Preparation of Sialic Acid-Imprinted Fluorescent Conjugated

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Preparation of Sialic Acid-Imprinted Fluorescent Conjugated Nanoparticles and Their Application for Targeted Cancer Cell Imaging Ronghua Liu, Qianling Cui, Chun Wang, Xiaoyu Wang, Yu Yang, and Lidong Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14320 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 4, 2017

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ACS Applied Materials & Interfaces

Preparation

of

Sialic

Acid-Imprinted

Fluorescent

Conjugated Nanoparticles and Their Application for Targeted Cancer Cell Imaging Ronghua Liu, Qianling Cui,* Chun Wang, Xiaoyu Wang, Yu Yang, and Lidong Li* State Key Lab for Advanced Metals and Materials, School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China Keywords: fluorescence, conjugated polymer, cellular imaging, phenylboronic acid, sialic acid Abstract Fluorescent conjugated polymer nanoparticles have attracted great interest for applications in biological imaging owing to their excellent optical properties and low cytotoxicity; however a lack of effective targeting limits their use. In this work, we design and synthesize a fluorescent conjugated polymer modified with a phenylboronic acid group, which can covalently bind with cis-diol-containing compounds, such as sialic acid

(SA), by forming a cyclic ester. However, the

obtained conjugated polymer nanoparticles failed to discriminate between cancer cells, with or without SA over-expressed surfaces (such as DU 145 and HeLa cells, respectively). To address this problem, we introduced SA template molecules into the polymer nanoparticles during the reprecipitation process and then removed the template by adjusting the solution pH. The SA-imprinted nanoparticles showed a uniform size around 30 nm and enhanced fluorescence intensity compared with unmodified polymer nanoparticles. The SA-imprinted nanoparticles exhibited 1

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selective staining for DU 145 cancer cells and did not enter HeLa cells even after long incubation times. Thus, we present a facile method to prepare fluorescent nanoparticles for applications in targeted cancer cell imaging.

Introduction Fluorescent conjugated polymers (CPs) possess excellent light-harvesting and strong fluorescence properties, making them promising for applications in biology, chemistry, and photoelectrical fields.1-11 For use in biological environments, CPs should be soluble in or dispersible in aqueous medium, in the form of water-soluble CPs or as CP nanoparticles (CPNPs). In recent years, CPNPs have aroused increasing interest owing to their potential applications in cell imaging, bio-sensing and photodynamic therapy.12-21 CPNPs have certain unique features such as their ease of preparation, low cytotoxicity, and good biocompatibility.22-27 Furthermore, CPNPs show clear advantages including good photostability and ease of surface functionalization compared with CPs in a molecular state. It is particularly important to control surface features for biological applications. Cancer is a major cause of death globally, accounting for about 13% of all deaths.28 Thus, specific recognition and early diagnosis of cancer are urgently required. To this end, fluorescent CPNPs conjugated with biomolecular probes have been developed for the recognition of cancer cells; suitable biomolecular targets include antibodies, aptamers, peptides, and lectins.29-32 However, some disadvantages of these biomolecules limit their practical applications, including their complicated 2


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preparation methods, poor stability, demanding storage conditions, and ease of degradation by protease. Therefore, the development of CPNPs with specific targeting abilities is still urgently required for selective cancer cell imaging. Cell surface glycans are involved in cellular communication including processes such as cell development and differentiation.33 The abnormal expression of specific glycans is a known marker for certain cancer cells. Sialic acid (SA) is an important component in glycans and its overexpression on cell surfaces is known to be an indicator for some cancers.34-37 The phenylboronic acid (PBA) group is widely used to covalently combine with cis-diol-containing compounds, such as glucose.38-40 The combination and dissociation of PBA and cis-diols-containing compounds is a reversible esterification, which can be controlled by changing the solution pH.41, 42 In recent years, PBA again attracts intense interests due to its interaction with SA molecules, which can be utilized for targeting cancer cells.43-46 However, it has been reported that PBA-modified nanoparticles (NPs) fail to target cancer cells because there is no preferential binding of PBA to SA over other monosaccharides at physiological pH.47, 48 Molecule imprinting techniques might provide an alternative way to resolve this problem. Imprinting methods involve introducing template molecules into the NPs. The template molecules are later removed from the NPs, which leaves holes in the structure that have a predictable recognition specificity for the template molecule.49-54 In this work, we prepared SA-imprinted fluorescent NPs based on a CP bearing PBA side chains, and applied this material for selective cancer cell imaging. First, a 3



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conjugated backbone, poly(fluorene-alt-benzothiadiazole) (PFBT), was modified with PBA groups as side chains, which have an affinity for SA molecules. Then, we used a facile reprecipitation process to embed SA molecules into the PFBT-PBA NPs. After acidifying the NP solution and dialysis, SA molecules were removed from the NP surfaces leaving SA-imprinted NPs. These NPs showed a uniform size around 30 nm as determined from dynamic light scattering (DLS) and scanning electron microscopy (SEM). The removal of SA molecules was also confirmed by Fourier transform infrared (FT-IR) spectroscopy. In addition, the SA-imprinted NPs showed more intense photoluminescence than the unmodified NPs. Finally, a cell imaging assay showed that the SA-imprinted NPs could selectively bind to over-expressed SA cancer cells (DU 145) and did not bind to HeLa cells, while the unmodified NPs failed to discriminate between these two cell types. The low cytotoxicity of the SA-imprinted NPs was also demonstrated, indicating good potential for these materials as fluorescent probes for targeted cancer cell imaging.

Experimental Section Materials.

2,7-Dibromo-9,9-bis(6′-bromohexyl)fluorene

[(11-diyl)bis(azanediyl))bis(ethane-2,1-diyl)]dicarbamate according to literature procedures.55,

56

(1)

(2)

and were

di-tert-butyl synthesized

Tetrakis-(triphenylphosphine)palladium(0)

[Pd(PPh3)4] and N-acetylneuraminic acid (sialic acid, SA) were purchased from Sigma-Aldrich

and

used

as

4,7-Bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,1,3-benzothiadiazole 4


received. was

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purchased from Beijing Allmers Chemical S&T Co., Ltd. Other reagents were purchased from J&K Chemical and used without further purification. All the solvents were purchased from Beijing Chemical Works and used as received unless otherwise stated. Tetrahydrofuran (THF) was distilled from Na/diphenylketone. Synthesis of Compound 2. The compound 1 (2.00 g, 3.08 mmol), N-boc-ethylenediamine (1.48 g, 9.23 mmol), and NaOH (0.25 g, 6.15 mmol) were placed into a 100 mL round-bottom flask. Acetonitrile (60 mL) was added to the flask and the solution was degassed. The reaction mixture was then heated to reflux under an argon atmosphere for 3 h. After cooling to room temperature, the reaction solvent was evaporated. The mixture was extracted with dichloromethane, and washed with water. The organic layer was dried over anhydrous sodium sulfate and the solvent was removed. The crude product was purified by column chromatography on silica gel with ethyl acetate: methanol (4:1 v/v) as an eluent to afford 2 (1.44 g, 58%). 1H NMR (400 MHz, CDCl3, δ): 7.46 (ddd, 6H), 3.33 (dd, 6H), 2.83 (ddd, 6H), 1.99‒1.81 (m, 5H), 1.70‒0.90 (m, 30H), 0.62 (d, 4H). MS (MALDI-TOF) m/z: M+ calcd: 808.73. Found: [M + Na]+ = 831.2. Synthesis of PFBT-Boc. Compound 1 (0.126 g, 0.193 mmol), compound 2 (0.174 g,

0.193

mmol),

and

4,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,1,3-benzothiadiazole (0.150 g, 0.386 mmol) were dissolved in 50 mL of toluene, and then 10 mL of 2.0 M potassium carbonate solution was added. After the solution was degassed with argon for 30 min, the catalyst Pd(PPh3)4 was added. The reaction mixture was stirred at 85 °C for 48 h 5



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under an argon atmosphere. The reaction mixture was extracted with CHCl3, and washed with brine. The organic layer was then dried over anhydrous sodium sulfate and the solvent was evaporated. The crude product was dissolved in chloroform (5 mL), and the precursor was then precipitated from methanol (150 mL) to afford a yellow powder (0.138 g, 50%). 1H NMR (400 MHz, CDCl3, δ): 7.34‒8.30 (br, aromatic backbone), 3.75‒4.28 (br, ‒CH2Br), 3.30‒3.62 (br, ‒CH2NHCO‒), 1.75‒2.27 (br, ‒CH2NHCH2‒), 1.57‒1.65 (s, ‒CH2CH2Br), 0.30‒1.65 (m, ‒(CH2)4CH2CH2Br, ‒(CH2)5CH2NH‒, ‒COOC(CH3)3). GPC: Mn = 4288, Mw = 8188, PDI = 1.91. Synthesis of PFBT-NH2. PFBT-Boc (0.10 g) was dissolved in CHCl3, and then a solution of trimethylamine (Me3N) in ethanol (30 wt%, 5 mL) was added. The reaction mixture was stirred for 48 h at room temperature. The solvent was evaporated and the product was dried under vacuum. A mixture of the polymers and trifluoroacetic acid (TFA) (4 mL) in dichloromethane (50 mL) was then stirred for 24 h at room temperature. The solvent was evaporated and residual TFA was removed under vacuum. The obtained polymer was dissolved in 50 mL of CHCl3, and triethylamine (Et3N) (4 mL) was added. The mixture was stirred for 24 h at room temperature. The solvent was removed by rotary evaporation and the product was dried under vacuum to yield a yellow powder (0.083 g, 100%). 1H NMR (400 MHz, CDCl3, δ): 7.39‒8.21 (br, aromatic backbone), 3.31‒3.72 (br, ‒N(CH3)3), 2.88‒3.23 (br, ‒CH2N(CH3)3), 2.13‒2.41 (br, ‒CH2NH2), 1.92‒2.11 (br, ‒CH2NHCH2‒), 0.92‒1.75 (m, ‒(CH2)5CH2N(CH3)3, ‒(CH2)5CH2NH‒). 6


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Synthesis of PFBT-PBA. PFBT-NH2 (50 mg) and 4-formylpenylboronic acid (0.0154 g) were dissolved in a mixture of CHCl3 (50 mL) and CH3OH (10 mL). The reaction mixture was then stirred for 24 h at 25 °C. The solvent was evaporated and the

obtained

solid

was

washed

with

methanol

to

remove

the

excess

4-formylpenylboronic acid, yielding a yellow powder (0.032 g, 52%). 1H NMR (400 MHz, CDCl3, δ): 7.55‒8.15 (br, aromatic rings), 7.38‒7.55 (br, fluorene ring), 7.30‒7.36 (s, ‒N=CH‒), 3.82‒3.98 (br, ‒CH2N=), 3.49‒3.78 (m, ‒N(CH3)3), 3.34‒3.49 (br, ‒CH2N(CH3)3), 2.44‒2.63 (br, ‒CH2NHCH2‒), 0.65‒0.93 (br, ‒(CH2)5CH2N(CH3)3, ‒(CH2)5CH2NH‒). Preparation of SA-Imprinted NPs, Polymer/SA NPs and Unmodified NPs. SA-imprinted NPs, polymer/SA NPs and unmodified NPs were prepared by a reprecipitation method.57, 58 Briefly, the polymer PFBT-PBA was dissolved in THF in the dark at a concentration of 2 mg/mL. The polymer solution was filtered through 0.22 µm poly(tetrafluoroethene) (PTFE) filters. Sialic acid (SA) was dissolved in methanol at a concentration of 0.87 mg/mL. Then, 500 µL of the PFBT-PBA solution in THF was added into 500 µL of the SA methanol solution (PFBT-PBA and SA at a molar ratio of 1:1) and stirred for 12 h, to ensure that the SA molecules were covalently linked to the phenylboronic acid groups of the PFBT-PBA side chain. For the preparation of the polymer/SA NPs, 250 µL of the mixture obtained was rapidly injected into 5 mL of water and subjected to ultrasonication for 3 min. In the case of SA-imprinted NPs, 250 µL of the PFBT-PBA/SA solution was rapidly injected into 5 mL of 0.1 M acetic acid solution (HAc, pH = 2.88) and subjected to ultrasonication 7



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for 3 min. After incubation for three hours to remove the SA template, SA-imprinted NPs were obtained by dialysis for 6 h. For the preparation of unmodified NPs, a 250 µL portion of the 2 mg/mL PFBT-PBA in THF solution was diluted with an equal amount of THF, confirming that the concentration of unmodified NPs would be the same as the concentration of the SA-imprinted NPs. We note that the concentrations of the NPs refer to the molar concentrations of the repeating units of polymer PFBT-PBA. Then, 250 µL of diluted PFBT-PBA THF solution was rapidly injected into 5 mL of 0.1 M HAc solution and subjected to ultrasonication for 3 min. After incubation for three hours, the unmodified NPs were collected by dialysis for 6 h. Cytotoxicity Assay by MTT Method. The cytotoxicity of SA-imprinted NPs and unmodified NPs for human prostate carcinoma cell line (DU 145) and cervical cancer cell

line

(HeLa)

was

evaluated

by

the

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. DU 145 cells were seeded into 96-well plates in Roswell Park Memorial Institute-1640 (RPMI-1640) medium containing 10% (v/v) fetal bovine serum (FBS). HeLa cells were seeded into 96-well plates in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) FBS. After incubation for 24 h at 37 °C in a 5% CO2 humidified atmosphere, DU 145 and HeLa cells were treated with various concentrations of SA-imprinted NPs or unmodified NPs (0‒14 µM) at 37 °C for 24 h. The concentrations of the SA-imprinted NPs and unmodified NPs were calculated from the initial concentration of PFBT-PBA molecules used in the synthesis. After that, the 8


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medium was poured out, and 100 µL of freshly prepared MTT (1 mg/mL) in phosphate buffered saline (PBS) solution was added to each well and further incubated for 4 h. The supernatant was removed, and the cells in the wells were lysed with 100 µL of DMSO. The plate was gently shaken for 5 min, and then the absorbance values of purple formazan were recorded at 570 nm using a Spectra MAX 340PC plate reader. Cell Imaging Assay. DU 145 and HeLa cells were seeded on 35 × 35 mm2 culture plates. DU l45 and HeLa cells were grown in RPMI-1640 medium containing 10% (v/v) FBS and DMEM containing 10% (v/v) FBS, respectively. Then the plates were incubated for 24 h at 37 °C in a 5% CO2 humidified atmosphere. A 100 µL portion of the SA-imprinted NPs, unmodified NPs, or polymer/SA NPs was added into 900 µL of medium containing DU 145 or HeLa cells in a 35 × 35 mm2 plate (final concentration of PFBT-PBA = 7 µM). After incubation at 37 °C for 4 h or 24 h, the medium was removed, and the cells were washed with PBS buffer (pH = 7.4). To observe the specimens, an Olympus FV1000-IX81 confocal laser scanning microscope with an oil immersion lens (100 × magnification, NA 1.4) was used. The excitation wavelength was set at 405 nm, and the fluorescence signals were collected in the range of 500‒550 nm. Characterization. The 1H NMR spectra were recorded on a 400 MHz AC Bruker spectrometer. Ultraviolet-visible (UV-vis) absorption and photoluminescence (PL) emission spectra were measured on a Hitachi U-3900H spectrophotometer and a Hitachi F-7000 fluorescence spectrophotometer, respectively. The hydrodynamic 9



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diameters of NPs in aqueous solution were determined by dynamic light scattering with a Malvern Zetasizer Nano ZS90 at room temperature. The molecular weight of compound 2 was measured on a Bruker Daltonics BIFLEX III MALDI-TOF analyzer in matrix-assisted laser desorption ionization time-of-flight (MALDI) mode. Gel permeation chromatography (GPC) analysis was performed on a Waters Styragel system using polystyrene as the calibration standard and tetrahydrofuran as eluent. Fourier transform infrared (FT-IR) spectra were recorded on a Bruker Hyperion 2000 spectrometer. Fluorescence microscopy images of the NPs were obtained with an Olympus FV1000-IX81 confocal laser scanning microscope through a 100× objective, with 325–375 nm excitation produced by a 100 W mercury lamp light source. Fluorescence images of cells were recorded with a confocal laser scanning microscope (CLSM; Olympus FV1000-IX81). Cell viability was detected with a Spectra MAX 340PC plate reader. The size and morphology of the NPs were measured with a high resolution field emission scanning electron microscope (HRSEM, JEOL JSM-7401F, accelerating voltage: 3.0 kV). The SEM samples were prepared by dropping a portion of the NP suspension onto silicon wafers and allowing the sample to dry naturally at room temperature. The absolute fluorescence quantum yields

were

determined

using

a

spectrofluorometer

(NanologR

FluoroLog-3-2-iHR320, Horiba Jobin Yvon) equipped with an integrating sphere. The excitation wavelength was set at 410 nm. The scattering spectral range was set from 405 nm to 415 nm, and the emission spectral range was from 430 to 800 nm. Results and Discussions 10


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Scheme 1 illustrates the design concept, preparation method, and operating principle of the SA-imprinted NPs for use as fluorescent probes. The fluorescent PFBT was selected as the macromolecular fluorescent probe. This polymer is widely used for fabrication of CPNPs owing to its strong fluorescence and ease of modification.59, 60 The PBA group is considered a classic binding site for cis-diol-containing compounds through the formation of stable cyclic esters. Although PBA acts as a recognition site for glucose, it has also been reported to combine with SA molecules, which are a glycan that is over-expressed on the surface of some cancer cells, such as DU 145 cells (a human prostate cancer cell).61 The binding behavior of PBA toward glucose and SA depends on the pH of the surrounding solution.62 Notably, even NPs that bear PBA groups on their surfaces fail to discriminate between cancer cells with and without SA groups.48 This is likely caused by the lower differential binding capacity of PBA for SA over other monosaccharides at physiological pH.47 Here, we choose the PBA group as a functional component to recognize SA molecules through a molecule-imprinting technique. The synthesis route of SA-imprinted NPs featured two main steps, namely, reprecipitation and template removal processes. Briefly, PFBT-PBA polymer and SA molecules were dissolved together in a good solvent to form covalent bonds with each other by formation of cyclic esters. After injection into water, a poor solvent, polymer NPs were immediately formed owing to the hydrophobic properties of the polymer. This step produced PFBT-PBA NPs embedded with SA molecules. To remove the SA molecules, the polymer NP dispersion was adjusted to an acidic pH range. Most of the SA was removed from the 11



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NPs’ surface by cleavage of the cyclic ester, and these free SA molecules were subsequently removed by dialysis. The SA-imprinted NPs featured pores, which had shapes and binding site arrangements that may be expected to exhibit recognition specificity toward SA or cancer cells with SA over-expression. To date, most of molecule imprinting polymers (MIP) were realized by the polymerization process.53 As far as we know, there was no report on the preparation of the molecule imprinted CPNPs. The polymerization process often brings some problems to the template molecules, especially fragile biomolecules, like the additional initiator and the elevated temperature which is necessary to the polymerization. These issues can be avoided in this work, where a simple one-step reprecipitation method is employed for the molecule imprinting. Furthermore, the rigidity of the conjugated backbones is also favorable for keeping the holes’ morphologies after the template molecules’ leaving, compared to other soft monomers/polymers.

Scheme 1. Schematic illustration of the preparation of SA-imprinted NPs and mechanism of their selectivity towards cancer cells. 12


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Synthesis and Characterization of PFBT-PBA. The chemical structure and synthesis of PFBT-PBA are illustrated in Scheme 2. The fluorescent π-conjugated backbone was composed of alternating fluorene and benzothiadiazole (BT) units. Fluorene units have strong blue photoluminescence and can be easily modified at the C9 position, and the BT unit has the ability to shift the fluorescence emission peak toward longer wavelengths.63, 64 Thus, conjugated polymers composed of fluorene and BT units with a molar ratio of 1:1 are often used as fluorescent probes for bio-sensing and bio-imaging.65,

66

The Boc-protected polymer precursor PFBT-Boc was

synthesized by a Suzuki cross coupling reaction with a Pd catalyst. After the deprotection reaction, PFBT-NH2 was obtained, which features reactive amino groups on side chains that allow for further functionalization of the polymer. A Schiff base catalyzed reaction between these amino groups and the aldehyde group of 4-formylpenylboronic acid, allowed the introduction of the PBA groups onto the side chains. The molar ratio of fluorene units modified with a PBA group was 50%, and the other units were transformed into quaternary ammonium salts. These positively charged groups helped to increase the hydrophilicity of the polymer, which is necessary for biological applications, and also improved electrostatic interactions with negatively charged SA molecules and cell membranes. Notably, although hydrophilic quaternary ammonium salts were introduced, the polymer PFBT-PBA did not dissolve in water due to its strongly hydrophobic π-conjugated backbone. Thus, the polymer remained hydrophobic enough to prepare polymer NPs by a reprecipitation method. 13



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Scheme 2. Synthesis of PFBT-PBA. The photophysical properties of the PFBT-PBA polymer were investigated in THF, which is a good solvent to dissolve this polymer. Its UV-vis absorption spectrum in Figure 1 displayed two absorption bands at 315 and 425 nm, which were assigned to typical absorptions of the fluorene and BT components, respectively. Upon excitation at 410 nm, the fluorescence spectrum of PFBT-PBA showed a PL emission peak at approximately 535 nm. This indicated that the introduction of BT units into the polymer red shifted the normally blue emission of PF to a green emission. A large Stokes shift of 110 nm was found, which is attractive for bioimaging applications because of the low fluorescent background.

14


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Figure 1. Normalized UV-vis absorption and fluorescence emission spectra of PFBT-PBA in THF. The excitation wavelength is 410 nm. Characterization of SA-Imprinted NPs. The SA-imprinted NPs were prepared in two steps, namely, reprecipitation to form polymer NPs and removal of the template, as illustrated in Scheme 1. Full details of the preparation process are described in the experimental section. To better investigate the effects of molecular imprinting, two control samples were also fabricated including the NPs without removing template SA molecules (denoted as polymer/SA NPs), and pure PFBT-PBA polymer NPs (named as unmodified NPs). The procedures for preparing the polymer/SA NPs were the same as those used to prepare the SA-imprinted NPs except for the pH adjusting procedure and followed dialysis. For the preparations of unmodified NPs, the procedure was similar to that of SA-imprinted NPs, without the introduction of SA molecules before the reprecipitation process. Other experimental conditions were maintained the same as those for the SA-imprinted NPs, including the pH adjusting process. Considering that PFBT-PBA polymer is positively charged and SA molecules carry negative charges, the variation in zeta potential might reveal the introduction and remove of the template molecules. Figure 2a shows the zeta potential values of the 15



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as-obtained three kinds of NPs. A high zeta potential of +61±2 mV was observed for the unmodified NPs, which can be resonably explained by the aboundant quaternary ammonium salts on PFBT-PBA polymers. After binding with negatively charged SA molecules, the polymer/SA NPs showed an obvious reduction in zeta potential values to +18±3 mV. Once the SA molecules were removed, a recovery in potential was found for SA-imprinted NPs, with a value of +48±2 mV. These observation clearly demonstrated the sucessful binding and leaving of the SA template molecules in the molecular imprinting process. It was noteworthy that the zeta potential of SA-imprinted NPs was not as high as that of unmodified NPs, possibly due to the presence of the residual SA molecules. The hydrodynamic size of the three kinds of NPs were also examined by dyanmic light scattering (DLS) technique, and the results are shown in Figure 2b. All the samples showed a unimodal and narrow distribution, indicating the good dispersity of the obtained NPs. The mean hydrodyanmic diameters were 41±5 nm, 57±3 nm, and 35±5 nm, for the unmodified NPs, polymer/SA NPs, and SA-imprinted NPs, respectively. The fluctuation in sizes was also probably caused by the binding and leaving of the SA molecules. To further explore the leaving process of SA molecules, the size change of the polymer/SA NPs druing the pH-adjusting process was also monitored by DLS, as shown in Figure 2c. After the polymer/SA NPs was injected into the acid medium, its average size rapidly decreased in 15 min and finally reached to a plateau in the following 30 min. This observation revealed a quick dissociation of SA molecules from the nanocomposites. It is well known that the stability of NPs is 16


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also an important factor for biological applications. To evaluate the stability of the SA-imprinted NPs, the aqueous dispersions were stored for 10 days, and the hydrodynamic diameters of the NPs were measured by DLS (Figure 2d). These results showed only a slight fluctuation in the mean NP size, and no obvious aggregation effects during storage, indicating that the obtained SA-imprinted NPs had good stability. Figure 2e and f show the SEM images of SA-imprinted NPs and unmodified NPs. It can be seen that the unmodified NPs was approximately spherical with a mean size of 28±6 nm (Figure 2e), which was comparable to the results obtained from DLS measurements. The SA-imprinted NPs also displayed spherical shapes with a mean size of 25±5 nm (Figure 2f). These results indicated that the mean size and shape of the SA-imprinted NPs were similar to those of the unmodified NPs. Furthermore, the small particle dimensions, uniform size distribution and good stability are all favorable characteristics for cell imaging applications.

17



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Figure 2. (a) Zeta potential and (b) DLS distribution of unmodified NPs, SA-imprinted NPs, and polymer/SA NPs. (c) Variation in average size of polymer/SA NPs in 0.1 M acetic acid medium within 30 min; (d) Colloidal stability of SA-imprinted NPs stored in aqueous solution for 10 days revealed by DLS result. SEM images of (e) unmodified NPs and (f) SA-imprinted NPs. Figure 3a and b show the UV-vis absorption and fluorescence spectra of the SA-imprinted NPs and unmodified NPs in an aqueous dispersion. Both dispersions showed two absorption bands centered at 319 and 435 nm, and a single emission peak around 538 nm, when excited at 410 nm. Compared to the PFBT-PBA polymer in 18


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THF (Figure 1), 4 nm and 10 nm red-shift were observed for the absorption band of the two fluorescent parts in these NPs, respectively. In addition, their emission peak displayed a 3 nm red-shift compared with the polymer. In this work, the red-shift phenomenon is not significant maybe ascribed to the small molecular weight of synthesized PFBT-PBA polymer. Its conjugated chain was not changed largely after it was transferred from well solved to aggregated state. Figure 3a shows the absorption intensity of the SA-imprinted NPs and unmodified NPs was nearly identical because the concentrations of PFBT-PBA polymer were maintained during their synthesis. However, the fluorescence emission intensity of the SA-imprinted NPs was stronger than that of unmodified NPs, i.e., the SA-imprinted NPs exhibited brighter fluorescence emission than that of the unmodified NPs at the same absorption intensity. The absolute quantum yields of the SA-imprinted NPs and unmodified NPs dispersions were determined to be 14.4% and 13.0%, respectively. The improved emission properties may be explained by that the presence of pores in the SA-imprinted NPs that formed after removal of template molecules. We know that self-quenching of fluorescence occurs as the solvation of a polymer is lowered and it forms aggregates. The pores in the SA-imprinted NPs reduced the degree of aggregation of PFBT-PBA polymers, as well as the aggregation-induced self-quenching phenomenon. Thus, the SA-imprinted NPs showed an enhanced fluorescence compared with that of the unmodified NPs loaded with same amount of PFBT-PBA polymers. Fluorescence microscopy was used to visualize the emission of the NPs in aqueous 19



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solution when irradiated by UV light (325‒375 nm) produced by a mercury lamp. The obtained fluorescence microscope images indicated that both the SA-imprinted NPs and unmodified NPs were uniformly dispersed and showed bright green emission (Figure 3c and d). Thus, the SA-imprinted NPs and unmodified NPs showed potential for use as probes in cell imaging.

Figure 3. (a) UV−vis absorption spectra and (b) fluorescence emission spectra of SA-imprinted NPs and unmodified NPs in aqueous solution. The excitation wavelength was 410 nm. Fluorescence microscopy images of (c) SA-imprinted NPs and (d) unmodified NPs dispersed in aqueous solution. To confirm that SA-imprinted NPs were successfully prepared, we used FT-IR spectroscopy to confirm the introduction and removal of SA molecules. The FT-IR spectra of SA, the unmodified NPs, polymer/SA NPs, and SA-imprinted NPs are shown in Figure 4. The FT-IR spectrum of polymer/SA NPs showed obvious speaks at 1735 cm−1 and 1380 cm−1, which were not observed in the unmodified NPs. These 20


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peaks were respectively attributed to C=O stretching vibrations of acetyl and C‒O stretching vibrations assigned to SA. This result demonstrated that SA was successfully linked to the polymer PFBT-PBA. The FT-IR spectrum of the SA-imprinted NPs showed an obvious reduction in absorption intensity at 1735 and 1380 cm−1 compared with that of the polymer/SA NPs. This finding indicated that most of the SA was successfully removed from the NPs. The remaining signals revealed the presence of some residual SA molecules, which were embedded deep inside the polymer NPs and less affected by changes in the environment. Together these results clearly show the successful linking and removal of the SA template molecules and suggest that SA-imprinted NPs were successfully prepared.

Figure 4. FT-IR spectra of SA, unmodified NPs, polymer/SA NPs, and SA-imprinted NPs. Cellular Imaging and Cell Viability Assay. To determine the selective binding behavior of the as-formed SA-imprinted NPs, two cell lines with different expression levels of SA glycans were chosen, DU 145 and HeLa cells. DU 145 cells are human prostate carcinoma cell line that are characterized by an overexpression of SA on the 21



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cell surface.61 HeLa cells, human cervical cancer cells, have a relatively lower level of SA expression on their sufaces.67 First, the two cell lines were cultured with the SA-imprinted NPs, unmodified NPs, or polymer/SA NPs at 37 °C for 4 h. The culture media was removed and washed with PBS buffer, and the cells were observed under a confocal laser scanning microscope (CLSM). The excitation laser wavelength was set at 405 nm. From Figure 5a, the CLSM images of DU 145 cells showed bright fluorescence with most of the signals derived from the cell membrane and a small number of signals from the cytoplasm. This phenomenon suggested that some SA-imprinted NPs entered the cell cytoplasm and other NPs were attached to the cell membrane owing to the affinity of the SA-imprinted NPs for SA. Conversely, the HeLa cells cultured with SA-imprinted NPs showed almost no fluorescence signals, indicating that the NPs were not taken up by the HeLa cells, which have lower expression of SA. These results showed that the SA-imprinted NPs selectively bound to the DU 145 cells. For the case of the unmodified NPs, fluorescence signals were observed both for DU 145 and HeLa cells (Figure 5b), indicating that the unmodified NPs failed to recognize the SA groups of the cancer cell surfaces. Figure 5c indicated that only a very weak fluorescence was observed for the DU145 cells incubated with polymer/SA NPs, while no any signal was found for HeLa cells. This phenomenon can be explained that most of the binding sites were taken up by the SA molecules, which affected the affinity of polymer/SA NPs towards the two cells.

22


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Figure 5. Confocal laser scanning microscopy (CLSM) images of DU 145 and HeLa cells incubated with (a) SA-imprinted NPs, (b) unmodified NPs and (c) polymer/SA NPs for 4 h at 37 °C. It has been reported that most NPs of an appropriate size (generally smaller than 200 nm) can enter into cells over time through an endocytosis process.68 To investigate if the SA-imprinted NPs would enter into HeLa cells over an extended time, with a loss of selectivity, we prolonged the incubation time to 24 h. These results are shown in Figure 6a, and again we only observed fluorescence from DU 145 cells with no signal from HeLa cells. However, over the longer incubation time more of the SA-imprinted NPs appeared to be internalized into the cytoplasm of DU 145 cells. In a control experiment for 24 h, the unmodified NPs again failed to recognize the SA expressing cells (Figure 6b). After 24 h incubation, the entrance of the polymer/SA NPs into DU 145 cells increased (Figure 6c), but still lower than that of SA-imprinted NPs. The polymer/SA NPs cannot enter the HeLa cells even after 24 h incubation, indicating it a weaker selectivity compared to SA-imprinted NPs. The above results indicate that SA-imprinted NPs could differentiate between DU 145 and 23



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HeLa cells even at longer incubation times of 24 h.

Figure 6. CLSM images of DU 145 and HeLa cells incubated with (a) SA-imprinted NPs, (b) unmodified NPs and (c) polymer/SA NPs for 24 h at 37 °C. For biological applications, cytotoxicity is a key parameter for evaluation of a probe’s potential effectiveness. Thus, we investigated the cytotoxicity of SA-imprinted

NPs

and

unmodified

NPs

using

a

standard

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. DU 145 and HeLa cells were incubated with different amounts of SA-imprinted NPs or unmodified NPs at 37 °C for 24 h. Figure 7 shows that neither of the NPs harmed either of the two cell lines. Nearly 100% of cells remained alive in the tested concentration range. These results indicate that SA-imprinted NPs have low enough toxicity and suitable biocompability for use as probes in living cell imaging.

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Figure 7. Cell viability results after incubation of DU 145 and HeLa cells with different concentrations of (a) SA-imprinted NPs and (b) unmodified NPs. The amounts of NPs are expressed as the concentration of conjugated polymer PFBT-PBA. Conclusions In summary, a novel fluorescent conjugated polymer NP cell imaging probe was successfully prepared. A classic conjugated polymer, PFBT, was modified with PBA groups as binding sites for SA molecules. The formation of a covalent bond between PBA and SA and a facile reprecipitation process resulted in SA molecules embedded in the conjugated polymer NPs. The SA molecules were then easily removed from the NP surfaces by adjusting the surrounding pH followed by dialysis. The insertion and removal of the SA template was clearly shown by FT-IR spectra. The imprinted NPs showed a uniform size of around 30 nm and a stronger fluorescence than that of the unmodified polymer NPs. A cell imaging assay clearly indicated that SA-imprinted NPs could selectively bind with SA over-expressed in DU 145 cancer cells. Thus, the small and uniform size, bright fluorescence, and low cytotoxicity of these SA-imprinted NPs indicate that these materials would be good potential for use as a suitable fluorescent probe for targeted cancer cell imaging. 25



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Author Information Corresponding Author *E-mail: [email protected] (L.L.), [email protected] (Q.C.) Notes The authors declare no competing financial interest.

Acknowledgments This work was supported by the National Natural Science Foundation of China (51503015, 51373022), the State Key Laboratory for Advanced Metals and Materials (2016Z-08) and the Fundamental Research Funds for the Central Universities (FRF-TP-15-003A1).

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Preparation of Sialic Acid-Imprinted Fluorescent Conjugated

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