Chemiluminescence of Conjugated Polymers Nanoparticles by Direct

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Chemiluminescence of Conjugated Polymers Nanoparticles by Direct Oxidation with Hypochlorite Beibei Zhu, Wei Tang, Yiqian Ren, and Xinrui Duan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04109 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 27, 2018

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Analytical Chemistry

Chemiluminescence of Conjugated Polymers Nanoparticles by Direct Oxidation with Hypochlorite Beibei Zhu,† Wei Tang,† Yiqian Ren,† Xinrui Duan*, † †

Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province and School of Chemistry and Chemical Engineering, Shaanxi Normal University, 620 Xi Chang’an Street, Xi’an, Shaanxi 710119, People’s Republic of China ABSTRACT: Chemiluminescence (CL) is an advantageous detection tool in vivo imaging due to its high signal-to-noise ratio optical signal readout without the requirement of an external excitation source. Conjugated polymers (CPs) now were used as an energy acceptor in CL nanoparticles to enhance the CL. Here, we demonstrated CL from the direct oxidation of CPs backbones in conjugated polymer nanoparticles (CPNs) by hypochlorite. Such CL CPNs completely avoided the involvement of small molecules CL donors. The strategy greatly simplified the CL probes preparation and increased the stability of CL nanoprobes by overcame the leakage problem of CL donors in nanoparticles. Hypochlorite can oxidize the vinylene bond (C=C) in polyfluorene-vinylene (PFV)/polyphenylene vinylene (PPV) via π2-π2 cycloaddition to form a PFV/PPV-dioxetane intermediate that are unstable and can spontaneously degrade into a PFV/PPV-aldehyde and generate photons. The dioxetane intermediate formation was confirmed by UVVis absorption, fluorescence, nuclear magnetic resonance spectroscopy (1H NMR), and Fourier-transform infrared spectroscopy (FTIR) spectroscopy. The CL quantum yield (QY) of brightest CL probe CPN-Poly[(9,9-di-(2- ethylhexyl)-9H-fluorene-2,7-vinylene)co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)] (90:10 mole ratio) (CPN-PFV-co-MEHPV) was 17.79 einsteins/mol (namely, photons per particle). CPN-PFV-co-MEHPV was size-stable, non-cytotoxic, selective, and sensitive for hypochlorite detection. The linear range and the LOD of CPN-PFV-co-MEHPV for ClO- detection are 2-30 µM and 0.47 µM. Thus, the CPN-PFV-coMEHPV was successfully applied for in vivo imaging of endogenously produced ClO- in the living animals. We expect that the represented strategy could be extended to construct other CL nanoprobes for bioimaging and disease diagnosis by simply optimizing and transforming the CPs backbone and such CL CPNs will have a profound impact in the field of bioimaging.

INTRODUCTION CPs have delocalized electronic structure in their backbone that have efficient coupling between optoelectronic segments. 1 Excitons are efficiently transferred to lower electron/energy acceptor sites along the backbone, which resulted in the superquenching of the fluorescence of CPs or amplification of the signals of acceptors.1-5 CPs have drawn more and more attention based on theirs optical signal amplification and light-harvesting properties for the applications in chemical and biological detections,2,4,6-14 such as DNA sensors, protein assays, cell imaging,15 in vivo imaging2 and photoacoustic imaging.10 CPNs inherit the advantageous properties of CPs and obtain many other virtues at the same time.7,16 They are easy to prepare and separate, have high brightness, high quantum yield, high photostability, and low cytotoxicity but also have the large surface area, small particle size, good in vivo biocompatibility. 11,16-23 For in vitro and vivo fluorescent sensing and imaging applications, an outer light source is needed to excite the CPNs, which may suffer from background interference, penetration limitation, and other problems. CL imaging is a widely applied approach for many biological analysis and disease diagnosis.24,25 Compared to fluorescence detection, the light emission of CL is activated by chemical reactions, and no light source is required to excite the luminescent probe. Thus, CL detection has a high signal-to-noise ratio optical signal readout and also avoid light damage and photobleaching.18 Therefore, CL is widely used as an advantageous detection tool in vivo imaging. Apart from imaging applications based on fluorescent property, CPs can also be used as an en-

ergy acceptor to enhance the CL by CL resonance energy transfer (CRET) for in vivo imaging due to its light-harvesting ability.10,26-28 CRET involves the nonradiative transfer of energy from a CL donor to a suitable acceptor molecule.29-31 The efficiency of CRET is highly distance depended. The CL donor and CP acceptor usually are packed into the hydrophobic core of CPNs. And the hydrophobic environment of the CPNs interior can further increase CL emission and prolong luminescence time. 32-35 Besides hydrophobic interaction, covalent binding of CL donor to the side chain of CPs acceptor is also used to reach a ready energy transfer and avoid the leakage of CL donors or small molecule fluorescent dyes encapsulated in nanoparticles. 36,37 Several CL molecules/systems have been used as donor in CRET nanoparticles, such as luciferase/luciferin,38 peroxalate derivatives/fluorescent dyes,39,40 luminol derivatives/H2O2,28,41 imidazopyrazinone derivatives,36 for various in vivo applications including reactive nitrogen species (RNS) / reactive oxygen species (ROS) imaging, anticancer and antifungal activities, targeted PDT, and lymph-node/tumor mapping. The above-mentioned CL probes are all based on CPs as energy receptors, realized by intermolecular CRET or CRET from non-conjugated side chain to conjugated backbone. It is worth to notice the covalent attachment of CL donor to the side chain of CPs results in a highly sensitive luminous probe.36 Inspired by the above excellent works, we speculate the CL directly emits from the backbone of CPs may open an alternative way to achieve CL emission. Such CL CPNs completely avoided the involvement of small molecules CL donors. The strategy greatly simplified the CL probes preparation and increased the

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stability of CL nanoprobes by overcame the leakage problem of CL donors in nanoparticles. The dioxetane motif, is a key structure in high energy intermediate of several kinds of CL molecules,42-44 can be synthesized by oxidation reaction from C=C bond. After careful examination of the structures of well-developed CPs with excellent optical properties, we speculate that PPV and PFV derivatives should produce CL under oxidization reaction via the generation of dioxetane intermediate. If we packed CPs into nanoparticles in the aqueous solution should separate unstable dioxetane intermediate with solvent molecules and increase the CL emission.27

EXPERIMENTAL SECTION Materials and Reagents Mouse colon carcinoma cell line CT26 WT was purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Dulbecco’s modified Eagle’s medium (DMEM), Poly[[[(2ethylhexyl)oxy]methoxy-1,4-phenylene]-1,2 ethenediyl] (MEH-PPV), Plutonic-F127, Poly(9,9-di-n-dodecylfluorenyl2,7-diyl) (PFD), Poly[(9,9-di-(2- ethylhexyl)-9H-fluorene-2,7vinylene)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)] (90:10 mole ratio) (PFV-co-MEHPV), Poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene] potassium salt solution (MPS-PPV), lipopolysaccharide (LPS), N-acetyl-Lcysteine (NAC), Xanthine Oxidase (XO), 2,2 ′ -Azobis(2methylpropionamidine)dihydrochloride (AAPH), Sodium hypochlorite solution were purchased from Sigma-Aldrich, Shanghai, China. Poly[(9,9-dioctyl-2,7-divinylenefluorenylene)-alt-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PFV-alt-MEHPV) and Poly[(9,9-dioctyl-9H-fluorene-2,7diyl)-(1E)-1,2-ethenediyl] (PFV) were purchased from Luminescence Technology Corp, Taiwan, China. Fetal bovine serum (FBS) was obtained from Hyclone Laboratories Inc. Sodium nitroprusside dehydrate (SNP), Ammonium iron(Ⅱ) sulfate hexahydrate, Sodium molybdate dihydrate were purchased from Aladdin Industrial Inc., Shanghai, China. Tert-Butyl hydroperoxide solution (TBHP), Taurine， Luminol (98%) were purchased from J&K Chemical, Beijing, China. 1 × PBS solution (1 × PBS solution contains 137 mM Sodium Chloride, 2.7 mM Potassium Chloride, 10 mM Phosphate Buffer, and 2 mM Potassium Phosphate), Xanthine (XA) was purchased from Sangon Biotech Co., Ltd., Shanghai, China. Analytical grade chemicals, including NaOH, NaNO2, 30% (v/v) H2O2 were purchased from Tianjin Chemical Reagent Co., Tianjin, China. Tetrahydrofuran (THF), chloroform (CHCl3), dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. Millipore Simplicity 185 purification unit purified water (18.2 MΩ cm) used for rinsing and preparing all aqueous solutions. All reagents were obtained commercially and used without further purification. Apparatus and Characterizations The UV-vis absorption spectra were obtained from a TU-1901 double-beam UV-vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China). The fluorescence spectra were measured using a Perkin Elmer LS-55 fluorescence spectrophotometer (PerkinElmer, Inc, Akron, Ohio, United States). In MTT experiment, the absorbance was measured using a BioTek Epoch Plate Readers (BioTek Instruments, Winooski, VT, USA). The CL measurements were performed on an MCDR-A chemiluminescence analyzer (Xi’an Ruimai Electronic Sci. Tech. Co. Ltd., Xi’an, China). Transmission electron microscope images were

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obtained on the FEI Tecnai G2 F20 system. Dynamic light scattering (DLS) was measured on Brookhaven BI-90 Plus laser particle size analyzer. FT-IR was performed on a Bruker Tensor 27 FT-IR Spectrometer. 1H NMR was performed on Bruker Ascend 600 NMR spectrometer. Rodent CL images were taken using a Bruker In-Vivo Xtreme II imaging system which was equipped with a sensitive charge-coupled device (CCD) camera and a VMR anesthesia machine (Matrx, USA). Molar particle concentration of CPNs can be determined by ZetaView® nanoparticle tracking analysis (Particle Metrix, Inning am Ammersee, Germany). All the experiments were carried out at room temperature unless otherwise specified. Preparation of CPN-PFV-co-MEHPV A THF solution (2 mL) containing PFV-co-MEHPV (0.25 g/L) was filtered through a poly(tetrafluoroethylene) (PTFE) syringe-driven filter (Millipore, 0.45 µm). Then, 1 mL of PFV-co-MEHPV solution was mixed with 1 mL of 20 μg/mL Plutonic-F127 in THF to make a solution that was used to prepare CPN-PFV-co-MEHPV by rapidly injecting into 9 mL of Milli-Q water under continuous sonication for 10 min. After sonication, THF was evaporated at 65°C under nitrogen atmosphere. The aqueous solution was filtered through a poly(ethersulfone) (PES) syringe-driven filter (Millipore, 0.22 µm) and the concentration of CPN-PFV-coMEHPV was adjusted at 50 μg/mL based on the mass extinction coefficient (εω) of PFV-co-MEHPV and stored in dark at 4°C. CPN-PFD, CPN-PFV, CPN-MEHPPV, and CPN-PFV-altMEHPV were prepared in the same procedure. All the mass concentrations of CPNs were based on the repeat unit mass of CPs using mass extinction coefficient (εω) in CHCl3 except MEH-PPV (in THF). Chemiluminescence Measurements CL signals were recorded at a static injection setup (Figure S1). In a typical experiment, 100 μL of 200 μM NaClO solution was injected into 100 μL of 20 μg/mL CPNs-PFV-co-MEHPV (in 1 × PBS, pH 7.4) solution. The voltage of PMT was -1000 V for the CL detection and integration time of the CL analyzer was set at 0.5 s at integration mode. In Vitro Chemiluminescence Imaging of CPN-PFV-coMEHPV CL imaging was performed on the Bruker in vivo imaging system under luminescence modes. Acquisition time was 5 min with an open filter at room temperature (RT). CL images were analyzed by ROI analysis using the Bruker MI SE Software. UV-Vis absorption, Fluorescence, 1H NMR, and FT-IR Spectroscopy of the Chemiluminescence Mechanism of CPNs UV-Vis absorption spectra and fluorescence Spectroscopy of CPN-PFV-co-MEHPV (10 µg/mL), CPN-PFV-altMEHPV (10 µg/mL), CPN-MEHPPV (10 µg/mL), CPN-PFV (10 µg/mL), and CPN-PFD (10 µg/mL) before and after induced by 5 mM NaClO in 1 × PBS buffer (pH = 7.4) at RT for 12 h. A mixed moderate amount THF solution containing PFVco-MEHPV (3 mg), PFV-alt-MEHPV (3 mg), MEH-PPV (3 mg), PFV (3 mg), and PFD (3 mg) was used to oxidative CPs by injecting it into NaClO (3 mmol) under continuous stirring with a magnetic stirring apparatus at RT for 12 h. Remove the aqueous phase from the mixture， the reaction was poured into fresh water (2 mL) and extracted with chloroform and dried in the vacuum. We next characterized the oxidized PFV-coMEHPV, PFV-alt-MEHPV, MEH-PPV, PFV, and PFD by 1H NMR and FT-IR spectra in CDCl3. At the same time, we characterized the PFV-co-MEHPV, PFV-alt-MEHPV, MEH-PPV, PFV, and PFD before induced by NaClO by 1H NMR and FTIR spectra in CDCl3.


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In Vivo Imaging of Inflammation Based CPN-PFV-coMEHPV All animal experiments were carried out with the approval of the university’s institutional animal care and use committee (Shaanxi Normal University). Female BALB/c Mice were obtained from Xi'an Jiaotong University Medical Experimental Animal Center (Xi'an, Shaanxi, China). BALB/c Mice (female) were housed in climate-controlled (24 °C/50-60% humidity) circumstance. Mice were fed with ordinary solid diet and distilled water and were subjected to 12 h light/12 h dark cycles. Health status of mice was monitored daily, and the weight was controlled at least once a week. After a week of acclimatization, mice were used for experimental research. Anesthetize mice under anesthesia with a 0.7 L/min oxygen/isoflurane stream. At the end of the study, the animals were sacrificed by diethyl ether inhalation. For in vivo imaging of externally added hypochlorite solution, female BALB/C mouse under anesthesia was given a subcutaneous injection of CPN-PFV-co-MEHPV (0.05 mg/mL, 0.095 mL) + ClO- (1 mM, 0.005 mL in 1 × PBS) into the dorsal area of anesthetized mice (2% isoflurane in oxygen). For the control experiment, the injection was performed with only CPN-PFVco-MEHPV (0.05 mg/mL, 0.095 mL) + 1 × PBS (0.005 mL) solution without NaClO. After injection, CL images were immediately acquired for 10 min acquisition time with an open filter using the Bruker in vivo imaging system. For in vivo imaging of endogenous NaClO in the mouse model of peritonitis, 250 μL of LPS (2 mg/mL in saline) was injected into the peritoneal cavity of mice. For the control experiment, mice were treated with taurine (200 mg/kg, a scavenger of hypochlorous acid) intraperitoneally 10 min before LPS treatment. For unstimulated mouse, 250 μL of saline was injected. Four hours later, mice were anesthetized by using 2% isoflurane in oxygen and followed by an intraperitoneal injection of CPN-PFV-co-MEHPV (0.05 mg/mL, 1 mL). CL images were captured with a 10 min acquisition time under an open filter by using the Bruker in vivo imaging system. For the NAC control experiment, animals were treated with NAC (400 mg/kg) intraperitoneally 1 h before LPS treatment, the remaining experimental steps are the same as taurine treated group. Imaging was performed by the Bruker in vivo imaging system with the following parameters: 8 × 8 binning, 19 cm field of view; -90 °C CCD working temperature; 300 mL/min anesthesia maintaining oxygen/isoflurane stream. X-ray images were taken with the same imaging system. Fusion of CL image and X-ray image was completed with the MI software equipped by the Bruker in vivo imaging system. The intensities of CL were measured by region of interest (ROI) analysis using the Bruker MI SE Software. Results were expressed as the mean ± standard deviation unless otherwise stated. Statistical comparisons were using student’s t-test. For all tests, p < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION Synthesis and Characterizations of CPNs We used five CPs including PFV-co-MEHPV, PFV-altMEHPV, MEH-PPV, PFV, and PFD to prepare CPNs by the emulsion process method (Figure 1a).7,19,45 All CPs contain C=C bond except for PFD, which was used as a control for next CL experiments. Plutonic-F127 was used to prepare all the CPNs. During the formation of the nanoparticles, the hydrophobic core backbone of Plutonic-F127 embedded into the CPs and

tightly integrated, while the hydrophilic corona of PEG block and functional hydroxyl groups were an affinity to the water environment. Due to the presence of Pluronic-F127, prepared CPNs had good water solubility and biocompatibility. Co-precipitation of the CPs and Plutonic-F127 led to a clear solution (Figure 1b) and exhibited strong fluorescence under UV (365 nm) illumination (Figure 1b). The size of CPNs kept the same even after 2 months of storage (Figure S2), suggesting very good stability in aqueous solution. The morphology of the CPNs was characterized by field emission transmission electron microscopy (FE-TEM) (Figure 1c), the CPNs were mostly spherical and dispersed rather evenly on the grid surface. The hydrodynamic diameters of the CPNs were measured by DLS (Figure 1d), showing the average particle size of CPN-PFD, CPN-PFV, CPN-MEHPPV, CPN-PFV-alt-MEHPV, and CPNPFV-co-MEHPV were 114.33  0.40 nm, 113.60  0.26 nm, 62.52  0.37 nm, 72.57  0.91 nm, and 52.13  0.34 nm, respectively (Figure 1d). The particles size measured by DLS is consistent with FE-TEM.

Figure 1. CPNs synthesis and characterization. (a) Chemical structures of PFV-co-MEHPV, PFV-alt-MEHPV, MEH-PPV, PFV, and PFD used for the preparation of CPNs. Schematic diagram of the preparation of CPNs through emulsion process and CL emission. (b) Photography of the solutions of CPN-PFD, CPN-PFV, CPNMEHPPV, CPN-PFV-alt-MEHPV, and CPN-PFV-co-MEHPV, respectively. left: White light; right: UV light excitation at 365 nm. [CPNs] =50 μg/mL. (c) High-resolution TEM images of CPN-PFD, CPN-PFV, CPN-MEHPPV, CPN-PFV-alt-MEHPV, and CPNPFV-co-MEHPV. (d) DLS size distribution histograms of CPNPFD, CPN-PFV, CPN-MEHPPV, CPN-PFV-alt-MEHPV, and CPN-PFV-co-MEHPV.

Chemiluminescence Response of CPNs to Hypochlorite Since fluorene-containing PFV-co-MEHPV copolymer has an excellent electroluminescence property even better than bright PFV or PPV polymers.46 We systematically studied the CL behaviors of CPN-PFV-co-MEHPV reacted with



physiological relevant oxidants in aqueous solution, the only hypochlorite can generate strong CL signals (detailed results are shown in Figure 4). Next, we speculated that any CPNs with vinylene bond should produce CL under oxidization reaction via the generation of dioxetane intermediate.42-44 CL properties of all CPNs were tested by reacting with hypochlorite. As shown in Figure 2, the CL signal of CPN-PFV-co-MEHPV is much higher than that of CPN-PFV-alt-MEHPV, CPNMEHPPV, and CPN-PFV. After quantification of the CL intensities of all five CPNs by using the Bruker in vivo imaging system, we can observe that the CL of CPN-PFV-co-MEHPV is 1.20, 2.27, and 9.97 times stronger than CPN-PFV-alt-MEHPV, CPN-MEHPPV, and CPN-PFV, while CL signal of CPN-PFD is neglectable at same condition (Figure 2a). Taken together, these results suggest that vinylene bond containing CPNs can produce CL via oxidation reaction, which is in full accordance with our previous assumption. We next investigated the CL kinetics of the CPN-PFV-co-MEHPV, CPN-PFV-alt-MEHPV, CPN-MEHPPV, CPN-PFV, and CPN-PFD at the same concentration after the addition of 100 μM ClO- in 1 × PBS buffer (pH = 7.4) using a CL analyzer (Figure S1), the CL signal of CPNPFV-co-MEHPV was still clearly higher than that of CPNPFV-alt-MEHPV, CPN-MEHPPV, and CPN-PFV (Figure 2b) and the results are consistent with the Bruker in vivo imaging system. The CL emission has a half-life of approximate 3.0 min, 3.2 min, and 2.4 min for CPN-PFV-co-MEHPV, CPN-PFV-altMEHPV, and CPN-MEHPPV respectively, which make them suitable for various in vivo imaging applications. Thus, CPNPFV-co-MEHPV was chosen as the ClO- sensing nanoprobe for the following in vitro and vivo experiments. Furthermore, we next determined the CL response of a water-soluble PPV-based conjugated polymer MPS-PPV for hypochlorite. The CL signal of MPS-PPV (100 µM in repeat units) and CPN-MEHPPV (25 µM in repeat units) were induced by the addition of NaClO (100 μM) in 1 × PBS buffer (pH = 7.4) and measured by using the Bruker in vivo imaging system. Figure S4 shows that we can barely detect the CL of MPS-PPV, but the CL signal of CPNMEHPPV is obvious. The results clearly indicate that MPSPPV cannot generate CL via oxidization. PPVs can only produce CL signals after the formation of nanoparticles, probably due to the nano-reactor effect.27

Figure 2． CL of CPNs. (a) Quantification of CL intensities of CPN-PFD (10 µg/mL), CPN-PFV (10 µg/mL), CPN-MEHPPV (10 µg/mL), CPN-PFV-alt-MEHPV (10 µg/mL), and CPN-PFV-coMEHPV (10 µg/mL) in 1 ×PBS buffer (pH = 7.4). CL was induced by the addition of NaClO (250 μM). The error bars represent the standard deviation (s.d.) (n = 3). Inset: representative CL images (pseudocolor) of CPNs. (b) CL dynamic curves of CPN-PFD (10 µg/mL), CPN-PFV (10 µg/mL), CPN-MEHPPV (10 µg/mL), CPN-PFV-alt-MEHPV (10 µg/mL), and CPN-PFV-co-MEHPV (10 µg/mL) in 1 × PBS buffer (pH = 7.4) with hypochlorite (100 μM). NaClO was injected at 100 s.

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Chemiluminescence Mechanism of CPN-PFV-co-MEHPV We assume that the CL mechanism of CPNs is due to ClOoxidation of vinylene bond in PFV/PPV segments. 47 Thus, we studied the effect of ClO- on the chemical structures of CPs by using UV-Vis absorption, fluorescence spectra, The 1H NMR, and FT-IR, results are shown in Figure 3 and Figure S5-8. Absorption and fluorescence spectroscopy of CPNs were measured in 1 × PBS buffer (pH =7.4). As indicated in Figure 3a,c and Figure S5, S6 (black lines), the absorption peaks of CPNs are located at 440 nm (CPN-PFV-co-MEHPV), 378 nm (CPN-PFD), 430 nm (CPN-PFV)， 512 nm (CPN-MEHPPV), and 450 nm (CPN-PFV-alt-MEHPV) respectively; While their fluorescence emission maxima are at 560 nm (CPN-PFV-coMEHPV), 433 nm (CPN-PFD), 474 nm (CPN-PFV), 592 nm (CPN-MEHPPV), and 506 nm (CPN-PFV-alt-MEHPV), respectively. Photophysical data of CPNs are summarized in Table S1. The absorption peak of CPN-PFV-co-MEHPV at 440 nm disappeared after treatment of ClO- (12 h) (Figure 3a), similar spectral changes were also observed for CPN-PFV-altMEHPV, CPN-MEHPPV, and CPN-PFV at 450 nm, 512 nm, and 430 nm, but not in PFD (Figure S5). The emission peak in the fluorescence spectrum of CPN-PFV-co-MEHPV at 560 nm completely disappeared after the addition of ClO- (12 h) (Figure 3c). similar spectral changes were also observed for CPN-PFValt-MEHPV, CPN-MEHPPV, and CPN-PFV at 506 nm, 592 nm, and 474 nm, but not in CPN-PFD (Figure S6). CPN-PFD still have the same fluorescence emission profile after ClOtreatment. The disappearance of absorption and fluorescence peak of CPN-PFV-co-MEHPV, CPN-PFV-alt-MEHPV, CPNMEHPPV, and CPN-PFV indicates a breakdown in the conjugation length and thus the decomposition of the polymer chain. In contrast, UV-vis absorption and fluorescence spectra of PFD after ClO- treatment suggest the integrity of conjugation structure of PFD still remains. The structure of oxidized PFV-coMEHPV, PFD, PFV, MEH-PPV, and PFV-alt-MEHPV was characterized by 1H NMR and FT-IR (Figure 3b, d, Figure S7, and Figure S8). 1H NMR spectrum of oxidized PFV showed two new peaks at 9.28 and 9.79 p.p.m. (Figure S7b). PFV-coMEHPV (Figure 3b) and PFV-alt-MEHPV (S7d) had two similar new peaks with PFV. These were assigned to the PFValdehyde and PFV-carboxyl peaks, respectively. 1H NMR spectrum of oxidized MEH-PPV showed two new peaks at 9.86 and 10.47 p.p.m. after induced by the NaClO (Figure S7c). These were assigned to the PPV-aldehyde and PPV-carboxyl peaks, respectively. Broadening and division of these peaks are observed, which may come from the different chemical environments and the formation of inhomogeneous fragments. The characteristic peaks of oxidized PFV fragments were also detected in FT-IR at 1750 cm−1. In addition, the peak at 3046 cm−1 corresponding to ethene-1,2-diyl groups, further proving the oxidation of vinylene bonds (Figure S8b). Similar spectral changes were also observed for PFV-co-MEHPV (Figure 3d), PFV-alt-MEHPV (Figure S8d), and MEH-PPV (Figure S8c). As expected, PFD didn’t show obvious changes in both 1 H NMR and FI-IR spectra (Figure S7a, and Figure S8a). These data clearly suggest that hypochlorite oxidizes vinylene bonds in PFV and PPV to break them into inhomogeneously oxidized fragments. Based on this observation, we propose a mechanism of CL of PFV/PPV-based CPNs (Figure 3e). Hypochlorite can oxidize the vinylene bond (C = C) of PFV and PPV via π2-π2 cycloaddition to form a PFV/PPV-dioxetane intermediates, which are unstable42 and can spontaneously degrade into a


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PFV/PPV-aldehyde and generate photons. Further oxidation of the PFV/PPV-aldehyde yields PPV-carboxyl as the final product of the oxidation reaction. Thus, the key step in CL is the hypochlorite-induced formation of PFV/PPV-dioxetanes,

which are depended on the oxidative sensitivity of the vinylene bonds in PFV and PPV. This explains why PPV/PFV-based CPNs have CL property, and PFD is not. Taken together, these data validate the proposed mechanism of CL of CPNs.

Figure 3. Characterization of the CL reaction products of CPN-PFV-co-MEHPV and PFV-co-MEHPV. UV-Vis absorption spectra (a) and fluorescence spectroscopy (λex= 440 nm) (c) of CPN-PFV-co-MEHPV (10 µg/mL) before (black line) and after (red line) induced by 5 mM NaClO in 1 × PBS buffer (pH = 7.4) at RT for 12 h; 1H NMR spectra of PFV-co-MEHPV (b) and FT-IR spectra (d) before (black line) and after (red line) oxidized by excess NaClO in THF; (e) Proposed mechanism for the CL of CPNs.

Selectivity of CPN-PFV-co-MEHPV for Hypochlorite Detection Selectivity is a vital feature of imaging hypochlorite over other biological oxidants that are mainly ROS. The selectivity CL of CPN-PFV-co-MEHPV was studied in 1 × PBS buffer (pH = 7.4) expect 1O2 that was in 0.1 M carbonate buffer of pH 10.5. Figure 4 shows that CPN-PFV-co-MEHPV has good selectivity for hypochlorite over other ROS. ClO- triggered a clear CL signal at 50 µM, while CL signal by other oxidants up to 1 mM were not discernible. Namely, at 50 µM hypochlorite,

CPN-PFV-co-MEHPV gave a CL intensity of 9.8×104 P/sec/cm/sq, which was 29 to 600 times higher than other biological oxidants (Figure 4). In short, these results indicate the high selectivity of the CPN-PFV-co-MEHPV for ClO-. This desirable selectivity may be ascribed to the oxidation-based mechanism. It is reasonable to assume that the weak oxidants could not oxidize CPs and the short-lived ROS produced in aqueous solution could hardly reach CPs that is at the hydrophobic core of CPNs. For example, hypochlorous acid is more oxidative than hydrogen peroxide in physiological pH (pH=7.40). 48,49



Most of the free radicals generally have a short half-life that could only react with reductant in a limited distance.50,51

Figure 4. CL response of CPN-PFV-co-MEHPV (10 μg/mL) to various biological oxidants: (1) PBS, (2) 200 µM •OH, (3) 200 µM •OtBu, (4) 1 mM 1O , (5) 1 mM NO•, (6) 1 mM H O , (7) 1 mM 2 2 2 TBHP, (8) 100 µM ONOO-, (9) 1 mM ROO•, (10) 1 mM O2•-, (11) 50 µM ClO-. The experiment was performed in 1 ×PBS buffer (pH = 7.4) expect 1O2 (in 0.1 M carbonate buffer of pH 10.5). The error bars represent the standard deviation (s.d.) (n = 3). Inset: representative CL images (pseudocolor).

Stability of CPN-PFV-co-MEHPV Such a high selectivity of CPN-PFV-co-MEHPV for ClO- is very suitable for the detection of hypochlorite in vitro and in vivo. Next, we examined the effect of pH on the CL responses of CPN-PFV-co-MEHPV toward ClO-. The data shows that CPN-PFV-co-MEHPV has good stability from pH 5.1 to pH 7.4 (Figure S9). The CL signal of CPN-PFV-co-MEHPV is the strongest at pH = 7.4 (in 1 × PBS buffer), which makes it very suitable for biological systems. Before applying CPN-PFV-coMEHPV for imaging the ClO- in biological systems, we investigated the cytotoxicity and stability of CPN-PFV-co-MEHPV. The cytotoxicity of CPNs were tested by the MTT assays (Figure S10), which show no obvious cytotoxicity up to 30 µg/mL. Results further confirmed the CPNs possessed good biocompatibility, which was vital for any potential biomedical applications. The stabilities of CPN-PFV-co-MEHPV (10 µg/mL), CPN-PFV-alt-MEHPV (10 µg/mL), CPN-MEHPPV (10 µg/mL), CPN-PFV (10 µg/mL), and CPN-PFD (10 µg/mL) before and after upon addition of excessive ClO- (5 mM) in 1 × PBS buffer (pH = 7.4) at 37 °C were test by using DLS. We have previously shown that when the NaClO is added to the CPN-PFV-co-MEHPV in 1 × PBS buffer (pH 7.4), the CL signal is generated immediately and the signal gradually weakens, which indicates the process of hypochlorite oxidation of CPNPFV-co-MEHPV is rapid and its CL is a flash type (Figure 2b). As shown in Figure S11, DLS data of CPNs before and after (10 min) upon addition of ClO- (5mM) demonstrated that the average diameters of CPNs are almost no change. These data suggest that CPNs did not disintegrate in excess of NaClO within ten minutes. Such a high stability of CPN-PFV-coMEHPV is very favorable for the detection of hypochlorite in vitro and in vivo. Next, we examined the stability of CPNs in

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1×PBS buffer (pH = 7.4) and DMEM medium (10% FBS) at 4 ºC in dark for 14 days, DLS data suggests no obvious change in average diameter (Figure S12), indicating that CPNs had sufficient long-term stability for biological imaging and sensing. We also investigated the sensitivity of CL probe for ClO- detection. Figure S3 demonstrates that the CL of CPN-PFV-coMEHPV has a linear correlation with ClO- concentration within the range of 2–30 µM with a LOD of 0.47 μM (3σ, n= 13). The concentration of the ClO- in body fluids is up to 200 µM.52 In diseased airways, its concentration increases even to millimoles.53 Such a limit of detection should be beneficial for in vivo imaging applications. Chemiluminescence Quantum Yield of CPN-PFV-coMEHPV We speculated that the luminous intensity of a single CPNs should significantly higher than single molecule luminophore. Since single CP chain contains at least dozens of luminophore (e.g. repeating units of PPVs or PFVs) and CPNs have more than one CPs chain. Furthermore, the light-harvesting property of tightly packed CPs can efficiently collect excitation energy from unstable high energy intermediates. And CPs in CPNs were separated by solvent molecules. Taken together, highly efficient CL emission should be expected from CPNs. The exact molar particle concentration of CPN-PFV-coMEHPV in aqueous solution was measured by nanoparticle tracking analysis (NTA) for fair comparison of CL QY with small molecule luminophore. For example, the molar particle concentration is 35.37 pM for 10.00 µg/mL CPN-PFV-coMEHPV. The CL QY of CPNs were determined by using the luminol-H2O2 system as a reference on a CL analyzer (Experimental Section and Table S1). The CL QY of CPN-PFV-coMEHPV based on the molar particle concentration is 17.79 einsteins/mol (namely, photons per particle), which is much higher than the single small-molecule luminophores.54,55 The CL QY date of other CPNs based on the molar particle concentration are shown in Table S1. The CL QY of CPN-PFV-co-MEHPV is close to some of the CRET based CL CPNs (Table S2). For fair comparison, CPNs’ CL QY based on the molar concentration of CPs’ repeated units were used in Table S2. The best CRET based CL CPNs are still much brighter due to the involvement of CL donor molecules in a single nanoparticle. However, by completely avoided the involvement of small molecules CL donors, such CL CPNs greatly simplified the CL probes preparation and increased the stability of CL nanoprobes by overcame the leakage problem of CL donors in nanoparticles. Such a bright and stable CL luminophore is very favorable for the detection of hypochlorite in vitro and in vivo. Detection of Exogenous and Endogenous ClO- in Live Mice After confirming the sensitivity, selectivity, biocompatibility, and size-stability of the CPNs and the CL response toward ClOin vitro, we next explored the potential application of CPNPFV-co-MEHPV for imaging ClO- in living mice. First, we performed in vivo imaging of ClO- by detecting the CL of CPNPFV-co-MEHPV by exogenous ClO- in live mice. After subcutaneous injection of a solution of CPN-PFV-co-MEHPV without ClO- (1) or with ClO- (2) into the dorsal area of BALB/C mice, optical images were acquired. As shown in Figure 5a and b, the value of the CL intensity of CPN-PFV-co-MEHPV with ClO- was much higher than that of the CPN-PFV-co-MEHPV with 1 × PBS. Quantification of CL intensities (Figure 5b) for the in vivo images showing that CPN-PFV-co-MEHPV with ClO- (50 uM) is approximately 1.70 times higher than CPN-


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PFV-co-MEHPV alone, suggesting robust CL response of CPN-PFV-co-MEHPV to ClO- in a complex biological environment.

Figure 5. In vivo imaging of exogenous ClO- and endogenous ClOin the mouse model of peritonitis inflammation. (a) In vivo imaging of exogenous ClO- using CPN-PFV-co-MEHPV. Representative CL image (pseudocolor) of mouse with the subcutaneous implantation of (1) CPN-PFV-co-MEHPV (0.05 mg/mL, 0.095 mL) + 1 × PBS (0.005 mL) and (2) CPN-PFV-co-MEHPV (0.05 mg/mL, 0.095 mL) + ClO- (1 mM, 0.005 mL in 1 ×PBS); (b) Histogram of CL intensities for the in vivo images of (a); (c) In vivo imaging of endogenous ClO- production from the peritoneal cavity of the mice with CPN-PFV-co-MEHPV during an LPS-mediated inflammatory response. Representative CL image (pseudocolor) of mice treated with saline (250 µL) (left), LPS (2 mg/mL, 250 µL in saline) (middle) or LPS (2 mg/mL, 250 µL in saline) with taurine (10 mg/mL, 500 µL in saline) (right), followed by intracerebral injection of CPN-PFV-co-MEHPV (0.05 mg/mL, 1 mL) 4 h later. CL images were acquired immediately after the injection of CPN-PFVco-MEHPV; (d) Histogram of CL intensities for the in vivo images of (c). The error bars represent the standard deviation (s.d.) (n = 3) *Statistically significant difference in the CL intensities between LPS treated and untreated or taurine remediation mice (n = 3, P < 0.05).

Subsequently, we examined the possibility of the CPN-PFVco-MEHPV for imaging of endogenously produced ClO - in the living mice. Hypochlorite anion (ClO-)/hypochlorous acid (HOCl) is a biologically significant ROS. Myeloperoxidase (MPO) catalyzes the peroxidation of chloride ions will produce ClO-,56-60 which participates in signal transduction, inflammation, atherosclerosis, neuron degeneration, arthritis, neurodegenerative injury, and cancers.61-63 To this end, we used the CPN-PFV-co-MEHPV to monitor the ClO- levels in a LPS induced acute inflammation. The ClO- was generated by activated macrophages and neutrophils in acute inflammation.57,64,65 The mouse model of peritonitis was induced by intraperitoneal injection of LPS, an endotoxin from the cellular surface of Gramnegative bacteria.66 To verify that the CL response from ClO- in mice, a control experiment was parallelly conducted by using high membrane permeabile ClO- scavenger (taurine)67,68 for CPNs-loaded mice along with LPS stimulation. In addition, another control experiment based on a powerful free-radical scavenger (NAC) was performed as well.57,65,69,70 The BALB/C mice were randomly divided into three groups to obtain reliable results. The mice were anesthetized and the abdominal fur was removed before imaging. The first group was given saline in the peritoneal cavity. The second group was given an i.p. injection of LPS. The

third group was given an i.p. injection of LPS after i.p. injection of taurine 10 min later. All groups were i.p. injection with CPNPFV-co-MEHPV after 4 h. CL imaged was taken immediately after CPN-PFV-co-MEHPV administration. As shown in Figure 5c, the mice injected with both LPS and CPN-PFV-coMEHPV exhibited a significantly higher CL intensity than the mice injected with only CPN-PFV-co-MEHPV. Histogram of the CL intensities from the abdominal area of the mice indicates that the mice loaded with LPS and CPN-PFV-co-MEHPV have approximately 2.4 times higher than the mice loaded with saline and CPN-PFV-co-MEHPV (Figure 5c). The injection of taurine to the mouse led to an obvious decrease of CL intensity upon remediation with taurine (Figure 5c and d). ClO- scavenging experiment suggested the taurine-treated mice had obviously lower CL intensity than that of the second group and no significant difference with the first group. Taken together, CPN-PFVco-MEHPV can be developed as a CL probe for imaging endogenously produced ClO- and the reducing repair of antioxidants in living mice. Results of NAC experiment were shown in Figure S13, the experimental procedure is the same as taurine treated group. As expected, no obvious CL was observed in the first group (Figure S13 a, b). The CL signal of the third group was injected both NAC and LPS is slightly stronger than the first group, but significantly weaker than the second group, indicating that NAC scavenged endogenously produced ClO- from mice and effectively inhibited the oxidation of CPN-PFV-co-MEHPV probe. The result further affirmed that NAC can effectively inhibit the activity of ClO-, thereby relieving peritonitis. These observations have demonstrated the capability of the CPN-PFV-coMEHPV to monitor ClO- in live mice. These results suggest the intensity increases in CL upon LPS treatment and its attenuation upon taurine and NAC remediation clearly prove that CPNPFV-co-MEHPV can monitor the variation in the endogenous levels of ClO- in living animals with high specificity and sensitivity, which suggest that CPN-PFV-co-MEHPV have the great potential for imaging hypochlorite-associated inflammatory diseases.

CONCLUSIONS In summary, we explored the CL ability of PFV/PPV based CPNs via the oxidation reaction of the vinylene bond (C=C) in CPs backbone. Hypochlorite can oxidize the vinylene bond (C=C) of PFV/PPV via π2-π2 cycloaddition to form a PFV/PPVdioxetane intermediate. Further oxidation of the PFV/PPValdehyde yields PFV/PPV-carboxyl as the final product. The CL mechanism of dioxetane intermediate formation via oxidation reaction was confirmed by using UV-Vis absorption, fluorescence spectra, 1H NMR, and FT-IR spectroscopy. CL kinetics study indicates CPN-MEHPPV, CPN-PFV-alt-MEHPV, and CPN-PFV-co-MEHPV a half-life of approximate 2.4 min, 3.2 min, and 3.0 min, respectively. After obtained the molar particle concentration of CPN-PFV-co-MEHPV by NTA, the CL QY of CPN-PFV-co-MEHPV was determined on a CL analyzer. The CL QY is 17.79 einsteins/mol, which is much higher than the small-molecule luminophores such as luminol (1.14 × 10−2 einsteins/mol). Later, we found the CL emission of CPNPFV-co-MEHPV is a highly specific response to hypochlorite over other biological oxidants. The linear range and the LOD of CPN-PFV-co-MEHPV for ClO- detection are 2-30 µM and 0.47 µM. Good stability after ClO- treatment as well as long-term (two weeks) incubation in PBS and cell culture medium were



confirmed by using DLS. No cytotoxicity of CPNs was observed in the MTT assay. After confirming the sensitivity, selectivity, biocompatibility, and size-stability of the CPN and the CL response toward ClO- in vitro, CL of CPN was successfully applied for in vivo imaging of endogenously produced ClO - in the living animals with high specificity and sensitivity. We demonstrated the bright CL of CPN-PFV-co-MEHPV via direct oxidation of CP backbone for in vivo imaging. Due to the excellent intramolecular energy transfer ability of the CPs, CL with a unique mechanism of oxidation of CPs backbone, have a high luminous efficiency and as little as 50 μg of the CPN-PFV-co-MEHPV was required to sensitively detect the elevated level of endogenous ClO- in the mouse abdominal cavity in real time without light excitation. This CPNs strategy decreased the essential synthesis steps of CL sensors. And this work provided a simple, effective, and rapid method in the investigations of the in ClO- status of diseases in living systems. We expect that the design strategy presented herein could be extended to construct other CPs based CL nanoprobes to the use of disease diagnosis and detection through optimization and transformation of CPs structure. We believe the CL CPNs will have a profound impact in the field of bioimaging.

ASSOCIATED CONTENT Supporting Information Experimental details, Figure S1-S13, and Table S1-2 are presented in Electronic Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.xxxxxxx.

AUTHOR INFORMAION Corresponding Author * E-mail: [email protected]

ORCID Xinrui Duan: 0000-0003-3776-6141

Author Contributions B. Z. synthesized and characterized all the CPNs, performed chemiluminescence assay and in vivo imaging. Y. R provided CT26 WT. cell line. W. T. and B. Z. designed in vivo experiments. X.D. designed and coordinated all the experiments. B. Z., W. T., and X. D. wrote the manuscript.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank the National Natural Science Foundation of China (Grant No. 21675108), Shaanxi province innovative talent promotion program (Grant No. 2017KJXX-86), and the Fundamental Research Funds for Central Universities (Grant No. GK201803025) for financial support.

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Chemiluminescence of Conjugated Polymers Nanoparticles by Direct

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