Bioorthogonal activation and mitochondrial targeting of a near-infrared-emitting iridium(III) nitrone complex via cyclooctynylated phosphonium cations for enhanced cellular imaging and photodynamic therapy

Abstract

In this work, we designed and synthesised a new cyclometallated iridium(III) nitrone complex [Ir(bpz)₂(bpy-nitrone)](PF₆) (1) (Hbpz = benzo[a]phenazine; bpy-nitrone = 4-((methyl(oxido)imino)methyl)-4′-methyl-2,2′-bipyridine) as a bioorthogonally activatable phototheranostic agent. Complex 1 displayed very weak emission and singlet oxygen (¹O₂) photosensitisation in solutions due to the quenching nitrone moiety. However, upon the strain-promoted alkyne–nitrone cycloaddition (SPANC) reaction with bicyclo[6.1.0]non-4-yne (BCN), which converted the nitrone unit to a non-quenching isoxazoline derivative, the complex exhibited a substantial increase in emission intensity in the near-infrared region and ¹O₂ generation efficiency. Given that mitochondria are a crucial target in cancer therapy, we prepared a series of BCN-functionalised phosphonium cations (BCN-Phos-n), each bearing different substituents, to serve as mitochondrial-targeting vectors for delivering complex 1 to the mitochondria via the bioorthogonal SPANC reaction. Notably, complex 1 exhibited more significant emission turn-on upon reaction with BCN-Phos-5 and BCN-Phos-6 (I/I_o = 24.7 and 14.1, respectively), attributed to their increased hydrophobicity resulting from the methylation or methoxylation of the phenyl rings on the phosphonium cation. Live-cell confocal imaging and flow cytometric analyses revealed that complex 1 showed larger emission enhancement in HeLa cells pretreated with BCN-Phos-5 or BCN-Phos-6 compared to other BCN-Phos-n analogues. Co-staining experiments confirmed that the resultant luminescent isoxazoline cycloadducts predominantly accumulated in the mitochondria. Additionally, both dark and light-induced cytotoxicity of complex 1 increased upon pretreatment of the cells with BCN-Phos-5 or BCN-Phos-6. Our results demonstrate that the theranostic potential of transition metal nitrone complexes can be significantly enhanced via strategic structural manipulation of their bioorthogonal reaction partners.

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Introduction

Over the past two decades, the development of bioorthogonal chemistry^1,2 has revolutionised the fields of chemical biology^3–5 and biomedicine.^6–8 Bioorthogonal ligation reactions between two abiotic functionalities have enabled the visualisation of specific biomolecules and associated biological events in live cells using fluorescence microscopy.⁹ The use of fluorescent probes, however, can lead to unavoidable background fluorescence due to non-specific covalent labelling and the entrapment of unreacted probes within the cellular environment. Thus, there has been significant interest in the development of fluorogenic bioorthogonal probes, whose fluorescence is quenched by the appended bioorthogonal group and can only be restored upon specific bioorthogonal reactions, thereby enhancing the precision of fluorescence imaging.^10–12 This design strategy has recently been extended to the development of activatable photosensitisers for targeted photodynamic therapy (PDT).^13,14 The controlled activation of the reactive oxygen species (ROS) photosensitisation capabilities of the photosensitisers through bioorthogonal reactions can minimise undesirable off-target photodamage often observed with traditional photosensitisers, which display “always-on” photosensitisation properties and lack target selectivity.

Luminescent and photofunctional transition metal complexes have gained significant attention as phototheranostics due to their attractive photophysical and photochemical properties, including high photostability, long-lived and environment-sensitive emission, as well as efficient ROS photosensitisation.^15–19 We have a long-standing interest in the development of these complexes as bioorthogonal reagents for various biological and biomedical applications.²⁰ In 2016, we demonstrated for the first time that nitrone, a 1,3-dipole that can selectively react with cyclooctynes via the strain-promoted alkyne–nitrone cycloaddition (SPANC) reaction,²¹ can serve as an emission quencher for transition metal complexes, providing a new avenue for the development of phosphorogenic bioorthogonal probes.^22,23 The nitrone-modified complexes are non-emissive in solutions, but exhibit significant emission enhancement upon reaction with bicyclo[6.1.0]non-4-yne (BCN) derivatives (Scheme 1). This modification also allows for the modulation of the ROS photosensitisation efficiencies of the complexes, enabling controlled activation of their emission and ROS generation behaviour in targeted cells.^24,25 Thus, transition metal nitrone complexes represent a promising scaffold for the development of bioorthogonally activatable probes and photosensitisers.

Scheme 1 The SPANC reaction of phosphorogenic iridium(III) nitrone complexes with BCN derivatives leading to the formation of luminescent isoxazoline cycloadducts.

Mitochondria are crucial subcellular organelles involved in a wide range of important biological processes including energy production,²⁶ biomolecular synthesis,²⁶ calcium signalling,²⁷ as well as cell proliferation and death.²⁸ Mitochondrial dysfunction can lead to various diseases such as cancer²⁹ and neurodegenerative disorders.³⁰ Given their pivotal role in maintaining cellular functions, mitochondria have become an important target for cancer therapy.³¹ Mitochondria possess a negative membrane potential (ca. −120 to −180 mV), and it has been reported that the mitochondria in cancer cells are more hyperpolarised than in normal cells due to their higher metabolic activity.³² Thus, lipophilic cations such as triphenylphosphonium cation (TPP⁺) preferentially accumulate in the mitochondria over other subcellular organelles, resulting in ca. 100–1000-fold higher intramitochondrial concentrations.³³ These moieties have been engineered with various bioorthogonal handles to precisely direct fluorescent/fluorogenic bioorthogonal probes to the mitochondria, facilitating the imaging of these organelles in live cells.^34–46 This approach enables the monitoring of mitochondrial membrane potential changes⁴⁷ and mitophagy,⁴⁸ as well as the activation of mitochondria-enriched prodrugs for applications in cancer therapy^49–53 and cardioprotection.⁵⁴ However, the use of a two-step bioorthogonal approach for delivering photoactive transition metal complexes to the mitochondria, and controlled activation of their emission and ROS photosensitisation properties for bioimaging and PDT applications remains unexplored.

In this work, we designed, synthesised and characterised a new cyclometallated iridium(III) nitrone complex [Ir(bpz)₂(bpy-nitrone)](PF₆) (1) (Hbpz = benzo[a]phenazine; bpy-nitrone = 4-((methyl(oxido)imino)methyl)-4′-methyl-2,2′-bipyridine) (Scheme 2) as a bioorthogonally activatable phototheranostic agent. The Hbpz ligand was selected because its metal complexes show near-infrared (NIR) emission and high singlet oxygen (¹O₂) generation efficiencies.⁵⁵ Additionally, we prepared a series of BCN-modified phosphonium cations (BCN-Phos-n) (Scheme 2) as mitochondrial-targeting vectors to direct the nitrone complex to the mitochondria via the SPANC reaction. Specifically, BCN-Phos-1–BCN-Phos-4 carried varying numbers of cyclohexyl (Cy) and phenyl (Ph) moieties on the phosphonium cation to tune their aromaticity;^56–58 while BCN-Phos-5–BCN-Phos-7 contained different substituents on the phenyl rings of the TPP⁺ unit, including two methyl (BCN-Phos-5) or methoxy groups (BCN-Phos-6) at the meta-positions to enhance their lipophilicity,^59–62 or a di(ethylene glycol) pendant at the para-position (BCN-Phos-7) to increase its aqueous solubility and biocompatibility.^57,58

Scheme 2 Structures of complex 1 and BCN-Phos-n.

Results and discussion

Synthesis and characterisation of complex 1 and BCN-Phos-n

The synthesis of complex 1 involved the reaction of the dichloro-bridged iridium(III) dimer [Ir₂(bpz)₄Cl₂] with the ligand bpy-nitrone in CH₂Cl₂/MeOH (Scheme 3a), followed by anion exchange with KPF₆ and purification by column chromatography and recrystallisation from CH₂Cl₂/Et₂O. The complex was characterised by high resolution (HR)-ESI-MS, ¹H and ¹³C NMR and IR spectroscopy, and gave satisfactory elemental analyses (ESI†).

Scheme 3 Synthetic routes of (a) complex 1 and (b) BCN-Phos-n.

The BCN-modified phosphonium cations BCN-Phos-n were synthesised using the corresponding phosphine precursors (Phos-n) (Scheme 3b), which were either purchased from commercial suppliers (for Phos-1–Phos-4) or prepared according to previously reported protocols (for Phos-5–Phos-7).^63,64 The phosphine precursors were reacted with 3-bromopropylamine hydrobromide in n-butanol under reflux for 3 days, in a procedure adapted from Zhou and co-workers.⁶⁵ The resultant amine-functionalised phosphines (H₂N-Phos-n) were obtained as ammonium salts in good yields (44–62%) after purification by recrystallisation from isopropanol/Et₂O, except for H₂N-Phos-1 which did not precipitate out of Et₂O and was, therefore, used in subsequent steps without further purification. Amide coupling of H₂N-Phos-n with (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (BCN–NHS) was achieved under mild basic conditions.^22–25 All BCN-Phos-n analogues were obtained in average to good yields (45–78%) after purification by silica gel column chromatography. These compounds were characterised by HR-ESI-MS and ¹H, ¹³C and ³¹P NMR spectroscopy (ESI†).

Photophysical and photochemical properties of complex 1

Complex 1 displayed intense spin-allowed intraligand (¹IL) (π → π*) (bpy-nitrone and bpz) absorption in the UV region (ca. 274–431 nm, ε on the order of 10⁴ dm³ mol⁻¹ cm⁻¹) and weaker spin-allowed metal-to-ligand charge-transfer (¹MLCT) (dπ(Ir) → π*(bpy-nitrone and bpz)) absorption features in the visible region (ca. 456–550 nm) (Fig. 1a and Table S1, ESI†).^25,55 The strong absorption in the region of 500–600 nm is an attractive feature because it allows for efficient photoexcitation using green light. The weaker absorption tailing beyond ca. 615 nm is assigned to spin-forbidden ³MLCT (dπ(Ir) → π*(bpy-nitrone and bpz)) transitions. Upon photoirradiation, the complex exhibited NIR emission in fluid solutions at 298 K (Table 1 and Fig. 1b). Additionally, it showed a vibronically structured emission band with an extraordinarily long emission lifetime (8.79 μs) in an alcohol glass at 77 K (Table 1 and Fig. 1b). These observations suggest that the emission of the complex originates from a predominant ³IL (π → π*) (bpz) excited state with possible mixing of some ³MLCT (dπ(Ir) → π*(bpy-nitrone/bpz)) character.^25,55 Similar emission features were observed when the complex was excited at 550 nm, indicating that the emission is largely independent of the excitation wavelength. Importantly, the emission quantum yields of the complex (Φ_em ≤ 0.0047; Table 1) were significantly lower than those of related bpz complexes,⁵⁵ indicating efficient emission quenching by the appended nitrone unit.^22–25 Additionally, the ¹O₂ generation quantum yield (Φ_Δ) of the complex was determined by monitoring the emission band of ¹O₂ centred at ca. 1270 nm in aerated CH₃CN. The small Φ_Δ value (0.05; Table 1) indicates strong suppression of the ¹O₂ photosensitisation capability of the complex by the quenching nitrone moiety.

Fig. 1 (a) Electronic absorption and (b) normalised emission spectra of complex 1 in CH₂Cl₂ (black) and CH₃CN (red) at 298 K and in alcohol glass at 77 K (blue).

Table 1 Photophysical data and ¹O₂ generation quantum yields (Φ_Δ) of complexes 1 and 1-BCN

Complex	Medium (T/K)	λem^a/nm	τo^b/μs	Φem^c	ΦΔ^d
a λex = 350 nm.b The lifetimes were measured at the emission maxima (λex = 355 nm).c The Φem values were determined in degassed solvents using [Ru(bpy)3]Cl2 (Φem = 0.040 in aerated H2O, λex = 455 nm)^66 as a reference.d The ΦΔ values were determined in aerated solvents using [Ru(bpy)3]Cl2 (ΦΔ = 0.57 in aerated CH3CN, λex = 450 nm)^67 as a reference.e EtOH/MeOH (4 : 1, v/v).
1	CH2Cl2 (298)	664	4.80	0.0047
	CH3CN (298)	668	2.97	0.0043	0.05
	Glass^e (77)	668, 730 sh	8.79
1-BCN	CH2Cl2 (298)	666	4.28	0.072
	CH3CN (298)	668	2.67	0.062	0.76
	Glass^e (77)	667, 729 sh	9.63

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Bioorthogonal reactivity and phosphorogenic response of complex 1

We utilised the strained alkyne (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN–OH) as a model substrate to examine the bioorthogonal reactivity of the nitrone complex. The second-order rate constant (k₂) of the SPANC reaction of the complex with BCN–OH in MeOH at 298 K was determined to be 0.3309 M⁻¹ s⁻¹ (Fig. S1, ESI†), which is 8.3 times greater than that of the free ligand bpy-nitrone (k₂ = 0.040 M⁻¹ s⁻¹).²² The accelerated reaction kinetics can be attributed to the direct coordination of the nitrone ligand to the cationic iridium(III) polypyridine unit, which enhanced its reactivity.^22–25 Importantly, upon the SPANC reaction with BCN–OH in aerated phosphate-buffered saline (PBS; pH 7.4)/MeOH (9 : 1, v/v), the complex showed substantial emission enhancement (I/I_o = 5.8; Table 2 and Fig. 2a) and lifetime extension (τ increased from 0.05 to 0.13 μs; Table 2), resulting from the conversion of the quenching nitrone moiety to a non-quenching isoxazoline derivative. The formation of the isoxazoline product 1-BCN was verified by ESI-MS analysis (Fig. S2, ESI†). Conjugate 1-BCN was isolated and purified, and its photophysical and ¹O₂-photogeneration properties were investigated. Both Φ_em (0.062–0.072; Table 1) and Φ_Δ (0.76; Table 1) values of conjugate 1-BCN are larger than those of complex 1 (Φ_em ≤ 0.0047, Φ_Δ = 0.05; Table 1). These results confirm that both the emission and ¹O₂-photosensitisation properties of complex 1 can be activated through the bioorthogonal SPNAC reaction with BCN derivatives, which effectively eliminates the nitrone-associated quenching pathway.

Fig. 2 (a) Emission spectra of complex 1 (10 μM) in the absence (black) and presence of BCN–OH (250 μM) (red) in aerated PBS (pH 7.4)/MeOH (9 : 1, v/v) upon incubation at 298 K for 24 h. (b) Emission spectra of complex 1 (10 μM) in the absence (black) and presence of BCN-Phos-1 (red), BCN-Phos-2 (blue), BCN-Phos-3 (magenta), BCN-Phos-4 (olive), BCN-Phos-5 (navy), BCN-Phos-6 (violet) and BCN-Phos-7 (orange) (250 μM) in aerated PBS (pH 7.4)/MeOH (9 : 1, v/v) upon incubation at 298 K for 24 h. Excitation wavelength = 350 nm.

Table 2 Emission wavelengths (λ_em), emission enhancement factors (I/I_o) and emission lifetimes (τ) of complex 1 (10 μM) upon reaction with BCN–OH or BCN-Phos-n (250 μM) in aerated PBS (pH 7.4)/MeOH (9 : 1, v/v) at 298 K for 24 h

Entry	λem	I/Io^a	τ/μs
a Io and I are the emission intensities of the complex (10 μM) in the absence and presence of BCN–OH or BCN-Phos-n (250 μM), respectively.
1	695	—	0.05
1 + BCN–OH	684	5.8	0.13
1 + BCN-Phos-1	687	4.2	0.15
1 + BCN-Phos-2	690	3.8	0.14
1 + BCN-Phos-3	703	3.1	0.12
1 + BCN-Phos-4	686	3.3	0.13
1 + BCN-Phos-5	674	24.7	0.33
1 + BCN-Phos-6	677	14.1	0.25
1 + BCN-Phos-7	706	4.5	0.19

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We also investigated the phosphorogenic response of complex 1 towards the BCN-Phos-n derivatives. Incubation of complex 1 with the BCN-Phos-n analogues in aerated aqueous buffers led to substantial emission enhancement (I/I_o = 3.1–24.7) and lifetime extension (τ = 0.12–0.33 μs) (Table 2 and Fig. 2b). Notably, the BCN-Phos-5 and BCN-Phos-6 treatment resulted in a larger increase in emission intensity (I/I_o = 24.7 and 14.1) and lifetime (τ = 0.33 and 0.25 μs), accompanied by a notable blue shift in the emission maximum from 695 nm to 674 and 677 nm, respectively (Table 2 and Fig. 2b). The more significant photophysical changes compared to other BCN-Phos-n analogues are likely due to the formation of a more hydrophobic pendant after reaction with BCN-Phos-5 and BCN-Phos-6, which feature two lipophilic methyl or methoxy groups on each of the phenyl rings of the TPP⁺ unit, resulting in a greater reduction in the polarity of the proximal environment of the complex. Such a result aligns with our previous observations that luminescent iridium(III) polypyridine complexes display higher emission intensities and longer lifetimes in less polar solvents or upon bioconjugation to proteins.^68–73

Cellular uptake, localisation and (photo)cytotoxicity of complex 1

We then studied the phosphorogenic response of the nitrone complex towards the BCN-Phos-n derivatives in live HeLa cells. The cells were first incubated with BCN-Phos-n (5 μM) for 2 h, washed with PBS (pH 7.4), and then treated with complex 1 (5 μM) for an additional 2 h prior to imaging. Laser-scanning confocal microscopy (LSCM) images reveal negligible emission from HeLa cells incubated with complex 1 (Fig. 3a). However, intense intracellular emission was observed upon pretreatment of the cells with BCN-Phos-5 or BCN-Phos-6 (Fig. 3a). Flow cytometric analysis confirmed that complex 1-treated cells exhibited a 2.15- and 2.94-fold increase in emission intensity when pretreated with BCN-Phos-5 and BCN-Phos-6, respectively (Fig. 3b and Table S2, ESI†). Additionally, ICP-MS analysis indicated that the cellular uptake of complex 1 remained similar without (1.83 fmol per cell) and with pretreatment of BCN-Phos-5 and BCN-Phos-6 (1.77 and 1.74 fmol per cell, respectively) (Table S3, ESI†). These results confirm that the observed intracellular emission enhancement is attributable to the SPANC reaction of complex 1 with BCN-Phos-5 and BCN-Phos-6, rather than an increase in cellular accumulation of the complex. However, similar emission enhancement was not observed for cells pretreated with other BCN-Phos-n derivatives (Fig. 3a and S3 and Table S2, ESI†), consistent with their smaller emission enhancement in solutions (I/I_o = 3.1–4.5; Table 2 and Fig. 2b). Co-staining experiments with MitoTracker Green showed that the luminescent isoxazoline cycloadducts formed from the reaction of complex 1 with BCN-Phos-5 and BCN-Phos-6 were enriched in the mitochondrial region of the cells, with Pearson's correlation coefficients (PCC's) of 0.79 and 0.81, respectively (Fig. 4). The mitochondrial accumulation of the isoxazoline cycloadducts is likely due to their cationic and lipophilic character.^74–80

Fig. 3 (a) LSCM images of HeLa cells incubated with complex 1 (5 μM, 2 h; λ_ex = 488 nm, λ_em = 650–750 nm) without or with pretreatment of BCN-Phos-n (5 μM, 2 h) at 37 °C. Scale bar = 25 μm. (b) Flow cytometric results of HeLa cells under different treatment. The cells were treated with blank medium (2 h) (grey); complex 1 (5 μM, 2 h) (black); BCN-Phos-5 (5 μM, 2 h) and then complex 1 (5 μM, 2 h) (red); and BCN-Phos-6 (5 μM, 2 h) and then complex 1 (5 μM, 2 h) (blue) at 37 °C.

Fig. 4 LSCM images of HeLa cells pretreated with BCN-Phos-5 or BCN-Phos-6 (5 μM, 2 h) and then incubated with complex 1 (5 μM, 2 h; λ_ex = 488 nm, λ_em = 650–750 nm) and MitoTracker Green (100 nM, 30 min; λ_ex = 488 nm, λ_em = 506–526 nm) at 37 °C. Scale bar = 10 μm. PCC = 0.79 (BCN-Phos-5) and 0.81 (BCN-Phos-6).

We also examined the (photo)cytotoxicity of complex 1 towards HeLa cells with or without BCN-Phos-n pretreatment using the Neutral Red uptake (NRU) assay. Complex 1 exhibited moderate dark cytotoxicity (IC_50,dark = 18 μM) and substantially enhanced photocytotoxic activity (IC_50,light = 0.37 μM) upon irradiation at 525 nm (10 mW cm⁻²) for 5 min (Fig. 5a and Table S4, ESI†). Notably, the (photo)cytotoxicity of the complex was further increased when the cells were pretreated with BCN-Phos-5 or BCN-Phos-6, with IC_50,dark values decreasing to 9.6 and 6.3 μM and IC_50,light values decreasing to 0.14 and 0.22 μM, respectively (Fig. 5b and c and Table S4, ESI†). The enhanced dark cytotoxicity of the complex can be attributed to its increased accumulation in the mitochondria after the SPANC reaction with BCN-Phos-5 or BCN-Phos-6, which probably interferes with mitochondrial functions.⁷⁹ Notably, the photocytotoxicity of the complex was further enhanced following the reaction, which is attributed to the increased ¹O₂ generation by the resultant isoxazoline cycloadducts. These results highlight that the therapeutic potential of the complex can be enhanced via a judicious selection of its bioorthogonal reaction partners.

Fig. 5 Viability of HeLa cells incubated with (a) blank medium, (b) BCN-Phos-5 (5 μM) or (c) BCN-Phos-6 (5 μM) for 2 h and then treated with complex 1 at different concentrations for 2 h, followed by incubation in the dark (blue) or irradiation at 525 nm (10 mW cm⁻²) (red) for 5 min, and then further incubated with blank medium for 20 h.

Conclusions

In summary, we developed a novel iridium(III) nitrone complex as a bioorthogonally activatable phototheranostic agent, and a series of BCN-modified phosphonium cations serving as mitochondrial-targeting vectors to direct the nitrone complex to the mitochondria via the bioorthogonal SPANC reaction for imaging and PDT applications. Notably, the complex displayed more pronounced emission turn-on upon reaction with BCN-Phos-5 and BCN-Phos-6 compared to other BCN-Phos-n analogues, attributed to the presence of additional hydrophobic methyl or methoxy groups on the phenyl rings of the TPP⁺ unit that resulted in a more hydrophobic pendant. Similar emission changes were observed in live cells pretreated with BCN-Phos-5 or BCN-Phos-6. Owing to their cationic and lipophilic character, the resultant luminescent isoxazoline cycloadducts were enriched in the mitochondria. Importantly, the (photo)cytotoxicity of the complex further increased when the cells were pretreated with BCN-Phos-5 or BCN-Phos-6. Our findings demonstrate that the theranostic potential of transition metal nitrone complexes can be enhanced through the strategic structural manipulation of their bioorthogonal reaction partners. We believe that our work will contribute to the development of effective mitochondria-targeting agents for diagnostic and therapeutic applications.

Author contributions

E. R. H. W., L. C.-C. L., P. K.-K. L., K. K.-W. L. and N. J. L. designed the project. E. R. H. W. carried out the synthesis and characterisation of the phosphonium cations. L. C.-C. L. carried out the synthesis and characterisation of the iridium(III) nitrone complex and the cellular studies. P. K.-K. L. carried out the photophysical measurements and cellular studies. E. R. H. W., L. C.-C. L., P. K.-K. L., K. K.-W. L. and N. J. L. analysed the data and wrote the manuscript.

Conflicts of interest

There are no conflicts to declare.

Data availability

We confirm that all the relevant research data is contained with the manuscript and ESI.† No databases have been used and no references to such databases are contained in the manuscript or ESI.†

Acknowledgements

We thank the Laboratory for Synthetic Chemistry and Chemical Biology (LSCCB) under the Health@InnoHK Programme launched by Innovation and Technology Commission, The Government of Hong Kong SAR, P. R. China.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5qi01139f

‡ Equal contribution from these authors.

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