In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired

Article pubs.acs.org/ac

In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes Pan Ding,‡ Houyu Wang,‡ Bin Song, Xiaoyuan Ji, Yuanyuan Su, and Yao He* Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano & Soft Materials (FUNSOM), and Collaborative Innovation Center of Suzhou Nano Science and Technology (NANO−CIC), Soochow University, Suzhou, Jiangsu 215123, China S Supporting Information *

ABSTRACT: Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing labelfree, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

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prepared extracellularly through complicated synthetic procedures, and requiring extra washing steps to remove untargeted probes. Nevertheless, annoying background fluorescence is still inevitable. Moreover, pristine nanomaterials (e.g., group II−VI semiconductor quantum dots (QDs), gold nanoparticles, carbon dots, and silicon nanoparticles) themselves are hard to specifically label nucleus, generally requiring additional surface modifications with nucleus-targeted peptides or proteins.38 In addition, the relatively poor antiphotobleaching of organic dyes/GFP,39,40 as well as the inevitable toxicity of group II−VI QDs containing heavy metals,35,36 makes them incompatible with real-time and long-term labeling in live cells. As such, there exist no labeling methods adequately suitable for label-free, in situ, long-term, and real-time nucleus labeling up to present, leading to difficulties in fully visualizing and unraveling nuclear structure and function in live cells. On the other aspect, dopamine (DA), which is a neurotransmitter of the catecholamine and phenethylamine families, plays a vital role in human brain functions and behavioral response, such as signal transduction, reward-motivated

ne of the biggest challenges in cellular imaging is the visualization of live cell nucleus with high spatial and temporal resolution, which plays a key role in exploring major cellular events, including DNA replication, mRNA synthesis and processing, ribosome subunit biogenesis, and so forth.1−3 Fluorescence imaging techniques have been major driving forces suitable for the visualization of organelle structure and function in live cells.4−21 A consensus has been reached that, to fully obtain the nucleus-relative information in live cells and meanwhile effectively alleviate negative effects of alien probes on cellular normal behaviors, the nucleus is expected to be labeled in label-free, in situ, real-time, and long-term manners. Extensive efforts have been devoted to developing various labeling strategies for nucleus labeling, including green fluorescent protein (GFP) and its variants-, organic dyes-, and organic/inorganic nanoparticles-based imaging techniques (Table S1 in the Supporting Information). Particularly, while established GFP-based methods can achieve in situ expression of genetically encoded fluorophores within nucleus, they are nevertheless involved in high-skilled biological operations, including the construction of gene carriers, transfection, and expression of GFP.22−24 On the other aspect, the conventional organic DNA dyes (e.g., propidium iodide (PI),25 SYTO 61,26 Vybrant DyeCycle Ruby,27 DRAQ5,28 Hoechst, and their variants)29−31 or nanomaterials-based probes32−37 are normally © 2017 American Chemical Society

Received: November 10, 2016 Accepted: June 16, 2017 Published: June 16, 2017 7861

DOI: 10.1021/acs.analchem.6b04427 Anal. Chem. 2017, 89, 7861−7868

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Analytical Chemistry behavior, and addiction.41 Notably, it is widely reported that DA can self-polymerize into polydopamine (PDA) through DA oxidation under rigorous conditions.42 Taking advantage of unique optical/electrical/magnetic properties, excellent water dispersibility, and favorable biocompatibility of PDA, it has been used for a myriad of biological and biomedical applications.43−45 As for bioimaging, unfortunately, the related reports are quite scarce, because the fluorescence intensity of PDA is relatively weak (e.g., ∼0.2% of quantum yield).44 Furthermore, in most cases, pH values required in synthetic process are still either too high (e.g., pH ∼10) or too low (e.g., pH ∼6), which limited their further bioapplications.46,47 More seriously, undecorated PDA prepared in current methods hardly label the nucleus ascribed to its larger size (e.g., ∼100− 200 nm).44 Therefore, it is desirable to develop a new methodology for live-cell nucleus labeling based on in situ generation of PDA nanoparticles under moderate conditions, in which the resultant PDA shows strong fluorescence coupled with robust photostability, benign biocompatibility, and high specificity to nucleus. To this end, we herein propose the first demonstration of in situ live-cell nucleus fluorescence labeling, which meets all merits mentioned above. Notably, compared to conventional fluorescence labeling methods, this new method can be readily performed via the in situ formation of fluorescent bioinspired polydopamine (PDA) nanoparticles within nucleus under physiological conditions (e.g., 37 °C, pH ∼7.4). Taking advantage of prominent merits of bioinspired PDA nanoparticles employed in such labeling strategy (e.g., in situ generation and work within nucleus, quantum yield of ∼35.8%, cell viability of ∼90%, good photostability, etc.), it can be utilized for real-time and long-term in situ imaging of the livecell nucleus.

Finally, the dried samples of PDA nanoparticles were characterized by a series of techniques. Confocal Microscopic Imaging of Different Cell Lines. Human lung cancer cells (A549), human retina epithelial cells (HREC), mouse melanoma cells (B16), and human breast cancer cells (MCF-7) were seeded (37 °C, 5% CO2) on different glass bottom dishes in RPMI-1640 and H-DMEM medium (for HREC cells) with 10% heat-inactivated FBS and antibiotics (100 μg/mL streptomycin and 100 U/mL penicillin) overnight. First, to label the nuclei in situ, cells were incubated with 20 mM EDC and 400 μM DA, using cell culture medium as the solvent for 3 h under physiological conditions (37 °C, pH ∼7.4). Then, for further staining, cells were washed three times with 1× PBS and fixed with 1× PBS containing 4% sucrose and 4% paraformaldehyde for 20 min, followed by blocking for 40 min in 1× PBS containing 4% BSA and 0.1% Triton X-100. Next, the fixed cells were washed three times with 1× PBS containing 0.1% Tween 20. To label microfilaments, the fixed and blocked cells were incubated with FITC-Phalloidin (100 nM) dispersed in 1× PBS containing 4% BSA for 1 h. To label nuclei with organic dyes, the stained cells were washed twice sequentially with 1× PBS containing 0.1% Tween 20 and Milli-Q water. They then were incubated with 3 μg/mL propidium iodide for 5 min. Finally, the stained cells were washed three times with Milli-Q water and mounted on slides in fluoromount (Sigma, F4680) with coverslips. Prepared samples were examined under a laser-scanning confocal fluorescent microscope (Model TCS-SP5, Leica, Germany) equipped with diode laser (405 nm), multiline argon laser (458, 476, 488, and 514 nm) and HeNe green laser (543 nm) employing a 63.0 × 1.40 oil objective. A cooled CCD camera was used to capture images that contained 1024 × 1024 pixels at a scan speed of 200 Hz. Nuclei labeled by fluorescent PDA nanoparticles and propidium iodide were respectively excited by irradiation of 405 nm (detection window = 420−480 nm) and 543 nm (detection window = 600−660 nm). Cellular microfilaments labeled by FITC-Phalloidin were excited by argon laser (λexcitation = 488 nm, detection window = 515−550 nm). Images were further processed with confocal image analysis software (Leica Application Suite Advanced Fluorescence Lite (LAS AF Lite)) and ImageJ (National Institutes of Health, http://imagej.nih.gov/ij/). Real-Time Observation of In Situ Formation Process of PDA Nanoparticles within Nuclei. A549 cells were uniformly seeded on a dish with a glass bottom in RPMI-1640 medium with 10% heat-inactivated FBS and antibiotics (100 μg/mL streptomycin and 100 U/mL penicillin) and incubated (37 °C, 5% CO2) overnight. Upon the addition of new cell culture medium containing 20 mM EDC and 400 μM DA, the treated A549 cells were immediately examined via confocal time lapse microscopy. Confocal images were recorded every 5 min for 3 h of observation. The images were collected between 420 nm and 480 nm with excitation at 405 nm (6% power of 50 mW, minimizing laser-induced cellular damage) using a cooled CCD camera through a 63 × 1.4 NA objective at a scan speed of 100 Hz and analyzed with image analysis software. Real-Time and Long-Term Live-Cell Nucleus Imaging with Confocal Time Lapse Microscopy. Taking commercial blue fluorescent organic dye Hoechst 33258 as a control group, the properties of PDA-based in situ live-cell nucleus fluorescence labeling strategy were evaluated within 3 h through confocal time lapse microscopy. The A549 and HREC cells were seeded (37 °C, 5% CO2) on different glass

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EXPERIMENTAL SECTION Preparation of Fluorescent PDA nanoparticles in Developed Labeling Strategy. To evaluate the reaction kinetics of dopamine hydrochloride (DA, Sigma−Aldrich, USA) self-polymerization activated by N-(3-(dimethylamino)propyl)-N-ethylcarbodiimde (EDC, Sigma−Aldrich, USA), a set of reacting systems were examined by preparing 1 mL 1× PBS solutions (pH ∼7.4) containing freshly prepared DA (0.4 mM) and EDC (1 mM, 5 mM, 10 mM and 20 mM). Timedependent evaluations of UV-vis absorption and fluorescence spectra of the solutions were recorded in situ, and another set of 0.5 mL freshly prepared DA (0.8 mM) solutions was induced with 0.5 mL of EDC (20 mM) at different temperatures (0, 25, 37, and 60 °C) and different pH values (4, 6, 7, 8, 10, which were achieved by using sodium hydroxide and hydrochloric acid). The unreacted DA and EDC were removed by using Nanosep centrifugal devices (Mw cutoff = 3 kDa, Millipore) through centrifugation at 8000 rpm/min for 15 min. The fluorescence spectra of them were measured after 3 h of reaction. Based on the optimized reaction conditions, a volume of 2 mL DA solutions (0.8 mM) in 1× PBS (pH ∼7.4) was polymerized by 2 mL of EDC (the final concentration in the system is 20 mM) for 3 h at room temperature (25 °C). Next, the resultant solution was filtered with a Millipore filter (0.22 μm). The solution then was purified using Nanosep centrifugal devices (Mw cutoff = 3 kDa, Millipore) through centrifugation at 8000 rpm/min for 15 min and sequentially dried with lyophilizer. It was calculated that the overall yield is ∼65%. 7862

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Figure 1. Characterizations and assessments of PDA nanoparticles generated in novel nucleus imaging strategy: (A) schematic illustration of the general reaction process for synthesis of PDA nanoparticles; (B) TEM image and (C) dynamic light scattering (DLS) data of PDA; (D) UV-PL (UV-vis absorption and photoluminescence) spectra of PDA; (E) linear relationships between integrated PL intensity and absorbance of standard quinine sulfate (QS) and PDA aqueous solutions (the relative photoluminescence quantum yield (PLQY) of PDA is calculated to be ∼35.8%); (F) electrophoresis image of the ladder DNA, PDA, ribosome RNA (rRNA), DNA, and their mixture; and (G) the photoluminescence intensity in the presence of 20 mM EDC and other intracellular species (100 μM dopamine (DA), glutamine (Glu), sucrose (Suc), alanine (Ala), glycine (Gly), lysine (Lys), histidine (His), arginine (Arg), urea, uric acid (UA), ascorbic acid (AA), bull serum albumin (BSA), and a mixture of all interfering chemicals (Mix)). The group that contains only 20 mM EDC is set as the blank. All error bars represent the standard deviation determined from three independent assays.

N-(3-(dimethylamino)propyl)-N-ethylcarbodiimde (EDC) for 3 h under physiological conditions (37 °C, pH ∼7.4), which can be directly employed for visualizing live-cell nucleus (Figure 1A). Comparatively, for conventional organic dyes and nanomaterials-based methods, the probes used are generally prepared in vitro through complicated synthetic procedures and ought to be fully incubated with cell lines, following extra washing steps to eliminate unloaded probes.25−37 As schematically indicated in Figure 1A, DA was first oxidized by EDC (which is a widely used coupling agent in biochemistry) to quinone, followed by rearrangement to form 5,6-dihydroxyindole (DHI).48 Next, a branching reaction of DHI spontaneously proceeded to form oligomers. Finally, PDA nanoparticles were created through chemical covalent bonding and physical self-assembly of primary oligomers.49 According to selectivity evaluation experiments, EDC could specifically induce the polymerization of DA to form fluorescent PDA against other intracellular species (Figure 1G). In addition, the fluorescent probes of PDA nanoparticles featured relatively strong binding affinity to DNA and ribosome RNA (rRNA), which was convincingly demonstrated in the agarose gel electrophoresis image of DNA and rRNA treated with PDA (Figure 1F).50 Thus, the high permeability of precursors, together with the strong interaction of PDA with DNA and rRNA, ensured that the PDA-based strategy presented could image the nucleus in label-free and specific manners. In sharp

bottom dishes respectively in RPMI-1640 and H-DMEM medium with 10% heat-inactivated FBS and antibiotics (100 μg/mL streptomycin and 100 U/mL penicillin) overnight. For the control group, to label live cell nucleus with Hoechst 33258, the cell culture media were removed and cells were washed by 1× PBS (pH ∼7.4). Cells then were incubated with cell culture media containing Hoechst 33258 (10.0 μg/mL). After 20 min of incubation, the media were removed and washed with 1× PBS (pH ∼7.4). Also fresh media were added to the dishes, which were transferred to the live cell station (37 °C, 5% CO2) to maintain the growth of cells and subsequently examined with confocal time lapse microscopy. Both fluorescent PDA nanoparticles and Hoechst 33258 were excited by the irradiation of 405 nm (6% power of 50 mW, minimizing laser-induced cellular damage) and detected between 420 nm and 480 nm. Images with 1024 × 1024 pixels were captured with a cooled CCD camera at 5 min intervals for 3 h through a 63.0 × 1.40 oil objective at a scan speed of 100 Hz and analyzed with image analysis software.

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RESULTS AND DISCUSSION Rational Design of In Situ Live-Cell Nucleus Fluorescence Labeling Strategy. In this method, the fluorescent bioinspired PDA nanoparticles are simply created in situ within the nucleus, when cells are incubated with dopamine (DA) and 7863

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features, there were two typical peaks in the absorption spectrum of PDA: strong absorption at ∼300 nm (assigned to phenol group of DA) and another broad peak at ∼420 nm (assigned to PDA). As such, PDA can be technically quantified with UV absorption at 420 nm (see Figure S5 in the Supporting Information). The emission spectrum indicated that maximal emission of fluorescent PDA was ∼465 nm under the excitation wavelength of 420 nm (Figure 1D). As revealed in Figure S3d, when the resultant PDA solution was excited with radiation of different wavelengths, ranging from 350 nm to 440 nm, the maximum emission wavelengths stabilized at ∼465 nm, indicating that the synthesized PDA displayed an excitation wavelength-independent manner, which possibly contributed to the homogeneous component of resultant PDA. In contrast, the emission of previously reported PDA nanomaterials was dependent on the excitation wavelength, demonstrating their inherent chemical heterogeneity.42 The relative photoluminescence quantum yield (PLQY) of such PDA nanoparticles was as high as ∼35.8% by setting quinine sulfate (QS) as a standard reference (Figure 1E), in contrast to a much lower PLQY value (e.g., ∼0.2%) of PDA prepared through other approaches under harsh conditions (e.g., pH of ∼10).44 Moreover, according to photostability assay of fluorescent PDA nanoparticles and organic dyes (PI, Hoechst and Rhodamine B isothiocyanate (RBITC)) during 3 h of continuous UV irradiation in Figure S6 in the Supporting Information, the fluorescence intensity of PDA was reduced by 82%) after 3 h of treatment. Notably, the cytotoxicity of EDC in 20 mM was significantly reduced by the addition of 400 μM DA and reached to >90% after 48 h of incubation, presumably due to the depletion of EDC in the polymerization of DA in this in situ synthesized PDA-based method (20 mM EDC and 400 μM DA, 37 °C, 3 h). Furthermore, no obvious cellular cytotoxicity of the synthesized PDA was observed after both

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b04427. Comparison of main methods of fluorescence labeling of cell nucleus (Table S1), confocal images of A549 cells stained with extracellularly prepared PDA nanoparticles (Figure S1), photographs of reaction precursors solutions (EDC and DA) and as-prepared PDA under ambient light and 365 nm irradiation (Figure S2), optimization of reaction conditions (Figure S3), dynamic light scattering (DLS) data of PDA solutions prepared in different EDC/ DA ratios (Figure S4), quantification of fluorescent PDA nanoparticles (Figure S5), photostability evaluation of PDA and organic dyes (Figure S6), FTIR spectra of EDC, DA and as-prepared PDA (Figure S7), 1H NMR spectrum of DA in DMSO (Figure S8), 1H NMR 7866

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spectrum of EDC in CDCl3 (Figure S9), 1H NMR spectrum of PDA in CDCl3 (Figure S10), quantification of fluorescence intensity in nuclei and cytosol of A549 cells collected from real-time Supplementary Video 1, optimization of concentration of Hoechst for live A549 cell staining (Figure S12), MTT assay of A549 and HREC cells treated with EDC (Figure S13), MTT assay of A549 and HREC cells treated with PDA (Figure S14) (PDF) Real-time observation of in situ formation process of PDA nanoparticles within live cell nuclei (Supplementary Video 1) (AVI) 3D confocal microscopy of A549 cells (Supplementary Video 2) (AVI)

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AUTHOR INFORMATION

Corresponding Author

*Fax: 86-512-65880946. E-mail: [email protected]. ORCID

Houyu Wang: 0000-0002-5134-9881 Yao He: 0000-0003-1672-4057 Author Contributions ‡

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We express our grateful thanks to Prof. Shuit-Tong Lee’s general help and valuable suggestion. The authors appreciate financial support from National Basic Research Program of C h i n a ( 9 7 3 Pr o g r a m , No s. 2 0 13 CB 9 3 4 4 0 0 an d 2012CB932400), the National Natural Science Foundation of China (Nos. 61361160412, 31400860, 21575096, and 21605109), the Natural Science Foundation of Jiangsu Province of China (Nos. BK20130052), the 111 project, and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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DOI: 10.1021/acs.analchem.6b04427 Anal. Chem. 2017, 89, 7861−7868