Silica-supported dual-dye nanoprobes for ratiometric hypoxia sensing

Abstract

Oxygen (O₂) levels provide an important parameter in the early-stage diagnosis of cancers and other diseases. Ratiometric optical O₂ sensing has attracted much interest due to its better calibration of oxygen levels at two wavelengths. Oxygen sensing nanoprobes are generally designed by the incorporation of a reference dye and phosphorescent indicator into various matrixes. Silica nanomaterials with intriguing characteristic properties are highly preferable in hypoxia sensing. However, physical entrapment of dyes in silica suffers from leaching and moderate sensitivity. Reported herein is a covalently encapsulated dual-dye silica nanoprobe for ratiometric hypoxia detection and imaging. The nanoprobe prepared in a one-pot approach has a core–shell nanostructure where silanized indicators and reference dyes are covalently grafted within the silica core, while poly(ethylene glycol) chains form an outer shell. To our delight, through the covelent bonding of the dyes for efficient energy transfer between the reference dye and the indicator, high phosphorescence brightness, long lifetime and excellent stability of the nanoprobe were achieved. More importantly, the designed nanoprobe can respond to different oxygen levels in both solution and living cells with impressive sensitivity and stability.

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Introduction

Molecular oxygen (O₂), one of the essential elements of all known higher-organisms, plays a vital role in living systems. Detecting and imaging O₂ levels in living cells represent an important and challenging issue in the biomedical field.^1–6 The lack of O₂, named as hypoxia, often provides an important parameter in the early-stage diagnosis of cancers and other diseases. It has been well established that quenching phosphorescence by O₂ is a simple, noninvasive method for imaging in vivo O₂ levels.^7–12 Transition-metal complexes such as Pt/Pd porphyrins have been widely employed as oxygen probes. Long lifetime and high quantum yield of the phosphorescence ensure the detection of oxygen at low levels. However, the utility of small molecules for this application is hampered by their poor water solubility, nonspecific binding, self-aggregation, and unfavorable photophysical properties. To address these issues, luminescent porphyrins were incorporated into various scaffolds for the design of oxygen nanoprobes. In particular, the co-encapsulations of the reference dye and phosphorescent indicator into the matrixes enable ratiometric optical O₂ sensing with better calibration of the oxygen levels at two wavelengths.^9–13

Silica nanomaterials are characteristic of optical transparency, photophysical inertness, chemical durability and biocompatibility, and are highly preferable for hypoxia sensing.^14–16 Immobilization of detection components in the supportive matrix is a critical step in the fabrication of silica-based optical sensors.¹⁷ Many efforts have been devoted to physical entrapment owing to its simple preparation. However, sensors designed in these ways suffer from leaching of the dyes out of the matrix and moderate sensitivity.

Motivated by our long-term interests in hypoxia sensing,^12a–c we attempted to design a ratiometric dual-dye covalently-bonded hypoxia silica nanoprobe. This probe has a core–shell nanostructure, in which a silanized oxygen sensitive indicator and oxygen-insensitive reference dye are covalently grafted within the silica core, while poly(ethylene glycol) (PEG) chains from Pluronic F-127 form an outer shell. The nanoprobe possesses preferable small particle size (10 ± 2 nm), excellent water-dispersibility, low biotoxicity and membrane-penetration ability. The chemical connection of the chromphores in silica matrixes effectively prevented their leakage from silica and facilitated their energy transfer, thus endowing the core–shell nanoprobe with excellent stability and brightness. The nanoprobe showed a lifetime up to 0.52 ms and a phosphorescence quantum yield up to 1% in aqueous solution, which is 10 times over that in organic solvent. Together with the excellent oxygen permeability of the mesoporous silica structure, sensitive ratiometric hypoxia detection both in solution and in living cells was achieved with the K_SV value of 243.1 and I₀/I₁₀₀ ratio of 153.

Results and discussion

Design, synthesis, characterization and photophysical properties of the silica nanoprobe

Schemes 1 and 2 show the one-pot preparation of the ratiometric hypoxia nanoprobe and synthetic routes to the silanized coumarin derivative (CM)^15d and silanized Pd(II) porphyrin derivative (TPP). CM and TPP were selected as the fluorescence reference and phosphorescence indicator precursors, respectively, owing to their easy synthesis, the distinct spectral separation between the fluorescence from CM and phosphorescence from TPP, and the substantial overlap between the emission spectrum of CM and the absorption spectrum of TPP. The efficient energy transfer from the fluorescent reference to the phosphorescent indicator led to the design of a ratiometric hypoxia sensor with enhanced phosphorescence intensity.¹⁸ After hydrolysis/condensation of tetraethoxysilane (TEOS) with CM and TPP in the presence of a micellar dispersion of Pluronic F-127 as a template, the nanoprobe was obtained with a silica core surrounded by a PEG shell (see details in the ESI†). Transmission electron microscope (TEM) images indicated that the nanoprobe is monodispersed with an average diameter of 10 ± 2 nm. The hydrodynamic diameter of the nanoprobe, shown by dynamic light scattering measurement (DLS), is 18 ± 2 nm (Fig. 1a and Fig. S1, ESI†). No aggregation was observed by DLS or UV-vis spectral changes even after several months’ storage, indicating outstanding stability of the nanoprobe.

Scheme 1 Schematic illustration of the one-pot preparation of ratiometric hypoxia nanoprobes based on covalent-bonded silica nanoparticles (NPs); the chemical structures of the oxygen-sensitive indicator (TPP) and oxygen-insensitive reference dyes (CM).

Scheme 2 (a) Synthetic routes to CM and TPP; (b) hydrolysis and condensation reactions of TEOS and silanized dyes.

Fig. 1 (a) The TEM diameter distribution and image of the silica nanoprobe. (b) Ratiometric emission spectral response of the silica nanoprobe in aqueous solution at different oxygen levels (0%, 0.1%, 0.2%, 0.5%, 0.8%, 1%, 2%, 3%, 5%, 7.5%, 15%, 21%), λ_ex = 410 nm. (c) Linear Stern–Volmer plot of R_N2/R as a function of oxygen partial pressure. (d) Phosphorescence decays at 704 nm of the nanoprobe in aqueous solution saturated with O₂, N₂ and air (λ_ex = 405 nm). The concentration of the nanoprobe is 1.4 × 10⁻⁷ M.²⁰

A collection of silica nanoparticles with different loading levels of CM and TPP, referred to as CM_x@SiO₂ and TPP_y@SiO₂, were prepared. The fluorescence of the silica nanoparticles (NPs) increased with increasing loading levels of CM from 0.1% to 0.25%, then decreased and broadened at the loading level of 0.5% (Fig. S2, ESI†). A 0.25% dye to TEOS ratio was initially taken as the preferable ratio. Then, silica nanoparticles of TPP at the ratio of 0.25% (TPP_0.25@SiO₂) were investigated, the phosphorescence quantum yield of which was measured to be 0.4% in aqueous solution, 4 times over that of TPP in chloroform (ϕ = 0.1%) (Table 1). Obviously, the nonradiative decay processes of the indicator induced by interaction with water and excited-state conformational mobility were inhibited through the covalent connection with the silica matrix and the rigid environments of the nanoparticles. Moreover, this method could ensure covalent-bonding of the dyes and polysiloxane chains quantitatively. During purification by dialysis, no significant trace of unreacted dyes was observed in the filtrate by spectrophotometric analysis, while the absorption of the silica NPs increased quantitatively with increasing loading levels of the CM monomer (Fig. S2a, ESI†).¹⁵

Table 1 Photophysical properties of CM and TPP in different media

Sample	λ abs [nm]	ε max [M^−1 cm^−1]	λ em [nm]	ϕ	τ
a Fluorescence quantum yield, λex = 370 nm, with 9,10-diphenylanthracene (DPA, ϕ = 90% in cyclohexane) as a fluorescence standard. b Phosphorescence quantum yield, λex = 410 nm, with [Ru(bpy)3]Cl2 (ϕ = 9.4% in acetonitrile) as a standard. c Fluorescence decays at maximum emission (λex = 405 nm). d Phosphorescence decays at 704 nm under air (λex = 405 nm). e Phosphorescence decays at 704 nm under N2 (λex = 405 nm). f In aqueous solution.
CM in CHCl3	418	4.1 × 10^4	449	92.7%^a	2.9 ns^c
TPP in CHCl3	416, 525	3.3 × 10^5, 3.0 × 10^4	704	0.1%^b	4.1 μs^d, 69.3 μs^e
CM0.25@SiO2^f	418	2.4 × 10^4	468	25.0%^a	3.0 ns^c
TPP0.25@SiO2^f	416, 524	1.7 × 10^5, 2.0 × 10^4	704	0.4%^b	6.9 μs^d, 370.5 μs^e
CM0.25TPP0.25@SiO2^f	416, 524	4.6 × 10^6, 4.1 × 10^5	468, 704	1.0%^b	1.3 ns^c, 7.4 μs^d, 516.1 μs^e

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Then, we prepared a series of dual-dye loaded silica nanoparticles at different ratios and examined the emission spectra carefully to confirm the final ratios for the nanoprobe (Fig. S2c, ESI†). Loading of CM and TPP at molar percentage ratios of x and y with respect to TEOS, was referred to as CM_xTPP_y@SiO₂. Considering the preferable fluorescence/phosphorescence intensity ratios and luminescence quantum yields, 0.25% of CM and 0.25% of TPP (CM_0.25TPP_0.25@SiO₂) in moles with respect to TEOS was selected for the nanoprobe (see ESI† for details).

The photophysical properties of the nanoprobe were carefully studied (Table 1, see details in the ESI†) The characteristic absorption bands of the nanoprobe at 416 nm (ε_max = 4.6 × 10⁶ M⁻¹ cm⁻¹) are attributed to CM and the Soret band of TPP, while the absorption band at 524 nm corresponds to the Q bands of TPP (ε_max = 4.1 × 10⁵ M⁻¹ cm⁻¹). Upon excitation at 410 nm, the dual-dye loaded nanoprobe showed both fluorescence at 430–600 nm and phosphorescence at 630–800 nm resulting from CM and TPP, respectively (Fig. S3, ESI†). The phosphorescence quantum yield of the nanoprobe in aqueous solution was enhanced 10 times to 1% compared to that of TPP in chloroform (ϕ = 0.1%). We attributed this further enhancement of phosphorescence to the efficient energy transfer between CM (donor) and TPP (acceptor) in the confined nanospace, which is evident by the significantly reduced fluorescence intensity of the nanoprobe compared to that of CM_0.25@SiO₂ under otherwise identical conditions (Fig. S4, ESI†). The energy transfer efficiency (η_ET) of the nanoprobe was calculated as 92% based on the fluorescenc quenching CM after the introduction of TPP in the nanoprobes, where the Förster radius R₀ was estimated to be 4.1 nm between the energy donor (CM) and acceptor (TPP). The phosphorescence lifetime of the nanoprobe at 704 nm was measured to be 516.1 μs in aqueous solution, which was extremely enhanced compared to that of TPP in chloroform under nitrogen atmosphere (69.3 μs) (Fig. S5, ESI†). This long lifetime would greatly improve the sensitivity of the sensors.^7d

Oxygen sensing in aqueous solution

To examine the hypoxia detection capability of the designed nanoprobe, the ratiometric emission spectral response in aqueous solution at different oxygen levels (0%, 0.1%, 0.2%, 0.5%, 0.8%, 1%, 2%, 3%, 5%, 7.5%, 15%, 21%) was investigated. Fig. 1b shows that the fluorescence at 465 nm in the spectra was almost completely independent of the oxygen partial pressure, while the phosphorescence at 704 nm was dramatically quenched with increasing oxygen partial pressure, indicating its ratiometric reponses to oxygen at 465/704 nm. Even 0.2% of oxygen partial levels could lead to over 50% decrease of the phosphorescence intensity, indicating its high sensitivity to extreme hypoxia environment.

To analyze the ratiometric oxygen sensing quantitatively, the Stern–Volmer quenching constant (K_SV) was calculated based on eqn (1)¹⁹ and the data in Fig. 1b, where R is defined as the ratio of phosphorescence intensity at 704 nm to that of the fluorescence intensity at 465 nm (R = I_P/I_F), R_N2 is the emission intensity ratio in fully deoxygenated solution, and pO₂ is the oxygen partial pressure.

R_N2/R = 1 + K_SVpO₂ (1)

As shown in Fig. 1c, there is a highly linear relationship between R_N2/R and pO₂, whose K_SV is obtained as 243.11 bar⁻¹. On the other hand, the ratio of the luminescence intensities in oxygen-free and in oxygen-saturated solution is as high as 153 (I_N2/I_O2 = 153), which is rather high among the reported silica oxygen probes (Table S2, ESI†).^7d In addition, the quenching efficiency (Q) was calculated as 99.3%, close to 100% (see details in the ESI†). The phosphorescence decays in oxygen, air and nitrogen were measured, respectively (Fig. 1d). All phosphorescence decay curves exhibited bi-exponential decay kinetics. The average lifetime of phosphorescence at 704 nm decreased from 516.1 μs in the nitrogen-saturated solution to 7.4 μs in the air-saturated solution, and to 4.1 μs in the oxygen-saturated solution. The phosphorescence lifetime under nitrogen atmosphere is 126 times over that of under oxygen atmosphere (τ_N2/τ_O2 = 126), indicating high sensitivity of the nanoprobe.

The detection of singlet oxygen

Owing to the triplet–triplet energy transfer (TTET) between the triplet excited state of the porphyrin in the nanoprobe and the ground state of molecular oxygen (³O₂), ¹O₂ would be generated accompanying the oxygen sensing process. This process was confirmed by spin trap experiments in conjunction with electron paramagnetic resonance (EPR) spectroscopy. When 2,2,6,6-tetramethylpiperidine (TEMP) was employed as a scavenger for ¹O₂, as shown in Fig. 2a, EPR signals attributed to ¹O₂ were detected in the aqueous solution of the nanoprobe (100 μL, 4 μM) upon irradiation. No significant EPR signal changes were detected when the sample was irradiated in the absence of nanoprobe under identical conditions, confirming that ¹O₂ was generated from the nanoprobe (Fig. S6, ESI†).

Fig. 2 (a) X-Band EPR spectra of the air-saturated aqueous solution of the nanoprobe (100 μL, 4 μM) with TEMP (3 μL) upon irradiation by a Hg lamp (100 mW). (b) Plots of the natural logarithm of the optical density of ABDA (at 378 nm) vs. irradiation time in the presence of nanoprobe (red line) and RB (black line) in H₂O. (c) Absorption spectra of the nanoprobe in aqueous solution (1.4 × 10⁻⁷ M), upon illumination by a medium pressure Hg lamp (300 W) with a glass filter (cutoff < 400 nm) at 6 cm distance. (d) Nanoprobe concentration dependence of the CCK-8 cell viability of HeLa cells under dark conditions and by a 532 nm laser at a power of 5.0 mW cm⁻² for 20 min. Nanoprobe in the concentration range of 0–0.56 μM.

To further assess the capability of the nanoprobe to generate ¹O₂, the singlet oxygen quantum yield (φ_Δ) of the nanoprobe in air-saturated solution was also measured by using the chemical trapping method, where 9,10-anthracenediyl-bi(methylene)dimalonic acid (ABDA) was employed as the trapping agent, and Rose Bengal (RB, φ_Δ = 0.75 in H₂O)²¹ was used as the standard. As illustrated in Fig. 2b and Fig. S7 (ESI†), the absorbance of the ABDA solution at 378 nm decreased gradually with prolonged irradiation time by a 532 nm laser in the presence of the nanoprobe and RB, in which a linear relationship between ln(A₀/A) and irradiation time was observed and the φ_Δ was measured to be 0.55 in air-saturated aqueous solution.

Ratiometric hypoxia sensing and imaging in living cells

With the above impressive results, we tested the capacity of the nanoprobe for hypoxia sensing and imaging in living cells by confocal laser scanning microscopy (CLSM) (Fig. 3).²² The cells were incubated at 37 °C for 1 h under 21% and 1% oxygen atmosphere, respectively, after introducing the nanoprobe into HeLa cells. The observed blue and red luminescence confirmed that the nanoprobe was cell-permeable. Upon excitation by a 405 nm laser, signals in the blue channel (430–470 nm, Fig. 3b and g) and red channel (660–740 nm, Fig. 3c and h) were collected. The blue emission channel was assigned to the fluorescence from CM segments; the fluorescence intensities from the cells were almost the same under 21% and 1% O₂ concentrations. In contrast, the red channel corresponding to the phosphorescent TPP segments shows a brighter image under 1% O₂ conditions than under 21% O₂ conditions. No obvious morphological changes of the cells were observed during the measurement. An evident change in the I_blue/I_red ratio from 3.4 under air to 1.7 under 1% O₂ conditions in living cells concluded that the silica nanoprobe is capable of visualizing the hypoxia status in living cells (Fig. 3e and j).

Fig. 3 Confocal luminescence, bright-field images and ratiometric luminescence images (λ_ex = 405 nm) of living HeLa cells incubated with the silica nanoprobe (1.4 × 10⁻⁹ M) at 21% (a–e) and 1% (f–j) oxygen partial pressure. In luminescence imaging, the blue (430–470 nm) and red (660–740 nm) channels were collected. In ratiometric imaging, the ratio of emission intensity at 430–470 nm to that at 660–740 nm (I_blue/I_red) was chosen as the detected signals. (Scale bar: 20 μm.)

Photostability and cytotoxicity are important parameters for a sensor. The UV-vis absorption spectra negligibly changed even after 6 h irradiation, indicating good stability of the nanoprobe (Fig. 2c). The dark and photocytotoxicity of the nanoprobe in HeLa cells was measured using a standard Cell Counting Kit-8 (CCK-8) assay at various concentrations (0–0.54 μM). As shown in Fig. 2d, a prominent photocytotoxicity was detected under the PDT process, while little effect on the survival of HeLa cells was observed under dark conditions at the same concentration. The investigations revealed that our prepared nanoprobe possesses low cytotoxicity, good biocompatibility and potential photodynamic cancer therapy capabilities.

Conclusions

In summary, we have developed silica-supported dual-dye nanoprobes for ratiometric hypoxia detection. The covalent-bonded phosphorescent indicator and fluorescent reference dye with silica in the nanoprobes led to enhanced phosphorescence brightness, long lifetime (0.52 ms), excellent stability and sensitivity to O₂ (K_SV = 243.11 bar⁻¹, Q = 99.3%). Together with the simple preparation, good biocompatibility and ratiometric hypoxia sensing in living cells, the covalently doped silica nanoprobe demonstrated its potential for future biomedical applications.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Financial support from the National Natural Science Foundation of China (Grants 22071258, 21871280 and 21861132004), the Ministry of Science and Technology of China (Grant 2017YFA0206903), the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant XDB17000000), the Key Research Program of Frontier Sciences of the Chinese Academy of Sciences (Grant QYZDY-SSW-JSC029), and K. C. Wong Education Foundation is gratefully acknowledged. The authors thank Dr Ye Tian, Si-Jia Gao and Na Tian from TIPC for their great help with the cell experiments.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0qm00653j

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