Improving the MR Imaging Sensitivity of Upconversion Nanoparticles

Jan 6, 2016 - In vivo T1-weighted MRI shows potent tumor-enhanced MRI with UCNPs–H@BSA·DTPAGd. An in vivo biodistribution study indicates that it i...
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Improving the MR Imaging Sensitivity of Upconversion Nanoparticles by an Internal and External Incorporation of the Gd3+ Strategy for in Vivo Tumor-Targeted Imaging Hongli Du,† Jiani Yu,§ Dongcai Guo,*,†,‡ Weitao Yang,∥ Jun Wang,§ and Bingbo Zhang*,§ †

School of Chemistry and Chemical Engineering and ‡Hunan Provincial Key Laboratory for Cost-effective Utilization of Fossil Fuel Aimed at Reducing Carbon-dioxide Emissions, Hunan University, Changsha 410082, China § Shanghai Skin Disease Hospital, The Institute for Photomedicine, The Institute for Biomedical Engineering & Nano Science, Tongji University School of Medicine, Shanghai 200443, China ∥ School of Materials Science and Engineering, School of Life Science, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin University, Tianjin 300072, China

ABSTRACT: Gd3+-ion-doped upconversion nanoparticles (UCNPs), integrating the advantages of upconversion luminescence and magnetic resonance imaging (MRI) modalities, are capturing increasing attention because they are promising to improve the accuracy of diagnosis. The embedded Gd3+ ions in UCNPs, however, have an indistinct MRI enhancement owing to the inefficient exchange of magnetic fields with the surrounding water protons. In this study, a novel approach is developed to improve the MR imaging sensitivity of Gd3+-ion-doped UCNPs. Bovine serum albumin (BSA) bundled with DTPA-Gd3+ (DTPAGd) is synthesized both as the MR imaging sensitivity synergist and phase-transfer ligand for the surface engineering of UCNPs. The external Gd3+ ion attachment strategy is found to significant improve the MR imaging sensitivity of Gd3+-ion-doped UCNPs. The relaxivity analysis shows that UCNPs@BSA·DTPAGd exhibit higher relaxivity values than do UCNPs@BSA without DTPAGd moieties. Another relaxivity study discloses a striking message that the relaxivity value does not always reflect the realistic MRI enhancement capability. The high concentration of Gd3+-ion-containing UCNPs with further surface-engineered BSA·DTPAGd (denoted as UCNPs−H@BSA·DTPAGd) exhibits a more pronounced MRI enhancement capability compared to the other two counterparts [UCNPs−N@BSA·DTPAGd and UCNPs−L@BSA·DTPAGd (−N and −L are denoted as zero and low concentrations of Gd3+ ion doping, respectively)], even though it holds the lowest r1 of 1.56 s−1 per mmol L−1 of Gd3+. The physicochemical properties of UCNPs are essentially maintained after BSA·DTPAGd surface decoration with good colloidal stability, in addition to improving the MR imaging sensitivity. In vivo T1-weighted MRI shows potent tumor-enhanced MRI with UCNPs−H@BSA·DTPAGd. An in vivo biodistribution study indicates that it is gradually excreted from the body via hepatobiliary and renal processing with no obvious toxicity. It could therefore be concluded, with improved MR imaging sensitivity by an internal and external incorporation of Gd3+ strategy, that UCNPs−H@BSA·DTPAGd presents great potential as an alternative in tumor-targeted MR imaging.

1. INTRODUCTION Molecular imaging is an upcoming promising technology in the accurate diagnosis of diseases in their early stages. Medical imaging has been classified into different specialties containing optical imaging,1 magnetic resonance imaging (MRI), 2 computed X-ray tomography (CT),3 positron emission tomography (PET) imaging,4 and so forth. Among them, MRI is one of the most commonly used diagnostic tools, with © XXXX American Chemical Society

the relaxation of water protons exposed to an external magnetic field, and morphological together with anatomical information can be obtained with unlimited tissue penetration yet high spatial resolution.5,6 With the aid of contrast agents, the Received: November 13, 2015 Revised: December 30, 2015

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biocompatibility and biodistribution of UCNPs@BSA·DTPAGd were also investigated.

contrast quality of MR imaging can be highly improved. In the past decade, nanoparticle-based contrast agents, called nanoprobes, have shown promising potential in medical imaging.7−10 Upconversion nanoparticles (UCNPs), which convert two or more low-energy pump photons from the NIR spectral region to a higher-energy output photon with a shorter wavelength, display some unique upconversion luminescence (UCL) advantages, such as no photobleaching and photoblinking, weak autofluorescence from living cells or tissues, deep light penetration depth in tissues, and minimal photodamage to living organisms,11 using UCNPs as contrast agents widely used for fluorescence imaging.12,13 Moreover, by being doped with Gd3+ ions, UCNPs combine their unique upconversion luminescence properties with magnetic performance to serve as dual modal contrast agents.14,15 UCNPs doped with paramagnetic Gd3+ ions have attracted increasing interest as promising molecular imaging contrast agents because the magical roles of Gd3+ ions can not only enhance the brightening performance of MRI to differentiate the signal from other pathogenic or biological conditions as T1 contrast agent but also control phase/size simultaneously, improve the upconversion luminescence efficiency, and tune upconversion emission spectra through energy migration trapping.15−17 Gd3+-ion-doped UCNPs can integrate the advantages of upconversion luminescence and MRI modalities to offer more imaging information for diagnosis. The combination of two or multiple molecular imaging techniques can therefore offer synergistic advantages over any modality alone.18 Many works have been published on the optical applications of UCNPs owing to their unique upconversion luminescence properties,19−22 whereas many fewer MR imaging applications of UCNPs are reported, which is mainly attributed to the insensitivity of Gd3+-ion-doped UCNPs found during MR imaging. The Gd3+ ions embedded in the UCNPs cannot efficiently exchange magnetic fields with surrounding water protons, resulting in low relaxivity and a poor MRI enhancement of Gd3+-ion-doped UCNPs.23,24 There is, therefore, a great need to search for new strategies to improving the MR imaging sensitivity of UCNPs for its applications in bioimaging. In this study, Gd3+ ions were integrated inside and outside of UCNPs to improve the MR imaging sensitivity of UCNPs. Gd3+-ion-doped UCNPs were synthesized as the basis of optical and MRI contrast agents, even though this internal Gd3+ ion embedding has limited relaxivity. Three different concentrations of Gd3+-ion-containing UCNPs were synthesized, namely, non-Gd3+-ion-doped UCNPs (denoted as UCNPs−N), a low concentration of Gd3+-ion-doped UCNPs (denoted as UCNPs−L), and a high concentration of Gd3+-ion-containing UCNPs (denoted as UCNPs−H) in a high-boiling-point solvent mixture.16 BSA·DTPAGd, a paramagnetic biomolecular complex with high relaxivity, was synthesized both as a synergist of the T1-weighted MRI contrast agent and a surface ligand for the phase transfer of Gd3+-ion-doped UCNPs. This external Gd3+ ion attachment is expected to contribute further to the enhancement of MR imaging sensitivity. The rate of contributions on MRI enhancement from internal and external Gd3+ ion attachments was systematically investigated in this study. The size and morphology, fluorescence property, and colloidal stability of UCNPs@BSA·DTPAGd were characterized by transmission electron microscopy (TEM), the UCL spectrum, and dynamic light scattering (DLS). The practical diagnostic application of UCNPs@BSA·DTPAGd was evaluated by in vivo imaging on living tumor-bearing mice. The

2. EXPERIMENTAL SECTION 2.1. Materials. All materials were obtained from commercial suppliers and used as received. YCl3·6H2O (99.99%), YbCl3·6H2O (99.99%), ErCl3·6H2O (99.99%), GdCl3·6H2O (99.99%), diethylenetriaminepentaacetic acid dianhydride (DTPAA, 95%), and oleic acid (OA, 90%, technical grade) were purchased from Alfa Aesar Co., Ltd., China. BSA and 1-octadecene (ODE, 90%, technical grade) were purchased from Aladdin Reagent Co., Ltd., China. NaOH, NH4F, ethanol, and chloroform were purchased from Sinopharm Chemical Reagent Co., Ltd., China. Ultrapure Millipore water (18.2 MΩ cm−1 resistivity at 25 °C) was used throughout the experiments. 2.2. Characterizations. The sizes and morphologies of UCNPs were obtained by using a Tecnai G2 F20 high-resolution TEM at 200 kV. Upconversion luminescence (UCL) spectra were recorded on an AvaSpec-2048 fluorescence spectrometer using an external 0−2 W adjustable 980 nm CW laser as the excitation source. Dynamic light scattering (DLS) and zeta potential analysis of aqueous UCNPs were carried out on a laser light scattering system (Nano ZS, Malvern). 2.3. Synthesis. 2.3.1. Synthesis of OA-Capped UCNPs. In this study, three types of OA-capped UCNPs, namely, NaYF4:Yb, Er (denoted as UCNPs−N), a high concentration Gd3+-ion-containing NaGdF4:Yb, Er (denoted as UCNPs−H), and a low concentration Gd3+-ion-doped NaGdF4:Yb, Er (denoted as UCNPs−L) were prepared via the thermal decomposition strategy reported previously.16 In a typical procedure for a high concentration Gd3+-ion-containing NaGdF4:Yb, Er nanoparticles, GdCl3·6H2O (0.78 mmol), YbCl3·6H2O (0.20 mmol), and ErCl3·6H2O (0.02 mmol) were mixed with 6 mL of OA and 15 mL of ODE in a three-necked round-bottomed flask and heated slowly to 120 °C to remove water under vacuum. Then, the system was maintained at 150 °C for 30 min until a homogeneous transparent yellow solution was formed. The mixture solution was then cooled to room temperature, and 10 mL of methanol solution containing NaOH (100.0 mg) and NH4F (148.1 mg) was slowly added with stirring for 30 min. The mixture solution was degassed at 100 °C for 10 min to remove methanol slowly, then heated to 300 °C at a heating rate of 10 °C min−1 until it became a bit turbid, and maintained at this temperature for 1 h under an argon atmosphere. The solution was then cooled naturally, and the nanoparticles were washed three times by precipitating in ethanol. The final products were dispersed in 10 mL of chloroform at 4 °C for ready use. For lowconcentration Gd3+-ion-doped UCNPs synthesis, GdCl3·6H2O (0.18 mmol), YbCl3·6H2O (0.80 mmol), and ErCl3·6H2O (0.02 mmol) were used. And for non-Gd3+-ion doped UCNPs, YCl3·6H2O (0.78 mmol), YbCl3·6H2O (0.20 mmol), and ErCl3·6H2O (0.02 mmol) were used instead. Other procedures were the same as mentioned above. 2.3.2. Synthesis of BSA·DTPAGd. The synthesis of BSA·DTPAGd was according to the literature with minor modifications.25 Briefly, 200.0 mg of bovine serum albumin (BSA) was added to 3 mL of NaHCO3 solution (pH 8.2−8.5) with constant stirring. DTPAA (200.0 mg) was dissolved in 1 mL of anhydrous dimethysulfoxide (DMSO) to form a homogeneous solution at room temperature and then added to the BSA solution dropwise, and the pH value of the mixture solution was adjusted to 8.2 via the addition of a 5.0 mol L−1 sodium hydroxide solution. The resulting mixture was magnetically stirred for 3 h at ambient temperature. After sufficient reaction, the solution was dialyzed against 0.1 mol L−1 citrate buffer with pH 6.5 for 3 days and then dialyzed against distilled water for another 2 days before GdCl3· 6H2O (100 mg) was added to the BSA·DTPA conjugate solution. After being stirred for 3 h, the newly formed solution was dialyzed against 0.1 mol L−1 citrate buffer at pH 6.5 for 2 days and then dialyzed against distilled water for another 2 days. Finally, the purified BSA·DTPAGd solid powder was collected via lyophilization. 2.3.3. Phase Transfer of UCNPs. The phase transfer of UCNPs was implemented according to our previously reported work with minor modifications.26 Briefly, the mole ratio of BSA·DTPAGd to UCNPs was kept in the range of 500−1000. UCNPs dispersed in chloroform were B

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Langmuir transferred to a clean syringe as the organic phase for injection. BSA· DTPAGd dissolved in 15 mL of deionized water was the aqueous phase. The organic phase of UCNPs was slowly injected into the aqueous phase of BSA·DTPAGd with ultrasonication by an ultrasonic cell crushing instrument. Upon injection was complete, the organic phase was completely evaporated on a rotary evaporator to form a transparent aqueous solution. After being centrifuged at 13 000g for 10 min to remove free BSA·DTPAGd, the final precipitation was redispersed in 2 mL of borate saline buffer (50 mmol L−1, pH 8.2) to obtain aqueous purified UCNPs@BSA·DTPAGd. 2.4. Property Characterization. 2.4.1. Colloidal Stability of UCNPs@BSA·DTPAGd. The UCNPs@BSA·DTPAGd samples were mixed with NaCl solution of different ionic strengths and a buffer solution with different pH values. The hydrodynamic diameters of nanoparticles were determined by DLS at different time intervals. 2.4.2. In Vitro Assessment of Relaxation Properties and T1Weighted MR Imaging of UCNPs@BSA·DTPAGd. The longitudinal (T1) and transverse (T2) relaxation times were determined on a 1.41 T mini spec mq 60 NMR analyzer (Bruker, Germany) at 37 °C. The relaxivity values of r1 and r2 were obtained by fitting the 1/T1 and 1/T2 relaxation time (s−1) versus Gd3+ ion concentration determined by inductively coupled plasma mass spectrometry (ICP-MS). The in vitro MR images were obtained using a Micro MR-25 mini MRI system (Meso MR23-060H-I; Shanghai Niumag Corporation, China). The measurement conditions were as follows: T1-weighted sequence, multislice spin echo, repetition time/echo time (TR/TE) = 1150/11.5 ms, matrix acquisition = 256 × 192, number of excitations (NEX) = 8, field of view (FOV) = 80 mm × 80 mm, FOV phase of 40%, thickness = 5.0 mm, 1.5 T, 32 °C. 2.4.3. In Vitro Cytotoxicity Assay. The cytotoxicity of UCNPs@ BSA·DTPAGd was evaluated by the typical 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Hela cells were seeds into a 96-well plate with 104/well and then cultured at 37 °C under 5% CO2 until adherent. After cells were washed twice with PBS, UCNPs@BSA·DTPAGd was dispersed into the culture media (DMEM) at concentrations of 12.5, 25, 50, 100, and 200 μg mL−1 and then added to the wells. The cells were subsequently incubated for 12 or 24 h at 37 °C under 5% CO2. At the end of the incubation, cells were treated with a 50 μL/well MTT solution, which was diluted in a culture medium to a final concentration of 5 mg mL−1 and incubated for another 4 h, and then the supernatant was removed and the precipitated formazan was dissolved in DMSO (150 μL/well) for 10 min. The absorbance was monitored by a microplate reader at a wavelength of 570 nm. The cytotoxicity was finally expressed as the percentage of cell viability of the treatment group relative to control group cells. 2.4.4. Tumor Model. BALB/c mice (20−25 g of body weight) were purchased from the Second Military Medical University (Shanghai, China). Briefly, Hela tumor models were inoculated by subcutaneous injection of 5 × 106 cells in 20 μL of the culture medium into the left leg of each nude mouse. The mice were treated when the tumor volumes approached ca. 50 mm3. Animals used for the following experiments were in agreement with the guidelines of the Institutional Animal Care and Use Committee of Tongji University. 2.4.5. In Vivo MR Imaging. The in vivo MR imaging was conducted on a 3.0 T clinical MR imaging system (GE Signa Excirte) with a small animal coil. For MR imaging, tumor-bearing mice were injected with an appropriate dosage of 200 μL of UCNPs@BSA·DTPAGd (200 μg mL−1) via the tail veins. Imaging was performed by a fast spin echo imaging sequence (TR/TE = 1000/23.8 ms, FOV = 60 mm × 60 mm, 7 slices, 2 mm thick, 0.5 mm space, acquisition time = 12 min) at three time intervals (preinjection, 2 h, and 4 h). 2.4.6. In Vivo Biodistribution Analysis. For in vivo biodistribution analysis of UCNPs@BSA·DTPAGd, the main organsheart, liver, spleen, lung, kidney, intestineand tumor were harvested from mice intravenously injected with UCNPs−H@BSA·DTPAGd (200 μL, 200 μg mL−1) at time intervals of 6 h, 24 h, and 7 days postinjection and from mice receiving no injections. The removed organs were treated with nitric acid and held at 80 °C before being filtered. The filtrates

were ready for inductively coupled plasma (ICP) analysis to measure the Gd3+ ion concentrations of the investigated organs quantitatively. 2.4.7. Histological Study. For hematoxylin and eosin (H&E) studies, healthy BALB/c mice were injected with UCNP@BSA· DTPAGd (200 μL, 200 μg mL−1) via the tail veins as the test group. The mice without injections were selected as the control group. Tissues were harvested from these completely anesthetized mice. The major organs including the heart, liver, spleen, lung, kidney, and intestine were collected and fixed with 4% paraformaldehyde, sliced, and stained with hematoxylin and eosin. Subsequently, the samples of organs were observed under an optical microscope, and the photographs obtained were magnified 200×.

3. RESULTS AND DISCUSSION 3.1. Need and Strategy for Relaxivity Improvement of UCNPs. Some advantages of Gd3+-ion-doped UCNPs are reported so that they can maintain their upconversion emission patterns and also produce MR signals, which favor multi/dual modal imaging. The relaxivity of Gd3+-ion-doped UCNPs, nevertheless, has been studied less than their optical properties.19−22 Previously published papers suggest that the embedded Gd3+ ions inside UCNPs have a limited exchange of magnetic fields with the surrounding water protons, resulting in low relaxivity and poor MRI enhancement.23,24 van Veggel’s group found that the relaxivity enhancement of the NaGdF4 nanoparticles depend on the S/V ratio, and smaller NaGdF4 nanoparticles always have a higher mass relaxivity and a lower nanoparticle relaxivity than do the larger ones.2 Guo et al. reported that the epitaxial growth of a gadolinium layer on an upconversion lanthanide seed can create a paramagnetic shell with improved magnetic resonance relaxivity.24 Similarly, such a strategy was also reported by Bu et al., who disclosed that the longitudinal relaxivity enhancement of Gd3+-ion-doped UCNPs was co-contributed by inner- and outer-sphere mechanisms for ligand-free probes and mainly by outer-sphere mechanism for silica-shielded probes.27 However, there is still lacking some solid data and competitive strategies to improve the relaxivities of Gd3+-ion-doped UCNPs. In this study, to address this issue, three different Gd3+-ion-containing UCNPs, namely, non-Gd3+ion-doped UCNPs (UCNPs−N), a low concentration of Gd3+ion-doped UCNPs (UCNPs−L), and a high concentration of Gd3+-ion-containing UCNPs (UCNPs−H) were synthesized for a comparison study on their relaxivities, aiming to disclose and provide solid evidence of the relaxivity profile of Gd3+-iondoped UCNPs. Furthermore, an internal and external Gd3+ ion incorporation strategy is developed in this study for MR imaging sensitivity enhancement. Generally, the synthesized Gd3+-ion-doped UCNPs by the internal incorporation approach hold a layer of hydrophobic organic OA molecules, hindering its dispersion in polar solvents. For biomedical applications, surface engineering of hydrophobic UCNPs is needed. Taking the relaxivity enhancement together with their phase transfer of UCNPs into account, in this study the surface modification of hydrophobic UCNPs is elaborately conducted with a highly paramagnetic water-soluble macromolecular protein consisting of Gd3+ ions chelated with BSA-DTPA. BSA is employed for the surface engineering of nanoparticles because of its low cost, excellent biocompatibility, and good water solubility.25,26,28,29 Our previous work discloses that BSA and its derivatives can be used for the phase transfer of hydrophobic nanoparticles from the organic to aqueous phase under ultrasonication.25,26,30 In this study, the BSA·DTPAGd derivative plays two roles, namely, a phase-transfer ligand and a relaxivity improvement synergist C

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Scheme 1. Schematic Illustration of the Relaxivity Enhancement Strategy, Typical Synthesis Process, and in Vivo TumorImaging Application of UCNPs@BSA·DTPAGd

UCNPs, with a surface charge of ca. −20.8 mV determined by a zeta potential analyzer. These differences in hydrodynamic diameters are mainly attributed to the BSA·DTPAGd ligand encapsulation. The negatively charged surface of UCNPs@ BSA·DTPAGd is also an indication of the successful surface modification on UCNPs because BSA·DTPAGd is carrying negatively charged carboxyl groups. 3.3. Optical, Stability, and Relaxivity Characterization of UCNPs@BSA·DTPAGd. 3.3.1. Optical and Stability Characterization. The UCL spectra of hydrophobic OAcapped UCNPs and its counterpart hydrophilic UCNPs@BSA· DTPAGd were collected under 980 nm diode laser excitation with a power density of ∼100 mW cm−2, as shown in Figure 3A. The characteristic emission peaks of the OA-capped UCNPs and UCNPs@BSA·DTPAGd are all located at ∼520, ∼ 540, and ∼656 nm, corresponding to the transitions of Er3+ ions: 2H11/2 → 4I15/2, 4S3/2 → 4I15/2, and 4F9/2→ 4I15/2, respectively. The hydrophobic OA-capped UCNPs present sharp and potent emissions at the three above-mentioned peaks. It is well known that decent fluorescent emission features are closely related to the high quality of crystallization of nanoparticles. The thermal decomposition strategy at high temperature in this study is of importance to the complete crystallization of UCNPs, which is very beneficial for obtaining very bright luminescence.16 The optical spectrum shows a fluorescence intensity decline in the aqueous UCNPs@BSA·DTPAGd sample, compared to that before surface engineering. This slight fluorescence quenching is mainly attributed to the microenvironmental disturbance on the surface of UCNPs. It could be inferred that the surface of UCNPs suffers from surface effects and other quenching factors induced by high-frequency hydroxyl (−OH) vibrational-energy-containing molecules such as water and proteins, which can influence the luminescence center.31 The energy-level diagram and transition processes of Yb3+/ Er3+-doped UCNPs are presented in Figure 3B. With 980 nm laser irradiation, the UCL emission occurs via two successive energy transfers from Yb3+ to Er3+. First, infrared photons are first absorbed by the strong transition from the ground-state 2 F7/2 to the first excited state 2F5/2 in the Yb3+ ions. At the same time, the transfer energy from neighboring Yb3+ to Er3+ can excite Er3+ ions from the ground state to 4I11/2 state. Subsequent radiative relaxations of 4I11/2−4I13/2 can also enrich the 4I13/2 levels. In the second-step excitation, the same infrared photons promote the excited-state electrons from 4I11/2 to 4F7/2 levels or from 4I13/2 to 4F9/2 states via phonon-assisted energy

for UCNPs. The mechanism of phase transfer of hydrophobic nanoparticles by BSA protein has been discussed in detail in our published paper.26 In this work, the relaxivity and its improvement of UCNPs are closely studied instead. The schematic illustration of the relaxivity enhancement strategy, synthesis process, and biomedical imaging application of UCNPs@BSA·DTPAGd is shown in Scheme 1. 3.2. Synthesis and Characterization of UCNPs@BSA· DTPAGd. Paramagnetic water-soluble BSA·DTPAGd is synthesized by the chemical reaction of cyclicanhydride of DTPA (DTPAA) with BSA and the subsequent addition of Gd3+ ions for chelation. DTPA was covalently bound to the amine groups of BSA, and the Gd3+ ions were chelated in the DTPA moieties. These chemical process and corresponding characterizations have been provided in our previously published paper.25 The prepared BSA·DTPAGd is highly water-soluble, owing to its abundant carboxyl and amino groups. With the unique composition and structure of BSA, it can serve as a capping ligands for the phase transfer of UCNPs. For the surface engineering of UCNPs, an “oil-in-water” nanoemulsion is triggered and formed by ultrasonication. And a stable aqueous solution of UCNPs@BSA·DTPAGd can be obtained after complete evaporation of the organic phase. The sizes, morphologies, and hydrodynamic diameters of hydrophobic OA-capped UCNPs and their surface-engineered hydrophilic UCNPs@BSA·DTPAGd were investigated via TEM and DLS techniques, respectively. As seen in Figure 1A, the

Figure 1. TEM photographs of (A) hydrophobic OA-capped UCNPs in chloroform and (B) hydrophilic UCNPs@BSA·DTPAGd in water.

three types of OA-capped UCNPs are found to be monodisperse, which is also confirmed by the DLS data (Figure 2A−2C). Upon phase transfer, the UCNPs show excellent water dispersion, which is reflected by TEM without obvious clusters (Figure 1B), and their corresponding DLS measurements (Figure 2C−2E) show that the effective hydrodynamic diameters of surface-engineered UCNPs, which are around 50 nm, are slightly larger than those of OA-capped D

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Figure 2. Hydrodynamic diameters measured by DLS: hydrophobic (A) UCNPs−N, (B) UCNPs−H, and (C) UCNPs−L in chloroform and their corresponding hydrophilic counterparts: (D) UCNPs−N@BSA·DTPAGd, (E) UCNPs−H@BSA·DTPAGd, and (F) UCNPs−L@BSA·DTPAGd in water.

Figure 3. (A) UCL spectra of hydrophobic OA-capped UCNPs and hydrophilic UCNPs@BSA·DTPAGd excited with a 980 nm laser,. (B) Schematic representation of the upconversion mechanism in Er3+/Yb3+/Gd3+ codoped UCNPs. Hydrodynamic diameters of UCNPs−H@BSA·DTPAGd in different solutions: (C) varying ionic strengths; and (D) varying pH values (the insets in C and D are the corresponding photographic images taken in sunlight) after 7 days of storage at room temperature; and (E) UCL images of UCNPs−H@BSA·DTPAGd under varying chemical conditions mentioned above, excited by a 980 nm laser in a dark room.

E

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Langmuir transfer from Yb3+. Subsequent 4F7/2−4S3/2 (2H11/2) occurred via nonradiative relaxations of Er3+ presented in the 4S3/2 (2H11/2) state. As a consequence, two-photon green emissions of 4 S3/2/2H 11/2− 4I15/2 and red emissions of 4F 9/2 −4I 15/2 occurred in different energy-transfer paths.32 The presence of some organic groups such as −COOH, −NH2, −SH, and −OH on the surface of particles may result in the nonradioactive relaxation across these energy gaps of 2H11/2 → 4F9/2 and 4S3/2 → 4F9/2, so the green emission is more prominent than the red emission for all samples synthesized via this procedure. The colloidal stability of UCNPs−H@BSA·DTPAGd was quantitatively investigated in vitro, in the biological solutions with different ionic strengths and pH values at room temperature, by a dynamic light scattering technique. The hydrodynamic diameters of UCNPs−H@BSA·DTPAGd and UCL images were recorded in Figure 3. As seen from Figure 3C,D, the sizes of UCNPs−H@BSA·DTPAGd have just a slight destabilization in all of the investigated situations, owing to the particle−particle and particle−interface repulsive electrostatic forces that are weakened upon the addition of salts and the structure of BSA being influenced by the pH value changes.33 Nevertheless, this fluctuation in size is acceptable for biomedical imaging. Moreover, as seen from the inserted images in Figure 3C,D, the aqueous sample solutions with different ionic strengths and pH values are transparent, indicating that UCNPs@BSA·DTPAGd is dispersed well in solutions with good colloidal stability. This could be attributed to the abundant amino and carboxyl groups on UCNPs@BSA· DTPAGd, which can afford buffering capability and prevent the nanoparticles from becoming aggregated in harsh chemical environments.26,34 The UCL photostability of UCNPs−H@BSA·DTPAGd is further qualitatively evaluated. The samples in different buffers with varying ionic strengths and pH values were excited by a 980 nm laser and recorded as digital photographs, as shown in Figure 3E. The UCL intensity of UCNPs−H@BSA·DTPAGd remains stable in most involved conditions without an obvious decrease in emissions, except at pH 3.45 and pH 12. It can be explained that the BSA is strong enough and stable in the pH range of 4−10,35 yet the structure of BSA could be changed in the more acidic or basic environments and therefore it affects the fluorescence emission.36 The good stability of UCNPs−H@ BSA·DTPAGd both in dispersion and fluorescence emission favors in vivo imaging applications. 3.3.2. Relaxivity Characterization. To the best of our knowledge, there are few reports on the study of UCNP relaxivity, even though increasing imaging applications have been demonstrated. Therefore, in this study we focus on the relaxivity investigation of UCNPs and its improvement approach development. To study the influence of the doping concentration of Gd3+ ions in UCNPs on relaxivity, three different amounts of Gd3+ ions are doped inside of UCNPs. Similar nanostructure samples denoted as UCNPs−N@BSA· DTPAGd, UCNPs−L@BSA·DTPAGd, and UCNPs−H@BSA· DTPAGd were synthesized, and their relaxivities were systematically studied in this work. The relaxivity values of the UCNPs−N/H/L@BSA·DTPAGd are listed in Table 1, and the plots of r1 and r2 versus Gd3+ ion concentrations and their stability assessment determined by the relaxation time at different time points are shown in Figure 4. The ionic relaxivity versus Gd3+ ion concentration is obtained from the slope of the linear regression fit by the relaxivity plots at 1.41 T. As seen from Table 1, the relaxivities of all three

Table 1. Relaxivity Values of Aqueous UCNPs Modified Either by BSA or BSA·DTPAGd with Different Concentrations of Gd3+ Ion Doping nanoparticles

r1 (mM−1 s−1)

r2 (mM−1 s−1)

r2/r1

UCNPs−N@BSA UCNPs−L@BSA UCNPs−H@BSA UCNPs−N@BSA·DTPAGd UCNPs−L@BSA·DTPAGd UCNPs−H@BSA·DTPAGd

0 1.02 0.52 10.8 2.27 1.56

0 2.87 1.84 22 4.36 4.16

2.81 3.54 2.04 1.92 2.67

types of UCNPs decorated by BSA·DTPAGd have higher values than those of the corresponding counterparts with pure BSA proteins, regardless of the Gd3+ ion doping concentrations. This indicates that synthetic BSA·DTPAGd is paramagnetic and successfully capped on the surface of UCNPs. Among the BSA· DTPAGd-decorated UCNPs, UCNPs−N@BSA·DTPAGd exhibits the highest r1 of 10.8 s−1 per mmol L−1 of Gd3+ ions, and UCNPs−H@BSA·DTPAGd holds the lowest r1 of 1.56 s−1 per mmol L−1 of Gd3+ ions. This experimental result discloses that the embedded paramagnetic Gd3+ ions inside the UCNPs do not completely contribute to the relaxivity because of the inefficient exchange of magnetic fields with surrounding water protons in Gd3+-ion-doped UCNPs.27 The same results are also visually given from the plots of r1 and r2 in Figure 4A,B. On the basis of the evidence presented above, it can be claimed that the external BSA·DTPAGd makes a greater contribution to the relaxivity than do the internal doped Gd3+ ions because the exposed Gd3+ moieties outside the UCNPs are more effective in the exchange of magnetic fields with surrounding water protons. Figure 4C shows that UCNPs@BSA·DTPAGd possess quite good stability with respec tot relaxivity. T1 and T2 relaxation times of UCNPs@BSA·DTPAGd do not vary much during the investigated period, suggesting that UCNPs@BSA· DTPAGd hardly leaks Gd3+ ions or aggregates and in return favors in vivo imaging. The relaxivities of UCNPs−L/‑H@BSA·DTPAGd are found to benefit from internal and external Gd3+ ion attachments. The above relaxivity study suggests that internal embedding Gd3+ ions make a smaller contribution to the relaxivity than does external Gd3+ ion labeling. Even so, some multi/dual-modality imaging based on internal Gd3+-ion-doped UCNPs have been regularly reported. The relaxivities of these nanoprobes are not promising and usually lead to dim MR imaging.37−39 In this study, UCNPs−N@BSA·DTPAGd is found to have the highest r1. For in vivo imaging, however, the internal doping of Gd3+ ions in UCNPs should be also appreciated because this internal doping certainly contributes to the MR imaging contrast, whether or not the combining of external Gd3+ ion attachment is used. Herein, to investigate and disclose the real MR imaging behaviors of UCNPs−N/H/L@BSA·DTPAGd, the experimental conditions for imaging are designed as follows: the same molar concentration of Gd3+ ions, the same mass concentration of the three types of UCNPs, and a typical sample of UCNP−H@BSA· DTPAGd with different Gd3+ ion concentrations. When the samples have the same molar concentration of Gd3+ ions, the sample of UCNPs−N@BSA·DTPAGd with the highest r1 relaxivity (10.8 s−1 per mmol L−1 of Gd3+ ions) exhibits the most pronounced MRI enhancement among all of the samples (Figure 5A). Nevertheless, when the samples have the same mass concentration of the nanoprobes, the sample of UCNPs−H@BSA·DTPAGd with the lowest r1 relaxivity (1.56 s−1 F

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Figure 4. (A) Plots of r1 versus Gd3+ ion concentrations of different samples. (B) Plots of r2 versus Gd3+ ion concentrations of different samples. (C) The relaxivity stability of samples evaluated by T1 and T2 relaxation times within 24 h.

Figure 5. In vitro T1-weighted MR images of samples: UCNPs−N/H/L@BSA and UCNPs−N/H/L@BSA·DTPAGd nanoprobes (A) under the same molar concentration of Gd3+ ions, (B) under the same mass concentration, and (C) UCNPs−H@BSA·DTPAGd under different Gd3+ ion concentrations. The images in a−c are the colored graphs corresponding to their counterparts.

per mmol L−1 of Gd3+ ions) shows the most significant MRI enhancement among the samples (Figure 5B). These results indicate that the realistic imaging capability of UCNPs is dependent on the means of concentration determination. A

striking message given in this study is that the relaxivity value does not always reflect the realistic MRI enhancement capability. It is different under the condition of the same mass concentration of nanoprobes. Although a high concenG

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Langmuir tration of Gd3+-ion-doped UCNPs makes a limited contribution to relaxivity, it really contributes to the enhancement of MR imaging. Bearing this fact in mind and in pursuit of a better MR imaging sensitivity, in this study an internal-and-external paramagnetic Gd3+ ion attachment strategy is put forward. And the above in vitro sample MR imaging shows that this strategy is effective and impressive. It is therefore important for the enhancement of MR imaging sensitivity by using UCNPs−H@BSA·DTPAGd nanoprobes, in which Gd3+ are labeled inside and outside. UCNPs−H@BSA·DTPAGd is then employed for in vivo tumor-targeted imaging. To the best of our knowledge, this is the first time we give such solid data to demonstrate and disclose the interplay between relaxivity and MR imaging. Figure 5C shows a set of UCNPs−H@BSA·DTPAGd with the Gd3+ ion concentrations ranging from 0 to 1.27 mmol L−1, imaged using T1-weighted spin−echo sequences. The signal intensity of UCNPs−H@BSA·DTPAGd is found to be dependent on the Gd3+ ion concentration. Even at the lowest concentration of Gd3+ ions (0.0102 mmol L−1), the signal of UCNPs−H@BSA·DTPAGd is observable, compared to that of water. This prominent MR imaging sensitivity is due to the developed internal-and-external Gd3+ attachment strategy for MR imaging. 3.4. Cytotoxicity Analysis of UCNPs@BSA·DTPAGd. When it comes to doing in vivo imaging with nanoparticles, it is encouraged to evaluate their cytotoxicity first to determine if they are biocompatible. The UCNPs−H@BSA·DTPAGd nanoparticles prepared in this study show good biocompatibility even at a high dose of 200 μg mL−1, with no significant cell proliferation differences observed between the experimental group and the control group as seen in the Figure 6. It is inferred that BSA protein encapsulation on UCNPs is beneficial to the biocompatibility of UCNPs.40

Figure 7. (A) In vivo T1-weighted images of tumor-bearing mice before and after injection of UCNPs−H@BSA·DTPAGd at 2 and 4 h time points. (B) Colored graphs correspond to their counterparts in (A).

the contrasts of two kidneys are also well enhanced compared to those before the injection of UCNPs−H@BSA·DTPAGd. The enhancement effect on the tumor and kidneys is found to be significant at 2 h and decreasing 4 h postinjection. This imaging pattern suggests that injected UCNPs−H@BSA·DTPAGd tends to accumulate in tumor lesions and then is gradually metabolized and finally excreted from body. Prospectively, imaging at the tumor site can be further enhanced by specific bioligand-mediated targeting, also called active targeting, leading to an increased accumulation of the administrated nanoprobes in the targeted area.25 UCNPs−H@ BSA·DTPAGd nanoprobes with BSA protein have abundant active chemical groups, which is favorable to covalent coupling with bioligands, such as antibodies, aptamers, peptides, and oligonucleotides. By doing so, UCNPs−H@BSA·DTPAGd would have more extensive applications in targeted imaging. In this study, the imaging work done at this stage is found to be accurate and effective and could be instructive for future biomedical translational applications. 3.6. Organ Distributions and Histology Toxicity Analysis. 3.6.1. Organ Distributions. The MR imaging of the tumor and kidney is discussed above. Biodistribution, via the inductively coupled plasma analysis of the Gd3+ ion technique, was conducted in this study to investigate the location, pharmacokinetics, and metabolism behavior of the

Figure 6. Relative viabilities of Hela cells incubated with different concentrations of UCNPs−H@BSA·DTPAGd.

3.5. In Vivo Tumor-Targeted MRI. The anatomical images acquired in the coronal planes can be used to determine the positive contrast enhancement of nanoparticles in vivo. Herein, the targeting strategy of optimized UCNPs−H@BSA·DTPAGd can be regarded as a passive targeting via the enhanced permeability and retention effect (EPR effect) with respect to the presence of fenestrations in the endothelium of tumor vessels with the entry of the nanoparticles into the tumor tissues.41,42 As seen in Figure 7, the tumor is highly contrasted in the coronal MR image 2 h postinjection. In the meantime, H

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Langmuir injected UCNPs−H@BSA·DTPAGd in the body. The biokinetics of UCNPs−H@BSA·DTPAGd is shown in Figure 8; after

samples; the glomerulus structure could be distinguished easily in the kidney samples; and also no necrosis is found in any group of samples. However, the spleen tissue is slightly affected by the UCNPs−H@BSA·DTPAGd, with slight hyperplasia in the periarteriolar lymphoid sheath of the white pulp. This may be caused by the involvement of UCNPs−H@BSA·DTPAGd, and this phenomenon has previously been observed in other nanoparticle-treated spleen tissues.44,45

4. CONCLUSIONS In this study, the strategy to enhance the MR imaging contrast of UCNPs benefits from internal Gd3+ ion doping and external paramagnetic BSA·DTPAGd capping of UCNPs. BSA·DTPAGd plays two roles in the construction of UCNPs−N/H/L@BSA· DTPAGd, namely, phase transfer as surface ligands and further MRI enhancement as a synergist. The relaxivity of Gd3+-iondoped UCNPs is found to be Gd3+-ion-concentration dependent. The values of ionic relaxivity are sorted by UCNPs−N@ BSA·DTPAGd > UCNPs−L@BSA·DTPAGd > UCNPs−H@BSA· DTPAGd. This sorted order indicates that external BSA· DTPAGd surface ligands contribute more to r1 enhancement than does internal Gd3+ ion doping in UCNPs. Nevertheless, once the mass concentration is consistent with UCNPs−N@ BSA·DTPAGd and UCNPs−L@BSA·DTPAGd, it is found that UCNPs−H@BSA·DTPAGd performs more potently upon MRI enhancement. This study for the first time discloses with solid evidence that the relaxivity does not reflect the realistic MRI enhancement capability under the condition of the same mass concentration of nanoprobes. And further in vivo MRI shows UCNPs−H@BSA·DTPAGd to be an eminent agent for tumortargeted imaging. Prospectively, it is encouraged to perform theranostic applications with this advanced nanoplatform upon conjugation with certain targeting bioligands and therapeutic moieties.

Figure 8. Biodistribution histogram of UCNPs−H@BSA·DTPAGd in the main organs and tumor.

intravenous injection, the UCNPs−H@BSA·DTPAGd is mainly accumulated in the liver and spleen. The concentration of UCNPs−H@BSA·DTPAGd accumulated in the liver is found to decrease along with the investigated time, while it is contrary in spleen organs. A relative smaller number of Gd3+ ions are present in the kidneys, intestines, and tumor. Considering the slightly larger size of UCNPs−H@BSA·DTPAGd, it has difficulty getting through the pores of glomerulus.43 Nevertheless, the enhanced MRI in kidneys indicates that UCNPs−H@BSA· DTPAGd could be metabolized in the liver, and the resulting degraded and smaller Gd3+ complex recirculates in the bloodstream and is shuttled in the kidneys. Taking the MRI profile and biodistribution pattern into consideration, it could be claimed that the clearance of UCNPs−H@BSA·DTPAGd in vivo follows hepatobiliary (HB) and renal processing. 3.6.2. Histological Toxicity Analysis. The biodistribution study shows that the injected UCNPs−H@BSA·DTPAGd is found in almost all of the main organs of mice at various levels. Furthermore, to determine whether UCNPs−H@BSA·DTPAGd causes negative effects in these tissues, an in vivo toxicity study of UCNPs−H@BSA·DTPAGd was undertaken. The micrograph images of H&E-stained tissue slices, including the heart, liver, spleen, lung, kidney, and intestine, harvested from the anesthetized mice are shown in Figure 9. The toxicity of nanoparticles in tissues is assessed through histological changes in such susceptible organs as the heart, liver, spleen, lung, kidney, and intestine. In this study, the mice injected with UCNPs−H@BSA·DTPAGd were dissected after 1 week of exposure. As seen in Figure 9, the muscle tissue in the heart samples does not present hydropic degeneration; the hepatocytes in the liver samples do not present inflammatory infiltrates either; no pulmonary fibrosis is observed in the lung

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AUTHOR INFORMATION

Corresponding Authors

*(D.G.) E-mail: [email protected]. Tel: +86-073188821449. *(B.Z.) E-mail: [email protected]. Tel: +86-02165983706-805. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial support of the National Natural Science Foundation of China (81571742, J1210040, 81371618, and 21341010), the Natural Science Foundation of Hunan Province (no. 11JJ5005), the Innovative Research Team in University (no. IRT1238), and the China Outstanding

Figure 9. Micrograph images of H&E-stained tissue slices harvested from the main organs of mice (magnification 200×). I

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