Facile Synthesis of Gadolinium Chelate-Conjugated Polymer

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Facile Synthesis of Gadolinium Chelate-Conjugated Polymer Nanoparticles for Fluorescence/Magnetic Resonance Dual-Modal Imaging Yi Pan, Wandi Chen, Jun Yang, Junhui Zheng, Mengsu Yang, and Changqing Yi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04078 • Publication Date (Web): 02 Jan 2018 Downloaded from http://pubs.acs.org on January 3, 2018

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Analytical Chemistry

Facile Synthesis of Gadolinium Chelate-Conjugated Polymer Nanoparticles for Fluorescence/Magnetic Resonance Dual-Modal Imaging ‡

‡

‡

Yi Pan1 , Wandi Chen1 , Jun Yang2 , Junhui Zheng2, Mengsu Yang3, Changqing Yi1∗

1. Key Laboratory of Sensing Technology and Biomedical Instruments (Guangdong Province), School of Engineering, Sun Yat-Sen University, Guangzhou, P. R. China 2. Guangdong General Hospital, Guangzhou, P. R. China. 3. Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, P. R. China

‡

These authors contributed equally to this work.

∗

Authors to whom any correspondence should be addressed. Email: [email protected];

Tel: 86-20-39342380.

ACS Paragon Plus Environment

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Fluorescence (FL)/magnetic resonance (MR) dual-modal imaging nanoprobes are significant not only for cutting edge research in molecular imaging, but also for clinical diagnosis with high precision and accuracy. However, synthesis of FL/MR dual-modal imaging nanoprobes that simultaneously exhibit strong fluorescent brightness and high MR response, long-term colloidal stability with uniform sizes, good biocompatibility and a versatile surface functionality has proven challenging. In this study, the well-defined core-shell structured Gd3+ chelate-conjugated fluorescent polymer nanoparticles (Gd-FPNPs) that consist of rhodamine B (RB)-encapsulated poly (methyl methacrylate) (PMMA) cores and Gd3+ chelate-conjugated branched polyethyleneimine (PEI) shells, are facilely synthesized via a one-step graft copolymerization of RB-encapsulated MMA from PEI-DTPA-Gd induced by tert-butyl hydroperoxide (TBHP) at 80 °C for 2 h. The mild synthesis route not only preserves the chemical environment for Gd3+ coordination, but also improves optical properties and chemo-/photo-stability of RB. A high local concentration of outer surface-chelated Gd3+ and their direct interactions with hydrogen protons endow Gd-FPNPs high longitudinal relaxivity (26.86 mM-1 s-1). The uniform spherical structure of Gd-FPNPs facilitates their bio-transfer, and their surface carboxyl and amine groups afford them both long-term colloidal stability and cell-membrane permeability. The excellent biocompatibility and FL/MR dual-modal imaging capability of Gd-FPNPs are demonstrated using HeLa cells and mice as models. All the results confirm that Gd-FPNPs fulfill the design criteria for a high-performance imaging nanoprobe. In addition, this study enables such probes to be prepared also by those not skilled in nanomaterial synthesis, and thus promoting the development of novel functional imaging nanoprobes. KEYWORDS: Dual-modal imaging nanoprobe, fluorescence imaging, magnetic resonance imaging, polyethyleneimine, rhodamine B, gadolinium chelates.


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INTRODUCTION Modern medical diagnosis relies to a great extent on different kinds of imaging techniques, such as ultrasound (US), fluorescence (FL), magnetic resonance (MR), X-ray computed tomography (CT), positron emission tomography (PET) and single photon emission CT (SPECT). However, relying on a single imaging mode, it is difficult to collect complete information for accurate diagnosis, because of the deficiency of each individual modality. Therefore, the development of multimodal imaging probes is significant not only for cutting edge research in molecular imaging, but also for clinical diagnosis with high precision and accuracy1-5. Out of the all multimodal imaging probes, the FL/MR probes which combine the single-cell sensitivity of FL imaging and the excellent 3D spatial resolution of MRI have attracted significant research attention and are already successfully implemented in clinical practice, since MRI is useful for surgical planning whereas FL imaging is useful to the operating surgeon during the procedure6-8. In particular, nanoparticles (NPs) have been proven to be ideal and robust framework to be integrated with diverse imaging modalities to generate novel properties and offer synergetic applications3-5. Therefore, in the past decade, much effort has been devoted to developing synthetic strategies for the preparation of the FL/MR dual-modal imaging nanoprobes, including heterostructure nanoparticle growth9-11, surface engineering of fluorescent NPs or magnetic NPs with complementary components12-15, co-encapsulation of magnetic and fluorescent components into organic and inorganic materials such as oil droplet, lipid micelle, block co-polymer and silica16-23. However, their practical application for biological processes monitoring and clinical translation for medical diagnosis are still limited. As a result, it is still highly desirable to develop simple and versatile synthetic routes for preparing multimodal imaging probes with enhanced performance in a more efficient way.


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To fully realize its potential and optimize its performance, an ideal FL/MR dual-modal imaging nanoprobe should have strong fluorescent brightness and high MR response, long-term colloidal stability with uniform sizes, good biocompatibility and low biotoxicity, and a versatile surface functionality. The adaptability of macromolecules for preparation of multimodal imging contrast agents can possibly fulfill the abovementioned design criteria, because they can be rationally designed as functinal materials by judicious incorporation of building blocks23-26. Especially, polymer nanoparticles with organic emitters as the fluorescent core and biocompatible macromolecules as the encapsulation matrix have emerged as a new generation of promising probes for in vivo applications. Thanks to its attractive characteristics including the highly positive charges and a large number of accessible reactive functional groups, polyethylenimine (PEI) has been extensively exploited for nucleic acid delivery and gene therapy and the NP stabilization and functionalization27-31. In this study, we report the simple and robust preparation of well-defined core-shell Gd-FPNPs that consist of rhodamine B (RB)-encapsulated poly (methyl methacrylate) (PMMA) cores and gadolinium chelate-conjugated branched PEI shells, and demonstrate their capabilities for FL/MR dual-modal imaging by the use of HeLa cells and mice as models. EXPERIMENTAL SECTION Synthesis of Gd-FPNPs. Scheme 1 illustrates the synthesis route of Gd-FPNPs. In order to prepare PEI-DTPA-Gd, cDTPAA is firstly prepared by mixing DTPA (7.9 g, 0.02 mol), acetic anhydride (8 mL, 0.085 mol) and pyridine (12 mL, 0.15 mol) under reflux at 50~60 °C for 24h.13 Then, the purified cDTPAA (0.28 g) is dissolved into a 6-mL-aqueous solution containing 0.035 g EDC and 0.035 g NHS for 30 min at room temperature, followed by reacting branched PEI


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(4.5 mL, 0.1 g·mL-1) for 2 h to prepare PEI-DTPA. Finally, 0.5 mL of GdCl3 (0.55 g·mL-1) is added to the solution and allowed to react for another 2 h to obtain PEI-DTPA-Gd. The resultant PEI-DTPA-Gd solution is diluted to 25 mL using H2O and adjusted to pH 7.0 using HCl solution (3 mL, 2 M). Then, the PEI-DTPA-Gd solution is purged with N2 for 30 min and heated to 80 °C, followed by adding a mixture solution containing 1.5 g MMA and 7 mg RB. Finally, 250 µL TBHP (10 mM) is added to induce the graft co-polymerization reaction, and the mixture is allowed to react at 80 °C for 2 h under N2 atmosphere. The resultant Gd-FPNPs (1 g·mL-1) are further incubated with purified γ-PGA (0.1 g mL-1) for 30 min to decrease their surface electropositivity. Biocompatibility and biotoxicity of Gd-FPNPs. Cytotoxicity of Gd-FPNPs is evaluated using a standard MTT assay, where the viability of HeLa cells, CT 26 cells, and HepG2 cells upon treatment with Gd-FPNPs at various concentrations (0, 0.055, 0.110, 0.165, 0.220, 0.275, 0.330, 0.385, 0.440 and 0.495 mg·mL-1) for 24 h is determined using MTT solution (dissolved in PBS, 5 mg mL-1)32. The blood compatibility of Gd-FPNPs is evaluated using the hemolysis assay. In brief, 0.5 mL blood cells suspension (1.0×108 cells mL-1) is mixed with 0.5 mL aqueous solution containing different amount of Gd-FPNPs (0.4, 0.6, 0.8 g L-1), and 0.5 mL PBS and ultrapure water are used as negative and positive control, respectively. After stew for 2h at RT under mild shake, the samples are centrifuged, and the upper supernatants are collected for measuring the absorbance at 541 nm to calculate the hemolysis percentage using the following equation32,50,51. Hemolysis (%) =

− × 100% −

The biotoxicity of Gd-FPNPs is evaluated using zebra fishes (gifts from Macau Univeristy) and Balb/c mice (Guangzhou Land-unicomed Biosciences Co., Ltd.). All experiments are


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approved by the Animal Experimentation Ethics Committee of Sun Yat-Sen University and conducted in accordance with guidelines for human care. 6 days old zebra fishes are added in 24 wells with 10-12 larvae per well, followed by incubation with 2 mL Gd-FPNPs solution (dissolved in E3 medium, 0.4 mg mL-1) for 8 h. Bright field and FL images of the zebrafish are then recorded using a fluorescent microscope (Eclipse Ti, Nikon) under excitation at 535 nm. In vivo toxicity of Gd-FPNPs is evaluated using Balb/c mice (18-22g, n=3). After intravenous injection of 0.8 mg mL-1 Gd-FPNPs, the mice anesthetized with 4 % chloral hydrate (6 mL kg−1) are dissected at 3 days, and the main organs are collected for H&E staining. The mice injected with 100 µL of normal saline are used as control. In vitro FL and MR imaging. For FL imaging assay, HeLa cells are treated with 0.05 mg·mL1

Gd-FPNPs suspension for 4 h and DAPI for 30 min, followed by being imaged under excitation

with 340-380 nm diode laser and 535 nm Ar-lase alternately. For MR imaging assay, HeLa cells are treated with and without 0.05 mg mL-1 Gd-FPNPs suspensions for 4 h, followed by in turn digested with trypsin-EDTA, centrifuged and re-dispersed in PBS (pH=7.4). MR imaging of the HeLa cells is performed using a clinical 1.5T MRI instrument.13,32 In vivo FL and MR imaging. The Balb/c mice (18-22 g) are injected with 100 µL of GdFPNPs at 0.8 mg mL-1 via tail vein. Then, the FL images are taken at 6 h, 9 h and 24 h after injection, and the MR images are taken at 6 h, 9 h and 12 h after injection. Ex vivo FL images of resected organs during necropsy at 24 h after injection, including heart, lung, kidney, liver, spleen, and intestines, are also acquired. All the FL imaging assays are performed on a small animal in vivo imaging system (IVIS Lumina XRMS series, PerkinElmer Inc), setting the excitation wavelength and emission wavelength to 520 nm and 570 nm, respectively.


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For in vivo MR imaging assays, the tumor bearing mice model is firstly established by inoculating 100 µL CT-26 cell (2×107 cells mL-1) solution to the right abdomen of the five-weekold female Balb/c mice (18-22 g). After 7-10 days of inoculation, mice bearing CT-26 tumors are injected with 100 µL 0.8 mg mL-1 Gd-FPNPs (0.05 mmol Gd3+ kg-1) or Gadobutrol (0.05 mmol kg-1) via tail vein, and then subjected to a 1.5T clinical MR instrument for imaging using the scanning parameters and sequences listed in Table S1. RESULTS AND DISCUSSIONS Synthesis and Characterization of the Gd-FPNPs. The robust and mild approach for facile synthesis of the dual-modal imaging nanoprobe is essential to integrate signal units efficiently. Scheme 1 illustrates the synthesis route and dual-modality imaging application of the Gd-FPNPs. RB which is a widely used dye for FL imaging and can be well dissolved in MMA, is employed as the fluorescent component of the nanoprobe, whereas Gd-DTPA which is the predominant clinical T1-MR contrast agents and can be easily conjugated to PEI via carbodiimide chemistry, is employed as the magnetic component of the nanoprobe. First of all, the cyclic DTPA dianhydride (cDTPAA) is covalently conjugated to PEI to form PEI-DTPA by carbodiimide chemistry, followed by chelated with Gd3+ to obtain PEI-DTPA-Gd. Then, well-defined coreshell Gd-FPNPs are facilely synthesized via a one-step graft copolymerization of RBencapsulated MMA from PEI-DTPA-Gd induced by tert-butyl hydroperoxide (TBHP) at 80 °C. Large-area transmission electron microscope (TEM) images revealed that the as-prepared spherical Gd-FPNPs have an average diameter of ~130 nm (Fig 1A). Dynamic light scattering (DLS) measurements give a hydrodynamic diameter of 150 ± 25.0 nm for water-dispersed GdFPNPs, confirming the uniform size distribution (Inset of Fig 1A). The well-defined core-shell


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nanostructures are clearly observed and easily differentiated at high levels of magnification, where a typical Gd-FPNP have a core diameter ~118 nm with a shell thickness ~6 nm (Fig 1B). The surface-chelated Gd on the nanoprobes is validated by the presence of the characteristic peaks corresponding to Gd 4d (142 eV) and Gd 3d (1188 eV) in the X-ray photoelectron spectroscopy (XPS) spectra of the Gd-FPNPs (Fig 1C-1E). The high-resolution XPS spectra reveal the coexistence of Gd 4d5/2, Gd 4d3/2 and Gd 3d5/2 lines at 141.8, 147.6 eV and 1188.4 eV, respectively (Fig 1D & 1E). The presence of both Gd 4d and Gd 3d peaks verifies that the oxidation state of the surface-chelated Gd remains +3 in Gd-FPNPs,12,32-34 indicating that our reported synthesis route is mild and chemical environment for Gd coordination is well preserved. This is quite important because MR imaging highly depends on the half-filled f orbital with seven unpaired electrons and symmetrical s ground state of Gd3+. In addition, the XPS analysis also confirms that Gd-FPNPs are composed of 70.92% C, 26.53% O, 2.4% N, as evidenced by the characteristic peaks corresponding to C 1s (285 eV), N 1s (399 eV), O 1s (532 eV) (Fig 1C). The deconvoluted XPS C 1s (Fig S1A), N 1s (Fig S1B), and O 1s (Fig S1C) spectra confirm the presence of surface carboxyl and amine group on Gd-FPNPs, affording Gd-FPNPs both good colloidal stability and cellmembrane permeability. Optical and MR properties of the Gd-FPNPs. Gd-FPNPs are highly dispersible in phosphate-buffered saline (PBS) buffer solution and cell culture medium, forming a transparent and homogeneous pink aqueous solution without visible precipitation for months (Inset of Fig 2A). The successful encapsulation of RB in Gd-FPNPs is validated by the characteristic emission peak at ~570 nm originating from RB when excited by 550 nm wavelength (curve a & b of Fig


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2A), and bright orange-yellow fluorescence under UV irradiation (Inset of Fig 2A). A blue shift of ca. 10 nm in the emission wavelength is observed for RB when being encapsulated into Gd-FPNPs (curve b & d of Fig 2A), possibly due to the weaker polarity of MMA than H2O35-37. The fluorescence lifetime of RB obviously lengthens ∼2 times (from 1.94 to 3.84 ns) (Fig 2B), because its molecular motion is restricted and its stretching and bond vibration are weakened when being polymerized with MMA. It has been widely documented that the polymer matrix provides an effective barrier keeping the dye from the surrounding environment, thus minimizing both photobleaching and photodegradation phenomena38-40. As expected and demonstrated in Fig 2C, no obvious photobleaching is observed for Gd-FPNPs over continuous UV irradiation for up to 5 hours. In contrast, the FL intensity of free RB in aqueous solution obviously decreased along with the increase of irradiation time, remaining only 60% of the original intensity after 5 h continuous irradiation. The excellent photostability makes Gd-FPNPs suitable for in vivo FL bioimaging applications which require high intensity or prolonged excitations. The improved chemical stability of Gd-FPNPs is validated by the no observable RB leakage and stable FL intensity in buffer solution with various pH values (Fig S2). All these results confirm that our reported synthesis route is mild, so that RB encapsulated into polymer matrix not only preserves its optical properties, but also improves its PL properties and chemo-/photo-stability. The surface-chelated Gd3+ on Gd-FPNPs is quantitated to be 0.39% (w/w) by inductively coupled plasma-mass spectrometry (ICP-MS) using HNO3 digestion, rendering Gd-FPNPs MRI modality. Inset of Fig 2D shows the gray-scaled T1-weighted MR images of Gd-FPNPs dispersions which exhibit a clear concentration-dependent positive contrast enhancement, confirming their capability to curtail the longitudinal relaxation time of hydrogen protons. The


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linearity of relaxation rates versus equivalent Gd concentration is plotted in Fig 2D, from which the r1 value of Gd-FPNPs is calculated to be 26.86 mM-1 s-1. The r1 value of Gd-FPNPs is higher than that of commercially available Gd contrast agents such as DTPA analogues Magnevists® (3.2 mM-1 s-1)42, ProHance®(4.3 mM-1 s-1)43, Omniscans® (3.3 mM-1 s-1)42, Gadobutrol

®

(2.5 mM-1 s-1), and Gadovist® (4.34 mM-1 s-1)44, because of their high content

of surface-chelated Gd3+ and nano-size24. It has been well documented that the direct interactions between Gd3+ and hydrogen protons largely determine longitudinal relaxivity13,33,41. In present study, Gd-FPNPs consist of RB-encapsulated PMMA cores and Gd3+ chelate-conjugated branched PEI shell, suggesting most of Gd3+ is presented on the outer surface of the nanoprobe and thus facilitating the direct interactions between Gd3+ and hydrogen protons. In addition, compared with small molecules, nano-size affords a slower tumble to increase their rotational correlation time45, further contributing to the increase of r1 value. In addition, the ratio of r2/r1 is calculated to be 1.06, confirming that Gd-FPNPs is a T1-contrast agent favoring positive contrast enhancement46. Clinically, a T1 -contrast agent with high relaxivity is much more desirable than T2-agent46,47. Biocompatibility of the Gd-FPNPs. It is essential for an ideal bioimaging probe to exhibit excellent biocompatibility. Since the Gd3+ leakage from Gd-FPNPs can induce serious toxicity46, 48

, Xylenol Orange is used as the indicator to detect Gd3+ leakage from Gd-FPNPs49. Results

confirm that no noticeable amount of Gd3+ is released into serum for up to 24 h (Fig S3A). This should be attributed to the well preserved chelation of Gd3+-DTPA on the PEI shell during the polymerization, thus again confirming not only the mild synthesis route but also the excellent chemical stability of Gd-FPNPs. Meanwhile, MTT results validate the good biocompatibility of


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Gd-FPNPs towards various cell lines including HeLa cells, CT 26 cells, and HepG2 cells, as evidenced by over 90% cell viability even with concentrations up to 0.5 mg mL-1 (Fig S3B). Since intravenous injection is the major route for contrast agent delivery, it is important to evaluate the hemolytic potential of Gd-FPNPs50,51. The presence of hemoglobin results in the red solutions, however, no visually red color is found with varied concentration of Gd-FPNPs from 0.2 to 0.4 g L-1 (Inset of Fig 3A), indicating their negligible hemolytic potential. The highest hemolytic efficiency of Gd-FPNPs is ~5.0% at its concentration up to 0.4 g L-1 (Fig 3A), inferring the excellent blood compatibility of Gd-FPNPs. In addition, Gd-FPNPs exhibit remarkable long-term colloidal stability in fetal bovine serum saline buffer (FBS) for up to 15 days without forming any aggregation (Fig S4). The biotoxicity of Gd-FPNPs is examined using zebrafish. After soaking with GdFPNPs for 8 h, bright orange-yellow FL signals are clearly observed at the yolk sac and esophagus of 6 days old zebrafish, confirming the successful absorption of Gd-FPNPs in zebrafish possibly via yolk membrane and swallowing behaviour (Fig 3B). Importantly, there is no obvious lesion and/or teratogenesis can be observed for Gd-FPNPs treated zebrafish, validating the low biotoxicity of Gd-FPNPs. The potential in vivo toxicity of Gd-FPNPs is evaluated by monitoring histological changes of the vital organs of mice including lung, liver, spleen, kidney, and heart. As shown in Fig 3C, there is no noticeable organ damage or inflammation observed in all major organs of the treated mice, suggesting no obvious side effect caused by Gd-FPNPs to the treated animals. All these results confirm excellent biocompatibility of Gd-FPNPs, laying a solid foundation for their applicability of dual-modal FL/MR bioimaging.


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In vitro dual-modal FL/MR imaging. Thanks to the bright fluorescence and high r1 value, Gd-FPNPs are expected to be effective dual-modal FL/MR imaging nanoprobes. In this study, HeLa cells are incubated with both DAPI and Gd-FPNPs (0.05 mg mL-1). It is clearly observed that bright orange-yellow and blue fluorescence signals are respectively concentrated in the cell cytoplasm and cell nuclei, suggesting that Gd-FPNPs are efficiently accumulated and evenly distributed only in the cell cytoplasm (Fig 4A-4C). These results confirm that Gd-FPNPs successfully cross the cell membrane barriers to enter the intracellular region, possibly due to the endosomolytic effects arisen by their free surface amine groups13,32,52. In addition, the brightfield image of HeLa cells confirm their intact morphology upon exposure to Gd-FPNPs for 4h, again verifying their biocompatibility (Fig 4D). Once they are internalized in HeLa cells, Gd-FPNPs can also illuminate cells upon exposure to a clinical MRI instrument. As shown in Fig 4F, significantly enhanced MR signals are observed in the cells upon treatment with 0.05 mg mL-1 Gd-FPNPs, indicating the obvious positive contrast enhancement. These results confirmed that the FL and MR efficiency of Gd-FPNPs remain after being internalized by cells, and can thereafter successfully illuminate cells with both FL and MR signals. In vivo dual-modal FL/MR imaging. In vivo bioimaging application of Gd-FPNPs is demonstrated using Balb/c mice as model. As shown in Fig 5A, though being acquired through intact skin and skull, FL signals are observed in liver after 6 hours of intravenous injection, and increased along with time. Through imaging of the single organ after dissection, the efficient accumulation of Gd-FPNPs in liver and intestines is confirmed, and no fluorescence is observed from the heart, lung, kidney and spleen (Fig. 5B). Different to previous studies12,24, the intestines instead of kidney exhibit the strongest fluorescence signal, suggesting that Gd-FPNPs are mainly


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eliminated by enteron excretion rather than by renal excretion. The MR images show similar imaging trend to FL imaging, where MR signals are clearly observed in liver and intestines (Fig. 6A) and increase along with time (Fig. 6C). Different to FL imaging, 4 times stronger MR signals (Fig. 6B & 6C) than control can be observed after 12 hours of intravenous injection of Gd-FPNPs, while clear and strong FL signal can only be collected after 24 hours of intravenous injection. The better tissue penetration capability of MR imaging than FL imaging should be responsible for this difference. No obvious FL (Fig. 5) and MR signals (Fig. 6A & 6B) can be observed from the control group. Cross-validation between FL and MR results reveals the synergistic metabolism of Gd-FPNPs through liver and intestines. In vivo MR imaging performance of Gd-FPNPs is further evaluated using CT-26 tumorbearing mice, and the commercially available MR contrast agent Gadobutrol is employed as the control to highlight the clinical practicality of Gd-FPNPs. Fig 7A and S5 reveal an obviously positive contrast enhancement of both Gd-FPNPs and Gadobutrol after 10 min of injection, suggesting their immediate entrance to the blood circulation. For Gadobutrol-treated mice, MR signals gradually transfer from the tumor site to the bladder after 30 min (red curve of Fig 7B & 7C). In contrast, for Gd-FPNPs treated mice, MR signals from the tumor site gradually increase for the first 120 minutes and thereafter remain constant up to 360 min (black curve of Fig 7B). ~37.1% and ~11.6% enhancement of MR signal are respectively observed at the tumor site for Gd-FPNPs treated mice and Gadobutrol-treated mice, suggesting significantly enhanced MR response by Gd-FPNPs which is ascribed to enhanced permeability and retention (EPR) effect. No significant increased MR signals are observed from the bladder of Gd-FPNPs treated mice (black curve of Fig 7C), indicating that Gd-FPNPs are not mainly excreted through the urine. In addition, T1-weighted imaging of muscle disclosed the low intensity signals without obvious


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enhancement effect for both Gd-FPNPs treated mice and Gadobutrol-treated mice (Fig 7D). Combining the FL and MR imaging results validates that Gd-FPNPs are sufficient for in vivo dual-modality applications. Conclusion In summary, a simple and robust synthesis method is reported to prepare the well-defined coreshell structured Gd-FPNPs that consist of RB-encapsulated PMMA cores and Gd3+ chelateconjugated branched PEI shells. The structural merits of Gd-FPNPs enable them bright orangeyellow FL and high MR contrast as high-performance FL/MR dual-modal imaging probes. The uniform spherical structure facilitates their bio-transfer, and their surface carboxyl and amine groups afford them both long-term colloidal stability and cell-membrane permeability, thus in favor of the in vitro and in vivo bioimaging applications of Gd-FPNPs. The dual response is confirmed with HeLa cells and mice as models, and complementary information provided by two imaging modalities demonstrates the single-cell sensitivity of FL imaging and the excellent 3D spatial resolution of MR imaging. Because of their simple synthesis under mild conditions, bright fluorescence and high longitudinal relaxivity, strong chemo-/photo-stability, good biocompatibility, and ease of functionalization, Gd-FPNPs possibly promote the future clinical translation of this material as safe and high-performance functional imaging probes. In addition, this work enables such imaging probes to be prepared also by those not skilled in the nanomaterial synthesis. Through rationally tailoring the encapsulated and outer surface conjugated components, it is feasible to use the approach reported in this study to prepare multimodal imaging probes with improved properties. Acknowledgement. The work was supported by grants from Guangdong-HongKong Technology Cooperation Funding Scheme (2016A050503027), Tip-top Scientific and Technical


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Innovative Youth Talents of Guangdong Special Support Program (2014TQ01R417), and the Fundamental Research Funds for the Central Universities (Grant No. 17lgjc09). Supporting Information Available: The more detailed experimental protocols, and additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.


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Scheme 1. Schematic illustration for the facile synthesis of Gd-FPNPs at 80 °C for 2 h and their FL/MR dual-modal bioimaging applications.


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Figure 1. (A) TEM image of Gd-FPNPs. Inset shows the histograms of particles size distribution of Gd-FPNPs. (B) High-resolution TEM image of a single nanoparticle reveals the well-defined core-shell structure of Gd-FPNPs. (C) XPS survey spectra, and (D, E) the deconvoluted XPS spectra of Gd-FPNPs: (D) Gd 4d, (E) Gd 3d.


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Figure 2. Optical and magnetic properties of Gd-FPNPs. (A) The fluorescence excitation and emission spectra of Gd-FPNPs (curve a and b) and RB (curve c and d). Inset shows the photograph of Gd-FPNPs under daylight (e) and 365 nm UV light (f). (B) Fluorescence decay profiles of Gd-FPNPs and RB. (C) Photostability comparison of Gd-FPNPs and RB. The solutions are under continuous exposure to UV light for different amounts of time. (D) T1weighted MR images of Gd-FPNPs with various concentrations (equivalent Gd concentration: 0.8, 1.7, 2.5, 3.3, 4.1 µM), and linear correlation between longitudinal relaxivity (r1) and equivalent Gd concentration of Gd-FPNPs.


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Figure 3. (A) Hemolytic percentage of Gd-FPNPs with various concentrations to human red blood cells. (B) Bright-field and fluorescence images of zebrafishes upon treatment with (GdFPNPs) and without (Blank) Gd-FPNPs (0.4 mg·mL-1) for 8 h. (C) Histological changes in the lung, liver, spleen, kidney and heart of mice after injection with saline solution (control) and GdFPNPs (Gd-FPNPs, 0.05 mmol Gd per kg).


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Figure 4. (A-D) The fluorescent microscopic images of HeLa cells incubated with Gd-FPNPs (0.05 mg·mL-1) and DAPI (0.1 µg·mL-1). Images are taken under (A) 340-380 nm excitation, (B) 535 nm excitation, (C) overlay of the fluorescence images, and (D) bright field image. Photograph (E) and T1-weighted MR images (F) of HeLa cells upon treatment with (Gd-FPNPs) and without (Blank) 0.05 mg·mL-1 Gd-FPNPs.


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Figure 5. (A) In vivo fluorescence images of mice treated with normal saline or Gd-FPNPs (0.8 mg mL-1) for 6 h, 9 h and 24 h, respectively. (B) Ex vivo fluorescence imaging of resected organs during necropsy at 24 h after injection.


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Figure 6. (A) In vivo T1-weighted MR images of mice treated with normal saline or Gd-FPNPs (0.05 mmol Gd3+ per kg) for 3 h, 6 h, 9 h and 12 h, respectively; (B) The average grey values of T1 weighted signals of the liver and the intestines at different time points after intravenously injected with normal saline; (C) The average grey values of T1 weighted signals of the liver and the intestines at different time points after intravenously injected with Gd-FPNPs.


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Figure 7. (A) In vivo T1-weighted MR images of CT-26 tumor-bearing mice before and after injection of Gd-FPNPs (0.05 mmol Gd3+ per kg) at different time points with Gadobutrol (0.05 mmol per kg) as positive control; (B-D) The average grey values of T1 weighted signals of the tumor (B), the bladder (C) and muscle (D) at different time points.


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SYNOPSIS TOC


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Facile Synthesis of Gadolinium Chelate-Conjugated Polymer

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