Photo-Cross-Linkable Polymer Dots with Stable Sensitizer Loading

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Photocrosslinkable Polymer Dots with Stable Sensitizer Loading and Amplified Singlet Oxygen Generation for Photodynamic Therapy Ying Tang, Haobin Chen, Kaiwen Chang, Zhihe Liu, Yu Wang, Songnan Qu, Hong Xu, and Changfeng Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14325 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 14, 2017

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ACS Applied Materials & Interfaces

Photocrosslinkable Polymer Dots with Stable Sensitizer Loading and Amplified Singlet Oxygen Generation for Photodynamic Therapy Ying Tang,1 Haobin Chen,2 Kaiwen Chang,2 Zhihe Liu,2 Yu Wang,1 Songnan Qu,3 Hong Xu,1* Changfeng Wu2* 1

Department of Gastroenterology, The First Hospital of Jilin University, Changchun 130021, China 2

State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, Changchun 130012, China

3

State Key Laboratory of Luminescence and Applications, Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130033, China

ACS Paragon Plus Environment

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ABSTRACT

Photodynamic therapy (PDT) is a promising treatment modality for clinical cancer therapy. However, the therapeutic effect of PDT is strongly dependent on the property of photosensitizer. Here, we developed photocrosslinkable semiconductor polymer dots doped with photosensitizer Chlorin e6 (Ce6) to construct a nanoparticle platform for photodynamic therapy. Photo-reactive oxetane groups were attached to the side chains of the semiconductor polymer. After photocrosslinking reaction, the Ce6-doped Pdots formed an interpenetrated structure to prevent Ce6 leaching out from the Pdot matrix. Spectroscopic characterizations revealed an efficient energy transfer from the polymer to Ce6 molecules, resulting in amplified generation of singlet oxygen. We evaluated the cellular uptake, cytotoxicity, and photodynamic effect of the Pdots in gastric adenocarcinoma cells. In vitro photodynamic experiments indicated that the Ce6-doped Pdots (~10 µg/mL) effectively killed the cancer cells under low dose of light irradiation (~60 J/cm2). Furthermore, in vivo photodynamic experiments were carried out in tumor-bearing nude mice, which indicated that the Pdot photosensitizer apparently suppressed the growth of solid tumors. Our results demonstrate that the photocrosslinkable Pdots doped with photosensitizer are promising for photodynamic cancer treatment.

KEYWORDS: semiconducting polymer; photocrosslinking; photosensitizer; energy transfer; photodynamic therapy


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1. INTRODUCTION Photodynamic therapy (PDT) has been extensively explored and translated to clinical cancer therapy.1-3 Under light excitation, a photosensitizer in PDT can transfer the energy to surrounding molecular oxygen (3O2) to generate cytotoxic reactive oxygen species (ROS) such as singlet oxygen (1O2), leading to destruction of cancer cells.4,5 In contrast with conventional cancer treatments, PDT possesses unique advantages including minimally invasive nature, tolerance of repeated doses, and fast treatment process,6 because the oxidative damage induced by PDT is limited to the target tissue, without additional damage to surrounding tissue. However, most photosensitizers used in PDT are hydrophobic in nature, making them easily form aggregates in aqueous solution.7 The aggregated photosensitizer results in low bioavailability, insufficient ROS yield, and unsatisfactory therapeutic effects.8,9 The tissue penetration depth of excitation light is another important factor that impacts the PDT.10,11 The light attenuation by tissue scattering and absorption can weaken the photodynamic effect in deep tissue and lead to tumor residue and recurrence in many cases. Additional factors including poor tumor selectivity and low absorption coefficients of photosensitizers can also limit the photodynamic effect in practical cancer treatment. Therefore, various nanoparticle based photosensitizers have been explored

extensively to

overcome the

above problems associated

with

molecular

photosensitizers.12 In order to enhance the photodynamic effect, photosensitizer loaded nanoparticles such as polymeric nanoparticles,13,14 upconversion nanoparticles,15-17 gold nanoparticles,18,19 mesoporous silica nanoparticles20-22 and quantum dots23,24 have been developed and demonstrated for photodynamic cancer treatments. Semiconductor polymer dots (Pdots) have attracted considerable attention due to their remarkable optical properties.25 Upon surface functionalization and biomolecular conjugation,


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Pdots have been successfully implemented for applications in specific cell labeling,26-28 in vivo fluorescence imaging,29-33 photoacoustic imaging,34,35 biosensing,36,37 drug delivery,38,39 and cancer therapy.40 For PDT applications, Pdots can be used as a carrier to load hydrophobic photosensitizer to overcome the problem of photosensitizer solubility. More importantly, Pdots serve as light harvesting matrix and transfer energy to the photosensitizer for amplified ROS generation.41 Beside the Pdot species, a variety of conjugated polyelectrolytes have also been demonstrated for photo-activated anticancer and antifungal applications.42 Despite the progresses, the absorptions of most currently available Pdot species are in the blue region that has a limited tissue penetration depth. In addition, the photosensitizer can probably leach out from Pdots that are physically doped with small molecule photosensitizer, especially in physiological environment. These issues need to be addressed to further implement the Pdots for PDT application. Here we synthesized a photocrosslinkable semiconductor polymer with the absorption band in green region, where the tissue penetration depth was increased compared to previous Pdots used for PDT.41 Furthermore, photocrosslinkable oxetanes groups were attached to the side chains of the semiconductor polymer, which was used as matrix to prepare Pdots doped with a photosensitizer Chlorin e6 (Ce6). Upon the nanoparticle formation, the side chains of the Pdots could react with each other to yield a polymeric network via photocrosslinking reaction, stably retaining the Ce6 molecules inside the Pdots in biological environment. In vitro cellular experiments indicated that the Pdot photosensitizer showed minimal cytotoxicity at high concentration in the dark, but apparent photodynamic effect at low photosensitizer concentration under low dose of light irradiation. In vivo experiments indicated that the Pdot photosensitizer apparently suppressed the tumor growth with less damage to surrounding normal tissues.


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2. EXPERIMENTAL SECTION 2.1. Materials. Ce6, 3-methyl-3-oxetanemethanol, 1,6-dibromohexane, 2,7-dibromofluorene, and (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene were obtained from J&K Chemical Ltd.

4,7-bis(5-bromo-4-hexylthiophen-2-yl)benzo[c][1,2,5]thiadiazole

was

obtained

from

Derthon. poly(styrene-co-maleic anhydride) (PSMA), 2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2dioxaborolane, tetrakis-(triphenylphosphine)palladium (Pd[PPh3]4), 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO),

acridine

orange

(AO),

ethidium

bromide

(EB),

9,10-anthracenediyl-

bis(methylene)dimalonic acid (ADMA) were purchased from Sigma-Aldrich. Lyso-tracker green was purchased from Invitrogen. Lactate dehydrogenase (LDH) release assay kit and Giemsa staining solution were purchased from Beyotime biotechnology. Annexin V and PI apoptosis detection kit was purchased from KeyGEN technology. 2.2. Synthesis of 9,9-Di-{6-[(3-methyloxetan-3-yl)methoxy]hexyl)-2,7-dibromofluorene (3). The synthesis was conducted according to a previous report.43 Step 1: A mixture of 3-methyl-3oxetanemethanol (1) (5.0 g, 0.049 mol), 1,6-dibromohexane (35.9 g, 0.147 mol), tetra-nbutylammonium bromide (TBAB, 0.2 g, 0.62 mmol), hexane (40 mL), and NaOH solution (50 mL, 40%) was stirred at room temperature (RT) for 24 h and then heated to reflux for 2 h. After the solution was cooled to RT, the mixture was poured into ultrapure water and extracted with hexane. The organic layer was dried over magnesium sulfate and the solvent was removed by evaporation under reduced pressure, the product was obtained after purification by column chromatography on silica gel (ethyl acetate/n-hexane =1/10) as a colorless liquid (10.1 g, 78%).13C NMR spectra were recorded on a 500 MHz Bruker Avance. 1H NMR spectra were recorded on a 300 MHz Varian Mercury, using CDCl3 as solvent and tetramethylsilane (TMS) as


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an internal standard (δ = 0.00 ppm). 13C NMR (125 MHz, CDCl3): δ 79.93, 75.86, 71.11, 39.70, 33.59, 32.52, 29.16, 27.74, 25.15, 21.19; 1H NMR (300 MHz, CDCl3, δ): 4.51 (d, 2H, J = 5.7 Hz), 4.35 (d, 2H, J = 5.7 Hz), 3.47 (t, 2H, J = 6.4 Hz), 3.46 (s, 2H), 3.41 (t, 2H, J = 6.9 Hz), 1.911.82 (m, 2H), 1.65-1.56 (m, 2H), 1.51-1.35 (m, 4H), 1.31 (s, 3H). Step 2: A mixture of 2,7dibromofluorene (1.3 g, 4 mmol), TBAB (0.165 g, 0.51 mmol), DMSO (35 mL), and NaOH solution (15 mL, 50%) was stirred for 5 min at 60 °C, then 3-[(6-bromohexyloxy)methyl]-3methyloxetane (2) (2.3 g, 8.8 mmol) in DMSO (15 mL) was added dropwise to the solution and refluxed for 12 h. After the solution was cooled to RT, the organic phases were washed with ultrapure water and extracted with ethyl acetate. The final product was obtained after purification by column chromatography on silica gel (ethyl acetate/n-hexane =1/5) as a colorless solid (2.2 g, 79%). mp: 63.2-64.0 °C;

13

C NMR (125 MHz, CDCl3): δ 152.33, 139.02, 130.18, 126.06,

121.45, 121.14, 80.16, 75.97, 71.43, 55.57, 40.08, 39.82, 29.60, 29.33, 25.68, 23.53, 21.33; 1H NMR (300 MHz, CDCl3, δ): 7.53-7.45 (q, 6H), 4.45 (d, 4H, J = 5.7 Hz), 4.32 (d, 4H, J = 5.7 Hz), 3.40 (s, 4H), 3.33 (t, 4H, J = 6.6 Hz), 1.94-1.88 (m, 4H), 1.43-1.34 (m, 4H), 1.26 (s, 6H), 1.151.05 (m, 8H), 0.63-0.50 (m, 4H). 2.3.

Synthesis

of

9,9-Di-{6-[(3-methyloxetan-3-yl)methoxy]hexyl)-2,7-Di-[boronicacid

bis(pinacol) ester]-fluorene (4). A three neck round bottom flask (100 mL) was fitted with a stir bar, stopper, septum, and argon inlet. This flask was flame-dried, and ~30 mL of anhydrous THF was added by cannula. In this flask, 9,9-di-{6-[(3-methyloxetan-3-yl)methoxy]hexyl)-2,7dibromofluorene (3) (2.0 mmol, 1.385 g) was added, and the flask was cooled to -78 °C. N-Butyl lithium (2.5 M, 12.5 mmol, 5 mL) was added by syringe and the solution was stirred for 2 h. 2Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (7.4 mmol, 1.5 mL) was added by syringe and the solution was stirred for 5 min. The solution was then allowed to warm to RT and stirred


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overnight. The solution was diluted with ~50 mL of diethyl ether, and extracted three times with water, once with brine. The organic fraction was then dried with magnesium sulfate, filtered, and dried by vacuum. The colorless crude product was then recrystallized from acetone. Finally, 1.12 g (71%) of a white crystalline solid was obtained. 13C NMR (125 MHz, CDCl3): δ 150.31, 143.90, 133.74, 128.85, 119.47, 119.17, 83.74, 80.24, 76.86, 71.56, 55.10, 40.12, 39.66, 29.80, 29.42, 25.75, 24.95, 23.64, 21.37; 1H NMR (300 MHz, CDCl3, δ): 7.86-7.69 (q, 6H), 4.44 (d, 4H, J = 5.7 Hz), 4.32 (d, 4H, J = 5.7 Hz), 3.38 (s, 4H), 3.30 (t, 4H, J = 6.6 Hz), 2.00 (m, 4H), 1.64 (m, 4H), 1.39 (s, 24H), 1.25 (s, 6H), 1.05 (m, 8H), 0.55 (m, 4H). 2.4. Synthesis of do-PFDTBT Polymer. In a 50 mL flask, monomer 4,7-bis(5-bromo-4hexylthiophen-2-yl)benzo[c][1,2,5]thiadiazole (5) (313.3 mg, 0.5 mmol) and monomer (4) (393.3 mg, 0.5 mmol) were dissolved in toluene (10 mL). Bu4NBr (6.4 mg, 0.02 mmol) and Na2CO3 (6 mL, 2 M) were also added to the solution. The mixture was degassed and refilled with N2 (repeated 6 times) before and after addition of Pd(PPh3)4 (10 mg, 0.008 mmol). The reactants were stirred at 90 °C for 48 h and (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene (30 mg) dissolved in toluene (1 mL) was added. After 5 h, bromobenzene (0.3 mL) was added and further stirred for 6 h. After the mixture was cooled to RT, it was poured into methanol (100 mL). The precipitate was filtered, washed with methanol, ammonia solution, deionized water, and acetone to remove monomers, small oligomers, and inorganic salts. The crude product was dissolved in THF (15 mL), filtered through 0.22 µm membrane and re-precipitated in methanol (100 mL). The powder was then stirred in acetone (150 mL) for 24 h and then collected by filtration, and dried in vacuum. (315 mg, 63%). 1H NMR (300 MHz, CDCl3, δ): δ = 8.08 (m, 2H), 7.93 (m, 2H), 7.79 (m, 2H), 7.52 (m, 4H), 4.44 (d, 4H, J = 5.7 Hz), 4.31 (d, 4H, J = 5.7 Hz), 3.39 (s, 4H), 3.34 (t, 4H, J = 6.6 Hz), 2.81 (m, 4H), 2.07 (m, 4H), 1.76 (m, 4H), 1.60-1.27 (m,


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16H), 1.24 (s, 6H), 1.22-1.07 (m, 8H), 0.89 (s, 6H), 0.77 (m, 4H). The molecular weight of doPFDTBT polymer was measured by gel permeation chromatography (GPC) with 515 HPLC pump, Waters, 2414 refractive index detector. GPC results: Mn =6505, Mw = 11951, PDI = 1.84 (Figure S1). The Fourier transform infrared (FTIR) spectra of do-PFDTBT polymer was collected on a Nicolet 6700 FTIR spectrophotometer (Figure S2). 2.5. Preparation of Ce6-doped Pdots and Photocrosslinking Reaction. do-PFDTBT, Ce6 and PSMA were respectively dissolved in THF to prepare the stock solutions (1mg/mL). 100 µL of do-PFDTBT was mixed with 20 µL of PSMA, 0-20 µL of Ce6, and a certain amount of THF was added to make 1 mL of the solution mixture. After that, the solution mixture was quickly added to 5 mL of Milli Q water under sonication. THF was removed and the solution was concentrated by nitrogen stripping under 100-120 °C. Finally, the solution was filtrated by 0.22 µm filter for further use. For photocrosslinking reaction, photoinitiator (triarylsulfonium hexafluorophosphate salts, 1 wt%) was added to the Pdot solution, which was further irradiated with a UV lamp at the power density of 50 mW/cm2 for 10 seconds. Finally, the resulting Pdots were purified with ultrafiltration centrifuge tubes for three times. 2.6. Characterizations of Ce6-doped Pdots. Absorption spectra were recorded by a spectrophotometer (Shimadzu UV-2550). Emission spectra were recorded by a fluorescence spectrometer (Hitachi F-4500). Particle size and Zeta potential of the Pdots were measured by dynamic light scattering (DLS, Malvern Zetasizer NanoZS). Morphology of the Pdots was measured by a transmission electron microscope (TEM, Hitachi H-600). ADMA was used to detect 1O2 generation by monitoring the absorption changes of ADMA. Pdots and ADMA were mixed and diluted to 5 µg/mL and 20 µg/mL with phosphate buffered solution (PBS; 0.01 M, pH 7.4). The mixed solution was irradiated by green light (520 nm) at 100 mW/cm2 and the


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absorption spectra was recorded every one minute. Absorbance of ADMA at 259 nm was recorded for analysis. For the stability studies, the uncrosslinked and crosslinked Pdots were treated with 0-20 wt% THF, and their particle size were measured by DLS. For the dye leaching study, the uncrosslinked and crosslinked Pdots were treated with 1 wt% Triton X-100 for 24 h and filtrated through an ultrafiltration centrifugation tube (MWCO, 100 KDa). The absorption spectra of the filtrate were measured to compare the Ce6 leaching from uncrosslinked and crosslinked Pdots. 2.7. Cell Culture and Fluorescence Imaging. Human gastric adenocarcinoma cells (SGC-7901) were obtained from the Key Laboratory of Blood Cancer Center in Jilin University. The cells were cultured by DMEM medium (high glucose) containing 10% fetal bovine serum (FBS), 1% penicillin and streptomycin. Cells were routinely cultured at 37 °C in an incubator containing 5% CO2. When cells reached 80% confluence, they were digested with 0.25% trypsin solution and collected for centrifugation (800 rpm for 5 min). For cellular imaging, 1×105 SGC-7901 cells were seeded into each well of 6-well plates. After culturing for 24 h, the cells were incubated with different concentrations of Ce6-doped Pdots for 0-8 h. Then the cells were washed with PBS, and cell imaging was performed on a fluorescence microscope (Olympus IX71). The cells with the same experimental treatments above were used for flow cytometry, which quantitatively determined the fluorescence intensity of cells (BD FACS Calibur, BD Accuri C6). To further investigate the intracellular localization of Ce6-doped Pdots, lyso-tracker green was used for lysosomes staining. The cells were incubated with 10 µg/mL Ce6-doped Pdots for 8 h and 24 h respectively. Then the pre-warmed (37 °C) probe-containing medium (300 µM) was added and incubated with cells for 30 min, and the cells were observed by a confocal laser scanning microscope (Olympus FV1000).


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2.8. In Vitro PDT Experiments. Cytotoxicity and photodynamic effect were evaluated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cytotoxicity of the Ce6doped Pdots without light irradiation was investigated before in vitro PDT study. Logarithmic phase SGC-7901 cells were collected and 1×104 cells were seeded to each well of 96-well plates. 24 h later, the cells were incubated with 0-100 µg/mL Ce6-doped Pdots for 24 h and 48 h respectively, then conventional process of MTT assay was carried out. For in vitro PDT study, logarithmic phase SGC-7901 cells were collected and 1×104 cells were seeded to each well of 96-well plates. 24 h later, the cells were incubated with 0-10 µg/mL Ce6-doped Pdots for 8 h, and were irradiated by green light (520 nm) at 100 mW/cm2 for 0-15 min. Conventional process of MTT assay was carried out after 16 h. MTT assay was measured by a microplate reader (BioTek Cytation3). To further evaluate the photodynamic effect, lactate dehydrogenase (LDH) release assay was carried out. 1×104 cells were seeded to each well of 96-well plates. 24 h later, the cells were incubated with 0-10 µg/mL Ce6-doped Pdots for 8 h, and were irradiated by green light (520 nm) at 100 mW/cm2 for 15 min. LDH release assay kit was used to detect LDH release (%) after 16 h. In addition, colony formation assay was carried out for the analysis of cell proliferation ability. 1×105 cells were seeded to each well of 6-well plates. 24 h later, the cells were incubated with 08 µg/mL Ce6-doped Pdots for 8 h, and were irradiated by green light (520 nm) at 100 mW/cm2 for 5 min. 16 h later, the cells after PDT treatment were collected and dispersed into single cell suspension through trypsin digestion. 1×103 cells were seeded into each culture dish and cultured for 2-3 weeks. Culturing was terminated until colony was observed, and appropriate Giemsa solution was added to stain the cells for 10-30 min. Surviving fraction (SF) and plating efficiency (PE) are calculated according to the literature.44 AO (acridine orange) / EB (ethidium bromide)


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staining was used to evaluate the photodynamic effect. 1×104 cells were seeded into each glass slide. 24 h later, the cells were incubated with 0-8 µg/mL Ce6-doped Pdots for 8 h, and were irradiated by green light (520 nm) at 100 mW/cm2 for 15 min. 16 h later, AO and EB (100 µg/mL) were added for staining, and the cells were observed by a confocal laser scanning microscopy. Annexin V and PI staining was used to quantitatively evaluate the photodynamic effect. 10×105 cells were seeded into each well of 6-well plates. 24 h later, the cells were incubated with 0-8 µg/mL Ce6-doped Pdots for 8 h, and then were irradiated by green light (520 nm 100 mW/cm2) for 15 min. 16 h later, the cells were collected and stained with Annexin V and PI apoptosis detection kit for flow cytometry. 2.9. In Vivo Photodynamic Therapy. Animal experiments were performed in accordance with relevant guidelines and regulations of Jilin University and China Association of Laboratory Animal Care. BALB/c-nu mice were purchased from Beijing HFK Bioscience Co, Ltd. Female BALB/c-nu mice aged 5-6 weeks (body weight, 18−20 g) were chosen for the experiments. To establish the tumor-bearing nude mice model, each mouse was subcutaneously injected with 2×106 SGC-7901 cells in left armpit, and maintained with regular food and water until the tumor volume reached to 55−100 mm3. To investigate the biodistribution of Ce6-doped Pdots, each tumor-bearing nude mouse was injected with 100 µg/mL Ce6-doped Pdots intratumorally or intravenously, and the mice without injection were used as controls. After 24 h, the fluorescence intensity of mice was detected by a small animal imaging system. Then tumors and major organs (heart, liver, spleen, lung and kidney) were collected for fluorescence imaging and biodistribution analysis.


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For in vivo PDT experiments, 20 mice were randomly divided into four groups: control group (without injection), intravenous injection group (intravenously injected with 100 µg/mL Ce6-doped Pdots), intratumoral injection low-dose group (intratumorally injected with 50 µg/mL Ce6-doped Pdots) and intratumoral injection high-dose group (intratumorally injected with 100 µg/mL Ce6-doped Pdots). The same volume of the Pdot solution (100 µL) was injected in the four groups. After 24 h of injection, the mice were irradiated by green light (520 nm) at 100 mW/cm2 for 30 min. This light irradiation was carried out at the first day and repeated once at the 8th day. Body weight and tumor volume of the mice were measured every four days and pictures were taken photos every eight days. Tumor volume was calculated as 1/2 × (tumor length) × (tumor width)2, and relative tumor volume was calculated as V/V0 (V0 was the tumor volume measured at 0th day). At the 24th day, mice were euthanized with chloral hydrate and their organs were collected for H&E staining. 3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterizations of Ce6-doped Pdots. We designed photocrosslinkable semiconductor polymer as a matrix to prepare photosensitizer doped Pdots. Oxetanes are commonly reactive groups for cationic photopolymerization, and they can react with each other induced by ultraviolet light irradiation in presence of photoinitiator. In our design, oxetanecontaining side chains were attached to polyfluorene backbone structure to develop the oxetanefunctionalized polymer. The synthetic route and chemical structure of do-PFDTBT polymer were shown in Scheme 1. We first synthesized the oxetane functionalized fluorene monomer according to the protocol described in a previous report.43 The oxetane functionalized doPFDTBT polymer was then synthesized by a Suzuki coupling. Ce6 is a widely used photosensitizer in clinical therapy because of its potency against a wide range of cancers,


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especially lung cancer, breast cancer and ovarian cancer.45 However, Ce6 is an amphiphilic compound comprised of three adjacent carboxyl groups on a lipophilic aromatic system. The small and amphiphilic nature result in the problem of leaching out from delivery vehicles. We employed the photocrosslinking strategy to overcome the problem of leaching and improve the stability of the photosensitizer inside Pdots. The preparation and photocrosslinking of Ce6-doped do-PFDTBT Pdots were shown in Scheme 1. The Ce6-doped Pdots in aqueous solution were prepared by using the nanoprecipitation method.46 The resulting Pdots were then photocrosslinked by UV light irradiation in presence of photoinitiator. The photocrosslinked Pdots were further characterized for photodynamic therapy applications.


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Scheme 1. Synthetic route of the photocrosslinkable polymer do-PFDTBT, preparation and photodynamic effect of the Ce6-doped Pdots.

Fluorescence spectroscopy indicates efficient energy transfer from the do-PFDTBT polymer to Ce6 photosensitizer. There was a reasonable spectral overlap between the emission of doPFDTBT and the absorption of Ce6 (Figure S3), indicating the possibility for energy transfer to occur inside the Pdot. We varied the Ce6 concentration to optimize optical properties of the Ce6doped Pdots. As shown in Figure 1a, the absorption intensity of the Ce6-doped Pdots at 660 nm was indicative of the increased doping fraction of Ce6 photosensitizer. The pure Pdots showed a broad band red emission with a fluorescence quantum yield of 0.8. As the Ce6 doping fraction was increased, the characteristic emission from Ce6 (~675 nm) was initially increased and then decreased, likely due to the aggregation of the Ce6 in the Pdots. At the doping fraction of 5 wt%, the doped Pdots showed the highest fluorescence intensity (Figure 1b). The absorption and emission spectra of the Ce6-doped Pdots (~5 wt% fraction) were shown in Figure 1c, which exhibited a broad absorption band in the green region and a major emission peak at 675 nm. The absorption band originated from the dominant do-PFDTBT polymer matrix while the major emission peak was due to the Ce6 dopant. The quenched fluorescence from the polymer donor and the dominant emission from the Ce6 upon exciting the polymer indicated the efficient energy transfer from the polymer to the Ce6 dopant. Fluorescence lifetime measurements provided additional evidence for the energy transfer inside Pdots (Figure 1d). The lifetime of the Ce6doped Pdots (5 wt% fraction) was determined to be 0.64 ns, which was shorter than that of the pure Pdots (1.11 ns), further confirming the occurrence of the energy transfer from the polymer to the Ce6 molecules.


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Figure 1. Optical characterizations of the Ce6-doped Pdots. (a) Absorption spectra of the Pdots doped with different Ce6 fractions. (b) Emission spectra of the Pdots doped with different Ce6 fractions. The emission spectra were obtained with an excitation at 520 nm. (c) Absorption and emission spectra of the Pdots doped with Ce6 fraction of ~5 wt%. The emission spectra were obtained with an excitation at 520 nm. (d) Fluorescence lifetime of the pure Pdots and the Ce6doped Pdots (~5 wt%). The lifetime was measured by monitoring the emission at 600 nm that originated from the polymer donor. The quenched fluorescence and reduced lifetime of the polymer donor indicated the occurrence of energy transfer.


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We characterized the colloidal stability and singlet oxygen generation of the Ce6-doped Pdots before and after photocrosslinking. Figure 2a showed the particle size distributions (20-30 nm) of the Pdots as measured by dynamic light scattering (DLS) which indicated the particle size was not affected by the photocrosslinking. However, the Zeta potential of the crosslinked Ce6doped Pdots (-31 mV) was slightly lower than that of the uncrosslinked Pdots (Figure 2b). The TEM results showed the spherical morphology of the uncrosslinked and crosslinked Pdots, which were similar to the pure Pdots (Figure 2c). Under light irradiation, the Ce6-doped Pdots efficiently generate singlet oxygen to kill cancer cells. Pure Pdots without photosensitizer doping can generate trace amount of ROS with low efficiency.47 Pure photosensitizers usually possess small absorption cross section that requires a relatively high dose of light irradiation for noticeable PDT effect. In the Ce6-doped Pdots, the large absorption cross section of the Pdots, together with the energy transfer to Ce6, resulted in an amplified effect in singlet oxygen generation as compared to the molecular photosensitizers and pure Pdots.41,48 We used ADMA as a probe for evaluation of 1O2 generation quantum yield of the Ce6-doped Pdots. The absorption intensity of ADMA is decreased due to the photodegradation induced by 1O2 generation.49 As a result, the absorbance change of ADMA at 259 nm is used for estimation of the 1O2 generation quantum field. For both the uncrosslinked and crosslinked Ce6-doped Pdots, the absorbance of ADMA at 259 nm was rapidly decreased under light irradiation at 100 mW/cm2 (Figure 2d and 2e). Since Rose Bengal (RB) was used as a standard substance (Φ (1O2) = 0.76) to quantitatively determine the ROS generation quantum yield, the 1O2 quantum yield for the pure Pdots was determined to be ~0.1, and the 1O2 quantum yield for the uncrosslinked and crosslinked Ce6doped Pdots were determined to be ~0.4, as shown in Figure 2f. These results indicated that the 1

O2 quantum yield of the Ce6-doped Pdots was not affected by photocrosslinking reaction. The


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high 1O2 quantum yield of the Ce6-doped Pdots was comparable to those of other good photosensitizers.50 A deliver vehicle is typically required because Ce6 is not fully water soluble due to the dominant aromatic structure. Photosensitizer leaching out from the delivery carrier results in negative

concerns

of

dark

toxicity

and

unsatisfactory

photodynamic

effect.

The

photocrosslinking property of the do-PFDTBT polymer can concretely retain the Ce6 molecules inside the Pdots. After photocrosslinking reaction, the absorption and emission spectra of the Ce6-doped Pdots occurred minor changes after a long-term storage (Figure S4). To further confirm the stability, we used harsh conditions to treat the uncrosslinked and crosslinked Ce6doped Pdots. The nanoparticle swelling is a primary reason for dye-leaching from the polymer matrix. We mimicked the swelling situation by adding different fractions (0-20 wt%) of organic solvent (THF), and their particle size distribution were measured by DLS (Figure 2g and 2h). With increasing the THF fraction to 20 wt%, the particle size of the crosslinked Ce6-doped Pdots showed minor changes from 21 nm to 28 nm. In contrast, the uncrosslinked Ce6-doped Pdots were significantly swelled by the THF treatment, as reflected by the large size changes from 21 nm to 50 nm. This observation indicated the photocrosslinking yielded stable and condensed nanoparticles. In addition, the Ce6-doped Pdots were treated with 1 wt% Triton X-100 for 24 h and filtrated through ultrafiltration centrifugation. The absorption spectra of the filtrate indicated the Ce6 leaching from the uncrosslinked Pdots was two times higher than the crosslinked Pdots (Figure 2i). These comparative results clearly indicated the important function of the photocrosslinkable polymer for obtaining the Ce6-doped Pdots that possessed excellent stability to overcome the photosensitizer leaching problem.


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Figure 2. Colloidal properties and singlet oxygen generation of the Ce6-doped Pdots before and after photocrosslinking. (a) Particle size distributions of the uncrosslinked and crosslinked Pdots. (b) Zeta potentials of the uncrosslinked and crosslinked Pdots (c) TEM images of the uncrosslinked and crosslinked Pdots. (d) Spectral changes of ADMA in presence of the uncrosslinked Pdots. (e) Spectral changes of ADMA in presence of the crosslinked Pdots. (f) Absorbance changes of ADMA at 259 nm with ADMA alone, Rose Bengal (RB), pure Pdots, uncrosslinked Pdots, and crosslinked Pdots. (g) Particle size changes of the uncrosslinked Pdots treated with different fractions of THF. (h) Particle size changes of the crosslinked Pdots treated


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with different fractions of THF. (i) Absorption spectra of the filtrate of the uncrosslinked and crosslinked Pdots treated with 1 wt% Triton X-100 for 24 h.

3.2. In Vitro Cellular Imaging and Photodynamic Study. Nanoparticle uptake is a necessary condition for effective photodynamic effect. The fluorescence property of the Ce6-doped Pdots enables monitoring the cellular uptake of Pdots. Figure 3a showed the dose-dependent fluorescence imaging of SGC-7901 cells incubated with Pdots. With the same incubation time for 8h, intracellular fluorescence was gradually enhanced with increasing the Pdot concentration to 10 µg/mL. Flow cytometry was carried out for quantitative evaluation of the Pdot uptake (Figure 3b), which showed that cellular uptake percentage and mean fluorescence intensity were increased as the incubation concentration was increased (Figure S5). The mean fluorescence intensity was determined to be ~60 times higher than that of the control without Pdot incubation. Figure S6 showed time-dependent fluorescence imaging of SGC-7901 cells incubated with Pdots. With the same incubation concentration of 10 µg/mL, nearly all the cells showed Pdot internalization after 2 h incubation with Pdots. These results indicated that Ce6-doped Pdots could easily enter into cancer cells and label cells with relatively high fluorescence intensity. To further explore the endocytosis process, we investigated the organelle location of Ce6-doped Pdots in SGC-7901 cells by lyso-tracking colocalization (Figure 3c). Figure 3c showed that majority of the Ce6-doped Pdots located in lysosomes, and others might locate in early endosome and late endosome. These results were helpful to understand the organelle location of the Pdots and explore the cell death pathways.


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Figure 3. Dose-dependent cellular uptake and intracellular localization of the Ce6-doped Pdots. (a) Bright field and fluorescence imaging of SGC-7901 cells incubated with 0-10 µg/mL Ce6doped Pdots for 8 h. (b) Flow cytometry of the cells incubated with Ce6-doped Pdots. (c) Intracellular localization of the cells incubated with 10 µg/ml Ce6-doped Pdots for 8 h and 24 h.

Biocompatibility is an important consideration for nanoparticles in biological applications. Ideal photosensitizer should have minimal cytotoxicity (dark toxicity) in absence of light illumination. We performed MTT assays with different Pdot concentrations and incubation time.


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As shown in Figure 4a, the Ce6-doped Pdots exhibited negligible toxicity for high incubation concentration (100 µg/mL) and long incubation time (48 h). However, the Pdots showed apparent phototoxic effect upon light irradiation from a green LED array with uniform illumination density (520 nm, 100 mW/cm2). The cell viability was decreased with increasing the Pdot concentration (0-10 µg/mL) and the irradiation time (0-15 min) (Figure 4b). Nearly all the cells were killed by using a Pdot concentration of 10 µg/mL and a light dose of 60 J/cm2. These results indicated the prominent photodynamic effect induced by the Ce6-doped Pdots even with low concentration and a light dose considerably less than that employed in clinical situation (typically > 200 J/cm2). Cell membrane damage caused by apoptosis or necrosis can lead to enzymes release, including lactate dehydrogenase (LDH) with relatively stable activity. LDH release is considered as an important indicator of cell membrane integrity and is widely used for evaluating cell damage. As shown in Figure 4c, the LDH release (%) was gradually enhanced with increasing the Pdot concentration (0-10 µg/mL) under the same light irradiation for 15 min, indicating that integrity of cell membrane was damaged due to the photodynamic effect of the Ce6-doped Pdots. Colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony, which is defined to consist of at least 50 cells. The colony formation assay can be used for quantitative analysis of cell proliferation ability.44 SGC-7901 cells were incubated with 0-8 µg/mL Ce6-doped Pdots for 8 h and then exposed to light irradiation for 5 min (520 nm, 100 mW/cm2), after which live cells were collected to detect the proliferation ability. As indicated by Figure 4d, the surviving fraction (SF) was apparently reduced as the incubation concentration was increased. For the Pdot concentration of 8 µg/mL, only a small


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fraction of the cells retained the capacity to produce colonies, indicating that the Ce6-doped Pdots effectively inhibited the cell proliferation.

Figure 4. In vitro cell toxicity and photodynamic effect of Ce6-doped Pdots. (a) Cell viability of SGC-7901 cells incubated with 0-100 µg/mL Ce6-doped Pdots for 24 h and 48 h without irradiation. (b) Cell viability of cells incubated with 0-10 µg/mL Ce6-doped Pdots under 520 nm irradiation for 0-15 min at 100 mW/cm2. (c) LDH release assay of cells incubated with 0-10 µg/mL Ce6-doped Pdots under irradiation for 15 min at 100 mW/cm2. (d) Colony formation assay of cells incubated with 0-8 µg/mL Ce6-doped Pdots under irradiation for 5 min at 100 mW/cm2.


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We carried out AO/EB staining, Annexin V and PI staining to further explore the mechanisms of cell death induced by the Ce6-doped Pdots. AO can pass through intact cell membrane and embed into nuclear DNA to produce bright green fluorescence. EB can pass through damaged cell membrane and embed into nuclear DNA to produce orange fluorescence. SGC-7901 cells were incubated with the Ce6-doped Pdots (0-8 µg/mL, 8 h) and irradiated by green light for 15 min (520 nm, 100 mW/cm2), and then observed by a fluorescence microscope. As showed in Figure 5a, nuclear chromatin of the cells in absence of Pdots was stained as green and showed normal structure, indicating that they were live cells. For the cells with Pdot incubation (2-4 µg/mL), nuclear chromatin was stained as green-yellow and some of them showed pyknosis-like and dead-like morphology, indicating that they might be early apoptotic cells. When the incubation concentration was increased to 8 µg/mL, nuclear chromatin was stained as orange, indicating that they were late apoptotic cells or non-apoptotic dead cells. In addition, Annexin V and PI staining can be used to detect cell apoptosis, even at early and late apoptosis.51 Annexin V is widely used for early apoptosis detection, because it can combine with phosphatidylserine that turns over from cell membrane inside to outside in early apoptotic cells. Propidium iodide (PI) as a nucleic acid dye, which cannot pass through the intact membrane, but enter into necrotic and late apoptotic cells due to increased permeability of cell membrane. Thus, the combined Annexin V and PI staining can distinguish different periods of cell apoptosis. As indicated by Figure 5b, the percentage of live cells was about 99.3% in absence of the Pdots. The fraction of live cells was apparently decreased with increasing the Pdot concentration. At the Pdot concentration of 8 µg/mL, nearly all the cells were late apoptotic and necrotic cells due to


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severe oxidative damage. These results indicated that the photodynamic effect induced by the Ce6-doped Pdots was remarkable and promising for PDT in vivo.

Figure 5. Fluorescence imaging and flow cytometry of photodynamic effect induced by Ce6doped Pdots. (a) AO/EB staining of SGC-7901 cells incubated with 0-8 µg/mL Ce6-doped Pdots under 520 nm irradiation for 15 min at 100 mW/cm2. (b) Annexin V and PI staining for flow cytometry of cells incubated with 0-8 µg/mL Ce6-doped Pdots under 520 nm irradiation for 15 min at 100 mW/cm2.

3.3. In Vivo Photodynamic Therapy. The fluorescence of the Ce6-doped Pdots is useful for monitoring the nanoparticles distributions in different organs. The Pdots were intravenously or intratumorally administered into each mouse. Biophotonic imaging was performed by a smallanimal imaging system. Different organs and tumors were also collected from mice for analysis of the Pdot biodistribution. As shown in Figure 6a, fluorescence signal was apparently observed


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from the tumor site of the mouse with intratumoral injection. However, no fluorescence signal was detected from the whole-body imaging when the Pdots were administered through tail-vein injection. Biophotonic imaging of tumors and different organs indicated that the Ce6-doped Pdots were mainly distributed in liver, and only a small amount of Pdots were distributed in tumor and other organs for the mouse receiving tail-vein injection (Figure 6b). The Ce6-doped Pdots were mainly distributed in tumor for the mouse receiving intratumoral injection. Statistical analysis of fluorescence intensity in tumors and different organs was shown in Figure 6c, which was helpful to investigate the biodistribution of the Ce6-doped Pdots.

Figure 6. Biodistribution of the Ce6-doped Pdots in tumor-bearing nude mice. (a) Bright field and fluorescence imaging of mice with intratumoral and intravenous injection. (b) Bright field and fluorescence imaging of tumors and major organs collected from mice. (c) Statistical analysis of fluorescence intensity in tumors and different organs. Con = Control, I.V. = intravenous injection, I.T. = intratumoral injection.


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In vivo PDT experiments were carried out to evaluate the photodynamic effect of the Ce6doped Pdots. Tumor-bearing nude mice were divided into four groups, including control group, intravenous injection group, intratumoral injection low-dose and high-dose groups, respectively. The body weights and tumor volumes of the mice were measured every four days. Figure 7a showed the pictures of representative mice at designated times. As seen from the Figure, tumor growth in the mice receiving both the Pdots and light treatment was apparently inhibited. Statistical analysis of the tumor volume was consistent with these observations (Figure 7b). The tumor volume of the control mice without treatment showed fast growth, while the tumor growth rate of the treatment group was obviously lower than the control group. The statistical analysis also indicated the high-dose Pdot treatment enhanced the therapeutic effect as compared to the low-dose group. Body weights of the mice in the four groups were monitored in the process of PDT, as shown in Figure 7c. The body weight of the treatment groups showed slight decrease, mostly likely due to the series of experimental operations such as anesthesia and light irradiation. Although the control group was carried out under the same operations, the fast tumor growth likely made contribution to the body weight so that the body weight of the control group showed relatively constant values. Finally, we collected the tumors and organs from mice in four groups and performed histological analysis by H&E staining (Figure 7d). For the intratumoral injection group, the tumors were damaged due to the photodynamic effect, as seen from the figure. Cancer cells necrosis, inflammatory cell infiltration and blood vessels destruction were clearly observed, indicating that the Pdots effectively killed cancer cells and damaged cancerous tissue by the pronounced photodynamic effect. Meanwhile, no obvious damages were observed in other


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organs, indicating that the Pdots by intratumoral injection did not produce side effects on other organs. However, punctate cell necrosis, ballooning degeneration, congestion and edema, and inflammatory cell infiltration could be occasionally observed in liver for the mice receiving intravenous injection. This was most likely due to the fact that most of the Ce6-doped Pdots were accumulated in liver by intravenous injection. Nevertheless, the effective PDT performance of the Ce6-doped Pdots provided a promising platform for nanomedicine and cancer therapy.


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Figure 7. In vivo photodynamic effect of Ce6-doped Pdots. (a) Pictures of representative tumorbearing nude mice during the process of PDT. Relative tumor volume (V/V0) (b) and body weight (c) of tumor-bearing nude mice in control group (Con), intravenous injection group (I.V.), intratumoral injection low-dose group (I.T. low-dose) and intratumoral injection highdose group (I.T. high-dose). (d) H&E staining of tumors and organs collected from tumorbearing nude mice after PDT.

4. CONCLUSION In summary, we synthesized photocrosslinkable semiconductor polymer dots (Pdots) with stable photosensitizer loading and amplified singlet oxygen generation for photodynamic therapy. The Ce6-doped Pdots were successfully prepared with particle size of 20-30 nm, exhibiting efficient ROS generation in aqueous solution. After photocrosslinking reaction, the Ce6-doped Pdots exhibited outstanding stability to prevent the photosensitizer leaching. We evaluated the cellular uptake, cytotoxicity, and photodynamic effect of the Pdots in SGC-7901 cells through cellular imaging, MTT assay, LDH release assay, and colony formation assay. In vitro experiments indicated that the Ce6-doped Pdots could be easily internalized by cells for in vitro imaging and display apparent photodynamic effect under low dose of light irradiation. Meanwhile, in vivo experiments were carried out in bearing-tumor nude mice to evaluate the photodynamic effect, which indicated that the Ce6-doped Pdots could effectively suppress solid tumor growth with little damage to important organs. Our results demonstrate that the photocrosslinkable Pdots hold promise for photodynamic cancer treatment.


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ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. GPC results and Fourier transform infrared (FTIR) spectra of the do-PFDTBT polymer, spectral overlap between the absorption spectra of Ce6 and the emission spectra of do-PFDTBT, absorption and emission spectra of the crosslinked Ce6-doped Pdots after two-week storage, dose-dependent and time-dependent cellular uptake of the Ce6-doped Pdots. (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Hong Xu) [email protected] (Changfeng Wu) Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Hong Xu acknowledges financial support from the National Science Foundation of China (Grant No.51173066). Changfeng Wu acknowledges financial support from the National Science Foundation of China (Grant No. 61222508 and 61335001), “Thousand Young Talents Program” and the project supported by State Key Laboratory of Luminescence and Applications.


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TOC Figure


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Photo-Cross-Linkable Polymer Dots with Stable Sensitizer Loading

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