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Preparation of Gemini Surfactant/Conjugated Polymer Aggregates for Enhanced Fluorescence and Bioimaging Application Hua Wang, Lingyun Zhou, Chengcheng Zhou, Weiwei Zhao, Jianwu Wang, Libing Liu, Shu Wang, and Yilin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07099 • Publication Date (Web): 19 Jun 2017 Downloaded from http://pubs.acs.org on June 26, 2017

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ACS Applied Materials & Interfaces

Preparation of Gemini Surfactant/Conjugated Polymer Aggregates for Enhanced Fluorescence and Bioimaging Application Hua Wang,†,§ Lingyun Zhou,‡,§ Chengcheng Zhou,†,§ Weiwei Zhao,†,§ Jianwu Wang,‡,§ Libing Liu,‡ Shu Wang,‡,§,* and Yilin Wang†,§,* †

Key Laboratory of Colloid and Interface Science, Beijing National Laboratory for Molecular Sciences (BNLMS), CAS

Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ‡

Key Laboratory of Organic Solids, Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Re-

search/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China §

University of Chinese Academy of Sciences, Beijing 100049, P. R. China

ABSTRACT: Conjugated polymers have great potential applications in bioimaging. However, the aggregation of conjugated polymers driven by electrostatic and hydrophobic interactions in aqueous media results in the reduction of photoluminescence quantum efficiency. In present work we synthesized a carboxylate gemini surfactant (SDHC) to adjust the aggregation behaviors and fluorescence properties of conjugated polymers (anionic MPS-PPV and cationic PMNT). This gemini surfactant shows very low critical micellar concentration (CMC) in aqueous solution and forms vesicles above CMC. In neutral and acidic conditions, MPS-PPV combines with the SDHC vesicles mainly via hydrophobic interactions and forms the aggregates, in which the photolumi1

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nescence quantum efficiency of MPS-PPV is highly enhanced from 0.1 % to 27 %. As to PMNT, the molecules are located in the bilayer of SDHC vesicles through both electrostatic and hydrophobic interactions, and this structure prevents the production and release of reactive oxygen species. Moreover, SDHC is nontoxic and can effectively decrease the dark- and photo-cytotoxicity of MPS-PPV and PMNT, laying a good foundation for their bioimaging application. The living cell imaging indicates that the surfactant/conjugated polymer aggregates can stain the MCF-7 cells with main location in the lysosome. This work provides insight into how to improve the fluorescence properties and bioimaging applications of conjugated polymers by surfactants. KEYWORDS: gemini surfactant, conjugated polymer, fluorescence property, cytotoxicity, bioimaging

INTRODUCTION With a large π-conjugated backbone and delocalized electronic structure, conjugated polymers (CPs) show great promise in optoelectronic devices, such as organic solar cells, and organic lightemitting diodes.1-7 By modifying the side chains with ionic functional groups, CPs exhibit a desired water solubility8-12 and display great potential applications in living cell imaging due to their unique light-harvesting abilities and high light-amplification effects.13-17 As a biosensor, CPs also possess high fluorescence brightness and excellent photostability.18-22 However, obstacles still remain in the progress of living cell imaging application. The aggregation of CPs in aqueous media results in the reduction of photoluminescence quantum efficiency (PLQE).23, 24 What’s more, CPs can act as photosensitizers to generate singlet oxygen and other reactive oxygen species (ROS) when exposed to light, which not only bleaches the fluorescence of polymers, but also is harmful to the living cells.25-29 Modification of CP structures is generally carried out by introducing hydro2


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philic or ionic side chains to polymers, which increases complexity and cost. Compared with the modification of CPs, fabrication of surfactant/CP aggregates is a more convenient and general strategy. Surfactants have been utilized to increase the sensitivity and water solubility of CPs.23, 24, 30-43 The interactions between CPs and surfactants mainly include electrostatic and hydrophobic interactions. The hydrophobic chains of nonionic or similarly charged surfactants can adhere to the conjugated backbone of CPs by hydrophobic interaction, and the surfactants can block π−π stacking of conjugated backbone and in turn reduce the non-radiative energy transfer among conjugated backbones, leading to the change of emission wavelength and intensity.30, 31 As to oppositely charged surfactants, the interaction process is more complex.32-34 At low concentration, surfactants can form ion pairs with CPs through strong electrostatic attraction which offsets the electrostatic repulsion among the side chains of CPs, and enhances the conjugation length and coplanarity of CPs, causing fluorescence quenching. When surfactant concentration is close to critical micelle concentration (CMC), CPs tend to be surrounded by surfactant molecules and the surfactant/CP complex forms "pearl necklace" structure. This structure decreases the coplanarity of CP, and leads to a blue-shift of absorption spectra of CPs. At the same time, the complex effectively reduces the distance of CP chains and the concentration of excimer, and brings about an enhancement of emission efficiency. Moreover, the influences of the interaction between CPs and surfactants depend on rigidity of conjugated backbones, chain length of CPs, hydrophobic chain length of surfactants, and even on the spacer length for ionic gemini surfactants and so on.35-37 Besides the traditional single-chain surfactant and gemini surfactant, phospholipid vesicles are used as model biological membranes to study the interaction with cationic, anionic and zwitterionic CPs. 42, 43


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In living cell imaging, ionic and nonionic surfactants are mostly used to form nanoparticles with conjugated polymers by ultrasonication, miniemulsion and evaporation.44-52 In the nanoparticles, the core is composed of the hydrophobic conjugated polymer covered by a layer of surfactant molecule with the hydrophilic head groups (e.g. -COOH, -SO4) orientating into the aqueous phase. These nanoparticles show high brightness, excellent photostability and low cytotoxicity, and they can sensitize oxygen molecules to generate ROS. In the conjugated polymer nanoparticles reported, surfactants are used as stabilizers. The enhanced fluorescence properties of conjugated polymer aggregates by surfactant self-assemblies have not yet been applied to bioimaging. Therefore, this work synthesized and applied a carboxylate gemini surfactant (SDHC, Scheme 1) to adjust the aggregation behavior, fluorescence property and bioimaging application of conjugated polymers. This surfactant exhibits low CMC and forms vesicles at different pH. The anionic polyphenylenevinylene (MPS-PPV) and cationic polythiophene (PMNT) derivatives (Scheme 1) were chosen to study their interactions with SDHC. At different pH, SDHC combines with MPSPPV and PMNT by different interaction forces, which leads to the enhancement of fluorescence and the decrease of cytotoxicity of CPs. The related mechanisms were also studied and discussed. The living cell imaging indicates that SDHC and CPs entered the cells simultaneously, and localized in the lysosome.

Scheme 1. Chemical structures of gemini surfactant (SDHC) and conjugated polymers (MPS-PPV and PMNT). ACS Paragon Plus Environment

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EXPERIMENTAL SECTION Materials and Measurements. 1-dodecylamine, 1,3-dibromo-2-propanol, methyl bromoacetate,

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride

(EDC·HCl),

4-

dimethylaminopyridine (DMAP), and MPS-PPV (Mw = (1.0-2.0) × 107 g/mol, ~5000 RU) were purchased from Acros, Aldrich Chemical Co., or Alfa-Aesar. All organic solvents were purchased from Beijing Chemical Works and were dried and distilled before use. PMNT (Mw = (2.0-4.0) × 104 g/mol, ~100 RU) was synthesized and purified as we reported previously.53 DCFH-DA was purchased from Sigma Chemical Co.. MCF-7 cells were obtained from Center for Cell, Institute of Basic Medical Science, Chinese Academy of Sciences. Phosphate buffered saline (PBS), and Dulbecco’s modified eagle medium (DMEM) were purchased from Hyclone (Beijing, China). Fetal bovine serum (FBS) was obtained from Sijiqing Biological Engineering Materials (Hangzhou, China). Cy-5 was purchased from Little-PA Sciences Co., Ltd. (Wuhan, China). Lyso Tracker Red DND-99 (1mM solution in DMSO) was purchased from Invitrogen/Thermo Fisher Scientific Inc. (USA). 1H NMR and 13C NMR were obtained from a Bruker Avance 400 MHz spectrometer. Mass spectra were recorded on a SHIMADZU LCMS-2010 spectrometer for ESI and elemental analysis was measured on Flash EA1112. Synthesis. Compound SDHC was synthesized according to Scheme 2 and characterized by 1H NMR, 13C NMR, mass spectra, and elemental analysis.


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Scheme 2. Synthetic procedure for SDHC. 1,3-Bis(dodecylamino)propan-2-ol (1). 1,3-dibromo-2-propanol (3.92 g, 18.0 mmol) was added to a solution of 1-dodecylamine (10.00 g, 54.0 mmol) in 100 mL of ethanol. This solution was stirred and refluxed at 90 °C for 2 days. Crystallization from ethyl acetate gave pure compound 1 as white solid (4.89 g, 64 % yield). 1H NMR (400 MHz, CDCl3, ppm): δ 0.88 (t, 6H, CH3, J = 6.5 Hz), 1.20-1.42 (m, 36H, CH2), 1.58-1.73 (m, 4H, CH2), 1.94-2.25 (m, 2H, NH), 2.85-2.97 (m, 4H, CH2), 4.65-5.53 (br, 5H, CH). 13C NMR (CDCl3, ppm): 14.5, 22.8, 26.8, 27.9, 29.4, 29.8, 30.4, 30.8, 31.7, 49.9, 53.7, 68.2. MS-ESI (m/z, [M+H] +): calcd, 427.5; found, 427.6. 2,6-didodecyl-4-hydroxy-2,6-diaza-1,7-heptanedicarboxylic Acid (2). Compound 1 (3.00 g, 7.0 mmol) and triethylamine (7.08 g, 70.0 mmol) were dissolved in 30 mL of anhydrous tetrahydrofuran, and methyl bromoacetate (2.35 g, 15.5 mmol) was added dropwise. After stirred at the room temperature for 4 h, the mixture was filtered to remove insoluble material and the filtrate was evaporated to dryness. The residue was extracted with diethyl ether. The precipitate was filtered off, and the filtrate was concentrated under vacuum. The residue was purified on a silica column by using dichloromethane/methanol (from 100:1 to 30:1) as the eluent to afford compound 2 as light yellow oil (2.99 g, 75 % yield). 1H NMR (CDCl3, ppm): δ 0.88 (t, 6H, CH3, J = 6.4 ACS Paragon Plus Environment

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Hz), 1.26-1.32 (m, 36H, CH2), 1.38-1.53 (m, 4H, CH2), 2.26-3.25 (m, 8H, CH2), 3.31-3.60 (m, 4H, CH2), 3.70 (m, 6H, CH3). 13C NMR (CDCl3, ppm): 14.2, 22.5, 27.7, 28.3, 28.8, 29.6, 30.3, 30.6, 31.1, 52.8, 57.6, 60.2, 61.3, 68.3, 174.3. MS-ESI (m/z, [M+H]+): calcd, 571.5; found, 571.6. Sodium 2,6-didodecyl-4-hydroxy-2,6-diaza-1,7-heptanedicarboxylate (SDHC). Compound 2 (3.00 g, 5.26 mmol) was dissolved in 10 mL of methanol, and a solution of NaOH (0.84 g, 21.0 mmol) in 10 mL of water was added dropwise. After stirred at room temperature for 4 h, the mixture was evaporated to dryness. The residue repeatedly recrystallized from acetone/ethanol (95:5) to give the pure SDHC as white solid (2.79 g, 90 % yield). The purity of SDHC was checked by surface tension until the surface tension curve did not have a minimum below CMC. 1H NMR (CD3OD, ppm): δ 0.90 (t, 6H, CH3, J = 6.5 Hz), 1.19-1.39 (m, 36H, CH2), 1.43-1.59 (m, 4H, CH2), 2.34-2.71 (m, 8H, CH2), 2.92-3.25 (m, 4H, CH2), 3.75-3.87 (m, 1H, CH), 5.32-5.38 (m, 1H, OH). 13C NMR (CD3OD, ppm): 14.4, 23.7, 27.4, 28.0, 28.3, 28.6, 28.7, 30.5, 30.8, 33.1, 56.9, 60.7, 61.0, 66.7, 179.7. HRMS-ESI (m/z, [M-2Na+H]-): calcd, 541.4581; found, 541.4586. Anal. Calcd for C31H60N2Na2O5·3H2O: C, 58.10; H, 10.38; N, 4.37. Found: C, 58.44; H, 10.03; N, 4.47. Surface Tension Measurement. The surface tension measurements of SDHC were conducted with a Pt/Ir plate method on a DCAT21 tensiometer (Dataphysics Co., Germany) at 25.00 ± 0.01 °C. The tensiometer was calibrated by measuring pure water before each set of measurements. The tests were repeated at least three times. Potentiometric pH Titration. SDHC was first dissolved in water at a concentration of 1.0 mM, and then the solution pH was adjusted to 12.0 with a small volume of 1 M NaOH. Then 1 M HCl was gradually added into this solution in small portions. The titration process was monitored with a pHS-2C acidity meter, and the temperature was kept at 25.0 ± 0.1 °C throughout the measurement. Three titrations were performed, and the mean pKa values were calculated.


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ζ-Potential Measurement. ζ-Potential measurements for the SDHC and SDHC/CP aggregates were carried out with a Nano-ZS instrument (ZEN3600, Malvern Instruments, Worcestershire, U.K.) equipped with a thermostated chamber and employing a 4 mW He-Ne laser (λ = 632.8 nm) at 25.00 ± 0.01 °C. The ζ-potential measurement of each sample was repeated for three times, and the average value was taken. Dynamic Light Scattering (DLS). DLS measurement for SDHC aggregates was carried out at 25.00 ± 0.05 °C by using an LLS spectrometer (ALV/SP-125) with a multi-τ digital time correlator (ALV-5000) and using the light of λ = 632.8 nm from a solid-state He-Ne laser (22 mW) as incident beam. The measurements were performed at a scattering angle of 90°. All the solutions were prepared after the triply distilled water had been filtered through a 0.45 μM membrane filter of hydrophilic PVDF. The correlation function was analyzed from the scattering data by the CONTIN method to obtain the distribution of diffusion coefficients (D) of the solutes. As a result, the apparent hydrodynamic radius Rh was calculated from the Stokes-Einstein equation Rh = kT/(6πηD), where T is the absolute temperature, k is the Boltzmann constant, and η is the solvent viscosity. DLS measurement for the SDHC/CP aggregates were studied at a scattering angle of 173° on a Nano-ZS instrument (ZEN3600, Malvern Instruments, Worcestershire, U.K.) equipped with a thermostated chamber and a 4 mW He−Ne laser (λ = 632.8 nm). The temperature was controlled at 25.0 ± 0.1 °C. The DLS measurement of each sample was repeated for three times, and the average value was taken. Cryogenic Transmission Electron Microscopy (Cryo-TEM). The SDHC and SDHC/CP solutions at different pH were embedded in a thin layer of vitreous ice on freshly carbon-coated holey TEM grids by blotting the grids with filter paper, and then they were plunged into liquid ethane cooled by liquid nitrogen. Frozen hydrated specimens were imaged by using an FEI Tecnai 20 electron microscope (LaB6) operated at 200 kV with the low dose mode (about 2000 e/nm2) and ACS Paragon Plus Environment

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the nominal magnification of 50000. For each specimen area, the defocus was set to 1-2 μm. Images were recorded on Kodak SO163 film and then digitized by Nikon 9000 with a scanning step 2000 dpi corresponding to 2.54 Å/pixel. Scanning Electron Microscopy (SEM). The morphologies of the SDHC/CP aggregates were imaged by a field-emission scanning electron microscope (Hitachi S-4300). The samples were prepared by freezing a small drop of the SDHC/CP solutions on a clean silica wafer with liquid nitrogen. Immediately afterward, the frozen samples were lyophilized under vacuum at about 58 °C. Finally, a 1-2 nm Pt coating completed the sample preparation. Fluorescence Spectrum and Absolute photoluminescence quantum efficiency. Fluorescence spectra were measured in a quartz cell (1 cm × 1 cm) with both the excitation and emission slits of 10 nm width by a Hitachi model F-4500 spectrofluorometer equipped with a xenon lamp excitation source. The excitation wavelengths of MPS-PPV and PMNT were both fixed at 450 nm. The fluorescence intensity is taken from the emission maxima of the fluorescence emission spectra. Absolute photoluminescence quantum efficiencies were gotten from HAMAMATSU absolute PL quantum yield spectrometer C11347. Reactive Oxygen Species (ROS) Measurement. 2,7-dichlorofluorescein diacetate (DCFHDA) was used to probe the generation of ROS. Under alkaline conditions, DCFH-DA was converted into 2,7-dichlorofluorescin (DCFH), which was followed by transforming into highly fluorescent 2,7-dichloro fluorescein (DCF, excitation 488nm, emission at 524 nm, quantum yield: 90 %) in the presence of ROS. CPs and SDHC/CP aggregates were added into the solutions of activated DCFH (40 μM), respectively. The final concentration of CPs was 10 µM in RU. The solutions were irradiated under white light (3 mW/cm2) for 10 min, and the emission intensity of DCF solution at 524nm was recorded every minute with the excitation wavelength of 488 nm.


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Cell Culture. Human breast cancer (MCF-7) cells were grown in DMEM with 10% FBS at 37 °C under a humidified atmosphere containing 5 % CO2. The cells were routinely passed by treatment with trypsin. Cytotoxicity Assay. MCF-7 cells were seeded in 96-well culture plates at a density of 5×103 cells per well. After 12 h incubation, the culture medium was replaced by fresh culture medium with various concentrations of SDHC, MPS-PPV, PMNT or their aggregates, and the cells were further cultured for 24 hours. For photo-cytotoxicity, the cells were exposed to 3 mW/cm2 white light for 15 min before further cultured. Then the drug was discarded and MTT (5 mg/mL in water, 10 μL per well) in culture medium was added to each well. After incubation for 4 h at 37 °C, the supernatant was abandoned and 100 μL of DMSO was added into each well to dissolve the produced formazan. After shaking the plates for 5 min, absorbance values of the produced purple formazan were recorded with a microplate reader at 570 nm. Cell Imaging and Localization. MCF-7 cells were seeded in 20 mm confocal dishes at a density of 8×104 cells per dish and further cultured in DMEM supplemented with 10 % FBS for 12 h. Then, the CPs with or without SDHC in fresh culture medium were added to the adherent cells. The cells were further cultured for 12 h. Then the culture medium was abandoned and the cells were washed thrice with phosphate buffered saline (PBS, pH 7.4). For cell imaging: after washing thrice with PBS, the images of the cells were taken by confocal laser scanning microscopy (CLSM). For dual-channel imaging: the above cells were directly used to take images by CLSM upon exciting at 488 nm and 559 nm. For localization: Lyso Tracker Blue DND-22 (the final concentration was 1 μM) was added and allowed to incubate with MCF-7 cells for 20 min. The cells were washed thrice with PBS, and their images were taken by CLSM.


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RESULTS AND DISCUSSION Surface Activity and Aggregation of Gemini Surfactant SDHC. Because gemini surfactant SDHC is a pH-sensitive compound, the property and aggregation behavior of SDHC in aqueous solution are highly dependent on pH. So at first, a potentiometric pH-titration was performed to determine the protonation constant pKa of SDHC at 25.0 °C. Figure 1a displays the titration curve at a SDHC concentration of 300 μM. From the titration curve, the protonation constant pKa is found to be 6.6 ± 0.1. Hence, the aggregation behavior of SDHC would be studied at pH 4.0, 7.4 and 10.0. The acid and basic conditions were chosen to ensure that most of the SDHC molecules are completely protonated or deprotonated, while the neutral condition of pH 7.4 was chosen to be consistent with the pH condition in the cell tests. (b)

10

pH

70

4.0 7.4 10.0

γ (mN/m)

60

8 6 4

50 40

(c) 10 ζ potential (mV)

(a) 12

pH

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


0 -10 -20 -30

30

2 0

100 200 300 400 500 600 700 VHCl (µL)

1.0

CSDHC (µM)

10.0

-40

2

4

6

pH

8

10

12

Figure 1. (a) pH titration curve of SDHC at 25.0 °C for a SDHC concentration of 300 μM; (b) Variations of the surface tension with the SDHC concentration at different pHs in buffer solutions (I = 10 mM) at 25.0 °C; (c) The ζ-potential of the SDHC solution plotted against pH at 25.0 °C. The surface tension measurement was employed to measure the CMCs of SDHC at different pH values. The surface tension is plotted against the SDHC concentration as shown in Figure 1b. The CMCs determined from the breaks in the surface tension curves are 1.8 μM, 2.3 μM and 3.2 μM at pH 4.0, 7.4 and 10.0, respectively, which is much lower than most of other ionic gemini surfactants.54 Moreover, the CMC increases with the increase of pH from 4.0 to 10.0. The dominant reason is that the protonation of carboxylic groups at lower pH reduces the electrostatic repulsion between them and induces the formation of the hydrogen bonds between the SDHC molecules,


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which enhances the self-assembling ability of the surfactant. This is similar to the situation of another carboxylate gemini surfactant we reported previously.55 In addition, the hydrogen bond between the hydroxyl groups in the spacer also promotes the self-assembly of the surfactant and in turn lowers the CMC and the surface tension. In order to know the properties of the SDHC aggregates, ζ-potential, DLS and Cryo-TEM were carried out. Figure 1c plots the ζ-potential of the SDHC aggregates against pH at the SDHC concentration of 10 μM, which is above CMC. When the pH is between 9.0 and 12.0, the ζ-potential value of the SDHC aggregates is around -36 mV, and then the ζ-potential value gradually becomes less negative with the decrease of pH. Below pH 4.0, the ζ-potential value becomes about 10 mV. That is, the SDHC aggregates change from negative charged surface to positive charged surface with the decrease of pH. The positively charged surface of the aggregates is attributed to the protonation of carboxylic groups and the formation of quaternary ammonium. The reversible process is shown in Scheme 3. The average hydrodynamic radius of the aggregates from DLS are around 100 nm, 75 nm and 50 nm at pH 4.0, 7.4 and 10.0, respectively, and do not show obvious change with the concentration above CMC (Figure 2a, 2b and 2c). The typical vesicles with diameters of 50~200 nm appear in the Cryo-TEM images (Figure 2d, 2e and 2f ), in line with the results performed by DLS. In brief, the SDHC molecules change from positively charged vesicles to negatively changed vesicles with the pH increasing from 4.0 to 10.0. The fusion of the vesicles56 was seen in the Cryo-TEM image (Figure S1), so to keep the uniformity of the vesicle morphologies, all the following measurements were taken with fresh vesicle solution.

Scheme 3. Chemical structural transformation of SDHC at different H+ concentrations. ACS Paragon Plus Environment

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Figure 2. Size distribution of the SDHC aggregates for different concentrations at (a) pH 4.0, (b) pH 7.4, and (c) pH 10.0. Cryo-TEM micrographs of the aggregates of 1000 μM SDHC at (d) pH 4.0, (e) pH 7.4, and (f ) pH 10.0.

Interaction between SDHC and CPs. The interactions of SDHC with MPS-PPV and PMNT and the resulting effects on the properties of these conjugated polymers were studied by SEM, Cryo-TEM, ζ-potential, DLS, fluorescence emission spectra, and PLQE and ROS measurements. The concentration of MPS-PPV or PMNT used is 10 μM in repeat units. Addition of the SDHC vesicles into MPS-PPV and PMNT solution induces obvious changes in the size, ζ-potential and the morphologies of the aggregates (Figure 3 and Table 1). Without the SDHC vesicles, the MPS-PPV and PMNT polymers separately associate into very large aggregates at micron scale (Figure 3a and 3f ) due to the hydrophobic effect and favorable π−π stacking between the conjugated backbones. Upon the additions of SDHC, the SDHC vesicles disperse the conjugated polymer aggregates through binding on the polymer chain by electrostatic and hydrophobic interactions. When SDHC and the conjugated polymers carry opposite charges, the binding is mainly driven by electrostatic interaction: negatively charged SDHC binds and disperses positively charged PMNT at pH 7.4 and 10.0 and the mixture forms smaller aggregates of about 200-300 nm (Figure 3h and 3i), while positively charged SDHC binds and disperses negatively charged MPS-PPV at pH 4.0 and forming smaller aggregates of about 300 nm (Figure 3b). ACS Paragon Plus Environment

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When SDHC and the conjugated polymers carry the same charges, they cannot effectively bind with each other, and thus the aggregates in the SDHC/MPS-PPV mixture at pH 10.0 (Figure 3d) are the same as those of MPS-PPV itself (Figure 3a) and the aggregates in the SDHC/PMNT mixture at pH 4.0 (Figure 3g) are the same as those of PMNT itself (Figure 3f ). However, although SDHC carries negatively charges at pH 7.4, it still binds and disperses the negatively charged MPS-PPV and leads to the smaller aggregates (Figure 3c). The possible reason is that both SDHC and MPS-PPV show weak electronegativity, and the binding may be driven by the hydrophobic interaction between their hydrophobic moieties and the hydrogen bonds between their ester and hydroxyl groups. As proved by the cryo-TEM images (Figure 3e and 3j, only showing the situations at pH 7.4 as representatives) and the size values from DLS (Table 1), either the SDHC/MPS-PPV aggregates at pH 4.0 and 7.4 or the SDHC/PMNT aggregates at pH 7.4 and 10.0 form vesicles about 250 to 400 nm, slightly larger than that of the SDHC vesicle. These SDHC/CP vesicles may keep the SDHC vesicles as the base and the conjugated polymers are attached to or inserted into the SDHC vesicles, which will be further discussed while discussing the results from the following fluorescence spectra. In brief, the SDHC vesicles can adjust the size, charge property and the morphologies of the conjugated polymer aggregates.

Figure 3. SEM micrographs of (a) MPS-PPV, (b-d) SDHC/MPS-PPV, (f ) PMNT and (g-i) SDHC/PMNT at different pH. Cryo-TEM micrographs of (e) SDHC/MPS-PPV and (j) SDHC/PMNT at pH 7.4. [SDHC] = 25 μM, [CPs] = 10 μM in RU. ACS Paragon Plus Environment

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Table 1. The DLS and ζ-potential values of SDHC, SDHC/MPS-PPV and SDHC/PMNT at pH 4.0, 7.4, and 10.0. [SDHC] = 25 μM, [CPs] = 10 μM in RU. SDHC

4.0 7.4 10.0

(a)

Intensity (a.u.)

25000

25000 pH 4.0 MPS-PPV

CSDHC (µM) 0 10 20 30 40

20000 15000 10000

15000

5000 0

(d)

1200 900

550 600 650 Wavelength (nm)

700 1000

pH 4.0 PMNT

CSDHC (µM) 0 10 20 30 40

600 300 500

500


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SDHC/MPS-PPV d/nm ζ/mV 353.7±19.9 1.2±0.3 234.4±11.9 -20.3±1.6 2334.3±49.4 -48.8±1.7

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Figure 4. Fluorescence emission spectra of (a, b, c) MPS-PPV and (d, e, f ) PMNT with SDHC of different concentrations at pH 4.0, 7.4, and 10.0. λex = 450 nm, [CPs] = 10 μM in RU. 30 25 Quantum Yield (%)

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Figure 5. Variations of absolute photoluminescence quantum efficiencies of MPS-PPV and PMNT against the concentration of SDHC at different pHs (25.0 °C). [CPs] = 10 μM in RU.


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(a) SDHC/MPS-PPV pH 4.0

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Figure 6. Schematic representation for the interaction of the gemini surfactant SDHC with (a, b, c) MPS-PPV and (d, e, f) PMNT at pH 4.0, 7.4, and 10.0. As shown in the fluorescence emission spectra (Figure 4) and the PLQE variations (Figure 5), the photophysical properties of MPS-PPV and PMNT are greatly dependent on the concentration of SDHC and the pH, and the two polymers show different responses to the additions of SDHC vesicles. The relevant mechanism is shown in the cartoon of Figure 6. As to the molecular structures of MPS-PPV and PMNT, the differences between them mainly include: (I) the charge of the side chain; (II) the number of the repeated units; and (III) the rigidity of the conjugated backbone. So the interaction of MPS-PPV with SDHC is far different from that of PMNT. The most significant changes induced by SDHC take place in the MPS-PPV solution at pH 4.0. The fluorescence emission spectra of MPS-PPV with and without SDHC are shown in Figure 4a, 4b and 4c. At pH 4.0, the emission maximum of MPS-PPV in the absence of SDHC is about 528 nm (Figure 4a). The electrostatic repulsion between the negatively charged side chains of MPSPPV may cause the benzene rings to twist, interrupting the conjugation of the delocalized π electrons, while the π-π stacking and hydrophobicity of the MPS-PPV backbone make the polymer ACS Paragon Plus Environment

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aggregate with each other, quenching the fluorescence. With the SDHC concentration (above CMC) increasing, the emission maximum shifts toward longer wavelengths (~543 nm), and the fluorescence intensity increases up to 62-fold at the corresponding emission maximum (Figure 4a). In this process, the absolute PLQE of MPS-PPV is lower than 1 %, and then rapidly rises to 27 % with the increase of the SDHC concentration (Figure 5). This situation can be explained by the effect of SDHC vesicles on the aggregate transition of MPS-PPV. With the addition of SDHC, the negatively charged MPS-PPV may be wrapped around the positively charged SDHC vesicles through both electrostatic attraction and hydrophobic interaction (Figure 6a). The electrostatic attraction offsets the electrostatic repulsion between the MPS-PPV side chains, permitting the coplanarity of the twisted backbone and the increase of conjugation length related to the observed red-shift. At the same time, the presence of the vesicles enhances the water solubility of MPS-PPV, prevents the π−π stacking of the conjugated backbone of MPS-PPV, and thus causes the increase of fluorescence intensity. At pH 7.4, the emission maximum and fluorescence intensity change in the same variation tendency (Figure 4b). The emission maximum shifts from 530 nm to 540 nm. But the varying extent of the absolute PLQE is much smaller, which climbs slowly from 2 % to 8 % with the addition of SDHC. At this pH, the SDHC molecules are close to charge neutrality, and the SDHC vesicles is weakly and negatively charged. In this situation, the dominant interaction between the SDHC vesicle and MPS-PPV is hydrophobic interaction, and MPSPPV backbone is possibly located in the bilayer of the SDHC vesicles (Figure 6b). The electrostatic repulsion and steric hindrance between the vesicles may increase the conjugation length and prevent the π−π stacking of the conjugated backbone of MPS-PPV. However, owing to the weak electrostatic repulsion between SDHC and MPS-PPV, the red-shift of emission maximum (~10 nm) and the increase of fluorescence intensity (~25-fold) are both smaller than in acidic condition. At pH 10.0, the SDHC molecules are fully deprotonated, so both SDHC and MPS-PPV carry negative ACS Paragon Plus Environment

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charges. Strong electrostatic repulsion between SDHC and MPS-PPV results that the SDHC vesicles cannot bind with MPS-PPV anymore (Figure 6c). Therefore, there is no change in the fluorescence spectra of MPS-PPV (Figure 4c) and in the absolute PLQE data (Figure 5). Next, see the fluorescence emission spectra and the absolute PLQE of PMNT as a function of the SDHC concentration (Figure 4d, 4e and 4f, and Figure 5). At pH 4.0, the strong electrostatic repulsion between the same charged SDHC and PMNT inhibits their incorporation. The fluorescence spectra show no difference with the addition of SDHC (Figure 4d). At pH 7.4, the fluorescence intensity decreases with the addition of SDHC, and then increases upon the addition of more SDHC molecules (Figure 4e). The dominant hydrophobic interaction may make PMNT enter the hydrophobic area of the bilayer of SDHC vesicles, while the weak electrostatic attraction causes PMNT to be close to the surface. Because of the short length of the PMNT backbone, PMNT binds with single SDHC vesicle and the SDHC/PMNT aggregates arrange randomly (Figure 6d, 6e and 6f ). The similar models are presented to explain the interaction between poly(thiophene) derivatives and phospholipid vesicles.42, 43 This kind of molecular packing promotes the interchain π−π stacking and the excimer formation of PMNT. Excimers are dimeric molecules formed at excited-state, which can emit at longer wavelength and quench the fluorescence.31, 33 Further increasing the SDHC concentration, the PMNT molecules are dispersed into more SDHC vesicles, resulting in a decrease of the π−π interaction and excimer as well as an increase of fluorescence intensity. At pH 10.0, the fluorescence intensity increases about 3-fold with adding the SDHC vesicles into the PMNT solution (Figure 4f ). In this situation, most anionic PMNT molecules may be bound at both the inside and outside surfaces of the negatively charged SDHC vesicles via strong electrostatic attraction, but the bilayer structure of the SDHC vesicles prevents the intermolecular association of the PMNT molecules. Although the fluorescence intensity of PMNT changes with the addition of SDHC at pH 7.4 and 10.0, the changing extent is ACS Paragon Plus Environment

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much smaller than that of MPS-PPV at pH 4.0 and 7.4. Moreover the absolute PLQE values also do not show obvious changes with the additions of SDHC at all the pH values used. As well known, conjugated polymers can sensitize oxygen molecules to singlet oxygen and other reactive oxygen species (ROS) under white light irradiation. The addition of SDHC also affects this property of MPS-PPV and PMNT. Figure 7 shows the variation of the fluorescence intensity of DCF at 525 nm with the irradiation time in the presence of MPS-PPV, PMNT or the aggregates of the two polymers with the SDHC vesicles. The fluorescence intensity for the SDHC/PMNT aggregates is much lower than that of PMNT. This result demonstrates that the ROS originated from the SDHC/PMNT aggregates is negligible in comparison with that from PMNT. As discussed above, PMNT is located in the bilayer of SDHC vesicles, thus, the SDHC molecules not only isolate PMNT from oxygen molecules, but also prevent the generated singlet oxygen escaping. However, the fluorescence intensity of the MPS-PPV and SDHC/MPS-PPV aggregates shows similar varying trends as the control group, which means that both the MPS-PPV and SDHC/MPS-PPV aggregates do not sensitize oxygen molecules. This is possibly because the long conjugated backbones and sulfonic groups quench the production of ROS. 700 DCFH PMNT PMNT/SDHC MPS-PPV MPS-PPV/SDHC

600 Intensity (a.u.)

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Figure 7. DCF emission intensity at 525 nm as a function of light irradiation time in phosphate buffered saline in the presence of CPs with or without SDHC. Light irradiation intensity: 3 mW/cm2, [SDHC] = 25 µM, [CPs] = 10 µM in RU. The excitation wavelength was 488 nm. The background emission of CPs was deducted.


19


Bioimaging. Imaging materials are expected to have excellent fluorescent properties but nontoxicity to mammalian cells. Thus, the cytotoxicities of these SDHC/CP aggregates were assayed against human breast tumor cell lines (MCF-7 cells) using a standard MTT method in which the conversion of MTT to formazan that is related to the cell viability. MCF-7 cells were incubated with SDHC up to 40 μM in phosphate buffered saline. After incubation with SDHC for 24 h, the cell viabilities remain above 95 % in the concentration range of 0~40 μM (Figure 8a), which indicates that SDHC has very less cytotoxicity against MCF-7 cells. The 25 μM of SDHC was chosen for further experiments. As depicted in Figure 8b, the cell viabilities decrease with the increases of the concentration of CPs, while the cell viabilities of CPs are higher than 80 % in the presence of 25 μM SDHC after 24 h incubation with MCF-7 cells in dark. That is to say, SDHC is beneficial to decrease the cytotoxicity of CPs against MCF-7 cells. It is possibly due to the fact that CPs may be wrapped by the surfactant molecules and the nontoxic surfactant molecules bring the low cytotoxicity to the SDHC/CP aggregates. (c) 120

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Figure 8. Cell viability of MCF-7 cells after incubation with various concentrations of (a) SDHC in dark, (b) MPS-PPV and PMNT with or without 25 μM SDHC in dark, and (c) MPS-PPV and PMNT with or without 25 μM SDHC upon exposure to 3 mW/cm2 white light for 15 min, then for 24 h at 37.0 °C and 5 % CO2. To investigate the photo-cytotoxicity of the SDHC/CP aggregates, photo-cytotoxicity experiments were performed upon exposure to 3 mW/cm2 white light for 15 min. As shown in Figure 8c, it is evident that PMNT shows a more severe photo-toxicity compared to the SDHC/PMNT aggregates, which is in good agreement with the above result that SDHC restricts the production and ACS Paragon Plus Environment

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release of ROS, and reduces the damage of the living cells. These results suggest that the aggregates of the CPs with the surfactant vesicles can be used in living cell imaging because of the low cytotoxicity, good biocompatibility and favorable anti-ROS effects. In order to confirm that the SDHC/CP aggregates could uptake into living cells, we synthesized a compound SDHC-Cy5 as a probe by connecting SDHC to a typical red-light excited sensor Cy-5 (Scheme S1). SDHC-Cy5 possesses the same hydrophobic chain and the same charge as SDHC, so the SDHC-Cy5 molecules are thought to assemble into the SDHC vesicles above CMC. The MCF-7 cells were incubated in DMEM containing 10 % FBS with CPs (10 μM in RU), SDHC (25 μM) and SDHC-Cy5 (1 μM) for 12 h before the cell imaging assay. Figure 9 shows bright green fluorescence emission for CPs and strong red fluorescence emission for SDHC-Cy5 under their corresponding excitation wavelengths. The confocal laser scanning microscopy (CLSM) images merge well, suggesting that both the CPs enter the cells with SDHC simultaneously. In the meanwhile, the CLSM images show that the two SDHC/CP aggregates are accumulated in the cytoplasm.

Figure 9. Cellular localization of CPs (10 μM in RU, excitation at 488 nm, and collection at 500 to 545 nm), SDHC (25 μM) and SDHC-Cy5 (1 μM; excitation at 559 nm, and collection at 570 to 670 nm) after 12 h incubation in MCF-7 cells.


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Figure 10. CLSM images of the CPs or the SDHC/CP aggregates with Lyso Tracker Red DND-99 in MCF-7 cells. (a) CPs channel (excitation: 488 nm; collection: 500 to 545 nm); (b) Lyso Tracker Red DND-99 channel (excitation: 559 nm; collection: 570 to 670 nm); (c) merged images of (a) and (b); (d) bright field images; (e) colocalization of CPs with Lyso Tracker Red DND-99. The false colors of CPs and Lyso Tracker Red DND-99 are green and red, respectively.

To further confirm their cellular localization, a lysosome specific dye (Lyso Tracker Red DND99) was used for colocalization. As shown in Figure 10, after incubation with the SDHC/MPS-PPV aggregates for 12 h and Lyso Tracker for 20 min with MCF-7 cells, the CLSM images for the aggregates and Lyso Tracker merges well, and a high Pearson’s coefficient (0.90) is obtained, while the Pearson’s coefficient for MPS-PPV and Lyso Tracker is only 0.75. These results indicate that the aggregates enter the cells mainly by endocytosis, and the SDHC vesicle wrapped by MPS-PPV prevents the aggregates deviating from lysosome. The environment in the lysosome is acidic (pH


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3.8~5.0), in which the photoluminescence quantum efficiency of the aggregates is significantly enhanced. The similar changes are also found for the SHDC/PMNT aggregates. The Pearson’s coefficient for the SDHC/PMNT aggregates and Lyso Tracker is 0.78, instead of 0.55 in the absence of SDHC, suggesting that the aggregates accumulate in the lysosome of MCF-7 cells. In brief, the CLSM images imply that the gemini surfactant SDHC can improve the performances of the CPs in the field of bioimaging, thus these SDHC/CP aggregates can be used as lysosome-specific imaging materials.

CONCLUSION The present work investigated the effects of a carboxylate gemini surfactant on the aggregation behavior, fluorescence property and bioimaging application of conjugated polymers. It is found that the gemini surfactant SDHC shows very low CMC, and forms positively charged vesicle with diameters of 200 nm at pH 4.0, and negatively charged vesicle with diameters of 100~150 nm at pH 7.4 and 10.0. The SDHC vesicles combine with anionic MPS-PPV at pH 4.0 and 7.4 to obtain aggregates. These aggregates lead to 62- and 25- fold increase of emission intensity as well as 15 nm and 10 nm red-shifting at pH 4.0 and 7.4, respectively. However, the SDHC vesicles bind with cationic PMNT molecules at pH 7.4 and 10.0, which slightly increases the fluorescence intensity of PMNT and prevents the production and release of ROS. Furthermore, the MTT assays reveal that nontoxic SDHC effectively reduces the dark- and photo-cytotoxicity of MPS-PPV and PMNT, laying a good foundation for their biological application. The living cell imaging indicates that the SDHC/CP aggregates can enter the MCF-7 cells simultaneously by endocytosis, and finally localize in the lysosome. Compared with the conjugated polymers themselves, the SDHC/CP aggregates have two advantages in bioimaging: enhancing the photoluminescence quantum effi-


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ciency, and weakening the dark and photo cytotoxicity. This work not only shows new efficient fluorescence probe systems for specific location imaging of living cells, but also provides a new perspective to improve bioimaging application of conjugated polymers by a vesicle-forming gemini surfactant.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Cryo-TEM image of the fusion process of the SDHC vesicles. The variations of maximum emission intensity and wavelength of MPS-PPV and PMNT with SDHC of different concentrations at different pH. Description about syntheses and purification of SDHC-Cy5. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (Shu Wang)；[email protected] (Yilin Wang). Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Note The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (21633002, 21327003). ACS Paragon Plus Environment

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Phenylene-[9,


9-Bis-(4-Phenoxybutylsulfonate)]

Fluorene-2,

7-Diyl}

Copolymer

in

N-

Dodecylpentaoxyethylene Glycol Ether Micelles. Macromolecules 2004, 37, 7425-7427. (38) Sun, L.; Hao, D.; Zhang, P.; Qian, Z.; Shen, W.; Shao, T.; Zhu, C. Indication of Critical Micelle Concentration of Nonionic Surfactants with Large Emission Change Using Water-Soluble Conjugated Polymer as Molecular Light Switch. J. Lumin. 2013, 134, 260-265. (39) Kim, S. I.; Jin, Y. J.; Lee, W. E.; Yu, R.; Park, S. J.; Kim, H. J.; Song, K. H.; Kwak, G. Microporous Conjugated Polymers with Enhanced Emission in Immiscible Two-Phase System in Response to Surfactants. Adv. Mater. Interfaces 2014, 1, 1300029. (40) Knaapila, M.; Evans, R. C.; Garamus, V. M.; Almásy L.; Székely, N. K.; Gutacker, A.; Scherf, U.; Burrows, H. D. Structure and “Surfactochromic” Properties of Conjugated Polyelectrolyte (CPE): Surfactant Complexes between a Cationic Polythiophene and SDS in Water. Langmuir 2010, 26, 15634-15643. (41) Evans, R. C.; Knaapila, M.; Willis-Fox, N.; Kraft, M.; Terry, A.; Burrows, H. D.; Scherf, U. Cationic Polythiophene−Surfactant Self-Assembly Complexes: Phase Transitions, Optical Response, and Sensing. Langmuir 2012, 28, 12348-12356. (42) Houston, J. E.; Kraft, M.; Scherf, U.; Evans, R. C. Sequential Detection of Multiple Phase Transitions in Model Biological Membranes Using A Red-Emitting Conjugated Polyelectrolyte. Phys. Chem. Chem. Phys. 2016, 18, 12423-12427. (43) Houston, J. E.; Kraft, M.; Mooney, I.; Terry, A. E.; Scherf, U.; Evans, R. C. Charge-Mediated Localization of Conjugated Polythiophenes in Zwitterionic Model Cell Membranes. Langmuir 2016, 32, 8141-8153. (44) Li, K.; Liu, B. Polymer Encapsulated Conjugated Polymer Nanoparticles for Fluorescence Bioimaging. J. Mater. Chem. 2012, 22, 1257-1264.


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