Biodegradable Polymer Nanoparticles for Photodynamic Therapy by

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Biodegradable Polymer Nanoparticles for Photodynamic Therapy by Bioluminescence Resonance Energy Transfer Yingkun Yang, Weiying Hou, Siyang Liu, Kai Sun, Minyong Li, and Changfeng Wu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01469 • Publication Date (Web): 06 Dec 2017 Downloaded from http://pubs.acs.org on December 9, 2017

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Biodegradable Polymer Nanoparticles for Photodynamic Therapy by Bioluminescence Resonance Energy Transfer Yingkun Yang1, Weiying Hou1, Siyang Liu1, Kai Sun1, Minyong Li2, Changfeng Wu3* 1

State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and

Engineering, Jilin University, Changchun, Jilin 130012, China 2

Department of Medicinal Chemistry, Key Laboratory of Chemical Biology of Natural Products

(MOE), School of Pharmacy, Shandong University, Jinan, Shandong 250012, China 3

Department of Biomedical Engineering, Southern University of Science and Technology,

Shenzhen, Guangdong 518055, China

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

ACS Paragon Plus Environment

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KEYWORDS Photodynamic therapy; Luciferase; Bioluminescence resonance energy transfer; Biodegradable polymer; Rose Bengal

ABSTRACT

Conventional photodynamic therapy is severely constrained by the limited light-penetration depth in tissue. Here, we show efficient photodynamic therapy (PDT) mediated by bioluminescence resonance energy transfer (BRET) that overcomes the light-penetration limitation. A photosensitizer Rose Bengal (RB) was loaded in biodegradable poly(lactic-coglycolic acid) (PLGA) nanoparticles, which were then conjugated with firefly luciferase. Spectroscopic characterizations indicated that BRET effectively activated RB to generate reactive oxygen species (ROS). In vitro studies of the cellular cytotoxicity and photodynamic effect indicated that cancer cells were effectively destroyed by BRET-PDT treatment. In vivo studies in a tumor-bearing mouse model demonstrated that tumor growth was significantly inhibited by BRET-PDT in the absence of external light irradiation. The BRET-mediated phototherapy provides a promising approach to overcome the light-penetration limitation in photodynamic treatment of deep-seated tumors.

INTRODUCTION Photodynamic therapy (PDT) is emerging as a noninvasive modality for a variety of cancer treatments.1-2 In typical PDT, photosensitizer is activated by external light to produce reactive


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oxygen species (ROS), which can damage cancer cells.3-5 For deep-seated tumors, the efficiency of PDT is decreased due to the limited penetration depth of external light in biological tissue. This problem has hindered further development of PDT in widespread clinical applications. In addition, skin photosensitivity and photothermal injury are also common concerns from the patients receiving PDT treatment.6-8 To overcome the light-penetration issue, considerable research has focused on the development of photosensitizers in the near-infrared (NIR) range.9 Up-conversion nanoparticles combined with photosensitizers have been explored because they can absorb NIR photons and emit visible light to activate the photosensitizer for PDT.10-11 Researchers have explored new approaches, for example, used Cerenkov radiation to activate photosensitizer for effective PDT.12-13 Alternatively, X-ray is currently widely used in clinical treatment, and X-ray activated photodynamic therapy is another way to overcome the light penetration limitation in deep-seated tumors.14-16 The use of internal light source is an intriguing solution for the light-penetration problem. For example, implanting fiber-optic light sources and emitting diodes could be a viable approach, but it still requires invasive operation.17-19 Alternatively, bioluminescence offers an attractive strategy because of the minimally invasive delivery of enzymes and substrates. Bioluminescence is a natural phenomenon that occurs in various organisms, such as marine organisms and some insects, in which visible light is produced via chemical reactions in vivo.20-21 Bioluminescence has been widely used in biological detection and optical imaging.22-24 Recently, researchers have recognized the therapeutic potential of bioluminescence. Bioluminescence is endogenous fluorescence that can be used to activate the photosensitizer inside the tumors. This process is not affected by the light penetration depth of tissue. Wang et al. reported that luminol has anticancer and antifungal activities through bioluminescence resonance energy transfer (BRET).25 Besides,


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quantum dots have also been combined with BRET system for in vivo imaging and PDT in recent reports.26-28 Despite these progresses, the BRET activated ROS generation and photodynamic effect is largely unexplored. In this study, we show effective photodynamic therapy mediated by the firefly luciferase bioluminescence system. Biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles were doped with photosensitizer Rose Bengal (RB). The PLGA-RB nanoparticles were then conjugated with luciferase, which produced a fluorescent signal in the presence of the luciferin substrate. The endogenous bioluminescence served as a light source to activate the photosensitizer for ROS generation. We evaluated cell toxicity, cellular fluorescence imaging, and therapeutic effect in cellular assays, which indicated that cancer cells were destroyed effectively. The antitumor effect of BRET-PDT was further investigated in tumor-bearing ICR mice, which showed that tumor growth was significantly inhibited. This study provides a promising approach for phototherapy of deep-seated tumors in the absence of external excitation. EXPERIMENTAL SECTION Materials and Preparation of Nanoparticles. Biodegradable polymer poly(lactic acid) (PLA; average Mn 4,000) and poly(lactic-co-glycolide acid) (PLGA; Mw: 7,000-17,000) were purchased from Sigma, and the photosensitizer Rose Bengal (Rose Bengal Sodium Salt) was purchased from J&K Chemical Ltd. All chemicals were dissolved in DMF (N,Ndimethylformamide). PLA and RB were mixed at a molar ratio of 1:1 with 50 mmol/L aqueous EDC (N-ethyl-N-(3-dimethylaminopropyl) carbodiimide). The mixture was stirred for 12 h under dark conditions. Then, PLGA was added to the mixture at a four times quality than the sum of PLA and RB. A 1-mL mixture was added immediately to 10 mL deionized water in a


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sonicator for 2 min. Nanoparticle formation was achieved when the solvent quickly changed from DMF to water. The obtained PLGA-RB nanoparticles were stored at 4°C. The nanoparticle solution was mixed with luciferase and EDC for conjugation (5 mg/mL in Milli-Q water) before use. Characterizations. Nanoparticle morphology was evaluated by TEM under a Hitachi H-600 microscope operated at 120 kV. The hydrodynamic diameter and surface potential were measured by DLS with a Zetasizer Nano ZS instrument (Malvern Instruments, Worcestershire, UK). Optical properties were analyzed using a Hitachi F-4600 fluorescence spectrophotometer, and UV-vis absorption spectra were obtained on a Schimadzu UV-2550 scanning spectrophotometer. Bioluminescence Studies. The components of the bioluminescence system in this study included 2 mmol/L ATP, 10 mmol/L magnesium ions, 60 µmol/L D-luciferin and 20 µg/mL firefly luciferase. Luciferin was purchased from J&K Scientific Ltd., firefly luciferase (Quantum Luciferase) was bought from Promega (stored at –80°C), and ATP (adenosine 5’-triphosphate disodium salt hydrate) and magnesium ions (from magnesium chloride) were purchased from Sigma. The substrate luciferin was dissolved in Tris solution (Trizma base; Sigma). The ATP solution was freshly prepared immediately before use. The oxidation reaction was allowed to occur in Tris buffer (pH 7.8, 50 mmol/L). Bioluminescence was clearly observed upon mixing with all of the above components at the given concentrations. The bioluminescence signal was measured on a Hitachi F-4600 fluorescence spectrophotometer, without excitation. Cellular Assays. Cancer cell lines MCF-7 and HeLa (American Type Culture Collection, Manassas, VA, USA) were cultured in T-75 culture flasks in Dulbecco’s modified Eagle’s


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Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were cultured in an atmosphere of 5% CO2 at 37°C, and cells were passaged 3 times per week with 0.25% trypsin. In vitro cytotoxicity of the materials was assessed by MTT assay. MCF-7 cells and HeLa cells were seeded into 96-well plates (4,000 cells/well) and cultured in 100 µL medium at 37°C for 24 h. Then, the cells were respectively cultured with PLGA-RB nanoparticles, luciferin and luciferase for another 24 h and 48 h. The organic solvent DMF was eliminated through dialysis of the nanoparticles dispersion against water for 24 h. The nanoparticles were used at a concentration gradient of 10, 20, 30, 40, 50 µg/mL, luciferin was at a concentration gradient of 10, 20, 40, 60, 80µmol/L, luciferase was at a concentration of 20 µg/mL. To determine the cytotoxicity of BRET-PDT, we prepared a mixture containing PLGA-RB nanoparticles conjugated with luciferase (NP-luciferase) and all of the components for bioluminescence (except substrate), and added it to the wells. The photodynamic process was triggered by adding luciferin at 5, 10, 20, 40, 60, 80µmol/L to the wells. After incubation for 48 h, cytotoxicity was determined using the MTT assay, measuring the absorbance at 490 nm on a Bio-Tek Cytation3 plate reader (SpectraMax M2e; Molecular Devices, Sunnyvale, USA). Fluorescence Imaging. One milliliter of a solution of 40 µg/mL PLGA-RB nanoparticles conjugated with 20 µg/mL firefly luciferase by EDC, 2 mmol/L ATP, 50 mmol/L Tris, and 10 mmol/L magnesium ions was added into each well of a 24-well plate. Fluorescence images were acquired on a Small Animal Imaging System after addition of the substrate luciferin at 0, 10, 20, 40, 60, 80 µmol/L. One healthy mouse with shaved back was subcutaneously injected with 100 µl of 40 µg/mL NP-luciferase in a solution including Tris, ATP, and Mg2+, and then subcutaneously injected with 40 µL luciferin (60 µmol/L), the time point of which was set as 0


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min. Fluorescence images were acquired every 2 min for 12 min using the Small Animal Imaging System. Besides, mice were subcutaneously injected with NP-luciferase in a solution including Tris, ATP, and Mg2+, followed by injection of luciferin at 0, 10, 20, 40, 60, 80, 100 µmol/L after nanoparticles stimulation by BRET and photographed with the Small Animal Imaging System. In Vivo PDT Studies. All animal experiments were performed strictly in accordance with the governmental guidelines of the Ministry of Science and Technology of the PR China for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Jilin University. All mice were maintained under suitable temperature and humidity, with free access to standard food and water. After three days of acclimation, 80µL of fresh H22 cells was subcutaneously injected into the back of the right waist to promote tumors. Tumor size was measured every 2 days until the tumors reached a volume of approximately 150 mm3. Tumor volume was calculated by the formula: V=1/2*L*W^2, where L is tumor length and W is tumor width. PDT treatments were initiated after tumors reached a volume of approximately 150 mm3, and we set this day as day 0. Mice were randomly divided into five treatment groups (5 mice per group). All mice were anaesthetized with isoflurane. Animals were respectively intratumorally injected with PBS (control group), 40 µg/mL NP-luciferase, 40 µg/mL PLGA-RB nanoparticles plus luciferin, 40 µg/mL PLGA-RB nanoparticles, and 40 µg/mL NP-luciferase and all components of bioluminescence except luciferin (at given concentrations in the previous subsection). Animals in the fifth group were immediately intratumorally injected 60 µmol/L luciferin to trigger the PDT process. In addition, mice in the fourth group were exposed to an LED lamp that emits 520 nm radiations at a power density of 200 mW/cm2 for 30 min to stimulate the PLGA-RB nanoparticles. The injected volume of the above mixture was 80 µL.


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PDT efficacy was evaluated by measuring tumor growth and body weight every 2 days over 14 days. The antitumor efficacy and organic damage were further evaluated by anatomy and histologic analyses after 14 days. Tumors and main organs, including heart, liver, spleen, lung, and kidney were excised, paraffin-embedded, sectioned, and stained with formalin for hematoxylin and eosin (H&E) for histological evaluation and to assess the apoptosis of tumor cells and organ damage by optical microscopy.


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Figure 1. (a) Schematic illustration of the preparation of PLGA-RB nanoparticles for BRETPDT and the related mechanism of reactive oxygen species (ROS) generation. (b) Firefly luciferase catalyzing the oxidation reaction of the native substrate D-luciferin for photon emission. RESULTS AND DISCUSSION We choose PLGA polymer to prepare the nanoparticles owing to its biodegradability and biocompatibility.29-30 RB is a typical photosensitizer that can generate singlet oxygen with a high quantum yield (~0.76).31 In addition, RB has an intense absorption band in the wavelength range of 480-550 nm, showing a good overlap with the bioluminescence spectrum of the luciferaseluciferin system. Figure 1a shows the schematic illustration for preparing the PLGA-RB nanoparticles. First, amine-terminated poly(lactic acid) (PLA) polymer was coupled with RB molecules via a carbodiimide catalyzed reaction.32 The RB-coupled PLA polymer was then blended with PLGA polymer to prepare PLGA-RB nanoparticles by reprecipitation.33 The carboxyl groups of PLGA polymer are available for bioconjugation of the firefly luciferase, which catalyzes the oxidation reaction of its native substrate, D-luciferin, to emit photons (Figure 1b). Characterizations. The amine-PLA, RB-PLA and PLGA polymers resolved in DMF solution was quickly mixed with water, resulting in nanoparticle formation driven by hydrophobic interactions. The resulting nanoparticles were dialyzed in water to remove unreacted RB molecules and DMF, yielding transparent pink nanoparticle solution (Figure 2a). The morphology of the PLGA-RB nanoparticles was investigated by transmission electron microscopy (TEM), which revealed the spherical shape with an average diameter of ~25 nm


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(Figure 2b). The hydrodynamic diameter of the PLGA-RB nanoparticles was characterized by dynamic light scattering (DLS), which revealed an average size of ~28 nm (Figure 2c), and was consistent with the TEM results. The zeta potential of the PLGA-RB nanoparticles was measured to be –26 mV, indicating good colloidal stability in aqueous solutions (Figure 2d).The particle size and zeta potential of PLGA-RB nanoparticles was further measured after bioconjugation of luciferase. As presented in Figure 2c, the PLGA-RB nanoparticles showed an increase in size to 33 nm, while the zeta potential changed from –26 mV to –24 mV after the luciferase bioconjugation (Figure 2d). These results demonstrated successful coating of the luciferase onto the PLGA-RB nanoparticles surface. A close proximity of luciferase and photosensitizer molecules is necessary for the occurrence of energy transfer. The biodegradability of the PLGA-RB nanoparticles was investigated. The nanoparticle morphology was assessed by TEM on day 1 and day 25 after preparation. As shown in Figure 2e, the PLGA-RB nanoparticles exhibited a spherical shape on day 1. However, the shape of the particles tended to be blurry and irregular on day 25, indicating slight decomposition. As reported in the literature, PLGA is one of the promising biodegradable polymers suitable for therapeutic applications, such as tissue regeneration and drug delivery systems.29-30 Besides, PLGA loses half of its tension by 6 months, and is completely absorbed in a few years.29 Optical Properties. The absorption and fluorescence properties of the PLGA-RB nanoparticles were further characterized by optical spectroscopy. The UV-vis absorption spectrum of the PLGA-RB nanoparticles is shown in Figure 2f. The absorption band of the PLGA-RB nanoparticles ranged from 480 to 550 nm, owing to the absorption of RB photosensitizer. There was an obvious spectral overlap between the absorption of RB molecule and the luciferase bioluminescence emission spectrum, which allowed for the occurrence of


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BRET. As shown by the emission spectrum in Figure 2f, the fluorescence emission peak of the PLGA-RB nanoparticles under light excitation was around 575 nm, which originated from the RB molecules. Figure 2g shows the bioluminescence spectra in absence and presence of BRET under the same luciferase and luciferin concentrations. The emission profile of BRET activated PLGA-RB nanoparticles can be well decomposed into two separate peaks, which are consistent with the pure luciferase bioluminescence and photo-excited RB fluorescence, respectively. These results indicated that the luciferin–luciferase system is effective to stimulate the PLGA-RB nanoparticles. By comparing the bioluminescence intensities in absence and presence of BRET, the BRET efficiency was estimated to be ~58%, which is consistent with similar BRET system.34 In addition, fluorescence images of the PLGA-RB nanoparticles conjugated with luciferase at different luciferin concentrations showed apparent dose-dependent fluorescence signal (Figure 2h), indicating the possibility of the PLGA-RB nanoparticles for simultaneous imaging and therapy applications.


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Figure 2. Characterizations of the PLGA-RB nanoparticles. (a) A room-light image of the nanoparticle solution. (b) Representative TEM image of NPs. (c) Size change in PLGA-RB NPs after coating with luciferase measured by DLS. (d) Zeta potential of the PLGA-RB NPs before and after coating luciferase measured by DLS. (e). Representative TEM images of PLGA-RB NPs on days 1 and 25. (f) UV-vis absorption, emission spectra of the PLGA-RB NPs and bioluminescence spectrum of the luciferase-luciferin system. (g) The bioluminescence spectra in


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absence and presence of BRET were measured under the same luciferase and luciferin concentrations. Inset: bioluminescence in a cuvette in the dark. (h) Representative image of fluorescence in a cell plate. Formulations: 40 µg/mL PLGA-RB NPs conjugated with 20 µg/mL luciferase, the concentration gradient of luciferin was from 0 to 80 µM.

In Vitro Photodynamic Studies. The anticancer performance of the PLGA-RB nanoparticles was investigated by cellular assays. MCF-7 cells and HeLa cells were seeded into culture plates and were then respectively incubated with PLGA-RB nanoparticles of different conditions. There was no obvious cytotoxicity observed for the pure PLGA-RB nanoparticles at a concentration up to 50 µg/mL after incubation with cells for 24 and 48 h, as indicated by the MTT assay (Figure 3a and Figure 3b), which indicated good biocompatibility of the PLGA-RB nanoparticles in the absence of light. In addition, nearly no cell death was observed after incubation with luciferin for 24 and 48 h (Figure 3c and Figure 3d). Moreover, as presented in Figure 3e, the 20 µg/mL luciferase alone showed no cellular toxicity after incubation with for 48 h. However, the cytotoxicity of PLGA-RB nanoparticles conjugated with luciferase was apparent in the presence of the substrate luciferin. As seen from Figure 3f, the cancer cells were killed in a luciferindependent manner from 0 to 60 µmol/L. Approximately 55% of the cells were killed when the concentrations of luciferin increased to 60 µmol/L. However, when the concentration of luciferin further increased to 80 µmol/L, the cell viability changed little. This is likely because the enzyme was saturated by the excess substrate and the ROS generation reached the maximum. The in vitro BRET-PDT effect was further assessed by fluorescence imaging. MCF-7 cancer cells with BRET-PDT treatment were viewed by confocal laser scanning microscopy (CLSM).


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Cancer cells were co-stained with acetoxymethyl ester (calcein-AM) and propidium iodide (PI), which are common fluorogenic probes for cell viability detection.35 Calcein-AM can penetrate the membranes of live cells and is hydrolyzed by intracellular ester enzyme to calcein, which fluoresces green light under 490-nm irradiation. PI is a nuclear dye that cannot permeate the intact cell membrane and it emits red fluorescence under 490-nm excitation, thus allowing the visualization of apoptotic or dead cells. As shown in Figure 3g, obvious green fluorescence could be observed when cells were cultured with PLGA-RB nanoparticles in the absence of light, while no red fluorescence was observed. Similar results were observed when cancer cells were incubated with PLGA-RB nanoparticles conjugated with luciferase or incubated with the PLGARB nanoparticles plus luciferin (no luciferase). In contrast, when cells were cultured with the PLGA-RB nanoparticles conjugated with luciferase plus luciferin, green fluorescence from the cytoplasm and red fluorescence from the nucleus were simultaneously observed. Nearly 50% of the cells showed red fluorescence in the image of BRET-PDT treated cells as compared to the untreated control group, which revealed that cancer cells were effectively killed by the BRETPDT mechanism. The cellular fluorescence imaging results were consistent with the MTT results, indicating an apparent cytotoxic effect owing to the BRET induced ROS generation.


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Figure 3. In vitro cytotoxicity studies of the PLGA-RB nanoparticles activated by BRET. (a), (b) Cell viabilities of MCF-7 and HeLa cancer cells cultured with PLGA-RB nanoparticles for 24 and 48 h at different concentrations. (c), (d) Cell viability of the cancer cells cultured with luciferin for 24 and 48 h at different concentrations. (e) Cell viability of the cancer cells cultured with 20 µg/mL luciferase for 48 h. (f) Therapeutic efficiency of the PLGA-RB nanoparticles conjugated with luciferase in presence of the luciferin at different concentrations. (g) Fluorescence images of MCF-7 cells by BRET-PDT treatment. Cells were stained with calceinAM and PI. Green fluorescence indicates living cells; red fluorescence shows dead cells. PLGA-


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RB nanoparticles (PLGA-RB NPs): 40 µg/mL, PLGA-RB NPs conjugated with 20 µg/mL luciferase (NP-luciferase), luciferin: 60 µmol/L. In Vivo Photodynamic Therapy. To demonstrate the usefulness of BRET-PDT in vivo, H22 tumor-bearing mice were used to assess the anticancer activity. Twenty-five mice were randomly divided into five groups after tumor volume reached approximately 150 mm3. The tumor-bearing mice were respectively treated with intratumoral injections of PBS, PLGA-RB nanoparticles conjugated with luciferase, PLGA-RB nanoparticles plus luciferin, PLGA-RB nanoparticles (with external light irradiation), PLGA-RB nanoparticles conjugated with luciferase plus luciferin. PLGA-RB NPs can be trapped in the tumor after intratumoral injection due to the enhanced permeability and retention (EPR) effect of tumor tissues. Since the nanocomposites do not have specific targeting against cancer cells, the nanoparticle distribution after intravenous injection would be higher in liver than that in tumor.36 Tumor volume and body weight were recorded every 2 days. Mice in the fourth group were exposed to a LED array that emits 520-nm radiations at a power density of 200 mW/cm2 for 30 min to stimulate the PLGA-RB nanoparticles. As presented in Figure 4a, the light-treated and BRET-PDT groups showed apparent tumor-growth inhibition, while the tumors in the control groups displayed rapid growth. Meanwhile, the BRET-PDT group showed the most remarkable inhibition. These results were consistent with the in vitro MTT results. On day 14 after treatment, the tumor volumes in the control groups were increased by approximately 6-fold, while the PDT groups showed obvious growth inhibition (Figure 4b). The mouse body weight was also used to evaluate the therapeutic effect. As shown in Figure 4c, no obvious body weight loss was observed in all five groups, suggesting the low side toxic effect of the treatment of BRET-PDT. Figure 4d shows representative tumors of the five groups excised after 14 days. The tumor sizes in the BRET-


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PDT group were apparently smaller than those in the control groups, which was attributed to the ROS generation of the PLGA-RB nanoparticles activated by the BRET-PDT mechanism. It is worth noting that the growth inhibition of subcutaneous tumors by the BRET-PDT process was comparable to the same PLGA-RB nanoparticles dose activated by external light excitation. This comparison confirmed the superior therapeutic effect by the BRET-PDT method, which might be particularly useful for treatment of deep-seated tumors as compared to the use of the external light source.


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Figure 4. In vivo animal studies of the PLGA-RB nanoparticles by BRET-PDT. (a) Tumor growth curves of five groups. Data are presented as the mean ± SD (n = 5). (b) Average tumor volume of five groups on day 0 and day 14. (c) Growth curves (body weight) of five groups. (d) Photographs of tumors excised from the BRET-PDT treatment and other groups on day 14. (e)


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Photographs of tumor-bearing mice from the five groups on day 14 and representative H&Estained histological sections of tumors in the five groups after treatment. Scale bar: 200 µm.

Bioluminescence signal was successfully detected from the PLGA-RB nanoparticles conjugated with luciferase at the subcutaneous site of the mice in the presence of the luciferin (Figure S1a and S1c). As shown in Figure S1b and S1d, the luminescence intensity was enhanced with increasing the substrate concentration. Additionally, the resected H22 tumors were subjected to hematoxylin and eosin (H&E) staining to evaluate apoptosis and necrosis of cells. As shown in Figure 4e, tumors in the BRET-PDT group showed severe cell death, with tissue disintegration and nuclear damage, while a large fraction of tumor cells in the untreated and control groups survived. These results demonstrated the good antitumor performance of PLGA-RB nanoparticles by BRET activation. Meanwhile, the main organs of mice, including heart, liver, spleen, lung, and kidney, were evaluated by H&E staining. Compared with the control group, the organs in BRET-PDT group did not show any pathological tissue damage or abnormality, indicating minimal toxicity of the PLGA-RB nanoparticles to the main organs (Figure S2). CONCLUSION In Summary, we developed a method that uses BRET for photodynamic treatment to overcome the light-penetration limitation. We used the biodegradable polymer PLGA to achieve biocompatibility of the nanoparticles, which were doped with the photosensitizer RB for efficient ROS production. Spectroscopic characterization indicated that BRET can effectively activate RB to generate ROS and destroy cancer cells. MTT assays demonstrated that the cancer cells were effectively killed by the BRET-PDT treatment. The antitumor efficacy in vivo was investigated


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in a tumor-bearing mouse model, which showed that tumors were inhibited by BRET-PDT, without the need for external light irradiation. Our results demonstrated that the BRET-PDT method has great potential for cancer therapy and provides a promising method to overcome the light-penetration limitation in PDT of deep-seated tumors.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Representative fluorescence images of mice (Supplementary Figure 1), H&E-stained tissue of main organs (Supplementary Figure 2). AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Changfeng Wu) Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the grants from the National Natural Science Foundation of China (Grant No. 61335001; Grant No. 81771930) ，and Shenzhen Science and Technology Innovation Commission (Grant No. JCYJ20170307110157501).


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TOC Figure


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Biodegradable Polymer Nanoparticles for Photodynamic Therapy by

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