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Conjugated polymer containing organic radical for optical/MR dual-modality bioimaging Meirong Hou, Xiaodan Lu, Zhide Zhang, Qi Xia, Chenggong Yan, Zhiqiang Yu, Yikai Xu, and Ruiyuan Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15052 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 9, 2017

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ACS Applied Materials & Interfaces

Conjugated polymer containing organic radical for optical/MR dual-modality bioimaging a

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a

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Meirong Hou , Xiaodan Lu , Zhide Zhang , Qi Xia , Chenggong Yan , Zhiqiang Yu , Yikai Xu* , Ruiyuan Liu* a

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Medical Imaging Center, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, P.R. China.

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Guangdong Provincial Key Laboratory of Medical Image Processing, School of Biomedical Engineering, Southern Medical University, Guangzhou 510515, P.R. China c

School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, P.R. China

KEYWORDS : Conjugated polymer, polyfluorene, organic radical, optical imaging, MRI imaging ABSTRACT: Optical/MRI bimodal probes have attracted much attention due to palmary soft tissue resolution and high imaging sensitivity. In this study, poly[fluorene-co-alt-p-phenylene] containing organic radical (PFPTEMPO+) is successfully developed for optical and MRI dual-modality bioimaging. PFPTEMPO+ displays advanced properties such as fluorescence emission, high photostablilty, reasonable T1 relaxation effect, low cytotoxicity, and good biocompatibility. Moreover, the ability of PFP-TEMPO+ for tumor tissues imaging confirms that it could be used as optical and MRI imaging probe for in vivo imaging. The results of the present

work disclose the potential applications of PFP-TEMPO+ as optical and MRI contrast agent. 1. INTRODUCTION Noninvasive imaging techniques such as optical imaging, X-ray computed tomography (CT), ultrasound imaging and magnetic resonance imaging (MRI) have played a growing role in disease diagnosis and imaging-guided treatment.1-4 However, each imaging modality is differ from the others and has its inherent limitations.5-8 For example, optical imaging possesses high imaging sensitivity while limited tissue penetration. Hence, multimodal imaging in a single system has been attracted considerable interests to conquer the inherent limitations of single imaging pattern. Very recently, combination of optical imaging and MRI has attracted more and more attention, which demonstrates palmary soft tissue resolution and high imaging sensitivity.9,10 A series of bimodal probes that act as fluorescent materials for optical imaging and gadolinium chelates for MRI have been widely reported.1113 To date, inorganic quantum dots and organic dyes have been broadly used for fluorescent part. However, inorganic quantum dots based fluorescent materials often suffer from heavy metals with potential cytotoxicity.14,15 Moreover, the poor photostablility and easily quenching in biological solutions of organic dyes also limit their applications.16,17 Compared with inorganic quantum dots and organic dyes, conjugated polymers (CPs) demonstrate great potential as a kind of materials with unique physiochemical properties.18-20 With broad absorption and emission spectra, high quantum yield, and good photostability, CPs not

only have been applied for detections of chemical and biological molecules,21-24 but also have been used for biomedical fields, such as cell imaging, gene delivery, drug delivery and release, and cell engineering.25-30 Polyfluorenes as one of the most common class of CPs, not only have the same excellent properties compared with other CPs, but also have a high charge-carrier mobility and good processability.31,32 As a consequence, polyfluorenes become an alternative choice for optical imaging application. On the other aspect, Gadolinium (Gd) based contrast agents (GBCAs) have been widely applied as T1 contrast agent for MR scans in clinical.33 However, the heavy metal toxicity of GBCAs can be problematic for patients with compromised kidney function. Furthermore, GBCAs show long-term deposition in brain tissue. Accordingly, the development of new-type T1 agents have attracted much attention.34-39 Since most of them are metal-based agents, a more comprehensive understanding of their biodistribution need to be performed. Compared with them, metal-free nitroxide stable free radicals (NSFR) are more promising. A series of related experiments in vitro and in vivo show that NSFR possess some favourable features similar to GBCAs, such as electron paramagnetism, fast renal elimination, long half-life and chemical versatility. Importantly, it is relatively low toxicity.40,41 MR contrast agents based on 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) have been successfully synthesized and evaluated in our previous work.42 What’s more, Chuan et al

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reported a new kind of nanoprobes based on core-shell NaYF4:Yb, Er/NaGdF4 nanocrystals containing more than one TEMPO to improve the T1 relaxivity of TEMPO. But the photostability of the fluorescent group still has great development space.43 In the paper, we described dualmodal imaging probe based on poly[fluorene-co-alt-pphenylene] containing TEMPO through polymerization of N-TEMPO-2,5-diiodobenzamide with 2,2'-(9,9-bis(6bromohexyl)-9H-fluorene-2,7-diyl)bis(4,4,5,5tetramethyl-1,3,2-dioxaborolane), followed by QAfunctionalization (named PFP-TEMPO+). This dual imaging agent showed substantial fluorescent emission and reasonable T1 relaxation effect with low cytotoxicity and high photostability. Furthermore, imaging of tumor tissues by PFP-TEMPO+ in vivo was monitored by MRI and histological examinations of A549 tumor-bearing mice. Ultimately, this manuscript highlighted conjugated polymer bearing TEMPO could be used in dual modual bioimaging in vitro and in vivo.

Scheme 1 Synthesis of PFP-TEMPO+ 2. MATERIALS AND METHODS 2.1. Materials. All reagents and solvents were purchased from commercial sources. Solvents were dried according to standard procedures. Deionized water (DI water) was used to prepare all the aqueous solutions. 2,5Diiodobenzoic acid, 4-amino-2,2,6,6tetramethylpiperidine-N-oxyl (4-NH2-TEMPO), 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl), and N-hydroxysuccinimide (NHS) were purchased from Adamas (China). 2,2'-(9,9-bis(6bromohexyl)-9H-fluorene-2,7-diyl)bis(4,4,5,5tetramethyl-1,3,2-dioxaborolane) was synthesized according to reference.44 Cell Counting Kits (CCK-8) were obtained from Dojindo (Japan). dimethyl sulfoxide (DMSO) and 4% paraformaldehyde were acquired from SigmaAldrich (USA). Roswell Park Memorial Institute (RPMI)1640 and Penicillin-Streptomycin Liquid were obtained from Gibco while fetal bovine serum (FBS) from Biological Industries(BI). 2.2. Characterization. 1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a Bruker Avance/DMX 400MHz NMR spectrometer with DMSO-d6, CDCl3,

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CD3OD or and tetramethylsilane as an internal reference. IR spectra were measured using a Shimadzu FTIR-8100 spectrophotometer. Absorbance detections were carried out on Shimadzu UV-2450 spectrophotometer. Fluorescence emission spectra were obtained at room temperature on a FLS-920 Edinburgh Fluorescence Spectrophotometer, with a Xenon lamp and 1.0 cm quartz cells. The number- and weight-average molecular weights (Mn and Mw, respectively) of the polymer were determined using gel permeation chromatography (GPC) on a Jasco Gulliver system (PU-980, CO-965, RI-930, and UV-1570) equipped with polystyrene gel columns (Shodex columns K804, K805, and J806) and calibrated by polystyrene standards at 30 °C using THF as the eluent. Dynamic light scattering (DLS) and zeta potential measurements were performed on a Malvern Zetasizer Nano-ZS90. High resolution mass spectra (HR-MS) were carried out on a Bruker spectrometer using ESI ionization. Transmission electron microscopy (TEM) were obtained from a Hitachi H-600. 2.3. Synthesis of 1. First, 2,5-diiodobenzoic acid (3.73g, 10mmol) , 4-NH2-TEMPO(1.71g, 10mmol) and CH2Cl2 (30mL) were mixed in a 100mL flask. Then EDC.HCl (1.91g, 10mmol) and NHS (1.15g, 10mmol) were added and the reaction was carried out overnight at room temperature. After the reaction, the mixture was washed with 1 M HCl, statured aq. NaHCO3 and aq. NaCl. And the organic layer was evaporated to dryness to obtain the crude mixture. The final product was purified via flash column chromatography (EtOAc: Hexane = 1:4 v/v). Red solid was obtained in 86% yield (4.53 g). 1H NMR (400 MHz, DMSOd6) δ (ppm): 8.29~8.31(d, 1H), 7.50(s, 1H), 7.48~7.49(d, 1H), 7.03~7.12(d, 1H), 1.81~1.84(d,1H), 1.43~1.49(t, 1H), 1.13~1.15(t, 12H). 13C NMR (100 MHz, DMSO-d6) δ(ppm): 166.67, 152.49, 145.14, 140.85, 139.11, 135.95, 128.52, 116.79, 111.58, 94.08, 93.26, 57.91, 44.58, 32.66, 19.68. IR：3297.35, 2992.14, 2937.89, 2854.33, 1662.75, 1537.25, 1453.58, 1357.52, 1320.34, 1278.50, 1232.02, 1173.92, 1073.21, 1018.98, 898.13, 868.69, 809.81, 722.27, 630.09, 601.42, 559.58, 471.27, 442.41. HR-MS (ESI): C16H21I2N2O2 m/z, 526.9692 for [M+Na]+ : 549.9581. 2.4. Polymerization. A solution of 1 (527 mg, 1 mmol) and 2,2'-(9,9-bis(6-bromohexyl)-9H-fluorene-2,7diyl)bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane) (742 mg, 1 mmol), Pd(PPh3)4 (57 mg, 50 µmol) in toluene (5 mL), statured aq. Cs2CO3 a.q.(1M, 2 mL), and EtOH (3mL) were stirred at 80 °C overnight. The organic phase was poured into MeOH to precipitate a polymer. Then the precipitate was redissolved in chloroform and reprecipitated three times by adding MeOH. GPC (THF, polystyrene standard): Mw:29317,Mn:12370, PDI:2.37, 1H NMR (400 MHz, CDCl3): δ (ppm) 8.24(s,1H, aromatic H), 8.12(s,1H, aromatic H), 7.89-7.16 (br, 7H,aromatic H), 3.323.30(br, 4H, alkyl side chain H), 3.10(s, 1H),1.99(br,8H, alkyl side chain H), 1.70(br, 8H, alkyl side chain H), 1.45(br, 4H, alkyl side chain H),1.26(br, 12H, methyl groups), 0.83(br, 4H, alkyl side chain H); IR (KBr, cm−1): 3439.12 ， 2975.86 ， 2929.37 ， 2854.23 ， 1716.98 ，


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1625.56， 1462.10, 1424.92, 1362.97, 1315.68, 1232.02, 1177.79, 1140.61, 1077.86, 1002.71, 885.73, 822.98, 785.02, 751.71, 685.09, 634.73, 563.46. 250 mg of polymer (PFP-TEMPO) was dispersed in THF (100mL) under stirring. Then 10mL Et3N was added and the mixture was kept stirring at room temperature for 5 d. During this period, the solubility of the polymer gradually reduced in THF and the polymer was precipitated. After that, DI water (10mL) was added and the precipitate was dissolved. Afterwards, the solution was evaporated then methanol was added to dissolve the residue. Finally, the ionic product (PFP-TEMPO+) was precipitated from ether and was characterized as dark yellow powders. 1H NMR (400 MHz, CD3OD): δ (ppm) 6.44-5.88(m, 9H,aromatic H), 3.01(br, 17H,alkyl side chain H, methylene groups of quaternized ammonium), 1.87(br,4H, alkyl side chain H), 1.68(br, 8H, alkyl side chain H), 1.21(br, 30H, methyl groups ), 1.05(br, 4H, alkyl side chain H), 0.74(br, 8H, alkyl side chain H). 2.5. Preparation of PFP-TEMPO+. PFP-TEMPO+ aqueous solutions were obtained based on the classical dialysis method and all the procedures were performed at room temperature. In brief, PFP-TEMPO+ dissolved in THF were stirred for 120 min. Then 5 mL of DI water was drop added. After that, the solution was transferred to dialysis bags and dialyzed to remove extra THF. And the final solution was being frozen, lyophilized, calculated, and redissolved in DI water for the follow works. 2.6. T1 Values Measurement and T1 Relaxation Rate Calculation. PFP-TEMPO+ were prepared at the concentrations of 0.355, 0.71, 1.42, 2.84, 5.68 mM in 5mL eppendorf tubes respectively. The longitudinal (T1) relaxation of these solutions were obtained using T1 mapping sequences on a Philips Achieva 3.0T MRI system. The scan parameters of the sequences were as follows: TR=3000 ms，TE=10 ms, IR=50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 ms; slices thickness=3mm; gap=0.5 mm; field of view=120×100×60mm; NSA=2. 1/T1 relaxation time of each concentration was calculated by defining the regions of interest (ROI) of each tube. Relaxivity (r1) was analysed via the fitting curve of the 1/T1 (s-1) relaxation times versus the PFP-TEMPO+ concentrations (mM). 2.7. Cell Culture and Incubation. Human lung cancer A549 cell line was gifted form the Research Center of Clinical Medicine in Nanfang Hospital (Guangzhou, China),and the cells were incubated with (RPMI)-1640 culture medium stored at 37℃ with 5% CO2 in the atmosphere. 2.8. Cytotoxicity Assay. A549 cells were seeded in a 96-well plate at 6000 cells/well and incubated at 37℃ in 5% CO2 incubator for 24h. Subsequently, the solution were replaced by 100 µL medium containing PFPTEMPO+ at different concentrations (0.355, 0.71, 1.42, 2.84, 5.68 mmol/L) and the cells were kept incubating for 24 h and 48h. Five replicate wells were taken per condi-

tion. After that, the medium was replaced with 100 µL culture medium containing 10% CCK-8 and incubated for 0.5 h. Then the medium were measured by enzyme-linked immunosorbent assay reader BIOTEK ELX80 at 450 nm wavelength. The cell viability (%) related to the blank control wells containing no nanoparticle was calculated by [[A]test-[A]blank control]/[[A]untreated-[A]blank control]×100%. 2.9. In vitro Fluorescence Imaging. A549 cells were seeded in two 35mm confocal culture dishes and incubated for 24h. Subsequently, 1.5 mL of 0.71 mM of PFPTEMPO+ in culture solution was added and co-incubated with cells for 4h while the blank control group was added the same volume of (RPMI)-1640 culture medium. After that, cells of each group were washed with phosphate buffered saline (PBS) three times and then fixed with 4% paraformaldehyde for 15 min. Image were processed using a confocal laser scanning microscopy(CLSM)(Olympus FV 1000). 2.10. Flow Cytometry. A549 cells were grown in 6-well culture plates and separately replaced with 0.71 mM of PFP-TEMPO+ in culture solution (experiment group) and (RPMI)-1640 culture medium (blank control group) to incubated for 4h. After incubation for 4h, the cells were washed with PBS to remove the extra PFP-TEMPO+. And the cells were digested and re-suspended in 300 μL PBS. The fluorescence intensity was measured by flow cytometry (BD FACSAria™ III, USA). 2.11. Photostability Study in Vitro. Under continuous laser excitation, CLSM pictures were taken at each 60 seconds time interval with a total of 10 cycle times to investigate the photostability of PFP-TEMPO+. In the end, the fluorescence intensity of each picture was calculated by the online available software ImageJ. The photostability of PFP-TEMPO+ was tested by the ratio of fluorescence intensity at different time intervals to the initial intensity. 2.12. Cellular MRI In vitro. A549 cells were grown and incubated with 0.71 mM of PFP-TEMPO+ in culture solution (experiment group) and (RPMI)-1640 culture medium (blank control group) for 4 h, respectively. And after washed with PBS three times, the cells was trypsinized, and resuspended in 150 μL PBS with 1% agarose. The cellular MRI signal of each group was acquired using T1weighted spin-echo sequence (TR/TE = 400/15ms, section thickness = 2 mm, FA =10o) in a 3.0T clinical MR system (Achieva 3.0T, Philips). 2.13. Animals. All animal studies were approved by the Animal Experimentation Ethics Committee of Nanfang Hospital and performed in line with current guidelines. All the procedures were conducted with male BALB/c nude mice (4-6 weeks old, Southern Medical University, Guangzhou, China) under general anaesthesia by injection of sodium pentobarbital (0.1-0.15ml/0.1 %). For A549 tumor model, cells (3 ×106) suspended in 100 µL of PBS were injected subcutaneously into the right flank of



BALB/c nude mice. In vivo experiments were carried out when the tumor diameters reached 0.8–1.2 cm. 2.15. In Vivo MR Imaging. Six Mice bearing A549 tumors were divided into two groups randomly as experiment group and blank control group. The MR imaging was obtained using a 3.0T MRI scanner based on a mouseimaging coil. Coronal spin-echo T1-weighted MRI images (TR = 500ms, TE = 10 ms, section thickness = 2 mm, field of view = 100 × 100 × 20 mm, number of excitation = 1) were obtained before and 30min after the intratumor injection of PFP-TEMPO+ or saline. Changes in the relative signal intensity were measured. 2.16. Fluorescence Immunohistochemistry. From this part, tumors were resected and frozen in an optimal cutting temperature compound for standard immunohistochemistry (with a thickness of 5 μm) ex vivo after MR imaging. Images were acquired under a fluorescence microscopy (BX51, Olympus, Tokyo, Japan). 2.17. Hematoxylin and Eosin (H&E) Stained Analysis. For acute toxicology study, three healthy BALB/c nude mice were intravenous injected 200μl of PFPTEMPO+ solutions (2.84mM) and kept following up for one week. Then cervical dislocation method was implemented to sacrifice the animals, and their livers, spleens, lungs, kidneys and hearts were quickly removed, fixed and stained with H&E staining. The H&E stained images were observed by using an upright microscopy. The same procedures were also performed on the blank control group.

cationic conjugated polymer PFP-TEMPO+. The chemical structures of PFP-TEMPO and PFP-TEMPO+ were analyzed with NMR spectroscopy or IR spectra (Figure S5S7). 3.2. Characterization of PFP-TEMPO+. The physicochemical properties of PFP-TEMPO+ were investigated in phosphate buffered saline (PBS). The UV-vis absorption spectrum of PFP-TEMPO+ from Figure 1A showed a maximum peak at 352 nm, while the emission spectrum exhibited a maximum peak at 450nm upon excitation at 390 nm. The zeta-potential of PPF-TEMPO+ was determined to be +46.7 mV, indicating enough positive charges on their surface. And the morphology of our as-prepared PFP-TEMPO+ were obtained by transmission electron microscopy (TEM), which suggests that the PFPTEMPO+ were spherical in shape with a mean diameter of 35.3 nm (the inset of Figure 1B). Because of the integration of hydrophilic and hydrophobic groups in PFPTEMPO+, it self-assembled into nanoparticles in aqueous solution. As shown in Figure 1B, dynamic light scattering (DLS) measurement results demonstrated that the average particle diameter was about 60 nm. Obviously the DLS result indicated a slightly higher mean size for the polymer as compared to that observed by TEM, which could be due to the drying process during the TEM sample preparation. The above physicochemical properties of PFP-TEMPO+ were in the appropriate range for im-

2.18. Statistical Analysis. The statistical significance of treatment outcomes were assessed using One-way ANOVA analysis or Paired-Samples T test or the independent sample t test. All dates were analysed using statistical software (SPSS version 20.0, IBM Corporation, Armonk, NY, USA), p < 0.05 was considered statistically significant. 3. RESULTS AND DISCUSSION 3.1. Synthesis. The synthesis of PFP-TEMPO+ was illustrated in Scheme 1. Compounds 1 was prepared by 2,5diiodobenzoic acid with 4-NH2-TEMPO (Scheme S1). After purification by silica gel chromatography, the final product was obtained in high yields (86%) and the monomer was red powder. Spectroscopic methods were used to characterize its molecular structure and satisfactory analysis data was observed in Figure S1-S4. Compounds 2 was prepared according to previously published methods.44 The precursor polymer PFP-TEMPO was synthesized from compound 1 and boronic ester 2 by Suzuki couplings. The weight-average molecular weight (Mw) and numberaverage molecular weight (Mn) of PFP-TEMPO obtained from the gel permeation chromatography (GPC) analyses were 29317 and 12370, respectively, and the polydispersity index (PDI) was 2.37. Then PFP-TEMPO mixed with excessive trimethylamine in THF was performed to obtain

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Figure 1 (A) Normalized absorption spectra and fluorescence spectra of PFP-TEMPO+ in PBS. The excitation wavelength is 390 nm. (B) Hydrodynamic diameter of PFP-TEMPO+ measured by DLS. (C) T1-weighed MR images of PFP-TEMPO+ in water solutions with various concentrations and (D) T1 relaxation rate curve of PFPTEMPO+ proved cell uptake.


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Figure 2 (A) Confocal Images of A549 cells incubated with (RPMI)-1640 culture medium and PFP-TEMPO+ (×1380). (B~C) Fluorescence intensities of A549 cells incubated with (RPMI)-1640 culture medium (B) and PFP-TEMPO+ (C) by flow cytometry 3.3. T1 Values Measurement and T1 Relaxation Rate Calculation. 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), as a kind of widely used nitroxide radical, have been explored as MR contrast agent for a long time. To evaluate the potential MRI properties of the obtained PFP-TEMPO+, PFP-TEMPO+ with varied concentrations was used. From Figure 1C and 1D we could see that there was a linear relationship between the T1 signal intensities and the PFP-TEMPO+ concentrations, and the r1 values were 0.28 mM-1s-1 for PFP-TEMPO+. 3.4. Cytotoxicity. A549 cells incubated with increasing amounts of polymer were used to assess the cytotoxicity of PFP-TEMPO+. The result of CCK-8 assay in Figure S8

showed that the cell viability and proliferation were not hindered by the presence of PFP-TEMPO+, and compared to the blank control group, the cell viability was larger than 77% after an exposure period of 24h and 48h at the concentration of 5.68mmol/L. This result indicated that PFP-TEMPO+ had well biocompatibility at the given concentrations (0-5.68mM). 3.5. Cellular Fluorescent Uptake In Vitro. Fluorescent uptake by cells was studied using CLSM and flow cytometry. From Figure 2A, a clear difference was visible between the uptake of PFP-TEMPO+ group and the blank control group. PFP-TEMPO+ with blue fluorescence were orientated in the cytoplasm within the incu-

Figure 3 (A) Series of confocal images of PFP-TEMPO+ stained A549 cells under continuous scanning at 352nm for 600s (×1800). (B) Normalized fluorescence intensities of PFP-TEMPO+ stained A549 cells under different scanning times



bation period without any detrimental effect on the cell morphological integrity, and it is noteworthy that no autofluorescence from the A549 cells itself could be detected on blank control group. All of these further confirmed the results of the cytotoxicity measurements and clearly showed that PFP-TEMPO+ had good cell membrane permeability. Apart from the A549 cell line, neurological cell line BV2 cells also showed similar uptake of PFPTEMPO+, which were confirmed by confocal image. (Figure S9) Moreover, the cellular uptake of PFP-TEMPO+ was also quantitatively tested by flow cytometry. As shown in Figure 2B, non-treatment A549 cells as blank control group showed only autofluorescence by itself. And the mean fluorescence intensity of cells incubated with PFPTEMPO+ (0.71 mM, Figure 2C) was significantly different from it (p＜0.05). This cytometry results matched well with the CLSM observation and both demonstrated the well cell membrane permeability of PFP-TEMPO+. 3.6. Photostability In Living Cells. Photostability is an important property of conjugated polymer. Thus the photostability of PFP-TEMPO+ was examined in living cells. As shown in Figure 3A and 3B, the intracellular fluorescent intensity of PFP-TEMPO+ decreased to some

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extent after continuous laser irradiated for 600 s, but the fluorescence intensity was still reach up to 80%. Evidently, PFP-TEMPO+ possessed a good photostability or relatively high resistance to photobleaching. As a comparison, the result was similar to those conjugated polymers reported before.45 The well optical properties made PFPTEMPO+ a promising candidate for bioimaging applications. 3.7. Cellular MR Imaging In Vitro. Besides the phantom study, cellular MR imaging in vitro was also used to access the T1-enhanced effect of PFP-TEMPO+ as illustrated in Figure 4A and 4B. Obviously, there was no statistical significance between the blank control group and the 1% agarose. In contrast, A549 cells incubated with PFP-TEMPO+ exhibited a brighter signal as compared to the blank control group and significant difference in the T1-weight signals between two groups were obtained (p＜ 0.05). The results demonstrated that PFP-TEMPO+ could be used as a good T1 contrast agent in vitro. For another, nitroxide radicals (TEMPO) have demonstrated their potential of enhanced cell permeability and favorable volume distribution as proved in our previous work.42

Figure 4 (A) T1-weighted MR images of A549 cells in vitro. (B) T1 Signal-to-noise Ratio intensities of A549 cells incubated with (RPMI)-1640 culture medium and PFP-TEMPO+. (C) T1 MR images of tumor-bearing mice before and post-injection of saline and PFP-TEMPO+. (D) Average signal intensity taken pre- and post-injection of PFPTEMPO+ or saline


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Figure 5 Hematoxylin and eosin stained histological images of major organs for different groups (×400). 3.8. MR Imaging In Vivo. Encouraged by the satisfied results in vitro, we subsequently assessed the MR imaging of PFP-TEMPO+ in vivo. Adult male BALB/c nude mice bearing A549 tumors in their right flank were intratumorly injected with PFP-TEMPO+ or saline. Figure 4C showed the T1-weighted MR imaging of the mouse anatomical section monitored pre-injection and 30min postinjection. As the PFP-TEMPO+ group, thanks to the potential of enhanced cell permeability and favourable volume distribution,42 obviously increased MRI signal intensity was observed in the tumor site. As compared to group receiving saline as blank control, there was no obvious signal intensity changes. And the average signal intensity of different groups in the tumor sites based on different time points was calculated in Figure 4D. In accordance with the MRI findings, the average signal intensity in the tumor sites of mice receiving PFP-TEMPO+ was significantly greater than that receiving saline (p＜0.05). Fluorescence microscopy ex vivo was further applied to assess the fluorescence imaging of PFP-TEMPO+ inside the tumor tissues. As shown in Figure S10, compared with the blank control groups, PFP-TEMPO+ groups demonstrated an enhanced accumulation of blue fluorescence within the tumor. All the above experiments validated that our PFP-TEMPO+ could be used for MRI and optical imaging in vivo. 3.9. Acute Toxicology. Histopathological assessment of tissues were used to access whether PFP-TEMPO+ would cause any tissue damage or inflammation. As shown in Figure 5, no obvious issue for the mice treated with PFP-TEMPO+ was observed while compared with the blank control mice, which further confirmed that PFP-TEMPO+ was low toxicity and suitable for in vivo applications.

sesses some advantages including good biocompatibility, strong fluorescence emission, high photostability and acceptable T1 enhanced effect. Furthermore, the tracking of PFP-TEMPO+ after injection in nude mice demonstrates that PFP-TEMPO+ can be used as optical and MRI imaging probe in vivo if sufficiently gathered in tumor site. Histological assessment of tissues treated with PFPTEMPO+ further confirms the low toxicity and good biocompatibility of PFP-TEMPO+. The results of the present work disclose the potential applications of PFPTEMPO+ as optical and MRI contrast agent.

ASSOCIATED CONTENT Supporting Information. Synthetic procedure, NMR, FTIR absorption spectroscopies and analysis characterization of 1 compound 1; H NMR and FTIR absorption spectroscopies of PFP-TEMPO and PFP-TEMPO+; Cytotoxicity, fluorescent imaging of BV2 cells, and fluorescence immunohistochemistry of PFP-TEMPO+ and saline (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected] Ruiyuan Liu: 0000-0002-6696-8351

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

4. CONCLUSIONS In summary, we reported a new optical/MR dual modual contrast probe (PFP-TEMPO+) based on conjugated polymer containing TEMPO. PFP-TEMPO+ pos-

ACKNOWLEDGMENT



This work was financially supported by National Natural Science Foundation of China (81271642, 31371009, and 81671749), National key research and development program of China (2016YFC0107104), Natural Science Foundation of Guangdong, China (2016A030313546), and Science and Technology Planning Project of Guangdong Province, China (2015B010131011 and 2015B020233019).

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Table of Contents Only Conjugated polymer containing TEMPO (PFP-TEMPO+) displays advanced properties such as fluorescence emission, high photostablilty, reasonable T1 relaxation effect, low cytotoxicity, and good biocompatibility, which could be used as optical and MRI imaging probe for bioimaging in vitro and in vivo.


Conjugated Polymer Containing Organic Radical ... - ACS Publications

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