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Near-infrared fluorescence probes to detect reactive oxygen species for keloid diagnosis† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc01865k

Development of molecular probes for the detection of reactive oxygen and nitrogen species (RONS) is important for the pathology and diagnosis of keloid diseases.


Introduction
Reactive oxygen and nitrogen species (RONS) are essential to regulate normal physiological processes, while abnormally high levels of RONS are associated with pathogenesis. In addition to cancer, inammation, cardiovascular diseases and organ brosis, 1 an abnormally high cellular level of RONS production has been reported in chronic wound healing and keloid scars. 2,3 Keloid is a type of tissue brosis aer initial lesion with characteristics of excess collagen neoplasm and invasiveness into surrounding tissues, causing severe cosmetic problems, pruritus, and pain. The current treatments of keloid including surgical incision, radiation, pressure and laser therapy suffer from high recurrence rate, not to mention that radiation therapy has the risk of carcinogenesis. 4 However, only a few diagnostic probes have been developed for keloids based on the overexpression of FAPa (broblast activation protein-alpha) 5 and CTGF (connective tissue growth factor). 6 In the scar formation process, an abnormal high RONS level could affect the processes of broblast epithelial to mesenchymal transition (EMT) required for ECM deposition, 7 broblast migration and adhesion, 8 and extra collagen deposition. 9,10 Abnormal expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), 11 and brogenic cytokine transforming growth factor b (TGF-b) 12,13 could account for this high RONS level. It yet still remains unclear which specic RONS plays a part in developing keloid pathogenesis. Thereby, understanding the molecular mechanism of RONS in keloid formation not only can provide useful information for early diagnosis of keloid but also may bring in new therapeutic interventions.
As compared to other imaging modalities, optical imaging has the advantages of rapid detection with high-throughput capacity, high sensitivity with detection at molecular and cellular levels, and relatively low instrumentation costs. 14 Molecular probes with high detection specicity and sensitivity have been readily developed for RONS such as O 2 c À , 15 H 2 O 2 , 16 ONOO À , [17][18][19][20] NOc, 21 HOc, 22 OCl À (ref. 23) and O 3 (ref. 24) and have been summarized in a number of nice reviews. [25][26][27] A natural extension of such efforts is the detection of RONS in living cells and living organisms with different disease models. Particularly, the development of near-infrared (NIR) probes have enabled deeper tissue penetration, minimized tissue auto-uorescence and enhanced signal-to-noise ratios. These RONS probes have been successfully applied in the evaluation of druginduced toxicity, 28 ischemia-reperfusion, 29 acute peritonitis inammation 30 and neuroinammation 31 models. However, optical molecular probes have not yet been used to study the role of RONS in keloids and to apply in its early diagnosis.
Here, we report the design and syntheses of two nearinfrared (NIR) uorescent activatable molecular probes for the detection of RONS in keloids. These two probes have different responses towards peroxynitrite (ONOO À ) in terms of specicity (Scheme 1a). CyTF is designed by linking a triuoromethyl ketone group to a NIR hemicyanine dye (CyOH), and it could undergo ONOO À -mediated oxidation in a highly-selective manner. The ketone unit forms a dioxirane intermediate, which selectively oxidizes the phenyl ring to afford a dienone product, releasing the caged oxygen atom. 17,18 Meanwhile, CyBA is designed by conjugating a boronic acid-based self-immolative group to CyOH. The boronate reaction site is Lewis acidic, and strong Lewis bases like ONOO À and H 2 O 2 could readily attack the boron atom, followed by aryl migration to oxygen and release of the boronate group. The self-immolative linker is then released through 1,4-immolation. 19,20,32 Both CyTF and CyBA are non-uorescent initially as the electron-donating oxygen atoms are caged by the ROS responsive group. Aer reaction with RONS, the caging groups are released, leading to free uorescent CyOH. This characteristic uorescence turn-on mechanism could be used to specically distinguish keloid scar cells from normal dermal broblasts, where an abnormally high oxidative stress is reported (Scheme 1b). 16

Syntheses of CyTF and CyBA
The probes were synthesized according to Scheme 2. 3-(4-Methoxyphenyl)propanoic acid (compound 1) was reacted with ethanol to give compound 2. Compound 2 was then reacted with trimethyl(triuoromethyl)silane to obtain compound 3, which was then reacted with tetra-n-butylammonium uoride to obtain compound 4. Compound 4 was then reacted with boron tribromide to give compound 5. CyOH was synthesized by reacting IR775-chloride with resorcinol via a retro-Knoevenagel reaction. CyOH was then reacted with synthesized compound 5 or bromomethylphenylboronic acid pinacol ester by caging the oxygen atom to afford CyTF and CyBA, respectively. The structures of puried probes were conrmed by NMR and mass spectroscopy (ESI †).

In vitro sensing of RONS
To validate and compare the RONS sensing ability between CyTF and CyBA, their absorption and uorescence spectra in the absence and presence of different RONS were measured (Fig. 1).
Both CyTF and CyBA had two absorption peaks at 605 and 654 nm and were initially non-uorescent; upon adding ONOO À , the absorption peak at 605 nm disappeared and a new peak at 690 nmthe ngerprint spectrum of free CyOH appeared. 33 Such a cleavage led to uorescence enhancement up to 15-fold at 717 nm ( Fig. 1a and b).
High performance liquid chromatography (HPLC) and liquid chromatography mass spectroscopy (LCMS) further conrmed that CyTF (HPLC retention time, T R ¼ 24 min) and CyBA (T R ¼ 24.1 min) were totally converted into free CyOH (T R ¼ 20.8 min) aer incubation with ONOO À (Fig. 1c and d). In addition, a gradual increase in uorescence intensity with ONOO À concentration could be observed, and linearity existed at concentrations ranging from 0-8 mM (Fig. 1e, S1 †). Under such conditions, the limits of detection (LODs) for CyTF and CyBA were determined to be 89.4 and 71.7 nM, respectively (LOD ¼ 3s/s, s: standard deviation of the blank, s: slope of the calibration curve).
The selectivity of CyTF and CyBA was tested against other RONS including O 2 c À , OCl À , cOH, and H 2 O 2 . As expected, CyTF was highly specic towards ONOO À . However, CyBA showed responses to both ONOO À and H 2 O 2 and the uorescence enhancement for H 2 O 2 was 5-fold (Fig. 1f). Moreover, the response rates of both CyTF and CyBA were tested by timecourse uorescence measurements (Fig. S2 †). Upon addition of ONOO À , the uorescence intensity of both probes at 717 nm increased drastically and reached a plateau in less than two minutes, indicating the rapid response of both probes to ONOO À . Thus, CyTF and CyBA showed similar uorescence enhancement and kinetics upon adding ONOO À , yet CyBA showed poorer selectivity as it was responsive towards both ONOO À and H 2 O 2 .

Imaging of RONS in cells
Aer conrming the low cytotoxicity of CyTF and CyBA against living cells by MTS assay (Fig. S4 †), these probes were used to detect endogenously generated RONS in cultured normal dermal broblasts (NDFs). NDFs without treatment served as control groups. NDFs treated with Transforming Growth Factor (TGF)-b1 alone were used as the positive control, whereas the cells treated with TGF-b1 along with a RONS scavenger (NAC: Nacetyl-L-cysteine) and the cells treated with NAC only were used as the negative control groups. All groups were then incubated with CyTF or CyBA for uorescence imaging.
As a whole, for control and negative control groups, weak uorescence signals were observed for both CyTF and CyBA ( Fig. 2a and b), indicating low RONS levels in NDFs. However, in the control group, CyBA had a slightly higher background signal than CyTF in the untreated cells (Fig. 2a). CyBA could react with both H 2 O 2 and ONOO À , implying a higher basal level of H 2 O 2 than ONOO À in untreated cells. This is reasonable because H 2 O 2 is a less reactive and hence more stable oxidative species than ONOO À . 34,35 Aer TGF-b1 stimulation, the uorescence of both CyTF and CyBA dramatically increased, and the uorescence enhancement for CyTF (3.76 fold) was 2.6-times higher than CyBA (1.43 fold) ( Fig. 2a and b). The overall increased uorescence signals aer TGF-b1 stimulation were caused by the fact that a high concentration of cytokine TGF-b1 could induce an inammatory response and in turn oxidative stress. Furthermore, upon NAC addition into these stimulated cells, the uorescence level decreased to basal levels similar to those untreated cells ( Fig. 2a and b), conrming the role of TGF-b1 in inducing oxidative stress. The great uorescence enhancement for CyTF and CyBA in TGF-b1 stimulated NDFs further implies that TGF-b1 could especially increase the intracellular ONOO À level. In the TGF-b1 signalling cascade (Fig. 2c), it rst binds to and phosphorylates the transmembrane TGF-b type II receptor (TbRII), and then trans-phosphorylates the TGF-b type I receptor (TbRI). This subsequently activates Smad signalling proteins, which then translocate into the nucleus to interact with other transcription factors to regulate transcription pathways. 36 In particular, TGF-b1 could increase the expressions of transmembrane proteins NADPH oxidase 4 (NOX4) 37 and endothelial nitric oxide synthase (eNOS). 38 eNOS generates nitric oxide, NOc from L-Arginine and the oxygen molecule; NOX4 produces superoxide anion, O 2 c À from the oxygen molecule. The generated O 2 c À could subsequently react with NOc to produce ONOO À (k z 10 10 M À1 s À1 ) or convert to H 2 O 2 by enzyme superoxide dismutase (k z 10 9 M À1 s À1 ). 35 As both reactants O 2 c À and NOc have high intracellular levels, and the reaction producing ONOO À is more kinetically favoured, the level of ONOO À should be higher than H 2 O 2 in TGF-b1 stimulated NDFs (Fig. 2c).
The NIR probes were then applied to detect keloid-derived broblasts (KFs) in culture, with NDFs as control groups. Compared with low basal uorescence levels in NDFs, both probes incubated in untreated KFs had shown enhanced uorescence levels. The uorescence enhancement in CyTF (2.26-fold) was 1.5-times higher than CyBA (1.5-fold) due to a higher background level of H 2 O 2 as explained earlier ( Fig. 3a and b). However, when KFs were further treated with a selective TbR1 inhibitor (RepSox), 39 the uorescence levels for both probes decreased to a large extent, suggesting the role of TGF-b1 signalling in causing keloid oxidative stress. As a result, by targeting the TGF-b1 signalling pathway, keloid therapeutic drugs like RepSox could be developed.
An uncontrolled TGF-b1 signalling was found in keloid pathogenesis, originating from both overproduction of the cytokine itself and higher expression levels of TbRI & TbRII in keloid cells. 3 Specically, the distinguishable uorescence enhancement from CyTF had demonstrated a much higher ONOO À level in KF cells. When RepSox was subsequently added to inhibit TGF-b1 signalling by blocking TbRII trans-phosphorylation, the intracellular ONOO À level was reduced (Fig. 3c).
Hence, ONOO À could become a specic biomarker for keloid disease, and CyTF has the potential to be applied in early diagnosis. Furthermore, identifying the signicance of TGF-b1 signalling in keloid pathogenesis has made developing TGFb signalling pathway-targeting drug treatment towards keloid disease plausible. By targeting the intracellular ONOO À level, CyTF could also become a potent drug screener.

In vivo keloid imaging
The ability of CyTF to distinguish keloid cells from normal skin cells was further tested in a xenogra mice model. The KF cell pellets (6 Â 10 5 cells), NDF cell pellets (6 Â 10 5 cells) separately dissolved in 200 mL solution (Matrigel/DMEM ¼ 1 : 1) and Matrigel (200 mL) only as the control group were injected into three points on the back of living mice, and 200 mL CyTF (0.2 mM in deionized water containing 20% ethanol) was intravenously injected (Fig. 4a). By virtue of its NIR uorescence with deep tissue penetration and minimized tissue auto-uorescence, uorescence signals from CyTF being turned on to CyOH could be readily observed in living mice (Fig. 4b).
The uorescence signal of CyTF at the keloid site was found to gradually increase from t ¼ 1 h and reached its maximum at t ¼ 3.5 h.
At this time point, the uorescence intensity was 3-fold compared to NDF cells and control (Fig. 4d). Aerwards, the uorescence signal at the keloid site decreased, until reaching a similar level to the background, suggesting that CyTF could be readily cleared over time. Aer 6 h, the mice were sacriced to study in vivo distribution, the organs were rst incubated in ONOO À solution to activate any remaining CyTF to uorescent CyOH, and most of the probes were found accumulated in the liver (Fig. 4c and e). The injected cell pellets were also extracted, and KF cell pellets showed stronger uorescence signals as compared to those of the NDF cell pellets and control group.
The in vivo experiments conrmed that CyTF could be activated by ONOO À and used to image implanted KF cells in living mice.

Conclusions
In summary, we have developed NIR molecular probes CyTF and CyBA (l ex /l em ¼ 640/717 nm) to detect ONOO À , and applied them to distinguish keloid broblasts from normal dermal broblasts. CyTF was highly specic towards ONOO À , while CyBA was responsive to both ONOO À and H 2 O 2 . In living cells, CyTF showed higher uorescence enhancement than CyBA, which were 3.76 and 2.26-fold in TGF-b stimulated normal dermal broblasts and keloid broblasts, respectively. In keloid broblasts treated with RepSox, the uorescence signals decreased to a large extent, proving CyTF to be a potent drug screener. This cellular imaging results proved that keloid broblasts had a higher intracellular level of ONOO À than H 2 O 2 , which could be explained by the rapid reaction between elevated NOc and O 2 c À from abnormal TGFb signalling. In the xenogra mice model, an obvious uorescence signal from CyTF was detected, which was 3-fold higher than that from normal tissues. Our study not only highlights the role of ONOO À in keloid pathogenesis, but validates the feasibility of applying optical molecular probes in keloid diagnosis.

Materials
All chemicals and reagents were purchased from Sigma-Aldrich unless otherwise stated. Commercially available reagents were used without further purication unless noted otherwise. Corning Matrigel Matrix was purchased from Corning Life Sciences.
Instrumentation UV-vis spectra were recorded on a Shimadzu UV-2450 spectrophotometer. Fluorescence measurements were carried out on a Fluorolog 3-TCSPC spectrouorometer (Horiba Jobin Yvon). HPLC analyses were performed on an Agilent 1260 system equipped with a G1311B pump, a UV detector and an Agilent Zorbax SB-C18 RP (9.4 Â 250 mm) column, with CH 3 CN (0.1% of TFA) and water (0.1% of TFA) as the eluent. NMR spectra were measured using a Bruker 300 MHz instruments (Germany). All 1 H chemical shis (d) are relative to residual protic solvent (CHCl 3 : d 7.26 ppm). Mass spectra were measured using a Thermo LCQ Fleet LC-MS in ESI mode (America). Fluorescence images of cells were acquired on a Laser Scanning Microscope LSM800 (Zeiss).

Synthesis
Synthesis of compound 2. To a solution of 3-(4-methoxyphenyl)propanoic acid (2.7 g, 15.0 mmol) in ethanol (20 mL) was added H 2 SO 4 (0.7 mL, con.) at room temperature under a nitrogen atmosphere. The resulting mixture was reuxed for 4 h. Then ethanol was evaporated under reduced pressure, and to the residue was added ethyl acetate (60 mL). The resulting solution was washed with H 2 O (3 Â 50 mL), saturated sodium bicarbonate (30 mL) and saturated sodium chloride (30 mL), respectively. The organic phase was dried with anhydrous Na 2 SO 4 , and then concentrated under reduced pressure, which led to ethyl 3-(4-methoxyphenyl)propanoate 2 (2.99 g, yield 96%) without purication. 1  Synthesis of compound 3. To a solution of ethyl 3-(4methoxyphenyl) propanoate 2 (2.91 g, 14.0 mmol) and TMSCF 3 (3.1 mL, 21.0 mmol) was added CsF (21.2 mg, 0.14 mmol). The mixture was stirred at room temperature under a nitrogen atmosphere for 18 h and then CH 2 Cl 2 (30 mL) was added to the resulting solution, and the mixture was treated with water (3 Â 10 mL) and saturated sodium chloride (10 mL). The organic phase was dried with anhydrous Na 2 SO 4 , and then was concentrated under reduced pressure. The residue was puried by silica gel column chromatography (ethyl acetate/hexane, 5%) to afford (2-ethoxy-1,1,1-triuoro-4-(4-methoxyphenyl)butan-2yl)trimethylsilane 3. Synthesis of compound 4. To a solution of compound 3 (4.0 g, 12 mmol) in THF (2 mL) was added TBAF (1 M in THF, 13.0 mL) dropwise under a N 2 atmosphere. The mixture was stirred at room temperature for 5 h and then was treated with hydrochloric acid (4.0 M, 4.0 mL) for 3 h. CH 2 Cl 2 (30 mL) was added to the resulting solution, and then washed with water and saturated sodium chloride, respectively. The organic phase was dried with anhydrous Na 2 SO 4 , and then concentrated under reduced pressure. The residue was puried by silica gel column chromatography (ethyl acetate/hexane, 5%) to obtain triuoro-4-(4-methoxyphenyl)butan-2-one (2.45 g, yield: 88%); 1  Synthesis of compound 5. To a solution of 1,1,1-triuoro-4-(4-methoxyphenyl) butan-2-one 4 (2.4 g, 10.3 mmol) in CH 2 Cl 2 (20 mL) was added BBr 3 (5.2 mL, 13.4 mmol) at À78 C under a nitrogen atmosphere. The resulting mixture was then warmed to room temperature and stirred for 10 h. Then ice-water was added slowly, and to the mixture was added ethyl acetate (50 mL). The resulting solution was washed with H 2 O (3 Â 20 mL), saturated sodium bicarbonate (30 mL) and saturated sodium chloride (30 mL), respectively. The organic phase was dried with anhydrous Na 2 SO 4 , and then concentrated under reduced pressure, which was puried by silica gel column chromatography (ethyl acetate/hexane, 25%) to obtain 1,1,1-triuoro-4-(4hydroxyphenyl)butan-2-one 5 (1.84 g, yield 82%); 1  Synthesis of CyOH. CyOH was synthesized according to a procedure reported in the literature. 25 Synthesis of CyTF. To a stirred solution of BTC (17.8 mg, 0.06 mmol) dissolved in dichloromethane (1 mL) and cooled to 0 C, compound 5 (17.4 mg, 0.08 mol) in dichloromethane (2 mL) was added drop-wise, and then triethylamine (8.0 mg, 0.08 mol) was added. During addition, the temperature was kept at 0 C. Aer the addition was completed, the mixture was allowed to rise to room temperature slowly and stirring was continued for 2 h. Then a solution of CyOH (30 mg, 0.06 mmol) in dichloromethane (2 mL) was added to the above mixture and stirred for 30 min. The resulting mixture was concentrated under vacuum, which was puried by silica gel column chromatography (ethyl acetate/hexane, 10%) to obtain CyTF (12.7 mg, yield 28%). 1  (3 mL) in a 15 mL round bottom ask was stirred at room temperature for 15 min. 4-Bromomethylphenylboronic acid pinacol ester (30 mg, 0.10 mmol) was then added and the reaction mixture was further stirred at room temperature overnight. Aer that, the mixture was washed with water (15 mL) and extracted by using dichloromethane. The dichloromethane layer was dried with anhydrous magnesium sulphate and concentrated under reduced vacuum to yield a crude product. The resulting crude product was puried by a silica gel column (CH 2 Cl 2 /MeOH ¼ 10 : 1) to afford compound CyBA as a blue solid (75%). 1

Cell culture and uorescence imaging
Normal dermal broblasts (NDFs) and keloid-derived broblasts (KFs) were cultured in DMEM (Dulbecco's Modied Eagle Medium) supplemented with heat-inactivated fetal bovine serum (10%) in a humidied environment containing 5% CO 2 and 95% air at 37 C, respectively. Cell media were replaced every three days. For cell uorescence imaging, NDFs and KFs (6 Â 10 4 cells per well in 1 mL) were seeded in the dishes (dia. 35 mm) and incubated overnight to reach 70% conuency. Cells in the negative control groups were treated with NAC alone or NAC followed by TGF-b1 and NAC. Cells in the positive control were treated with TGF-b1. Cells in the control group underwent the same treatment procedure with DMEM (10% FBS, 1% PE). Aer incubation, the medium was removed, and the cells were washed three times with PBS buffer. Then the cells were stained with Hoechst 33342 (NucBlue Live ReadyProbes Reagent, Thermo Fisher) for nuclei and xed with 4% polyformaldehyde solution. Fluorescence images of the live cells were acquired on a Laser Scanning Microscope LSM800 (Zeiss). The excitation and emission wavelengths for cell imaging were 640/655-710 nm for CyTF/CyBA and 405/410-470 nm for Hoechst. ImageJ soware was utilized to remove signal background and quantify cellular uorescence intensity.

Cytotoxicity assay
Normal dermal broblasts (NDFs) and keloid-derived broblasts (KFs) were seeded in 96-well plates (2 at 10 4 cells per well) for 24 h, and then CyTF or CyBA (nal concentration: 5, 10, 15, and 20 mM) was added to the cell culture medium. Cells were incubated with or without (control) CyTF or CyBA, followed by the addition of MTS assay (Promega Cat. no. G3581, 100 mL, 0.1 mg mL À1 ) for 4 h. The absorbance of MTS at 490 nm was measured by using a microplate reader. The cytotoxic effects (VR) of CyTF or CyBA were assessed using the following equation: VR ¼ A/A 0 Â 100%, where A and A 0 are the absorbance of the experimental group and control group, respectively. The assays were performed in ve sets for each concentration.

In Vivo imaging
All animal studies were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of the Nanyang Technological University-Institutional Animal Care and Use Committee (NTU-IACUC) and approved by the Institutional Animal Care and Use Committee (IACUC) for Animal Experiment, Singapore. NDF and KF cell suspensions in DMEMdiluted Matrigel were freshly prepared. Then the mixtures were injected at three positions on the back under the skin of nude 8 week-old female mice with equal volume. CyTF was intravenously injected. Fluorescence imaging was performed using an IVIS imaging system (exposure time: 60 s, IVIS-CT machine, PerkinElmer).

Statistics analysis
The uorescence images were processed with ROI analysis using Living Image 4.0 Soware. The results are expressed as the mean AE standard deviation unless otherwise stated. Statistical comparisons between the two groups were determined by Student's t-test. For all tests, p < 0.05 was considered as statistically signicant. All statistical calculations were performed using GraphPad Prism v.6 (GraphPad Soware Inc., CA, USA).

Conflicts of interest
There are no conicts to declare.