with the control group with one-way ANOVA (p<0.05). aRefers to statistically significant differences within group with LSD Duncan (p<0.05). bRefers to statistically significant differences within group with LSD Duncan (p<0.05). cRefers to statistically significant differences within group with LSD Duncan (p<0.05). dRefers to statistically significant differences within group with LSD Duncan (p<0.05). A 3.0 T human magnetic resonance scanner was used.

Biodistribution of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex in the mouse

To show the biodistribution and residual persistence of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex in different organs, ICP-MS was employed to determine the Gd concentration in the heart, liver, kidneys, and lungs after 1 h and 24 h following injection of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex. As shown in Figure 10, the Gd concentration in the above organs all decreased, except in the kidney. The residual Gd concentrations in heart, liver, kidneys, and lungs were 1.90±0.12, 2.41±0.06, 2.15±0.08, and 2.27±0.1 μg/g at 1 h post-injection and 1.54±0.11, 1.94±0.12, 2.46±0.14, and 1.69±0.17 μg/g at 24 h post-injection, respectively.

Figure 10 The concentrations of Gd in the heart, lung, liver, and kidney at 1 h and 24 h post-intravenous injection of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex as determined by inductively coupled plasma-mass spectrometry (n=3, *p<0.05, **p<0.01).

In vivo toxicity evaluation

The in vivo toxicity of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex was evaluated with standard hematologic and biochemical analyses and by referring to the literature.⁴⁵ Compared with the normal saline group, the HCT, HGB, RBC, WBC, MCV, MCH, and MCHC levels in the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex group showed no statistically significant differences, except for PLT (Figure 11). The ALT, AST, BUN, CREA, TP, ALB, and TBIL levels displayed no statistically significant difference between the normal saline group and the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex group (Figure 12).

Figure 11 Hematologic analysis of mice at 1 week post-injection of normal saline (black bar) and the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex (red bar). Notes: (A) White blood cells (WBC), (B) red blood cells (RBC), (C) platelets (PLT), (D) mean corpuscular volume (MCV), (E) hemoglobin (HGB), (F) mean corpuscular hemoglobin concentration (MCHC), (G) mean corpuscular hemoglobin (MCH), and (H) hematocrit (HCT) levels in the blood. ***Refers to statistically significant differences compared with the normal saline group (p<0.001, n=3).

Figure 12 Biochemical analysis of mice at 1 week post-injection of normal saline (black bar) and the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex (red bar). Notes: (A) Alanine transaminase (AST), (B) aspartate transaminase (ALT), (C) total protein (TP), (D) albumin (ALB), (E) total bilirubin (TBIL), (F) blood urea nitrogen (BUN), and (G) creatinine (CREA) levels in the blood; (n=3).

In vivo MRI

For in vivo MR imaging, T₁ and T₂ dual-mode images were obtained pre- and post-injection of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex using a 3T MR scanner at room temperature. MRI images were collected at scheduled intervals of 24 h post-injection. As shown in Figures 13 and S7, compared with the pre-injection reference, a darker signal in T₂-weighted images and a brighter signal in T₁-weighted images in the kidney at 15 min after administration were observed. However, at the 6-h time point, such signal contrasts in the kidney brought by the nanocomplex injection became weaker and the signal contrasts in the bladder became stronger, indicating that the excretion of the nanocomplex was mainly via the kidneys and bladder. These results showed the potential of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex as an effective T₁–T₂ dual-mode CA for in vivo MRI with self-confirmation.

Figure 13 (A) T1-weighted in vivo magnetic resonance imaging (MRI) images of mice post-injection of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex at different time points (0, 15 min, 6 h, and 24 h). The signal intensities in the kidneys (B) and bladder (C) at different time points after intravenous injection of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex. The red arrow indicates the kidney.

Discussion

T₁–T₂ dual modal MR imaging avoids the false-positive signals caused by a single imaging mode and provides more accurate and complementary information. The development of T₁–T₂ dual CAs favors T₁–T₂ dual modal MR imaging. However, interferences between the T₁ and T₂ CAs when they are in proximity would reduce their MRI relaxivity. To avoid this influence, a 20-nm mSiO₂ nanoshell was employed to increase the distance between T₁ CA, BSA-Gd₂O₃ NP and the T₂ CA, Fe₃O₄ NP in this study, because the interference of T₂ CA on the contrast enhancement of paramagnetic T₁ CAs might be weakened when their distance exceeds 12 nm. As expected, our fabricated Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex presented a comparable T₁ relaxivity of 11.5 s⁻¹ mM⁻¹ to BSA-Gd₂O₃ (12.2 s⁻¹ mM⁻¹), which was three times that of the commercial MRI CAs, Gd–DTPA (r₁=4.3 s⁻¹ mM⁻¹). As reported, in a certain range, the larger the size of NPs, the stronger the saturation magnetization and relaxation rate of the nanomaterials.⁴⁶ In our work, the existence of PDDA favored more BSA-Gd₂O₃ NPs loading on the surface of Fe₃O₄@mSiO₂ NPs. Therefore, the T₁ relaxivity of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex should be higher than that of pure BSA-Gd₂O₃ NPs. However, there was little difference of T₁ relaxivity between the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex and BSA-Gd₂O₃ NPs, which might arise from the interference of the Fe₃O₄ core, more or less. Besides the excellent MR imaging behaviors, the good biocompatibility and good stability in the cell culture medium or different pH solutions further provide the basis for their use to fabricate an mMRI nanoprobe. For hematologic analysis, all of the hematology markers displayed no statistically significant differences between the normal saline-treated group and the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex-treated group, except for PLTs. PLTs play a key role in hemostasis and thrombosis. Nanomaterials interact with PLTs and affect their count and function, which might result in severe clinical outcomes.⁴⁷ For example, Radomski et al found multi- or single-walled carbon nanotubes (MWCNTs or SWCNTs) induced PLT activation and aggregation whereas C60 fullerences (nC60) had no effect.⁴⁸ Reinish et al observed a reduction in the PLT blood count in the first 5 min after the injection of negatively charged liposomes to rats, but noted a recovery of PLT count after 60 min post-injection.⁴⁹ Nanomaterial properties such as size, shape, chemical composition, and surface physicochemical property influence their effect on PLT, obviously. For biochemical analysis, the commonly used serum biochemical parameters including AST, ALT, TP, ALB, TBL, BUN, and CREA were measured. ALT and AST are commonly used indicators of liver function. Creatinine and BUN are important indicators of kidney function.⁴⁵ Compared with the normal saline-treated group, no statistically significant difference was observed in the above parameters in the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex-treated group, showing the good biocompatibility of Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex in vivo. Then, after conjugation with the AS1411 aptamer as the targeting molecule, the 786-0 cells could be specifically targeted with the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃-AS1411 nanoprobe by MRI. Furthermore, the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex could produce a signal enhancement in the kidney, suggesting their potential use as CAs in vivo. The biodistribution and clearance of nanomaterials in vivo is related to the characteristics of nanomaterials and organ specificity, such as hydrodynamic size, the surface charge, surface modification, and so on.^50–52 It was reported that NPs with a hydrodynamic diameter less than 5.5 nm could be excreted by renal clearance, and those with a hydrodynamic diameter more than 15 nm were usually captured and eliminated through the liver into bile and into feces.^53,54 However, in our case, the increase of Gd concentration in the kidney with time indicated the possible excretion route of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex from the body through the kidneys, as even its hydrodynamic size was approximately 345.6 nm. This result was similar with that reported in Mekuria et al’s report; they prepared G4.5-Gd₂O₃-PEG NPs with a hydrodynamic diameter of approximately 50 nm. They concluded that the kidneys removed NPs from the vascular compartment to the gall bladder.⁵⁵ This means the clearance route of nanomaterials was not only related to the hydrodynamic size, but also to other factors. The Gd concentrations in the heart, liver, and lung all decreased with time, as indicated by the gradual clearance of the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex from the body.

Conclusion

The introduction of CAs has improved the sensitivity of MRI greatly, stimulating its wider application in disease diagnosis. However, single-mode CAs are not yet perfected. The combination of T₁ and T₂ CAs can integrate the high tissue resolution of T₁ mode contrast imaging and the high feasibility of softer tissue detection of T₂ mode contrast imaging. Herein, to get a T₁–T₂ dual MRI CA with better signal enhancement effect, a Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex was synthesized for better relaxivity of nano-CAs. The obtained nanocomplex displayed high longitudinal (r₁=11.47 mM s⁻¹ Gd) and transverse (r₂=195.1 mM s⁻¹ Fe) relaxivities, enabling its use as a T₁–T₂ dual CA for MRI. To further apply their use as mMR imaging nanoprobes for the specific recognition of tumor cells, the AS1411 aptamer which can specifically combine with the nucleolin of the surface of the tumor cell was covalently coupled with the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex. When intravenously injected into the mice, the Fe₃O₄@mSiO₂/PDDA/BSA-Gd₂O₃ nanocomplex could produce a kidney contrast-enhancement and was excreted via the bladder, thereby showing promise as a T₁–T₂ dual-mode CA.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant no 81771904), the Natural Science Foundation of Jiangsu Province for the Excellent Young Scholars (grant no BK20170054), China Postdoctoral Science Foundation (grant nos 2016M601890 and 177607), Qing Lan Project, the Peak of Six Talents of Jiangsu Province (grant no WSN-112), Jiangsu Provincial Medical Youth Talent (grant no QNRC2016776), and the Natural Science Fund for Colleges and Universities in Jiangsu Province (grant no 16KJB310019).

Disclosure

The authors report no conflicts of interest in this work.

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Supplementary materials

Figure S1 Transmission electron microscopy characterizations of Fe3O4 nanoparticles (NPs) (A) and Fe3O4@mSiO2 NPs (B). The insert in (A) is the distribution of Fe3O4 NP size.

Figure S2 X-ray powder diffraction patterns of the Fe3O4, Fe3O4@mSiO2 and Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex.

Figure S3 T1-weighted magnetic resonance (MR) images and T1-map images as well as T2-weighted MR images and T2-map images of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex prepared with different concentrations of Fe3O4 nanoparticles. A 3.0 T human MR scanner was used.

Table S1 T1 and T2 relaxation time of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex prepared with different concentrations of Fe3O4 nanoparticles (±S) (n=4)

Figure S4 T1-weighted magnetic resonance (MR) images as well as T2-weighted MR images of the mSiO2/PDDA/BSA-Gd2O3 nanocomplex, Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex, and Fe3O4@mSiO2 nanocomplex. A 3.0 T human MR scanner was used.

Table S2 T1 and T2 relaxation time of the mSiO2/PDDA/BSA-Gd2O3 nanocomplex, Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex and Fe3O4@mSiO2 nanocomplex (±S) (n=4) Notes: *Refers to statistically significant differences when compared with the other two groups using one-way ANOVA (p<0.05). #Refers to statistically significant differences when compared with the other two groups using one-way ANOVA (p<0.05). aRefers to statistically significant differences when compared with the other two groups using one-way ANOVA (p<0.05).

Figure S5 T1-weighted magnetic resonance (MR) images and T1-map images as well as T2-weighted MR images and a T2-map images of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex and Fe3O4@mSiO2/PDDA/BSA-Gd2O3-AS1411 nanoprobe. No significant difference of T1 and T2 relaxation time could be observed after the AS1411 aptamer modification. A 3.0 T human MR scanner was used.

Figure S6 T1-weighted magnetic resonance (MR) images and T1-map images (A) as well as T2-weighted MR images and T2-map images (B) of NIH-3T3 cells treated with 1 mg mL−1 Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex (−) and Fe3O4@mSiO2/PDDA/BSA-Gd2O3-AS1411 nanoprobe (+). A 3.0 T human MR scanner was used.

Table S3 T1 relaxation time of NIH-3T3 mouse fibroblast cells and 786-0 renal carcinoma cells treated with 1 mg mL−1 Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex (−) and Fe3O4@mSiO2/PDDA/BSA-Gd2O3-AS1411 nanoprobe (+) (±S) (n=4) Notes: *Refers to statistically significant differences when compared with control group using one-way ANOVA (p<0.05). #Refers to statistically significant differences when compared with the control group using one-way ANOVA (p<0.05). aRefers to statistically significant differences between NIH-3T3 and 786-0 cells with LSD Duncan (p<0.05).

Table S4 T2 relaxation time of NIH-3T3 mouse fibroblast cells and 786-0 renal carcinoma cells treated with 1 mg mL−1 Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex (−) and Fe3O4@mSiO2/PDDA/BSA-Gd2O3-AS1411 nanoprobe (+) (±S) (n=4) Notes: *Refers to statistically significant differences when compared with the control group using one-way ANOVA (p<0.05). #Refers to statistically significant differences when compared with the control group using one-way ANOVA (p<0.05). aRefers to statistically significant differences NIH-3T3 and 786-0 cells with LSD Duncan (p<0.05).

Figure S7 (A) T2-weighted in vivo magnetic resonance imaging (MRI) images of mice post-injection of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex at different time points (0, 15 min, 6 h, and 24 h). The signal intensities in the kidneys (B) and bladder (C) at different time points after intravenous injection of the Fe3O4@mSiO2/PDDA/BSA-Gd2O3 nanocomplex. The red arrow indicates the kidney.