Int J Clin Exp Med 2017;10(8):11442-11453 www.ijcem.com /ISSN:1940-5901/IJCEM0053708 Original Article Gadopentetic acid-doped, multifunctional, potentially targeted mesoporous silica nanoparticles as a novel MRI nano-contrast agent: synthesis, characterization and MRI study Xue Zhou1, Wei Jiang1, Jiuling Zheng2, Jiannong Zhao1, Wei Wu1, Dajing Guo1, Xinjie Liu1, Haitao Ran3, Xiaojing He1 Departments of 1Radiology, 2Hepatobiliary, The Second Affiliated Hospital of Chongqing Medical University, No. 74 Linjiang Rd, Yuzhong District, Chongqing 400010, China; 3Institute of Ultrasound Imaging, Department of Ultrasound, The Second Affiliated Hospital of Chongqing Medical University, No. 74 Linjiang Rd, Yuzhong District, Chongqing 400010, China Received March 24, 2017; Accepted July 2, 2017; Epub August 15, 2017; Published August 30, 2017 Abstract: Gadolinium-doped mesoporous silica nanoparticles (Gd-MSNs) with potential molecular targeting capa- bility have drawn worldwide attention as a magnetic resonance imaging (MRI) contrast agent. However, to date, Gd-MSNs have exhibited less-than-ideal properties. In this study, a reverse microemulsion method was used to dope gadopentetic acid (GA) into the mesopores of mesoporous silica nanoparticles (MSNs) to synthesize, for the first time, novel Gd-MSNs with high gadolinium content (GC). These Gd-MSNs were characterized using transmis- sion electron microscopy (TEM) and a Malvern Zetasizer. The GCs and stability of the Gd-MSNs were determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). Cell viability was tested via the Cell Counting Kit-8 (CCK-8) assay. Finally, cellular and rat MRI was performed to determine the in vitro and in vivo MR enhancing effects of the Gd-MSNs. We observed a clearly visible pore structure with a large number of doped GA molecules on the Gd-MSNs. The globular Gd-MSNs had higher GC and good stability in a simulated in vitro environment. No significant cellular toxicity was observed in either human umbilical vein endothelial cell (HUVEC) or human prostate adenocarcinoma cell (PC-3) lines at Gd-MSN concentrations of 25, 50, and 100 μg/mL. According to the cellular and rat MRI data, Gd-MSNs had strong MR enhancing effects both in vitro and in vivo. Gd-MSN is a promising novel MRI contrast agent for its multifunctional and highly efficient properties. Keywords: Mesoporous silica nanoparticles, gadopentetic acid, nano-contrast agent, magnetic resonance imag- ing, reverse microemulsion Introduction fore, a novel carrier that can combine MR con- trast agents and targeting groups is needed, Magnetic resonance imaging (MRI) is one of the and many researchers have recently conducted most powerful techniques for clinical diagno- studies in this field [6-8]. ses [1], especially for the early diagnosis of tumors, because of its high soft tissue resolu- Commonly used carriers include carbon nano- tion [2, 3]. The application of MR contrast tubes [9], superparamagnetic iron oxide nano- agents has further improved the diagnostic particles (SPIONs) [10], liposomes and mesopo- rate of tumors. Currently, MR contrast agents rous silica nanoparticles (MSNs) [11]. All these are mainly small-molecular gadolinium (Gd) nanoparticles (NPs) have several advantages chelates that are difficult to combine with bio- over conventional imaging agents [12]. Load- logical macromolecules [4]. Thus, these con- ability is one advantage, wherein the concentra- trast agents do not meet the requirements of tion of the imaging agent can be controlled modern theranostics, which requires contrast within each NP during the synthesis process. agents with good targeting ability to achieve Another advantage is the tunability of the sur- molecular diagnosis and treatment [5]. There- face of the NPs, which can potentially prolong Gadopentetic acid-doped mesoporous silica nanoparticles as MRI contrast agent the circulation time of the agent in the blood or Many previous studies have used the classical target a specific location within the body [13]. Stöber method to synthesize Gd-containing Of course, NPs also have some of their own MSNs [26-28]. However, NPs synthesized by characteristics. SPIONs are widely used as neg- this method usually have less-than-ideal prop- ative imaging agents, which produce a signal- erties, such as large particle sizes (> 100 nm), decreasing effect [14]. Carbon nanotubes are low gadolinium content (GC) (< 10%), irregular both excellent contrast agents and efficient sphericity and poor dispersity [29, 30]. These drug carriers, and they can be designed as properties may be caused by adding the Gd positive or negative imaging agents, or both. complex after synthesizing the MSNs, which However, carbon nanotubes are not hydrophilic could lead to the Gd complex attaching only to and easily agglomerate in aqueous solutions, the NP surface. Thus, the attachment of the resulting in biological toxicity [15]. Liposomes complex to the surface would decrease the can solve this problem because of their bio- regularity of both the dispersity and morpholo- compatibility and are thought to have no toxic gy of the NPs. Moreover, the preparation pro- or injurious effects on biological systems [16]. cess of Gd-MSNs in many previous studies was However, the lipid composition of liposomes divided into several complex steps to combine influences the relaxivity produced by the con- the Gd and NPs with poor reproducibility. To trast agents encapsulated or incorporated overcome these shortcomings, in this study, we within them. Compared with other NPs, MSNs improved the reverse microemulsion method possess larger surface areas and pore vol- [31] to simplify the preparation process and umes; thus, they can be doped with larger optimize the Gd-MSNs properties. amounts of contrast agent [17]. In addition, these NPs can be combined with antibodies, Reverse microemulsion can produce hydrophil- siRNA, aptamers or peptides for MR-targeted ic and fairly uniform-sized NPs and allows easy molecular diagnosis and treatment because of modulation of the NP surfaces for various appli- the rich abundance of easily modifiable hydrox- cations [32-34]. This method had long been yl groups on their surfaces [18-21]. Additionally, applied in the preparation of silica NPs but rare- they can increase the sensitivity and specificity ly been used during the synthesis of Gd-MSNs. and decrease the dosage and toxic side effects In this study, we innovatively introduced gado- of MR contrast agents [17]. pentetic acid (GA) solution into the microemul- sion reaction system when synthesizing MSNs. MR contrast agents synthesized with MSNs as GA was doped into Si-O-Si cages that were cre- a carrier include magnetic iron oxide NPs, man- ated by hydrolyzing tetraethylorthosilicate (TE- ganese (Mn), Gd, and others. Kim J et al. found OS) into the MSNs through this method. Thus, that core-shell NPs consisted of a single Fe3O4 GA is one component of this reverse micro- nanocrystal core and a dye-doped mesoporous emulsion reaction system. As a result, the silica shell with a poly (ethylene glycol) coating; amount of gadolinium loaded into the MSNs these NPs can be used as MRI and fluores- was greatly increased. The amount of GC within cence imaging agents and as drug delivery the MSNs and particle size could be controlled vehicles [22]. Kim T et al. synthesized mesopo- by adjusting the content of GA solution in the rous silica-coated hollow Mn oxide NPs as posi- reaction system. Furthermore, this improved tive T1 contrast agents for labeling and MRI method ensures good spherical structure and tracking of adipose-derived mesenchymal stem dispersion of Gd-MSNs, simplifies the experi- cells [23]. Gd-based contrast agents are the mental steps and increases reproducibility. most widely used due to their higher r1 relax- ation rate. Hsiao et al. found that Gadopentetate In previous studies, Gd chloride and Gd oxide dimeglumine (Gd-DTPA) -modified MSNs were were most widely used in the synthesis of ideal T1 contrast agents for tracking stem cells Gd-MSNs [35, 36]. As a simple compound, the in MRI [24]. Shao et al. incorporated Gd2O3 mol- Gd ions contained therein leached easily, caus- ecules into the MSNs of Mobil Composition of ing toxicity [37]. Other studies used stable Matter No. 41 (MCM-41) to obtain Gd2O3@ Gd chelates with safe performance, such as MCM41 and demonstrated that this was supe- Gd-DTPA and Gd-DOTA [38-40]. Gd chelates rior to current contrast agents and could be have a large molecular weight and are more dif- used for tumor imaging in preclinical studies [25]. 11443 Int J Clin Exp Med 2017;10(8):11442-11453 Gadopentetic acid-doped mesoporous silica nanoparticles as MRI contrast agent Synthesis of Gd-MSNs The Gd-MSNs and MSNs were synthesized by a reverse micr- oemulsion method [41]. Gd- MSNs with different GCs were synthesized first. A mixture of Triton X-100 (1.77 mL), cyclo- hexane (7.5 mL) and 1-hexa- nol (1.8 mL) was stirred for 10 min. A GA aqueous solution at a concentration of 0.274 g/ Figure 1. The fabrication process and a sche- mL was prepared. Different matic illustration of the structure of Gd-MSNs. amounts (0.5, 1, 2, 3, 4, and 5 mL) of GA aqueous solution were then separately added to lar weight of Gd-DTPA is approximately 938, the mixture. The resulting mixtures were stirred and Gd-DOTA is approximately 580. Thus, a for an additional 30 min followed by the addi- relatively small-molecular-weight Gd chelate is tion of TEOS (100 μL) and an additional 30 min needed. We innovatively utilized GA (molecular of stirring. Ammonia solution (1.5 mL) was weight of approximately 547), an important added, and the reaction mixture was stirred for intermediate during the synthesis of Gd-DTPA, 6 h until a microemulsion was obtained. Finally, as the imaging agent to synthesize a new type acetone (4 mL) was added to break the emul- of Gd-MSNs. sion. The particles were isolated by centrifuga- tion and washing with water and ethanol.