Amplified Fluorescence from Polyfluorene Nanoparticles with Dual

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Amplified Fluorescence from Polyfluorene Nanoparticles with Dual state Emission and Aggregation-Caused Red Shifted Emission for Live Cell Imaging and Cancer Theranostics Balakrishnan Muthuraj, Sudip Mukherjee, Chitta Ranjan Patra, and Parameswar Krishnan Iyer ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11373 • Publication Date (Web): 04 Nov 2016 Downloaded from http://pubs.acs.org on November 4, 2016

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ACS Applied Materials & Interfaces

Amplified Fluorescence from Polyfluorene Nanoparticles with Dual state Emission and Aggregation Caused Red Shifted Emission for Live Cell Imaging and Cancer Theranostics Balakrishnan Muthuraj,a,ǂ Sudip Mukherjee,b,c,ǂ Chitta Ranjan Patra*b,c and Parameswar Krishnan Iyer*a,d a

Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati–781039,

India. b

Biomaterials Group, CSIR–Indian Institute of Chemical Technology, Uppal Road, Tarnaka,

Hyderabad-500007, Telangana State, India. c

Academy of Scientific and Innovative Research (AcSIR), Academy of Scientific and

Innovative Research (AcSIR), Taramani, Chennai – 600 113, India. [email protected]; [email protected] d

Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati-781039,

India. [email protected] ǂThese authors contributed equally.

KEYWORDS: polyfluorene; aggregation-induced enhanced emission; fluorescence imaging; cancer therapy; theranostics.

Abstract A newly synthesized polyfluorene derivative with pendant di(2–picolyl)amine (PF–DPA) shows dual state emission and aggregation caused red shifted emission that was utilized for cell imaging and cancer theranostics. PF–DPA was nontoxic to normal cells but showed cytotoxicity against cancer cells, suggesting its utility for cancer therapy. PF–DPA exhibits a large and unique red shifted emission at 556 nm at higher water ratio of THF:H2O (10:90) due to the formation of polymer nanoparticles or PDots spontaneously by intra and intermolecular self–assembly induced aggregation. Dual state emission and aggregation caused red shifted emission (>100 nm) in PF–DPA homopolymer nanoparticles is very unique and attributed to the combined effect of intramolecular planarization and J–type aggregate formation in the PDots (25 ± 5 nm). The PF–DPA PDots exhibit bright green and

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orange fluorescence with exceptional live cell imaging properties and potential applications in cancer theranostics due to their selective cytotoxic nature towards cancer cells.

Introduction Fluorescent probes1,2 comprising small molecule dyes, quantum dots and nanoparticles are widely used in living cell fluorescent bioimaging because of their size and biocompatibility. Yet, poor photo stability of these probes and leakage of heavy metals in quantum dots limits their broad applicability in long–term monitoring of living cells. Hence, the development of efficient probes for live cell imaging that are easy to synthesize, non-toxic, provide high sensitivity and selectivity are receiving attention. Conjugated polymers (CPs) are attractive materials since they fulfill the above requirements and have demonstrated imaging applications as well.3,4 CPs are known to have high fluorescence quantum yield, large extinction coefficients, efficient optical signal transduction and the synthetic versatility of CPs allows incorporation of a wide selection of functional groups for biological applications.3 Herein, we introduce a new CP that is capable of fluorescence imaging in live cells due to its unique multicolor imaging ability in aqueous media. The multicolor fluorescent imaging behavior in living cells are observed due to the formation of conjugated polymer nanoparticles (CNPs), also known as semiconducting polymer dots (PDots).5-11 The PDots exhibit strongly enhanced fluorescence emission, attributed to the combined effect of planarization and J–aggregation due to the aggregation induced emission enhancement (AIEE) phenomenon.12 The propeller–shaped molecules and CPNs exhibit the phenomenon of AIE, due to the restricted intramolecular rotation (RIR) of the multiple phenyl rotors in the aggregate state.13 These tetraphenylethylene and silolefluorophores have attracted enormous attention in chemo– and biosensing, imaging and therapy since they emit strong fluorescence in the aggregated state due to which they have been incorporated into polymers and macromolecules either as a pendant or as a copolymerizing unit.14-18 Hence, the development of bright multicolor fluorescent PDots remains interesting for widespread biological applications and cancer research. However, the use of highly specific AIE fluorogens, expensive multistep synthesis involving costly catalysts and purification steps, limits their practical utility. To encompass the benefits of AIE effect at the same time reduce the overall reaction economy, alternate structures overcoming the drawbacks in existing systems are required. We present here a polyfluorene (PF) homopolymer appended with a flexible di(2– picolyl)amine pendant unit on the PF side chains (PF–DPA) that spontaneously displayed aggregation caused red shifted emission phenomenon (in contrast to blue emitting planar 2


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polyfluorene). This PF–DPA has no restricted rotation in organic solvent (THF) whereas, at higher water ratio the RIR behavior likely dominates, resulting in bright and remarkable red shifted emission.

Experimental Section All reagents and solvents were purchased from commercial sources and solvents used were of spectroscopic grade. UV-vis absorption spectra were recorded on a PerkinElmer Lambda-25 spectrometer. Fluorescence spectra were carried out on a FluoroMax-4 SpectrofluorometerHoriba Scientific equipped with a Quanta-ϕ integrating sphere for solid state fluorescence quantum yield. A 10 × 10 mm quartz cuvette was used for solution spectra and emission was collected at 90° relative to the excitation beam. Deionized water was obtained from Milli-Q system (Millipore). 1H NMR (400 and 600 MHz) and

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C NMR (150 MHz) spectra were

obtained with a Varian-AS400NMR and Bruker spectrometer. The PF-DPA NPs were examined using an ultrahigh resolution transmission electron microscope (TEM; JEM 2100; Jeol, Peabody, MA, USA). DLS were measured by Zetasizer Nano series Nano-ZS90 instrument. Cell lines: Human ovarian cancer cells (SKOV3); mouse melanoma cell line (B16F10); primary mouse embryonic fibroblast cells (3T3); and CHO-Chinese hamster ovarian cell were purchased from ATCC, USA.

Synthesis of monomer Synthesis of 9,9-Bis-(6-bromohexyl)-fluorene: Fluorene (4 gm, 24.064 mmol), 50% aq. NaOH and a catalytic amount of tetrabutylammonium iodide (1.7 gm, 3.248 mmol) were added to a flask. The flask was degassed 3 times by applying freeze-thaw cycles. 1, 6 dibromohexane (35.22 gm, 215.13 mmol) was added through a syringe (degassed) and the mixture stirred continuously for 4 h at 70 °C (Scheme 1). The reaction mixture was cooled to room temperature and extracted with chloroform. The organic layer was washed with water and dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the crude was purified using column chromatography over a small pad of silica gel using pure hexane as an eluent to give the desired fluorene dialkylated product. Yield = 3.68 g (92 %). ¹H NMR (400 MHz, CDCl3): δ (ppm): 7.68 (m, 2H), 7.28 (m, 6H), 3.26 (t, 4H), 1.96 (m, 4H), 1.63 (m, 4H), 1.17 (m, 4H), 1.07 (m, 4H), 0.60 (m, 4H). ¹³C NMR (150 MHz, CDCl3) δ (ppm): 150.50, 141.32, 127.29, 127.05, 122.95, 119.92, 55.10, 40.42, 34.12, 32.83, 29.23, 27.95, and 23.71. Synthesis of PF-Br 3



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In a 100 mL three-necked round-bottom flask equipped with a nitrogen inlet, anhydrous ferric chloride (2.25g, 13.86 mmol) was dissolved in 20 mL of nitrobenzene. 9,9-Bis-(6bromohexyl)-fluorene (1.0 g, 6.09 mmol) dissolved in 15 mL nitrobenzene was added to the flask using a syringe. The reaction mixture was stirred at room temperature for 36 h, followed by precipitation from methanol (Scheme 1). This was stirred for 1 h, centrifuged and washed repeatedly with methanol. The resulting polymer was dried under reduced pressure to obtain 1.39 g (70%) as dark brown powder. ¹H NMR (400 MHz, CDCl3), δ (ppm): 7.65-7.79 (ArH, broad), 7.33 (ArH, broad), 3.26 (broad), 2.02 (broad), 1.54 (broad), 1.23 (broad), 0.78 (broad). ¹³C NMR (150 MHz, CDCl3) δ (ppm): 151.68, 141.30, 127.29, 127.04, 122.94, 119.91, 40.41, 34.15, 32.82, 31.13, 29.25, 27.95, and 23.86. Mw = 2.04 × 104, PDI-1.95 (GPC in THF, polystyrene standard). Synthesis of PF-DPA Polymer PF-Br (67 mg), di(2-picolyl)amine (DPA) (60 mg, 0.3 mmol), potassium carbonate (70 mg, 0.5 mmol) were mixed and heated at 150 °C for 36 hrs in dry DMF (Scheme 1). The mixture was cooled to room temperature and poured into methanol (100 mL). The brown precipitant was collected and washed with acetone and dried overnight in a vacuum desiccator (68 mg, 86.8%). ¹H NMR (600 MHz, CDCl3), δ (ppm) : 8.46 (ArH, broad), 7.80 (ArH, broad), 7.65 (ArH, broad), 7.56 (ArH, broad), 7.43 (ArH, broad), 7.32 (ArH, broad), 7.07 (ArH, broad), 3.70 (–NCH2–), 2.39 (–CH2-N–, broad), 2.04 (–CH2–, broad), 1.35 (– CH2–, broad), 1.10 (–CH2–, broad), 1.03 (–CH2–, broad), 0.74 (–CH2, broad). ¹³C NMR (150 MHz, CDCl3) δ (ppm): 160.40, 151.43, 149.02, 139.55, 136.48, 125.76, 122.99, 121.99, 121.46, 120.31, 114.26, 60.60, 54.53, 40.44, 34.01, 32.11, 29.55, 27.20, and 22.88. UV-vis (THF:H2O (90:10), 5.0 × 10-6 mol L-1): max (nm): 374; Emission (THF:H2O (90:10), 0.33 × 10-6 mol L-1) : 412 nm. Mw = 2.96 × 104, PDI-1.43 (GPC in THF, polystyrene standard). Preparation of PF-DPA nanoparticles or PDots PF-DPA (10 µM) polymer was regularly injected into THF: H2O (10:90) with vigorous stirring at room temperature, using a syringe. After the injection of PF-DPA, the solution was filtered by membrane filter with 0.2 µm pore size. The collected PF-DPA nano particles or PDots were characterized by TEM, FESEM, AFM and DLS (Figure S1-S4) and then used for all other studies. Concentration dependent UV−visible spectra of PF-DPA in THF (100%) The UV−vis spectrum of PF-DPA was observed in THF (100%) with different concentrations of PF-DPA (from 1 to 5 µM). This isolated PF-DPA polymer shows maximum absorption 4


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band at 370 nm without any shift, on increasing the concentration from 1 to 5 µM. The absorbance of PF–DPA enhanced linearly (R2 = 0.9997) on increasing the concentration of PF-DPA over the range 1–5 µM. Concentration dependent UV−visible spectra of PF-DPA in THF: H2O (1:9) ratio The UV−vis spectra of PF-DPA in THF: H2O (10: 90) ratio was observed with different concentrations (1 to 10 µM). The isolated PF-DPA polymer absorption band at 370 nm is red shifted by 8 nm to 378 nm and is enhanced with the increase in concentration from 1 to 10 µM. This red-shifted phenomenon can be interpreted as a result of J-aggregation by noncovalent interactions between intermolecular polymers. The absorbance of PF–DPA increased linearly (R2 = 0.999) on increasing the concentration of PF-DPA over the range 1– 10 µM. Fluorescence images study of PF-DPA in THF:H2O (10:90) ratio 100 µL of PF-DPA (1.65 mg in THF (100 µL) + H2O (900 µL)) were taken in 96 well plate and images were captured using fluorescence microscopy in red, green and blue filter respectively using fluorescence microscope (Nikon Eclipse TE2000-E). Subsequently, the observed multicolor fluorescence images greatly support the formation of J- aggregate PFDPA nanoparticles via AIEE behavior. Cell culture experiments All cancer (SKOV-3: human ovarian cancer cell; B16F10: mouse melanoma cancer cell line) and normal cells (NIH-3T3: mouse fibroblast cell line; CHO: Chinese hamster ovarian cell) lines were cultured in DMEM (Dulbecco’s Modified Eagle Medium) media supplemented with 5% L-glutamine, 1% antibiotics (penicillin-streptomycin) and 10% fetal bovine serum (FBS), in a humidified 5% CO2 incubator at 37 °C for in vitro experiments. All samples were sterilized under UV irradiation for 10 minutes before doing in vitro treatment. In vitro cell viability assay using MTT reagent Initially, 1×104 cells were seeded in 96 well plates and allowed to incubate for 24 h to grow. Cell viability assay of NIH-3T3, CHO, B16F10 and SKOV-3 was carried out after 24 h incubation with PF-DPA in a dose dependent manner using MTT reagents according to our published protocol.19 Briefly, all cells treated with PF-DPA were washed extensively by PBS to remove surface attached nanoparticles. Later the cells were incubated with 100 µL of MTT solution in DMEM media (0.5 mg/mL) and allowed to incubate for 4 h under dark condition. After 4 h of incubation, the MTT solution was replaced by fresly prepared 100 µL of DMSO+MeOH (1:1) in each well to solubilise the formazan dye. Absorbance reading of each 5



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well were recorded at λ = 575 nm and results were expressed as percent cell viability = {[A570 (treated cells) - background] / [A570 (untreated cells) - background]} ×100. Cell imaging study using fluorescence microscopy All normal and cancer cells (NIH-3T3, CHO, SKOV-3 and B16F10) cells were incubated with PF-DPA for 24 h. All the treated cells were washed extensively by PBS for 4-5 times and finally HBSS buffer (Hank's Balanced Salt Solution, pH = 7.4) was added.

The

fluorescence images were collected by fluorescence microscope (Nikon Eclipse TE2000-E). The orange emission (λem = 605 nm) was collected with a 20X microscope objective after excitation at λex = 510-560 nm. Similarly, the green fluorescence emission (λem = 525 nm) was collected with a 20X microscope objective after excitation at λex = 420-495 nm.

Results and discussion

Scheme 1. Synthesis scheme of polymer PF–DPA. Herein, the design and synthesis of a new PF–DPA fluorophore based on poly(9,9–Bis–(6– bromohexyl)–fluorene) (Scheme 1) is presented.20-23 PF-DPA emits in the blue region (355 nm excitation) in THF:H2O (90:10) (HEPES buffer, pH 7.4). Additionally, the presence of DPA also facilitates in realizing the unique AIEE effect at room temperature which is unusual and has never been observed previously in blue emitting PF derivatives possessing alkyl 6


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chains.24 The bulky DPA molecule on the polymer pendant chain restricts the intramolecular rotation in higher water ratio (THF:H2O, 10:90) that results in red shifted emission from PF– DPA due to the formation of PDots via intermolecular self–assembly behavior. This observation of bright orange emission from a PF homopolymer is unique and served as a motivation to utilize PF-DPA in cellular imaging and cancer therapeutics.

Aggregation Caused Red Shifted emission or Dual State Emission behavior in PF–DPA The PF–DPA exhibited blue emission at 412 nm (355 nm excitation) in THF (Figure 1a) since no PET (photo induced electron transfer) is involved here due to the longer spacer unit between fluorophore (polyfluorene) and receptor (DPA). In this state, the PF–DPA may likely exist in an isolated state and no emission at 556 nm was observed. In contrast, PF–DPA exhibited strong red shifted emission at 556 nm (Figure 1a) in THF:H2O (10:90) (HEPES buffer, pH 7.4) solution at higher PF–DPA concentration (50 µM) (355 nm excitation). To investigate the unique 144 nm shift from 412 to 556 nm in 100% THF to 90% H2O solution, (Figure 1a and 1c) we recorded the PL spectra in various THF and water fraction ratio with constant concentration of PF–DPA (50 µM). When the water fraction was increased to 60% there is a change at 412 nm with a minor decrease in the peak intensity. Upon increasing water up to 70% the 412 nm peak is red shifted to 556 nm which suggests the formation of Jtype aggregation. The peak intensity was enhanced on increasing the water fraction from 70% to 90%, due to the formation of polymer nano particles (Figure 1b and 1c). These combined results imply that the PF–DPA exhibited an unusual aggregation caused red shifted emission behavior. Moreover, in aqueous medium PF–DPA spontaneously formed nanoparticles (25 ± 5 nm) (PDots) and demonstrates strong intermolecular self–assembly behavior (Figure 1b). At low concentration (0.33 µM) the fluorescence intensity of PF–DPA at 412 nm showed no significant changes till the ratio of water in THF:H2O was (40:60) (HEPES buffer, pH 7.4), however, when the ratio of water increased from 30:70 to 10:90 (THF:H2O) (HEPES buffer, pH 7.4) the fluorescence intensity of PF–DPA got quenched [Figure S5 (a, b) ] due to the formation of intermolecular self–assembly induced aggregation in the polymer. Though, fluorescence quenching occurred at 412 nm (blue region) in THF:H2O (10:90), under UV light it showed orange color emission [Figure S5 (b)] due to the formation of polymer nano particles by intermolecular self–assembly behavior, suggesting that PF-DPA polymer does not behave like other ACQ materials. This unusual behavior is described as aggregation 7



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caused red shifted emission or dual state emission. Furthermore, at higher concentration of PF–DPA (50 µM), the fluorescence intensity of PF–DPA appeared at 412 nm and was similar as above, with no significant changes till the THF:H2O ratio was (40:60) (HEPES buffer, pH 7.4), however, when the ratio of water was increased from 30:70 to 10:90 (THF:H2O) (HEPES buffer, pH 7.4) the fluorescence intensity of PF–DPA showed a remarkable red shift from 412 nm and a new intense emission appeared at 556 nm [Figure 1(c, d)] due to the formation of intermolecular polymer self–assembly induced aggregation and is in agreement to the earlier observation. This emission shift is further confirmed under UV lamp where the solution color changed from blue to bright orange, indicating no quenching in PF-DPA polymer due to the formation of emissive polymer nanoparticles or PDots via noncovalent interactions. This confirms that, the polymer PF-DPA is an aggregation caused red shift emissive material (homopolymer) which is an exceptional example not known among existing systems.

Figure 1. (a) Fluorescence spectra of PF–DPA (0.33 µM) in THF:H2O (100:0) and PF–DPA (50 µM) THF:H2O (10:90) (HEPES buffer, pH 7.4). (b) TEM images of PF–DPA NPs (PDots). (c, d) Aggregation caused red shifted emission spectra of PF–DPA (50 µM) at 556 nm in THF:H2O (100:0) to THF:H2O (10:90) (HEPES buffer, pH 7.4). In fig. d, the inset photos taken under UV light shows the color change of PF–DPA from blue [THF: H2O (100:0)] to intense yellow orange [THF:H2O (10:90), (HEPES buffer, pH 7.4)]. (e) Absorbance spectra of PF–DPA (5 µM) in THF: H2O (100:0) and THF:H2O (10:90) (HEPES buffer, pH 7.4). 8


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This decrease in emission at 412 nm could be either due to H or J–aggregation (Scheme 2). When the water fraction was lower than 70%, the molecules are more emissive at 412 nm compared to 556 nm. However, in a system with higher water, THF:H2O (10:90), the PF– DPA agglomerated spontaneously in a random way to form PDots that were less emissive in the blue region (412 nm) but highly fluorescent in the orange region (556 nm). The inset photos taken under UV light illumination depict the color change from blue to intense yellow orange (Figure 1d) assigned to the PF–DPA PDots formation spontaneously in aqueous solution that display strong fluorescence due to the intermolecular self–assembly behavior. This unique and yet unexplored observation of the Aggregation caused red shifted emission phenomenon in an inherent blue emitting fluorene homopolymer derivative has never been observed previously and presents immense application potential in cell imaging, cancer therapy, sensors and optical devices. The aggregation caused red shifted emission effect could be explained in terms of intra– and intermolecular effects on PF–DPA polymer in aqueous and solid state. In the THF solution of PF–DPA, the alkyl chain restricts the aggregate formation due to the parallel–type intermolecular interactions of polymer chains thereby exhibiting blue–shifted emission bands compared to the isolated state (H–aggregate formation). However, PF–DPA at 10:90, THF:H2O ratio shows remarkable red shifted emission band at 556 nm indicating that the prevention of H–aggregate formation compelled the formation of J–aggregation by likely end–to–end arrangements which induces a huge bathochromic shift and enhancement in the emission maximum at 556 nm (Scheme 2).25-27 This emission maximum shift at 556 nm, is assigned to the combined effects of aggregation induced planarization and J–aggregate formation in the PF–DPA polymer. Moreover, the bulky chelating side chains (DPA) are one of the main reasons to form supramolecular aggregates.28 Consequently, the appropriate size of substituent is required at 9,9 position in polyfluorene to enhance the aggregation via intermolecular self–assembly behavior. Moreover, this bulky group may not allow them to form the detrimental aggregates instead it facilitated to form intermolecular self–assembly via noncovalent interactions. Furthermore, this aggregation caused red shifted emission was confirmed by quantum yield (Φ) calculation (Table S1). However, in THF solvent at 412 nm the isolated polymers quantum yield was found to be 57.68%. Moreover, no significant emission band appeared at 556 nm in THF, whereas, in case of H2O medium (at 556 nm), the aggregated polymer quantum yield was calculated to be only 2.88% that confirmed the aggregation caused red shifted emission in aqueous medium due to the intermolecular self–assembly. However, the observed 9



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fluorescence efficiency (Φ = 2.88%) does not confirm that PF-DPA is an ACQ polymer instead shows it as a dual state emissive polymer. Recently, Tang et al29 discussed the differences between ACQ and AIE compounds and their advantages that created highly efficient luminogens in both solution and solid states, which are likely to have broader applications.30-33 It is expected that the solution and solid state as well as the dual state emitting luminogens have to simultaneously fulfill the requirements of considerable rigidity with limited intramolecular motions in solution13,14,34,35 and considerable twisting conformations in solid to prevent detrimental exciton interactions.36 Higher rigidity in solution state along with twisted conformations in the solid state within a single molecular system was thought to be contradictive and fundamentally impossible. However, it is found that triphenylamine (TPA) fluoresces in THF solution and in solid state with photoluminescence (PL) efficiencies of 13.0% and 10.2%, respectively.35 Herein, the PF-DPA homopolymer also displayed unique optical behavior, as a solution and solid dual state emitting luminogen by aggregation caused red shifted emission that fulfills the requirements of substantial rigidity with limited intramolecular motions in solution and considerable twisting conformations in solid to prevent detrimental exciton interactions.

Scheme 2. Schematic diagram for the formation of PF–DPA PDots showing aggregation caused red shifted emission or Dual state emission behavior. Figure 1(e) displays the UV−vis absorption spectra of PF-DPA (5 µM) in THF:H2O (100:0) and THF:H2O (10:90) ratio. In THF:H2O (100:0), 5 µM PF-DPA shows a band at 370 nm which indicates it to be in isolated state (Figure 1e). Similarly, in THF:H2O (10:90), the isolated PF-DPA (5 µM) band at 370 nm is 8 nm red shifted to 378 nm and the absorption decreases, indicating that PF-DPA is aggregated in aqueous medium (Figure 1e). Moreover, 10


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this red-shifted absorption confirms the J-aggregate formation by π−π stacking between intermolecular PF-DPA polymers through end-to-end type assembly.37,38 Herein, the effect of solvent and pH on PF-DPA fluorescence spectra were also studied (Figure S6 and S7). In addition the fluorescence lifetime experiment shows that the fluorescence lifetime decay profile for the PF-DPA in THF solution exhibited a mono-exponential decay with a lifetime value of 0.327 ns attributable to the solvated monomer species present under this condition (Figure S8). However, in case of water, PF-DPA showed biexponential decay with the species having higher lifetimes [T1 = 0.592 ns (35.31 %) and T2 = 2.819 ns (64.69 %)]. Further, we performed the fluorescence study of PF-Br (Figure S9) in organic [THF:H2O (100:0)] and aqueous solution [THF:H2O (10:90)]. The resultant spectra showed the red shifted emission at 556 nm in aqueous solution due to the bulky Br atom in the pendent chain that restricts the formation of detrimental aggregates in higher water ratio (THF:H2O, 10:90) and instead, forms intermolecular self–assembly via noncovalent interactions between fluorene backbones. Moreover, this result confirmed that the resultant PF-Br emission spectra also showed the red shifted emission at 556 nm in aqueous solution without DPA group which validates the J-aggregates are formed via fluorene-fluorene noncovalent interaction and not by fluorene-DPA interaction.

Cell viability assay of PF-DPA Cell viability or cytotoxicity study is one of the basic studies for any drug or compound before successful applications in biomedical sciences. Accordingly, cell viability assay of different normal (NIH-3T3 & CHO) and cancer cells (SKOV-3 & B16F10) was carried out in presence of PF-DPA (in THF:H2O = 10:90 solvent system) in a dose dependent manner (from 40 µg/mL and above) for 24 h of incubation using MTT reagents (Figure 2).39 The cell viability results show that PF–DPA is biocompatible to NIH-3T3 (upto ∼1.6 mg/mL) and CHO cells (upto 500 µg/mL). However, the PF-DPA exhibits significant inhibition of proliferation in cancerous cells (SKOV-3 and B16F10) even at low concentration of PF-DPA (~200 µg/mL or below this concentration) after 24 h of incubation. Doxorubicin (2.5 µM), FDA approved chemotherapeutic drug was used as positive control cytotoxic.

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Figure 2. Cell viability assay of different cell lines using PF-DPA (a) NIH-3T3; (b) CHO; (c) SKOV-3; and (d) B16F10. Doxorubicin (DOX) was used as positive control experiment.

The solvent system [THF:H2O (10:90)] was used as vehicle control in both B16F10 and CHO cells in a dose dependent manner (1 to 10µL). The results show no significant toxicity of the solvent system (Figure S10). Further, we calculated the IC50 values of PF-DPA in both cancer cells (B16F10 & SKOV-3) as well as in normal cells (CHO & NIH-3T3).40 The IC50 values are tabulated in the Table S2. The results show that, IC50 values of PF-DPA are lower (B16F10: 411 µg/mL & SKOV-3: 766 µg/mL) than that in normal cells (CHO: 1851 µg/mL & NIH-3T3: >2000 µg/mL). The lower IC50 values of PF-DPA in cancer cells indicate the anticancer activity of the material. The high IC50 values of PF-DPA in normal cells suggest that PF-DPA is biocompatible even at very high concentrations.

Cell imaging and cytotoxicity against cancer cell studies Figure 1 (a-e) confirms PF-DPA to exist as PDots due to the π−π stacking that led to the Jaggregates formation thereby exhibiting multi-color imaging ability as confirmed by fluorescence microscopy (Figure S11). Since PF–DPA spontaneously forms PDots (25±5 nm) in aqueous solution (Figure 1b), presenting huge application potential in cell imaging and labeling. PF–DPA showed intense orange (λem = 605 nm) and green (λem= 525 nm) fluorescence inside live cells under a fluorescence microscope (at λex = 510–560 nm and λex = 420–495 nm) due to the aggregation caused red shifted emission which was exceptional and 12


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further motivated us to check its fluorescence labelling ability in few other live cells. This tunable orange and green fluorescence of any probe inside the live cells are critical requirements to examine even small fold changes in a cell which are difficult to observe in fluorophores emitting in the UV–vis range. In order to demonstrate this unique tuneable fluorescence property of PF–DPA PDots in cellular level, both cancer (SKOV-3 and B16F10) and normal cells (NIH-3T3) were incubated with PF–DPA (40 µg/mL and 80 µg/mL) (Figure 3 and S12). From fluorescence microscopic data, it is visible that the intensity of green and orange fluorescence intensities in cancer cells (SKOV-3 and B16F10) is higher than that of normal cells (NIH-3T3 and CHO) based on the normalized data (Figure S13). Quantification of uptake of PF-DPA was carried out in both normal cells (CHO) as well as in cancer cells (B16F10) using spectrofluorimeter (Figure S14). Fluorescence results confirm more uptake of PF-DPA in cancer cell (B16F10) as compared to normal (CHO) cell based on the quantitative data. These results corroborate well with the fluorescence microscope data (Figure 3). Thus the uptake of PF–DPA is more specific towards cancer cells than normal cells. These results together supports the in vitro cytotoxicity data (Figure 2) where PF–DPA shows potent cytotoxicity to cancer cells compared to the normal cells under identical concentration.

Figure 3. Fluorescence images of cancer cells (a1-a3: SKOV3; b1-b3: B16F10) and normal cells (c1-c3: NIH-3T3; d1-d3: CHO) treated with PF–DPA (40 µg/mL) at 20X optical zoom. First, second and third row indicates the corresponding phase images (a1-d1), green (a2-d2) and red (a3-d3) fluorescence images of cells treated with PF-DPA [Scale bar: 50 µm].

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Additionally, we performed the fluorescence emission spectroscopy of PF-DPA in DMEM media upon excitation at different wavelength (λex = 420/510/530/560 nm) (Figure S15). The intensity of orange fluorescence is high (~20,000 a.u.) when excited at 420 nm which decreased (~5000 a.u.) when PF-DPA was excited at 510-530 nm. However, this intensity is still high enough (~5000 a.u.) to be visible by fluorescence microscopy. Again, a broad hump of spectra in the range from 550 nm to 700 nm was observed. Thus, both green and orange fluorescence are observed upon excitation at various wavelengths (λex = 420/510/530/560 nm). We performed additional experiments that helped to explain the detailed mechanistic studies of the anticancer potential of PF-DPA in cancer cells and biocompatibility in normal cells. Chick embryo angiogenesis (CEA) assay, a standard in vivo assay shows time dependent (0-4 h) decrease of blood vessels formation (angiogenesis) in embryos after treatment with PFDPA [Figure 4(a-d)] compared to control untreated embryo. The results show the antiangiogenic properties of PF-DPA that could be useful for cancer therapeutics. Anticancer potential of any drug/nanomaterials is also associated to some extent of apoptosis by these molecules. Apoptosis assay with FITC Annexin-V staining of B16F10 cells treated with PFDPA showed higher apoptosis in cancer cells (~80%) compared to control untreated B16F10 cells [Figure 4(e-h)]. These results together demonstrate the high anticancer activity of PFDPA. Cell cycle arrest in any of the four phases namely sub-G1, G0/G1, S and G2/M is also linked with anticancer potential of nanomaterials, that can be monitored by FACS.41 Cell cycle results imply the arresting of B16F10 cells in G2/M phase while the cells were treated with PF-DPA and DOX compared to untreated cells [Figure S16 (a-d)].

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Figure 4. In vivo CEA assay and apoptosis assay. (a-d) CEA assay of embryos incubated with PF-DPA (400 µg) reveals that the vascular sprouting was damaged from 0 h to 4 h (c-d) (marked by green arrows at 0 h & black arrows at 4 h) compared to vehicle control i.e. VC (ab) (150 µL THF:H2O in 10:90 ratio) treatment after 4 h. Results showed anti-angiogenic properties of PF-DPA. (e-g) Flow cytometric analysis of B16F10 cells treated with PF-DPA using Annexin V FITC staining and (h) quantification of apoptosis. (e) UT, (f) DOX (2.5 µM), (g) PF-DPA (500 µg/mL). This study reveals the increased population of the late apoptotic cells (Q2) in presence of PF-DPA for 30 h compared to untreated control cells.

It is a well-known that, reactive oxygen species (ROS) especially H2O2 or superoxide radicals (O2-) can play a crucial role in cancer cell death.42 In this context, we quantified the amount of ROS produced in cancer cell (B16F10) as well as in normal cells (CHO) after treatment with PF-DPA. Results showed that PF-DPA produced significant amount of both H2O2 and O2- in B16F10 melanoma cancer cells compared to untreated B16F10 cells [Figure 5(a,b)]. DOX was used as positive control experiment. Thus, we hypothesized that ROS might be one 15



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of the possible mechanisms for the anticancer effect of PF-DPA. However, in case of normal cells (CHO), generation of ROS is almost same for untreated control cells and cell treated with PF-DPA [Figure 5(c,d)]. However, the ROS produced in cancer cells are significantly higher than (~3-5 times) ROS produced in normal cells. Based on the combined data from uptake study and ROS generation in both cancer and normal cells, the PF-DPA is biocompatible to normal cells (less uptake and low ROS generation) and cytotoxic to the cancer cells (more uptakes and more ROS generation). Additionally, to check the possible role of structural moiety towards the generation of ROS, we measured the amount of ROS generated in B16F10 cancer cell after treatment with DPA (400 µg/mL) and fluorene (400 µg/mL) using DCFDA dye with the help of spectrofluorimeter. The result confirms that B16F10 cells treated with DPA (400 µg/mL) produced enhanced amount of H2O2 compared to untreated cells and cells treated with fluorene (400 µg/mL) (Figure S17).

Figure 5. (a-d) Quantification of intracellular ROS (H2O2 and O2.-) formation in untreated B16F10 and CHO cells and cells treated with PF-DPA (400 µg/mL). DOX (2.5 µM) was kept as positive control. Importantly, it is evident that one molecule of DPA which contains three atoms of nitrogen, may trigger the production of ROS. Earlier published reports suggest that amine and 16


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polyamine compounds can induce ROS at an apoplastic site from peroxidase- or amine oxidase-type enzymes.43 Furthermore, it has been demonstrated that polyamines or polyamine analogues could directly contribute to programmed cell death through their regulatory effect on ion channels through K+/Ca2+ efflux, causing the ROS mediated cell death.44 Moreover, the potential anticancer activity of manganese complexes of N-substituted di(picolyl)amine (DPA) in various cancer cells has also been reported.40 The mechanistic studies reveal that these complexes increased ROS level in cancer cells. Thus, the enhanced ROS level is probably the key reason for cell death through apoptosis. It is also reported that, cancer cells are more vulnerable to H2O2 induced cell death than normal cells. The earlier reports and our experimental results together suggest the role of DPA and their complexes for the generation of ROS and consequently anti-cancer activities. Additionally, the nuclear damage of both untreated control cells (CHO and B16F10) and cells treated with PF-DPA, were observed after staining with Hoechst dye.45 Results indicate that B16F10 cells treated with PF-DPA shows nuclei with chromatin condensation and apoptotic bodies (marked by white arrows) (Figure S18), whereas, CHO cells treated with PF-DPA do not exhibit any nuclei damage. The results support the biocompatible nature of PF-DPA towards normal cells but cytotoxic nature in cancer cells. These results together demonstrate that uptake of PF–DPA in cancer cells is more than that in normal cells that lead to increased toxicity and specificity of PF– DPA towards cancer cells compared to normal cells. All the above bioassay results suggest the high potential of PF-DPA for cancer theranostics. This PDots provide an efficient working platform as compared to other molecules in addition to overcoming the aggregation caused quenching (ACQ) effect usually observed in PF derivatives. Furthermore, PF-DPA provides a simpler structure for the rapid multi-color imaging of cancer cells and presents immense opportunities for the development of newer probes with multiple applications in cancer theranostics as well as for optoelectronics.

Conclusion In summary, a new fluorophore based on poly(9,9–Bis–(6–bromohexyl)–fluorene) homopolymer (PF-DPA) was designed, synthesized and its unusual aggregation caused red shifted emission or dual state emission properties were carefully examined. This uncharacteristic aggregation caused red shifted emission or dual state emission property of PF-DPA homopolymer was utilized as a multi-color probe for cancer cell imaging and therapy. The spontaneously formed PF–DPA PDots exhibited orange and green emission in 17



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cellular environment indicating the future potential application of bio-imaging for cancer diagnostics. Since the PF–DPA PDots were biocompatible in normal cells and cytotoxic to cancer cells, PDots could be useful for cancer theranostics, ROS generation and quantification, multicolor bioimaging and labeling. Together, these results demonstrated that appropriate functionalization of polyfluorene homopolymer derivative such as PF–DPA could be utilized to tune the photophysical properties with potential applications in cell imaging, delivery of bioactive molecules or anticancer drugs in cells as well as theranostics agents. This conjugated homopolymer PF-DPA due to their inherent film forming ability are also suitable to be used directly as a free standing membrane or a paper strip based sensor probe for diagnosis on-site to work at ambient and physiological conditions.

ASSOCIATED CONTENT Supporting Information It contains the synthesis and characterization of PF-DPA, instrumentation details, photophysical studies, pH studies, lifetime, toxicity and cell uptake of PF-DPA and its application in bioimaging with various cells, ROS studies and bioassay experimental details. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected], [email protected] *E-mail: [email protected] (P.K.I.). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS Financial support from the DST, India (No. DST/SERB/PC-20/2014/000034), (No. DST/TSG/PT/2009/23), DST−Max Planck Society, Germany (No. INT/FRG/MPG/FS/2008), DIT, India, DeitY project No. 5(9)/2012–NANO (Vol.II), Ramanujan Fellowship Grant, DST (SR/S2/RJN–04/2010; GAP0305) and CSIR, New Delhi (Advanced Drug Delivery System: CSC0302) is gratefully acknowledged. The CIF, IIT Guwahati is thanked for instrument facilities. SM is thankful to CSIR, New Delhi for his Research Fellowship.

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