Intraparticle Energy Level Alignment of Semiconducting Polymer

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Intraparticle Energy Level Alignment of Semiconducting Polymer Nanoparticles to Amplify Chemiluminescence for Ultrasensitive In Vivo Imaging of Reactive Oxygen Species Xu Zhen,† Chengwu Zhang,‡ Chen Xie,† Qingqing Miao,† Kah Leong Lim,‡ and Kanyi Pu*,† †

School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457, Singapore National Neuroscience Institute, Singapore 308433, Singapore

‡

S Supporting Information *

ABSTRACT: Detection of reactive oxygen species (ROS), a hallmark of many pathological processes, is imperative to understanding, detection and treatment of many lifethreatening diseases. However, methods capable of realtime in situ imaging of ROS in living animals are still very limited. We herein report the development and optimization of chemiluminescent semiconducting polymer nanoparticles (SPNs) for ultrasensitive in vivo imaging of hydrogen peroxide (H2O2). The chemiluminescence is amplified by adjusting the energy levels between the luminescence reporter and the chemiluminescence substrate to facilitate intermolecular electron transfer in the process of H2O2-activated luminescence. The optimized SPN can emit chemiluminescence with the quantum yield up to 2.30 × 10−2 einsteins/mol and detect H2O2 down to 5 nM, which substantially outperforms the previous probes. Further doping of this SPN with a naphthalocyanine dye creates intraparticle chemiluminescence resonance energy transfer (CRET), leading to the near-infrared (NIR) luminescence responding to H2O2. By virtue of high brightness and ideal NIR optical window, SPN-NIR permits ultrasensitive imaging of H2O2 in the mouse models of peritonitis and neuroinflammation with the minute administration quantity. Thus, this study not only provides a category of optical probes that eliminates the need of external light excitation for imaging of H2O2, but also reveals the underlying principle to enhance the brightness of chemiluminescence systems. KEYWORDS: molecular imaging, chemiluminescence, reactive oxygen species, organic nanoparticles based on boronic-acid-caged firefly luciferin8 and peroxalate nanoparticles,9 respectively. Both approaches eliminate the need for external light excitation, minimizing tissue interference. However, the bioluminescence probe requires the enzyme to trigger the signal and thus is limited to the firefly luciferase expressed transgenic mouse models.10 In contrast, the chemiluminescence design is a nonenzymatic approach that takes advantage of the specific reaction between H2O2 and peroxalates to chemically excite the luminescent reporter.9 However, current chemiluminescence probes mainly rely on small-molecule dyes as the reporters,11−14 which are low emissive and unstable in the presence of highly oxidative ROS such as hypochlorite (ClO−) and hydroxyl radical (•OH).15 As

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eactive oxygen species (ROS) derived from metabolism of oxygen by living organisms play critical roles in both physiological and pathological processes.1 A major ROS generated in living organisms is hydrogen peroxide (H2O2), overproduction of which is closely associated with the onset and progression of many diseases such as cancer, arthritis, chronic obstructive pulmonary diseases and neurodegenerative diseases.2 To decipher the oxidation biology of H2O2 in living organisms, molecular imaging probes including boronated fluorophores,3 fluorescent protein reporters,4 gold nanodots5 and single-wall carbon nanotubes6 have been developed. However, most of them are devised to function in cultured cells, and thus are less effective in imaging of H2O2 in living mammalian animals because of their short excitation wavelengths that can cause strong tissue autofluorescence.3,7 Recent advance in in vivo imaging of H2O2 consists in the bioluminescence design and the chemiluminescence design © 2016 American Chemical Society

Received: May 2, 2016 Accepted: June 14, 2016 Published: June 14, 2016 6400

DOI: 10.1021/acsnano.6b02908 ACS Nano 2016, 10, 6400−6409

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Figure 1. Synthesis and characterization of SPNs. (a) Chemical structures of PFO, PFVA, PFPV, PFBT, PFO-DBT, TCPO and PEG-b-PPG-bPEG used for the preparation of SPNs. (b) Schematic of the preparation of SPNs through nanoprecipitation. (c) Photography of the solutions of SPN-PFO, SPN-PFVA, SPN-PFPV, SPN-PFBT and SPN-PFODBT, respectively. [SPN] = 18 μg/mL (d) Representative TEM image of SPN-PFPV. (e) Representative DLS profiles of SPNs.

reporters to respectively pair with the substrate peroxalate bis(2,4,6-trichlorophenyl) oxalate (TCPO), affording a series of SPNs that selectively emit chemiluminescence in response to H2O2. The optical properties of SPNs were systematically studied and compared to reveal the impact of energy level alignment on chemiluminescence efficiency. To facilitate the in vivo applications, the SPN with the highest chemiluminescence efficiency was doped with a naphthalocyanine dye to induce intraparticle chemiluminescence resonance energy transfer (CRET)29,30 and in turn near-infrared (NIR) luminescence. The sensitive and high chemiluminescence brightness of the SPN ultimately allowed for imaging of H2O2 in various inflammation-related diseases including peritonitis and neuroinflammation in living mice.

ROS are ubiquitous in living animals, small-molecule dyes are likely to undergo ROS-induced bleaching, potentially resulting in the false negative signals for the chemiluminescence probes. Thereby, both luminescence designs have their respective flaws that partially constrain their imaging applications. Semiconducting polymer nanoparticles (SPNs) have emerged as a category of optical nanomaterials for molecular imaging.16−20 As SPNs are transformed from semiconducting polymers (SPs) that are originally synthesized for application of optoelectronic devices, they naturally possess excellent photostability and high brightness.21−23 In addition to a variety of biological applications including cell imaging and tracking,24 targeted tumor imaging,25,26 hemodynamic imaging27 and photoacoustic imaging,18−20,28 we recently revealed that SPNs are inert to ROS and thus can serve as a versatile nanoplatform to develop activatable probes for imaging of ROS in living mice.17 Particularly, chemiluminescent feature was integrated into the SPN to enable simultaneous and differential imaging of peroxynitrite (ONOO−) and hydrogen peroxide (H2O2) for evaluation of drug toxicity.29 However, the mechanism that governs the chemiluminescence process has been overlooked and remains elusive not only for this SPN-based probe but also for other small-molecule dye based probes, which should play a pivotal role in determining sensitivity and define the scope of their imaging applications. We herein report the importance of the energy level alignment between SP and peroxalate in determining the chemiluminescence of SPNs for ultrasensitive detection of H2O2, and demonstrate the proof-of-concept in vivo imaging applications. Five polyfluorene derivatives with different optoelectronic properties were chosen as the luminescent

RESULTS AND DISCUSSION The chemiluminescence SPNs were synthesized via nanoprecipitation. Five polyfluorene-based SP including poly(9,9′dioctylfluorenyl-2,7-diyl) (PFO), poly[(9,9′-dioctyl-2,7-divinylenefluorenylene)-alt-(9,10-anthracene)] (PFVA), poly[(9,9′dioctyl-2,7-divinylene-fluorenylene)-alt-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PFPV), poly[(9,9′-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazol-4,7-diyl)] (PFBT) and poly[2,7-(9,9′-dioctylfluorene)-alt-4,7-bis(thiophen-2-yl)benzo2,1,3-thiadiazole] (PFODBT) (Figure 1a) were chosen to prepare SPNs. A hydrophobic peroxyoxalate, bis(2,4,6-trichlorophenyl) oxalate (TCPO) (Figure 1a), was chosen as a chemiluminescent substrate which could specifically react with H2O2 to generate photons. An amphiphilic triblock copolymer (PEG-b-PPG-b-PEG) was used as the matrix to encapsulate the SP and TCPO meanwhile to endow the SPN with good water6401

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Figure 2. Spectral characterization, stability, sensitivity and specificity of SPNs. (a) UV−visible absorption spectra of SPNs. (b) Fluorescence spectra of SPNs. (c) Chemiluminescence spectra of SPNs. Chemiluminescence was induced by the addition of H2O2 (10 mM). (d) Fluorescence intensities of SPNs at the same mass concentration of SPs (1 μg/mL, 1 mL). Excitation wavelength: 387 nm for SPN-PFO, 440 nm for SPN-PFVA, 452 nm for SPN-PFPV, 455 nm for SPN-PFBT and 536 nm for SPN-PFODBT; Fluorescence intensities of SPNs collected at their respective emission maxima: 436 nm for SPN-PFO, 566 nm for SPN-PFVA, 507 nm for SPN-PFPV, 534 nm for SPN-PFBT and 698 nm for SPN-PFODBT. (e) Chemiluminescence intensities of SPNs (18 μg/mL, 1 mL) detected by luminometer. Chemiluminescence was induced by the addition of H2O2 (10 mM). (f) Representative fluorescence images and chemiluminescence images of SPNs (18 μg/mL, 0.1 mL) in the presence of H2O2 (10 mM). (g) Chemiluminescence response of SPN-PFPV (18 μg/mL, 1 mL) to various ROS at 10 mM. OCl− solution was prepared by directly diluting commercially available NaOCl. •OH were generated by reacting Fe2+ with H2O2. ONOO− stock solution was prepared by directly diluting commercially available NaONOO. 1O2 was produced from the H2O2-molybdate ions (Na2MoO4) system. (h) Chemiluminescence intensities of SPN-PFPV (18 μg/mL, 1 mL) as a function of H2O2 concentration. (i) Chemiluminescence decay of SPN-PFPV (18 μg/mL, 1 mL) in the presence of H2O2 (10 mM). Error bars (d, e, g, h, i), Standard deviation (s.d.) (n = 3 replicates).

solubility and biocompatibility. Coprecipitation of the SP, TCPO and PEG-b-PPG-b-PEG led to the clear nanoparticle solutions (Figure 1c) and the nanoparticles were spherical as indicated by transmission electron microscopy (TEM) (Figure 1d). The hydrodynamic diameters of the SPNs were measured by dynamic light scattering (DLS), showing the similar sizes within the range of 15−25 nm (Figure 1e). The slightly larger size measured by DLS relative to that by TEM (∼10 nm) is due to the shrinkage of nanoparticles in the sample preparation of TEM. No precipitation and change in size were observed for the SPNs after storage for two months, suggesting their excellent stability in aqueous solution (Figure S1, Supporting Information). The optical properties of SPNs were measured in PBS (pH = 7.4). Due to the difference in band gap, the absorption maxima of SPNs (Figure 2a) are differently located at 387 nm (SPNPFO), 440 nm (SPN-PFVA), 452 nm (SPN-PFPV), 455 nm (SPN-PFBT) and 536 nm (SPN-PFODBT), respectively; while their emission maxima (Figure 2b) are at 436 nm (SPN-PFO),

566 nm (SPN-PFVA), 507 nm (SPN-PFPV), 534 nm (SPNPFBT) and 698 nm (SPN-PFODBT), respectively. At the same mass concentration of SPs, SPN-PFPV has the highest fluorescence intensity, which is followed by SPN-PFO, SPNPFODBT, SPN-PFBT and SPN-PFVA (Figure 2b). Due to the difference in the mass absorption coefficients of SPNs, the ranking for the fluorescence quantum yields (QYs) of SPNs are different from that in Figure 2d. The fluorescence QYs are 36.6% (SPN-PFO), 2.56% (SPN-PFVA), 19.2% (SPN-PFPV), 11.1% (SPN-PFBT) and 2.24% (SPN-PFODBT), respectively. In general, the fluorescence quantum yields (QYs) of SPNs are close to those reported in the literatures.21,24 The slight difference should be caused by the presence of the additional component (TCPO) and the different amphiphilic polymer used for the preparation of SPNs, which is also observed in our previous report.29 The chemiluminescence spectra of SPNs were measured by adding excessive H2O2 to the nanoparticle solutions. As shown in Figure 2c, they closely resemble their fluorescence spectra, 6402

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Figure 3. (a) Illustration of the chemically initiated electron exchange luminescence (CIEEL) mechanism of SPNs. (b) The LUMO level of 1,2-dioxentanedione and the HOMO levels of SPs. (c) The fluorescence and chemiluminescence QYs of SPNs as a function of the energy intervals between the LUMO of 1,2-dioxentanedione and the HOMO of SPs. HOMO: −4.4 (PFPV),33 −5.0 (PFVA), −5.4 (PFODBT),34 −5.6 (PFO)35 and −5.9 ev (PFBT);36 the LUMO of 1,2-dioxetanedione is −3.2 ev.32

of H2O2, the chemiluminescence of SPN-PFPV is ∼800-fold higher as compare to that in the presence of other ROS (Figure 2g). This confirms that SPN-PFPV has a high selectivity toward H2O2, which is a general characteristic for peroxalate-based chemiluminescene design.9 Additionally, the chemiluminescence of SPN-PFPV responses linearly to the concentration of H2O2 (Figure 2h), showing the feasibility of quantification. The detection limit for H2O2 is estimated to be ∼5 nM, which is much lower than other fluorescence and chemiluminescence probes.9,11,13 The half-life of chemiluminescence for SPN-PFPV is measured to be ∼80 min and only decreases by two-third even at t = 300 min (Figure 2i). Such a long luminescence should be beneficial for in vivo imaging applications. The underlying mechanism for the different chemiluminescence QYs of SPNs are discussed according to the H2O2/ peroxalate reaction and the frontier molecular orbital theory. The luminescence produced by the reaction between H2O2 and peroxalate is also known as chemically initiated electron exchange luminescence (CIEEL) as shown in Figure 3a.31 The oxidation reaction between the peroxalate compound (TCPO) and H2O2 spontaneously and instantaneously affords the high energy intermediate1,2-dioxetanedione. 1,2-Dioxetanedione first undergoes the reduction reaction by obtaining an electron from the SP, giving rise to the SP radical cation and the carbon dioxide radical anion. Then, the back electron transfer occurs between the cation and the anion to produce the excited SPs, ultimately leading to the luminescence of SP. Accordingly, the key step of the CIEEL that determines the chemiluminescence efficiency of SPNs is the intermolecular electron transfer from the SP to 1,2-dioxetanedione.32 The degree of freedom for such an intermolecular electron transfer process can be qualitatively evaluated according to the energy interval between the highest occupied molecular orbital (HOMO) of SP and the lowest unoccupied molecular orbital (LUMO) of 1,2-dioxetanedione. Among these SPNs, the

conforming that TCPO is in close proximity with SP and is able to chemically excite SP in the presence of H2O2. Besides, their particle sizes remain the same before and after reaction with H2O2 (Figure S2, Supporting Information). According to the absorption efficiencies of SP and TCPO, the doping concentration of TCPO is estimated to be ∼7900 TCPO molecules per nanoparticle for all the SPNs. Although the fluorescence intensities of all the SPNs are in the same order of magnitude (Figure 2d), the chemiluminescence of SPN-PFPV is ∼2−3 orders of magnitude stronger than the other four SPNs (Figure 2e). The substantially stronger chemiluminescence of SPN-PFPV makes it easier to be detected, while other SPNs are not detectable under the same intensity scale bar (Figure 2f and Figure S3, Supporting Information). In addition, the ranking for the chemiluminescence intensities of SPNs is different from that for the fluorescence intensities at the same mass concentration of SPs (Figures 2b vs 2c). The discrepancy indicates that other mechanism is probably involved to govern the chemiluminescence of SPNs. In fact, the chemiluminescence QYs are measured to be 8.16 × 10−5, 7.88 × 10−4, 2.30 × 10−2, 2.18 × 10−5 and 2.07 × 10−4 einsteins/mol for SPN-PFO, SPN-PFVA, SPN-PFPV, SPN-PFBT and SPN-PFODBT, respectively. The chemiluminescence QY of SPN-PFPV is the highest among these SPNs, which is much higher than the small-molecule dye based chemiluminescent nanoparticles (10−3 ∼ 10−4 einsteins/mol)11,13 and luminol (1.29 × 10−2)31 as reported previously. In addition, the UV-absorption and fluorescence spectra of SPN-PFPV show no obvious change after incubation in serum at 37 °C for 24 h (Figure S4, Supporting Information). These results indicate that PFPV is the best luminescent reporter among five SPs and thus SPNPFPV is used as the representative for the following in vitro and in vivo experiments. The selectivity and sensitivity of chemiluminescence of SPNPFPV were further studied in PBS (pH = 7.4). In the presence 6403

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Figure 4. Synthesis and characterization of SPN-NIR. (a) Schematic of SPN-NIR. (b) Chemiluminescence and fluorescence spectra of SPNNIR. Chemiluminescence was induced by the addition of excessive H2O2 (10 mM). (c) Representative chemiluminescence and fluorescence images of SPNs (18 μg/mL, 0.1 mL) in the presence of H2O2 (10 mM). The fluorescence signals were detected at 520 or 780 nm; the chemiluminescence signals were detected with open filter or at 780 nm. (d) In vivo imaging of exogenous H2O2 using SPN-NIR. Representative chemiluminescence image of mouse with the subcutaneous implantation of (1) H2O2 (8 nM) + SPN-NIR (0.1 mg/mL, 0.1 mL) and (2) SPN-NIR (0.1 mg/mL, 0.1 mL). (e) The in vivo chemiluminescence intensities of the subcutaneous inclusion of SPN-NIR as a function of the concentration of H2O2. Values are the mean ± s.d. for n = 3 mice.

plateau at the doping concentration of 2.7 w/w%, probably due to the self-quenching. Thus, this optimal doping concentration (2.7 w/w%) was used for the in vivo imaging application and the corresponding SPN is termed as SPN-NIR. The size and morphology of SPN-NIR are similar to other SPNs (Figure S6, Supporting Information). The energy transfer was studied by comparing the emission spectra of SPN-PFPV, SPN-NIR and NIR775 nanoparticles by excitation of PFPV at 452 nm. Owing to the weak absorption of NIR775 at 452 nm (Figure S5a, Supporting Information), NIR775 nanoparticles show very weak fluorescence (Figure S6d, Supporting Information). In contrast, the fluorescence intensity of SPNNIR at 775 nm is very strong, and its emission peak at 507 nm is obviously lower than that for SPN-PFPV at the same concentration of PFPV (Figure S6d, Supporting Information). These spectral differences indicate that the energy transfer from PFPV to NIR775 occurs to sensitize the NIR emission of NIR775. The chemiluminescence spectrum of SPN-NIR is nearly identical to its fluorescence spectrum (Figure 4b), confirming that the luminescence spectrum is resulted from the CRET from PFPV to NIR775. The fluorescence and chemiluminescence images of SPN-NIR in Figure 4c further illustrate that the efficient luminescence energy transfer occurs to make the optical signals detectable in the NIR region for both fluorescence and chemiluminescence. In addition, SPNNIR has the chemiluminescence QY of 2.12 × 10−2 einsteins/ mol, the chemiluminescence half-life of ∼50 min (Figure S6e, Supporting Information) and good cytocompatibility (Figure S6f, Supporting Information). All these data imply that SPNNIR is promising for in vivo imaging of H2O2.

HOMO of PFPV is the closest to the LUMO of 1,2dioxetanedione (Figure 3b), and thus the intermolecular electron transfer between them is most facilitated, leading to the highest chemiluminescence QY. As shown in Figure 3c, the chemiluminescence QYs of SPNs are proportional to the energy interval between the HOMO of SP and LUMO of 1,2dioxetanedione, while their fluorescence QYs do not comply in such a way. The fact that the ranking for the five SPNs in terms of the chemiluminescence quantum yields does not follow the order of their fluorescence quantum yields indicates that the chemiluminescence efficiencies of SPNs are not mainly determined by their fluorescence QYs; rather, they are more closely related to the HOMOs of SPs. To endow the SPN with the ability to emit light in the NIR optical window with minimized tissue autofluorescence and deep tissue penetration, a NIR dye, silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NIR775), was doped as the energy acceptor in the SPN-PFPV to create the CRET (Figure 4a). The spectral overlap between the emission of PFPV and the absorption of NIR775 exists from 600 to 700 nm (Figure S5, Supporting Information), confirming the feasibility of energy transfer between them. The absorption spectrum of the doped SPN-PFPV has two peaks at 452 and 773 nm, corresponding to PFPV and NIR775, respectively (Figures S5 and S6, Supporting Information). Similarly, the fluorescence spectrum of SPN-NIR shows a new peak at 775 nm from NIR775 in addition to the peak of PFPV at 507 nm. The doping concentration of NIR775 in SPN was optimized (Figure S5, Supporting Information). Along with increasing the doping concentration of NIR775, the absorption peak of NIR775 gradually increases; and the emission peak of NIR775 gradually increases and reaches its 6404

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Figure 5. In vivo imaging of endogenous H2O2 in the mouse model of peritonitis. (a) Representative chemiluminescence and fluorescence images of mice intraperitoneally treated with saline, LPS (8 mg/kg) or LPS (8 mg/kg) with GSH (200 mg/kg), followed by an intraperitoneal injection of SPN-NIR (0.3 mg/mL, 0.2 mL) at t = 4 h later. Chemiluminescence was acquired at t = 1 min after SPN-NIR administration, while fluorescence was acquired at t = 30 min after SPN-NIR administration (n = 3 mice per group). Quantification of chemiluminescence (b) and fluorescence (c) intensities for the in vivo images in (a). Values are the mean ± s.d. for n = 3 mice. *Statistically significant difference in the chemiluminescence intensities between LPS treated and untreated or GSH remediation mice (n = 3, P < 0.05).

The feasibility of SPN-NIR for in vivo imaging of H2O2 was first evaluated by detecting the chemiluminescence of SPN-NIR activated by exogenous H2O2 in living mice. The optical images were acquired after subcutaneous implantation of SPN-NIR solution with or without H2O2 into the dorsal area of anaesthetized nude mice. As shown in Figure 4d and Figure S7 and S8 (Supporting Information), the chemiluminescence intensity of SPN-NIR in the presence of H2O2 (8 nM) is ∼1.5fold higher than SPN-NIR alone. A linear correlation between the H2O2 concentration and the chemiluminescence intensity of SPN-NIR is observed for the tested H2O2 concentrations ranging from 8 nM to 10 μM (Figure 4e). Such a detection window well includes the biologically relevant concentrations of H2O2 in the range from nanomolar to micromolar, suggesting the ability of SPN-NIR to detect the abnormal variation of H2O2 in living mice. According to the previous literatures,9,11−13 few probes can detect the H2O2 in vivo at nanomolar level as SPN-NIR does. In vivo imaging of endogenous H2O2 using SPN-NIR was then tested in living mice. The mouse model of peritonitis was induced by intraperitoneal injection of lipopolysaccharide (LPS), an endotoxin found in the surface of Gram-negative bacteria.37 At t = 4 h after the injection of LPS or saline, SPNNIR (0.3 mg/mL, 0.2 mL) was administered by intraperitoneal injection and chemiluminescence was acquired in 1 min. As shown in Figure 5a,b, the chemiluminescence for LPS-treated mice is 2.5-folder higher as compared to that for the control mice, while a ∼51% signal reduction is observed upon remediation with glutathione (GSH), an antioxidant and nucleophilic scavenger of reactive metabolites and ROS. In contrast, the fluorescence intensities are almost the same for all the groups (Figure 5a,c), reflecting the structure resistance of SPNs toward ROS. The intensity increases in chemiluminescence upon LPS treatment and its attenuation up GSH

remediation clearly prove that SPN-NIR can monitor the variation in the endogenous levels of H2O2 in living animals. The ability of SPN-NIR to detect H2O2 in vivo was further explored in the mouse model of neuroinflammation induced by LPS. Neuroinflammation is associated with neurodegenerative diseases and characterized by the activation of brain glial cells, primarily microglia and astrocytes that release various soluble factors that include ROS, cytokines and lipid metabolites.38 Intracelebral injection of LPS is one of the methods that are widely used to induced neuroinflammation.39 The key challenge in imaging neuroinflammation is the limited volume of the probe that is permitted to be administered via intracerebral injection into mouse brain, which is in the consideration of safety.40 The restriction that only 5 μL solution at most is allowed requires the high brightness and sensitivity of the probe. To image H2O2 in neuroinflammation, SPN-NIR (10 mg/mL, 2 μL) was administered via intracerebral injection at t = 4 h after the injection of LPS or saline. Chemiluminescence images were acquired 1 min after administration of SPN-NIR (Figure 6a). Despite the minute administration quantity of SPN-NIR, the chemiluminescence intensity for the LPS-treated mice is ∼1.7 times higher than that for the saline-treated mice (Figure 6a), which is reduced by 21% upon GSH remediation (Figure 6b). In contrast, the fluorescence intensities remain the same for all the groups (Figure S9, Supporting Information). Given that ROS in the brain is predominantly derived from activated microglia and astrocytes,41 immunofluorescent staining was carried out and the cells specific markers CD11b and GFAP were used to stain to activated microglia and astrocytes, respectively. As compared to the slices of the control mice, a much larger population of activated microglia and astrocytes can be detected for the LPS-treated mice (Figure 6c). This confirms that the microglia and astrocytes are activated by LPS to generate H2O2, leading to the higher in vivo 6405

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Figure 6. In vivo imaging of endogenous H2O2 in the mouse model of neuroinflammation. (a) Representative chemiluminescence images of mice treated with saline, LPS (10 mg/mL, 2 μL) or LPS with GSH (500 mg/mL, 1 μL), followed by intracerebral injection of SPN-NIR (10 mg/mL, 2 μL) at t = 4 h later. Chemiluminescence was acquired at t = 1 min after SPN-NIR administration (n = 3 mice per group). Quantification of chemiluminescence (b) signals calculated from the in vivo images in (a). (c) Mouse brain slice immunofluorescence staining with cells specific markers, GFAP and CD11b, respectively. GFAP were labeled with anti-GFAP and FITC-conjugated antimouse IgG (Green). CD11b were labeled with anti-CD11b and Alexa Fluor 594-conjugated antirat IgG (Red). Values are the mean ± s.d. for n = 3 mice. *Statistically significant difference in the chemiluminescence intensities between LPS treated and untreated or GSH remediation mice (n = 3, P < 0.05).

previously reported chemiluminescence systems including dyedoped peroxalate nanoparticles and luminol. The structural flexibility of SPNs allowed for doping a hydrophobic 2,3-naphthalocyanine dye into the nanoparticle to create intraparticle CRET, resulting in the NIR light emission at 775 nm without comprising the chemiluminescence efficiency. The proof-of-concept application using SPN-NIR for in vivo imaging of ROS was demonstrated in the mouse models of peritonitis and neuroinflammation. By virtue of the high NIR chemiluminescence (2.12 × 10−2 einsteins/mol) and low detection limit (∼8 nM), only a minute quantity administration of the SPN (2 μL) was required to sensitively detect the elevated level of endogenous H2O2 in the mouse brain in real time with no need of external light excitation. These data demonstrate the great potential of our probe for in vivo imaging H2O2 in the neurodegenerantive diseases such as Parkinson’s disease and Alzheimer’s disease. Thus, this study not only introduces an advanced category of organic probes for ultrasensitive imaging of H2O2 in living mice, but also reveals the underlying mechanism to improve the chemiluminescence efficiency, which should be applicable to other organic systems.

chemiluminescence signals (Figure 6a). These data show that SPN-NIR successfully detects H2O2 in mouse brain at the minute administration quantity, which benefits from its high chemiluminescence QY and NIR luminescence. To the best of our knowledge, this is the first example using optical probe to detect H2O2 in mouse brain in real-time.

CONCLUSIONS Five polyfluorene-based SPs with different molecular orbitals were respectively paired with peroxalate and transformed into chemiluminescent SPNs. Systematic investigation of the optical properties of SPNs revealed for the first time the importance of aligning the molecular orbitals between the luminescent reporter (SP) and the chemiluminescence substrate (TCPO) for enhanced chemiluminescence. Rather than the fluorescence brightness of SP, the energy interval between the HOMO of SP and the LUMO of the high-energy intermediate (dioxetanedione) was found to play a major role in determining the H2O2activated chemiluminescence of SPNs. With minimized energy interval, the electron transfer could be facilitated to amplify chemiluminescence. Such a molecular orbital optimization eventually led to a SPN with the chemiluminescene QY up to 2.30 × 10−2 einsteins/mol, which was much higher than the 6406

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METHODS

fphoto =

Chemicals. All chemicals were obtained from Sigma-Aldrich unless otherwise stated. Poly(9,9′-dioctylfluorenyl-2,7-diyl) (PFO) (Mw = 73912 g/mol; Polydispersity = 4.10), poly[(9,9′-dioctyl-2,7-divinylenefluorenylene)-alt-(9,10-anthracene)] (PFVA) (Mw = 53906 g/mol; Polydispersity = 3.45), poly[(9,9′-dioctyl-2,7-divinylene-fluorenylene)alt-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PFPV) (Mw = 75842 g/mol; Polydispersity = 3.62), poly[(9,9′-dioctylfluorenyl-2,7diyl)-alt-(benzo[2,1,3]thiadiazol-4,7-diyl)] (PFBT) (Mw = 11842 g/ mol; Polydispersity = 3.62) and poly[2,7-(9,9′-dioctylfluorene)-alt-4,7bis(thiophen-2-yl)benzo-2,1,3-thiadiazole] (PFODBT) (Mw = 30350 g/mol; Polydispersity = 2.55) were purchased from Luminescence Technology Corp. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) solution were purchased from Promega, USA. Milli-Q water was supplied by Milli-Q Plus System (MilliporeCorporation, Breford, U.S.A.). Instrumentation. Dynamic light scattering (DLS) was performed on the Malvern Nano-ZS Particle Size. TEM images were obtained on a JEM 1400 transmission electron microscope with an accelerating voltage of 100kV. UV−vis spectra were recorded on a Shimadzu UV2450 spectrophotometer. Fluorescence and chemiluminescence spectra were recorded on a Fluorolog 3-TCSPC spectrofluorometer (Horiba JobinYvon). Fluorescence and chemiluminescence images of SPNs solutions were acquired with the IVIS spectrum imaging system. Chemiluminescence intensities of SPNs were recorded using luminometer (Promega, USA). The fluorescence QYs of SPNs were directly measured using a Fluorolog 3-TCSPC spectrofluorometer (Horiba JobinYvon). Synthesis of SPNs. PEG-b-PPG-b-PEG (35 mg), TCPO (6 mg) and SP (0.25 mg) were dissolved into a THF (1.0 mL) solution. The mixture was then rapidly injected into Milli-Q water (9 mL) under continuous sonication at 110 W output with a sonicator bath (Branson) for 2 min. After THF was evaporated with a gentle nitrogen flow, the aqueous solution was filtered through a 0.22 μm PVDF syringe driven filter (Millipore). The formed SPNs suspension was finally concentrated to different concentrations by ultrafiltration and used immediately for experiments. The concentration of SPs and TCPO were quantified through a Shimadzu UV-2450 spectrophotometer. Employing the molar absorption coefficients of TCPO (ε290 = 5.7 × 103 M−1 cm−1) and SPN (ε = ∼4.5 × 107 M−1 cm−1), the doping concentration of TCPO was calculated to be ∼7900 TCPO molecules per nanoparticle. Synthesis of SPN-NIR. PEG-b-PPG-b-PEG (35 mg), TCPO (6 mg), PFPV (0.25 mg) and NIR775 (7 μg) were dissolved into a THF (1.0 mL) solution. The mixture was then rapidly injected into Milli-Q water (9 mL) under continuous sonication at 110 W output with a sonicator bath (Branson) for 2 min. After THF was evaporated with a gentle nitrogen flow, the aqueous solution was filtered through a 0.22 μm PVDF syringe driven filter (Millipore). The formed SPN-NIR suspension was finally concentrated to different concentrations by ultrafiltration and used immediately for experiments. Chemiluminescence QYs of SPNs. The chemiluminescence QYs of SPNs were measured using luminol with H2O2 as oxidant and hemin as catalyst as the standard with a known QY of 1.29 × 10−2 einsteins/mol at pH = 11.6 according to the literature method.30 Briefly, a solution of SPNs (18 μg/mL, 1 mL) was placed in a tube, and then hydrogen peroxide (0.1 M, 100 μL) was added to the solution. Chemiluminescence spectra of the mixture was measured at every 10 s from 10 s after H2O2 addition using luminometer. The chemiluminescence QYs of SPNs were calculated according to the following equations ϕCL =

flum =

Q × flum × fphoto n

(einsteins/mol)

(3)

where ϕCL is the chemiluminescence QYs of SPNs, Q is the total light emission obtained by integration of emission intensity under time curves. f lum is obtained by measuring the emission kinetics of the luminol reaction performed in standard conditions. f photo is obtained from the sensitivity at the emission wavelength (λmax = 431 nm) of the luminol standard, f(λlum), and the emission maximum of the SPNs, f(λs). n is the number of moles of luminol (nlum) or the number of moles of SPs (n). Optical Responses of SPN-PFPV toward Different ROS in Solution. H2O2, ONOO− and OCl− stock solutions were prepared by directly diluting commercially available H2O2, NaONOO and NaOCl, respectively. •OH was generated from Fenton reaction between H2O2 and Fe(ClO4)2. 1O2 was produced from the H2O2-molybdate ions (Na2MoO4) system. Chemiluminescence intensities were measured by luminometer. In Vitro Characterization of SPN-PFPV and SPN-NIR. For the stability measurement, SPN-PFPV (18 μg/mL, 1 mL) or SPN-NIR (18 μg/mL, 1 mL) was placed in a tube. After addition of excess H2O2 (10 mM), the chemiluminescence was continuously acquired using luminometer. Both fluorescence and chemiluminescence imaging were performed using an IVIS spectrum imaging system. Chemiluminescence images were acquired for 30 s with open filter or emission at 780 ± 10 nm, and fluorescence images were acquired for 0.1 s with excitation of 465 ± 10 nm, and emission at both 520 ± 10 and 780 ± 10 nm. Cell Culture and Cytotoxicity Test. HeLa cervical adenocarcinoma epithelial cells were purchased from the American Type Culture Collection (ATCC). HeLa cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium) (GIBCO) supplemented with 10% FBS (fetal bovine serum) (GIBCO) in a humidified environment containing 5% CO2 and 95% air at 37 °C. Cells were seeded in 96 well plates (5000 cells in 200 μL per well) for 24 h, and then SPN-NIR (final concentration 1, 5, 10, and 18 μg/mL) was added to the cell culture medium. Cells were incubated with or without (control) SPNNIR for 24 h, followed by the addition of MTS (100 μL, 0.1 mg/mL) for 4 h. The absorbance of MTS at 490 nm was measured by using a microplate reader. Cell viability was expressed by the ratio of the absorbance of the cells incubated with SPN-NIR solution to that of the cells incubated with culture medium only. In Vivo Chemiluminescence Imaging. All animal studies were performed in compliance with the guidelines set by the Institutional Animal Care and Use Committee (IACUC), Sing Health. Female nude mice were used for in vivo imaging. For subcutaneous in vivo imaging, SPN-NIR (0.1 mg/mL) were mixed with or without H2O2 (8 nM), and the nanoparticle suspension (0.1 mL) was injected subcutaneous into the dorsal of anaesthetized mice (2% isoflurane in oxygen). Chemiluminescence image was captured with a 3 min acquisition time with open filter using the IVIS spectrum imaging system. Fluorescence image was captured with excitation of 465 ± 10 nm, and emission at 780 ± 10 nm using the IVIS spectrum imaging system. For in vivo imaging of endogenous H2O2 in the mouse model of peritonitis, LPS (8 mg/kg) was injected into the peritoneal cavity of mice. For inhibitor study, animals were treated with GSH (200 mg/ kg) intraperitoneally 5 min before LPS treatment. Four hours later, mice were anesthetized using 2% isoflurane in oxygen, and followed by an intraperitoneal injection of SPN-NIR (0.3 mg/mL, 0.2 mL). Chemiluminescence images were captured with a 3 min acquisition time with open filter using the IVIS spectrum imaging system. Fluorescence images were captured with a 0.1 s acquisition time with excitation of 465 ± 10 nm, and emission at 780 ± 10 nm using the IVIS spectrum imaging system. For in vivo imaging of endogenous H2O2 in the mouse model of neuroinflammation, LPS (10 mg/mL, 2 μL) was intracerebral injected into the mice. For inhibitor study, animals were treated with GSH (500 mg/mL, 1 μL) 10 min before LPS treatment. Briefly, after anesthetization with Ketamine and Xylazine, the mouse was mounted

(1)

ϕlum × nlum Q lum

f (λs) f (λlum)

(2) 6407

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ACS Nano on stereotaxic frame (Narishige). A small hole was drilled in the skull with coordinates AP + 0.5 mm to Bregma, ML 2.0 mm right to midline. LPS or saline (as control) was delivered to the striatum with a Hamilton syringe. Once injection is complete, the needle is left in place for 5 more min to allow the pressure of the injected volume to dissipate and prevent backflow along the needle tract. The needle was then slowly retracted. Four hours later, mice were anesthetized, and followed by an intracerebral injection of SPN-NIR (10 mg/mL, 2 μL) in the same way as mentioned above. Chemiluminescence images were captured with a 3 min acquisition time with open filter using the IVIS spectrum imaging system. Fluorescence images were captured with a 0.1 s acquisition time with excitation of 465 ± 10 nm, and emission at 780 ± 10 nm using the IVIS spectrum imaging system. Immunofluorescence Staining. The mice were given a lethal overdose of anesthesia and perfused through the heart with cold saline, followed by 4% paraformaldehyde. Brains were collected, postfixed overnight and transferred to a 15% and then a 30% sucrose solution overnight. The brain was sectioned (20 μm) on CM3050S cryostat (Leica Biosystem Inc., Buffalo Grove, IL, USA). After sectioning, the slices were mounted on the poly-D-lysine coated slides and dry for 1 h to prevent slices dropping off. The slices were washed in PBS with 0.1% Triton-X100 (PBST) and blocked for 1 h in PBST with 5% goat serum. The sections were incubated in ant-GFAP (1:500; Cell Signaling Technology) and anti-CDllb (1:200; Biolegend) at 4 °C overnight. After washing with PBS for 3 times, brain sections were then incubated in secondary antibody, FITC-conjugated antimouse IgG and Alexa Fluor 594-conjungated antirat IgG (1:400; Molecular Probes) for 1 h at room temperature and then washed with PBS. The staining results were observed under confocal microscope (Olympus Fluoview FV1000). Data Analysis. The intensities of fluorescence or chemiluminescence were measured by region of interest (ROI) analysis using IVIS living imaging system. Results were expressed as the mean ± standard deviation unless otherwise stated. Statistical comparisons between two groups were determined by t test, and between 3 or more groups by one-way ANOVA followed by a posthoc Tukey’s HSD test. For all tests, p < 0.05 was considered statistically significant. All statistical calculations were performed using GraphPad Prism v. 5 (GraphPad Software Inc., CA, USA).

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b02908. Figures S1−S6 (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by Nanyang Technological University start-up grant (NTU-SUG: M4081627.120) and Academic Research Fund Tier 1 from Singapore Ministry of Education (M4011559.120, RG133/15). REFERENCES (1) Fransen, M.; Nordgren, M.; Wang, B.; Apanasets, O. Role of Peroxisomes in ROS/RNS-Metabolism: Implications for Human Disease. Biochim. Biophys. Acta, Mol. Basis Dis. 2012, 1822, 1363− 1373. (2) Medzhitov, R. Origin and Physiological Roles of Inflammation. Nature 2008, 454, 428−435. 6408

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