HSA-based phosphorescent probe for two-photon in vitro visualization

Journal of Inorganic Biochemistry 149 (2015) 108–111

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Short communication

HSA-based phosphorescent probe for two-photon in vitro visualization Pavel S. Chelushkin a,b,⁎, Natalia V. Nukolova c,d, Alexei S. Melnikov e,f, Pavel Yu. Serdobintsev e,f, Pavel A. Melnikov d, Dmitry V. Krupenya a, Igor O. Koshevoy g, Sergey V. Burov a,b, Sergey P. Tunik a,⁎⁎ a

Institute of Chemistry, Saint Petersburg State University, Universitetskii Pr. 26, Saint Petersburg 198504, Russia Institute of Macromolecular Compounds, Russian Academy of Sciences, Bolshoi Pr. V.O., 31, Saint Petersburg 199004, Russia Serbsky State Scientific Center for Social and Forensic Psychiatry, Kropotkinsky Per, 25, Moscow 119991, Russia d Pirogov Russian National Research Medical University, Ostrovitianov St. 1, Moscow 117997, Russia e Department of Physics, Saint Petersburg State University, Ulianovskaya St. 3, Saint Petersburg 198504, Russia f Research Institute of Nanobiotechnologies, Saint Petersburg State Polytechnical University, 29, Polytechnicheskaya St., Saint Petersburg 195251, Russia g Department of Chemistry, University of Eastern Finland, Joensuu 80101, Finland b c

a r t i c l e

i n f o

Article history: Received 31 October 2014 Received in revised form 25 March 2015 Accepted 26 March 2015 Available online 1 April 2015 Keywords: Imaging agents Luminescence Non-linear optics Non-covalent interactions Human serum albumin

a b s t r a c t Two-photon microscopy reveals several advantages over conventional one since it provides higher spatial resolution as well as deeper penetration into the sample under study. The development of suitable two-photon probes is one of the most challenging tasks in this area. Here we present phosphorescent non-covalent adduct of human serum albumin and Au-Ag alkynyl-diphosphine complex, [Au14Ag4(C2Ph)12(PPh2C6H4PPh2)6][PF6]4, which exhibits high cross section of two-photon-induced luminescence (δTPE) within large near-infrared excitation wavelength region (700–800 nm) with maximum δTPE about 38 GM at 740 nm. This feature makes it a promising probe for multiphoton bioimaging as demonstrated by successful visualization of glioma C6 cells and various tissues by two-photon confocal microscopy both in planar and z-stacking modes. Additionally, the broad excitation region enables optimization of the signal-to-background auto-fluorescence ratio via variation of excitation wavelength. © 2015 Elsevier Inc. All rights reserved.

Implementation of two-photon excitation into the practice of confocal microscopy is one of the most important innovations in the field of optical bioimaging over recent decades [1]. The main features of a two-photon confocal microscopy are as follows: (a) fluorescence intensity depends on the square of the incident light intensity thus providing localization of the excitation in a limited space area [2], and (b) excitation wavelengths are almost twice longer than those in one-photon imaging offering the use of near-infrared (NIR) window of tissue transparency for excitation [3]. The former feature provides spatial resolution of about 250 nm [2], while the latter enables to visualize tissues at depths of up to a millimeter with excellent resolution along the z-axis [4]. The development of suitable two-photon probes is one of the most challenging tasks in this area [3]. Many commercially available fluorescent probes exhibit relatively low two-photon absorption cross-sectional (δTPA) values scarcely reaching 102 GM [5–7] (although some tailor-made organic fluorophores possess δTPA up to 103 GM [8,9])

⁎ Correspondence to: P.S. Chelushkin, Institute of Chemistry, Saint Petersburg State University, Universitetskii Pr. 26, Saint Petersburg 198504, Russia. Tel./fax: + 7 812 3241258. ⁎⁎ Corresponding author. Tel./fax: +7 812 3241258. E-mail addresses: [email protected] (P.S. Chelushkin), [email protected] (S.P. Tunik).

http://dx.doi.org/10.1016/j.jinorgbio.2015.03.014 0162-0134/© 2015 Elsevier Inc. All rights reserved.

and do not emit in the NIR region. Alternatively, various inorganic nanomaterials (e.g., quantum dots [10], gold [11] and carbon [12] nanoparticles, to list just a few) overcome the above limitations of organic probes revealing δTPA values up to 105 GM [10] but show lack in water solubility and biocompatibility or display high toxicity [13–15]. One of the promising classes of probes combining water solubility, biocompatibility and remarkable luminescent properties (such as high δTPA accompanied with excitation and/or emission in the NIR region) is covalent or non-covalent conjugates of phosphorescent organometallic complexes and endogenous proteins, e. g., human serum albumin (HSA) [16,17]. Recently, we have obtained the heterometallic Au-Ag alkynyldiphosphine complex, [Au14Ag4(C2Ph)12(PPh2C6H4PPh2)6][PF6]4, 1 (See Fig. S4 or Ref. [18] for schematic structure of 1), which demonstrates high two-photon absorption in the NIR region and wide emission band with the maximum at 650 and lasting up to 800 nm [18]. Although water insoluble, it forms water-soluble adduct with HSA (here and below designated as 1-HSA), thus acquiring both water solubility and biocompatibility [16]. The 1-HSA adduct reveals remarkable photophysical properties under one-photon excitation [16]: (a) an intense oxygen-insensitive phosphorescence with large Stokes shift with almost the same characteristics of emission band compared to that of 1, and (b) lifetimes ca. 0.14 and 1.35 μs that enable use of this adduct as phosphorescent probe for time-resolved one-photon optical

P.S. Chelushkin et al. / Journal of Inorganic Biochemistry 149 (2015) 108–111

imaging. Since the complex 1 does not change significantly its onephoton phosphorescent properties while forming the adducts, we suggested that pronounced non-linear optical properties of the parent compound are retained in the adduct, too. Here we present investigation of non-linear optical properties of the 1-HSA adduct together with illustration of its utility as two-photon probe in confocal studies. Table 1 and Fig. 1 summarize photophysical properties of 1 and 1HSA under one- and two-photon excitation. The emission spectra of 1 and 1-HSA are very similar to each other and do not depend on the excitation mode (that is typical case for both fluorophores [5,6] and phosphors [19,20]; Fig. 1A), suggesting that the same excited states are reached regardless of the excitation mechanism. Indirectly, this also indicates that there is no degradation of the luminophor under intense laser irradiation. δTPA value of 1 reaches 500 GM at 740 nm (Fig. 1B). This value is relatively large compared to those of commercially available organic luminophors (i.e. ~ 250 GM for Rhodamine B [21]). It is in reasonable agreement with the previously reported δTPA = 206 ± 10 GM for 1 in CH2Cl2 at 800 nm using Z-scan technique [18] since the difference between δTPA values obtained by Z-scan and two-photon-induced fluorescence methods may typically differ by a factor of several times [21]. Surprisingly, the 1-HSA adduct revealed significantly larger δTPA values compared to 1 (Fig. 1B, Table 1). Such an increase in δTPA cannot be ascribed to HSA luminescence because its δTPA does not exceed 0.2 GM in a wide region of excitation wavelengths. Hence, there is an effective interaction between electronic shells of 1 and some amino acid residue(s) in HSA leading to excitation transfer from the HSA matrix onto chromophoric center of 1 in the adduct. This assumption is also supported by a strong decrease of quantum yield of 1-HSA compared with 1 (Table 1), implying the appearance of an effective channel for energy transfer in both directions upon adduct formation. Direct resonance energy transfer is believed to be one of the most probable mechanisms of δTPA increase; this mechanism is very likely to be operative because of substantial overlap between the fluorescence emission spectrum of HSA and the absorption spectrum of 1 [22]. Deeper insight into this subject can be achieved using information on the binding site of 1. Although any structural evidence is not available, we speculate that the most probable binding site is hydrophobic cavity in the domain IIA, which is either probable or proven binding site for many different organometallic complexes [22–24]. The maximum cross section of two-photon-induced luminescence (δTPE = δTPA · QY where QY is quantum yield) for 1-HSA is 38 GM at 740 nm. It exceeds typical δTPE values of commercially available organic luminophors but is lower compared to those of tailor-made two-photon probes [8–12]. Note that the 1-HSA adduct reveals lower δTPE values compared to the parent compound 1. Nevertheless, 1-HSA retains pronounced non-linear optical properties, thus implying its high potential in bioimaging applications. This is demonstrated by glioma C6 cells staining with 1-HSA and visualization using confocal microscopy (Fig. 2). These experiments showed that untreated reference cells and cells treated with HSA reveal background auto-fluorescence of nuclei in blue (425 nm b λEm b 475 nm) and green (500 nm b λEm b 550 nm) channels, presumably generated by DNA [3] (Fig. 2A and B; splitting of channels is presented in Fig. S5). In the cells treated with 1-HSA (Fig. 2C), additional signal appears in red (570 nm b λEm b 620 nm) channel that can be only ascribed to the

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Fig. 1. (A) Luminescence spectra of 1 (curve 1) and 1-HSA adduct (curve 2) under twophoton excitation at 760 nm. (B) Dependencies of δTPA on excitation wavelength for 1 (curve 1) and 1-HSA adduct (curve 2). Relative uncertainties of the measurements are shown in the graph.

emission of 1-HSA. Furthermore, the latter signal does not co-localize with the auto-fluorescence signals from nuclei. These observations clearly indicate that the red emission originates from the adduct resided within cytoplasm. A wide wavelength range suitable for two-photon excitation enables optimization of the signal-to-background (auto-fluorescence) ratio through the variation of the excitation wavelength. Indeed, background auto-fluorescence of nuclei slightly falls down with the increase of λex from 700 to 800 nm while the adduct signal intensity goes through the maximum at 760 nm (Fig. 2C–F; splitting of channels is presented in Fig. S5), which seems to be the optimal excitation wavelength.

Table 1 Photophysical data for 1 and 1-HSA, λEx = 800 nm, 296 K. Compound

Solvent

λEm, nm

QY

Lifetimea, µs

δTPE, GM

δTPA, GM

1 1-HSA

acetone water

640 647

0.69 ± 0.07 0.029 ± 0.005

4.9 ± 0.5 0.14 ± 0.02 (94%) 1.35 ± 0.20 (6%)

92 ± 28 15 ± 4

130 ± 40 500 ± 150

a

Lifetime values were published in our previous work [16]. Figures in parentheses show contribution of the corresponding exponent to emission decay.

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P.S. Chelushkin et al. / Journal of Inorganic Biochemistry 149 (2015) 108–111

Fig. 2. Combined confocal images of glioma C6 cells: nontreated (A), treated with HSA (B) and treated with 1-HSA (C–F). Two-photon excitation at 700 (D), 730 (E), 760 nm (A–C) and 800 nm (F). Scale bar 50 μm.

The most important advantage of the two-photon microscopy is a combination of its ability to provide a very good spatial resolution [2] and to perform deep scanning [4], that is extremely useful for tissue visualization. We carried out 3D reconstructions of heart tissue obtained in one-photon (Fig. 3A) and two-photon (Fig. 3B) modes. Comparison of the pictures reveals that the two-photon mode provides much better resolution for myocardial muscle (right and lower parts of pictures); moreover, full reconstruction of a coronary vessel (upper left part of pictures) was possible in the two-photon mode only. Detailed z-stack investigation reveals that heart tissue can be visualized with high resolution up to the depth of 40–50 μm in the two-photon mode while one-photon mode provided penetration only up to 20 μm. Higher penetration depths (up to 140 μm) were achieved for brain tissue (Fig. S6), but in this case, the penetration depths were comparable for both modes. Most probably this is a result of lower light scattering and consequently deeper penetration because of less structured brain tissue compared to that of heart [3]. Nevertheless, only the two-photon mode allowed obtaining of the details and high contrast of the image.

Altogether, the results presented, as well as high photostability (Fig. S1), oxygen insensitivity and low cytotoxicity of 1-HSA adduct (Fig. S7) clearly demonstrate its high potential in two-photon bioimaging. Abbreviations GM HSA NIR QY δTPA δTPE

Goeppert–Mayer units Human serum albumin Near infrared Quantum yield Two-photon absorption cross section Cross section of two-photon-induced luminescence

Acknowledgements This research has been supported by the St. Petersburg State University research grant (nos. 1.37.153.2014, 0.37.169.2014) and the

Fig. 3. Three-dimensional reconstructions of heart tissue obtained in (A) one-photon (λEx 405 nm) and (B) two-photon (λEx760 nm) modes. Picture dimensions: 290 × 290 × 74 μm.

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