Dual-Modality Imaging Probes with High Magnetic Relaxivity and Near-Infrared Fluorescence Based Highly Aminated Mesoporous Silica Nanoparticles

1Department of Medical Imaging, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, China 2Department of Medical Imaging, The First Affiliated Hospital with Nanjing Medical University, Nanjing 210029, China 3Department of Medicine, Nanjing University, Nanjing 210093, China 4Department of Pathology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, China


Introduction
Magnetic resonance (MR) and near-infrared (NIR) fluorescence have been widely used as powerful imaging tools in biomedical areas [1][2][3][4][5][6].However, due to the inherent limitations of MR and NIR, it is still impossible to get molecular, functional, and anatomical information by signal imaging method [1,2,7,8].In order to take the synergistic advantages of each imaging modality, great efforts have been conducted to explore MR/NIR dual-modal imaging tools [3,[9][10][11][12][13].For example, NIR dye has been modified onto MRdetectable nanomaterials such as superparamagnetic iron oxide nanoparticles (SPIONs) to build MR/NIR bimodal imaging probe [14][15][16].However, the direction modification dyes on the SPIONs indeed do not have many disadvantages.First, the black SPIONs can directly quench the fluorescence of the dye.Second, the emitted fluorescence can also be absorbed by the SPIONs [14,16].In addition, surface modifications are commonly needed to connect functional molecules, which makes the SPION based dual-modality probes even more complicated [14].Another method is conjugating MR agents with semiconductor quantum dots (QDs) [1,[17][18][19]].Yet, the drawback of the toxicity of heavy cadmium metal makes QD based imaging probes potential clinical usage limited [1].
Recently, mesoporous silica nanoparticles (MSNs) have attracted increasing attention as an ideal matrix to integrate imaging tags to develop imaging probes, due to the high surface area, uniform pore size, excellent biocompatibility, and no absorption of NIR light or interference with magnetic fields [11,17,20,21].There have been several reports about MSN based imaging probes [17,[21][22][23].Taylor et al. [23] synthesized MSN based nanoparticulate  1 weighted MR contrast agents with a high  1 relaxivity by refluxing MSNs and Gd-Si-DTTA complex in toluene.Although rodent experiment confirmed the MR imaging ability of the particles, the authors failed to evaluate the optical fluorescent property in vivo [23].Sharma et al. [22] produced gadolinium-doped silica nanoparticles by encapsulation indocyanine green with a reverse-microemulsion-based synthesis protocol; both in vitro and in vivo studies showed fine NIR and MR contrast capabilities.However, the protocol is complex and the  1 relaxivity elevation was only 7.3 mM −1 s −1 on a 4.7 T MR.Till now, there very few reports on the high MR  1 relaxivity and NIR imaging probes have been documented.
In this study, we designed and synthesized a novel NIR/MR dual-modality imaging probe (NIR/MR-MSNs) by facile conjugation of NIR fluorescent heptamethine dyes IR-808 and MR contrast agents Gd-DTPA within highly aminated MSNs (MSNs-NH 2 ) via a one-step procedure.The NIR/MR-MSNs not only were easy to produce, but also possessed uniform particle size, high surface area, and excellent biocompatibility.In vitro cell experiment illustrated high cellular uptake, retention abilities, and excellent biocompatibility of the NIR/MR-MSNs.Excellent NIR fluorescence property and highly efficient MR  1 contrast effect were also found in in vitro and in vivo imaging studies.Importantly, the NIR/MR-MSNs provide simultaneously clear anatomical border of enhanced tumor region and high sensitive fluorescence imaging.These all suggested that the NIR/MR-MSNs are suitable for potential applications as a useful dualmodality biomedical imaging probes.

Synthesis of MSNs-NH 2 .
MSNs-NH 2 were synthesized according to the previously reported method [24].In brief, 7.04 mL of ethyl acetate, 24.24 mL of ammonium hydroxide, and 0.8 g of CTAB were added to 1135 mL deionized water and stirred at room temperature until CTAB completely dissolved.Afterward, 1.8 mL of TEOS and 2.2 mL of APTES were added under vigorous stirring.After 24 h, the products were centrifuged and washed with deionized water.Finally, the CTAB was removed by three extractions in 240 mL of ethanol and 480 L of concentrated hydrochloric acid at 60 ∘ C for 3 h.

Synthesis of
Gd/NIR-MSNs. 1 mL of MSNs-NH 2 solution (50 mg/mL), 0.7 mg IR-808, and 4 mL Gd-DTPA aqueous solution (0.1 M) were mixed in MES-NHS buffer and shattered by an ultrasonic cell disrupter for 30 min in dark.Then, EDC⋅HCl was added in the solution and stirred in dark overnight.The sample was washed with water and redispersed in deionized water.

Characterization of Gd/NIR-MSNs.
Transmission electron microscopy (TEM) images were obtained with a Tecnai G2 F20 microscope (FEI, Hillsboro, OR) at 200 KV.Nitrogen sorption isotherms were obtained with a V-Sorb 2800P physisorption instrument (Gold APP Instrument Corporation, China).Surface areas were calculated according to the Brunauer-Emmett-Teller (BET) method.The pore size distributions were calculated from the adsorption branches of the isotherms according to the nonlocal density functional theory (NLDFT) method.The absorption curve of Gd/NIR-MSNs and loading efficiency of IR-808 were determined by UV-vis spectrum with a UV-vis Lambda 35 (PerkinElmer, USA).To confirm the presence of Gd-DTPA in Gd/NIR-MSNs, electron dispersive X-ray (EDX) was performed using a Tecnai G2 F20 electron microscopy with an EDX detector system.Gd concentration was determined with a Perkin-Elmer OPTIMA 5300 DV (PerkinElmer, USA) inductively coupled plasma atomic emission spectroscopy (ICP-AES).MR examination was performed with a 3 T clinical MR scanner (MAGNETOM Trio, SIMENS, Germany) at room temperature.After acquiring the  1 map MR images,  1 values were measured by manually drawn regions of interest for each sample.Relaxation rates  1 ( 1 = 1/ 1 ) were calculated from  1 values at different Gd concentrations.

Cellular Experiments
2.5.1.Cell Culture.Human embryonic kidney HEK 293T cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, California, USA) supplemented with 1 mmol/L L-glutamine and 10% fetal bovine serum (FBS; Life Technologies, Inc. Burlington, Canada).Human glioblastoma cell line U87-MG-GFP cells were maintained in Dulbecco's modified eagle medium (DMEM; KeyGEN Bio TECH, Nanjing, China) supplemented with 10% FBS.All media were added with antibiotics (100 U/mL of penicillin G and 100 g/mL of streptomycin).All cells were incubated at 37 ∘ C in a humidified atmosphere of 95% air and 5% CO 2 .

In Vitro Fluorescent Microscopy and MR Imaging of U87-MG Cell
Labeling with Gd/NIR-MSNs.Green fluorescent protein (GFP) labeling human glioblastoma cell line U87-MG were passaged 1 day before adding Gd/NIR-MSNs and inoculated in a 24-well chamber slides.After the cells attached to the chamber slides, stock solutions of Gd/NIR-MSNs were centrifuged and redispersed in fresh growth media and added to cultured cells to a final concentration of 0.1 mg/mL.Cells were incubated at 37 ∘ C overnight.The next day, label-containing media were aspirated carefully.Cells were washed five times with phosphate buffered saline solution (PBS) to remove excess nanoparticles and fixed with 10% formaldehyde at 4 ∘ C.After supernatant removed, 4  ,6diamidino-2-phenylindole (DAPI) was added to each chamber and incubated for 20 min.The slides were then washed three times with PBS and covered with glass coverslips.
Fluorescent images were recorded by a fluorescent inverted microscopy (Olympus, Japan).Imaging of DAPI was carried out with excitation of 330∼385 nm and emission at 420 nm, while imaging of GFP was performed with excitation of 460∼490 nm and emission of 510 nm.Gd/NIR-MSNs were excited by the external laser at 950 nm, while the emissions were collected in the ranges of 684 to 719 nm.To identify the Gd/NIR-MSNs uptaken by cells, fluorescent microscopy images were overlaid.
To further evaluate the dual-modality imaging ability of the Gd/NIR-MSNs, we designed an in vitro phantom experiment.In brief, U87-MG cells were incubated with Gd/NIR-MSNs or MSNs-NH 2 containing medium at a density of 5 × 10 6 cells/mL in 6 cm culture dishes at 37 ∘ C for 4 h, respectively.And then, the labeled cells were harvested and washed five times by centrifuge to remove additional nanoparticles.NIR imaging was performed using an IVIS Lumina XR system (Xenogen, USA), with the emission collected 808 nm and excited by the external laser at 778 nm.MR  1 map imaging was performed with a 3.0 T clinical MR scanner and a custom, premade four-channel mouse coil.The parameters were as follows: TR = 15.0 ms, TE = 1.73 ms, Average = 2, Slice Thickness: 2.0 mm, FOV = 113 mm, and Flip Angle = 5, 26.

Cell Cytotoxicity Evaluation of Gd/NIR-MSNs. HEK
293T cells were passaged when they are at confluence of 70% and inoculated to a 96-well plate (cell density 1 × 10 4 cells/well) in growth media (100 L).Cells were cultured for 24 h before experiments.For the dose-dependent assay, Gd/NIR-MSNs were centrifuged and redispersed in fresh growth media and added to cultured cells to a final concentration of 0, 20, 40, 100, and 200 g/mL.After 24 h of incubation at 37 ∘ C, supernatant was carefully removed and 100 L medium and 20 L MTT solution (5 mg/mL) were added and incubated for 4 additional hours.The supernatant in each well was aspirated and 150 L dimethyl sulfoxide (DMSO) was added to solubilize cells and MTT crystals.After 4 h of shaking on a shaking table at room temperature and dark to dissolve all crystals, the blue color was read in a multiwall scanning spectrophotometer (ELx800, BioTek, USA) at a wavelength of 540 nm.

In Vivo Dual-Modal NIR and MR Imaging. U87-MG
tumor bearing mice were anesthetized with 2% pentobarbital sodium and received an intratumor injection of 15 L of Gd/NIR-MSNs PBS solution (5 mg/mL).MRI was conducted on a 3.0 T clinical MR scanner and a custom, premade four-channel mouse coil, using a coronal and transversal  1 weighted sequence (TR = 670 ms, TE = 11 ms, Average = 4, Slice Thickness = 2.0 mm, FOV = 60 mm, and Flip Angle = 150).The nude mice were scanned before and after the intratumor injection of Gd/NIR-MSNs PBS solution (50 L, 5 mg/mL).NIR imaging was performed using an IVIS Lumina XR system, with the emission collected at 808 nm and excited by the external laser at 778 nm.MR and NIR imaging were performed at different time points after injection (0, 4, 8, 24, and 48 h).

Results and Discussion
The synthesis procedures of the Gd/NIR-MSNs are shown in Figure 1.Firstly, MSNs-NH 2 were synthesized via a CTABdirected sol-gel procedure by using TEOS and APTES as coprecursors.Secondly, CTAB was removed with ethanol and concentrated hydrochloric acid.Thirdly, NIR dye IR-808 and MR contrast agents Gd-DTPA were conjugated to MSNs-NH 2 simultaneously with condensation reaction between -NH 2 moieties of MSNs-NH 2 and -COOH groups of IR-808 as well as Gd-DTPA, resulting in the dual-modality imaging probes Gd/NIR-MSNs.
TEM images show that MSNs-NH 2 have a truncatedoctahedral shape with a mean diameter about 120 nm (Figures 2(a)-2(c)).It was also observed that the particles consist of highly ordered mesostructure (Figure 2(b)).To further determine the porous features, nitrogen adsorptiondesorption isotherm assay was conducted and uncovered a type IV isotherm according to the IUPAC nomenclature (Figure 2(d)).The sharp capillary condensation step and large hysteresis loop in the / 0 range of 0.45-1.0reveal characteristics of mesoporous materials with a narrow pore size distribution.The pore size distribution calculated based on the nonlocal density functional theory (NLDFT) illustrated that MSNs-NH 2 have uniform mesopore size of 3 and 6 nm and a total pore volume of 0.53 cm 3 g −1 .
After conjugation of IR-808 dyes and Gd-DTPA to MSNs-NH 2 , BET results uncovered the nanoparticle surface area reduction from 473 m 2 /g to 200 m 2 /g, while the zeta potential is reduced from 32 ± 5 mV to 7.35 ± 5 mV.These features suggested the successful conjugation of the NIR and MR agents with MSN-NH 2 .UV-vis absorption spectroscope and EDX spectrum measurement were also employed to confirm the presence of IR-808 and Gd-DTPA on Gd/NIR-MSNs particles.The absorption curve of Gd/NIR-MSNs illustrated a peak at 779 nm (Figure 2(e)), which is similar to the absorption peak of IR-808 and suggested the successful connection of IR-808 dyes with MSN-NH 2 [25].Another peak documented by UV-vis absorption at 680 nm was also observed, which is attributed to Gd-DTPA on the particles.UV-vis absorption spectroscope analysis also showed that Gd/NIR-MSNs particles have an IR-808 dye loading amount of 0.3 wt%.EDX spectrum result provided direct evidence of Gd 3+ presence on Gd/NIR-MSNs particles (Figure 2(f)), while ICP-AES measurements demonstrated 0.045 wt% conjugation amount for Gd-DTPA.The conjugation rate of Gd 3+ is lower than previous report (3.42 wt%) [23,26].
Strong fluorescence signals from NIR Gd/NIR-MSNs in deionized water were also identified (Figure 3(a)).The fluorescence emission spectra of the Gd/NIR-MSNs demonstrated a peak at 794 nm, which was similar with NIR dye IR-808 under the same condition [25].These features suggesting the spectral properties of IR-808 are unchanged during the synthesis procedure, which will validate the potential ability of the bimodal probes for NIR imaging.As shown in Figure 3(b), in vitro phantom MRI study demonstrated a clear Gd 3+ concentration dependent  1 signal enhancement effect with a high  1 value of 14.54 mM −1 s −1 .The relaxivity value is much larger than Gd-DTPA (4.75 mM −1 s −1 ) under the same condition.The high magnitude relaxivities of Gd/NIR-MSNs are attributed to Gd 3+ chelates attached to the mesochannels of the MSNs-NH 2 , which makes readily accession and efficient relax of water protons through a reduction in the tumbling rate [10,23].
Before proceeding to in vivo applications for NIR and MR dual-modal imaging, the uptake and retention ability of Gd/NIR-MSNs were evaluated in vitro with human glioblastoma cells U87-MG, which were stably transfected with green fluorescence protein.Figures 4(a)-4(d) demonstrated that the particles (red) distributed in the cytoplasm of U87-MG cells.TEM (Figure 4(e)) image further confirmed the uptake of the Gd/NIR-MSNs by the U87-MG cells.Biocompatibility is one of the most important prerequisites of nanoprobers for in vivo imaging.We chose human embryonic kidney cells HEK 293T to assess the cytotoxicity of Gd/NIR-MSNs with a MTT method.The results showed that no significant differences in the proliferation of the HEK 293T cells after 24 h of incubation with 20-200 g/mL of Gd/NIR-MSNs (Figure 4(f)), suggesting the Gd/NIR-MSNs, have good biocompatibility.
To further investigate the biomedical dual-modality imaging ability of the probes, U87-MG cells were incubated with the Gd/NIR-MSNs before unndergoing NIR fluorescent and MR  1 map imaging.U87-MG cells labeled with MSNs-NH 2 were treated as control.Strong NIR signal fluorescence was clearly visible in the optical image of tumor cells labeled with Gd/NIR-MSNs but completely absent in the image of cells incubated with MSNs-NH 2 (Figure 5(a)).Moreover, significant longitudinal relaxation time reduction was observed of cells labeled with Gd/NIR-MSNs on MR  1 map image compared with control (Figure 5(b)), which suggested that robust  1 enhancement can be achieved with the bimodal probes.These results validated the potential NIR and MR in vivo imaging ability of the Gd/NIR-MSNs.
In vivo experiments were performed on a rodent subcutaneous U87-MG glioblastoma modal to evaluate the dualmodal imaging effects of Gd/NIR-MSNs.NIR fluorescence and MR  1 imaging were acquired at different time points (0, 4, 8, 24, and 48 h) after 50 L of Gd/NIR-MSNs were intratumor injected (Figure 6).At 4 h after injection, the enhanced region on MR images were slightly augmented and showed a more homogeneous distribution with a distinction

Conclusions
In summary, we have successfully prepared a mesoporous silica nanoparticle based dual-modality probe (Gd/NIR-MSNs) by a facile protocol and carried out in vivo NIR fluorescence and MR imaging on a rodent tumor modal.
The Gd/NIR-MSNs have uniform particle size about 120 nm and ordered mesostructure.Excellent biocompatibility and high intracellular retention ability of Gd/NIR-MSNs make it possible to be used as an in vivo imaging tool.Remarkable  NIR fluorescence property and highly MR  1 relaxivity observed both in vitro and in vivo confirm the feasibility of Gd/NIR-MSNs as dual-modality imaging contrast agents in tumor detection.In addition, high sensitive images with distinct anatomical information are obtained by the Gd/NIR-MSNs, making the nanoprobes more potential for biomedical research.

2. 6 .
Animal Experiment 2.6.1.Animal Model.The animal study protocol was approved by the Animal Ethics Committee of Jinling Hospital.Female BALB/c nude mice (3-4 weeks old, 20-25 g) were purchased from the Comparative Medical Department of Jinling Hospital.U87-MG cells were harvested at 70% confluence and resuspended in PBS.Mouse tumor xenografts were established by U87-MG cells (2.0 × 10 6 ) inoculation into the right hind limbs of nude mice.

Figure 1 :
Figure 1: Schematic illustration of the synthesis procedures of the dual-modality imaging probes Gd/NIR-MSNs.

Figure 2 :Figure 3 :
Figure 2: (a) and (b) TEM images of the highly aminated MSNs prepared by CTAB-directed sol-gel processes by using TEOS and APTES as coprecursors.(c) The particle size distribution of Gd/NIR-MSNs by counting about 100 particles on TEM images.(d) Nitrogen sorption isotherms and pore size distribution curve of the MSNs-NH 2 .(e) The UV-vis absorption spectrum of the Gd/NIR-MSNs.(f) EDX spectrum of the Gd/NIR-MSNs.

Figure 6 :
Figure 6: In vivo MR (a) and NIR florescence (b) dual-modality imaging of U87-MG tumor bearing mice model (coronal position) after intratumoral injection of Gd/NIR-MSNs at 0, 4, 8, 24, and 48 h.Quantitative analysis of the MR  1 value (c) and NIR fluorescence intensity (d) in different time points.
).At 48 h after injection, only slightly hyperintense  1 signal remained in the tumor.The MR signal reduction is attributed to the diffusion of the nanoparticles.