Photoluminescent Eu-containing polyoxometalate/gemini surfactant hybrid nanoparticles for biological applications

Abstract

In water, Eu-containing polyoxometalate/gemini surfactant hybrid spheres with long emission timescale (3.758 ms) and high fluorescence quantum yield (25.17%) behaviors were synthesized. The hybrid spheres can be used as a bioprobe to image living cells.

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Biological fluorescent probes are emerging as a powerful, bottom-up approach tool to study cellular behaviors and various biological processes.^1,2 Typical fluorescent probes usually used in cell imaging include natural fluorescent proteins, organic fluorescent dyes and quantum dots.^3–6 However, the fluorescence signal of fluorescent proteins and organic fluorescent dyes are easily overwhelmed by background noise from the cell matrix and nonspecific scattering of light. Although quantum dots show high quantum yield and photo-stability, their synthetic methods are complicated.⁷ In this regard, lanthanide-containing polyoxometalates (POMs) have attracted much attention owing to their photoluminescent properties with narrow emission bands, large Stokes shift, high fluorescence quantum yield and long emission timescale (micro- to milliseconds).^8–11 Thus the fluorescence measurements related to lanthanide-containing POMs, utilized as a highly sensitive biochemical assay technique, have significant prospect in labeling and imaging of biological molecules. However, practical application of lanthanide-containing POMs materials in bioimaging is quite scarce because of their inherent limitations, e.g. poor processability in the solid state and weakened functionality in aqueous solution. The Eu-containing POMs Na₉EuW₁₀O₃₆·32H₂O (Eu-POM) displays only weak photoluminescence in water in comparison to the solid state due to fluorescence quenching by water molecules. Therefore, it is very important for the application of Eu-POM to protect and utilize their photochemical characteristics in aqueous solution by supramolecular methodologies. Herein, we employed one-step synthetic method, namely ionic self-assembly (ISA) strategy,¹² to fabricate nanostructure materials consisted of Eu-POM and an oppositely charged ionic liquid-type gemini surfactant, 1,2-bis(3-hexadecylimidazolium-1-yl) butane bromide ([C₁₆-2-C₁₆im]Br₂) (Fig. S1a†). The self-assembled [C₁₆-2-C₁₆im]Br₂/Eu-POM hybrid nanoscale materials possess excellent fluorescence properties: long emission timescale and high fluorescence quantum yield. As far as we know, there are still no reports on the emission and lifetime enhancement of Eu-POM in aqueous solution through ISA method in the presence of ionic liquid-type gemini surfactant. Given the appropriate hydrophilicity, long emission timescale, high fluorescence quantum yield and avoidance of fluorescence quenching by water molecules, self-assembled nanospheres were applied as promising agents for cell imaging by presenting high contrast.

A single phase approach was adopted to prepare [C₁₆-2-C₁₆im]Br₂-encapsulated hybrid materials using Eu-POM and gemini imidazolium surfactant [C₁₆-2-C₁₆im]Br₂ in water. Fig. 1 shows the forming process of the as-prepared hybrid materials. First, Eu-POM and [C₁₆-2-C₁₆im]Br₂ form supramolecular complexes through electrostatic interactions. Subsequently, the supramolecular complexes can form larger supramolecular aggregates due to hydrophobic interactions. Eventually, the larger supramolecular aggregates assemble into hybrid spheres. As shown in Fig. 2a, hybrid spheres with the diameter of 50–100 nm can be obtained after 1 h under ultrasound condition.

Fig. 1 Schematic illustration of formation mechanism for [C₁₆-2-C₁₆im]Br₂/Eu-POM supramolecular materials.

Fig. 2 (a) TEM image of the [C₁₆-2-C₁₆im]Br₂/Eu-POM hybrid spheres self-assembled at room temperature in water. (b) FTIR spectra of Eu-POM (top) and [C₁₆-2-C₁₆im]Br₂/Eu-POM hybrids (bottom), respectively.

Composition of the hybrid spheres was investigated to understand how they formed in water. FTIR bands at 925, 856, 830 and 771 cm⁻¹ occur for structures of Eu-POM (Fig. 2b top). However, for the [C₁₆-2-C₁₆im]Br₂/Eu-POM hybrid spheres, the characteristic peaks show red shift (Fig. 2b bottom) compared to Eu-POM. This can be ascribed to electrostatic interaction between [C₁₆-2-C₁₆im]Br₂ and Eu-POM.¹³ To further explore the self-assembling behavior of the hybrid materials, hydrophobic interaction was analyzed. When the alkyl chains are highly ordered (all-trans conformation), the bands appear at 2915–2920 cm⁻¹ and 2846–2850 cm⁻¹, respectively. However, when the frequencies shift upward to 2924–2929 cm⁻¹ and 2854–2856 cm⁻¹, the alkyl chains are relatively disordered (gauche conformer). In the FTIR spectra of the complex (Fig. S2†), the ν_as (CH₂) and ν_s (CH₂) bands appear at 2920 cm⁻¹ and 2850 cm⁻¹, respectively. This indicates that all-trans conformation exists in hybrid materials and alkyl chains are highly ordered.¹⁴

The emission of Eu³⁺ is dependent on its coordinated water molecules, because radiationless deactivation of the ⁵D₀ excited state through weak coupling with the vibrational states of high-frequency OH oscillators of water ligands. So the fluorescence can be quenched.¹⁵ In the present work, the remarkably enhanced emission of Eu³⁺ illustrates that Eu-POM is protected in a relatively hydrophobic environment where the cationic [C₁₆-2-C₁₆im]Br₂ has a strong enough affinity to the anionic Eu-POM to replace water molecules through strong electrostatic interaction. The fluorescence quantum yield of [C₁₆-2-C₁₆im]Br₂/Eu-POM reaches 25.15% and fluorescence lifetime becomes τ₁ = 1.039 ms and τ₂ = 3.758 ms (Table 1). To confirm effect of [C₁₆-2-C₁₆im]Br₂, several contrast experiments were carried out. Firstly, we chose sing-chained ionic liquid-type surfactant, 1-hexadecyl-3-methylimidazolium bromide ([C₁₆im]Br) (Fig. S1b†) and traditional surfactant, hexadecyl trimethyl ammonium bromide (CTAB) (Fig. S1d†) to combine with Eu-POM, respectively. Obviously, compared to [C₁₆-2-C₁₆im]Br₂/Eu-POM hybrids, both fluorescence quantum yield and fluorescence lifetime (Fig. 3a and b and Table 1) of the newly prepared luminescent materials decrease dramatically, which suggests that gemini surfactant can distinctly improve the fluorescence properties of materials. This may be attributed to the dense arrangement and identical orientation of Eu-POM. As is well known, gemini surfactant connects two positively charged amphipathic groups at or near the terminals of a spacer group. This can lead to the more compact arrangement of Eu-POM molecules, and then cause the big π-conjugated bonds in supramolecules. Subsequently, the ISA complexes incorporating of gemini surfactant [C₁₂-2-C₁₂im]Br₂ (Fig. S1c†) and Eu-POM were also fabricated. As is shown in the Fig. 3a and b, the fluorescence quantum yield and fluorescence lifetime of [C₁₂-2-C₁₂im]Br₂/Eu-POM hybrids decrease remarkably, compared to [C₁₆-2-C₁₆im]Br₂/Eu-POM system. Obviously, the hydrophobic effect can improve the photoluminescent efficiency of the hybrid materials. It can further prevent the fluorescence of Eu-POMs from quenching in water environment. To further explain the hydrophobic effect for improvement of fluorescence, the small angle X-ray scattering (SAXS) experiment was investigated (Fig. 4). The SAXS diffractogram of [C₁₂-2-C₁₂im]Br₂/Eu-POM and [C₁₆-2-C₁₆im]Br₂/Eu-POM shows two scattering peaks, indicative of the highly ordered supramolecular structures. The ratio of the two peaks is 1 : 2, suggesting a typical lamellar structure of nanoparticles. The calculated interplanar distance (d) of [C₁₂-2-C₁₂im]Br₂/Eu-POM and [C₁₆-2-C₁₆im]Br₂/Eu-POM is 3.08 and 3.65 nm, respectively. The increasing of interplanar distance can further prevent the fluorescence of Eu-POM from quenching in water environment. In addition, the photographs also indicate the change of photoluminescence in the different supramolecules.

Table 1 Summary of I_{(⁵D0–⁷F2)}/I_{(⁵D0–⁷F1)}, lifetimes τ₁ and τ₂, and quantum yield η

Sample	I^5D0–^7F2/I^5D0–^7F1	τ1 [ms]	τ2 [ms]	η
Eu-POM	2.32	0.24	2.201	0.91%
[C12-2-C12im]Br2/Eu-POM	1.72	—	3.040	19.73%
[C16im]Br/Eu-POM	1.29	0.855	3.399	12.94%
[C16-2-C16im]Br2/Eu-POM	1.77	1.039	3.758	25.17%
CTAB/Eu-POM	1.35	0.607	1.391	23.37%

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Fig. 3 Fluorescent emission spectra (a), lifetimes (b) and photos taken under illumination with 254 nm UV light (c) of Eu-POM (1), [C₁₆im]Br/Eu-POM (2), [C₁₂-2-C₁₂im]Br₂/Eu-POM (3), [C₁₆-2-C₁₆im]Br₂/Eu-POM (4) and CTAB/Eu-POM (5), respectively.

Fig. 4 SAXS of [C₁₂-2-C₁₂im]Br₂/Eu-POM and [C₁₆-2-C₁₆im]Br₂/Eu-POM, respectively.

The integral intensity ratio of ⁵D₀–⁷F₂ to ⁵D₀–⁷F₁ (I_{(⁵D0–⁷F2)}/I_{(⁵D0–⁷F1)}) is often used to evaluate the degree of variation of Eu³⁺ symmetry in different environment. As shown in Table 1, the I_{(⁵D0–⁷F2)}/I_{(⁵D0–⁷F1)} value of hybrid materials is lower than that of Eu-POM system. As is well known, the Eu-POM molecules in aqueous solution coordinate with four water molecules and have C₄ symmetry. Compared with Eu-POM, the lower I_{(⁵D0–⁷F2)}/I_{(⁵D0–⁷F1)} value for the as-prepared hybrid materials reflects the increasing symmetry of the microenvironment around Eu³⁺, which can be attributed to the facts that surfactant segments have completely replaced water molecules and Eu-POM molecules are located in the relatively homogenously distributed surfactant molecules in the core.

As is shown in Table 1, compared to Eu-POM, the hybrid materials have increasing fluorescence quantum yield and luminescent lifetime, which exhibits their excellent luminescent efficiency. To the best of our knowledge, this is the first work to fabricate hybrid materials to enhance luminescent efficiency through ISA of gemini surfactants and Eu-POM with a time-gated luminescence function, which enables the hybrid materials to be a promising bioprobe for time-gated luminescence bioassays. To show the feasibility of hybrid materials as a bioprobe in cell imaging, Hela cells were coincubated with the [C₁₆-2-C₁₆im]Br₂/Eu-POM hybrid materials, and then subjected to luminescence imaging detection. Nanoparticles with diameter of 50–100 nm can easily enter cells, and the hybrid materials with strong luminescent property prepared here just have such suitable size.¹⁶ The images were acquired at the excitation wavelength of 405 nm. After being incubated under experimental conditions for 12 h, the Hela cells display intense red emission (Fig. 5a and b and inset in Fig. 5a), implying that the hybrid materials have already entered into the interior of the cells. After 36 h, the Hela cells still manifest intense red emission (Fig. 5c and d). Compared to QDs and organic dyes,^5,17 hybrids have a stable fluorescence property in bioimaging. The images of Fig. 5 show the high biocompatibility of the [C₁₆-2-C₁₆im]Br₂/Eu-POM hybrid materials. In addition, cytotoxicity of the supramolecular materials was evaluated by standard 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It is found that the hybrids exhibit low cytotoxicity and excellent biocompatibility (Fig. 6).

Fig. 5 Cell viability of [C₁₆-2-C₁₆im]Br₂/Eu-POM at various concentrations as determined with the MTT assay.

Fig. 6 Bright-field (a and c) and luminescence (b and d) images of Hela cells stained by the [C₁₆-2-C₁₆im]Br₂/Eu-POMs hybrid materials for 12 h (a and b) and 36 h (c and d) in the concentrated water environment. Scale bar: 20 μm.

In summary, the [C₁₆-2-C₁₆im]Br₂/Eu-POM hybrid materials were synthesized which present intense luminescent emission and their biological application. Electrostatic interactions and hydrophobic interactions are the main driving forces for the formation of the hybrid spheres. Of particular interest is that gemini ionic liquid-type surfactants can improve the fluorescence lifetimes and fluorescence quantum yield compared with single-chained ionic liquid-type and conventional surfactants. In addition, the hybrid materials can exhibit strong luminescence in Hela cell. This study reveals the potential of [C₁₆-2-C₁₆im]Br₂/Eu-POM hybrid materials in the biological applications.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 21373128), the Scientific and Technological Projects of Shandong Province of China (No. 2014GSF117001) and the Natural Science Foundation of Shandong Province of China (No. ZR2011BM017).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra21544g

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