Oxygenic Hybrid Semiconducting Nanoparticles for Enhanced

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Oxygenic Hybrid Semiconducting Nanoparticles for Enhanced Photodynamic Therapy Houjuan Zhu, Jingchao Li, Xiaoying Qi, Peng Chen, and Kanyi Pu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b04759 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 9, 2017

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Oxygenic Hybrid Semiconducting Nanoparticles for Enhanced Photodynamic Therapy Houjuan Zhu,#, † Jingchao Li,#, † Xiaoying Qi,§ Peng Chen,*, † and Kanyi Pu*, † †

School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457, Singapore

§

Singapore Institute of Manufacturing Technology (SIMTech), A*STAR (Agency for Science Technology and Research), 71 Nanyang Drive, Singapore 638075, Singapore

ABSTRACT: Photodynamic nanotheranostics has shown great promise for cancer therapy; however, its therapeutic efficacy is limited due to the hypoxia of tumor microenvironment and the unfavorable bioavailability of existing photodynamic agents. We herein develop hybrid coreshell semiconducting nanoparticles (SPN-Ms) that can undergo O2 evolution in hypoxic solid tumor to promote photodynamic process. Such oxygenic nanoparticles are synthesized through a one-pot surface growth reaction and have a unique multilayer structure cored and coated with semiconducting polymer nanoparticles (SPNs) and manganese dioxide (MnO2) nanosheets, respectively. The SPN core serves as both NIR fluorescence imaging and photodynamic agent, while the MnO2 nanosheets act as a sacrificing component to convert H2O2 to O2 under hypoxic and acidic tumor microenvironment. As compared with the uncoated SPN (SPN-0), the oxygenic nanoparticles (SPN-M1) generate 2.68-fold more 1O2 at hypoxic and acidic conditions under NIR laser irradiation at 808 nm. Due to such an oxygen-evolution property, SPN-M1 can effectively eradicate cancer cells both in vitro and in vivo. Our study thus not only reports an insitu synthetic method to coat organic nanoparticles but also develops a tumor-microenvironmentsensitive theranostic nanoagent to overcome hypoxia for amplified therapy. KEYWORDS: polymer nanoparticles, photosensitizer, photodynamic therapy, cancer

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Phototheranostics combines photodiagnosis and phototherapy and potentially provides a remote-controlled way to treat cancer.1-3 As a result of high temporospatial resolution of light, phototheranostics has the advantages such as minimal invasiveness, low cumulative toxicity, reduced long-term mortality, and high therapeutic selectivity and efficacy.1,

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In particular,

photosensitizers have often been incorporated into near-infrared (NIR) fluorescence systems for imaging-guided photodynamic therapy (PDT).5-7 Upon light irradiation, photodynamic theranostic systems not only can induce NIR fluorescence for identification of ideal therapeutic window, but also can excite photosensitizer to generate cytotoxic reactive oxygen species (ROS) to eradicate cancer cells.8-9 However, there are two major challenges in photodynamic theranostics that hamper its clinical applications. First, PDT inherently requires O2 to convert to ROS, but solid tumors often encounter hypoxia, and thus the therapeutic efficacy of PDT theranostics systems is still limited.10-12 Secondly, conventional organic photosensitizers include porphyrin, phthalocyanines and bacteriochlorin derivatives sometimes suffer from low hydrophilicity and poor photostability, leading to unfavorable bioavailability.13 Therefore, there is an urgent need to develop alternative photodynamic theranostic systems. As an emerging category of organic nanoagents for optical imaging and therapy,14-19 semiconducting polymer nanoparticles (SPNs) have gained more and more attention because of excellent optical properties and good biocompatibility.20 SPNs are made from optically and electronically active semiconducting polymers (SPs) that are organically synthesized for electronic applications.21 The luminescent properties of SPNs have led to fluorescence,22-25 chemiluminescence26-27 and afterglow imaging,28-29 while their high photothermal conversion ability30 has resulted in photoacoustic imaging,6,

14, 31-35

photothermal therapy36-37 and

photothermal control of cellular behaviors.38-39 However, their photodynamic properties of SPNs


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are less exploited and mainly focused on those that can only be excited at visible region with the shallow tissue penetration for antibacterial applications.40-41 In this study, we report the synthesis of near-infrared (NIR) light excitable semiconducting hybrid nanoparticles (SPN-Ms) that can undergo O2 evolution in the tumor microenvironment for enhanced PDT. These oxygenic nanoparticles comprise two key components (Scheme 1): an organic SPN core and an inorganic shell formed by manganese dioxide (MnO2) nanosheets. Poly(cyclopentadithiophene-alt-benzothiadiazole) (PCPDTBT) with a high NIR absorption and a triple energy over 0.98 eV42-43 is used as both NIR fluorescence imaging agent and photosensitizer, while MnO2 nanosheets are able to react with H2O2 in the acidic tumor microenvironment to supply O2 for PDT. It should be noted that although MnO2 nanosheets have been applied as a catalyst for enhanced PDT, most systems are based on inorganic nanoparticles and often utilize visible light excitation.44-46 In contrast, our work utilizes a one-pot straightforward method to in-situ grow the nanosheets from the surface of an organic nanoparticle and this is the first organic-inorganic hybrid MnO2 nanosystem with a core-shell structure for NIR PDT. In the following, the synthesis and characterization of the MnO2-coated SPNs (SPN-Ms) are first described, followed by the study on the coating effect on their optical and physical properties. Then, the photodynamic properties of SPN-Ms are discussed at both neutral and acidic pHs. After validation of in vitro PDT performance of SPN-Ms along with non-coated SPNs, their proof-of-concept application as the new optical nanotheranostics are demonstrated at last in the mouse model of xenograft tumor.


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Scheme 1. (a) Schematic illustration for the H2O2-responsive mechanisms of SPN-M1. (b) The detailed mechanism of SPN-M1 for amplified photodynamic therapy in tumor. The oxygenic nanoparticles with a multilayer structure were synthesized through four steps, as shown in Figure 1b. An amphiphilic triblock copolymer (PS-b-PAA, Figure 1a) were firstly used to coprecipitate with PCPDTBT (Figure 1a) at the weight ratio of 4:1. Such a method led to the formation of nanosized micelles, in which the hydrophobic PS segment of PS-b-PAA and PCPDTBT formed the inner core and the hydrophilic PAA segment of PS-b-PAA constituted the outer arms. Then, a silane coupling agent (MPTMS, Figure 1a) was added in the in the presence of NH3•H2O to cross-link with the polymeric micelles, resulting in the formation of the middle silica layer (the nanoparticles are termed as SPN-Si).47 Subsequently, KMnO4 was introduced to form the third MnO2 shell through an in-situ reduction reaction catalyzed by mercapto groups on the surface of silica at room temperature. At last, another amphiphilic triblock copolymer (PEGb-PPG-b-PEG, Figure 1a) was utilized to coat the nanoparticles via strong hydrophobic interaction, leading to the homogeneous SPN-Ms with good stability under physiological relevant conditions. The synthetic yields in each step were closed to 90 %, which was confirmed by measuring the concentration of PCPDTBT in products. The silica coating was necessary for the synthesis of SPN-Ms, because it protected PCPDTBT from the oxidation by KMnO4 and allowed for the in-situ growth of MnO2 nanosheets on the surface of organic nanoparticles. As


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compared with the reported methods that generally involved extra reducing agent such as 2-(NMorpholino)ethanesulfonic acid (MES) to catalyze the formation of MnO2 nanosheets,48-49 our one-pot synthetic approach was simpler and more controllable. Besides, the mercapto groups on the surface of precursor nanoparticles ensured the in-situ formation and adhesion of MnO2 nanosheets, reducing the probability of forming free MnO2 nanosheets. To optimize the coating amount, SPNs with the different coating amount of MnO2 were prepared and termed as SPN-0, SPN-M0.5, SPN-M1, SPN-M1.5, SPN-M2 and SPN-M2.5, which had the weight percentages of MnO2 of 0, 0.5, 1, 1.5, 2, and 2.5

w/w%,

respectively.

Transmission electron microscopy (TEM) images showed that SPN-0 had a spherical structure with smooth surface and an average diameter of 35 nm (Figure 1c). In contrast, SPN-Ms had a rough surface assembled with MnO2 nanosheets (Figure 1d). Using SPN-M1 as an example, scanning TEM (STEM) clearly showed the nanosheets on the surface of nanoparticles (Figure 1e). The corresponding energy-dispersive X-ray spectroscopy (EDX) (Figure 1h) and elemental mapping images (Figure S1a, Supporting Information) detected the presence of carbon, oxygen, silica, sulfur, and manganese, further confirming the coating of MnO2 nanosheets on the surface of SPN-M1. In addition, X-ray photoelectron spectroscopy (XPS) showed that SPN-M1 had two typical binding-energy peaks at 653.1 and 642.3 eV, corresponding to Mn(IV)2p1/2 and Mn(IV)2p3/2, respectively. This further confirmed the generation of MnO2 nanosheets (Figure S1b, Supporting Information). Both SPN-0 and SPN-M1 nanoparticles were negatively charged with an average zeta potential of -18 and -20 mV, respectively, due to the presence of carboxyl groups of PS-b-PAA (Figure 1g). The effect of MnO2 nanosheets on the optical properties of SPN-Ms was studied. With increased amount of MnO2, the characteristic peak of PCPDTBT within SPN-0 at 650 nm


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remained unchanged, while the absorption peak at 404 nm gradually increased (Figure 2a). Such an increase at 404 nm was caused by the d-d band transitions between the lower energy (3d t2g) and higher energy (3deg) levels of manganese ions in MnO2 crystal lattices. The color of nanoparticle solution was thus changed from cyan to brownish yellow (Figure S2a, Supporting Information). The fluorescence intensity at 840 nm of SPN-Ms decreased with increased coating amount of MnO2 (Figure 2b), which was attributed to the electron transfer process from PCPDTBT to MnO2 nanosheets.


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Figure 1. Synthesis and characterization of SPN-Ms. (a) Chemical structures of PCPDTBT, PSb-PAA, MPTMS and PEG-b-PPG-b-PEG used for the preparation of SPN-Ms. (b) Illustration of the synthesis of the SPN-Ms. Representative TEM images of SPN-0 (c), SPN-M1 (d), and STEM image of SPN-M1 (e). (f) DLS of SPN-0 and SPN-M1. (g) Zeta potential of SPN-0 and SPNM1. (h) EDX patterns of SPN-M1. To study the coating effect on the stability, the average diameters of SPN-Ms were measured by dynamic light scattering (DLS) in deionized (DI) water and PBS. The average diameters of SPN-Ms gradually increased from 40 nm to 76 nm when the weight percentage of MnO2 increased from 0 to 2.5

w/w%

(Figure 2c). No obvious size change was observed for all SPN-Ms

in DI water (Figure S2b, Supporting Information). However, only SPN-Ms with the weight percentages of MnO2 of 0, 0.5, and 1

w/w%

remained stable in PBS (Figure 2c), and a higher

percentage of MnO2 led to rapidly aggregation with an obvious increase in the size from ~140 to 600 nm. Therefore, SPN-M1 with the weight percentages of MnO2 of 1

w/w%

was used for the

following studies. Note that the fluorescence intensity of SPN-0 was 4.2 times of that for SPNM1. To evaluate the degradation behavior of SPN-M1 in the presence of H2O2, we carried out the time-dependent H2O2 assays for SPN-M1 at both neutral and acidic conditions (Figure 2d). The degradation of MnO2 was quantified by the change in absorption. Correspondingly, the kinetics of O2 generation at both pHs was quantified (Figure S3a, Supporting Information) based on the degradation of MnO2 nanosheets (molar absorption coefficient of MnO2 at 380 nm is 9.6 × 103 M-1 cm-1).50-51 The absorbance of MnO2 in SPN-M1 decreased over time at both pH = 7.4 and 6.5 upon addition of H2O2, and the decrease at pH 6.5 was 3-fold faster than that at pH 7.4. By contrast, no change was observed at both pHs in the absence of H2O2. Moreover, the average diameters and zeta potential of SPN-M1 were respectively changed from 55 nm and -20 mV to


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45 nm and -17 mV (Figure S2d, e&f, Supporting Information). These data confirmed the H2O2induced degradation of MnO2 on the surface of SPN-M1. To investigate the effect of MnO2 on the photodynamic properties of SPN-Ms, generation of 1

O2 for SPN-0 and SPN-M1 under NIR laser irradiation (808 nm) and normoxic condition at

both neural (pH = 7.4) and acidic (pH = 6.5) was monitored and quantified by a 1O2 indicator, singlet oxygen sensor green (SOSG) (Figure 2e). Under NIR irradiation, the fluorescence of SOSG remained unchanged in the absence of SPN-Ms with and without addition of H2O2, allowing to attribute the fluorescence changes to the generation of 1O2 by SPN-Ms. However, the fluorescence of SOSG increased over time for both SPN-0 and SPN-M1 with and without addition of H2O2. In particular, in the absence of H2O2, the fluorescence enhancement of SOSG (F/F0) for SPN-M1 and SPN-0 was similar at both neutral and acidic pHs. In contrast, in the presence of H2O2, F/F0 for SPN-M1 was 2-fold higher than that for SPN-0 at pH = 7.4, which was 2.8-fold higher than that for SPN-0 at pH = 6.5. These data indicated the presence of MnO2 coating in SPN-M1 elevated the generation of 1O2 in response to H2O2. Using indocyanine green (ICG, Φ△ = 0.2) as the reference % (Figure S3b, Supporting Information), the 1O2 generation efficiency (Φ△) of SPN-M1 and SPN-0 was measured to be 7.28% and 7.64% , respectively.52-54 To further understand H2O2-regulated photodynamic behaviors, SOSG was used to monitor the generation of 1O2 for SPN-0 and SPN-M1 in response to the addition of H2O2 under hypoxic conditions (Figure 2f). In the absence of H2O2, NIR laser irradiation of SPN-0 and SPN-M1 led to the fluorescence enhancement of SOSG (F/F0) only by 1.4- and 1.2-fold, respectively, at both pHs. This minimal fluorescence increment indicated that 1O2 was not efficiently generated due to the lack of O2. In the presence of H2O2, the fluorescence of SOSG under laser irradiation of SPN-0 remained slightly increased by only 1.3-fold at both pHs; by contrast, it dramatically


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increased by 2.8 (pH = 7.4) and 3.8-fold (pH = 6.5) under laser irradiation of SPN-M1. Thus, different from SPN-0, SPN-M1 was able to generate 1O2 under hypoxic conditions in the presence of H2O2. These data indicated that with the MnO2 coating, SPN-M1 could efficiently generate 1O2 in tumor microenvironment, wherein the exuberant metabolism of cancer cells and insufficient blood supply inside solid tumors resulted in acidification and increased H2O2 level inside tumors.55-56 The underlying mechanism that governed such H2O2- and pH-dependent photodynamic properties is associated with the different oxygen-generated level of MnO2 at neutral and acidic conditions.57-58 At pH = 7.4, MnO2 only acts as a catalyst to promote the disproportionation reaction of H2O2 for producing O2 and H2O. However, at pH = 6.5, MnO2, still serving as a catalyst can be reduced to form water-soluble Mn2+, and H2O2 can be oxidized to generate oxygen. Thus, MnO2 can theoretically produce 2-fold more oxygen under acidic condition than that under neutral condition. In fact, in the presence of H2O2, SPN-M1 generated 2.26 and 1.34-fold more 1O2 at pH 6.5 than that at pH 7.4 under normoxic and hypoxic conditions, respectively. Besides, SPN-M1 generated 2.68-fold more 1O2 as compared with SPN0 at pH = 6.5 under hypoxic conditions. Therefore, SPN-M1 should be a better photodynamic agent than SPN-0 for cancer therapy due to its tumor-microenvironment-responsive photodynamic properties.


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Figure 2. In vitro optical spectral characterization of SPN-Ms. (a) UV-vis absorption (b) and fluorescence spectra excited at 630 nm of SPN-Ms. [SPN] = 5 µg/mL in 1 × PBS (pH 7.4). (c) Size stability of SPN-Ms with different amount of MnO2 for 20 min in water or 1 × PBS (pH = 7.4). (d) Degradation behaviors of SPN-M1 dispersed at pH = 7.4 or 6.5 with or without H2O2 measured by absorption. The generation of 1O2 determined by the increased fluorescence of SOSG, for SPN-0 or SPN-M1 with or without addition of H2O2 under normoxic (e) or hypoxic conditions (f) upon NIR laser irradiation at 808 nm (0.44 W/cm2). [SPN] = 10 µg/mL in 1 × PBS (pH 7.4). The error bars represent the standard deviations (SD) of three separate measurements. To study the cellular uptake efficiency of SPN-Ms, the confocal fluorescence imaging of murine breast cancer 4T1 cells was conducted after incubation of SPN-0 or SPN-M1 (Figure 3a). LysoTraker Green and Hoechst were utilized to stain lysosome and nuclei, respectively. After incubation for 8 h, red fluorescence was detected in the cytoplasm of cells for both SPN-0 and SPN-M1 treated cells, suggesting efficient cellular uptake of both nanoparticles. The overlaid images further showed that the red fluorescence from SPN-Ms overlapped well with the green


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fluorescence from LysoTraker Green, indicating the localization of both SPN-Ms in the lysosomes (the control in Figure S4a, Supporting Information). To examine the photodynamic effect of SPN-Ms on cell death, the cytotoxicity of SPN-Ms was evaluated under NIR laser irradiation. Note that no obvious cytotoxicity of SPN-Ms on 4T1 cells was observed even at a high concentration up to 25 µg/mL under dark (Figure 3b). Under laser irradiation at 808 nm for 5 min, the cell viabilities decreased with increased concentration of both SPN-Ms. It should be noted that the SPN-Ms also generated heat under 808 nm laser irradiation and the photothermal conversion efficiencies were calculated to be 24.5% for SPN-0 and 22.3% for SPN-M1 (Figure S5a&b, Supporting Information). Therefore, the maximum temperature under such laser irradiation was controlled to be less than 43 °C (Figure S2c, Supporting Information), which was the threshold temperature to induce cell apoptosis.59-60 This controlled laser irradiation could minimize the photothermal effect of SPNs. At 25 µg/mL, SPNM1 led to 80 ± 1.6% of cell death, which was 2.6-fold higher than that of SPN-0 (30 ± 1%). The better PDT efficiency of SPN-M1 should be attributed to the higher H2O2 level and acid pH (4.55.0) in cancer cells.58, 61-63 This result was also confirmed by confocal fluorescence imaging (Figure 3c & Figure S4b, Supporting Information), wherein green (calcein AM) and red (PI) fluorescence showed live and dead cells, respectively. Furthermore, the intracellular 1O2 generation of SPN-Ms was detected using a green fluorescent ROS indicator, CM-H2DCFDA. Upon NIR laser irradiation, the fluorescence intensity of CM-H2DCFDA from SPN-M1-treated 4T1 cells was much stronger than that for SPN-0-treated cells (Figure 3d). This data verified that SPN-M1 generated more 1O2 relative to SPN-0, leading to a higher PDT efficacy in vitro.


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Figure 3. In vitro PDT using SPN-Ms. (a) Confocal fluorescence images of 4T1 cancer cells incubated with SPN-0 or SPN-M1 for 24 h, [SPN] = 10 µg/mL. The cells were co-stained by LysoTracker and DAPI. Scar bar: 50 µm. (b) Relative cell viability of 4T1 cells incubated with SPN-0 or SPN-M1 at different concentrations for 48 h without or with laser irradiation at 808 nm (0.44 W/cm2) for 5 min. Error bars were based on SD of three samples per group. (c) Confocal fluorescence images of SPN-incubated 4T1 cancer cells after laser irradiation at 808 nm (0.44 W/cm2) for 5 min, [SPN] = 10 µg/mL. The cells were co-stained by calcein AM and propidium iodide (PI). Scar bar: 200 µm. (d) Confocal fluorescence images of SPN-Ms-incubated 4T1 cancer cells after laser irradiation at 808 nm (0.44 W/cm2) for 5 min [SPN] = 10 µg/mL. The cells were stained by CM-H2DCFDA before imaging. Scar bar: 100 µm.

To validate the tumor-microenvironment response of SPN-Ms, nanoparticles were intratumorally injected into 4T1 tumor bearing mouse and their NIR fluorescence was monitored over time. Due to the quenching effect of MnO2 nanosheets, the fluorescence of SPN-M1-


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injected mice was 21-fold lower than that of SPN-0 at t = 5 min post-injection. However, the fluorescence of SPN-M1-injected mice gradually increased and reached the maximum at 24 h post-injection (Figure 4a&b), while it decreased a bit for SPN-0. At 24 h, the fluorescence intensities for SPN-M1 and SPN-0 injected mice were similar. The fluorescence recovery for SPN-M1 confirmed that the degradation of MnO2 nanosheets in SPN-M1 could be induced by H2O2 (from 50 to 100 µM) in the tumor microenvironment,63-64 which should be similar to in vitro (Figure S5c, Supporting Information). Moreover, this data suggested that O2 should be generated in situ as the downstream product of MnO2 degradation to overcome the challenge of PDT under hypoxic condition. The ability of SPN-Ms for in vivo PDT was performed on xenografted 4T1 tumor mouse models at first 5 min post-injection of nanoparticles. After intratumoral injection of SPN-0, SPNM1 or saline at t = 5 min, the 4T1-tumor bearing mice were irradiated at 808 nm for 5 min in a discontinuous manner, allowing to limit the maximum temperature below 43 °C and thus to minimize the photothermal effect (Figure 4c&d). Note that the body weights of the mice for all groups showed no significant weight loss for 15 days after PDT (Figure 4f), indicating the low toxicity for all treatments. To evaluate the in vivo therapeutic effect of PDT using SPN-Ms, the volumes of tumor were monitored continuously for 15 days after treatment (Figure 4e). Without laser irradiation, the tumor for SPN-treated mice showed the similar growth rate as that for saline-treated-tumor, suggesting no significant therapeutic effect of SPN-M1. Among the PDT groups, the tumor growth of SPN-M1-treated groups was effectively inhibited, which was not observed for SPN-0. Such a therapeutic difference should be attributed to the presence of MnO2 coating in SPN-M1, validating that SPN-M1 could serve as oxygen-generating photodynamic agent for better PDT.


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Figure 4. In vivo NIR fluorescence imaging and PDT of tumor using SPN-Ms. (a) Real-time fluorescence images of 4T1 tumor-bearing mice after intratumoral injection of SPN-M1 and SPN-0 (50 µL, 120 µg/mL). (b) Quantification of fluorescence intensity as a function of postinjection time of SPN-Ms. (c) IR thermal images of 4T1 tumor-bearing mice under laser irradiation at 808 nm (0.3 W/cm2) for 5 min after intratumoral injection of SPN-Ms or saline. (d) Mean tumor temperature as a function of laser irradiation time after intratumoral injection of SPNs or saline. Error bars were based on standard error of mean (SEM) (n = 3). (e) Tumor growth curves of different groups of mice after intratumoral injection of SPN-0, SPN-M1 or saline with and without laser irradiation. Error bars were based on standard error of mean (SEM). (f) Body weight data of mice after intratumoral injection of SPN-0, SPN-M1 and saline with and without laser irradiation. Error bars were based on standard error of mean (SEM) (n = 3) *statistically significant difference in relative tumor volume at day 15 after the treatment with SPN-M1 or SPN-0 under NIR laser irradiation at 808 nm (p < 0.001, n = 3). (g) Histological H&E staining for tumors at day 15 after the treatment with SPN-M1, SPN-0 and saline with and


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without laser irradiation (0.3 W/cm2). All the scale bars are 100 µm. (h) Immunofluorescence caspase-3 staining of tumor slices at day 15 after the treatment with SPN-M1, SPN-0 and saline with and without laser irradiation (0.3 W/cm2). All the scale bars are 50 nm. Green fluorescence indicates the signal from caspase-3 staining, while the blue fluorescence is from the nucleus staining. To further confirm the therapeutic effects of SPN-Ms, tumor tissues were retrieved 15 days after treatments and their histological analysis were performed using hematoxylin and eosin (H&E) staining as well as immunofluorescence caspase-3 staining. With laser irradiation, more cell necrosis in the tumor tissue was noticed for SPN-M1-treated mice as compared with SPN-0treated mice (Figure 4g). In contrast, no typical nucleus dissociation was found for the tumor tissue of SPN-treated mice without laser irradiation. Moreover, immunofluorescence caspase-3 staining images revealed that more fluorescent spots were observed for the tumor tissues of SPNM1-treated mice relative to that of SPN-0-treated mice (Figure 4h), indicating severer apoptosis caused by SPN-M1. It should be noted that both H&E and immunofluorescence caspase-3 staining for saline-treated mice showed no obvious apoptosis and necrosis. Therefore, these histological and immunofluorescence data verified at cellular level that SPN-M1 was a better PDT agent than SPN-0, which was consistent with the in vivo results. In summary, we developed hybrid semiconducting nanoparticles (SPN-Ms) that can react with H2O2 to generate O2 to promote photodynamic process in hypoxic solid tumor. Such oxygenic nanoparticles were synthesized through a one-pot in-situ surface growth reaction and have a unique multilayer structure cored and coated with SPN and MnO2 nanosheets, respectively. The SPN core served as the NIR fluorescence imaging and photodynamic agent, while the MnO2 nanosheets acted as a sacrificing component to convert H2O2 to O2 under hypoxic and acidic tumor microenvironment. As compared with the uncoated SPN (SPN-0), the oxygenic


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nanoparticles (SPN-M1) generated 2.68-fold more 1O2 at hypoxic and acidic conditions under NIR laser irradiation, and thus SPN-M1 was proven to be a better PDT agent for cancer therapy in vitro, in vivo and ex vivo. Our study thus not only reports an in-situ synthetic method to coat organic nanoparticles but also develops a tumor-microenvironment-sensitive theranostic nanoagent to overcome hypoxia for amplified PDT.

ASSOCIATED CONTENT Supporting Information. Experimental details, synthetic procedures of SPN-Si, SPN-Ms, in vitro characterization of nanoparticles including EDX elemental mapping, XPS spectrum, TEM, Zeta potential, DLS, photothermal heating spectra and in vitro fluorescence imaging of 4T1 cells can be found in the Supporting Information Available online. (Figure S1−S5) AUTHOR INFORMATION Corresponding Author *Email: [email protected]; *Email: [email protected]; Author Contributions #

Both authors contributed equally to this work.

Funding Sources Any funds used to support the research of the manuscript should be placed here (per journal style). Notes


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The authors declare no competing financial interest. ACKNOWLEDGMENT K.P. thanks Nanyang Technological University (Start-Up grant: NTU-SUG: M4081627.120) and Singapore Ministry of Education (Academic Research Fund Tier 1: RG133/15 M4011559 and Academic Research Fund Tier 2 MOE2016-T2-1-098) for the financial support. P.C. thanks Singapore Ministry of Education (MOE2014-T2-1-003) for the financial support. REFERENCES 1. Ng, K. K.; Zheng, G., Molecular Interactions in Organic Nanoparticles for Phototheranostic Applications. Chem. Rev. 2015, 115 (19), 11012-42. 2. Tao, W.; Ji, X.; Xu, X.; Islam, M. A.; Li, Z.; Chen, S.; Saw, P. E.; Zhang, H.; Bharwani, Z.; Guo, Z.; Shi, J.; Farokhzad, O. C., Antimonene Quantum Dots: Synthesis and Application as Near-Infrared Photothermal Agents for Effective Cancer Therapy. Angew. Chem. Int. Ed. Engl. 2017, 56 (39), 11896-11900. 3. Liang, C.; Xu, L.; Song, G.; Liu, Z., Emerging nanomedicine approaches fighting tumor metastasis: animal models, metastasis-targeted drug delivery, phototherapy, and immunotherapy. Chem. Soc. Rev. 2016, 45 (22), 6250-6269. 4. Mitsunaga, M.; Ogawa, M.; Kosaka, N.; Rosenblum, L. T.; Choyke, P. L.; Kobayashi, H., Cancer cell-selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules. Nat. Med. 2011, 17 (12), 1685-91. 5. Jang, B.; Park, J. Y.; Tung, C. H.; Kim, I. H.; Choi, Y., Gold nanorod-photosensitizer complex for near-infrared fluorescence imaging and photodynamic/photothermal therapy in vivo. Acs Nano 2011, 5 (2), 1086-94. 6. Liu, K.; Liu, X.; Zeng, Q.; Zhang, Y.; Tu, L.; Liu, T.; Kong, X.; Wang, Y.; Cao, F.; Lambrechts, S. A.; Aalders, M. C.; Zhang, H., Covalently assembled NIR nanoplatform for simultaneous fluorescence imaging and photodynamic therapy of cancer cells. Acs Nano 2012, 6 (5), 4054-62. 7. Zhu, H.; Fang, Y.; Miao, Q.; Qi, X.; Ding, D.; Chen, P.; Pu, K., Regulating Near-Infrared Photodynamic Properties of Semiconducting Polymer Nanotheranostics for Optimized Cancer Therapy. Acs Nano 2017, 11 (9), 8998-9009. 8. Lucky, S. S.; Soo, K. C.; Zhang, Y., Nanoparticles in photodynamic therapy. Chem. Rev. 2015, 115 (4), 1990-2042. 9. Lovell, J. F.; Liu, T. W.; Chen, J.; Zheng, G., Activatable photosensitizers for imaging and therapy. Chem. Rev. 2010, 110 (5), 2839-57. 10. Liu, Y.; Bu, W.; Cheng, C.; Zuo, C.; Xiao, Q.; Sun, Y.; Ni, D.; Zhang, C.; Liu, J.; Shi, J., Hypoxia Induced by Upconversion-Based Photodynamic Therapy: Towards Highly Effective Synergistic Bioreductive Therapy in Tumors. Angew. Chem. Int. Ed. Engl. 2015, 54 (28), 81059. 11. Schulze, A.; Harris, A. L., How cancer metabolism is tuned for proliferation and vulnerable to disruption. Nature 2012, 491 (7424), 364-73.


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TOC for Oxygenic Hybrid Semiconducting Nanoparticles for Enhanced Photodynamic Therapy


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