Study of the Fluorescence Based Applications of Pyrene-Tagged Poly

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Study of the fluorescence based applications of pyrene-tagged poly(N-Vinyl-2-pyrrolidone) Kheyanath Mitra, Shikha Singh, Sumit Kumar Hira, Vijay Kumar Patel, Deovrat Singh, Sambhav Vishwakarma, Rajshree Singh, Archana Kumari, Partha Pratim Manna, and Biswajit Ray ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00397 • Publication Date (Web): 17 Aug 2016 Downloaded from http://pubs.acs.org on August 19, 2016

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ACS Biomaterials Science & Engineering

Study of the fluorescence based applications of pyrenetagged poly(N-Vinyl-2-pyrrolidone)

Kheyanath Mitra,† Shikha Singh,† Sumit Kumar Hira, ‡,§ Vijay Kumar Patel,† Deovrat Singh,† Sambhav Vishwakarma,† Rajshree Singh,† Archana Kumari,† Partha Pratim Manna, ‡* Biswajit Ray†* †

‡

Department of Chemistry, Institute of Science, Banaras Hindu University, Varanasi – 221 005, India Immunobiology Laboratory, Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi –

221 005, India

ABSTRACT: In this study we have explored the fluorescence based applications of luminescent pyrene-tagged PNVP (PyPNVP) reported in our previous work (Int. J. Polym. Mater. Polym. Biomater., 2016, vol.65, p.269-276). PyPNVP has successfully acted as ‘turn off’ chemosensor for metal ions Cu2+, Hg2+, and Pb2+. It has also successfully acted as fluorescent probe for critical micellar concentration (CMC) determination of amphiphilic block copolymer of poly(D,L-Lactide) and poly(N-vinylpyrrolidone) (PDLLA42-b-PNVP120) (Mn = 19,400 g/mol and PD = 1.52). It has also successfully showed interaction with both plasmid and calf thymus (CT) deoxyribonucleic acid (DNA)s as evidenced by its fluorescence quenching. Different magnitude and type of quenching has been observed for both the cases which may be useful in distinguishing different kinds of DNAs. In order to further understand the potential of PyPNVP in various biotechnological processes, binding property of it with bovine serum albumin (BSA) has also been studied. The efficient quenching of intrinsic fluorescence of BSA by PyPNVP through binding and the 2

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occurrence of the fluorescence resonance energy transfer (FRET) type of interaction have been studied using steady state, synchronous and 3D fluorescence spectroscopies. Moreover, fluorescence microscopic cell imaging study has revealed the significant uptake of PyPNVP in the nucleus of HEPG2 and U87 cells compared to free Py. In addition, cytotoxicity study showed the tolerance of PyPNVP in all the cell lines tested with no significant cytotoxicity at lower concentrations. Key words: Fluorescence, Pyrene-tagged PNVP, Turn-off chemosensor, CMC, DNA, BSA, Cell tracker

1. INTRODUCTION Poly-(N-Vinyl-2-pyrrolidone) (PNVP) has been used in both chemical and biomedical fields due to its low toxicity, high water solubility and bio-compatibility. It has wide applications in drug delivery, cosmetics, stabilization, phase transfer catalyst and selective chelating agent for separation.1-7 Different controlled/living polymerization methods has been applied for welldefined synthesis of it. Amongst the polymerization methods reversible addition-fragmentation chain transfer (RAFT) polymerization has been evolved as one of the most successful method for controlled synthesis of PNVP.1-4 Earlier, from our group xanthate based RAFT agents has been successfully exploit for controlled polymerization of PNVP with tunable chain ends for postpolymerization modifications.1-4 In recent past azide-alkyne click chemistry has been involved as smart atom economy group for designing materials with tunable properties.8-9 From the constant efforts of our groups, in previous publication, an azide terminated RAFT agent [(S)-2-(4-azidobutyl propionate)-(O-ethyl 3


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xanthate)] for controlled polymerization of NVP have been reported.4 This RAFT agent has evolved as very efficient chain transfer agent for controlled polymerization of NVP with azide containing chain end. Thereof, we have functionalized azide-terminated PNVP with pyrene via click chemistry with 1-prop-2-ynyloxymethyl-pyrene. The resultant pyrene-tagged PNVP (PyPNVP) showed good luminescent property and enhanced solution processability in water.4 Pyrene is a well-known fluorescent, hydrophobic, polycyclic, aromatic hydrocarbon (PAH). Pyrene and its derivatives have been exploited widely in the field of chemical, environmental and biomedical sciences as fluorescent sensor and probe.5,10-12 Pyrene functionalized chemosensors fabricated via click chemistry has been used for successful sensing of chemically and biologically significant and very toxic transition metals like Cu, Hg, Pb etc. via binding with triazole group.12-15 Cu2+ has crucial role in various biological processes and the presence of free ion can damage live cells by generating reactive oxygen species, whereas Hg is very toxic towards living organisms. So, in the present study, we have used PyPNVP as ‘turn off’ fluorescence based chemosensor for Cu2+, Hg2+, and Pb2+ ions. In addition, we have also explored for the first time the use of PyPNVP as fluorescent probe for the determination of critical micellar concentrations (CMC) of amphiphilic block copolymers. It is to be noted here the studies of the interaction of pyrene tagged polyethylene glycols with amphiphilic surfactants are well documented in the literature.16-18 Further, PAHs itself have widespread as consistent organic pollutants. Deposition of PAHs as metabolites in-vivo can result significant adverse effects.19,20 So, interactions of this kind of metabolite with biomacromolecules (e. g. proteins, DNA etc.) have studied widely to develop various biotechnological processes.19-22 Serum albumins are most abundant proteins in plasma. It plays crucial role in binding and uptake of various metabolites. Further, due to their 4



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excellent binding properties, these globular proteins are used as functional product in various biomedical processes. For protein binding studies, bovine serum albumin (BSA) has universally been used as model owing to its close resemblance with human serum albumin, abundance and cost effectively. 19-25 So far to the best of our knowledge, mainly small molecule pyrene derivatives with high hydrophobicity have been used for the binding study with DNA and BSA.19-22, 26 However, Haldar et. al has reported interaction of pyrene tagged polyethylene glycol with BSA and human serum albumin (HAS) by steady state fluorescence studies.27 Here, for the first time, we have also explored the binding properties of PyPNVP with DNA and BSA using fluorescence spectroscopy. The efficient quenching of intrinsic fluorescence of BSA by PyPNVP have been revealed by steady state, synchronous and 3D fluorescence spectroscopy studies. Moreover, we have explored the efficacy of PyPNVP as a cell tracker in the imaging of HEPG2 (human hepatocellular carcinoma), U87 (human glioblastoma) and RAW264.7 (mouse macrophage) cells. Further, in order to check its biocompatibility, we have also studied its direct cellular toxicity in above mentioned cell lines, and compared the same with PNVP and pyrene.

2. EXPERIMENTAL SECTION 2.1 Materials. Synthesis of pyrene-tagged PNVP (Mn = 1400 g/mol and PD = 1.2) has been reported previously by our group.4 Calf Thymus (CT) DNA (Merck, India) was received as gift from Prof. Daya Shankar Pandey, Dept. of Chemistry, Banaras Hindu University. pUC 12 plasmid DNA was received as gift from Prof. J. K. Roy, Dept. of Zoology, Banaras Hindu University. Bovine serum albumin (Sigma-Aldrich, USA) was received as gift from Dr. R. Gundampathy, Post-doc fellow, Molecular Biology Unit, Institute of Medical Science, Banaras

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Hindu University. AR grade CuSO4, HgCl2 and Pb(NO3)2 of Loba chemi, India were used as received. 2.2 General Methods. Fluorescence studies were recorded in Cary-Eclipse fluorescence spectrophotometer (Agilent Technologies) using solutions of requisite concentrations in deionized water at ambient temperature. 2.3. CMC Determination. Detailed method is published elsewhere.5 Typically, a series of aqueous block copolymer solutions with concentrations ranging from 5 x 10-4 to 1 mg/mL were prepared by dilution with deionized water. A PyPNVP stock solution in acetone was transferred to a series of vials, the acetone was evaporated under nitrogen, and the block copolymer solutions were added to the vials to get a final pyrene tagged PNVP concentration of 6 x 10-7 M in each vial. The excitation spectra (300-360 nm) of the solutions were recorded at an emission wavelength of 394 nm using a slit width of 5 nm. The ratio of the peak intensities of the excitation spectra of pyrene at 344 nm (I344) and 337 nm (I337) was plotted as a function of polymer concentration. The critical micelle concentration (cmc) value was considered as the interception point of the two tangent straight lines at low concentration.5,6 2.4. Cell Culture. HEPG2 (human hepatocellular carcinoma), U87 (human glioblastoma) and RAW264.7 (mouse macrophage) cells were cultured in T75 flasks with RPMI-1640 medium containing 10% fetal calf serum (FCS) and supplemented with final concentrations of 1% Lglutamine (200 mM), 0.5% penicillin/streptomycin (10,000 IU mL−1/10,000 mg mL−1). Cells were trypsinized (2.5% trypsin solution) in phosphate buffered saline (PBS), washed in culture medium and were cultured in 6 well tissue culture plates for 24 hours. Cells were washed 3 times with 1 mL Hank’s Balanced Salt Solution (HBSS) and cultured in presence or absence of the substrate 1-prop-2-ynyloxymethyl-pyrene (Py), PNVP and PyPNVP for 8 hs at 37 oC, 5% CO2 in

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culture medium. The final concentration of the substrate added to the cells was 10µM. The cells were washed and were stained with Hoechst for nuclear staining. The cells were washed 3 times with 1 mL of HBSS prior to imaging using fluorescence microscope EVOS® FL Cell Imaging System (Life Technologies, USA). 2.4.1. Quantification of Cellular Uptake. Quantification of fluorescence in HEPG2, U87 or RAW264.7 cells was determined following the protocol from our earlier studies.28 Cells were treated with Py, PNVP or PyPNVP conjugate (25 mg/mL) and were incubated at 37 oC for 2, 4, 6, 8, 10, and 12 hs. The culture medium was removed and cells were washed three times with cold PBS. The cells were lysed in 200 mL of lysis buffer (50 mM Tris-HCL, 150 mM NaCl, 0.1% NP40, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100). For fluorimetric analysis, total cellular uptake was determined by measuring the fluorescent emission of the solution (λex = 485 nm, λem = 528 nm) in the cell lysate with Synergy HT Multi-Mode Micro plate Reader (BioTek, USA). 2.4.2. Cytotoxicity Study. The lytic activity of Py, PNVP or PyPNVP cojugate was measured by 18 h non-radioactive cytotoxicity assay using the CytoTox 96 Non-Radioactive Cytotoxicity assay kit from Promega, USA, which quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme released upon cell lysis. Percent lytic activity was calculated as described before.28 2.4.3. Statistical Analysis. The mean ± SD was calculated for each experimental group. n represents the number of times the experiment was performed. Differences between the groups were analyzed by unpaired Student’s t-test and one- or two-way ANOVA analysis of variance depending on the requirement.

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3. RESULTS AND DISCUSSION Synthesis and characterization of pyrene-tagged PNVP (PyPNVP) [ Mn = 1400 g/mol, PD = 1.2] via RAFT polymerization of NVP using (S)-2-(4-azidobutyl propionate)-(O-ethyl xanthate) RAFT agent followed by click chemistry with 1-prop-2-ynyloxymethyl-pyrene has recently been reported by our group (Scheme 1).4 Herein, we focused on different fluorescence based uses of this end functionalized polymer.

Scheme 1. Synthetic procedure of PyPNVP.4

3.1. ‘Turn off’ Chemosensing of Metal Ions. Pyrene based derivatives synthesized via click chemistry have already been reported to serve as ‘turn off’ fluorescence based sensors for transition metal ions.12-15 Here, we have used the relatively higher (with respect to pyrene) water soluble pyrene-tagged PNVP polymer for the

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study of its interaction with Cu2+, Hg2+ (transition metal ions) and Pb2+ (p-block metal ion) ions. In steady state fluorescence titration, with increasing concentration of Cu2+ ion, both emission [Figure 1(a)] and excitation [Figure 1 (b)] intensities of PyPNVP have been quenched gradually. Maximum quenching (65.3%) was observed at saturation in 1: 1.92 molar ratio of PyPNVP and Cu2+. Titration using Hg2+ ion has also been shown the same trend of quenching phenomenon [Figure 1(c) for emission and Figure 1(d) for excitation, respectively]. However, in comparison to Cu2+ ion, higher magnitude (81.8 %) of quenching has been observed with the saturation at higher molar ratio (1 : 2.7) of PyPNVP and Hg2+. We have also carried out fluorescence quenching study with Pb2+ [Figure 1(e) for emission and Figure 1(f) for excitation, respectively]. Here, quenching of fluorescence has also been observed but magnitude (38 %) was much less compared to Cu2+ or, Hg2+ and the corresponding molar ratio (1 : 3.96) of PyPNVP and Pb2+ at saturation was much higher. This fluorescence ‘turn off’ phenomena in the presence of metal ions probably resulted from the interaction of the metal ion with the triazole ring and the thioester group present at the polymer chain ends, which eventually led to the quenching of the fluorescence via the separation of the fluorofore followed by electron transfer. Here, during quenching, plausibly a reverse photoinduced electron transfer (PET) mechanism is occurring where metal ion bound triazole groups acted as electron acceptor and pyrene unit as electron donor.12-15 The observed higher quenching efficiency of transition metal cations, Cu2+ or, Hg2+, is probably due to their partially filled d-orbital and easily reducible nature which facilitate the electron transfer compared to p-block Pb2+ cation.15 Therefore, we can conclude that triazole containing PyPNVP is showing better fluorescence quenching effectivity towards transition metal ions.

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Figure 1. Steady state fluorescence titration of PyPNVP (1 × 10-5 mol/mL) using Cu2+ [(a) emission and (b) excitation spectra], Hg2+ [(c) emission and (d) excitation spectra], and Pb2+ [(e) emission and (f) excitation spectra]at excitation wave length 342 nm for emission spectra and emission wave length 400 nm for excitation spectra. 10



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3.2. Critical Micellar Concentration (CMC) Determination. In order to study the usefulness of PyPNVP, in comparison to pyrene, as fluorescence probe for CMC determination, we have determined the CMC of PDLLA42-b-PNVP120 block copolymer (Mn = 19,400 g/mol and PD = 1.52) using PyPNVP as fluorescene probe. The observed CMC of this block copolymer using peyrene was 0.006 mg/mL.6 It is to be noted here that the characteristic fluorescence peaks of PyPNVP in block copolymer solution have been red shifted [Figure 2(b)] compared to that of pyrene.6 So, for CMC determination, we have plotted [Figure 2(a)] here the intensity ratio of I344/I337 [in place of I337/I333 (using pyerene as probe)] against the log of the concentration of block copolymer.

Figure 2. CMC measurement of PDLLA42-b-PNVP120 block copolymer: (a) I344/I337 vs. log (conc.) plot and (b) corresponding excitation spectra (emission wave length 400 nm).

The corresponding CMC was found to be 0.0042 mg/mL. This value is little lower than that obtained using pyrene.6 Amphiphilic nature of the PyPNVP probe is plausibly assisting the micelle formation of the amphiphilic block copolymer at lower CMC value. Similar type of observation is also reported for surfactant systems using pyrene tagged polyethylene glycols as 11


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probe.16-18 Therefore, PyPNVP has worked successfully as the probe for the CMC determination of amphiphilic block copolymer using fluorescence spectroscopy.

3.3. Deoxyribonucleic Acid (DNA) Sensing. In order to check the interactions and binding property of PyPNVP with DNA, we have also studied its interaction with two types of DNAs: cyclic plasmid DNA and CT DNA using fluorescence spectroscopy.

Figure 3. Fluorescence emission spectra [Excitation wave length 342 nm] (a) and excitation spectra [emission wave length 400 nm] (b) of PyPNVP (0.1 mg/mL) with increasing amount of plasmid DNA.

During steady state fluorescence titration study with plasmid DNA, both emission spectra [Figure 3 (a)] and excitation spectra [Figure 3(b)] of PyPNVP was gradually quenched in almost

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equal magnitude with increasing amount of DNA up to saturation (PyPNVP : Plasmid = 1 :0.15 wt. ratio). Similar type of quenching phenomenon was also observed for CT DNA. In steady state fluorescence titration study, both emission and excitation spectra [Figures 4(a) and 4(b), respectively] showed gradual quenching up to saturation. Here, saturation has been reached at 1 : 0.27 wt ratio of PyPNVP and CT DNA, respectively, indicating that PyPNVP has interacted with greater amount of CT DNA compared to plasmid DNA. Further, in the excitation spectra, the two peaks at 240 nm and 275 nm have showed much greater amount of quenching compared to other excitation and emission peaks. This phenomenon indicates that CT DNA hinders the S0→S3 transitions more compare to others.29

Figure 4. Fluorescence emission spectra [excitation wave length 342 nm] (a) and excitation spectra [emission wave length 400 nm] (b) of PyPNVP (0.2 mg/mL) with increasing amount of CT DNA.

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In order to get deeper insight of the interaction phenomenon, the fluorescence emission data of both the systems have been analyzed by Stern-Volmer plot ( I0/I vs. [Q] plot; where [Q] is the concentration of quencher, Io and I are the fluorescence intensities of PyPNVP in the absence and presence of quencher). Very interestingly, the quenching process involving plasmid DNA showed a linear relationship following the Stern-Volmer equation (Eq. 1) with the value of Stern-Volmer constant (Ksv) = 31.82 [Figure 5(a)].

1 ……. Eq. (1)

On the other hand, in CT DNA system, I0/I vs. [Q] plot [Figure 5(b)] showed a nonlinear relationship with an upward deviation. This result indicates that here both static and dynamic quenchings are occurring simultaneously following the modified Stern-Volmer equation (Eq. 2).

1 1 ……. Eq. (2)

Here, Ksv and Ka are Stern-Volmer quenching constant and static quenching constant, respectively, with the values of each found to be 3.25 (using non-linear regression method). This result indicates that both type of quenching occurred in almost equal magnitude.

Figure 5. Stern-Volmer plot of fluorescence titration study of PyPNVP using (a) Plasmid and (b) CT DNA. 14



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Thus, the decrease in fluorescence intensity of PyPNVP in the presence of DNAs indicates clearly their interactions. The fluorescence quenching occurs most probably because of the hydrophobic interactions between pyrene and DNA as well as wrapping of the hydrophilic PNVP chain upon DNA segments leading to its micro-environmental changes and eventually hindering the electronic transition. Both the DNAs showed difference in magnitude and quenching pattern during interaction. This phenomenon may be useful to differentiate among different kind of DNAs. 3.4. Protein Binding Study. BSA is globular type of protein. Its primary structure consist of approximately 600 amino acids residues. Secondary structure of it consists of mostly α-helix loops and disulphide bridges, which united in heart shaped 3D structure. Its tertiary structure is composed of 3 domains each of which contained around 190 amino acids residues and subdivided into two subdomains. Three intrinsic fluorophore residues [tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) ] are present in BSA. Trp and Tyr residues have corresponding emission peaks at 348 nm and 303 nm, respectively, whereas Phe residue has very low quantum yield which could not be excited in most of the cases.19-20 The intrinsic fluorescence of BSA is easily affected by local microenvironmental changes due to binding with other molecules.19,20,23-25

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Figure 6. Steady state fluorescence emission spectra of BSA (0.03 mg/mL) with increasing amount of PyPNVP (excitation wave length 280 nm) at ambient temperature (27 oC) (a) and at elevated temperature (37 oC) (b).

In order to check the binding of BSA with PyPNVP, the quenching of its steady states fluorescence emission spectra upon addition of PyPNVP has been studied [Figure 6(a)]. The titration study showed very efficient fluorescence quenching of BSA up to saturation (at BSA : PyPNVP = 1 : 0.26 wt ratio) as well as gradual increase in peaks at 370 and 400 nm corresponding to pyrene residue. This phenomenon suggests successful binding of PyPNVP with BSA and most probably a FRET mechanism has been occurring from Trp residue to pyrene.19,20,30

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Figure 7. Stern-Volmer plots of fluorescence titration study of BSA with PyPNVP at ambient

temperature (27 oC) (a) and at elevated temperature (37 oC) (b).

This quenching phenomenon is further analyzed by Stern-Volmer plot (Figure 7). The I0/I vs. [Q] plot, where [Q] is concentration of quencher PyPNVP, Io and I are the fluorescence intensities of BSA in the absence and presence of quencher, showed linear relationship (Eq. 1). The value of Stern-Volmer constant (Ksv) was found to be 87.1 at ambient temperature (27 oC) [Figure 7(a)]. To further analyze the nature of quenching interactions, the titration has also been performed at elevated temperature (37 oC). The corresponding Stern-Volmer plot [Figure 7(b)] also showed linear relationship with Ksv value of 68.9. The decrease in the value of Ksv with increasing temperature suggest a static type of quenching phenomenon is predominant here.25 The synchronous fluorescence emission spectra of BSA at ∆λ = 15 nm [Figure 8(a)] and ∆λ = 60 nm [Figure 8(b)] have also been recorded in order to get further inside of this quenching phenomenon. At ∆λ = 60 nm fluorescence intensity is much greater than that at ∆λ = 15 nm. This result indicates that intrinsic fluorescence of BSA is mainly contributed by Trp residues.23-25For

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both ∆λ = 15 and 60 nm, fluorescence intensities have been decreased with increasing amount of PyPNVP. However, the magnitude of quenching is greater at ∆λ = 60 nm. This result shows that PyPNVP interacted with both Trp and Tyr residues but in greater magnitude with Trp residues. Eventually, it buried the fluorophores towards hydrophobic subdomains via significant conformational change during interaction.

Figure 8. Synchronous fluorescence spectra of BSA (0.03 mg/mL) with increasing amount of PyPNVP at (a) ∆λ =15 nm and (b) ∆λ = 60 nm.

For further investigation of micro-environmental changes in BSA during interaction with PyPNVP, 3D fluorescence spectroscopy has also been performed. The corresponding fluorescence spectra of BSA have shown four characteristic peaks [Figure 9(a)]. Peaks 1 and 4 correspond to first and second order Rayleigh scattering, peak 2 corresponds to fluorophores and peak 3 for polypeptide backbone chains.25 After reaching the saturation point [Figure 9(b)] the peaks 3 and 4 is almost quenched. These indicate that during binding, fluorophores have been

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buried into hydrophobic subdomains with conformational distortion of polypeptide backbone, probably led by insertion of pyrene and wrapping by corresponding PNVP chains around the helical structure of BSA. Whereas for the same excitation regions corresponding to peaks 2 and 3, two new peaks 5 and 6 corresponding to emission of pyrene appeared indicating the occurrence of the FRET type of interaction during complexion process.19,20,30 Like BSA protein system, PyPNVP may find useful in studying the binding properties of any other protein systems.

Figure 9. (a) 3D fluorescence spectra of BSA (0.03 mg/mL). (b) 3D fluorescence spectra of BSA and PyPNVP after saturation (BSA: PyPNVP 1:0.26 wt. ratio).

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3.5. Cellular Uptake Study. In order to find the possible application of PyPNVP, we have also explored the efficacy of it as a tracker in cell imaging. HEPG2 (human hepatocellular carcinoma), U87 (human glioblastoma) and RAW264.7 (mouse macrophage) cells were cultured in the presence and absence of 10 µM of each Py, PNVP and PyPNVP separately. Then, the cells are stained with Hoechst for nuclear staining. The fluorescence microscopy cell imaging of these cells indicates differential intracellular distribution based on cell types [Figure 10(a)-(c)]. Colocalisation studies suggest that PyPNVP and Py are distributed in the nucleus of HEPG2 and U87 cells besides its presence in the cytoplasm. Moreover, HEPG2 and U87 show considerable uptake of PyPNVP compared to Py alone [Figure 10(a) and (b)]. On the other hand, RAW264.7 does not show any uptake under similar experimental condition [Figure 10(c)]. The differential uptake pattern could be attributed to cell types and their specific characteristics. Both HEPG2 and U87 are with epithelial morphology compared to the immune cells RAW264.7, a BALB/c mouse derived peritoneal macrophages. This indicates that immune cells may behave differentially compared to cells of other types. Significant uptake of PyPNVP by HEPG2 and U87 cells over Py suggests that the conjugation of PNVP accentuate Py transport to nucleus significantly higher compared to free Py.

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Figure 10. Fluorescence Microscopic analysis of the uptake of PyPNVP in HEPG-2 (a), U87 (b) and RAW264.7 (c) cells. Cells were treated with free Py, PNVP, or, PyPNVP of 10.0 µM concentration in complete RPMI 1640 medium for 8 h at 37 °C. Representative images (scale bar = 200 µm) shown were obtained with green and blue filters for Py and Hoechst, respectively. n=4, where n represents the number of times experiment was performed.

We have also performed quantitative assessment of fluorescence in all three cell lines following treatment with Py, PNVP or PyPNVP conjugate. As shown in Figure 11, PyPNVP treatment in HEPG2 and U87 cells significantly increases the fluorescence compared to either Py or PNVP treatment. In contrast, no enhanced fluorescence was observed in RAW264.7 cells, supporting the imaging data as documented in Figure 10.

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Figure 11. Quantitative analysis for the uptake of the Pyrene-PNVP in HEPG2 (a), U87 (b) and RAW264.7 (c) cells following treatment with Pyrene, PNVP or PyPNVP conjugate (25 mg/mL each). Uptake of Pyrene was assessed using fluorescence plate reader. Intracellular Pyrene concentration (µg/mL) was measured in triplicate at different time points up to 12 hours. Data presented as Mean ± SD, n = 4.

3.6. Cytotoxicity Study. The cytotoxicity of PyPNVP has also been studied and compared with PNVP and pyerene (Figure 12) Assessment of direct cellular toxicity by PyPNVP on HEPG2, U87 and RAW264.7 cells suggest that the above compounds are tolerant to all the cell lines tested with no significant cytotoxicity in lower concentrations. In higher concentration (100 mg/mi), a moderate loss of cell death (< 20%) was observed which appears to be not significant (Figure 12). Therefore, PyPNVP could be useful as a tracking device in specific cellular organelles.

We could also speculate that PyPNVP would be useful as effective machinery for

the delivery of cargo like doxorubicin, non-fluorescent imatinib and other therapeutic agents in the heart of the nucleus for maximal chemotherapeutic efficacy against the tumor cells.

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Figure 12. Cytotoxic effect of Pyrene, PNVP or PyPNVP conjugate against HEPG2 (a), U87 (b) and RAW264.7 (c) cells was determined by 18 hours LDH release assay. Cells were incubated with indicated concentrations of the compounds for 18 hours followed by measurement of the released LDH according to the manufacture’s protocol. Data represents mean ± SD, n = 3.

4. CONCLUSION PyPNVP has evolved as fluorescent polymeric probe for multipurpose uses, owing to its enhanced solution proccessibility in water. It has successfully been used for the interaction with transition metal ions Cu2+ and Hg2+ through efficient fluorescence quenching. It has also been successfully used as fluorescence probe for CMC determination of amphiphilic block copolymer. It has also showed excellent interactions with biomacromolecules. It has interacted with CT 24



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DNA and cyclic plasmid DNA, as evidenced from quenching of its fluorescence during interaction process. Different magnitude and type of quenching can find usefulness in identifying the different type of DNAs. Further, it has showed excellent binding properties with model protein BSA. The quenching of the intrinsic fluorescence of BSA has been studied in terms of steady state, synchronous and 3D fluorescence spectroscopy, which suggests that during binding process a FRET type of interaction is occurring and also the fluorophores are buried into hydrophobic subdomains with conformational changes in polypeptide backbones. Such protein and DNA binding properties can make PyPNVP suitable for the therapeutic delivery purpose and other biotechnological uses like biosensors assay, biomaterial separations etc. As revealed by cellular uptake study, conjugation of PNVP accentuates Py transport to the nucleus of the HEPG2 and U87 cells significantly higher compared to free Py. Cytotoxicity study showed that PyPNVP is tolerant to all the cell lines tested with no significant cytotoxicity in lower concentrations.

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (PPM); [email protected] (BR). Present Addresses §

Department of Zoology, TheUniversity of Burdwan, Burdwan – 713104, West Bengal, India.

Funding Sources Department of Biotechnology (DBT), Govt. of India [Nos. BT/PR889/NNT/28/570/2011 dt. 2308-2013 (BR&PPM)]. Notes

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Authors declare no competing financial interest.

ACKNOWLEDGEMENTS Authors acknowledge Department of Biotechnology, Govt. of India, for financial support (Grant no.BT/PR889/NNT/28/570/2011).

Abbreviations RAFT, Reversible addition-fragmentation chain transfer; PNVP, poly(N-vinyl-2-pyrrolidone); Py, Pyrene; PyPNVP, Pyrene-tagged poly(N-vinyl-2-pyrrolidone); CMC, Critical miceller concentration; CT DNA, Calf thymus deoxyribonucleic acid; BSA, Bovine serum albumin.

REFERENCES (1) Patel, V. K.; Mishra, A. K.; Vishwakarma, N. K.; Biswas, C. S.; Ray, B. (S)-2-(Ethyl propionate)-(O-ethyl xanthate) and (S)-2-(Ethyl isobutyrate)-(O-ethyl xanthate)-mediated RAFT Polymerization of N-vinylpyrrolidone. Polym. Bull, 2010, 65(2), 97-110. (2) Patel, V. K.; Vishwakarma, N. K.; Mishra, A. K.; Biswas, C. S.; Ray, B. (S)-2-(Ethyl propionate)-(O-ethyl xanthate)- and (S)-2-(Ethyl isobutyrate)-(O-ethyl xanthate)Mediated RAFT Polymerization of Vinyl Acetate. J. Appl. Polym. Sci., 2012, 125(4), 2946–2955. (3) Patel, V.K., Viswakarma, N. K., Mishra, A. K., Biswas, C. S., Maiti, P.; Ray B. Synthesis of Alkyne-Terminated Xanthate RAFT Agents and Their Uses for the Controlled Radical Polymerization of N-Vinylpyrrolidone and the Synthesis of Its Block Copolymer Using Click Chemistry. J. Appl. Polym. Sci., 2013, 127(6), 4305–4317. (4) Patel, V. K.; Viswakarma, N. K.; Singh, S.; Mitra, K.; Ramesh, K.; Misra, N.; Ray, B.Synthesis of Fluorescence Poly(N-vinylpyrrolidone) via Click Chemistry using Azideterminated Xanthate Mediator (S)-2-(4-azidobutyl propionate)-(O-ethyl xanthate). Int. J. Polym. Mater. Polym. Biomater.2016, 65(6), 269–276.

26



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 30

(5) Mishra, A. K.; Patel, V. K.; Vishwakarma, N. K.; Biswas, C. S.; Raula, M.; Mishra, A.; Mandal, T. K.; Ray, B. Synthesis of Well-Defined Amphiphilic Poly(ε-caprolactone)-bpoly-(N-vinylpyrrolidone) Block Copolymers via the Combination of ROP and XanthateMediated RAFT Polymerization. Macromolecules, 2011, 44(8), 2465-2473. (6) Ramesh, K.; Mishra A. K.; Patel, V. K.; Vishwakarma, N. K.; Biswas, C. S.; Paira, T. K.; Mandal, T. K.; Maity, P.; Mishra, N.; Ray, B. Synthesis of Well-defined Amphiphilic Poly(D,L-lactide)-b-poly(N-vinylpyrrolidone) Block Copolymers Using ROP and Xanthate Mediated RAFT Polymerization. Polymer, 2012, 53(25), 5743-5753. (7) Hira, S. K.; Ramesh, K.; Gupta, U.; Mitra, K.; Misra, N.; Ray, B.; Manna, P. P. Methotrexate-Loaded Four-Arm Star Amphiphilic Block Copolymer Elicits CD8+ T Cell Response against a Highly Aggressive and Metastatic Experimental Lymphoma. ACS Appl. Mater. Interfaces, 2015, 7(36), 20021–20033. (8) S. Binauld, and M. H. Stenzel, Acid-degradable Polymers for Drug Delivery: a Decade of Innovation, Chem. Commun., 2013, 49(21), 2082-2102. (9) Tsarevsky N. V.; Bencherif, S. A.; Matyjaszewski K. Graft Copolymers by a Combination

of

ATRP

and

Two

Different

Consecutive

Click

Reactions.

Macromolecules,2007, 40(13), 4439-4445. (10)

Strutt, N. L.;Forgan, Ross S.; Spruell, J. M.;Botros, Y. Y.; Stoddart,J. F.;

Monofunctionalized Pillar[5]arene as a Host for Alkanediamines. J. Am. Chem. Soc., 2011, 133(15), 5668–5671. (11)

Bains, G.; Patel, A. B.; Narayanaswami, V. Pyrene: A Probe to Study Protein

Conformation and Conformational Changes. Molecules, 2011, 16(9), 7909-7935. (12)

Hung, H.; Cheng, C.; Wang, Y.; Chen, Y.; Chung, W. Highly Selective

Fluorescent Sensors for Hg2+ and Ag+ Based on Bis-triazole-Coupled Polyoxyethylenes in MeOH Solution. Eur. J. Org. Chem. 2009, 2009(36), 6360–6366

27


Page 27 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(13)

Chen, K.; Lu, C.; Cheng, H.; Chen, S.; Hu, C.; Wu, A. A Pyrenyl-appended

Triazole-based Ribose as a Fluorescent Sensor for Hg2+ ion. Carbohydrate Research, 2010, 345(17), 2557–2561. (14)

Hung, H.; Cheng, C.; Ho, I.; Chung, W. Dual-mode Recognition of Transition

Metal Ions by Bis-triazoles Chained Pyrenes. Tetrahedron Letters,2009, 50(3), 302–305. (15)

Park, S. M.; Kim, M. H.; Choe, J.-I.; No, K. T.; Chang, S.-K. Cyclams Bearing

Diametrically Disubstituted Pyrenes as Cu2+- and Hg2+-Selective Fluoroionophores. J. Org. Chem. 2007, 72(9), 3550-3553. (16)

Chen, S.; Duhamel, J.; Peng, B.; Zaman, M.; Tam, C. K. Interactions between a

Series of Pyrene End-Labeled Poly(ethylene oxide)s and Sodium Dodecyl Sulfate in Aqueous Solution Probed by Fluorescence. Langmuir, 2014, 30(44), 13164−13175. (17)

Haldar, B.; Chakrabarty, A.; Mallick, A.; Mandal, M. C.; Das, P.; Chattopadhyay,

N. Fluorometric and Isothermal Titration Calorimetric Studies on Binding Interaction of a Telechelic Polymer with Sodium Alkyl Sulfates of Varying Chain Length. Langmuir, 2006, 22(8), 3514-3520. (18)

Haldar, B.; Chakrabarty, A.; Chattopadhyay, N.Photophysics of Pyrene-end-

capped Poly(ethyleneoxide) in Aqueous Micellar Environments.

J. Mol. Liq. 2004,

115(2-3), 113– 120. (19)

Zhang, J.; Chen, W.; Tang, B.;, Zhang, W.; Chen, L.; Duan, Y.; Zhu, Y.; Zhu,

Y.; Zhang, Y.Interactions of 1-hydroxypyrene with Bovine Serum Albumin: Insights from Multi-spectroscopy, Docking and Molecular Dynamics Simulation Methods. RSC Adv., 2016, 6(28), 23622–23633.

28



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(20)

Page 28 of 30

Xu, C.; Gu, J.; Ma, X.; Dong, T.; Meng, X. Investigation on the Interaction of

Pyrene with Bovine Serum Albumin using Spectroscopic Methods. Spectrochim. Acta A: Mol. Biomol. Spectrosc. 2014, 125, 125391–125395. (21)

Li, L.; Lu, J.; Xu, C.; Li, H.; Yang, X. Investigation on the Interaction of Pyrene

with Bovine Serum Albumin using Spectroscopic Methods.Molecules,2012, 17(12), 14159-14173. (22)

C.,Youqiang; Shi, G. Fluorescence Detection and Discrimination of ss- and ds-

DNA with a Water Soluble Oligopyrene Derivative. Sensors, 2009, 9(6), 4164-4177. (23)

Geng, F.; Zheng, L.; Yu, L.; Li, G.; Tung, C. Interaction of Bovine Serum

Albumin and Long-chain Imidazolium Ionic Liquid Measured By Fluorescence Spectra and Surface Tension. Process Biochemistry, 2010, 45(3), 306–311. (24)

Kumari, M.; Maurya , J. K.; Singh, U. K.; Khan, A. B.; Ali, M.; Singh,P.; Patel,

R. Spectroscopic and Docking Studies on the Interaction between Pyrrolidinium based Ionic Liquid and Bovine Serum Albumin. Spectrochim. Acta A: Mol. Biomol. Spectrosc. 2014, 124, 349–356. (25)

Mukhopadhyay, S.; Gupta, R. K.; Paitandi, R. P.; Rana, N. K.; Sharma, G.; Koch,

B.; Rana, L. K.; Hundal, M. S.; Pandey, D. S.Synthesis, Structure, DNA/Protein Binding, and Anticancer Activity of Some Half-Sandwich Cyclometalated Rh(III) and Ir(III) Complexes. Organometallics, 2015, 34(18), 4491−4506. (26)

Matsushita, Y.; Moriguchi, I.; Binding of Pyrene-1-butyric Acid to Serum

Albumin: Species Differences. J. Pharmacobio-Dyn. 1989, 12(12), 762-770. (27)

Haldar, B.; Chakrabarty, A.; Chattopadhyay, N.Interaction of Pyrene-end-capped

Poly(Ethylene Oxide) with Bovine Serum Albumin and Human Serum Albumin in Aqueous Buffer Medium: A Fluorometric Study. J. Photochem. Photobiol. B, 2005, 80(3), 217–224.

29


Page 29 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(28)

Hira, S. K.; Mishra, A. K.; Ray, B.; Manna P. P. Targeted Delivery of

Doxorubicin-Loaded

Poly

(ε-caprolactone)-b-Poly

(N-vinylpyrrolidone)

Micelles

Enhances Antitumor Effect in Lymphoma. Plose One, 2014, 9(4), e94309. (29)

Turro,

N.J.;Ramamurthy,

V.;

Scaiano.J.

C.

Principles

of

Molecular

Photochemistry: An Introduction,University Science Books:USA, 2009. (30)

Maity, A.; Mukherjee, P.; Das, T.; Ghosh, P.; Purkayastha,P.Forster Resonance

Energy Transfer between Pyrene and Bovine Serum Albumin: Effect of the Hydrophobic Pockets of Cyclodextrins. Spectrochim. Acta A: Mol. Biomol. Spectrosc.,2012, 92, 382– 387.

30



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For Table of Contents Use Only:

Study of the fluorescence based applications of pyrenetagged poly(N-Vinyl-2-pyrrolidone) Kheyanath Mitra,† Shikha Singh,† Sumit Kumar Hira,‡, § Vijay Kumar Patel,† Deovrat Singh,† Sambhav Vishwakarma,† Rajshree Singh,† Archana Kumari,† Partha Pratim Manna,‡* Biswajit Ray†*

Successful uses of pyrene-tagged PNVP (PyPNVP) as multipurpose fluorescent probe have been explored for (i) ‘turn off’ chemosensing of metal ions Cu2+, Hg2+ etc; (ii) critical micellar concentration (CMC) determination of amphiphilic molecules; (iii) binding property study of DNAs and proteins, and (iv) preferential uptake in the nucleus of HEPG2 and U87 cells compared to free Py.

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Formatted: Line spacing: Double

Study of the Fluorescence Based Applications of Pyrene-Tagged Poly

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