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Amplified Singlet Oxygen Generation in Semiconductor Polymer Dots for Photodynamic Cancer Therapy Shouying Li,†,§ Kaiwen Chang,† Kai Sun,† Ying Tang,§ Ni Cui,‡ Yu Wang,‡ Weiping Qin,† Hong Xu,*,§ and Changfeng Wu*,† †

State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and Engineering and ‡College of Clinical Medicine, Jilin University, Changchun 130012, China § Department of Gastroenterology, The First Hospital of Jilin University, Changchun 130021, China S Supporting Information *

ABSTRACT: This paper described the energy-transfer amplified singlet oxygen generation in semiconductor polymer dots (Pdots) for in vitro and in vivo photodynamic therapy. Hydrophobic photosensitizer tetraphenylporphyrin was facilely doped in the nanoparticles consisting of densely packed semiconductor polymers. Optical characterizations indicated that the fluorescence of Pdots was completely quenched by the photosensitizer, yielding an energy transfer efficiency of nearly 100% and singlet-oxygen generation quantum yield of ∼50%. We evaluated the cellular uptake, dark toxicity, and photodynamic therapy of the Pdot photosensizer in human gastric adenocarcinoma cells. The in vitro studies indicated that cancer cells were efficiently destroyed at very low dose of the Pdots such as 1 μg/mL by using the light dose of 90 J/cm2, which is considerably less than that in clinical practice. The antitumor effect of the Pdots was further evaluated in vivo with human gastric adenocarcinoma xenografts in Balb/c nude mice, which show that the xenograft tumors were significantly inhibited and eradicated in some cases. Our results indicate the energy transfer amplified Pdot platforms have great therapeutic potential for treating malignant cancers. KEYWORDS: semiconducting polymer, nanoparticle, photosensitizer, energy transfer, photodynamic therapy

1. INTRODUCTION Photodynamic therapy (PDT) is a minimally invasive approach for treatment of various malignant cancers and other diseases.1,2 In a typical PDT process, a photosensitizer upon light excitation generates reactive oxygen species (ROS) such as singlet oxygen,3 which attacks the cells and tissues locally exposed to light with minimal damage to the surrounding tissue.4,5 Photosensitizer plays an essential role in the development and applications of modern PDT treatment. Current photosensitizers are mainly based on porphyrin, chlorin, and their derivatives.6−8 A major drawback for these photosensitizers is their poor solubility in aqueous solution. Because most photosensitizer molecules comprise the highly conjugated ring structures, they are intrinsically hydrophobic and thus difficult to deliver. Design of new photosensitizers and chemical modification of existing ones to achieve water solubility have been explored, which was synthetically complex and time-consuming.9,10 Another issue is that the photosensitizers usually have small absorption cross-section comparable to small molecule dye;11 a sufficient dosing is thus required to yield effective photodynamic action. However, the toxicity of photosensitizers limits the amount of the sensitizer that can be administered. Inadequate selectivity and photo© XXXX American Chemical Society

sensitizer leaching from the delivery carriers can cause severe concerns about the toxicity.12 In fact, photosensitizer drugs used in PDT make patients very sensitive to light for an extended period of time, and special precautions must be taken after the drugs are administered. It would be of great benefit to overcome the solubility issue of hydrophobic photosensitizers and simultaneously minimize their dosage in modern PDT. Recently, nanoparticle-based photosensitizing agents have attracted considerable interest due to the improved properties as compared to single dyes and the capability of nanoparticle bioconjugation with enhanced selectivity toward cancerous tissue.13−16 Nanoparticles consisting of photosensitizer encapsulated in liposome, polymer, and silica matrix been developed in PDT to overcome the solubility issue.17 Alternative strategies involve association of photosensitizer to the surface of luminescent nanoparticles such as quantum dots or upconversion nanoparticles,18−20 resulting in singlet oxygen generation by indirect excitation through energy transfer from the Special Issue: Applied Materials and Interfaces in China Received: August 27, 2015 Accepted: October 16, 2015

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anthracenediyl-bis(methylene)dimalonic acid (ADMA), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), and the solvent tetrahydrofuran (THF, anhydrous, 99.9%) were purchased from SigmaAldrich. 2.2. Preparation of the TPP-Doped PFBT Pdots. The photosensitizer TPP-doped PFBT nanoparticles were prepared using the reprecipitation method as described previously.42 In a typical preparation, the PFBT polymer, functional polymer PSMA, and photosensitizer TPP were dissolved in THF, respectively. The solutions of PFBT, PSMA, and TPP in THF were mixed to make a solution mixture with a PFBT concentration of 100 μg/mL, a PSMA concentration of 20 μg/mL, and a TPP concentration from 0 to 20 μg/mL. The mixture was sonicated to form a homogeneous solution, and a 2 mL quantity of the solution mixture was quickly added to 10 mL of Milli-Q water in a bath sonicator. THF was removed by nitrogen stripping, and solution was filtered through a 0.22 μm filter. The resulting Pdots dispersions are obtained for further characterizations. 2.3. Characterizations of the TPP-Doped PFBT Pdots. The nanoparticle size was characterized by the dynamic light scattering (DLS, Malvern Zetasizer NanoZS). The size and morphology of the nanoparticles were measured by a Hitachi H-600 transmission electron microscopy (TEM). Optical absorption spectra were recorded by a Shimadzu UV-2550 scanning spectrophotometer. Fluorescence spectra were measured by a Hitachi F-4500 fluorescence spectrometer. An organic dye, ADMA, was used to detect singlet oxygen generation from TPP-doped Pdots by monitoring the absorption changes of ADMA. Briefly, the Pdots and ADMA were mixed 4 mL of phosphatebuffered saline (PBS; 0.01 M, pH 7.4), and the resulting mixture contains 20 μg/mL of Pdots was and 10 μg/mL ADMA. The absorption spectra were recorded at 1 min intervals under irradiation at 460 nm (50 mW/cm2). 2.4. Cell Culture and in Vitro Experiments. Human gastric adenocarcinoma cells (SGC-7901) and human gastric mucosal epithelial cells (GES-1) were obtained from the Key Laboratory of Blood Cancer Center at Jilin University. The two types of cells were cultured in Dulbecco modified eagle medium with 10% fetal bovine serum and 100U/mL penicillin/streptomycin. The cells were maintained at 37 °C in humidified environment with 5% CO2. For cellular uptake, SGC-7901 cells were seeded into six-well plate, treated with Pdots at various concentrations for 8 h and washed twice with PBS before imaging. Fluorescence microscopy (Olympus IX 71) and flow cytometry (BD FACS Calibur) were used to characterize the nanoparticle uptake in SGC-7901 cells. The in vitro dark toxicity of Pdots was assessed by a standard MTT assay in SGC-7901 cells and GES-1cells. In a typical MTT assay, 2 × 104 SGC-7901 cells or 1 × 104 GES-1 cells were seeded and cultured for 24 h. The cells were incubated with variable concentrations of TPP-Pdots for 24, 48, and 72 h, respectively. MTT (20 μL, 5 mg/mL) was introduced to the cell culture well and incubated for 4 h. Then, the culture medium was removed, and 150 μL of DMSO was added to each plate well. After the plate rocked for 10 min, the absorbance of each plate well was measured by a Bio-Tek Cytation 3 cell imaging multimode reader. For in vitro PDT study, 2 × 104 SGC-7901 cells were plated and cultured for 24 h. After 8 h of incubation with TPP-Pdots, the cells were irradiated by a blue light-emitting diode (LED) array (460 nm, 100 mW/cm2) for 5, 10, or 15 min, respectively. The cells treated with TPP-Pdots in absence of light irradiation were used as a control. After 16 h, MTT assay was performed according to the above-described procedure. DCFH-DA probe was used to detect singlet oxygen generation from Pdots inside cells. The SGC-7901 cells were seeded to six-well plate and then treated with Pdots at various concentrations for 8 h. Then, the cells were irradiated at 460 nm for 15 min (100 mW/ cm2). After staining with DCFH-DA probe, the cells were viewed by a fluorescence microscopy (Olympus IX71). To further confirm the photodynamic effect, the cells were stained with propidium iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI), AO, and EB, respectively, to observe the changes in cell membrane permeability, nuclear morphology, and cell apoptosis and necrosis after PDT. Analyses of

nanoparticle to photosensitizer. Particularly, there is a great deal of effort in the development of lanthanide upconversion nanoparticles that can absorb deep-penetrating near-infrared light and produce UV−visible light to activate photosensitizers for PDT.21,22 However, these strategies encounter severe limitations such as low absorption cross section of the nanoparticle probes and less efficient energy transfer to the surface photosensitizers. Conjugated polymers have been widely demonstrated for biological applications because their light-harvesting and energy transfer properties can lead to high brightness and signal amplification in fluorescence detection.23−27 Efficient energy transfer in conjugated polymers has been employed for development of a variety of chemsensors and biosensors.27−34 We and other groups developed semiconductor polymer nanoparticles for biological imaging,23−26 and sensitive detection of oxygen,34,35 pH,29 temperature,33 ions,36 reactive oxygen species,26 and biomolecules.37 A dye-doped conjugated polymer platform has recently showed persistent near-infrared luminescence that is promising for in vivo optical imaging.38 Particularly, the energy transfer strategy provides a promising approach for designing multichromophoric photosensitizers for antimicrobial and anticancer applications.39 In these systems, the conjugated polymer efficiently absorbs and transfers the excitation energy to the photosensitizer units, resulting in singlet oxygen generation under both one-photon or twophoton excitations.5,40 Conjugated polymers with large twophoton absorption cross sections can act as light-harvesting materials to significantly enhance the two-photon induced emission and two-photon PDT via energy transfer.41 The energy transfer amplified singlet oxygen generation in conjugated polymer nanoparticles can potentially reduce the photosensitizer dosing for a given light dosage and simultaneously overcome the solubility issue of most hydrophobic photosensitizers. Despite the substantial achievements in the field, clinical PDT applications encounter certain limitations such as lack of an ideal photosensitizer, challenges in formulating photosensitizer, and choosing the right light dosimetry for a complete and effective treatment.17 Conjugated polymer-based energytransfer systems provide new opportunities to address these limitations in clinical PDT.39 In this work, we developed an energy-transfer mediated Pdot platform for in vitro and in vivo PDT studies. Highly fluorescent Pdots were completely quenched by doping a photosensitizer molecule, yielding an energy transfer efficiency of nearly 100% and singlet oxygen generation quantum yield of ∼50%. We evaluated the cellular uptake, dark toxicity, and therapeutic effect of the Pdot photosensizer in human gastric adenocarcinoma cells (SGC7901). In vitro PDT studies indicated that cancer cells were effectively destroyed at very low photosensitizer concentration and light dose that are considerably less than those in literature. In addition, the antitumor effect of the Pdots was evaluated in vivo with human gastric adenocarcinoma xenografts in Balb/c nude mice.

2. EXPERIMENTAL SECTION 2.1. Materials. The conjugated polymer poly[(9,9-dioctylfluorenyl2,7-diyl)-co-(1,4-benzo-{2,1′,3}-thiadazole)] (PFBT) was purchased from ADS Dyes, Inc.. The photosensitizer tetraphenylporphyrin (TPP), poly(styrene-co-maleic anhydride) (PSMA), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), acridine orange (AO), ethidium bromide (EB), 9,10B

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Figure 1. Preparation and characterizations of TPP-doped Pdots. (a) Schematic illustration for the preparation of TPP-doped PFBT Pdots. (b) DLS particle size distribution of TPP-doped PFBT Pdots. (c) Typical TEM image of TPP-doped PFBT Pdots. (d) Normalized absorption and emission spectra of TPP-doped PFBT Pdots. (inset) A fluorescence picture of pure PFBT Pdots and TPP-doped PFBT Pdots under a 365 nm UV-lamp excitation.

energy transfer from the polymer to photosensitizers.45,46 Selection of appropriate conjugated polymer and photosensitizer is crucial for establishing an efficient energy-transfer system for ROS generation. According to Förster theory,47 the energy transfer efficiency is highly dependent on the donor− acceptor distance and their spectral overlap. We choose a highly fluorescent PFBT polymer as donor and TPP as acceptor to prepare Pdots (Figure 1a) because the emission spectrum of PFBT shows a good overlap with the Q bands absorption of TPP molecules (Figure S1). Moreover, the hydrophobicity of both the polymer and the photosensitizer can produce densely packed Pdots and thus the close proximity of donor−acceptor that can greatly facilitate the energy transfer from the PFBT polymer to the TPP photosensitizer compared to conjugated polyelectrolytes.30 The TPP-doped PFBT Pdots were prepared by using a method similar to the re-precipiation as described previously.15 A particle size analysis obtained from dynamic light scattering (DLS) shows that most particles possess diameters in the range of 25 ± 10 nm (Figure 1b). The Pdot dispersions were further drop-cast onto copper grid for analysis of particle size and morphology by TEM. A representative TEM image of the Pdots is shown in Figure 1c, indicating spherical shape with a particle diameter that is comparable to the DLS results. The TPP doping showed little effect on the particle size for the doping concentration less than 20 wt %. For these doping concentrations, the majority of the TPP molecules were doped inside the Pdots. A small portion of the TPP molecules might form aggregates and can be removed by a membrane filter (∼0.2 μm). UV−vis absorption measurements indicate the resulting TPP-Pdots were consistent with the initial doping concentration. However, dye-doping at a few percent fraction can significantly quench the donor’s fluorescence in the hydrophobic Pdots due to the combined effects of energy diffusion and energy transfer.15 Figure 1d shows the absorption and emission spectra of TPP-doped PFBT Pdots at 10 wt % doping fraction. As expected, the doped Pdots present a red fluorescence (∼650 nm) from the TPP dopant alone, while the fluorescence from PFBT is completely quenched. The Pdots show a dominant absorption in the range of 400−500 nm, which can be a limited factor for treatment of deep-seated

variance (ANOVA) and Student’s t-tests were used for MTT assays, and statistical significance was reported for p < 0.05. 2.5. In Vivo Photodynamic Therapy. All animal experiments were conducted in agreement with all relevant guidelines and regulations set by Jilin University and with approved institutional protocols set by the China Association of Laboratory Animal Care. BALB/c-nu mice were purchased from Beijing HFK Bioscience Co, Ltd. Female or male Balb/c-nu mice (18−20g), aged five to six weeks, were subcutaneously injected with 2 × 106 SGC-7901 cells in left armpit. The mice were maintained for 10 d with regular food and water, until the tumor volumes approached 55−100 mm3. For wholeanimal biophotonic imaging and in vivo PDT, the tumor-bearing nude mice were intravenously or intratumorally injected with TPP-Pdots (2 mg/kg). The mice without Pdot administration were used as controls. After 24 h, the mice were then anesthetized for whole-animal fluorescence imaging. Finally, tumor masses and major organs (heart, liver, spleen, kidney, lung, and heart) were collected for imaging and biodistribution analysis by a custom-built animal imaging system. For in vivo PDT, tumor-bearing mice were divided into three experimental groups, which are the control group (without any treatment), intravenous injection group (intravenously injected 2 mg/ kg TPP-Pdots), and intratumoral injection group (intratumorally injected 2 mg/kg TPP-Pdots), with five mice in each group. After the TPP-Pdot administration (24 h) , the mice were anesthetized by intraperitoneal injection of 0.2 mg/kg chloral hydrate. The tumors were then irradiated with the LED array (100 mW/cm2) for 30 min. The same dose of light irradiation was repeated again after 7 d. Tumor volumes were measured weekly, and volume (mm3) of tumors was calculated as (tumor length) × (tumor width)2/2. Relative tumor volume was calculated as V/V0 (V0 was the corresponding tumor volume when the treatment was initiated). The mice were euthanized, and tumor masses were collected after 28 d. Then tumor and major organs (heart, liver, spleen, kidney, lung, and heart) were collected, fixed in 10% neutral buffered formalin, embedded in paraffin, cut into 4 μm sections, and stained with hematoxylin and eosin (H&E). The histological tissue sections were observed by optical microscope.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterizations of TPP-Doped Pdots. Pristine conjugated polymers upon light excitation can directly sensitize oxygen molecules to produce ROS for antimicrobial and anticancer applications.43,44 However, the ROS generation yield of pure conjugated polymer is generally low. Incorporation of photosensitizer into the Pdots can significantly improve the ROS generation efficiency due to C

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Figure 2. Fluorescence spectroscopy and singlet oxygen generation of TPP-doped Pdots. (a) Concentration-dependent absorption spectra of TPPdoped PFBT Pdots. (b) Concentration-dependent fluorescence spectra of TPP-doped PFBT Pdots. (c) Fluorescence intensity change of the PFBT green emission and TPP red emission as a function of the TPP concentration in the doped Pdots. (d) Absorption intensity of ADMA as a function of irradiation time in the presence of TPP-doped Pdots. (e) Absorption intensity of ADMA as a function of irradiation time in absence of TPP-doped Pdots. (f) ADMA depletion by 1O2 at 259 nm in phosphate buffer with TPP-PFBT Pdots (▲), PFBT Pdots (●), RB (▼), and ADMA alone (■) under the same light irradiation.

oxygen and thus can be used as an indicator for 1O2 generation. Figure 2d,e shows the absorption intensity of ADMA as a function of irradiation time in presence and absence of TPPdoped Pdots, respectively. In the former case, the prompt drop in optical density of ADMA revealed efficient 1O2 generation from the TPP-Pdots. In absence of the Pdots, the ADMA shows little change, confirming the bleaching is induced by singlet oxygen and not by the irradiation light. In another control, the pure Pdots under the same irradiation conditions resulted in much less photodestruction as compared to the TPP-Pdots, further indicating the low 1O2 generation in pure Pdots but the significantly enhanced 1O2 generation by TPP doping. We further estimate the singlet oxygen quantum yield by a spectrophotometric method as described in a previous report.5 In this method, the 1O2 quantum yield is known for a standard such as Rose Bengal (Φ(1O2) = 0.76)51 and can be determined for Pdots by comparing the slopes of the ADMA bleaching kinetics (Figure 2f). The 1O2 quantum yield for the TPP-doped PFBT Pdots is determined to be ∼0.5, slightly lower than that of RB molecule and comparable to those of porphyrin molecules and other good photosensitizers.51 The pure PFBT Pdots were estimated to have an 1O2 quantum yield of ∼0.1, which is similar to other undoped conjugated polymer nanoparticles and conjugated polyelectrolytes.5,52 For comparison, TPP molecules were doped into an optically inert matrix such as PSMA nanoparticles. The amplified effect by conjugated polymer can be estimated by comparing the absorption cross section and singlet oxygen quantum yield of TPP-Pdots to those of TPP-PSMA nanoparticles. On the basis of our measurements, the TPP-Pdots presented at least 10-fold enhancement in singlet oxygen generation as compared to pristine TPP or TPP-doped PSMA nanoparticles. Taken together, these results indicated that TPP-doped semiconductor Pdots greatly amplified singlet oxygen generation due to efficient energy transfer from the polymer to the TPP molecules. 3.2. In Vitro Cellular Imaging and Photodynamic Study. Besides the photodynamic effect, it is greatly beneficial

tumors. However, there is a great need for treating superficial tumors such as skin, esophagus, bladder, and so on.48−50 PDT in the blue-green wavelength region is still very useful for treating the superficial tumors.1 We investigated spectroscopic changes versus dopant concentration to find the optimal composition for subsequent PDT experiments. Figure 2a,b shows the TPP-dependent absorption and emission spectra of the composite Pdots, respectively. The absorption spectra of the doped Pdots are consistent with the broad-band absorption of the PFBT polymer, overlaid with the sharp peak originates from the TPP molecule. Highly efficient energy transfer is clearly indicated by the decreased fluorescence intensity with increasing the TPP concentration. The fluorescence lifetime of the PFBT donor was measured by a time-correlated single photon counting system (Supporting Figure S2), which indicated the lifetime of PFBT was significantly decreased as the TPP concentration increased. The PFBT polymer is completely quenched as the TPP content is increased to 5 wt %, indicating the energy transfer efficiency approaches unity at this concentration. The fluorescence from TPP also reaches a maximum at ∼5 wt %, after which a slight reduction in fluorescence intensity was observed due to the formation of minor aggregation species. On the basis of the spectroscopic feature, we conclude that the TPP molecules are likely uniformly distributed in the Pdot host, without significant segregation for the doping fraction of 5 wt %. The lack of significant TPP aggregation and highly efficient energy transfer are important for both the TPP fluorescence and amplified ROS generation in the doped Pdots. At this doping concentration, the fluorescence quantum yield (∼650 nm) of the TPP-PFBT Pdots was determined to be ∼4%, comparable to that of the TPP molecules in other conjugated polymer nanoparticles.5 The fluorescence function is desirable for monitoring nanoparticle uptake and tumor imaging. The singlet oxygen generation from TPP-Pdots was evaluated by monitoring the absorption of the ADMA probe. The ADMA dye was bleached by oxidation reaction with singlet D

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Figure 3. (a) Fluorescence imaging of SGC-7901 cells incubated with variable concentrations of TPP-Pdots for 8 h. (b) Flow cytometry results of SGC-7901 cells treated with TPP-Pdots at the same concentrations as those in (a). (c) Cellular uptake efficiency of SGC-7901 cells determined by the flow cytometry.

Figure 4. In vitro dark toxicity and photodynamic effect of TPP-doped Pdots. (a) Cell viability of SGC-7901 cells incubated with TPP-doped PFBT Pdots at variable concentrations for 24, 48, and 72 h. (b) Cell viability of GES-1 cells incubated with TPP-doped PFBT Pdots at variable concentrations for 24, 48, and 72 h. (c) Photodynamic effect on SGC-7901 cells treated with TPP-Pdots and immediately irradiated with light without preincubation time. (d) Photodynamic effect on SGC-7901 cells treated with TPP-Pdots and incubated for 8 h before light irradiation.

Pdots in SGC-7901 cells by fluorescence imaging and flow cytometry. Figure 3a shows fluorescence imaging of the cells incubated with TPP-Pdots at variable concentrations from 0 to 4 μg/mL. After 8 h of incubation, intracellular fluorescence was

for photosensitizer to have fluorescence that can be used to monitor nanoparticle uptake and tumor distribution by fluorescence imaging. As the TPP-Pdots show a relatively strong red fluorescence, we evaluate the cellular uptake of the E

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Figure 5. Photodynamic toxicity and cell staining. Fluorescence images of SGC-7901 cells incubated with TPP-doped Pdots (0, 0.5, and 1 μg/mL), treated with light irradiation, and further stained by different dyes. (a) Intracellular singlet oxygen level indicated by DCFH-DA staining. (b) Dead cells indicated by PI nuclear staining. (c) DNA damage indicated by DAPI nuclear staining. (d) Apoptotic and necrotic cells indicated by AO&EB staining.

determined to be limited to a spherical radius of ∼100 nm in cellular environment.53 Therefore, efficient nanoparticle uptake is essential for intracellular drug delivery and therapy, which ensure the TPP-Pdots to execute their therapeutic functions. Negligible dark toxicity is required for a clinically useful photosensitizer. The dark toxicity of TPP-Pdots was evaluated by using MTT assay. Figure 4a,b shows the cell viability results of the TPP-Pdot-treated SGC-7901 cells and GES-1 cells, respectively. The two types of cells were incubated with TPPPdots at variable concentrations (1−4 μg/mL) for designated incubation times (24−72 h). As indicated from the cell viability results, the TPP-Pdots did not induce noticeable cytotoxicity as

clearly observed for cells with Pdot incubation as compared with control in absence of TPP-Pdots. The cells show concentration-dependent uptake, as indicated by the brighter cellular fluorescence at higher Pdot concentration. We used flow cytometry to quantify the cellular uptake in the SGC-7901 cells and the percentage of the labeled cell by TPP-Pdots (Figure 3b). The SGC-7901 cells show brighter fluorescence and higher uptake efficiency as the Pdot concentration is increased. For the cells treated with TPP-Pdots at 4 μg/mL, the uptake efficiency was determined to be ∼95% (Figure 3c), indicating the Pdot internalization is highly efficient in SGC7901 cells. The diffusion range of singlet oxygen was F

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Figure 6. In vivo fluorescence imaging by TPP-doped Pdots. (a) Bright-field and fluorescence images of whole-animal imaging for tumor-bearing mice with intravenous injection (upper) or intratumoral injection of TPP-Pdots (lower). (b) Ex vivo bright-field and fluorescence images of different mouse organs including tumor, liver, spleen, kidney, lung, heart. The upper panel showed the control group without Pdot administration, the middle panel showed the organs collected from mice with intravenous injection, and the lower panel showed the organs collected from mice with intratumoral injection. I. V. = intravenous injection, I. T. = intratumoral injection.

moderate decreases for the cells treated with the TPP-Pdots and light illumination immediately. However, when the cells were preincubated with TPP-Pdots for 8 h and then illuminated with light the cell viability showed apparent decreases (Figure 4d), indicating significant photodynamic effect induced by the TPP-Pdots. The photodynamic killing of cancer cells was dependent on the preincubation time, irradiation time (light dose), and Pdot concentration. As indicated in Figure S3, light irradiation alone did not induce any phototoxicity for the light dose used in our experiment (⩽90 J/cm2). With the same irradiation time and Pdot concentration, the cell viability decreases with increasing the preincubation time, again indicating the importance of cellular uptake of the Pdots in PDT. For the same preincubation time (8 h) and light irradiation (5−15 min, 30−90 J/cm2), the cell viability decreases with increasing the Pdot concentration from 0.25 to 4.0 μg/mL (Figure 4d). The majority of the cancer cells were killed by using a Pdot concentration of 1.0 μg/mL and a light dose of 90 J/cm2, indicating the excellent photodynamic effect of the TPP-Pdots. Table S1 presented the treatment conditions of the photosensitizer and light dose for effective PDT in recent literatures where other nanoparticles carriers were employed. It is worth noting that both the photosensitizer concentration and the light dose in our experiments are much lower than those used in

compared with control cells without Pdot incubation. These findings are in good agreement with previous reports that the Pdots are biocompatible and nontoxic to a variety of cells such as macrophage (J774A.1), mouse fibroblast (3T3), and human breast cancer cells (MCF-7).54−56 The TPP-Pdots efficiently kill cancer cells at very low nanoparticle concentration using a light dose that is less than that used in clinical PDT. The SGC-7901 cells were incubated with TPP-Pdots at 1−4 μg/mL concentration (∼0.5−2 nM) and further illuminated with a blue LED array with uniform light illumination density (100 mW/cm2, ⩽15 min). The light dose (⩽90 J/cm2) in our PDT experiments is considerably less than that employed in clinical situation (typically >200 J/cm2) where porphyrin and chlorophyll molecules were used as photosensitizer.57 Because of the ultralow Pdot concentration and highly localized effect of singlet oxygen (∼100 nm), we expect nanoparticle uptake and intracellular 1O2 generation would be much more effective for killing cancer cells. To verify this point, light illumination was applied to the cells with 8 h of preincubation so that the TPP-Pdots can be sufficiently internalized. For a comparison, light illumination was immediately applied to the cells after adding the same amount of TPP-Pdots (Figure 4c). The photodynamic effects were subsequently investigated by the MTT assay. As seen from Figure 4c, the cell viability of SGC-7901cells show only G

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Figure 7. In vivo PDT. (a) Representative photographs of tumor-bearing mice on the 28th day after various treatments. (upper) The control mice with light irradiation only. (middle) The mice treated with I.V. injection of TPP-Pdot and light irradiation. (lower) The mice treated with I.T. injection of TPP-Pdot and light irradiation. (b) Representative photographs of tumors collected from the mice after various treatments. (c) Tumor growth curves in the tumor-bearing mice after different treatment. Tumor volumes were normalized to their initial sizes. Error bars represent the standard deviations of eight mice per group. (d) Histological H&E staining of various organs from the mice of control group, I. V. group, and I. T. group.

compared to control, indicating that ROS destroyed nucleic acids and resulted in severe DNA damage in cancer cells. AO/ EB staining was further performed to confirm the photodynamic toxicity of Pdots. AO can pass through integrated cell membrane and bind with DNA to produce green fluorescence, but EB can only pass through damaged cell membrane and bind with DNA to produce red fluorescence. Thus, live cells (green), early apoptotic cells (yellow-orange), and late apoptotic/ necrotic cells (red) can be simultaneously identified by fluorescence microscope. The AO/EB staining results (Figure 5d) showed that the cells treated with Pdots and light irradiation presented three types of states, where the number of green living cells was decreased, the number of orange-red apoptotic cells and red necrotic cells were increased. Taken together, the cell-staining results indicated that TPP-Pdots as a potent nanoparticles photosensitizer could cause cell apoptosis and necrosis due to the efficient ROS generation. 3.3. In Vivo Distribution and Photodynamic Therapy. The fluorescence properties of the TPP-Pdots can be employed to monitor the in vivo distribution of Pdots in tumor-bearing nude mice before PDT treatment. Each tumor-bearing nude mouse was intravenously or intratumorally injected with TPPPdots (2 mg/kg in PBS). The animals were imaged by a small animal imaging system at 24 h post injection. As shown in Figure 6a, fluorescence signal can be clearly detected from the liver for the mouse intravenously injected, while the control animal without intravenous injection presented no fluorescence. Similarly, the intratumorally injected mouse presented strong fluorescence from the tumor as compared to the control.

most literature, indicating that the amplified ROS generation by Pdots can significantly reduce the photosensitizer dosing for a given light dosage. We performed cellular staining to investigate the photodynamic toxicity induced by TPP-Pdots. Generation of intracellular ROS is critical to kill cancer cells in PDT. We used a fluorescent probe DCFH-DA to detect ROS in living cells.58 DCFH-DA can pass through cell membrane into the cell, and ROS can transform DCFH into fluorescent DCF. The SGC-7901cells were incubated with TPP-Pdots of different concentrations and irradiated with identical light dose (90 J/ cm2). As shown in Figure 5a, the cells treated with Pdots and light irradiation showed increased fluorescence signal as compared to control without Pdots incubation, indicating the high intracellular ROS level induced by TPP-Pdots. Propidium iodide (PI) dye was further used in cell labeling to show the dead cells relative to the surviving cells. The PI dye can enter into the damaged cell membrane rather than the integrated membrane,59 so the PI fluorescence in cell nucleus can be used as a marker for late apoptosis and necrosis. As shown in Figure 5b, the number of dead cells was apparently increased as the Pdot concentration varied from 0 to 1 μg/mL, consistent with the effective photodynamic cell destruction by the TPP-Pdots. In addition, DAPI dye, binding to DNA of both living cells and dead cells,60 was used to observe changes in nuclear morphology (Figure 5c). As indicated by the images, the cells treated with Pdots and light irradiation showed nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation with increasing concentration of Pdots H

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that the distribution and excretion of Pdots from the body must be resolved for using the TPP-Pdot as photosensitizing drugs via systemic injection. However, no obvious liver damage was found in intratumoral injection group because no Pdots were found in the liver, indicating local administration of photosensitizer drug can be an option for effective PDT and minimal damage to other organs.

These successful whole-animal imaging indicated the usefulness of TPP-Pdots for simultaneous tumor imaging and treatment, although the absorption wavelength was not optimized to the near-infrared region. The tumor and various organs were further collected from the Pdot-treated mice and analyzed by fluorescence imaging (Figure 6b). As indicated by the imaging, the majority of TPP-Pdots were accumulated in the liver, spleen, and tumor tissue via intravenous injection, while minor distributions were distributed in kidney, lung, and heart. However, nearly all the TPP-Pdots were trapped in the tumor after intratumoral injection, and there was no detectable fluorescence in the liver, spleen, kidneys, lung, and heart, as compared to control group. The accumulation of Pdots intravenously injected in the liver and spleen may be related to reticuloendothelial system (RES) of liver and spleen, which was consist with previous results on Pdots and other nanoparticles.61 The dominant accumulation of Pdots intratumorally injected is obviously due to the local injection, and the enhanced permeability and retention effect (EPR) of tumor can help to trap the Pdots in the tumor site. We evaluated the therapeutic effect in the mice intravenously or intratumorally injected with TPP-Pdots by measuring tumor growth rate after light irradiation. Figure 7 shows representative pictures of the tumor-bearing nude mice on 28th day after light irradiation. Additional pictures of the treatment results were provided in Supporting Figure S4. As shown in Figure 7a, the mice treated with TPP-Pdots and light irradiation, either injected intratumorally or intravenously, showed obvious effects of tumor-growth inhibition, whereas the control mice without Pdots and irradiation displayed rapid tumor growth. For the intravenous injection group and intratumoral injection group, effects of inhibiting tumor growth are apparent but different between the two groups. As indicated in Figure 7b, the tumors in the mice with intratumoral injection showed much smaller tumor volume than those in the intravenous injection group, which was likely due to the dominant distribution of Pdots in tumor as compared to the intravenous injection group. It is worth noting that the tumors were eradicated in 3 of 10 mice with intratumoral injection. Additionally, skin damage was observed in both groups, probably due to the accumulation or leakage of Pdots into the skin tissue nearby the tumors. Nevertheless, these results provide strong evidence that Pdots platforms induced irreversible damage to cancer cells, apparent tumor inhibition, and complete tumor eradication in some cases, revealing a promising therapeutic effect for treatment of malignant cancers. Finally, the mice were euthanized, and organs including tumor, liver, spleen, kidneys, lung, and heart were collected for staining with hematoxylin and eosin (H&E). By analyzing the H&E results of tumor tissues, we find that there are more necrotic tissues, more inflammatory cells, and less vascular tissue in tumor tissue of treatment groups as compared to control without treatment. In certain cases that the tumors were significantly inhibited, some amount of remaining tumor tissue and surrounding fibrous tissue were observed by optical microscope in tissue section. For both the intravenous injection group and intratumoral injection group, no pathological changes were observed in the spleen, kidney, lung, and heart, as compared to control group. Minor liver damage was found in the intravenous injection group, such as hepatocellular necrosis, ballooning degeneration, congestion, and inflammatory cell infiltration (Figure 7c), which is likely due to a significant accumulation of Pdots in the liver. This observation indicated

4. CONCLUSION In summary, we developed semiconductor polymer-based nanoparticles for effective PDT. By doping a photosensitizer TPP, the Pdots exhibited a strong emission from the TPP acceptors due to efficient energy transfer from the PFBT polymer to the TPP molecules. Optical spectroscopic characterizations indicated an energy transfer efficiency of nearly 100% and singlet-oxygen generation quantum yield of ∼50% in the TPP-doped Pdots. We evaluated the cellular uptake, cytotoxicity, singlet oxygen generation, and photodynamic effects of the Pdot photosensizer in SGC-7901 cells by a variety of assays. The in vitro results indicated that cancer cells were efficiently destroyed at very low dose of the Pdots such as 1 μg/mL by using the light dose of 90 J/cm2 that is considerably less than that in clinical practice. The antitumor effect of the TPP-Pdots was further evaluated in vivo with human gastric adenocarcinoma xenografts in Balb/c nude mice, which showed that the xenograft tumors were significantly inhibited or eradicated in certain cases. Our results indicate the energy transfer amplified Pdot platforms have great therapeutic potential for treating malignant cancers.

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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/acsami.5b07995. A summary of recently published results on nanoparticles for PDT, normalized absorption and emission spectra of PFBT Pdots and TPP molecules, fluorescence lifetime decay traces of TPP doped PFBT Pdots, concentration dependent lifetime change of TPP doped PFBT Pdots, preincubation time dependent cell viability of SGC-7901, representative photographs of tumor-bearing mice on the 28th day after various treatments. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (C.W.) *E-mail: [email protected]. (H.X.) Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS H.X. acknowledges financial support from the National Science Foundation of China (Grant No. 51173066). C.W. acknowledges financial support from the Thousand Young Talents Program. This work is also supported by the National Science Foundation of China (Grant Nos. 61222508 and 61335001).

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