Fabrication of photostable PEGylated polymer nanoparticles from AIE monomer and trimethylolpropane triacrylate

Abstract

Biocompatible and stable fluorescent polymer nanoparticles play significant roles in bioimaging and biomedical applications due to their convenient preparation strategies and unique properties. However, the construction methods are still limited now. Herein novel water dispersible and bright fluorescent polymer nanoparticles (PhE-TT-PEG FPNs) with great biocompatibility and stability have been facilely fabricated, and their cellular imaging applications were successfully demonstrated. The PhE-TT-PEG FPNs were readily prepared by self-assembly of amphiphilic copolymer PhE-TT-PEG, which was synthesized through free radical polymerization in one pot from AIE monomer PhE, trifunctional cross-linker TT, and biofavorable monomer PEGMA. A series of characterizations have been conducted to confirm the successful synthesis of PhE-TT-PEG, including gel permeation chromatography, X-ray photoelectron spectroscopy, FTIR spectroscopy, and ¹H NMR spectroscopy. The morphology and optical properties of PhE-TT-PEG FPNs have been tested by transmission electron microscopy, dynamic light scattering, UV-Visible absorption spectroscopy and fluorescence spectroscopy. The PhE-TT-PEG FPNs can emit strong fluorescence with a high quantum yield of 40%, and they also demonstrate superb water dispersibility, morphology stability, photostability and biocompatibility. Finally, the fluorescence imaging of HeLa cells with PhE-TT-PEG FPNs is investigated in detail.

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Introduction

Fluorescent probes play significant roles in sensing, imaging, and biomedical applications owning to their facile operation, great sensitivity, prompt response, and unique fluorescent properties.^1–6 By now, the most utilized fluorescent probes include small-molecule organic fluorophores, inorganic nanoparticles, and organic polymer nanoparticles. Normally, compared with small-molecule fluorophores, nanoparticles demonstrated more superiority in bioapplications because of their stability and capacity for simultaneous, multiple applications due to their high surface area: volume ratio.^7–11 Moreover, the good permeability and enhanced permeability and retention (EPR) effect of nanoparticles makes them a more desirable system for drug delivery and biomedical-related imaging than small-molecule dyes.¹² As for the inorganic nanoparticles, such as quantum dots and up-conversion nanoparticles, they can emit bright and tunable luminescence in a large range of wavelength.^13–19 However, on account of the heavy metals existing in the preparation procedure, the biocompatibility of these inorganic nanoparticles becomes the biggest concern and challenge in their bioapplications.^20–22 Therefore, organic polymer nanoparticles are considered to have more potential for bioimaging and biological applications owning to their numerous fabrication strategies, great biocompatibility, water dispersibility, and so on.^23–28

The organic polymer nanoparticles can be prepared by self-assembly technique through introducing hydrophobic fluorophores and hydrophilic side chains into the backbone of polymers; or by encapsulating fluorophores within biocompatible amphiphilic polymers.^29–33 In both cases, the fluorophores are indispensible. Conjugated polymers (CPs), as the macromolecular emitter, have emerged with distinguished advantages.^5,34–37 As a consequence, many CPs-based nanoparticles have been constructed and have captured much interest.^38–41 Nevertheless, to date the number of CPs is limited, and more fluorophores are still in highly demanded. But most commercial dyes usually have hydrophobic planar structures, which will encounter the notorious problem of aggregation-caused quenching (ACQ).^42–44 To solve this problem, aggregation induced emission (AIE) dyes were fabricated by Tang et al. in 2001, which can emit much intenser fluorescence in their aggregation states.^43,45 Since then, diverse AIE fluorogens and their nanoparticles have been rapidly developed.^46–56 Besides the chosen of fluorophores, the hydrophilic part in the polymer and the cross-linkers also play very significant roles in the successful fabrication of stable fluorescent polymer nanoparticles (FPNs). In the previous reports, glucose is often used to prepare fluorescent glycopolymer nanoparticles in view of their great biocompatibility and diverse biological functions.^57–60 Polyethylene glycol (PEG) is another nontoxic amphiphilic polymer, which is approved by Food and Drug Administration to supplement in plenty of health and beauty aids.^61–63 Therefore, PEGylated polymer would be of great scientific interest to prepare biocompatible FPNs for imaging and other bioapplications. On the other hand, as the nanoparticles fabricated from self-assembly of linear polymers are often unstable, crosslinking technique is also very important in the preparation of stable FPNs. In order to attach more fluorophores and PEG in the polymer, trimethylolpropane triacrylate (TT) is especially picked up as the cross-linker, which is a trifunctional monomer and often be used as plastic components for the medical industry. Accordingly, it would be very interesting and promising to prepare PEGylated, cross-linked FPNs from AIE dyes and TT with great stability and biocompatibility for imaging and biomedical applications.

Herein, a new PEGylated, cross-linked amphiphilic copolymer was facilely synthesized through free radical polymerization of AIE monomer (PhE), trifunctional cross-linker (TT) and poly(ethylene glycol) monomethyl ether methacrylate (PEGMA) in one pot (Scheme 1), which can self-assemble into nanoparticles (PhE-TT-PEG FPNs) with great stability due to their cross-linked structures. It was the first time that TT was employed to construct AIE dyes based FPNs, which provided not only a new product but also a new strategy to fabricate stable cross-linked FPNs. The resulted PhE-TT-PEG FPNs demonstrated high water dispersibility because of the PEGylation of polymer, and their bright fluorescent intensity can be ascribed to the AIE monomer aggregated in the core of FPNs. A series of characterizations have been conducted to confirm the successful synthesis of PhE-TT-PEG, including gel permeation chromatography, X-ray photoelectron spectroscopy, FTIR spectroscopy, and ¹H NMR spectroscopy. The morphology and optical properties of PhE-TT-PEG FPNs have been tested by transmission electron microscopy, dynamic light scattering, UV-Visible absorption spectrum and fluorescence spectrum. At last, the biocompatibility and cellular imaging behavior of PhE-TT-PEG FGNs were investigated in order to evaluate their cellular imaging applications.

Scheme 1 Fabricating route of PhE-TT-PEG copolymers through free radical polymerization of AIE monomer PhE, cross-linker TT and PEGMA in one pot, and subsequent self-assembly of the amphiphilic copolymers to afford PhE-TT-PEG FPNs.

Experimental section

Materials and method

Phosphoryl chloride, 2-(4-bromo-phenyl)acetonitrile, tetrabutylammonium bromide, tetrabutylammonium hydroxide, azobisisobutyronitrile (AIBN), ethyl acetate, N,N-dimethylformamide, 1,2-dichloroethane, and trimethylolpropane triacrylate were purchased from J&K Scientific Ltd and used as received. PEGMA (M_n = 950 Da) was bought from Aldrich company. All other agents were used directly without further purification.

The composition of resulted PhE-TT-PEG FPNs was recorded by X-ray photoelectron spectra (XPS) using a VGESCALAB 220-IXL spectrometer. The chemical structures were characterized by ¹H NMR spectroscopy and FTIR spectroscopy with a JEOL 400 MHz spectrometer and a Perkin-Elmer Spectrum 100 spectrometer (Waltham, MA, USA) in transmission mode, respectively. Gel permeation chromatography (GPC) analyses were performed on a Shimadzu LC-20AD pump system. The optical properties were characterized with UV/Vis/NIR Perkin-Elmer lambda750 spectrometer and PE LS-55 spectrometer (Waltham, MA, USA). The morphology and size distribution of PhE-TT-PEG FPNs were characterized by transmission electron microscopy (TEM) and a zetaPlus apparatus (ZetaPlus, Brookhaven Instruments, Holtsville, NY). The detailed conditions were the same with those described in our earlier reports.

Preparation of PhE-TT-PEG FGNs

PhE was synthesized according to the literature methods.⁶⁴ To prepare PhE-TT-PEG, free radical polymerization in one pot can be adopted to afford the cross-linked amphiphilic polymer. PhE (34 mg, 0.050 mmol), TT (15 mg, 0.50 mmol), a PEG monomer (PEGMA, 475 mg, 0.50 mmol), AIBN (5.0 mg), and ethyl acetate (6 mL) were mixed together, which was then maintained in a 70 °C oil bath for 12 h after being purged with nitrogen flow. The product can be harvested from above mixture after thorough dialysis against water and further lyophilization to remove any undesired molecules. The obtained product was 358 mg with the yield be calculated as about 68.3%.

Cytotoxicity of PhE-TT-PEG FGNs

The effects of PhE-TT-PEG FPNs to HeLa cells were determined by examining the changes of cell morphology according to our previous methods. After cell attachment, PhE-TT-PEG FPNs were employed to incubate with HeLa cells. Then, all samples were carefully washed with PBS, and the cell morphology can be identified by an optical microscopy (Leica, Germany).

Cell counting kit-8 (CCK-8) assay was used to evaluate the cell viability of PhE-TT-PEG FPNs on HeLa cells. Briefly, after cell attachment on 96-well microplates, PhE-TT-PEG FPNs of different concentrations in DMEM solution were added to the wells for incubation of 8 and 24 h. Subsequently, 10 μL of CCK-8 dye with DMEM was added to each well and the plate was incubated at 37 °C for another 2 h. The absorbance at 450 nm was measured on a microplate reader (VictorШ, Perkin-Elmer).

Cellular imaging of PhE-TT-PEG FGNs

The cellular imaging of PhE-TT-PEG FPNs was recorded based on our earlier reports. PhE-TT-PEG FPNs of 20 μg mL⁻¹ were incubated with HeLa cells for a period of time at 37 °C. Then, the cells were fixed with 4% paraformaldehyde, and stained with Hoechst 33 258 (5 μg mL⁻¹) for 5 min in the dark. After extensive washes, the cells were imaged by a confocal laser scanning microscope (CLSM) Zeiss 710 3-channel (Zeiss, Germany). The photostability test of PhE-TT-PEG FPNs in live cells was conducted on Zeiss 710META (Zeiss, Germany) with a live cells culture system. After HeLa cells were incubated with PhE-TT-PEG FPNs for three hours and washed with PBS, the dish was put into the incubator of CLSM with fresh added culture medium. The laser power at 458 nm used in the photostability test was about 30 times of that utilized in the imaging experiments.

Results and discussion

Synthesize and characterization of PhE-TT-PEG

In order to prepare FPNs with intense fluorescence emission, great stability and favorable biocompatibility, the fluorogen PhE with AIE characteristic, cross-linker TT with trifunctional groups, and PEGMA were especially selected for the polymerization reaction. PhE with a high fluorescence quantum yield in its aggregated states was synthesized as described in the earlier report.⁶⁴ Due to the trifunctional groups existed in TT, it can cross-link with plenty of monomers to obtain a stable structure. More importantly, the PEGylation of the polymer would endow them with great water dispersiblity and excellent biocompatibility. As expected, the three monomers can facilely be reacted in one pot via free radical polymerization to afford amphiphilic polymer PhE-TT-PEG (Scheme 2). After the resulted PhE-TT-PEG copolymers were purified, the number average molecular weight (M_n) values of PhE-TT-PEG were determined by GPC and showed as 43 678 Da with a polydispersity index (PDI) of 1.17 (Fig. S2†). In order to characterize the chemical composition of PhE-TT-PEG, X-ray photoelectron spectra (XPS) studies was adopted to determine the elemental information of the polymer. From the raw XPS data of PhE-TT-PEG demonstrated in Fig. 1A, it can be found that carbon and oxygen were the major components along with other two minor components of nitrogen and sulfur. To be specific, the overall wt% of elements present in PhE-TT-PEG was calculated as C : O N : S ∼ 81 : 16 : 2: 1. The main peak of carbon was at 285 eV with a shoulder peak at 286 eV, which were attributed to the sp² and sp³ atoms. The N 1s, O 1s and S 2p XPS spectra of PhE-TT-PEG showed peak at around 398.5, 531.6 and 163.5 eV, respectively (Fig. S1†). These results demonstrated that monomers were successfully incorporated into the PhE-TT-PEG.

Scheme 2 Synthetic route of PhE-TT-PEG via free radical polymerization in one pot.

Fig. 1 (A) XPS spectrum of PhE-TT-PEG indicating the presence of carbon, nitrogen, oxygen, and sulfur. (B) ¹H NMR spectra of PhE, TT, PEGMA and PhE-TT-PEG and (C) magnified ¹H NMR spectrum of PhE-TT-PEG from 6.0 to 8.5 ppm. (D) FTIR spectra of PhE, TT, PEGMA, and PhE-TT-PEG.

The ¹H NMR spectra of monomers and PhE-TT-PEG were conducted using d₆-DMSO as a solvent, which were shown in Fig. 1B. It was found that the characteristic peaks of monomers can be clearly observed in as-prepared PhE-TT-PEG, which demonstrated the successful polymerization of PhE, TT, and PEGMA. To be specific, the characteristic chemical shifts of methylene group in TT and PEGMA located at around 4.1 and 3.6 ppm, respectively, which can be obviously detected at about 3.5 ppm in PhE-TT-PEG. The chemical shift of methylene groups (1.2 ppm) and the multiple sharp peaks of aromatic hydrogen (6.5 to 8.0) from PhE can be also observed in the same region in NMR spectrum of PhE-TT-PEG. Amplified ¹H NMR spectrum of PhE-TT-PEG was shown in Fig. 1C, in which the peaks of aromatic hydrogen were evidently broadened. Meanwhile, the peaks of double bonds from the monomers (5.8 and 5.3 ppm for PhE; 5.6–6.3 ppm for TT and PEGMA) almost disappeared in PhE-TT-PEG, which further proved the successful synthesis of PhE-TT-PEG. According to the integration of methylene groups and PhE, the ratio of PEG segments and PhE (z/y) could be calculated approximately as 11.6.

The successful synthesis of PhE-TT-PEG can also be confirmed by FTIR spectroscopy. As demonstrated in Fig. 1D, the characteristic peaks of C–H stretching vibration from CH₃ and CH₂ groups located at around from 2850 to 2925 cm⁻¹, which could be observed in all the monomers and the resulted PhE-TT-PEG. Meanwhile, the clear characteristic peak at 1750 cm⁻¹ can be observed in both PEGMA and PhE-TT-PEG, which were ascribed to the stretching vibration of C=O band. Furthermore, the peak located at 1040 cm⁻¹ meant the C–O stretching vibration band, which can be clearly observed in PEGMA and PhE-TT-PEG; and another peak located at 1110 cm⁻¹ could be attributed to the C–N stretching vibration band.^27,30 All these results confirmed the successful formation of PhE-TT-PEG.

Preparation of PhE-TT-PEG FPNs

It has been proved that the amphiphilic polymers could readily turn into nanoparticles when they were dispersed in aqueous solution through self-assembly technique, which has become a particularly facile and promising strategy to form nanomaterials for various biomedical applications.^65–67 Thus the obtained PhE-TT-PEG copolymers facilely self-assembled into nanoparticles when they were dispersed in phosphate buffer solution (PBS, pH 7.4), with the hydrophobic PhE in the core and hydrophilic PEG on the surface. Due to the PEGylation of polymer, these obtained PhE-TT-PEG FPNs demonstrated great water dispersibility, which was favorable for bioapplications. In order to clearly identify their morphology in PBS, PhE-TT-PEG FPNs were characterized by transmission electron microscopy (TEM). Results of PhE-TT-PEG FPNs in PBS. From Fig. 2A, it can be obviously indicated that the PhE-TT-PEG copolymers had been successfully self-assembled into nanoparticles of 20 to 70 nm, which were not quite uniform. In addition, it was determined by zetaPlus particles size analyzer that PhE-TT-PEG FPNs in PBS demonstrate a size distribution of 88.9 ± 1.6 nm with a polydispersity index (PDI) of 0.207 (Fig. 2B), which was larger than those sizes observed through TEM owning to the hydrodynamic effects and drying-causing shrinkage.

Fig. 2 (A) TEM image of PhE-TT-PEG FPNs dispersed in water, scale bar = 200 nm. (B) Dynamic light scattering results of PhE-TT-PEG FPNs in PBS.

Fluorescence property and stability

After the amphiphilic PhE-TT-PEG copolymers self-assembled into nanoparticles, the AIE monomer PhE was aggregated into the hydrophobic cores of FPNs, which endowed the obtained PhE-TT-PEG FPNs with intense fluorescence. Before the research on their luminescent emission, the UV-Vis absorption spectrum of PhE-TT-PEG FPNs was investigated as shown in Fig. 3A, in which two peaks located at around 330 and 430 nm can be evidently observed. The absorption signals were collected from the 800 nm to 250 nm, it could be found that the absorption spectrum began to grow from the starting wavelength of 800 nm due to the Mie effect, which also indicated the existence of nanoparticles in the solution. To study the fluorescence property of PhE-TT-PEG FPNs in water, their fluorescence spectrum was harvested and shown in Fig. 3B, demonstrating the excitation wavelengths at around 330 nm and 438 nm with maximum emission wavelength at around 580 nm. In order to avoid the influence from other emission, the excitation wavelength used for the acquisition of fluorescence spectrum was set as 488 nm, which was far from the first absorption peak around 330 nm but in the range of the second absorption peak. Owning to the aggregation of the AIE components in the core, these PhE-TT-PEG FPNs emitted strong orange fluorescence with a high quantum yield value of about 40%, being determined using quinine sulphate as the reference dye. It was studied in our previous papers that the quantum yields value would decrease if the relative ratio of AIE monomer in the self-assembled polymer was reduced.^27,67 Thanks to the great water dispersibility and strong fluorescence emission, these PhE-TT-PEG FPNs were considered to have prominent advantages for bioimaging applications.

Fig. 3 (A) The UV-Vis absorption spectra of PhE-TT-PEG FPNs dispersed in water. (B) Fluorescence excitation (ex) and emission (em) spectra of PhE-TT-PEG FPNs dispersed in water (λ_ex = 488 nm).

Moreover, the stability of PhE-TT-PEG FPNs also played very significant roles in their bioapplications. First of all, the photostability of PhE-TT-PEG FPNs was measured to be quite satisfactory with almost no fluorescence intensity decrease even after 30 min continuous eradiation (Fig. 4A), which was an indispensible quality required in durable tracing and imaging inspections. Secondly, the AIE dye leakage study was carried out to test their structure stability. Thanks to the stable cross-linked linkage, there was no dye leakage even after dialysis against THF or PBS solution for three days using 7000 Da M_w cut-off membranes (Fig. S3†). Besides, their hydrodynamic size distribution and fluorescence intensity can also remain stable in PBS of different pH values (Fig. S4†). To further investigate the morphology stability of PhE-TT-PEG FPNs in water, the fluorescence intensity vs. the logarithm of their concentration was conducted to determine the critical micelle concentration (CMC). To be specific, the fluorescence spectra of PhE-TT-PEG FPNs with different concentrations in water were measured under the excitation wavelength of 488 nm, and then the emission intensities at 580 nm were collected to plot the intensities vs. the logarithm of these different concentrations. At the concentration below CMC, the detected emission intensity was very low since the copolymer had not been self-assembled into nanoparticles. However, when the concentration of PhE-TT-PEG reached above CMC, the aggregated emission increased abruptly. Therefore, two straight lines can be drawn up according to the different growth rates of fluorescence intensities as shown in Fig. 4B, and the traced intersection of the two lines was corresponding to the CMC. Thanks to the cross-linked structure of PhE-TT-PEG, the CMC was calculated as low as 0.0057 mg mL⁻¹. Therefore, the obtained PhE-TT-PEG FPNs would demonstrate great stability in both morphology and luminescence.

Fig. 4 (A) The fluorescence time traces of PhE-TT-PEG FPNs monitored at 580 nm (λ_ex = 488 nm) for 30 min, suggesting the durable photostability of the PhE-TT-PEG FPNs. (B) Intensity of the fluorescence emission vs. logarithm of the concentration of PhE-TT-PEG FPNs (λ_ex = 488 nm, λ_em = 580 nm) emission when the concentration of PhE-TT-PEG in aqueous solution above CMC.

Biocompatibility of PhE-TT-PEG FPNs

To apply PhE-TT-PEG FPNs in biological applications, biocompatibility was a pre-requirement which has to be met. In this case, the biocompatibility of PhE-TT-PEG FPNs to HeLa cells was determined prior to the cellular imaging applications. Optical microscopy was employed to examine the influences of PhE-TT-PEG FPNs to HeLa cells morphology after the cells were incubated with different concentrations of PhE-TT-PEG FPNs for 24 h. It can be seen from Fig. 4 that the cells were still in good condition and adhered to the cell plate well. Being compared with the control cells (Fig. 5A), there was no obvious changes in the morphology and number of cells incubated with PhE-TT-PEG FPNs, even when the concentration of PhE-TT-PEG FPNs was as high as 80 μg mL⁻¹ (Fig. 5C). Furthermore, in order to quantitatively evaluate the cytocompatibility of PhE-TT-PEG FPNs, cell counting kit-8 (CCK-8) assay was used to test the cell viability of HeLa cells against PhE-TT-PEG FPNs. As demonstrated in Fig. 5D, there was no evident cell viability decrease after the cells were incubated with PhE-TT-PEG FPNs for 8 and 24 hours, respectively. Even when the concentration of these PhE-TT-PEG FPNs reached up to 120 μg mL⁻¹, the cell viability value was still better than 90% after incubation of 24 hours. All these results indicated that the PhE-TT-PEG FPNs are biocompatible to HeLa cells, which made them have great potential for bioapplications.

Fig. 5 Biocompatibility evaluation of PhE-TT-PEG FPNs. (A–C) optical microscopy images of HeLa cells incubated with different concentrations of PhE-TT-PEG FPNs for 24 h, (A) control cells, (B) 40 μg mL⁻¹, (C) 80 μg mL⁻¹, (D) cell viability of PhE-TT-PEG FPNs with HeLa cells for 8 and 24 h, respectively.

Cellular imaging and photostability

In view of the excellent biocompatibility, water dispersibility, stability and fluorescence property of PhE-TT-PEG FPNs, their cellular uptake behavior and cellular imaging were investigated by CLSM observation to evaluate their potential biomedical applications. Hoechst 33 258 was used to stain the cell nuclei before cellular imaging studies. First of all, the cellular uptake of PhE-TT-PEG FPNs was studied by incubation FPNs with HeLa cells for different periods of time. As demonstrated in Fig. 6, after incubation with HeLa cells for two hours using PhE-TT-PEG FPNs of 20 μg mL⁻¹, only a small quantity of PhE-TT-PEG FPNs were uptaken by HeLa cells with weak fluorescence emission. However, after incubation with HeLa cells for four hours using PhE-TT-PEG FPNs of the same concentration under laser irradiation of same intensity, it can be obviously identified that plenty of PhE-TT-PEG FPNs lightened HeLa cells with bright luminescence. Meanwhile, from Fig. 6, it can also be found that abundant PhE-TT-PEG FPNs localized in the cytoplasm of HeLa cells with intense orange fluorescence while Hoechst 33 258 localized in the nuclei. In order to clearly reveal the localization of PhE-TT-PEG FPNs in HeLa cells, a magnified fluorescent image of HeLa cells incubated with 20 μg mL⁻¹ of PhE-TT-PEG FPNs for four hours was demonstrated in Fig. 7, from which it can be evidently seen that the FPNs did localize in the cytoplasm of HeLa cells. These images meant that PhE-TT-PEG FPNs could be facilely uptaken by HeLa cells, which was assumed through endocytosis of the cells, and also demonstrated their excellent tracing ability.^39,40

Fig. 6 CLSM images of HeLa cells incubated with PhE-TT-PEG FPNs for 2 h (A, B and C), 3 h (D, E and F), and 4 h (G, H and I), respectively. (A, D and G) The cell nuclei were stained by Hoechst 33 258 under excitation wavelength of 405 nm; (B, E and H) fluorescent images were taken after incubation with PhE-TT-PEG FPNs of 20 μg mL⁻¹ under the excitation wavelength of 458 nm; (C, F and I) the corresponding overlay images. Scale bar = 50 μm.

Fig. 7 CLSM images of HeLa cells. (A) the cell nuclei were stained by Hoechst 33 258; (B) fluorescent image taken after incubation with PhE-TT-PEG FPNs for 4 h; (C) differential interference contrast image; (D) the corresponding overlay image. Scale bar = 20 μm.

Although the photostability of PhE-TT-PEG FPNs in water was satisfactory as revealed above in Fig. 4A, the photostability of PhE-TT-PEG FPNs in cells was also particularly crucial in practical imaging applications. Therefore, the fluorescence change of PhE-TT-PEG FPNs in HeLa cells upon continuous laser irradiation at 458 nm for 20 min was tested using the same intensity of laser power as that in the imaging experiments (Fig. 8). After irradiation, the PhE-TT-PEG FPNs still possessed about 95% of their initial fluorescence intensity, which indicated their excellent photostability in cells and that would greatly benefit their long-term cell tracing applications. The photostability of PhE-TT-PEG FPNs in live cells was also determined using increased laser intensity, about 30 times of above laser power that employed in imaging experiments. After such continuous and violent laser irradiation for more than 8 minutes, the fluorescence intensity of PhE-TT-PEG FPNs reduced to about 55% of their initial intensity (Fig. S6†), when the live cells had been burned to death by the powerful laser for a long time. Actually, even at the first minute after the irradiation, the cells began to shown signs of rupture at the edge of membrane. After 8 min, the cells were completely dead but the fluorescence intensity of PhE-TT-PEG FPNs can still remain 55%, which proved their powerful photostability in live cells (Fig. S7†).

Fig. 8 Photostability of PhE-TT-PEG FPNs in HeLa cells under continuous scanning at 458 nm. Insets show confocal images of PhE-TT-PEG FPNs stained cells before (0 min, left) and after the laser irradiation for 20 min (right). Scale bar = 20 μm.

Conclusions

In conclusion, we facilely fabricated water dispersible, stable, bright and biocompatible PhE-TT-PEG FPNs, and successfully demonstrated their cellular imaging applications. The PhE-TT-PEG FPNs were readily prepared by self-assembly of amphiphilic copolymer PhE-TT-PEG, which was synthesize through free radical polymerization in one pot from AIE monomer PhE, trifunctional cross-linker TT, and biofavorable monomer PEGMA. The introduction of AIE monomer makes these PhE-TT-PEG FPNs emit strong fluorescence with a high quantum yield of 40%, and the PEGylation of the copolymer endows the resulted self-assembled PhE-TT-PEG FPNs with superb water dispersibility and biocompatibility. Meanwhile, thanks to the cross-linking of the monomers with the trifunctional TT, the obtained PhE-TT-PEG FPNs demonstrated excellent stability with low CMC of 0.0057 mg mL⁻¹. More importantly, the PhE-TT-PEG FPNs also demonstrated excellent photostability in HeLa cells, which made them highly potential for durable imaging applications. This work provides a new construction method for preparation of florescent polymer nanoparticles with great stability and biocompatibility. Further efforts will be made for further introduction of other functional moieties and their biomedical applications.

Acknowledgements

This research was supported by the National Science Foundation of China (No. 21134004, 21201108), and the National 973 Project (No. 2011CB935700), and the China Postdoctoral Science Foundation (2015M571018).

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Footnote

† Electronic supplementary information (ESI) available: Detailed information about XPS spectra, GPC chart, absorption spectra of the filtrate after dialysis, hydrodynamic size changes and fluorescence intensity changes of PhE-TT-PEG FPNs in PBS of different pH values and concentrations, photostability in live HeLa cells. See DOI: 10.1039/c5ra16258k

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