Yan Cui,Ruowen Zhang,Li Yang,Shijie Lv*

a Jilin Medical University, Jilin 132013, China

b Beihua University, Jilin 132013, China

c First Hospital of Jilin University, Changchun 130021, China

Keywords:

AIE

Nanoparticles

Self-assembly

Non-invasive

Long-term bio-imaging

ABSTRACT

Fluorescent organic nanoparticles(NPs)based on aggregation-induced emission(AIE)-active molecules,are widely applied as non-invasive tools in bio-imaging and demonstrate great potential for studying physiological and pathological processes.In this paper, we report the synthesis of highly emissive AIEgen-based NPs(SCA NPs)via reprecipitation without any carriers as long-term cell trackers.Uniformly sized SCA NPs enjoy the advantages of high brightness, good stability, large Stokes shift, good biocompatibility,and high photostability.The SCA NPs were successfully applied for in vitro bio-imaging of HeLa cells with an excellent cancer cell uptake.In addition,a strong fluorescence from SCA NPs can still be clearly observed in HeLa cells after incubation for six generations over 15 days.Thus,the SCA NPs could be ideal fluorescent probes for non-invasive long-term cellular imaging.The AIE-active NPs display superior performance and provide a basis for the development of fluorescent organic probes for monitoring biological processes.

Bio-imaging has become a powerful tool in biomedical research for making the process of diagnosis easy and providing information with physiological and pathological relevance details of cells and tissues [1-3].In recent years, various imaging techniques have been extensively studied,such as magnetic resonance imaging[4],computed tomography [5], ultrasound scanners, single-photon emission computed tomography, fluorescence imaging [6-8],positron emission tomography[9],and photoacoustic tomography[5].Among various imaging tools, fluorescence techniques have become powerful tools for analytical sensing and optical imaging in terms of their convenience, high contrast, sensitivity, direct visualization, minimal invasiveness, good spatiotemporal resolution, and quantitative information at subcellular levels [10-15].Research on nanomaterials [16-29]brings new developments to bio-imaging.

Over the past decades,a large variety of optical agents including organic dyes,fluorescent proteins,inorganic quantum dots(QDs),up-conversion NPs,carbon dots and semiconducting polymer NPs have been extensively studied for fluorescence imaging [30-37].Unfortunately, before wide clinic applications, further research is needed to addresses some issues such as limited molar absorptivity and low photobleaching thresholds for organic dyes [38],possible cytotoxicity caused by the heavy metal ions for QDs[34],and complicated and time-consuming transfection procedures for fluorescent proteins [37].In particular, fluorescent organic NPs(FONPs) have showed several advantages such as broader absorption spectra, tunable size and emissions, larger Stokes shifts,excellent photostability,better biocompatibility,and surface functionality over organic dyes and other fluorescent materials[7].However,most FONPs remain unexplored for biomedical application,mainly due to fluorescent intensity of many molecules would be decreased or quenched during aggregation to form NPs,commonly known as concentration quenching [8].

Fortunately,fluorogens with AIE characteristics(AIEgens)has a potential to overcome this limitation[39-43].AIEgens are a special type of molecules, which have no emission in dissolved state but possess high fluorescence in the aggregated state[44,45].Since the concept of AIE was originally reported by Tang et al.[46],many AIE fluorophores with highly twisted structures have been synthesized and explored for potential application in optical sensing, bioimaging,optoelectronic devices,and other fields[47].In addition,high cellular retention, large Stokes shift, resistance to photobleaching, robust biocompatibility, and long-term non-invasive cellular imaging make them perfect for bio-imaging [48].FONPs based on AIEgens have shown advanced features of tunable emission and brightness,excellent biocompatibility,superb photoand physical stability,potential biodegradability,and facile surface functionalization [49,50].These features enable cancer cell detection, long-term cell tracing, and tumor imaging in a noninvasive and high contrast manner [51].Two ways are commonly used to generate AIE NPs including: (1) physical cladding AIEgen with polymer matrix or other nanocarriers; (2)covalent conjugated AIEgen on polymer or other NPs [52].These strategies can efficiently improve AIEgens cellular uptake,stability,blood circulation time, accumulation in target sites, etc.[52]However, in these nanosystems, the carriers themselves are generally the major components and have no therapeutic efficacy,which can raise concerns regarding their possible toxicity and biodegradation.Therefore, development of an alternative type of AIE-active nanosystems with minimum use of inert materials as carriers is highly desirable.

Recently, self-assembly of organic molecules without surfactants has been successfully applied to fabricate various functional nanomaterials, which received more attention [53-57].In this work, we propose a new strategy for preparing new FONPs via reprecipitation method based on an AIE compound without adding any external inert agents or further surface modification, and showed excellent imaging ability with a large Stokes shift about 140 nm.These endow the nanoprobe with high fluorescent brightness and high signal-to-noise ratio.On the other hand, the nanoprobe also shows low cytotoxicity, good stability, superior resistance against photobleaching.Endowed with such merits in term of optical performance,biocompatibility,and stability,the asprepared SCA NPs is demonstrated to be an ideal fluorescent probe for applications in direct long-term cellular imaging with superior photostability.

Herein, SCA NPs were prepared in aqueous solution via reprecipitation.Their morphology and size distribution were characterized by transmission electron microscope (TEM) and dynamic light scattering (DLS), respectively.As shown in Fig.1A,TEM images indicated that SCA NPs had a spherical shape and a smooth surface and the dark dots are due to the high electron density of AIE dyes.SCA NPs showed an average size of about 153 nm(Fig.1B)with a PDI value of 0.161.The absorption spectra of SCA NPs are shown in Fig.1C.AIE dyes showed two absorption peaks at 340 nm and 435 nm, while those of SCA NPs were blueshifted, appearing at 325 and 420 nm.The emission peak of SCA NPs appears at 585 nm with intense emission tails extending to 725 nm (Fig.1D).Their Stokes shifts were larger than 140 nm,which greatly minimize the self-absorption effect that is universally observed in conventional organic dyes.In addition, AIE dyes dissolved in THF solution showed no emission under UV light,whereas SCA NPs in water emitted an intense yellow fluorescence.These results indicate that AIE-active NPs have been successfully prepared.The aggregation-induced fluorescence of AIE dyes(Fig.S1 in Supporting information) was demonstrated using a UV lamp with a wavelength of 365 nm, and the corresponding fluorescent spectra are showed in Fig.S2(Supporting information).In pure THF, no emission from AIE molecules can be observed visually.Addition of different fraction of water in THF, the fluorescence intensity of AIE dyes dramatically increased.Intense emission from AIE dyes was observed when the fraction of water was 70%,and the intensity dramatically increased to nearly 1.5-fold when the water fraction reached 95%.These results confirm the AIE characteristics of AIE dyes.

Fig.1.(A)TEM image of SCA NPs.Scale bar in TEM image:500 nm.(B)DLS intensity-weighted diameter of SCA NPs.(C)Absorbance spectra and(D)PL(excited at 450 nm)spectra of AIE dyes and SCA NPs.Insets: Photographs of AIE dyes in THF, and SCA NPs in water under UV light (365 nm), respectively.

Fig.2.(A)The stability of size distribution of SCA NPs in FBS for 48 h.The data are shown as the mean values±standard deviation(SD)(n=3).(B)Photostability of SCA NPs under continuous scanning at 488 nm.Insets show confocal images of the cells stained by SCA NPs before(0 min,left)and after the laser irradiation for 30 min(right).I0 is the initial PL intensity,while I is that of the corresponding sample after a designated time interval.Cell viability of HeLa cells after incubation with various concentrations of AIE dyes (C) or SCA NPs (D) for 48 h or 72 h.Data represent mean values±SD, n=3.

Excellent stability of NPs in various conditions is essential for maintaining their structure and functions.Most NPs prepared via reprecipitation are quite unstable in water and easily precipitate or aggregate.Here,we studied the stability of SCA NPs by monitoring the size distribution in various conditions for 48 h.As shown in Fig.S3 (Supporting information), SCA NPs dispersed in water had unchanged size and size distributions for 48 h.Moreover, to simulate the physiological conditions, 20% fetal calf serum (FBS)was added into the SCA NPs aqueous solution.In Fig.2A,SCA NPs maintained their initial size and size distribution for 48 h.Then,we used TEM to monitor the morphology of SCA NPs.After 48 h, SCA NPs maintained a spherical morphology with no aggregation(Fig.S4 in Supporting information).These results revealed that the SCA NPs are stable at room temperature.In addition,as one of the key factors for evaluating a fluorescent bio-imaging agent, the photostability of SCA NPs were assessed by continuous excitation.As depicted in Fig.2B, the fluorescent intensities of SCA NPs changed slightly within 30 min of irradiation,and maintained 83%of their initial values in this process unlike traditional organic dyes,which suffer from severe photobleaching after several minutes of laser irradiation.The photostability of the SCA NPs in human cervical cancer (HeLa) cells were also studied under continuous laser scanning upon excitation at 488 nm.Confocal laser scanning microscopy(CLSM)images in Fig.2B show the fluorescence change of SCA NPs in cells upon continuous laser irradiation for 30 min.After irradiation, the SCA NPs possess 90% of their initial fluorescence intensity.These data indicate the excellent photostability of SCA NPs in a biological environment,which will greatly benefit the studies on in vitro long-term cell imaging.

The biocompatibility of nanomaterials is very important for their use as bio-imaging agents.To study the biocompatibility of SCA NPs, the in vitro cytotoxicity of SCA NPs in HeLa cells was investigated using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide assay.As shown in Fig.S5 (Supporting information) and Fig.2C, after incubation with different concentration of AIE dyes for 24 h,48 h or 72 h,no significant decrease in cell viability was observed.For SCA NPs (Fig.S6 in Supporting information and Fig.2D),the cell viability remains above 90%after treatment with 9μg/mL SCA NPs within the tested periods,demonstrating that AIE-active SCA NPs has low cytotoxicity or good biocompatibility to HeLa cells.In addition, we studied the biocompatibility of SCA NPs with cell live/dead kit.As displayed in Fig.S7(Supporting information),after incubation with AIE dyes or SCA NPs for 24 h, no obvious red fluorescence (dead cells) was detected, and only green calcein fluorescence(live cells) could be detected.These results demonstrate that SCA NPs have low cytotoxicity toward cancer cell lines, which makes SCA NPs promising for bio-imaging applications.

Cellular uptake is necessary for nanomaterials to exert their functions.Further cell imaging study employed HeLa cells as a cell model to verify the cellular uptake and localization of SCA NPs with CLSM.4,6-Diamidino-2-phenylindole(DAPI)was used to stain the cell nucleus.As presented in Fig.S8 (Supporting information), an intense homogeneous cytoplasmic yellow fluorescence around nucleus can be clearly observed after cultured with the SCA NPs for 1 h, indicating the SCA NPs can be successfully taken up and localize in the cytoplasm and the perinuclear region within the cells.In addition,we used Lyso-tracker to stain the endosome after incubation with SCA NPs for 1 or 3 h at 37°C in the culture medium.As presented in Fig.3, yellow fluorescence from SCA NPs was observed in HeLa cells and well merged with red fluorescence from Lyso-tracker,indicating the SCA NPs localize with the endosome in the cytoplasm.Moreover,SCA NPs exhibit internalization by living cells with a time-dependent manner.These results manifest that SCA NPs could be applied as an effective fluorescent bioprobe for cellular imaging with high signal-to-noise ratio.

To investigate the non-invasive long-term cellular tracking capability of SCA NPs,we captured fluorescence images at different incubation periods (Fig.4).HeLa cells were first incubated with SCA NPs for 6 h at 37°C (labeled as day 0).The treated cells were then subcultured for designated time intervals and each sample was detected by CLSM.For each cell passage, the old culture medium was extracted and HeLa cells were washed with PBS twice to remove the existing SCA NPs in the culture medium.At the initial stage (day 0), strong yellow fluorescence from SCA NPs can be clearly observed.With the increase of incubation time(from day 3 to 15),the yellow fluorescence gradually decreases because of cell proliferation, in which SCA NPs divide into daughter cells.Importantly, after 15 days of subculture, the yellow fluorescence from SCA NPs was still clearly observed in HeLa cells, which indicates that SCA NPs can act as fluorescent probes for long-term cellular tracing and imaging.More importantly, this long-term tracking strategy is based on cellular proliferation containing endogenous organic nanoprobes rather than continuous exogenous addition of imaging agents during long-term monitoring.This non-invasive nature makes SCA NPs suitable for potential clinical bio-imaging.

In summary, we have developed facile AIE-active NPs that are one-step formulated, which can effectively stain living cells with low cytotoxicity.SCA NPs exhibited several advantages including uniform size, efficient fluorescence in aqueous solution, large Stokes shift,high emission efficiency,superior photostability,good biocompatibility,and ability to successfully enter HeLa cancer cells via endocytosis.More importantly,the yellow signal from SCA NPs can still be clearly observed after 15 days(cells were incubated for six generations in these days),exhibiting superior performance in long-term cell tracing.These incredible properties make AIE-based SCA NPs promising as ideal alternative fluorescent probes for noninvasive long-term cellular tracing and imaging.Furthermore,this study would significantly promote new strategies in the development of AIE study for fluorescent imaging and therapeutic applications.

Fig.4.Long-term cell tracing images of SCA NPs at 37°C for 6 h, which were then subcultured for designated time intervals including day 0,day 3,day 6,day 9,day 12,and day 15.Scale bars represent 20μm in all images.

Acknowledgments

This work was supported by Health Technology Innovation Project of Jilin Province (No.2016J064) and National Natural Science Foundation of China (No.81703136).

Appendix A.Supplementary data

Supplementary material related to this article can be found,in the online version, at doi:https://doi.org/10.1016/j.cclet.2018.10.017.