In recent years,conjugated polymers (CPs) with unique opto-electronic properties have attracted much attention as prevalent fluorescence materials^[1]. Owing to the delocalized π-electronic structure,CPs display outstanding light-harvesting and light-amplifying properties,and have been applied in biological and chemical detections as an ideal optical probes^[2,3,4]. In order to meet the requirements of biological applications,water soluble conjugated polymers were prepared by modifying CPs with hydrophilic side chains^[5,6]. Among these water soluble conjugated polymers,poly(fluorene)s are one of the most common species in biological applications because of their facile substitution at the fluorene C9 position and excellent optical properties^[7]. Besides side-chain modifications,many other CPs-based systems (such as dendritic conjugated polymers) have been developed to improve the water-solubility^[8,9]. Despite the many efforts,there are still some problems existed such as complicated preparation and purification processes,which limit their further application.

Very recently,conjugated polymer nanoparticles (CPNs) have been prepared and developed rapidly because of their versatility and outstanding properties,such as high brightness,good photostability,biocompatibility and non-toxicity^[6]. In the meanwhile,CPNs have many unique features such as facile chemical synthesis,tunable spectral properties,and versatile surface modification^[10]. Owing to these excellent characteristics,CPNs have been developed in cross-disciplinary areas of chemistry,biology,materials and medicine. Particularly in the biological field,many multifunctional CPNs have been prepared and used in cellular imaging,gene and drug delivery,and photodynamic therapy^{[11,12,13,14]}. It is noted that pH-responsiveness of biophysical environment has been utilized in tumor diagnosis or antitumor drug delivery due to the various pH values in different tissues and cellular compartments^{[15, 16]}. Owing to the high sensitivity and low background noise,fluorescence pH sensing has been achieved in imaging cancer cells and other biological applications with good selectivity^{[17, 18]}. In this contribution,we designed and synthesized a new conjugated polymer PFPA and the nanoparticles were prepared subsequently. The PFPA nanoparticles disperse very well in water and exhibit good optical properties,low cytotoxicity and pH responsive ability. Moreover,the PFPA nanoparticles can easily enter cytoplasm and serve as cell imaging probes,which exhibit potential value in biological applications.

All chemicals were procured from Aldrich Chemical Company,Alfa-Aesar or Acros and used as received. All organic solvents were purchased from Beijing Chemical Works and used without further purification. 2,7-dibromo-9,9-bis(2′-(2″-(2'''-bromoethoxy)ethoxy)ethyl)fluorene was prepared according to the reported procedures^[22]. Dulbecco's modified Eagle's medium (DMEM) was purchased from HyClone/Thermo Fisher (Beijing,China). Neonatal bovine serum (NBS) was purchased from Sijiqing Biological Engineering Materials(Hangzhou,China).(3-(4′,5′-Dimethylthiazol-2′-yl)-2,5-diphenyl-2H-tetrazolium hydrobromide) (MTT) was obtained from Xinjingke Biotech. (Beijing,China) and dissolved in 1×PBS before use.

The ¹HNMR and ¹³CNMR spectra were recorded on Bruker ARX 400 or ARX 500 instrument with tetramethylsilane as the internal standard. Mass spectrometry (MS) (electron impact (EI)) was recorded on a Shimadzu GCMS spectrometer. Elemental analysis was carried out on a Flash EA1112 instrument. GPC analysis was performed on a Waters Styragel system using polystyrene as a calibration standard with THF as the eluent. The UV-Vis absorption spectrum was taken on a JASCO V-550 spectrophotometer. The fluorescence spectrum was measured on a Hitachi F-4500 fluorometer equipped with a Xenon lamp excitation source. The MTT assay was performed on a BIO-TEK Synergy HT microplate reader.

2,7- dibromo- 9,9- bis(2′- (2″- (2'''- bromoethoxy) ethoxy) ethyl)fluorene 1 (0.8 mmol,0.57 g),sodium azide (2.2 mmol,0.144 g) and thionyl chloride (20 mL) were mixed together in a 50 mL flask. The reaction mixture was kept in the temperature of 70 ℃ for 12 h to ensure the completeness of the reaction. The resulting mixture was cooled to room temperature. After adding 30 mL distilled water,the mixture was extracted with CHCl₃ three times. The combined organic layer was washed with distilled water and then dried with anhydrous NaSO₄. 2,7-dibromo-9,9-bis(2′- (2″- (2'''-azidoethoxy)ethoxy)ethyl)fluorene (compound 2 ) was obtained after the organic solvent was removed. Compound 2 was applied to the synthesis of compound 3 without further characterization or purification. Compound 2 (obtained from aforementioned steps) and triphenylphosphine (2.2 mmol,0.577 g) were added to the mixed solvent of THF/H₂O (22 mL/3 mL). The mixture was kept in the temperature for 12 h,followed by adding di-tert-butyldicarbonate (2.2 mmol,0.480 g) in THF (4 mL). After reacting for another 12 h,the reaction was terminated and cooled to room temperature. The solvent was removed and the residue was purified by silica gel chromatography using petroleum ether/ethyl acetate (2∶1) as the eluent to afford the white crystal (0.515 g,82.1%). ¹HNMR (400 MHz,CDCl₃) δ=7.50 (M,J=9.6,7.7,1.3 Hz,6H),4.91 (s,2H),3.42 (t,J=5.1 Hz,4H),3.38-3.30 (m,4H),3.25 (d,J=4.7 Hz,4H),3.20-3.13 (m,4H),2.79 (t,J=7.2 Hz,4H),2.35 (t,J=7.2 Hz,4H),1.57 (s,3H),1.44 (s,18H); ¹³CNMR (100 MHz,CDCl₃ ) δ: 155.94,150.94,138.47,130.71,126.75,121.68,121.25,79.19,77.30,77.05,76.79,70.22,70.07,69.99,66.88,51.98,40.29,
39.43,28.46; EI-MS (m/z): 809.0 [Na⁺]; Anal. alcd for C₃₅H₅₀Br₂N₂O₈: C 53.44,H,6.41.N 3.56. Found: C 52.86,H 6.32,N 3.51.

A mixture of monomer 3 (0.38 mmol,300 mg) and monomer 4 (0.38 mmol,93.4 mg) in toluene (6.0 mL) and 2 mol L -1 aqueous sodium carbonate (3.0 mL) was degassed with nitrogen for 30 minutes. Afterwards,PdCl₂(dppf) (10 mg) was added under nitrogen atmosphere. The resulting mixture was stirred at 90℃ for 48 h. After cooled to room temperature,50 mL chloroform was added and the mixture was filtrated to remove the solid impurities. The organic solution obtained was washed three times with diluted water (30 mL) and dried with anhydrous NaSO₄. After the organic solvent was removed,the residue was precipitated into methanol to afford the product as a dark-coloured solid (198 mg,73.6%). Gel permeation chromatography (GPC) (THF,polystyrene standard),M_w:11921; polydispersity:2.32; ¹HNMR (400 MHz,CDCl₃) δ =7.65 (d,J=132.8 Hz,10H),4.93 (s,2H),3.30 (d,J=62.3 Hz,16H),2.92 (s,4H),2.45 (d,J=76.9 Hz,4H),1.70-0.87 (m,18H).

PFP-Boc (0.28 mmol,198 g) was solved into the chloroform (5 mL),and hydrochloric gas was bubbled into it to eliminate the tert -butoxycarbonyl (—Boc) group. Methanol was added when there was some solid precipitated out from the solvent during this process to make sure that the reaction was kept in a homogeneous phase. After about 6 h,the BOC group was almost eliminated and the solvent was removed to get the brown solid (0.146 g,90.7%).¹HNMR (400 MHz,CDCl3) δ=8.10-7.64 (m,10H),3.54 (s,4H),3.37 (d,J=18.9 Hz,4H),3.31 (s,16H),3.00 (s,8H),2.62 (s,4H),1.28 (s,0.33H).

PFPA (32.8 mg) was solved in methanol (6 mL),followed by filtrating with filter (0.2 μL) to afford the matrix solution. The matrix solution (1.0 mL) was rapidly added into 15 mL ultrapure water under ultrasonication and kept in the ultrasonic state for another 5 min. The resulted solution was heated to 110 ℃ under nitrogen atmosphere. After the methanol and part of the water were evaporated,the homogeneous nanoparticles dispersion was obtained. The size distribution of PFPA nanoparticles was measured by the dynamic light scattering (DLS) experiment.

The photophysical properties of PFPA nanoparticles were investigated in aqueous solution with standard quartz cells. The fluorescence quantum yield of PFPA nanoparticles in water was measured using quinine sulfate as the standard. The photostability of PFPA nanoparticles was also investigated. The solution of PFPA nanoparticles was dropped on a glass plate and covered with a coverslip. The sample was continuously irradiated for 2 min by a mercury lamp (100 W) with an excitation filter of 380/30 nm and an emission filter of 460/50 nm. The exposure time was 100 ms. The emission intensity of PFPA nanoparticles was recorded at 5 s intervals.

Human cervical carcinoma cells (HeLa cells) were grown in Dulbecco′s modified Eagles medium (DMEM) with 10% fetal bovine serum at 37 ℃ under 5% CO₂ atmosphere. The cells were seeded in a 96-well culture plate with the concentration of 8×10⁴ cell/mL and grown for 24 h. When the cells were approximately 80% confluent,PFPA nanoparticles with different concentrations were added to the culture medium respectively. A control group in the absence of PFPA nanoparticles was also carried out. After treatment for 24 h,the culture medium was replaced with 100 μL of 1 mg·mL -1 MTT in phosphate-buffered saline (PBS,pH 7.4) solution each well followed by incubation at 37℃ for 4 h. The supernatant was abandoned,and DMSO (100 μL) per well was added to dissolve the produced formazan and the plates were shaken for an additional 5 min. The absorbance values of the wells were then read with microplate reader at a wavelength of 520 nm. The cell viability rate (VR) was calculated according to the following equation:

The pH-responsiveness of PFPA nanoparticles was investigated in the phosphate-buffered saline (PBS) with the polystyrene cuvette. The fluorescence spectra were measured on a Hitachi F-4500 fluorometer equipped with a Xenon lamp excitation source. The pH of the 1×PBS was adjusted with 1 mol·L -1 HCl aqueous solution and 1 mol·L -1NaOH aqueous solution to different pH values. The PFPA nanoparticles with the same concentration were added to the buffer solutions. The fluorescence spectra of these samples were measured after stabilized in room temperature for 10 min,respectively. The excitation wavelength and the excitation voltage of the PFPA nanoparticles were 390 nm and 700 V.

PFPA nanoparticles were added into 1 mL of medium containing HeLa cells in 35 mm×35 mm plates (the final concentration of PFPA nanoparticles in medium was 17 μmol·L -1). A control group in the absence of PFPA nanoparticles was also carried out. After incubating at 37 ℃ for 24 h,the DMEM medium was replaced and washed twice with PBS (pH 7.4),and then the fluorescence images of the cells were recorded with fluo rescence microscopy. The phase-contrast images were taken at 100 ms exposure time for both experimental and control groups. The fluorescence images were taken at 500 ms exposure time for PFPA nanoparticles and the type of light filter was a D380/30 nm exciter,and D460/50 nm emitter.

The synthesis procedure of PFPA is outlined in Scheme 1. 2,7- dibromo- 9,9- bis(2′-(2″-(2'''-azidoethoxy)ethoxy)ethyl)fluorene ( 2 ) was prepared by reacting 2,7-dibromo- 9,9- bis(2′-(2″-(2'''-bromoethoxy)ethoxy)ethyl) fluorene ( 1 ) with NaN₃ in dimethyl sulfoxide (DMSO). Compound 3 was obtained through reduction of compound 2 by triphenylphosphine (PPh₃) followed by protecting with di-tert-butyldicarbonate with a yield of 82%. PFP-Boc was synthesized by Suzuki coupling of monomers 3 and 4 (molar feed ratio was 1∶1) and purified by repeated precipitation of chloroform solution into methanol with 74% yield. Gel permeation chromatography (GPC) analyses show that PFP-Boc has a weight-average molecular weight (M_w) of 11920 with a polydispersity index (PDI=M_w/M_n) of 2.32. The targeted polymer PFPA was achieved in 91% yield by eliminating reaction with hydrochloric gas to PFP-Boc . The ¹HNMR spectrum indicated that the elimination of Boc group was completely.

Fig .1

The synthesis route of PFPA

PFPA has a hydrophobic backbone and hydrophilic side chains. The nano-precipitation method was used to prepare PFPA nanoparticles^[11]. Methanol was selected as the "good" organic solvent with the ability of dissolution of PFPA and miscibility with water. The procedure involves a rapid injection of PFPA in methanol solution into water under ultrasonication. A significant change of solvent polarity leads to the aggregation of polymer chains,resulting in the formation of PFPA nanoparticles. Dynamic light scattering (DLS) measurement shows that the average size is approximately 8 nm (Figure 1).

Fig. 1

Dynamic light scattering analysis of PFPA nanoparticles in aqueous solution

The UV-Vis absorption and fluorescence emission spectra of PFPA nanoparticles were measured in water solution (Figure 2). As shown in Figure 2A,the absorption spectrum of PFPA nanoparticles in water exhibits a maximum peak at 379 nm corresponding to the π-π^* transitions of the conjugated polyflorene backbone. The molar absorption coefficient is 2.1×10⁶ L ·mol -1·cm -1. The emission spectrum shows a maximum peak at 422 nm. The fluorescence quantum yield (QY) of PFPA nanoparticles in water is 35 % using quinine sulfate as the standard.The high QY benefits from the polyflorene backbone. The photostability of PFPA nanoparticles was also investigated. As shown in Figure 2B,upon exposure to a mercury lamp (100 W) for 120 s,53 % of the fluorescence intensity of PFPA nanoparticles still remains. Compared with common fluorescence dyes,the photostability of PFPA nanoparticles is rather high and desired for cell imaging application^[19].

Fig. 2

Optical properties of PFPA nanoparticles: (A) Normalized UV-Vis absorption and fluorescence emission spectra in water. (B) Photostability of PFPA nanoparticles upon irradiation under a mercury lamp (100 W),the excitation filter is 380/30 nm

Cytotoxicity is an important consideration of fluorescent materials for cell imaging and other biological applications. Cell cytotoxicity of PFPA nanoparticles was evaluated via a standard assay,in which the conversion of 3-(4′,5′-dimethylthiazol-2′-yl)-2,5-diphenyl-2H-tetrazolium hydrobromide (MTT) into formazan is related to mitochondrial activity and thereby reflecting the cell viability. In this experiment,HeLa cells were firstly incubated with different concentrations of PFPA nanoparticles in a 96-well cell culture plate for 24 h. After the incubation,the cell viability was measured. As shown in Figure 3,the PFPA nanoparticles do not show obvious cytotoxicity even at the concentration of 128 μmol·L -1. The result indicates that these nanoparticles possess good biocompatibility and could be used for cell imaging^[6].

Fig. 3

Cell viability of HeLa cells after incubated with various concentrations of PFPA nanoparticles by a typical MTT assay

The pH values in variant tissues and cellular environment are always different,for example the relatively acidic environment of cancer cells compared to normal cells. This physiological feature inspires the design of fluorescence pH responsive PFPA nanoparticles which may be applied in distinctive imaging the cancer cells from the normal cells. As shown in Figure 4,PFPA nanoparticles display a stable and bright fluore- scence signal in the acidic buffer solution. With the increase of pH value from 5.3 to 9.5,the fluorescence intensity decreases from 6100 to 1600 and keep the low intensity in the basic buffer solution. The pH responsive ability of the nanoparticles is attributed to the protonation/deprotonation of the primary amine groups in the side chain of PFPA . The amine groups exist in the form of ammonium in an acidic environment. With the increase of pH value,the amine groups are deprotonated gradually and the aggregation of PFPA nanoparticles causes the reduction of fluorescence intensity. The pH-responsive property can be applied in cancer diagnosis because of the acidic environment of cancer cells. Except for the fluorescence signal variation responding to the pH change,the primary amine groups of PFPA can be modified with anhydrides or other moieties^[20,21].

Fig. 4

Fluorescence pH responsive property of PFPA nanoparticles: (A) the fluorescence spectra of PFPA nanoparticles in different PBS solution. (B) The maximum emission wavelength with the variation of pH values. The excitation wavelength is 390 nm

The advantages such as high brightness,satisfactory photostability,and nontoxicity make PFPA nanoparticles a competitive candidate for the cellular imaging application. In this study,the PFPA nanoparticles were applied in fluorescence imaging for Hela cells. The HeLa cells were incubated in the DMEM medium containing PFPA nanoparticles (the final concentration of PFPA nanoparticles was 17 μmol·L -1). After incubating at 37℃ for 24 h,the DMEM medium was replaced and washed twice with PBS (pH 7.4). The phase-contrast images (Figure 5A) and fluorescence images (Figure 5B) were recorded using a fluorescence microscopy. According to the overlap image (Figure 5C) of phase contrast and fluorescence,we can learn that PFPA nanoparticles were mainly located in cytoplasm. Thus,the PFPA nanoparticles can be endocytosed by cells and mainly distribute in the cytoplasmic region.

Fig. 5

Cell imaging of HeLa cells using PFPA nanoparticles: (A) phase-contrast image; (B) fluorescence image;(C) overlap image of phase contrast and fluorescence of HeLa cells incubated PFPA nanoparticles. [PFPA nanoparticles]=17 μmol·L -1. Fluorescence images were taken with a D380/30 nm excitation filter and D460/50 nm emitter with 500 ms exposure time. Magnification of the objective lens was 10 ×

In conclusion,a new conjugated polymer PFPA was synthesized and the nanoparticles were prepared using a nano-precipitation method. The obtained PFPA nanoparticles were applied to fluorescence imaging for HeLa cells. This new-designed fluorescent probe has several features. Firstly,PFPA nanoparticles display outstanding optical properties,good biocompatibility and high quantum yield because of π-conjugated polyfluorene backbone. Secondly,PFPA nanoparticles have terminal amine groups leading to the pH-responsive behavior reflected by the changes of fluorescence signal. The pH-responsive property can be applied in tumor diagnosis because of the acidic environment of cancer cells. Moreover,the free primary amine groups can be modified with many chemical or biological elements. Thus,the PFPA nanoparticles show great potential as probe for imaging and also vehicle for drug or gene delivery.