benzene Derivative and Its Inclusion Complexes with Cyclodextrins

Jul 1, 2010 - Departments of Nanofusion Technology and Cogno-Mechatronics Engineering (WCU), Pusan National. UniVersity, Miryang 627-706, Republic ...
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J. Phys. Chem. B 2010, 114, 9684–9690

Two-Photon Absorption Properties of Cationic 1,4-Bis(styryl)benzene Derivative and Its Inclusion Complexes with Cyclodextrins Okhil Kumar Nag,†,‡ Rati Ranjan Nayak,†,‡ Chang Su Lim,| In Hong Kim,§ Kwangseuk Kyhm,§ Bong Rae Cho,*,| and Han Young Woo*,†,‡ Departments of Nanofusion Technology and Cogno-Mechatronics Engineering (WCU), Pusan National UniVersity, Miryang 627-706, Republic of Korea, Department of Physics Education, Pusan National UniVersity, Busan 609-735, Republic of Korea, and Department of Chemistry, Korea UniVersity, Seoul 136-701, Republic of Korea ReceiVed: March 25, 2010; ReVised Manuscript ReceiVed: May 12, 2010

Two-photon absorption properties of 1,4-bis{4′-[N,N-bis(6′′-trimethylammoniumhexyl)amino]styryl}benzene tetrabromide (C1) and its inclusion complexes (ICs) with cyclodextrins (CDs) have been studied. Upon complexation with CDs, the absorption spectra of C1 showed a slight red shift, whereas the emission spectra showed a blue shift with concomitant increase in the fluorescence quantum efficiency. A Stern-Volmer study using K3Fe(CN)6 as a quencher revealed significant reduction in the photoinduced charge transfer quenching, in accord with the IC formation. Comparison of the spectroscopic results reveals that C1 forms increasingly more stable ICs in the order C1/β-CD < C1/γ-CD < C1/(3γ:β)-CD (γ-CD/β-CD 3:1, mole ratio). Moreover, the two-photon action cross section of C1 increased from 200 GM for C1 to 400 GM for C1/β-CD, 460 GM for C1/γ-CD, and 650 GM for C1/(3γ:β)-CD, respectively. Furthermore, the two-photon microscopy images of HeLa cells stained with C1 emitted strong two-photon excited fluorescence in the plasma membrane. These results provide a useful guideline for the development of efficient two-photon materials for bioimaging applications. Introduction Recently, the development of efficient two-photon (TP) materials has attracted considerable interest because of their various applications such as frequency upconverted lasing,1 optical power limiting,2 3D optical data storage,3 fabrication of photonic band gap structures,4 photodynamic therapy,5 and particularly biological imaging by using two-photon microscopy (TPM).6 TPM has been used as the best noninvasive bioimaging tool in biological researches and medical diagnosis. It has several advantages including larger penetration depth, reduced photodamage, better spatial resolution, ability to image turbid samples, and negligible background cellular autofluorescence compared with its single-photon counterpart.7 However, the lack of suitable water-soluble TP fluorophores has limited the utility of TPM.8 To develop a useful TP probe, it is crucial to optimize the photoluminescence (PL) quantum efficiency (η) and two-photon absorption (TPA) cross section (δ), in addition to biocompatibility and photostability. It is well known that the quasi linear D-π-A-π-D molecules (where D and A are an electron donor and an acceptor moiety, respectively, and π represents a π-conjugated bridge) with large intramolecular charge transfer (ICT) character show large δ values exceeding 1000 GM (GM ) 1 × 10-50 cm4 · photon-1 · molecule-1).9 Unfortunately, the ICT interaction often induces serious fluorescence quenching * Corresponding authors. (H.Y.W.) Tel: 82-55-350-5300. Fax: 82-55350-5279. E-mail: [email protected]. (B.R.C.) Tel: 82-2-3290-3129. Fax: 82-2-3290-3121. E-mail: [email protected] (for correspondence regarding the two-photon properties). † Department of Nanofusion Technology, Pusan National University. ‡ Department of Cogno-Mechatronics Engineering (WCU), Pusan National University. § Department of Physics Education, Pusan National University. | Department of Chemistry, Korea University.

in polar media such as water to result in a significant decrease in the ηδ values, hampering the imaging performance. Therefore, the development of a proper TP fluorophore in aqueous medium has been a challenge.10 Cyclodextrins (CDs) can provide a nonpolar microenvironment around a chromophore in water by forming an inclusion complex (IC). CDs are cyclic oligomers of dehydroglucopyranose, consisting of six (R-CD), seven (β-CD), and eight (γCD) units of 1,4-linked glucose (Scheme 1a). They have a torusshaped structure with a hydrophobic inner cavity and hydrophilic outer surface. The cavity diameters of R-, β-, and γ-CDs are 5.7, 7.8, and 9.5 Å, respectively, although the depth of the cavities for the three types of CDs is similar (∼5 Å) (Scheme 1b).11 Because of these structural features, CDs can include guest molecule/molecules of similar size and form host-guest ICs via hydrophobic interaction.12 Mechanically interlocked ICs are known as rotaxanes,13 and the structures with quarternary ammonium end groups form pseudorotaxanes, which are nearly stable as rotaxanes.11,14,15 There are several mechanisms for the formation of ICs: clipping, threading, and slippage, as described in detail elsewhere.16 The slippage mechanism, in which the terminal groups (E) of the guest molecule slip into the CD cavity, is favorable when the diameter of the terminal group is smaller or comparable to that of the CD cavity (Scheme 1d).11,17,18 By enclosing the guest molecules in the ICs, PL quantum efficiency, chemical stability, photostability, biocompatibility, and solubility in water can be significantly increased.19 Indeed, the ICs of CDs and their derivatives have been successfully utilized in various biological applications such as sustained release of peptide and protein drugs,20 oral bioavailability of the drugs with prolonged therapeutic effects on the target, and gene delivery.21

10.1021/jp102682m  2010 American Chemical Society Published on Web 07/01/2010

Cationic 1,4-Bis(styryl)benzene Derivative SCHEME 1: (a) Chemical Structures of r-, β-, and γ-Cyclodextrins, (b) Approximate Size of r-, β-, and γ-cyclodextrins, (c) Chemical Structure of C1, (d) Slippage Mechanism for the Formation of Inclusion Complex, and (e) 1:1 and (f) 2:1 Inclusion Complexes

J. Phys. Chem. B, Vol. 114, No. 29, 2010 9685 solutions containing C1 (6.00 µM), and the resulting mixtures were sonicated for 40 min at ∼35 °C. All sample solutions were kept overnight to reach the equilibrium before the measurements. Synthesis of C1. We synthesized C1 by modifying the previous method.22 Neutral precursor of C1, 1,4-bis{4′-[N,Nbis(6′′-bromohexyl)amino]styryl}benzene (N1), was synthesized by the Horner-Emmons-Wittig reaction between 4-N,N-bis(6′bromohexyl)aminobenzaldehyde and 1,4-bis[(diethylphosphoryl)methyl]benzene in the presence of t-BuOK in dry THF under reflux (yield: 54.7%). We obtained C1 by treating N1 with 30% Me3N(aq) in THF/MeOH (yield: 80.3%). All intermediates and the products were characterized by 1H and 13C NMR spectroscopy (Supporting Information). Measurement of Two-Photon Action Cross Section. The TP action spectra of C1 and its ICs were measured by using the two-photon excited fluorescence (TPEF) technique with femtosecond laser pulses.23,24 The TPEF spectra of a standard and the samples ([C1] ) 6.00 µM) in water were recorded at the same excitation wavelength (710-860 nm). Fluorescein (in water, pH ∼11) was used as a reference, whose TP property has been well characterized in previous studies.25 The TPA cross section was calculated using the following equation

δ ) δr(Isηrφrcr)/(Irηsφscs) In this work, we have synthesized 1,4-bis{4′-[N,N-bis(6′′trimethylammoniumhexyl)amino]styryl}benzene tetrabromide (C1, Scheme 1c) and studied its photophysical properties in the absence and presence of R-, β-, and γ-CDs and (3γ:β)-CD (γCD/β-CD 3:1, mole ratio). The formation of the ICs changed the microenvironment around C1 to result in appreciable spectral shifts, three-fold increase in PL quantum efficiency, and threefold increase in the TP action cross section (ηδ). Moreover, the TPM images of HeLa cells labeled with C1 revealed a bright image, indicating the possibility of using C1 as a TPM molecular tag. Experimental Section General. All chemicals were purchased from Aldrich Chemical and used as received unless otherwise mentioned. R- and β-CDs were purchased from Aldrich Chemical, and γ-CD was obtained from Junsei Chemical. The 1H and 13C NMR spectra were recorded on a JEOL (JNM-AL300) FT NMR system. The UV/vis absorption spectra were measured using a Jasco (V630) spectrophotometer. The PL spectra were obtained on a Jasco (FP-6500) spectrofluorometer with a Xenon lamp excitation source, using 90° angle detection for the solution samples. All of the UV/vis and PL spectra were measured at room temperature, and the fluorescence quantum efficiency was measured relative to a freshly prepared aqueous solution of fluorescein at pH 11. The fluorescence decay was measured by a time-correlated single-photon counting (TCSPC) technique using a picosecond (ps) diode pulse laser as a light source with 4 ps resolution. The PL decay was detected without a detection polarizer to increase the signal-to-noise ratio. We also measured the same temporal traces of the normalized fluorescence intensity at various emission polarization angles for C1 and its CD complexes. We confirmed that both horizontal and vertical polarization angles gave the same decay time in the normalized intensity in comparison with the case with the magic angle (Figure S4 of the Supporting Information). For inclusion studies, the desired amount of cyclodextrins ([CD] ) 1.00 × 10-5 to 1.58 × 10-2 M) was added to aqueous

where the subscripts s and r denote the values related to sample and reference measurements, respectively. The intensity of the signal collected by a CCD detector was denoted as I. η is the fluorescence quantum efficiency and φ is the overall fluorescence collection efficiency of the experimental apparatus. The number density of the molecules in solution was denoted as c, and δr is the TPA cross section of the reference molecule. In all cases, the output intensity of TPEF was linearly dependent on the square of the input laser intensity, thereby confirming the occurrence of TPA (Figure S5 of the Supporting Information). Photostability. We determined photostability by measuring the decrease in the TPEF of C1 and C1/(3γ:β)-CD in the phospholipid vesicles composed of DOPC/sphingomyelin/ cholesterol (1:1:1, raft mixture, probe/lipid 1/100) as a function of time. The TPEF of C1 and C1/(3γ:β)-CD remained nearly the same for 1 h (Figure S6 of the Supporting Information). This outcome indicates the high photostabilities of C1 and C1/ (3γ:β)-CD in the phospholipid vesicles. Cell Culture and Imaging. HeLa human cervical carcinoma cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured in DMEM (WelGene, Seoul, Korea) supplemented with heat-inactivated 10% FBS (WelGene), penicillin (100 units/mL), and streptomycin (100 µg/mL). All cell lines were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Two days before imaging, the cells were detached and were replated on glass-bottomed dishes (MatTek). For labeling, the growth medium was removed and replaced with DMEM without FBS. The cells were incubated with C1 or C1/(3γ:β)-CD (6 µM) for 10 min, followed by incubation with Hoechst 33342 (1 µM) for an additional 10 min, washed three times with DMEM without FBS, and imaged. Cell Viability. To confirm that C1 does not affect the viability of HeLa cells under our incubation condition, we used CCK-8 kit (cell counting kit-8, Dojindo, Japan) according to the manufacture’s protocol. C1 showed negligible toxicity up to 6 µM and appreciable toxicity at 9 µM (Figure S7 of the Supporting Information).

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Figure 1. (A) Absorption spectra of C1 in the absence and presence of CDs in water. [C1] ) 6.00 µM and [CD] ) 8.80 × 10-3 M. (B) Spectral shift of C1 in absorption as a function of [CD] (∆λabs ) λabs (C1/CDs) - λabs (C1)). [CD] ) 1.00 × 10-5 M ∼ 1.58 × 10-2 M. Each sigmoid curve shows the guidance line for easy recognition.

Two-Photon Fluorescence Microscopy. TP fluorescence microscopy images of probe-labeled cells were obtained with spectral confocal and multiphoton microscopes (Leica TCS SP2) with a × 100 (NA ) 1.30 oil) and ×20 (NA ) 0.30 dry) objective lens, respectively. The TP fluorescence microscopy images were obtained with a DM IRE2Microscope (Leica) by exciting the probes with a mode-locked titanium-sapphire laser source (Coherent Chameleon, 90 MHz, 200 fs) set at wavelength 730 nm and output power 1230 mW, which corresponded to ∼10 mW average power in the focal plane. Results and Discussion Absorption and Photoluminescence Spectra. It is well known that CDs produce ICs with guest molecules of similar size to their internal cores. Inclusion of C1 inside the cavity would not only change the polarity of the microenvironment around C1 but also influence the electron density and effective conjugation of the π orbitals.26 Figures 1 and 2 show the UV/vis and PL spectra of C1 in the absence and presence of CDs. The spectra were obtained with an aqueous solution of [C1] ) 6.00 µM with increasing [CD] ) 1.00 × 10-5 to 1.58 × 10-2 M. The spectral data reveal that the absorption spectrum of C1 was not altered by the R-CD, although the PL intensity increased slightly with a small blue shift at higher [R-CD] (Figures 1A and 2A and Figures S1A and S2A of the Supporting Information). This indicates that C1 does not form an IC with R-CD, presumably because of the small cavity of R-CD [diameter (r) ) 5.7 Å]. It was reported that the slippage of trimethylammonium groups (r ) ∼5 Å) into the R-CD cavity required a high activation energy because of the steric hindrance.27 Moreover, R,ω-bis(trimethylammonium)hexane did not form an IC with R-CD.28 The small changes

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Figure 2. (A) Normalized PL spectra of C1 in the absence and presence of CDs in water ([CD] ) 8.80 × 10-3 M). (B) Spectral shift of C1 in emission as a function of [CD] (∆λPL ) λPL(C1/CDs) λPL(C1)). [CD] ) 1.00 × 10-5 M ∼ 1.58 × 10-2 M. All spectra were obtained by exciting at 405 nm. Each sigmoid curve shows the guidance line for easy recognition.

in the PL spectra at higher [R-CD] are most likely due to the increased hydrophobicity of the solution (Figure S2A of the Supporting Information). When β-CD was added to C1, the maximum absorption wavelength (λabs) remained nearly the same, and the molar absorptivity increased slightly (Figure 1 and Figure S1B of the Supporting Information). This suggests that the conjugated core of C1 is not enclosed by β-CD, although there may be a slight alteration in the transition moment. The PL intensity increased with a hypsochromic shift (34 nm), indicating an appreciable decrease in the polarity of the microenvironment around C1 (Figure 2 and Figure S2B of the Supporting Information). Because the cavity of β-CD (r ) 7.8 Å) is larger than the diameter of the trimethylammonium group, the terminal alkyl group of C1 can slip into the β-CD (Scheme 1e). Therefore, the observed results can be explained if β-CD produced ICs with C1 by enclosing the trimethylammoniumhexyl groups into its cavity forming a 1:4 complex while leaving the conjugated core exposed to the exterior (Scheme 2). A similar interpretation was put forward to the ICs between an alkyl trimethylammonium bromide (similar to the end groups of C1) surfactant and β-CD.29,30 γ-CD induced substantial changes in both absorption and emission spectra. When γ-CD was added to C1, the absorbance initially decreased with a hypsochromic shift with ∆λabs {∆λabs ) λabs (C1/CD) - λabs (C1)} ) -9 nm at [γ-CD] ) 1.00 × 10-5 M ≈ 4.60 × 10-4 M (Figure 1B and Figure S1C of the Supporting Information). At higher [γ-CD], the absorbance was recovered, accompanied by a bathochromic shift; the ∆λabs began to increase and reached near-zero at [γ-CD] ) 1.96 × 10-3 M, after which a red shift was observed until it reached a saturation point (∆λabs ) ∼14 nm). This indicates that the formation of

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SCHEME 2: Schematic Structures of C1 and the ICs of C1/β-CD, C1/γ-CD, and C1/(3γ:β)-CDa

Figure 3. PL quantum efficiency (η) of C1 with increasing [CD]. Quantum efficiency was measured relative to fluorescein in water at pH ∼11.

a The picture (P) shows the PL emission of C1 and its ICs. From left to right: C1 itself, C1 in the presence of R-, β-, γ-, and (3γ:β)CDs upon UV illumination at 356 nm. [C1] ) 6.00 × 10-6 M and [CD] ) 8.80 × 10-3 M in water.

ICs between C1 and CDs are concentration-dependent dynamic equilibrium processes. A similar result with absorption was observed in the PL spectra. The PL intensity decreased first and then increased with a gradual blue shift in the maximum PL wavelength (λPL), as shown in Figure 2 and Figure S2C of the Supporting Information. Because γ-CD has the cavity (r ) 9.5 Å) almost twice the diameter of the trimethylammonium group, it can enclose two terminal trimethylammonium groups (Schemes 1f and 2).30 This would increase the electrostatic repulsion between the two ammonium ions and the angle strain between the two N-alkyl groups, thereby perturbing the molecular geometry and the effective π-conjugation. It would also decrease the polarity of the microenvironment by partially blocking the solvation by water. A combination of these two effects would induce a blue shift in the absorption and emission maxima (Figures 1B and 2B). At higher [γ-CD], γ-CDs would enclose the conjugated core of C1 (Scheme 2). This would flatten the core, provide a more hydrophobic environment, and result in the recovery of absorbance and emission, accompanied by a red shift in λabs and a further blue shift in λPL (Figures 1 and 2 and Figures S1C and S2C of the Supporting Information). The spectral properties of C1/(3γ:β)-CD were similar to those of C1/γ-CD, except that the change from the hypsochromic to bathochromic shift in absorption occurred at [(3γ:β)-CD] ) 9.12 × 10-4 M, the saturation point was reached at [(3γ:β)-CD] ≈ 8.80 × 10-3 M with ∆λabs ) ∼16 nm, and the molar absorptivity and PL quantum efficiency were higher (Figures 1B, 2, and 3). It is to be noted that the mixture of [γ-CD]/[β-CD] 3:1 showed the optimum CD ratio to achieve the maximum PL spectral shift along with the highest PL intensity (Figure S3 of the Supporting Information). The higher PL quantum efficiency for C1/(3γ: β)-CD can be attributed to the formation of a strain-free pseudorotaxane, where the whole parts (terminal alkyl chains and conjugated core) of C1 are encapsulated by the CDs. When (3γ:β)-CD is added to the aqueous solution, γ-CDs will dissolve faster than β-CDs because of the higher solubility. γ-CDs will enclose the conjugated core first, and then β-CDs will encapsulate the terminal alkyl groups (Scheme 2). Consequently, the

core will be more flattened and the microenvironment will become more hydrophobic than those of C1/γ-CD. This would predict a larger molar absorptivity and a higher PL quantum efficiency, as observed. The normalized PL spectra of C1 showed gradual blue shifts in the maximum emission wavelength (λPL) in the presence of CDs in the order R-CD , β-CD < γ-CD < (3γ:β)-CD (Figure 2A). The spectral shifts, |∆λPL| {∆λPL ) λPL (C1/CDs) - λPL (C1)}, increased with increasing [CD] until it reached at the maximum at [CD] ≈ 8.80 × 10-3 M. The maximum |∆λPL| measured with β-, γ-, and (3γ:β)-CD was 34, 40, and 45 nm, respectively (Figure 2B). In contrast, the λPL remained nearly the same when R-CD was added to [R-CD] ) 8.80 × 10-4 M, after which a slight blue shift (∼6 nm) was observed. Upon addition of increasing amount of CDs, the PL quantum efficiency (η) increased gradually from 0.32 until it reached the maximum values of 0.64, 0.71, and 0.91 in the presence of excess β-CD, γ-CD, and (3γ:β)-CD, respectively (Figure 3). This is a strong evidence of the formation of ICs. By forming the ICs, the microenvironment around C1 would become more hydrophobic, which would destabilize the emitting states and reduce the ICT quenching, thereby causing a blue shift in the emission spectra with enhanced PL efficiency. The combined results reveal that the IC formation is increasingly more favorable with the nature of the CDs in the order R-CD , β-CD < γ-CD < (3γ:β)-CD. Unfortunately, all attempts to determine the exact stoichiometry of C1/β-CD and C1/γ-CD ICs by the continuous variation technique (Job’s method) failed.31 However, one major structure seems to exist as the predominant species (Scheme 2), as demonstrated by the single-exponential PL decay (vide infra, Figure 4). If more than one types of ICs existed, then multiple exponential decays should have been observed. Moreover, the structures in Scheme 2 are in good agreement with the spectroscopic data and the geometrical requirements of C1 and CDs. Time-Resolved Fluorescence Spectroscopy. Time-resolved PL decay was monitored by the TCSPC technique. In all cases, the PL showed a single-exponential decay, as depicted in Figure 4, indicating the existence of a single major emitting species. The PL lifetime increased from 1.3 (C1) to 1.60 ns for C1/βCD, 1.77 ns for C1/γ-CD, and 1.60 ns for C1/(3γ:β)-CD (Table 1). This outcome is consistent with the formation of the ICs, because the inclusion of C1 in the CDs would reduce the PL quenching by blocking the ICT interactions with water and restricting the rotational and torsional relaxation pathways.

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Figure 4. Fluorescence decay curves of C1, C1/β-CD, C1/γ-CD, and C1/(3γ:β)-CD in water. [C1] ) 6.00 × 10-6 M and [CD] ) 8.80 × 10-3 M. Excitation was provided at 405 nm, and decay was monitored at 556 nm for C1, at 520 nm for C1/β-CD, at 513 nm for C1/γ-CD, and at 510 nm for C1/(3γ:β)-CD, respectively.

Figure 5. Stern-Volmer plot of C1 quenched by K3Fe(CN)6 in 1 mM phosphate buffer (pH 8.0) at 25 and 60 °C. Inset shows the linear region of the plot.

TABLE 1: Linear and Nonlinear Spectroscopic Properties in Water a

λabs (nm) λPL (nm)a ηb max. δ (GM)c max. ηδ (GM)d τ (ns)e

C1

C1/β-CD

C1/γ-CD

C1/(3γ:β)-CD

405 556 0.32 630 200 1.30

405 520 0.64 630 400 1.60

420 513 0.71 650 460 1.77

422 510 0.91 710 650 1.60

a

Single-photon absorption and emission maximum wavelengths. Fluorescence quantum efficiency, (10%. c TP cross section (GM ) 1 × 10-50 cm4 · s · photon-1 · molecule-1), (15%. d TP action cross section, (15%. e Fluorescence lifetime, (10%.

b

Figure 6. Stern-Volmer plot for the complexes C1/β-CD, C1/γ-CD, and C1/(3γ:β)-CD quenched by K3Fe(CN)6 in 1 mM phosphate buffer (pH 8.0) at 25 °C. Inset shows the linear region including that of C1 for comparison.

Stern-Volmer Quenching Study with Potassium Ferricyanide. To provide additional evidence of the formation of ICs, we have conducted a Stern-Volmer (SV) quenching experiment using K3Fe(CN)6 as the PL quencher (Q). The SV equation is shown below

PL0 /PL ) 1 + KSV[Q] where PL0 and PL are the fluorescence intensity in the absence and presence of the quencher (Q), respectively, and KSV is the SV quenching constant that can be used to quantify the quenching efficiency.24 At a given [K3Fe(CN)6], the PL quenching decreased at higher temperature. Moreover, the KSV calculated from the linear region of the SV plots decreased from 2.18 × 105 M-1 at 25 °C to 9.38 × 104 M-1 at 60 °C (Figure 5, inset). This suggests that the quenching between C1 and K3Fe(CN)6 is a static process, which occurs via photoinduced charge transfer (PCT) in the electrostatic complex between cationic C1 and anionic Fe(CN)63-. At higher temperature, the static quenching would be reduced because of the dissociation of the electrostatic complex.32,33 Furthermore, the PL quenching measured in the linear regions of the SV plots decreased substantially in the presence of β-, γ-, and (3γ:β)-CD (Figure 6, inset). Because the rate of PCT decreases exponentially with the donor-acceptor distance and the intermolecular separation between C1 and K3Fe(CN)6 would be increased upon complexation with CDs, this result provides additional evidence of the formation of the ICs.24 At higher [K3Fe(CN)6], however, the SV plot for C1/β-CD showed upward curvature, indicating the onset of additional quenching phenomena,32,33 whereas those

Figure 7. Spectral shift in λPL for C1/β-CD, C1/γ-CD, and C1/(3γ: β)-CD complexes upon dilution in water. The solution containing [C1] ) 6.00 × 10-6 M and [CD] ) 8.80 × 10-3 M was diluted by 5, 10, and 15 times.

for C1/γ-CD and C1/(3γ:β)-CD retained the linearity.34 This can be attributed to the smaller pore size of β-CD than others, which may have induced the partial encapsulation of C1. The combined results reveal that C1 does not form ICs with R-CD but forms increasingly more stable ICs with other CDs in the order β-CD , γ-CD < (3γ:β)-CD. Dissociation of Inclusion Complexes upon Dilution. The probe stability upon dilution is one major concern for biological applications. To assess the stability of ICs, we measured the PL spectral shift upon dilution in water (Figure 7). The spectral shifts (in λPL) measured for C1/β-CD was 20 nm upon 15-fold dilution, whereas those for C1/(3γ:β)-CD and C1/γ-CD were only 7 and 8 nm, respectively. This indicates a higher stability

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Figure 8. TPA action spectra of C1 and C1/CD ICs in water. [C1] ) 6.00 × 10-6 M and [CD] ) 8.80 × 10-3 M.

of C1/γ-CD and C1/(3γ:β)-CD than that of C1/β-CD against dissociation; the more stable the IC is, the more resistant it is to the dissociation upon dilution. The inclusion of the conjugated core of C1 by γ-CD would give the better stability against dissociation. Two-Photon Excited Fluorescence Spectroscopy. To assess the utility of C1 and ICs in bioimaging applications, we have determined the TP action cross sections (ηδ; η is the fluorescence quantum yield and δ is the TPA cross section). For the optimum TPM signal-to-noise ratio, the ηδ values should be maximized. A previous study reported that the ηδ values of bis(styryl)benzene derivatives decreased substantially in water compared with those in organic solvents.22 The result has been attributed to the ICT-related PL quenching and solvent interaction via hydrogen bonding. Consistently, the ηδ value of C1 increased from 200 to 400 GM for C1/β-CD, 460 GM for C1/ γ-CD, and 650 GM for C1/(3γ:β)-CD in water, respectively, as the microenvironment became more hydrophobic by forming increasingly more stable ICs in water (Figure 8 and Table 1). Also, a parallel increase in η was observed. The δ value ranged from 630 to 710 GM in all of the ICs (Table 1). The values are very similar to that of closely related 1,4-bis(p-dibutylaminostyryl)benzene in toluene (635 GM),23 indicating that they represent intrinsic TPA cross section for such compounds. Hence, the gradual increase in the ηδ values can be attributed to the larger η in more hydrophobic environment. Moreover, the ηδ value of C1/(3γ:β)-CD (650 GM) is close to the maximum possible ηδ value of 710 GM (η ) 1.0); the maximum ηδ value is fully recovered in water by forming a stable IC. This suggests a useful guideline for the development of efficient water-soluble TP materials. Furthermore, the ηδ values are much larger than those of commonly used fluorescent reporters (ηδ ) ∼1-50 GM).35 Moreover, C1 and C1/(3γ:β)CD showed high photostability in the phospholipid vesicles composed of DOPC/sphingomyelin/cholesterol (1:1:1, raft mixture, probe/lipid 1/100) (Figure S6 of the Supporting Information). These results indicate the potential utility of C1 and ICs in bioimaging applications. We then tested C1 and C1/(3γ:β)-CD as TP fluorescent tags for biological imaging. The TPM image of HeLa (human cervical epithelioid carcinoma) cells costained with C1 and Hoechst 33342 shows bright TPEF emission in the plasma membrane in addition to the one-photon emission in the nucleus (Figure 9a-c). This indicates that C1 is predominantly located in the plasma membrane probably because it cannot internalize into the cytoplasm because of the presence of four polar end groups. Moreover, the emission is strong, presumably because of the hydrophobic environment of the plasma membrane.36 However, when the cells were stained with C1/(3γ:β)-CD, the

Figure 9. (a) TPM images of live HeLa cells incubated with 6 µM of C1 for 10 min at 37 °C. (b) Image of HeLa cells in panel a after incubation with Hoechst 33342 (1 µM) for an additional 10 min at RT. The emission was collected at 500-700 nm (C1) (a) and 400-500 nm (Hoechst 33342) (b), respectively. (c) Co-localized images. (d) Bright field image. The wavelengths for one- and two-photon excitation were 350 and 730 nm, respectively. Scale bar, 20 µm. Cells shown are representative images from replicate experiments (n ) 5).

emission was bright at the beginning, and the intensity decayed rapidly during the imaging (data not shown). This may be due to the presence of uncomplexed or dissociated CDs from the ICs, which destroy the hydrophobic domain (lipid rafts) by forming ICs with cholesterol and washing out cholesterol in the lipid rafts. This would increase the polarity of the membrane, reduce the ηδ of C1, and decrease the TPEF intensity. The bright-field images indicate the cell viability during the imaging (Figure 9d). Furthermore, C1 showed negligible toxicity up to 6 µM, indicating that it is suitable for imaging live cells (Figure S7 of the Supporting Information). The combined results reveal that C1 is a useful TP fluorescent tag to image the cell membrane, whereas C1/(3γ:β)-CD may be useful for applications that require large ηδ in water. Conclusions In summary, we have developed a water-soluble TP fluorophore, 1,4-bis{4′-[N,N-bis(6′′-trimethylammoniumhexyl)amino]styryl}benzene tetrabromide (C1) and studied the linear and nonlinear optical properties of C1 and its CD ICs. The optical properties were substantially influenced by forming ICs with CDs due to the changes in the microenvironments and perturbation of the molecular structures and effective π-conjugation. The absorption showed a slight red shift, and the emission displayed a blue shift with concomitant increase in the PL quantum efficiency in the presence of CDs. The ηδ value increased from 200 GM for C1 to 400 GM for C1/β-CD, 460 GM for C1/γ-CD, and 650 GM for C1/(3γ:β)-CD. Moreover, the ηδ value of C1/(3γ:β)-CD in water is close to the maximum possible ηδ value of 710 GM (η ) 1.0). This suggests a useful guideline for the development of efficient water-soluble TP materials. Moreover, the TPM images of C1-labeled HeLa cells showed a bright image in the plasma membrane that persisted for more than 1 h. When the cells were stained with C1/(3γ:

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β)-CD, however, the TPEF intensity decreased rapidly with time, probably because of the destruction of the lipid rafts by CDs. These results indicate that C1 is a useful TP fluorescent tag for the plasma membrane, whereas C1/CD may be useful for applications that require large TP action cross section in water. Acknowledgment. This research was supported by the World Class University program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education, Science and Technology, Korea (grant no. R31-2008-00020004-0 and 2009-0083078). This work was also supported by Basic Science Research Program through the NRF funded by the Ministry of Education, Science and Technology (20090085182). Supporting Information Available: Synthetic details and additional UV/vis and PL spectra of C1 with changing CD concentration. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) (a) Wang, H. Z.; Lei, H.; Wei, Z. C.; Zhao, F. L.; Zheng, X. G.; Xu, N. S.; Wang, X. M.; Ren, Y.; Tian, Y. P.; Fang, Q.; Jiang, M. H. Chem. Phys. Lett. 2000, 324, 349. (b) Mukherjee, A. Appl. Phys. Lett. 1993, 62, 3423. (2) (a) He, G. S.; Bhawalkar, J. D.; Zhao, C. F.; Prasad, P. N. Appl. Phys. Lett. 1995, 67, 2433. (b) Fleitz, P. A.; Sutherland, R. A.; Strogkendl, F. P.; Larson, F. P.; Dalton, L. R. SPIE Proc. 1998, 3472, 91. (3) Hunter, S.; Kamilev, F.; Esener, S.; Parthenopoulos, D. A.; Rentzepis, P. M. Appl. Opt. 1990, 29, 2058. (4) (a) Parthenopoulos, D. A.; Rentzepis, P. M. Science 1989, 245, 843. (b) Pudarvar, H. E.; Joshi, M. P.; Prasad, P. N.; Reinhardt, B. A. Appl. Phys. Lett. 1999, 74, 1338. (5) (a) Stiel, H.; Teuchner, K.; Paul, A.; Freyer, W.; Leupold, D. J. Photochem. Photobiol., A 1994, 80, 289. (b) Chen, W. R.; Jeong, S. W.; Lucroy, M. D.; Wolf, R. F.; Howard, E. W.; Liu, H.; Nordquist, R. E. Int. J. Cancer 2003, 107, 1053. (c) Chen, W. R.; Adams, R. L.; Carubelli, R.; Nordquist, R. E. Cancer Lett. 1997, 115, 25. (6) (a) Xu, C.; Zipfel, W.; Shear, J. B.; Williams, R. M.; Webb, W. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10763. (b) Kim, H. M.; Yang, P. R.; Seo, M. S.; Yi, J. S.; Hong, J. H.; Jeon, S. J.; Ko, Y. G.; Lee, K. J.; Cho, B. R. J. Org. Chem. 2007, 72, 2088. (c) Kim, H. M.; Jung, C.; Kim, B. R.; Jung, S. Y.; Hong, J. H.; Ko, Y. G.; Lee, K. J.; Cho, B. R. Angew. Chem., Int. Ed. 2007, 46, 3460. (d) Kim, H. M.; Kim, B. R.; Hong, J. H.; Park, J. S.; Lee, K. J.; Cho, B. R. Angew. Chem., Int. Ed. 2007, 46, 7445. (e) Kim, H. M.; Kim, B. R.; An, M. J.; Hong, J. H.; Lee, K. J.; Cho, B. R. Chem.sEur. J. 2008, 14, 2075. (f) Kim, H. M.; Cho, B. R. Acc. Chem. Res. 2009, 42, 863. (7) (a) Pawley, J. B. Handbook of Biological Confocal Microscopy; Plenum: New York, 1995. (b) Prasad, P. N. Introduction to Biophotonics; Wiley: Hoboken, NJ, 2003. (c) Denk, W.; Strickler, J. H.; Webb, W. W. Science 1990, 248, 73. (d) Helmchen, F.; Denk, W. Nat. Methods 2005, 2, 932. (8) (a) Chung, S.-J.; Lin, T.-C.; Kim, K.-S.; He, G. S.; Swiatkiewicz, J.; Prasad, P. N.; Baker, G. A.; Bright, F. V. Chem. Mater. 2001, 13, 4071. (b) Reinhardt, B. A.; Brott, L. L.; Clarson, S. J.; Dillard, A. G.; Bhatt, J. C.; Kannan, R.; Yuan, L.; He, G. S.; Prasad, P. N. Chem. Mater. 1998, 10, 1863. (9) (a) Zojer, E.; Beljonne, D.; Kogej, T.; Vogel, H.; Marder, S. R.; Perry, J. W.; Bre´das, J.-L. J. Chem. Phys. 2002, 116, 3646. (b) Albota, M.; Beljonne, D.; Bre´das, J.-L.; Ehrlich, J. E.; Fu, J.-Y.; Heikal, A. A.; Hess, S. E.; Kogej, T.; Levin, M. D.; Marder, S. R.; McCord-Maughon, D.; Perry, J. W.; Ro¨ckel, H.; Rumi, M.; Subramaniam, G.; Webb, W. W.; Wu, X.-L.; Xu, C. Science 1998, 281, 1653. (10) (a) Woo, H. Y.; Korystov, D.; Mikhailovsky, A.; Nguyen, T.; Bazan, G. C. J. Am. Chem. Soc. 2005, 127, 13794. (b) Kim, H. M.; Cho, B. R. Chem. Commun. 2009, 153. (c) Kim, S.; Pudavar, H. E.; Bonoiu, A.; Prasad, P. N. AdV. Mater. 2007, 19, 3791. (11) Nepogodiev, S. A.; Stoddart, J. F. Chem. ReV. 1998, 98, 1959. (12) (a) Schneider, H. J.; Sangwan, N. K. Angew. Chem., Int. Ed. Engl. 1987, 26, 896. (b) Purkayastha, P.; Chattopadhyay, N. J. Mol. Struct. 2001, 570, 145. (c) Uyar, T.; El-Shafei, A; Hacaloglu, J.; Tonelli, A. E. J. Inclusion Phenom. Macrocyclic Chem. 2006, 55, 109. (d) Uyar, T.; Rusa, C. C.; Hunt,

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