Enhancing Resonance Imaging Sensitivity and Upconversion Emission of Gd-based Nanoscale Coordination Polymers by Combination With Au Nanoparticles

Chen Xiao Research Center for Ultra ne Powder Materials, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang 330022, China. Tuck Yun Change Breast Disease Center, The First A liated Hospital, Sun Yat-Sen University, Guangzhou 510080, China Yin Li Research Center for Ultra ne Powder Materials, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang 330022, China. Hualan Xu Research Center for Ultra ne Powder Materials, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang 330022, China. Shengliang Zhong (  slzhong@jxnu.edu.cn ) Research Center for Ultra ne Powder Materials, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang 330022, China. https://orcid.org/0000-0001-5900-9884


Introduction
Magnetic resonance imaging (MRI) is a kind of non-radioactive imaging technology [1,2]. It is an extremely important clinical diagnosis model for diseased tissues, including solid tumours, tissues, organs, and blood vessels. MRI has several advantages over other diagnosis models because it does not require ionizing radiation, can provide high spatial resolution and high-contrast three-dimensional images [3][4][5]. It functions as a contrast imaging by changing the longitudinal and transverse relaxation times of the body's water environment [6]. At present, there are many nanocarriers that can be used for MRI, such as noble metal nanoparticles, silica, and rare earth nanomaterials [7].
In particular, Gd3 + has a large magnetic moment due to its seven unpaired electrons, its compounds are widely used as magnetic contrast agents for clinical diagnosis [8]. For example, Gd-DTPA is one of the most used MRI agents. However, it is limited by the low relaxation rate and nephrotoxicity [9]. To address these problems, various efforts that focus on enhancing the relaxation rate of contrast agents and reducing their toxicity have been developed [10]. Among the techniques used, surface modi cation is an effective method to enhance MRI signals. For example, carbon quantum dots with good optical properties are used to modify Gd-based materials to improve relaxation rates [11]. Contrast agent materials coated with SiO 2 have been reported to improve the relaxivity information [12]. Other strategies have also emerged. For example, a radionuclide-Cu-64 labelled doxorubicin (DOX)-loaded polydopamine (PDA) gadolinium-metallofullerene core-satellite nano-theragnostic agent is developed for multimodal imagingguided combination cancer therapy [13]. It was found that the compound showed good biocompatibility, strong near infrared (NIR) light absorption, and high relaxation rate. Hai et al. designed a Gd-based probe through γ-glutamytraspetidase cleavage and intracellular condensation reaction to enhance the T 2weighted MR contrast [14]. Another strategy for enhancing MRI signals is to control the internal structure of the nanomaterials, such as the formation of core-shell structures and heterojunction structures. Adah Almutairi et al. reported a modulation of the interfacial layer thickness to enhance the MRI signal [15].
Recently, Ye et al. exploited an MRI material through liposome surface modi ed (cRGD@MLP-Gd) exhibiting T 1 and T 2 weighted contrast enhancement in vivo [16]. Compared to the single modal imaging, multimodal imaging probe is signi cant not only for the great improved detection sensitivity, but also for decreasing the toxicity risk and other side effect [15]. Thereby providing accurate diagnosis and precise therapy.
The most developed nanostructured multimodal imaging probe is the combination of MRI and optical imaging [17], which can overcome the limitations of each modal to generate more comprehensive imaging information with high spatial resolution, in nite tissue penetration and high sensitivity. Gd-based nanomaterials have been proved to be an ideal candidate for multifunctional materials, which combine with upconversion luminesces and MRI [18]. Lanthanide-doped CP upconversion nanoparticles demonstrate an anti-Stokes behavioural process, which converts continuous low energy (such as NIR light or IR light) into high energy light (visible light) by continuous multiphoton absorption or energy transfer processes [19,20], providing a broad range of applications, from electrochemistry to biomedicine [21,22]. One of the major attractions for biomedical application of lanthanide-doped upconversion nanoparticles (UCNPs) is that the anti-Stokes process can eliminate background auto uorescence and light scattering effects of the biological material [23]. The other conspicuous attraction is the irradiation light can achieve high tissue penetration depth and have low toxicity to the tissue [24]. Nevertheless, the overall upconversion e ciency is relatively low, and their practical application for biomedical are still limited [25]. As a result, it is desirable to develop simple and versatile synthetic routes for UCNPs with enhanced performance.
In recent years, nanoscale CPs have aroused extensive research interest in the elds of chemistry, physics, medicine, nanotechnology, and materials [26][27][28][29]. Particularly, in biomedical, they are excellent contrast agents for bioimaging bene ting from the highly tunable composition and structures, they are intrinsically biodegradable and easy to combine multiple modalities [30]. Furthermore, by combining with suitable materials, the properties of CPs can be improved, with enhanced functionality/stability, ease of preparation, and selectivity of operation [31]. It is expected that CP composites may nd extensive applications in near future [32][33][34][35]. However, to date, few reports have appeared on the preparation and application of GdCP composites, and rare work studying GdCPs with enhanced MRI effects. Based on the localized surface plasmon resonance (LSPR) effect of Au nanoparticles (AuNPs) and the limitations of its low relaxation rate and potential biotoxicity of Gd-based nano-compounds [36].
In this work, we present the synthesis and characterization of a new MRI/upconverstion luminescence multimodal contrast agent, the as-obtained GdCP nanospheres have good uniformity and monodispersity, the effect of Au NPs used for enhancing upconversion luminesces, relaxation information, and T1-weight MRI and biocompatibility of Au@GdCP has also been explored. Au@GdCP may offer great potential for future biomedical applications.

Synthesis of Au@GdCP nanocomposites
The preparation method of Au@GdCP composite nanoparticles contains the following processes. Firstly, AuNPs were synthesized by reduction of chloroauric acid (HAuCl 4 , 1 mL, 0.025 mol/L) under sodium citrate solution (3 mL, 0.035 mol/L) and hexadecyl trimethyl ammonium bromide (CTAB, 20 mL, 0.125 mol/L) was used as stabilizer, the gold nanoparticles (AuNPs) with an average diameter of about 20 nm was diluted with DI water to 5 mL. Afterwards, 0.2 mmol rare earth ions solution which contains Gd(NO 3 ) 3 , Yb(NO 3 ) 3 and Er(NO 3 ) 3 (molar ratio of Gd: Yb: Er = 0.16: 0.03: 0.01) were add into allantoin solution (0.3 mmol) stirring for 15 min at room temperature. Subsequently, the above solution was mixed with together and transferred into a Te on lined stainless-steel autoclave with capacity of 50 mL, heated at 160 ℃ for 15 h. As the autoclave was cooled to room temperature naturally. Finally, the product was isolated by centrifugation and washed with deionized water and absolute ethanol for three times, and dried in vacuum at 60 ℃ for 12 h. The products marked as Au@GdCP. Meanwhile, the GdCP prepared with same method without Au NP introduced.

Characterization
The morphologies and sizes of the samples were obtained through scanning electron microscope (SEM, HITACHI S-3400N, 30 kV) and transmission electron microscope (TEM, Hitachi JEM2100, 200 kV). XRD pattern were performed on a Rigaku X-ray diffractometer with Cu Kα radiation (λ = 154178 Å) with the range of 5-90°, FT-IR data was obtained with Nicolet 6700 FT-IR spectrometer using KBr pellet between 4000 cm-1 and 500 cm-1 at room temperature. Photoluminescence (PL) emission spectra were recorded with an FLS 980 (Edinburgh Instruments, England). Elemental analysis was carried out on an EA3000 elemental analyzer. Magnetic properties were measured using a SQUID magnetometer equipment. The relaxation information was performed on a 0.5T NMI20-Analyst NMR system (Niumag Corporation, Shanghai, China).

Characterization of GdCP and Au@GdCP
The morphologies and sizes of the as-obtained GdCPs were initially characterized by scanning electron microscopic (SEM). As can be seen in Figure. 1(a), the products were discrete and spherical, with a mean diameter of ~ 179 nm and relative narrow size distribution, as determined by dynamic light scattering (DLS, insert of Figure. 1a). TEM images under different magni cations ( Figure. 1b and c) further verify that GdCP is uniformly round and monodispersed. Interestingly, after combination with Au nanoparticles, A decrease in the particle size was obtained, with an average size of 150 nm ( Figure. 1d) for Au@GdCP, which is helpful for the biomedical applications. The reduction in diameter probably due to the Au particles acting as the nuclei, leading to a decrease in the size of the nal product, which is always observed in the growth of the crystal. The TEM image in Figure. 1e shows the internal structure of Au@GdCP. It can be clearly observed that each nanosphere encapsulating several Au nanoparticles.
Magni cation of the TEM image in Figure. 1f demonstrate that the Au@GdCP nanosphere has an outer layer with thickness of approximately 50 nm. In addition, the energy dispersive spectroscopy (EDS) mapping analysis of a single nanosphere are illustrated in Figure. 1g, the C, O, N, Gd, Yb, and Er elements coexist and are distributed homogeneously throughout the particle. However, the Au element is aggregated and located in the internal part of the Gd-based coordination polymers. All this evidence veri es that AuNPs have successful embedded within GdCP.
To assess the structure of the compounds, powder X-ray diffraction (XRD) patterns of GdCP spheres and Au@GdCP nanocomposites are presented in Figure.

Magnetic properties
The magnetic properties of GdCP and Au@GdCP nanocomposites were measured by SQUID magnetometer. Figure. 3 shows the magnetization of the magnetic eld (M-H) curves of samples at 300 K. The magnetization of GdCP and Au@GdCP are 5.19 emu/g and 683.52 emu/g at 300 k, respectively.
With the introduction of AuNPs, the magnetization was enhanced remarkably. No hysteresis loop was observed. The magnetization curve of the samples displayed typical paramagnetic properties. These results show that even after introduction of AuNPs, the particle keep well paramagnetic properties.

Luminescence properties of Au@GdCP
The upconversion emission spectrum of Au@GdCP under 980 nm laser excitation is shown in Figure. 4.
Simultaneously, GdCP was used as the control under the same conditions. In this system, Yb 3+ acts as a sensitizer and transfers the absorbed excitation energy to the activator ions of Er 3+ . First, Yb 3+ is excited from the state level ( 7 F 7/2 ) to the exciting level ( 5 F 5 /2) under the continuous 980 nm laser excitation.
Subsequently, Er 3+ ions are excited to the intermediate state ( 4 I 11 /2) and then transfer energy to the excited state ( 4 F 7/2 ), which resulting the energy transfer of Er 3+ ions from 4 F 7/2 levels to the levels of 2 I 11/2 , 4 S 3/2 , and 4 H 9/2 with nonradioactive process, respectively [37][38][39]. The emission spectra exhibited three bands, attributed to green emission (525 nm and 545 nm) and red emission (655 nm). Compared with GdCP, an obvious enhanced emission intensity of 38.4% was obtained after Au ions modi cation, coupled with a slight blue shift for 4 nm, mainly attributed to the LSPR effect of the AuNPs [38]. LSPR strengthens the local crystal eld, resulting in the enhancement of excitation eld intensity [41].
Meanwhile, coupling between the broad LSPR peaks of AuNPs and the upconversion luminescence enhances the energy transfer effect, improving the decay rate of luminescent centres and the e ciency of the emission intensity [42,43]. Therefore, AuNPs play a role in enhancing the uorescent intensity in Au@GdCP.

θ
Relaxation information and T 1 -weighted MRI maps Gd-based composites have good kinetic and thermodynamic stabilities. They can accelerate the T 1 relaxation of water protons, and improve the brightness of MRI signals. Approximately 40% of MRI scans employ Gd 3+ chelates for contrast agent in clinical [44]. However, free Gd 3+ ions are known to have serious toxic effect due to the replacement of Ca 2+ ions in the body, further improvement is required. Thanks to the biocompatibility and tuneable morphology, AuNPS have been frequently used in clinical applications. In recent years, they have been exploited to create very promising tools for multimodal imaging and MRI-guided therapies [45]. To estimate the performance of GdCP and Au@GdCP as MRI contrast agent, the relaxation rate and T 1 -weighted MRI were investigated in vitro at the low eld of 0.5T.
As shown in Figure.  The T1-weighted MRI of Au@GdCP and GdCP nanocomposites were also obtained (Fig. 3b, d), in which T1-weighted MRI signals got brighter and brighter with the increasing concentration of Gd 3+ . Compared with the T 1 -weight MRI signal of both nanocomposites, the contrast effect of Au@GdCP is much better than GdCP, demonstrating that the existence of AuNPs greatly enhances the MRI signal intensity and the contrast of the MRI signal.
It is well known that reducing the inversion rate of the contrast agent in the aqueous proton solution can reduce the average spin-nuclear spin and the dipole-dipole interaction between the contrast agent, and it can prolong the inversion time, which can improve the relaxation e ciency of the contrast agent [25].
With the introduction of AuNPs, the LSPR of the AuNPs may strengthen the external magnetic eld, which results in intermolecular dipole-dipole interactions, increasing the contrast relaxation rate and the T 1weighted MRI signal [47][48]. Moreover, the strong light scattering of AuNPs translates the absorbed light to the plasmon wavelength, resulting in enhancement of T 1 -weighted MRI signals [49].

Biocompatibility of the Au@GdCPs
As is well known, good biocompatibility is an important prerequisite and crucial factor for biological applications. In this work, the in vitro biocompatibility of Au@GdCP was evaluated through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with incubated HeLa cells.
Meanwhile, GdCP was tested as a control under the same conditions. Figure. 6 shows that with the increasing concentration of Au@GdCP and GdCP, the relative cell viability of HeLa gradually decreased. When the concentration of Au@GdCP and GdCP reached 1.0 mg/mL, the relative cell viability is 80% and 82.2%, respectively. The MTT results indicate that Au@GdCP exhibit low toxicity and good biocompatibility.

Conclusions
In summary, Au@GdCP nanocomposites were fabricated through a facile hydrothermal method. With the increasing concentration of GdCP and Au@GdCP, the longitudinal relaxation time and lateral relaxation time exhibited an excellent linear relationship, and T 1 -weight MRI displayed brighter and brighter. When AuNPs were introduced into GdCP, based on the LSPR of AuNPs, the longitudinal relaxation rate of Au@GdCP was much higher than that of GdCP. Meanwhile, Au@GdCP displayed good upconversion luminesces and excellent biocompatibility. These properties demonstrated that Au@GdCPs can be effectively applied to MRI/ upconverstion luminescence multimodal contrast agent and are promising for biomedical applications.   The magnetic properties of GdCP (a) and Au@GdCP(b).

Figure 5
Relaxation information and T1-weighted MRI maps with different concentrations of GdCP (a, b) and Au@GdCP (c, d).

Figure 6
Relative cell viability of HeLa incubated with various concentrations of GdCP and Au@GdCP at 37℃ for an incubation time of 24 h.