Selenium Incorporated Cationic Organochalcogen: Live Cell

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Selenium Incorporated Cationic Organochalcogen: Live cell Compatible and Highly Photostable Molecular Stain for Imaging and Localization of Intracellular DNA Pankaj Gaur, Ajay Kumar, Gourab Dey, Rajendra Kumar, Shalmoli Bhattacharyya, and Subrata Ghosh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00675 • Publication Date (Web): 12 Apr 2016 Downloaded from http://pubs.acs.org on April 15, 2016

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ACS Applied Materials & Interfaces

Selenium Incorporated Cationic Organochalcogen: Live cell Compatible and Highly Photostable Molecular Stain for Imaging and Localization of Intracellular DNA Pankaj Gaur,a,1 Ajay Kumar,b,1 Gourab Dey,a Rajendra Kumar,c Shalmoli Bhattacharyya,b,* Subrata Ghosha,*

a

School of Basic Sciences, Indian Institute of Technology Mandi, Mandi-175001, H.P, India. Department of Biophysics, Post Graduate Institute of Medical Education and Research, Chandigarh-160012 c UGC Centre of Excellence in Applications of Nanomaterials, Nanoparticles &Nanocomposites Panjab University, Chandigarh-160014 b

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ABSTRACT: Successful integration of selenium unit into a newly designed cationic chemical architecture led to the development of a highly photostable molecular maker PA5 to be used in fluorescence microscopy as cellular nucleus staining agent for longer duration imaging under continuous laser illumination. Adaptation of a targeted single-atom modification strategy led to the development of a series of proficient DNA light up probes (PA1-PA5). Further, their comparative photophysical studies in the presence of DNA revealed the potential of electron rich heteroatoms of chalcogen family in improving binding efficiency and specificity of molecular probes toward DNA. The findings of cell studies confirmed the outstanding cell compatibility of probe PA5 in terms of cell permeability, biostability and extremely low cytotoxicity. Moreover, the photostability experiment employing continuous laser illumination in solution phase as well as in cell assay (both fixed and live cells) revealed the admirable photobleaching resistance of PA5. Finally, while investigating the phototoxicity of PA5, the probe was found not to exhibit light-induced toxicity even when irradiated for longer duration. All these experimental results demonstrated the promising standing of PA5 as a futuristic cell compatible potential stain for bioimaging and temporal profiling of DNA.

KEYWORDS: Nuclear stain; Fluorescence microscopy; Selenium organochalcogen; DNA detection; Cell imaging.

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1. INTRODUCTION According to the central dogma of molecular biology, DNA forms the platform for cellular development, evolution as well as transfer of genetic information in living organisms. The broad insights in structural elucidation and determination of its importance at the molecular level outline the principles of molecular pathology, which in turn provides the novel approaches for diagnosis and prognostic assessments of human diseases.

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Therefore, specific detection and

molecular imaging of DNA in cellular system have been proven to be extremely crucial. In recent time, fluorescence microscopy is being used as a powerful tool for optical imaging which allows direct visualization of biological analytes at the molecular level and offers useful insights into the complex biological structures and events.

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Henceforth, the development of promising

nuclear stains with advanced optical properties are highly desirable. Through decades of efforts, conjugates of thiazole orange, oxazole yellow with nucleic acids and heterocyclic cyanine dyes have been prepared and applied for molecular imaging, which are efficient as DNA-RNA traffic lights. 6,7 Though currently used π-conjugated cationic stains such as TOTO or YOYO 8 are well known for their specificity and excellent optical properties on interaction with phosphate backbone of nucleic acids, 6, 7they require cell fixation and permeabilization. 9 Among few recent developments, piperazine containing, cyano at methane bridge

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twisted thiazole orange based cyanine containing

and cyanine-indole-quinolinium.12 DNA targeting stains with good

membrane permeability and live cell compatibility have been reported. However, despite the accessibility of optical nuclear stains, the shortcomings such as photobleaching inside the cell, 13

poor membrane permeability, cytotoxicity and most importantly the phototoxicty are still the

major issues which restrict their utilities. Hence, during the formulation of potential optical nuclear stains, one needs to consider the aforementioned parameters to ensure their promising applicability in the field of fluorescent bioanalytics and molecular cell imaging of nucleic acids.14 Herein, we report our newly developed DNA-targeting ‘turn on’ probes engineered by strategic targeted-atom specific modification of carbon through oxygen, sulfur and selenium. The central 3



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motive behind the atomic modification was to evaluate their structure-interaction relationship for the binding and optical properties of the probes upon interaction with DNA. Structurally, the probes were characterized using FT-IR, HRMS and NMR spectroscopic techniques. The comparative analysis of their optical properties was examined in the presence of DNA by UV-vis and emission spectroscopy. The probes were found to exhibit the advantage over often used commercial stains by discarding the chance of cellular deterioration caused by phototoxicity under continuous exposure of light.15-18 Their potential and utility as the nuclear stains for molecular imaging of nucleic acids in live cells were demonstrated using confocal imaging, flow cytometry and most importantly photostability inside the live cells for real time analysis. Our findings strongly reflected the attractive candidature of probe PA5 over other probes (PA1-PA4) in all the requisite aspects of a promising nuclear stain for selective molecular imaging of DNA in live cells. It has been investigated and reported that the incorporation of selenium unit into molecular architecture of fluorescent dyes with insignificant structural perturbation improves their biophysical and biochemical properties.19-30We have established for the first time through delicate structural modification that selenium incorporation may improve photostability inside live cells remarkably.

2. EXPERIMENTAL SECTIONS

2.1. Synthesis The probes were synthesized by adopting a three-step synthetic pathway. In short, synthesis of azoles 33

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from their precursors followed by the synthesis of their corresponding iodide salts,

and finally Knoevenagel condensation

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of iodide salts with indole-3-carboxyaldehyde to get

the desired probes (Scheme S1). Complete synthetic and characterization detail has been given in supporting information.

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2.2. Materials Solvents and chemicals were purchased from commercial resources and used without further purification. Spectroscopic grade solvents were used for photophysical studies. Doubly ionized water used in all experiments is from Milli-Q systems. Phosphate buffer saline (pH = 7.34, 10 X, 0.1M) was prepared using doubly ionized water. DNA and RNA solutions were prepared in phosphate buffer saline (PBS). Stock solutions of 5 mM for each probe were prepared in DMSO and the aliquots of DNA stock solutions (0-3.6 mg/mL) were incubated with 10 µL (16.5 µM) solution of each probe for absorption and emission titration experiments.

2.3. Instruments and Measurements 1

H and

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C NMR spectra were recorded on Jeol JNM ECX 500 MHz spectrometer in CDCl3,

CDOD3 and DMSO-d6. FT-IR spectra were recorded on a Perkin Elmer Spectrum 2 spectrophotometer. HRMS-ESI spectra were recorded on Bruker Maxis Impact HD instrument. UV–vis and fluorescence spectra were recorded on Simadzu UV-2450 and Cary Eclipse spectrophotometer respectively, using 1 cm quartz cell with 5/5 slit widths. All the spectral studies were performed at 25 °C. The complete description of methods used for evaluation of photophysical parameters has given in the supplementary information. Fluorescence intensity profile was determined using software package NIS- Elements D4.13.00 provided with the microscope (Nikon Eclipse TS100). Confocal microscopy was done with Laser Scanning Confocal Microscope (Nikon C2 plus) using NIS element software. Femtosecond spectrometer (Spectra physics) was used to evaluate quantification data (half life time of stains) in solution assay.

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2.4. Cell Study HeLa cell line was procured from National Centre for Cell Science (NCCS), Pune, India and cultured in α-MEM media (Sigma) supplemented with 10% Fetal Bovine Serum (Sigma, US origin), penicillin/streptomycin and gentamycin. Cells were maintained at a 5% CO2 and 95% air atmosphere at 37°C in a humidified CO2 incubator. Culture media was changed every third day. The complete detail of methods used for cell experiments has been given in supporting information.

3. RESULTS AND DISCUSSIONS 3.1. Photophysical Properties To explore the optical behavior of these newly developed probes in the presence of nucleic acids, their absorption and emission properties were evaluated under physiological conditions (PBS buffer, 10X, pH = 7.34). We started our studies with PA1 and PA2 which exhibited major peaks at 456 (Extinction coefficient ≈ 46900 M-1cm-1) and 465 nm (Extinction coefficient ≈ 42800 M-1cm-1) respectively in their absorption spectra. Upon mixing with DNA, small red shifts (17 and 15 nm respectively) in absorption maxima were observed. Both probes PA1 and PA2 were weakly fluorescent in PBS buffer (ɸ0 = 0.07% for both), but 11 and 13-fold enhancement in their emission intensities (λem = 522 and 560 nm, ɸDNA= 0.8 and 0.9% respectively) was observed in the presence of 3.6 mg/mL DNA (see Fig. S1). Though the optical response of PA1/PA2 was not very promising, the emission results revealed that fine tuning in chemical structure of this newly designed cationic architecture may lead to strong optically responsive molecular materials for DNA. We then became interested to embed hetero atoms of chalcogen family into the cationic part to replace geminal dimethyl unit of PA1/PA2. Carefully employed synthetic routes resulted in the production of three new probes, PA3/PA4/PA5, containing oxygen, sulphur and selenium atoms respectively in place of gem-dimethyl unit (see Scheme S1). The absorption

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maxima of PA3 at 420 nm (Extinction coefficient ≈ 42800 M-1cm-1) displayed red shift of 34 nm and approximately 17-fold fluorescence (ɸDNA/ɸ0= 1/0.058 %) enhancement at 490 nm upon interaction with DNA (see Fig. S1). Interestingly, weakly fluorescent PA4 and PA5 (ɸ0 = 0.09 and 0.24% respectively) showed remarkable enhancement in fluorescence intensity (32 and 29-fold with ɸDNA = 3 and 7% respectively) at 520 and 534 nm with large stokes shifts (᷉ 72 nm for both) upon interaction with DNA, whereas they exhibited clear bathochromic shifts of 37 and 48 nm in their absorption maxima at 447 (Extinction coefficient ≈ 44500 M-1cm-1) and 456 nm (Extinction coefficient ≈ 22800 M-1cm-1) respectively (see Fig. S1 and Fig. 1b, 1c). Looking at their chemical structure it is quite obvious that intramolecular rotation in the excited state of these probes in PBS buffer makes them weak fluorescent.

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Upon binding with DNA, the

intramolecular rotation process becomes non-functional largely due to the interactions of cationic unit of probes with phosphate backbone of nucleic acids. Hence, the enhancement in emission spectra and bathochromic shift in absorption maxima of the probes while interacting with DNA were attributed to the intramolecular charge transfer (ICT) from electron rich indole to corresponding cationic azoles.36-42 Moreover, the variation in properties from probe PA1-5 was attributed to the difference in interactions between the electron rich heteroatoms (O, S and Se) and electrophilic centre (phosphorus) of phosphate group.43, 44Next, the binding efficacy of the probes was evaluated by association constant calculation which validated the strong binding efficiencies of PA4 and PA5 (Ka = 2.4×103 M-1 and 7.4×103 M-1 respectively) over rest of the markers (see Table S1). Likewise, the limit of detection calculation also favored the proficiency of PA4 and PA5

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Figure 1. a) General molecular framework showing ‘Turn on’ mechanism due to ICT from indole to cationic scaffold, b) Molecular structure of the probes PA1-PA5, c, d) Optical response of PA5 (16.5 µM) in presence of DNA (0 – 3.6 mg/mL) in PBS (pH=7.34).

(LOD = 0.88 and 0.48 µM respectively) as illustrated in Table S1. Their visible excitation and green emission properties coupled with efficient brightness (B = 1476, 2072 M-1cm-1 for PA4 and PA5) upon interaction with DNA made it quite logical to think that PA4/PA5 could be the promising nuclear stains for fluorescence imaging of DNA in live cells. Before we started our investigation on their potential evaluation in imaging nucleus in live cells, we examined their 8


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affinity toward RNA and various types of proteins. Both the probes PA4/PA5 were found to be more selective for DNA as compared to RNA and proteins (see Fig. S2 and Fig. S3). However, all these experimental findings clearly demonstrated that PA4/PA5 could be the efficient ones of choice as molecular optical materials for fluorescence imaging of native DNA.

3.2. Theoretical Analysis To get deep insight into the optical behavior of these probes, the geometries of the probes (PA1-PA5) were optimized using density functional theory (DFT) and HOMO-LUMOs were also calculated. The ground state optimized geometry of the probes illustrated that the electronic cloud is concentrated over whole molecular framework in their corresponding HOMOs.

In

contrast, a shifting of the electronic cloud in the center of the probes was observed in their LUMOs which clearly revealed the existence of intramolecular charge transfer from indole to the corresponding azolium units (see Fig. S18-22). In continuation, the time dependent-DFT (TDDFT) study was carried out to simulate their UV-vis absorption profile. Their simulated absorption spectra matched well with the experimentally observed absorption spectra (see Fig. S18-22). Further, the natural transition orbitals (NTO) corresponding to first exited state of probe PA5 were calculated to study its excited state behavior. As evidenced from Figure S22, the existence of hole and electron in a similar fashion as observed in the ground state validated the occurrence of intramolecular charge transfer (ICT) characteristic in the probes.

3.3. Fluorescence Life Time Measurements Further to explore the excited state dynamics of PA4/PA5, the fluorescence lifetime measurements were made in the presence and absence of DNA. In the cases of both PA4/PA5, very fast fluorescence decay (100 ps) was observed in their buffered solutions due to the intramolecular torsional relaxation in the excited state. In contrast, on probing with DNA, the life

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time of the probes enhanced dramatically as shown in Figure 2a and Figure S23. One can clearly see from the decay profiles of the probes that on probing with DNA, the probes PA4/PA5 exhibited the slower decay traces with fourth-exponential fitting that possibly could be attributed to the complex charge transfer state.45-47 The average life time and decay constants were calculated from fluorescence decay traces and tabulated in Table S3.

Figure 2. a) Fluorescence decay profile of PA5 (16.5 µM) in absence and presence of DNA (3.6 mg/mL). L is the excitation profile of lamp, λexc = 441 nm. b) Emission spectra of PA5 upon subsequent addition of glycerol. 3.4. Viscosity Dependence of Fluorescence Emission To comment on the presence of intramolecular rotation and charge transfer (CT) state in the probes, viscosity dependent emission behavior of the probes PA4/PA5 was investigated in various glycerol-water mixtures.48 Interestingly, the enhancement in emission intensities of the probes PA4/PA5 was observed with the increase in glycerol percentage in glycerol-water system as depicted in Figure 2b and Figure S23. The enhancement in the emission intensity of the probes was attributed to viscous microenvironment surrounded the probes which led to the

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freezing of intramolecular rotation of the probes and facilitated the intramolecular charge transfer.48 Therefore, based upon theoretical and experimental analysis, it was concluded that the binding of the probes in restrictive microenvironment of DNA groove led to the deactivation of nonradiative decay due to the restriction of their intramolecular rotations (RIM). Moreover, RIM may have provided the geometry with planar configuration which have emissive ICT process attached with it and hence the reason for enhancement in the emission.47-49

3.5. Intensity Profiling and DNA Localization Inside Cell Temporal profiling of staining intensity and nuclear specificity (bleeding to cytoplasm) was done in fixed cells at 1 min (see Fig. S25a) and 30 min (see Fig. 3, 4 & S25b). The probes got accumulated primarily into the nucleus after penetrating the live cells which revealed that permeabilization and fixing were not mandatory for these probes to enter into the nucleus (see Fig. 3). This is a useful feature for a bioprobe since fixation of the cells often produces undesired artifacts that affect the final analysis. All these five probes varied in their nuclear binding capabilities as observed from their fluorescence intensity. We observed that PA5, PA4 and PA2 showed the highest fluorescence intensity in live cells while in fixed cells the flourescence intensity of PA2 was reduced significantly (see Fig. 4 and 5). Thus, PA4/PA5 could be used as optical probe forimaging both live and fixed cells without any loss of intensity. The decrease in intensity of PA2 might be due to leaching out of the probe in fixed cell. These probes also varied in terms of their specific localization in the nucleus, and it was measured as the spillage of probe into the cytoplasm of cell apart from binding to nucleus. For assessing the exact spillage, we counterstained the cells with DAPI, and determined the area of spillage into cytoplasm for a particular probe. It allowed us to assess the exact nucleus specificity of these newly developed fluorescent markers. Based on the area calculations, the highest spillage was observed for PA1, then for PA3, PA2, PA4, and negligible spillage for PA5 (see Fig. 5). Thus,

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PA4 and PA5 showed almost no

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bleeding from the nucleus to the cytoplasm. These

experimnets provided additional information on counterstain compatibility of these probes. Fig. 4 shows the levels of compatibility of PA1-5 with DAPI as the counterstain. Intensity surface plot (ISP) was generated by the microscope software based on the pixel intensities of the probe luminosity. We plotted the ISP for all the probes and found that PA1, PA4 and PA5 have the highest pixel intensities while PA2 showed the least pixel intensity generated after binding to DNA inside the cells (see Fig. S26). Though PA1 showed strong fluorescence intensity upon binding with DNA, at the same time it also showed highest cytoplasmic spill during imaging. Therefore, PA4 and PA5 can undoubtedly be considered as much superior DNA dyes than PA1.

Figure 3. Fluorescence staining by PA1-PA5 in live HeLa cells. Live cells were observed under fluorescence microscope after incubation with PA1-PA5. Bar diagram shows the quantification of mean fluorescence intensity of all probes in live cells. Results are represented as mean ± SD.

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Figure 4. Staining in fixed HeLa cells for 30 min without permeabilization. Cells were incubated with respective probes (16.5 µM, PA1-PA5) for 30 min and DAPI (0.3 µM) for 5 min. Magnification 20X, Scale bar 50 µm.

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Figure 5. Staining in fixed HeLa cells for 30 min after permeabilization. Cells were incubated with respective probes (16.5 µM, PA1-PA5) for 30 min and DAPI (0.3 µM) for 5 min after fixation. Magnification 20X, Scale bar 50 µm. Bar diagrams F shows the quantification of mean fluorescence intensity (MFI) of all probes in live cells. MFI was calculated after background normalization using the NIS- Elements D4.13.00 inbuilt in Nikon Eclipse TS100.Bar diagram in lower panel shows the area of cytoplasmic spillover, measured in px2. Statistical comparison for intensity was made between PA1 and all other probes (PA2-PA5), comparison for cytoplasmic spillage was made between DAPI and respective probes (PA1-PA5)-P value ≤0.05*, P≤0.005**, P≤0.001***

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3.6. Cell-Viability Assay Cytotoxicity is one among those several factors which is seriously considered while developing any fluorescent dyes for biological applications. It is well documented that commonly used vital DNA dyes can be genotoxic and reduce the cell viability over a period of time.50-53The fluorescent probes used for long term cell tracing and imaging need to have minimal cell toxicity. To assess the cytotoxicity of these probes (PA1-PA5) at the concentration used during imaging studies (16.5 µM), we incubated cells in growth medium with each probe in separate experiments for 24 h. We observed highest cell viability (96%) with almost no cell death in the presence of PA5 after 24 h, while all other probes showed reduced cell viability (see Fig. S27). These results indicated that the incorporation of selenium into cationic architechture not only stopped cytoplasmic spill through improving binding efficiency, but also reduced the cytotoxicity in larger extent. Thus, PA5 can be employed for real time studies of live cells for longer period without any significant effect on cell viability.

3.7. Resistance toward Photobelaching and Phototoxicity In the area of fluorescence imaging, development of highly photostable dyes has always been challenging54-59 as many newly developed otherwise efficient flourescent probes get photobleached inside live cells under continuous laser illumination. Therefore, development of fluorescent materials those survive with strong emission inside living cells under laser illumintion without any photoinduced toxicity have garnered substantial interest from the researchers. Given the aforementioned challanges, we then became curious to determine the photobeaching resistance of PA4/PA5. The photostablity of DNA solutions incubated with PA4/PA5 were quantitatively investigated by continuous exposure of the samples using 488 nm high power pulsed laser (140 mW, 34×1013 photons/pulse, FWHM˜50 fs). The intensity vs time plot provided

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the quantification of photostability in term of their half-life time (t1/2) as 47 and 77 min for PA4/PA5 respectively (see Fig.S17). To compare their photo-exposure tolerance with commercial known dye, the photostability of propidium iodide (PI) was also evaluated under similar set of conditions and it exhibited 71 min as its half-life time (t1/2) (see Fig.S17). The results demonstrated amazing photostability of PA4/PA5 in solution assays. However, a newly developed fluorescent probe for biological application can be claimed as a photostable optical material only when the probe shows good photostability inside cellular milieu under continuous laser light illumination.

Figure 6. Photostability of PA4 & PA5. a, b,c) Confocal microscopic images of HeLa cells (fixed) after 2 h incubation with PA4 (16.5 µM), PA5 (16.5 µM) and PI (16.5 µM) respectively. d) Graph showing mean fluorescence intensity retained for PA4, PA5 and PI in fixed HeLa cells 16


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after 20 min of laser exposure. e, f) Confocal microscopic images of HeLa (live) incubated with PA4 and PA5 respectively. g) Graph showing the mean fluorescence intensity retained for both PA4 and PA5 in live HeLa cells after 10 min of laser exposure. Fluorescence intensity of Images captured at 0 min was considered 100%. Images are representative images only. Magnification 20X for PA4 and PA5 in both live and fixed cells, and 30X for PI in fixed cells. Scale bar 50 µm. Results are mean of three independent experiments. Values are represented as mean ± SD. P-value, *

Selenium Incorporated Cationic Organochalcogen: Live Cell

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