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Preparation of conjugated polymer nanoparticles with white emission and their application for cell imaging
Accepted Manuscript Title: Preparation of conjugated polymer nanoparticles with white emission and their application for cell imaging Authors: Ronghua Liu, Qianling Cui, Yu Yang, Rui Peng, Lidong Li PII: DOI: Reference:
S1010-6030(17)30772-4 http://dx.doi.org/doi:10.1016/j.jphotochem.2017.07.042 JPC 10768
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date: Revised date: Accepted date:
4-6-2017 18-7-2017 31-7-2017
Please cite this article as: Ronghua Liu, Qianling Cui, Yu Yang, Rui Peng, Lidong Li, Preparation of conjugated polymer nanoparticles with white emission and their application for cell imaging, Journal of Photochemistry and Photobiology A: Chemistryhttp://dx.doi.org/10.1016/j.jphotochem.2017.07.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation of conjugated polymer nanoparticles with white emission and their application for cell imaging
Ronghua Liu, Qianling Cui,* Yu Yang, Rui Peng, Lidong Li*
School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China
Graphical abstract
Highlights
A conjugated polymer bearing blue-, green-, and red-emitting components was synthesized.
White-emissive polymer nanoparticles were prepared successfully.
These polymer nanoparticles are efficient probe for cellular imaging.
Abstract: Conjugated polymer nanoparticles have been regarded as promising fluorescent material for biological application, due to their high emission efficiency, 1
good photostability and low cytotoxicity. In this contribution, we synthesized a conjugated polymer which integrates blue-, green- and red-emitting components into the backbone in an appropriate molar ratio. After being transferred from solution to nanoparticles, its fluorescence color turns from blue to pink with enhanced fluorescence quantum yield. After incorporation of an amphiphilic polymer as matrix, white-emissive polymer nanoparticles are obtained as a result of optimized energy transfer degree among the three components. Finally, their good performance on cell imaging are demonstrated by incubation with HeLa cells, after being proved to be safe for biological application.
Keywords: fluorescence, conjugated polymer nanoparticles, cell imaging, white emission, multi-color
1. Introduction In the past decades, conjugated polymers have attracted numerous interests because of their promising photophysical properties, such as strong light harvesting ability, high emission efficiency, good photostability, molecular wire effect and low cytotoxicity [1]. These features make them suitable for wide application fields including solar cells, polymer light-emitting diode, field effect transistor, chemical or biological sensing, cellular imaging, photodynamic therapy and antibacterial [2-9]. For biological application, it requires the conjugated polymer processable in aqueous medium. To this end, water-soluble conjugated polymer modified with hydrophilic moieties and 2
conjugated polymer nanoparticles are the two commonly used solutions [10-13]. Compared with water-soluble conjugated polymer which requires complicated chemical modification and elaborate purification, the preparation of conjugated polymer nanoparticles is much easier. Reprecipitation technique is the most often employed method to fabricate the conjugated polymer nanoparticles, which is simple, reliable, and more importantly, no need of assistance from additional surfactants [14]. In recent years, conjugated polymer nanoparticles have become among the most attractive fluorescent materials for biological application [11]. For instance, by using the conjugated polymer nanoparticles as the carrier, our group realized the gene delivery into the cells and the whole process can be monitored by variation in fluorescence signals [15]. Taking advantage of the molecule imprint technique, targeted cancer cell imaging was achieved by sialic acid-imprinted conjugated polymer nanoparticles, which can selectively bind with DU 145 cell line having sialic acid over-expressed surfaces [16]. Very recently, conjugated polymer nanoparticles with extremely small size had been reported as efficient photoblinking probes for super-resolution imaging of living cells [17]. However, there are still some limitations of conjugated polymer nanoparticles, most important of which is the significant aggregation caused quenching problem possibly as a result of the aggregation-induced depopulation of the excitons [18,19]. Thus, decrease in emission efficiency is often observed for conjugated polymer when it is transferred from solvated state to solid state like nanoparticles or films [20]. To resolve this problem, organic or inorganic matrix have been chosen to disperse the 3
conjugated polymer which could reduce their aggregation degree and thereby could decrease the quenching effect [21-23]. An alternative way is to introduce aggregation-induced emission (AIE) structures into the conjugated backbone, which has an enhanced photoluminescence at solid state, possibly due to the restricted molecular rotation and increased radiative decay rate [24-27]. On the other hand, although a variety of conjugated polymer nanoparticles with multi-color emission have been developed, which are useful for fluorescent coding and multi-channel cellular imaging [28,29]. Nevertheless, fabrication on the white-emissive conjugated polymer nanoparticles has been merely reported. In this work, a conjugated polymer integrating three parts with blue, green and red emission into its backbone was designed and synthesized. Fluorescence resonance energy transfer (FRET) could take place between these three parts, which could be facilely adjusted to acquire the emission color of the whole polymer. The conjugated polymer solution displayed intense blue fluorescence, while its nanoparticles gave rise to pink signals, which turned to white emission after introduction of a polymer matrix. Their photophysical behaviors and further understanding on the FRET efficiency were explored and discussed. Finally, the toxicity of the white-emissive conjugated polymer nanoparticles against HeLa cells were evaluated, after which their good performance on cellular imaging via three channels was demonstrated.
2. Experimental Section 2.1. Materials 4
1,2-Bis(4-bromophenyl)-1,2-diphenylethene (TPE-DBr) was synthesized according to literature procedures [30]. Tetrakis-(triphenylphosphine)palladium(0) [Pd(PPh3)4], zinc powder, and amphiphilic functional polymer poly(styrene-co-maleic anhydride) (PSMA, cumene terminated, average Mn ~ 1,700) were purchased from Sigma-Aldrich and used as received. 9,9-Dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol)ester (monomer A), 9,9-dioctyl-2,7-dibromofluorene (monomer B), and 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole (DBT-Br) were purchased from Beijing Allmers Chemical S&T Co., Ltd. Titanium tetrachloride (TiCl4) and 4-bromobenzophenone 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. Ultrapure Millipore water (18.6 MΩ·cm) was used throughout the experiments. 2.1.1. Synthesis of TPE-DBr 4-Bromobenzophenone (5.00 g, 19.1 mmol) and Zn powder (2.50 g, 38.3 mmol) were placed into a 250 mL two-necked round-bottom flask. 150 mL of THF was added to the flask and the solution was thoroughly degassed. The mixture was cooled to -78 °C and TiCl4 (5.32 mL, 38.3 mmol) was added dropwise by a syringe. The mixture was slowly warmed to room temperature. After stirring for 30 min, the reaction mixture was then heated to reflux under an argon atmosphere for 24 h. After cooling to room temperature, the mixture was quenched with saturated NaHCO3 solution and filtered. The filtrate was evaporated to remove solvent, and the mixture 5
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 petroleum ether as an eluent to afford 7.51 g of white solid with a molar yield of 80%. 1H NMR (400 MHz, CDCl3, δ): 7.10 (d, 7H), 6.97 (s, 5H), 6.92‒6.44 (m, 6H). 13C NMR (100 MHz, CDCl3, δ): 142.92, 142.84, 142.40, 142.30, 140.32, 133.07, 132.70, 131.39, 131.27, 131.07, 131.00, 130.95, 130.74, 128.22, 128.03, 127.86, 127.63, 127.14, 127.04, 126.75, 126.66, 120.80, 120.66. MS (MALDI-TOF) m/z: M+ calcd: 490.23. Found: M+ = 490.0. 2.1.2. Synthesis of PFTD (PF0.95TPE0.0495DBT0.0005) Monomer A (0.30 g, 0.54 mmol), monomer B (0.26 g, 0.48 mmol), TPE-DBr (0.026 g, 0.053 mmol), and DBT-Br (492 μL, 0.5 mg/mL, 0.00054 mmol) were dissolved in toluene and placed into a 100 mL two-necked round-bottom flask. 40 mL of toluene and 10 mL of potassium carbonate aqueous solution (2.0 M) was added to the flask. After the solution was degassed with argon for 30 min, the catalyst Pd(PPh3)4 was added. The reaction was stirred at 85 °C for 48 h under an argon atmosphere. The 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 5 mL of chloroform, and the polymer was then precipitated by 150 mL of methanol. 0.228 g of light pink powder was collected and the molar yield was estimated to be about 55%. 1H NMR (400 MHz, CDCl3, δ): 8.00‒7.56 (m, aromatic backbone), 2.30‒1.88 (br, ‒CCH2‒), 1.26‒1.02 (br, 6
‒CCH2(CH2)6‒), 0.95‒0.81 (br, ‒CH2CH3). EA calculated: C, 89.82%; H, 10.16%. Found: C, 88.64%; H, 10.25%. GPC: Mn = 18 204, Mw = 37 146, PDI = 2.04. 2.2. Preparation of PFTD NPs and PFTD/PSMA NPs PFTD NPs and PFTD/PSMA NPs were prepared by a reprecipitation method reported in literature [15, 31]. Briefly, the polymer PFTD and PSMA were separately dissolved in THF to make stock solution at a concentration of 1 mg/mL. The polymer solution was filtered through 0.22 μm poly(tetrafluoroethene) (PTFE) filters. Then, a 200 μL portion of the 1 mg/mL PFTD solution in THF was diluted to 4 mL, obtaining 50 μg/mL PFTD solution in THF. Then 1 mL of 50 μg/mL PFTD solution in THF was rapidly injected into 10 mL of water and subjected to ultrasonication for 3 min. THF was removed by nitrogen stripping. The obtained nanoparticle was denoted as PFTD NPs. For the preparation of PFTD/PSMA NPs, a 200 μL portion of the 1 mg/mL PFTD solution in THF was mixed with 200 μL, or 1400 μL, or 2000 μL of 1 mg/mL PSMA solution in THF, and the above solution was diluted to 4 mL. Then 1 mL of the PFTD/PSMA solution was rapidly injected into 10 mL of water and subjected to ultrasonication for 3 min. THF was removed by nitrogen stripping. According to the different molar ratio, the obtained nanoparticles were named PFTD/PSMA 1:1 NPs, PFTD/PSMA 1:7 NPs and PFTD/PSMA 1:10 NPs, respectively. 2.3. Cytotoxicity Assay by MTT Method The cytotoxicity of PFTD/PSMA 1:7 NPs for cervical cancer cell line (HeLa) was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 7
method. HeLa cells were seeded into 96-well plates in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS). After incubation for 24 h at 37 °C in a 5% CO2 humidified atmosphere, HeLa cells were treated with various concentrations of PFTD/PSMA 1:7 NPs (0‒2.6 μM) at 37 °C for 24 h. The concentrations of PFTD/PSMA 1:7 NPs were calculated from the initial concentration of PFTD molecules used in the synthesis. Thereafter, the 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. 2.4. Cell Imaging Assay HeLa cells were seeded on 35 × 35 mm2 culture plates. HeLa cells were grown in DMEM containing 10% (v/v) FBS. Then the plates were incubated for 12 h at 37 °C in a 5% CO2 humidified atmosphere. A 100 μL portion of PFTD/PSMA 1:7 NPs was added into 900 μL of medium containing HeLa cells in a 35 × 35 mm2 plate (final concentration of PFTD = 1.3 μM). After incubation at 37 °C for 12 h, the medium was removed, and the cells were washed with PBS buffer (pH = 7.4) for three times. 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 separately through three channels, including the range from 425 nm to 485 nm (blue 8
channel), 490 nm to 560 nm (green channel), and 575 nm to 675 nm (red channel). 2.5. Characterization The 1H NMR spectra and 13C NMR were recorded on 400 MHz and 100 MHz AC Bruker spectrometer, respectively. Ultraviolet−visible (UV−vis) absorption spectra were measured on a Hitachi U-3900H spectrophotometer. Fluorescence emission spectra were recorded using a Hitachi F-7000 fluorescence spectrophotometer. The molecular weight was performed on a Bruker Daltonics BIFLEX III MALDI-TOF analyzer in matrix-assisted laser desorption ionization time-of-flight (MALDI) mode. Element analysis (EA) was conducted on a FLASH EA 1112 element analyzer. Gel permeation chromatography (GPC) analysis was measured on a Waters Styragel system using polystyrene as the calibration standard and THF as eluent. The zeta potentials of nanoparticles dispersions were determined on Malvern Zeta-sizer Nano ZS90. 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 field emission scanning electron microscope (SEM, ZEISS SUPRA55). The absolute fluorescence quantum yields were determined using a spectrofluorometer (NanologR FluoroLog-3-2-iHR320, Horiba Jobin Yvon) equipped with an integrating sphere. Time-domain lifetime measurements were performed using an ultrafast lifetime spectrofluorometer (Delta flex) according to time-correlated single-photon counting technique. For the samples of PFTD solution in THF, PFTD NPs, and PFTD/PSMA NPs, the excitation wavelength was set at 380 nm with 9
scattering spectral range at 375-385 nm, and emission spectral range for PF units, TPE units, and DBT units were calculated from 395-484 nm, 484-565 nm, and 565-750 nm, respectively. 3. Results and Discussions 3.1. Design concept and synthesis of PFTD The chemical structure and synthetic route of the conjugated polymer are shown in Scheme 1. In the conjugated polymer backbone, fluorene (F), tetraphenylethene (TPE), and 1,4-dithienylbenzothiadiazole (DBT) were employed as the blue, green, and red-emitting units, respectively. Their molar ratio is needed to be elaborately adjusted so as to generate appropriate fluorescence resonance energy transfer (FRET) efficiency and thereby wanted emission behavior. Blue-emitting polyfluorene (PF) are selected as the energy donor, in virtue of its high emission efficiency and easy modification on its C-9 position with long alkyl groups to increase the polymer’s solubility and operability [32-34]. TPE is a typical aggregation-induced emission (AIE) fluorophore, which is widely used to build up various sorts of solid-state emissive materials showing enhanced emission [35,36]. Herein, TPE acts as the green-emitting units, and more importantly, plays a crucial role in reducing the aggregation-induced quenching problem. In this designed polymer, DBT with a narrow band-gap serves as the red-emitting component and energy acceptor for FRET. It is well known that efficient FRET requires spectroscopic overlap and suitable distance (less than 10 nm) between donor and acceptor. In this case, aggregation of the polymer chains is supposed to lead to efficient intermolecular FRET via several 10
paths, including PF→TPE, PF→DBT, and TPE→DBT. As shown in Scheme 1, the polymer was synthesized by Suzuki cross coupling reaction using fluorene monomer A and B, TPE-Br, and DBT-Br, in the presence of Pd catalyst. The initial molar ratio of fluorene, TPE, and DBT was set as 95 %, 4.95 % and
0.05
%,
respectively.
The
obtained
polymer
could
be
named
as
PF0.95TPE0.0495DBT0.0005 based on the initial molar ratio of each component precursor, which was simply denoted as PFTD in this work. Its chemical structure was characterized by 1H NMR and element analysis (EA). The EA results confirmed that the final composition of PFTD was close to the initial molar ratio of each component. The polymer’s average molecular weight (Mw) was 37 146 g mol-1 with a polydispersity index (PDI) of 2.04, determined by GPC. Based on these results, we can speculate that less than one DBT unit on each PFTD chain, which means the polymer might be a mixture of polymer PF-TPE and PF-TPE-DBT. The obtained polymer dissolved well in common organic solvents such as chloroform, dichloromethane, tetrahydrofuran (THF), and N,N-dimethylformamide (DMF), but not in methanol and acetone. 3.2. Photophysical properties of PFTD The photophysical properties of the as-prepared PFTD polymer were firtstly determined in THF solution, a good solvent for it. It has been reported that TPE has a chacteristic absorption band of 280-350 nm and a cyan emission around 470 nm, which would had a red shift after conjugation with fluorene units [35, 36]. For PF, it always possesses an intense absorption band around 380 nm, and gives rise to blue 11
fluorescence with broad emission band showing maximum around 420 nm and several shoulder peaks [20]. The UV-vis spectrum in Fig. 1a shows a strong absorption band between 335 and 430 nm with a peak centered at 390 nm, which is attributed to both the PF and TPE units. Due to its trace amount in the PFTD backbone, the absorption of DBT structures are hardly visible. By magnification, a maximum absorption around 512 nm was observed for DBT component, as seen in Fig. 1a inset. Upon excitation with 380 nm wavelength, the PFTD in THF solution gererates a strong blue fluroescence. A emission band from 400 to 500 nm was found in emission spectrum of Fig. 1b, showing a strongest peak at 418 nm companied with several shoulder peaks, similar to that of PF in organic solvent. This observation revealed the fluorescence of PFTD were mainly from fluorene units, reasonably explained by the well solvated state of PFTD in THF showing neglectable aggregation and any subsequent aggregation induced FRET. Considering the TPE units features AIE effect, the emission behavior of PFTD in THF/H2O mixtures with different water fraction were recorded, and the corrresponding fluorescent photographs were captured, as shown in Fig. 1c and 1d respectively. As the water fraction increased, the emission band of PF (400-500 nm) decreased quickly while the red emission band from DBT rose up slowly. It should be noted that the shoulder peak at 440 nm became to the strongest emission when the water fraction was over 20%, due to the formation of the β-phase conformation of PF as reported previously [37, 38]. On the other hand, Fig. 1d suggested the fluorescence signal of the mixtures turned from pure blue to purple then to pink finally. The 12
variation in emission color implied the occurrence of aggregation-induced FERT from PF (or/and TPE) to DBT upon water was added. It is understandable that addition of poor solvent caused the aggregation of the hydrophobic conjugated polymer, leading to increased local concentration of DBT part which were surrounded by lots of donors. Thereby, efficient energy transfer took place, and DBT acceptor dominated the whole emission resulting in pink color when water fraction was 99%. 3.3. Preparation and characterization of PFTD NPs and PFTD/PSMA NPs To fabricate the nanoparticles of the as-prepared polymer, a reprecipitation technique was employed, which is a facile and efficient method widely used for developing conjugated polymer nanoparticles [14]. The schematic illustration of the preparation process is shown in Scheme 2. Briefly, the PFTD polymer was firstly dissolved in THF, which was then quickly injected into a large amount of water, followed by 3 min ultrasonication. Residual THF was removed by nitrogen stripping. Eventually, PFTD nanoparticles dispersion was obtained, showing pink fluorescence upon excitation. To prepare the white-emissive nanoparticles, other polymer matrix is needed to help PFTD adjust the FRET to an appropriate extent. Here, amphiphilic polymer poly(styrene-co-maleic anhydride) (PSMA) was selected, which is often used as the matrix material to reduce the aggregation caused quenching of the conjugated polymers [17]. The PFTD and PSMA were dissolved in THF, whose molar ratio was varied at 1:1, 1:7, and 1:10, respectively. The following reprecipitation process was the same as stated above. The acquired nanoparticles were denoted as PFTD/PSMA 1:1 NPs, PFTD/PSMA 1:7 NPs, PFTD/PSMA 1:10 NPs, respectively. 13
The size and morphology of the as-obtained PFTD NPs and PFTD/PSMA 1:7 NPs were examined by scanning electron microscopy (SEM). Fig. 2a revealed that PFTD NPs had an approximately spherical shape with a mean diameter around 80 nm. After incorporation of PSMA, the PFTD/PSMA 1:7 NPs showed a quite spherical shape with an average size down to around 40 nm. This difference can be explained by the well self-assembly ability of the amphiphilic PSMA, which even can reduce the size of conjugated polymer nanoparticle down to below 10 nm [17]. These SEM images clearly demonstrated the successful preparation of PFTD NPs and PFTD/PSMA NPs, and their sizes are suitable for biological application. Moreover, the zeta potential of PFTD NPs and PFTD/PSMA 1:7 NPs were determined as -27.4 ± 2.5 mV and -21.8 ± 3.0 mV, respectively. This observation indicated these nanoparticles were negatively charged, which keep them stably dispersed in aqueous medium. 3.4. Photophysical properties of PFTD NPs and PFTD/PSMA NPs The photophysical properties of these polymer nanoparticles were determined in water dispersion. First of all, we tried to figure out the possibility of FRET between the three components in the PFTD backbone. Thus, the excitation spectra at three emissions of PFTD/PSMA 1:7 NPs were recorded and displayed in Fig. 3a. It showed that both the excitation spectra of emission at 625 nm and 498 nm were well overlapped with that at 418 nm, suggesting that all these emissions were related to the excitation of PF and TPE parts. This phenomenon indicated the occurrence of FRET from PF/TPE to DBT. Then, the absorption spectra of the as-prepared nanoparticles were determined and shown in Fig. 3b, which were similar to PFTD in THF solution 14
in Fig. 1a. Fig. 3c displayed the fluorescence spectra of these nanoparticles under excitation at 380 nm, and their corresponding fluorescence photographs were shown in Fig. 3d. PFTD NPs gave rise to bright pink fluorescence, indicating a significant FRET efficiency and the DBT emission dominated the fluorescence. As the PSMA content increased, the relative ratio of the red emission part decreased, implying the reduction of FRET degree from the PF/TPE to DBT. Accordingly, their fluorescence color changed from light pink to white finally. When the molar ratio of PFTD/PSMA was 1:7 and 1:10, their nanoparticles kept white emission. In order to futher understand the emission efficiency and FRET degree in these nanoparticles, their absolute quantum yield and fluorescence lifetime experiments were carried out. The absolute quantum yields were determined using a spectrofluorometer equipped with an integrating sphere, which were summarized in Table 1. The excitation wavelength was set at 380 nm, and the emission spectral range for PF units, TPE units, and DBT units were calculated from 395 nm-484 nm, 484 nm-565 nm, and 565 nm-750 nm, respectively. At free state, PFTD showed a total quantum yield of 36.5 %, possessing the highest quantum yield value for PF (31.8 %) and the lowest for DBT (1.50 %), which can be explained by the low FRET efficiency in solution. After being tansferred to nanoparticles, the total quantum yield of PFTD NPs rose up to 50.0 %, revealing its AIE features. Specifically, the quantum yield of PF decreased to 13.6 %, while that of TPE and DBT increased, suggesting the FRET of PF→TPE and PF→DBT took place. By incorpration of PSMA as the matrix materials, the total quantum yield of PFTD/PSMA NPs decrased to 23.6-26.6 % with 15
little differences in contribution of each part, suggesting a weaker AIE feature and lower FRET efficiency than that of PFTD NPs. Moreover, the quantum yield of DBT in PFTD/PSMA 1:1 NPs is a little higher than the other two parts, which led to the apparent pink fluorescence. In the case of PFTD/PSMA 1:7 NPs and PFTD/PSMA 1:10 NPs, comparable quantum yield from each part was observed which resulted in white emission. Fig. 4 shows the fluorescence decay curves of each component of PFTD in THF solution, PFTD NPs and PFTD/PSMA 1:7 NPs, respectively, and the average lifetimes were calculated and displayed in Table 1. As the energy donor, PF showed a faster decay rate and shorter lifetime in nanoparticles than in solution, consisitent with the observation for the donor when FRET occured [33]. On the contrary, the TPE and DBT part showed an increased lifetime in the nanoparticles than at solvated state. This can be explained by their actor of energy acceptor, AIE features and change in the environment. These observation in lifetime was coincidence with the findings on the fluorescence behavior and quantum yield.
3.5. Cell viability and cell imaging of PFTD/PSMA 1:7 NPs Taking advantage of its high brightness and suitable size, the PFTD/PSMA nanoparticle might be suitable for multi-color cell imaging. It is well known that cytotoxicity is a key parameter to evaluate the probe materials in biological application. Before applying it in cellular environment, the cytotoxicity of PFTD/PSMA 1:7 NPs against HeLa cell line, cervical cancer cell, was tested using a 16
standard MTT assay. HeLa cells were incubated with the PFTD/PSMA 1:7 NPs dispersions at various amonut, which was represented by the unit molar concentration of PFTD used. After 24 h incubation, the the medium was poured out and MTT in PBS solution was added, whose absorbance at 570 nm is correlated with the viability of HeLa cells. Fig. 5 displays the cell viability of HeLa cells as a function of PFTD/PSMA 1:7 NPs amounts. The cell viability of HeLa cells in the absence of nanoparticles was defined as 100 %. The result clearly showed that nearly 100 % of cells were alive in the presence of PFTD/PSMA 1:7 NPs at tested concetrantion. Thus, we can conclude that this PFTD/PSMA NPs had almost no cytotoxicity or side effects towards HeLa cells, and they are safe to be used for biological application. Then, the performance of PFTD/PSMA NPs on cellular imaging was examined using HeLa cells. Briefly, a 100 μL portion of PFTD/PSMA 1:7 NPs dispersion was added into the medium containing HeLa cells, and the final concentration of PFTD was calculated to be 1.3 μM. After cocultured with these nanoparticles for 12 h at 37 ºC, the HeLa cells were washed with PBS buffer and observed under a confocal laser scanning microscope. A 405 nm laser was used as the excitation source, and the fluorescence images were captured through blue channel (425-485 nm), green channel (490-560 nm) and red channel (575-675 nm). The HeLa cells incubated without nanoparticles were employed as the control example, proving no fluorescence background at the same condition (Fig. 6a). Fig. 6b shows that all the three channels gave rise to clear fluorescence cell imaging with high brightness, and the well-overlapped fluorescent signals in the merged image confirmed these signals came from the nanoparticles. Although the PFTD/PSMA 1:7 NPs possessed negatively charged surface, they were taken well by the HeLa cells and finally located at the cytoplasm. By viture of their small size, these polymer nanoparticles were internalized by cells most likely via an endocytosis process [39]. Moreover, the 17
shapes of these HeLa cells also indicated their good condition, confirming the low cytotoxcity of these nanoparticles. These results proved that the as-preprared PFTD/PSMA 1:7 NPs were ideal fluorescent materials for multi-color cellular imaging. 4. Conclusions In summary, a new conjugated polymer was synthesized bearing fluorene, TPE and DBT component as the blue, green, and red emitting part, respectively, where FRET can take place from fluorene to TPE and DBT. This conjugated polymer showed intense blue fluorescence in organic solution, and turned to pink emitting after being transferred to nanoparticles. By incorporation of amphiphilic PSMA as the matrix to adjust the FRET efficiency, white-emissive nanoparticles were obtaiend as PFTD/PSMA NPs. The photophysical experiments including emission spectra, quantum yield and lifetime demonstrated the AIE feature and variation in FRET degree of PFTD in solution and nanoparticles. Taking advantage of their high brightness and low cytotoxicty, these hybrid PFTD/PSMA NPs showed well performance on cell imaging which can be observed by three channels.
Acknowledgements The authors thank the financial support from the National Natural Science Foundation of China (51503015, 51373022), and the Fundamental Research Funds for the Central Universities (FRF-TP-15-003A1).
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Accepted Manuscript Title: Preparation of conjugated polymer nanoparticles with white emission and their application for cell imaging Authors: Ronghua Liu, Qianling Cui, Yu Yang, Rui Peng, Lidong Li PII: DOI: Reference:
S1010-6030(17)30772-4 http://dx.doi.org/doi:10.1016/j.jphotochem.2017.07.042 JPC 10768
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date: Revised date: Accepted date:
4-6-2017 18-7-2017 31-7-2017
Please cite this article as: Ronghua Liu, Qianling Cui, Yu Yang, Rui Peng, Lidong Li, Preparation of conjugated polymer nanoparticles with white emission and their application for cell imaging, Journal of Photochemistry and Photobiology A: Chemistryhttp://dx.doi.org/10.1016/j.jphotochem.2017.07.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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24
Fig. 1. (a) Absorption spectra of PFTD in THF solution. Inset is the magnified spectra over the range of 450-700 nm. (b) Emission spectra of PFTD in THF solution upon exciated at 380 nm. (c) Emission spectra of PFTD in THF/H2O mixtures with different water fraction. (d) Fluorescent photographs of the PFTD in THF/H2O mixtures with different water fraction under a hand-held UV lamp illumination (365 nm).
25
Fig. 2. SEM images of (a) PFTD NPs and (b) PFTD/PSMA 1:7 NPs.
26
Fig. 3. (a) Excitation spectra of PFTD/PSMA 1:7 NPs in aqueous dispersion at emission of 437 nm, 498 nm, 625 nm, respectively. (b) Normalized absorption spectra of PFTD NPs and PFTD/PSMA NPs with various ratios. (c) Fluroescence spectra of PFTD NPs in aqueous solution, as well as the PFTD/PSMA NPs with various ratios excitated by 380 nm. (d) Fluorescent photographs of the as-acquired nanoparticles in aqueous solutions under a hand-held UV lamp illumination (365 nm).
27
Fig. 4. Decay curves of (a) 418 or 437 nm emission wavelength, (b) 498 nm emission wavelength, and (c) 625 nm emission wavelength of PFTD in THF solution, PFTD NPs and PFTD/PSMA 1:7 NPs.
28
Fig. 5. Cell viability of HeLa cell as a function of PFTD/PSMA 1:7 NPs concentration.
29
Fig. 6. Confocal laser scanning microscopy images of HeLa cells incubated (a) without and (b) with PFTD/PSMA 1:7 NPs for 12 h. The excitation laser wavelength is 405 nm.
30
Scheme 1. Synthetic route of the conjugated polymer PFTD. The ratio of each component was based on the initial molar ratio of precursor before polymerization, which was set as 95 %, 4.95 % and 0.05 % for fluorene, TPE and DBT, respectively.
31
Scheme 2. Schematic illustration of the preparation of PFTD NPs and PFTD/PSMA 1:7 NPs.
32
Table 1. Absolute quantum yield (ΦF) and average lifetime (τ) of each part of PFTD in solution and nanoparticles. ΦF (%)a τ (ns)b PF TPE DBT PF TPE DBT Total units units units units units units PFTD in THF 31.8 3.20 1.50 36.5 0.18 0.14 0.39 PFTD NPs 13.6 6.97 26.4 50.0 0.14 0.24 4.19 PFTD/PSMA 1:1 NPs 6.50 6.67 10.4 23.6 0.10 0.29 3.50 PFTD/PSMA 1:7 NPs 6.88 8.32 9.11 24.3 0.15 0.60 3.45 PFTD/PSMA 1:10 NPs 7.70 9.57 9.36 26.6 0.19 0.64 3.40 a The absolute quantum yields ΦF were determined using a spectrofluorometer equipped with an integrating sphere. The excitation wavelength was set at 380 nm with scattering spectral range at 375-385 nm, and the emission spectral range for PF units, TPE units, and DBT units were calculated from 395 nm-484 nm, 484 nm-565 nm, and 565 nm-750 nm, respectively. The total quantum yield was the sum value of the three parts. b The average lifetimes were calculated from the decay curves of PF units, TPE units, and DBT units at 437 nm (418 nm for solution), 498 nm, and 625 nm, respectively.
33
S1010-6030(17)30772-4 http://dx.doi.org/doi:10.1016/j.jphotochem.2017.07.042 JPC 10768
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date: Revised date: Accepted date:
4-6-2017 18-7-2017 31-7-2017
Please cite this article as: Ronghua Liu, Qianling Cui, Yu Yang, Rui Peng, Lidong Li, Preparation of conjugated polymer nanoparticles with white emission and their application for cell imaging, Journal of Photochemistry and Photobiology A: Chemistryhttp://dx.doi.org/10.1016/j.jphotochem.2017.07.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation of conjugated polymer nanoparticles with white emission and their application for cell imaging
Ronghua Liu, Qianling Cui,* Yu Yang, Rui Peng, Lidong Li*
School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China
Graphical abstract
Highlights
A conjugated polymer bearing blue-, green-, and red-emitting components was synthesized.
White-emissive polymer nanoparticles were prepared successfully.
These polymer nanoparticles are efficient probe for cellular imaging.
Abstract: Conjugated polymer nanoparticles have been regarded as promising fluorescent material for biological application, due to their high emission efficiency, 1
good photostability and low cytotoxicity. In this contribution, we synthesized a conjugated polymer which integrates blue-, green- and red-emitting components into the backbone in an appropriate molar ratio. After being transferred from solution to nanoparticles, its fluorescence color turns from blue to pink with enhanced fluorescence quantum yield. After incorporation of an amphiphilic polymer as matrix, white-emissive polymer nanoparticles are obtained as a result of optimized energy transfer degree among the three components. Finally, their good performance on cell imaging are demonstrated by incubation with HeLa cells, after being proved to be safe for biological application.
Keywords: fluorescence, conjugated polymer nanoparticles, cell imaging, white emission, multi-color
1. Introduction In the past decades, conjugated polymers have attracted numerous interests because of their promising photophysical properties, such as strong light harvesting ability, high emission efficiency, good photostability, molecular wire effect and low cytotoxicity [1]. These features make them suitable for wide application fields including solar cells, polymer light-emitting diode, field effect transistor, chemical or biological sensing, cellular imaging, photodynamic therapy and antibacterial [2-9]. For biological application, it requires the conjugated polymer processable in aqueous medium. To this end, water-soluble conjugated polymer modified with hydrophilic moieties and 2
conjugated polymer nanoparticles are the two commonly used solutions [10-13]. Compared with water-soluble conjugated polymer which requires complicated chemical modification and elaborate purification, the preparation of conjugated polymer nanoparticles is much easier. Reprecipitation technique is the most often employed method to fabricate the conjugated polymer nanoparticles, which is simple, reliable, and more importantly, no need of assistance from additional surfactants [14]. In recent years, conjugated polymer nanoparticles have become among the most attractive fluorescent materials for biological application [11]. For instance, by using the conjugated polymer nanoparticles as the carrier, our group realized the gene delivery into the cells and the whole process can be monitored by variation in fluorescence signals [15]. Taking advantage of the molecule imprint technique, targeted cancer cell imaging was achieved by sialic acid-imprinted conjugated polymer nanoparticles, which can selectively bind with DU 145 cell line having sialic acid over-expressed surfaces [16]. Very recently, conjugated polymer nanoparticles with extremely small size had been reported as efficient photoblinking probes for super-resolution imaging of living cells [17]. However, there are still some limitations of conjugated polymer nanoparticles, most important of which is the significant aggregation caused quenching problem possibly as a result of the aggregation-induced depopulation of the excitons [18,19]. Thus, decrease in emission efficiency is often observed for conjugated polymer when it is transferred from solvated state to solid state like nanoparticles or films [20]. To resolve this problem, organic or inorganic matrix have been chosen to disperse the 3
conjugated polymer which could reduce their aggregation degree and thereby could decrease the quenching effect [21-23]. An alternative way is to introduce aggregation-induced emission (AIE) structures into the conjugated backbone, which has an enhanced photoluminescence at solid state, possibly due to the restricted molecular rotation and increased radiative decay rate [24-27]. On the other hand, although a variety of conjugated polymer nanoparticles with multi-color emission have been developed, which are useful for fluorescent coding and multi-channel cellular imaging [28,29]. Nevertheless, fabrication on the white-emissive conjugated polymer nanoparticles has been merely reported. In this work, a conjugated polymer integrating three parts with blue, green and red emission into its backbone was designed and synthesized. Fluorescence resonance energy transfer (FRET) could take place between these three parts, which could be facilely adjusted to acquire the emission color of the whole polymer. The conjugated polymer solution displayed intense blue fluorescence, while its nanoparticles gave rise to pink signals, which turned to white emission after introduction of a polymer matrix. Their photophysical behaviors and further understanding on the FRET efficiency were explored and discussed. Finally, the toxicity of the white-emissive conjugated polymer nanoparticles against HeLa cells were evaluated, after which their good performance on cellular imaging via three channels was demonstrated.
2. Experimental Section 2.1. Materials 4
1,2-Bis(4-bromophenyl)-1,2-diphenylethene (TPE-DBr) was synthesized according to literature procedures [30]. Tetrakis-(triphenylphosphine)palladium(0) [Pd(PPh3)4], zinc powder, and amphiphilic functional polymer poly(styrene-co-maleic anhydride) (PSMA, cumene terminated, average Mn ~ 1,700) were purchased from Sigma-Aldrich and used as received. 9,9-Dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol)ester (monomer A), 9,9-dioctyl-2,7-dibromofluorene (monomer B), and 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole (DBT-Br) were purchased from Beijing Allmers Chemical S&T Co., Ltd. Titanium tetrachloride (TiCl4) and 4-bromobenzophenone 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. Ultrapure Millipore water (18.6 MΩ·cm) was used throughout the experiments. 2.1.1. Synthesis of TPE-DBr 4-Bromobenzophenone (5.00 g, 19.1 mmol) and Zn powder (2.50 g, 38.3 mmol) were placed into a 250 mL two-necked round-bottom flask. 150 mL of THF was added to the flask and the solution was thoroughly degassed. The mixture was cooled to -78 °C and TiCl4 (5.32 mL, 38.3 mmol) was added dropwise by a syringe. The mixture was slowly warmed to room temperature. After stirring for 30 min, the reaction mixture was then heated to reflux under an argon atmosphere for 24 h. After cooling to room temperature, the mixture was quenched with saturated NaHCO3 solution and filtered. The filtrate was evaporated to remove solvent, and the mixture 5
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 petroleum ether as an eluent to afford 7.51 g of white solid with a molar yield of 80%. 1H NMR (400 MHz, CDCl3, δ): 7.10 (d, 7H), 6.97 (s, 5H), 6.92‒6.44 (m, 6H). 13C NMR (100 MHz, CDCl3, δ): 142.92, 142.84, 142.40, 142.30, 140.32, 133.07, 132.70, 131.39, 131.27, 131.07, 131.00, 130.95, 130.74, 128.22, 128.03, 127.86, 127.63, 127.14, 127.04, 126.75, 126.66, 120.80, 120.66. MS (MALDI-TOF) m/z: M+ calcd: 490.23. Found: M+ = 490.0. 2.1.2. Synthesis of PFTD (PF0.95TPE0.0495DBT0.0005) Monomer A (0.30 g, 0.54 mmol), monomer B (0.26 g, 0.48 mmol), TPE-DBr (0.026 g, 0.053 mmol), and DBT-Br (492 μL, 0.5 mg/mL, 0.00054 mmol) were dissolved in toluene and placed into a 100 mL two-necked round-bottom flask. 40 mL of toluene and 10 mL of potassium carbonate aqueous solution (2.0 M) was added to the flask. After the solution was degassed with argon for 30 min, the catalyst Pd(PPh3)4 was added. The reaction was stirred at 85 °C for 48 h under an argon atmosphere. The 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 5 mL of chloroform, and the polymer was then precipitated by 150 mL of methanol. 0.228 g of light pink powder was collected and the molar yield was estimated to be about 55%. 1H NMR (400 MHz, CDCl3, δ): 8.00‒7.56 (m, aromatic backbone), 2.30‒1.88 (br, ‒CCH2‒), 1.26‒1.02 (br, 6
‒CCH2(CH2)6‒), 0.95‒0.81 (br, ‒CH2CH3). EA calculated: C, 89.82%; H, 10.16%. Found: C, 88.64%; H, 10.25%. GPC: Mn = 18 204, Mw = 37 146, PDI = 2.04. 2.2. Preparation of PFTD NPs and PFTD/PSMA NPs PFTD NPs and PFTD/PSMA NPs were prepared by a reprecipitation method reported in literature [15, 31]. Briefly, the polymer PFTD and PSMA were separately dissolved in THF to make stock solution at a concentration of 1 mg/mL. The polymer solution was filtered through 0.22 μm poly(tetrafluoroethene) (PTFE) filters. Then, a 200 μL portion of the 1 mg/mL PFTD solution in THF was diluted to 4 mL, obtaining 50 μg/mL PFTD solution in THF. Then 1 mL of 50 μg/mL PFTD solution in THF was rapidly injected into 10 mL of water and subjected to ultrasonication for 3 min. THF was removed by nitrogen stripping. The obtained nanoparticle was denoted as PFTD NPs. For the preparation of PFTD/PSMA NPs, a 200 μL portion of the 1 mg/mL PFTD solution in THF was mixed with 200 μL, or 1400 μL, or 2000 μL of 1 mg/mL PSMA solution in THF, and the above solution was diluted to 4 mL. Then 1 mL of the PFTD/PSMA solution was rapidly injected into 10 mL of water and subjected to ultrasonication for 3 min. THF was removed by nitrogen stripping. According to the different molar ratio, the obtained nanoparticles were named PFTD/PSMA 1:1 NPs, PFTD/PSMA 1:7 NPs and PFTD/PSMA 1:10 NPs, respectively. 2.3. Cytotoxicity Assay by MTT Method The cytotoxicity of PFTD/PSMA 1:7 NPs for cervical cancer cell line (HeLa) was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 7
method. HeLa cells were seeded into 96-well plates in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS). After incubation for 24 h at 37 °C in a 5% CO2 humidified atmosphere, HeLa cells were treated with various concentrations of PFTD/PSMA 1:7 NPs (0‒2.6 μM) at 37 °C for 24 h. The concentrations of PFTD/PSMA 1:7 NPs were calculated from the initial concentration of PFTD molecules used in the synthesis. Thereafter, the 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. 2.4. Cell Imaging Assay HeLa cells were seeded on 35 × 35 mm2 culture plates. HeLa cells were grown in DMEM containing 10% (v/v) FBS. Then the plates were incubated for 12 h at 37 °C in a 5% CO2 humidified atmosphere. A 100 μL portion of PFTD/PSMA 1:7 NPs was added into 900 μL of medium containing HeLa cells in a 35 × 35 mm2 plate (final concentration of PFTD = 1.3 μM). After incubation at 37 °C for 12 h, the medium was removed, and the cells were washed with PBS buffer (pH = 7.4) for three times. 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 separately through three channels, including the range from 425 nm to 485 nm (blue 8
channel), 490 nm to 560 nm (green channel), and 575 nm to 675 nm (red channel). 2.5. Characterization The 1H NMR spectra and 13C NMR were recorded on 400 MHz and 100 MHz AC Bruker spectrometer, respectively. Ultraviolet−visible (UV−vis) absorption spectra were measured on a Hitachi U-3900H spectrophotometer. Fluorescence emission spectra were recorded using a Hitachi F-7000 fluorescence spectrophotometer. The molecular weight was performed on a Bruker Daltonics BIFLEX III MALDI-TOF analyzer in matrix-assisted laser desorption ionization time-of-flight (MALDI) mode. Element analysis (EA) was conducted on a FLASH EA 1112 element analyzer. Gel permeation chromatography (GPC) analysis was measured on a Waters Styragel system using polystyrene as the calibration standard and THF as eluent. The zeta potentials of nanoparticles dispersions were determined on Malvern Zeta-sizer Nano ZS90. 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 field emission scanning electron microscope (SEM, ZEISS SUPRA55). The absolute fluorescence quantum yields were determined using a spectrofluorometer (NanologR FluoroLog-3-2-iHR320, Horiba Jobin Yvon) equipped with an integrating sphere. Time-domain lifetime measurements were performed using an ultrafast lifetime spectrofluorometer (Delta flex) according to time-correlated single-photon counting technique. For the samples of PFTD solution in THF, PFTD NPs, and PFTD/PSMA NPs, the excitation wavelength was set at 380 nm with 9
scattering spectral range at 375-385 nm, and emission spectral range for PF units, TPE units, and DBT units were calculated from 395-484 nm, 484-565 nm, and 565-750 nm, respectively. 3. Results and Discussions 3.1. Design concept and synthesis of PFTD The chemical structure and synthetic route of the conjugated polymer are shown in Scheme 1. In the conjugated polymer backbone, fluorene (F), tetraphenylethene (TPE), and 1,4-dithienylbenzothiadiazole (DBT) were employed as the blue, green, and red-emitting units, respectively. Their molar ratio is needed to be elaborately adjusted so as to generate appropriate fluorescence resonance energy transfer (FRET) efficiency and thereby wanted emission behavior. Blue-emitting polyfluorene (PF) are selected as the energy donor, in virtue of its high emission efficiency and easy modification on its C-9 position with long alkyl groups to increase the polymer’s solubility and operability [32-34]. TPE is a typical aggregation-induced emission (AIE) fluorophore, which is widely used to build up various sorts of solid-state emissive materials showing enhanced emission [35,36]. Herein, TPE acts as the green-emitting units, and more importantly, plays a crucial role in reducing the aggregation-induced quenching problem. In this designed polymer, DBT with a narrow band-gap serves as the red-emitting component and energy acceptor for FRET. It is well known that efficient FRET requires spectroscopic overlap and suitable distance (less than 10 nm) between donor and acceptor. In this case, aggregation of the polymer chains is supposed to lead to efficient intermolecular FRET via several 10
paths, including PF→TPE, PF→DBT, and TPE→DBT. As shown in Scheme 1, the polymer was synthesized by Suzuki cross coupling reaction using fluorene monomer A and B, TPE-Br, and DBT-Br, in the presence of Pd catalyst. The initial molar ratio of fluorene, TPE, and DBT was set as 95 %, 4.95 % and
0.05
%,
respectively.
The
obtained
polymer
could
be
named
as
PF0.95TPE0.0495DBT0.0005 based on the initial molar ratio of each component precursor, which was simply denoted as PFTD in this work. Its chemical structure was characterized by 1H NMR and element analysis (EA). The EA results confirmed that the final composition of PFTD was close to the initial molar ratio of each component. The polymer’s average molecular weight (Mw) was 37 146 g mol-1 with a polydispersity index (PDI) of 2.04, determined by GPC. Based on these results, we can speculate that less than one DBT unit on each PFTD chain, which means the polymer might be a mixture of polymer PF-TPE and PF-TPE-DBT. The obtained polymer dissolved well in common organic solvents such as chloroform, dichloromethane, tetrahydrofuran (THF), and N,N-dimethylformamide (DMF), but not in methanol and acetone. 3.2. Photophysical properties of PFTD The photophysical properties of the as-prepared PFTD polymer were firtstly determined in THF solution, a good solvent for it. It has been reported that TPE has a chacteristic absorption band of 280-350 nm and a cyan emission around 470 nm, which would had a red shift after conjugation with fluorene units [35, 36]. For PF, it always possesses an intense absorption band around 380 nm, and gives rise to blue 11
fluorescence with broad emission band showing maximum around 420 nm and several shoulder peaks [20]. The UV-vis spectrum in Fig. 1a shows a strong absorption band between 335 and 430 nm with a peak centered at 390 nm, which is attributed to both the PF and TPE units. Due to its trace amount in the PFTD backbone, the absorption of DBT structures are hardly visible. By magnification, a maximum absorption around 512 nm was observed for DBT component, as seen in Fig. 1a inset. Upon excitation with 380 nm wavelength, the PFTD in THF solution gererates a strong blue fluroescence. A emission band from 400 to 500 nm was found in emission spectrum of Fig. 1b, showing a strongest peak at 418 nm companied with several shoulder peaks, similar to that of PF in organic solvent. This observation revealed the fluorescence of PFTD were mainly from fluorene units, reasonably explained by the well solvated state of PFTD in THF showing neglectable aggregation and any subsequent aggregation induced FRET. Considering the TPE units features AIE effect, the emission behavior of PFTD in THF/H2O mixtures with different water fraction were recorded, and the corrresponding fluorescent photographs were captured, as shown in Fig. 1c and 1d respectively. As the water fraction increased, the emission band of PF (400-500 nm) decreased quickly while the red emission band from DBT rose up slowly. It should be noted that the shoulder peak at 440 nm became to the strongest emission when the water fraction was over 20%, due to the formation of the β-phase conformation of PF as reported previously [37, 38]. On the other hand, Fig. 1d suggested the fluorescence signal of the mixtures turned from pure blue to purple then to pink finally. The 12
variation in emission color implied the occurrence of aggregation-induced FERT from PF (or/and TPE) to DBT upon water was added. It is understandable that addition of poor solvent caused the aggregation of the hydrophobic conjugated polymer, leading to increased local concentration of DBT part which were surrounded by lots of donors. Thereby, efficient energy transfer took place, and DBT acceptor dominated the whole emission resulting in pink color when water fraction was 99%. 3.3. Preparation and characterization of PFTD NPs and PFTD/PSMA NPs To fabricate the nanoparticles of the as-prepared polymer, a reprecipitation technique was employed, which is a facile and efficient method widely used for developing conjugated polymer nanoparticles [14]. The schematic illustration of the preparation process is shown in Scheme 2. Briefly, the PFTD polymer was firstly dissolved in THF, which was then quickly injected into a large amount of water, followed by 3 min ultrasonication. Residual THF was removed by nitrogen stripping. Eventually, PFTD nanoparticles dispersion was obtained, showing pink fluorescence upon excitation. To prepare the white-emissive nanoparticles, other polymer matrix is needed to help PFTD adjust the FRET to an appropriate extent. Here, amphiphilic polymer poly(styrene-co-maleic anhydride) (PSMA) was selected, which is often used as the matrix material to reduce the aggregation caused quenching of the conjugated polymers [17]. The PFTD and PSMA were dissolved in THF, whose molar ratio was varied at 1:1, 1:7, and 1:10, respectively. The following reprecipitation process was the same as stated above. The acquired nanoparticles were denoted as PFTD/PSMA 1:1 NPs, PFTD/PSMA 1:7 NPs, PFTD/PSMA 1:10 NPs, respectively. 13
The size and morphology of the as-obtained PFTD NPs and PFTD/PSMA 1:7 NPs were examined by scanning electron microscopy (SEM). Fig. 2a revealed that PFTD NPs had an approximately spherical shape with a mean diameter around 80 nm. After incorporation of PSMA, the PFTD/PSMA 1:7 NPs showed a quite spherical shape with an average size down to around 40 nm. This difference can be explained by the well self-assembly ability of the amphiphilic PSMA, which even can reduce the size of conjugated polymer nanoparticle down to below 10 nm [17]. These SEM images clearly demonstrated the successful preparation of PFTD NPs and PFTD/PSMA NPs, and their sizes are suitable for biological application. Moreover, the zeta potential of PFTD NPs and PFTD/PSMA 1:7 NPs were determined as -27.4 ± 2.5 mV and -21.8 ± 3.0 mV, respectively. This observation indicated these nanoparticles were negatively charged, which keep them stably dispersed in aqueous medium. 3.4. Photophysical properties of PFTD NPs and PFTD/PSMA NPs The photophysical properties of these polymer nanoparticles were determined in water dispersion. First of all, we tried to figure out the possibility of FRET between the three components in the PFTD backbone. Thus, the excitation spectra at three emissions of PFTD/PSMA 1:7 NPs were recorded and displayed in Fig. 3a. It showed that both the excitation spectra of emission at 625 nm and 498 nm were well overlapped with that at 418 nm, suggesting that all these emissions were related to the excitation of PF and TPE parts. This phenomenon indicated the occurrence of FRET from PF/TPE to DBT. Then, the absorption spectra of the as-prepared nanoparticles were determined and shown in Fig. 3b, which were similar to PFTD in THF solution 14
in Fig. 1a. Fig. 3c displayed the fluorescence spectra of these nanoparticles under excitation at 380 nm, and their corresponding fluorescence photographs were shown in Fig. 3d. PFTD NPs gave rise to bright pink fluorescence, indicating a significant FRET efficiency and the DBT emission dominated the fluorescence. As the PSMA content increased, the relative ratio of the red emission part decreased, implying the reduction of FRET degree from the PF/TPE to DBT. Accordingly, their fluorescence color changed from light pink to white finally. When the molar ratio of PFTD/PSMA was 1:7 and 1:10, their nanoparticles kept white emission. In order to futher understand the emission efficiency and FRET degree in these nanoparticles, their absolute quantum yield and fluorescence lifetime experiments were carried out. The absolute quantum yields were determined using a spectrofluorometer equipped with an integrating sphere, which were summarized in Table 1. The excitation wavelength was set at 380 nm, and the emission spectral range for PF units, TPE units, and DBT units were calculated from 395 nm-484 nm, 484 nm-565 nm, and 565 nm-750 nm, respectively. At free state, PFTD showed a total quantum yield of 36.5 %, possessing the highest quantum yield value for PF (31.8 %) and the lowest for DBT (1.50 %), which can be explained by the low FRET efficiency in solution. After being tansferred to nanoparticles, the total quantum yield of PFTD NPs rose up to 50.0 %, revealing its AIE features. Specifically, the quantum yield of PF decreased to 13.6 %, while that of TPE and DBT increased, suggesting the FRET of PF→TPE and PF→DBT took place. By incorpration of PSMA as the matrix materials, the total quantum yield of PFTD/PSMA NPs decrased to 23.6-26.6 % with 15
little differences in contribution of each part, suggesting a weaker AIE feature and lower FRET efficiency than that of PFTD NPs. Moreover, the quantum yield of DBT in PFTD/PSMA 1:1 NPs is a little higher than the other two parts, which led to the apparent pink fluorescence. In the case of PFTD/PSMA 1:7 NPs and PFTD/PSMA 1:10 NPs, comparable quantum yield from each part was observed which resulted in white emission. Fig. 4 shows the fluorescence decay curves of each component of PFTD in THF solution, PFTD NPs and PFTD/PSMA 1:7 NPs, respectively, and the average lifetimes were calculated and displayed in Table 1. As the energy donor, PF showed a faster decay rate and shorter lifetime in nanoparticles than in solution, consisitent with the observation for the donor when FRET occured [33]. On the contrary, the TPE and DBT part showed an increased lifetime in the nanoparticles than at solvated state. This can be explained by their actor of energy acceptor, AIE features and change in the environment. These observation in lifetime was coincidence with the findings on the fluorescence behavior and quantum yield.
3.5. Cell viability and cell imaging of PFTD/PSMA 1:7 NPs Taking advantage of its high brightness and suitable size, the PFTD/PSMA nanoparticle might be suitable for multi-color cell imaging. It is well known that cytotoxicity is a key parameter to evaluate the probe materials in biological application. Before applying it in cellular environment, the cytotoxicity of PFTD/PSMA 1:7 NPs against HeLa cell line, cervical cancer cell, was tested using a 16
standard MTT assay. HeLa cells were incubated with the PFTD/PSMA 1:7 NPs dispersions at various amonut, which was represented by the unit molar concentration of PFTD used. After 24 h incubation, the the medium was poured out and MTT in PBS solution was added, whose absorbance at 570 nm is correlated with the viability of HeLa cells. Fig. 5 displays the cell viability of HeLa cells as a function of PFTD/PSMA 1:7 NPs amounts. The cell viability of HeLa cells in the absence of nanoparticles was defined as 100 %. The result clearly showed that nearly 100 % of cells were alive in the presence of PFTD/PSMA 1:7 NPs at tested concetrantion. Thus, we can conclude that this PFTD/PSMA NPs had almost no cytotoxicity or side effects towards HeLa cells, and they are safe to be used for biological application. Then, the performance of PFTD/PSMA NPs on cellular imaging was examined using HeLa cells. Briefly, a 100 μL portion of PFTD/PSMA 1:7 NPs dispersion was added into the medium containing HeLa cells, and the final concentration of PFTD was calculated to be 1.3 μM. After cocultured with these nanoparticles for 12 h at 37 ºC, the HeLa cells were washed with PBS buffer and observed under a confocal laser scanning microscope. A 405 nm laser was used as the excitation source, and the fluorescence images were captured through blue channel (425-485 nm), green channel (490-560 nm) and red channel (575-675 nm). The HeLa cells incubated without nanoparticles were employed as the control example, proving no fluorescence background at the same condition (Fig. 6a). Fig. 6b shows that all the three channels gave rise to clear fluorescence cell imaging with high brightness, and the well-overlapped fluorescent signals in the merged image confirmed these signals came from the nanoparticles. Although the PFTD/PSMA 1:7 NPs possessed negatively charged surface, they were taken well by the HeLa cells and finally located at the cytoplasm. By viture of their small size, these polymer nanoparticles were internalized by cells most likely via an endocytosis process [39]. Moreover, the 17
shapes of these HeLa cells also indicated their good condition, confirming the low cytotoxcity of these nanoparticles. These results proved that the as-preprared PFTD/PSMA 1:7 NPs were ideal fluorescent materials for multi-color cellular imaging. 4. Conclusions In summary, a new conjugated polymer was synthesized bearing fluorene, TPE and DBT component as the blue, green, and red emitting part, respectively, where FRET can take place from fluorene to TPE and DBT. This conjugated polymer showed intense blue fluorescence in organic solution, and turned to pink emitting after being transferred to nanoparticles. By incorporation of amphiphilic PSMA as the matrix to adjust the FRET efficiency, white-emissive nanoparticles were obtaiend as PFTD/PSMA NPs. The photophysical experiments including emission spectra, quantum yield and lifetime demonstrated the AIE feature and variation in FRET degree of PFTD in solution and nanoparticles. Taking advantage of their high brightness and low cytotoxicty, these hybrid PFTD/PSMA NPs showed well performance on cell imaging which can be observed by three channels.
Acknowledgements The authors thank the financial support from the National Natural Science Foundation of China (51503015, 51373022), and the Fundamental Research Funds for the Central Universities (FRF-TP-15-003A1).
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Accepted Manuscript Title: Preparation of conjugated polymer nanoparticles with white emission and their application for cell imaging Authors: Ronghua Liu, Qianling Cui, Yu Yang, Rui Peng, Lidong Li PII: DOI: Reference:
S1010-6030(17)30772-4 http://dx.doi.org/doi:10.1016/j.jphotochem.2017.07.042 JPC 10768
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date: Revised date: Accepted date:
4-6-2017 18-7-2017 31-7-2017
Please cite this article as: Ronghua Liu, Qianling Cui, Yu Yang, Rui Peng, Lidong Li, Preparation of conjugated polymer nanoparticles with white emission and their application for cell imaging, Journal of Photochemistry and Photobiology A: Chemistryhttp://dx.doi.org/10.1016/j.jphotochem.2017.07.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fig. 1. (a) Absorption spectra of PFTD in THF solution. Inset is the magnified spectra over the range of 450-700 nm. (b) Emission spectra of PFTD in THF solution upon exciated at 380 nm. (c) Emission spectra of PFTD in THF/H2O mixtures with different water fraction. (d) Fluorescent photographs of the PFTD in THF/H2O mixtures with different water fraction under a hand-held UV lamp illumination (365 nm).
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Fig. 2. SEM images of (a) PFTD NPs and (b) PFTD/PSMA 1:7 NPs.
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Fig. 3. (a) Excitation spectra of PFTD/PSMA 1:7 NPs in aqueous dispersion at emission of 437 nm, 498 nm, 625 nm, respectively. (b) Normalized absorption spectra of PFTD NPs and PFTD/PSMA NPs with various ratios. (c) Fluroescence spectra of PFTD NPs in aqueous solution, as well as the PFTD/PSMA NPs with various ratios excitated by 380 nm. (d) Fluorescent photographs of the as-acquired nanoparticles in aqueous solutions under a hand-held UV lamp illumination (365 nm).
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Fig. 4. Decay curves of (a) 418 or 437 nm emission wavelength, (b) 498 nm emission wavelength, and (c) 625 nm emission wavelength of PFTD in THF solution, PFTD NPs and PFTD/PSMA 1:7 NPs.
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Fig. 5. Cell viability of HeLa cell as a function of PFTD/PSMA 1:7 NPs concentration.
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Fig. 6. Confocal laser scanning microscopy images of HeLa cells incubated (a) without and (b) with PFTD/PSMA 1:7 NPs for 12 h. The excitation laser wavelength is 405 nm.
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Scheme 1. Synthetic route of the conjugated polymer PFTD. The ratio of each component was based on the initial molar ratio of precursor before polymerization, which was set as 95 %, 4.95 % and 0.05 % for fluorene, TPE and DBT, respectively.
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Scheme 2. Schematic illustration of the preparation of PFTD NPs and PFTD/PSMA 1:7 NPs.
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Table 1. Absolute quantum yield (ΦF) and average lifetime (τ) of each part of PFTD in solution and nanoparticles. ΦF (%)a τ (ns)b PF TPE DBT PF TPE DBT Total units units units units units units PFTD in THF 31.8 3.20 1.50 36.5 0.18 0.14 0.39 PFTD NPs 13.6 6.97 26.4 50.0 0.14 0.24 4.19 PFTD/PSMA 1:1 NPs 6.50 6.67 10.4 23.6 0.10 0.29 3.50 PFTD/PSMA 1:7 NPs 6.88 8.32 9.11 24.3 0.15 0.60 3.45 PFTD/PSMA 1:10 NPs 7.70 9.57 9.36 26.6 0.19 0.64 3.40 a The absolute quantum yields ΦF were determined using a spectrofluorometer equipped with an integrating sphere. The excitation wavelength was set at 380 nm with scattering spectral range at 375-385 nm, and the emission spectral range for PF units, TPE units, and DBT units were calculated from 395 nm-484 nm, 484 nm-565 nm, and 565 nm-750 nm, respectively. The total quantum yield was the sum value of the three parts. b The average lifetimes were calculated from the decay curves of PF units, TPE units, and DBT units at 437 nm (418 nm for solution), 498 nm, and 625 nm, respectively.
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