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Carbon Dots Decorated Carbon Nitride Nanoparticles for Enhanced Photodynamic Therapy against Hypoxic Tumor via Water Splitting Di-Wei Zheng, Bin Li, Chu-Xin Li, Jin-Xuan Fan, Qi Lei, Cao Li, Zushun Xu, and Xian-Zheng Zhang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.6b04156 • Publication Date (Web): 17 Aug 2016 Downloaded from http://pubs.acs.org on August 17, 2016
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Carbon
Dots
Nanoparticles
Decorated for
Carbon
Enhanced
Nitride
Photodynamic
Therapy against Hypoxic Tumor via Water Splitting Di-Wei Zheng1,2,#, Bin Li1,#, Chu-Xin Li1, Jin-Xuan Fan1, Qi Lei1, Cao Li2, Zushun Xu2, Xian-Zheng Zhang1,*
1
Key Laboratory of Biomedical Polymers of Ministry of Education, Institute for Advanced Studies (IAS), Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China.
2
Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Key Laboratory for the Green Preparation and Application of Functional Materials of Ministry of Education, Hubei University, Wuhan, Hubei 430062, P. R. China
* Corresponding author. Email:
[email protected] #
These authors contributed equally to this work
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ABSTRACT: Hypoxia, a typical feature of solid tumors, remarkably restricts the efficiency of photodynamic therapy (PDT). Here, a carbon nitride (C3N4) based multi-functional nano-composite (PCCN) for light driven water splitting was used to solve this problem. Carbon dots were first doped to C3N4 to enhance its red region absorption since red light could be used to trigger the in vivo water splitting process. Then, a polymer containing protoporphyrin (PpIX) photosensitizer, polyethylene glycol (PEG) segment and targeting Arg-Gly-Asp (RGD) motif was synthesized and introduced to carbon dots doped C3N4 nanoparticles. In vitro study showed that PCCN, thus obtained, could increase the intracellular O2 concentration and improve the reactive oxygen species (ROS) generation in both hypoxic and normoxic environments upon light irradiation. Besides, cell viability assay demonstrated that PCCN fully reversed the hypoxia triggered PDT resistance, presenting a satisfactory growth inhibition of cancer cell in an O2 concentration of 1%. In vivo experiments also indicated that PCCN had superior ability to overcome tumor hypoxia. The use of water splitting materials exhibited great potential to improve the intratumoral oxygen level and ultimately reverse the hypoxia triggered PDT resistance and tumor metastasis. KEYWORDS: water splitting, photodynamic therapy, carbon nitride nanoparticle, hypoxic tumor
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Photodynamic therapy (PDT), which utilizes O2 to generate cytotoxic reactive oxygen species (ROS), is becoming a promising method in cancer treatment owning to its noninvasive feature, high efficiency and ideal accuracy.1-3 However, PDT process highly relies on the local oxygen level. Unfortunately, hypoxia is a key feature of tumor microenvironment (pO2 ≤ 2.5 mmHg) and the low O2 level restricts the efficiency of PDT.4 Owing to the PDT-induced oxygen consumption, worsened hypoxia would cause irreversible tumor metastasis or drug resistance.5 To solve this problem, clinical trials used hyperbaric oxygen therapy (HBO) to improve the therapeutic effect of PDT. However, serious side effects, including hyperoxic seizures and barotrauma, greatly limit the efficiency of HBO.6,7 Recent reports have indicated that O2 generation materials, such as perfluorohexane, catalase and MnO2 could improve intratumoral O2 supplement and enhance the efficiency of PDT, radiotherapy and chemotherapy.8-14 Perfluorohexane is able to enrich intratumoral O2 and induces oxygen self-enriching PDT. However, the use of perfluorohexane has limited effect in reducing intratumoral hypoxia. Catalase or MnO2 could catalyze H2O2 decomposition and produce O2, whereas the low intracellular raw materials concentration (H2O2 < 50 µM) greatly limits the O2 generation amount of catalase and MnO2. In nature, plants integrate light-harvesting, charge generation/separation and catalytic reaction into chloroplast to produce O2 from abundant H2O with high efficiency. Inspired from this, nano-composite based on inorganic, organic, 3
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macromolecular or hybrid materials have been used as solar-driven water splitting materials to produce H2 and O2 directly from water.15,16 In the past decade, water splitting materials have attracted extensive concern for its promising applications in solving energy and environmental issues. Nevertheless, the biomedical use of water splitting materials has not been explored. Since water is also the most abundant compound in living organism, and compared with previous O2 generating materials, using water as source would provide unlimited row materials for in vivo O2 generation. Among various types of water splitting materials, carbon nitride (C3N4) has attracted considerable attention for their adjustable band gap and band position. After the modification, water splitting can be driven under high penetrable red light ( > 600 nm), which makes C3N4 suitable for in vivo therapy.17,18 Most importantly, due to the absence of metal elements, C3N4 was considered as a highly biocompatible material for biomedical applications. Keeping all these issues in mind, we hypothesize that enhanced PDT to fight against hypoxic tumor could be achieved by using water splitting materials. Here, metal-free carbon nitride (C3N4) was chosen as a promising water splitting material. Since C3N4 has limited water splitting efficiency in red light region, carbon dots was prepared and decorated into C3N4. After a ball-milling process, carbon dots decorated C3N4 nanocomposite (CCN) with an enhanced red light absorption was synthesized.19 In addition, an amphipathic polymer, PpIX-PEG-RGD consisted of photosensitizer protoporphyrin IX (PpIX) and tumor targeting sequence RGD using the PEG as the 4
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linker was assembled with CCN based on π-π stacking between PpIX and C3N4 to obtain polymer modified, carbon dots doped carbon nitride nanoparticles (PCCN). When PCCN arrives and accumulates at the tumor tissue through active RGD targeting and enhanced permeability and retention (EPR) effect, 630 nm laser is performed to trigger PCCN to split water for generating O2. Besides, photosensitizer could further transmit the energy to the produced O2 to generate cytotoxic singlet oxygen (1O2) for cancer treatment under 630 nm laser irradiation. The design of PCCN is diagramed in Figure 1 and this strategy would overcome the restriction of hypoxia in PDT.
RESULTS AND DISCUSSION Characterization of PCCN. In this study, a simple thermal decomposition method was performed to synthesize CCN derived from urea and carbon dots. Then, a ball-milling process was used to prepare CCN nanoparticles. Amphipathic polymer PpIX-PEG-RGD was synthesized and its molecular weight was determined to be 1313 by electrospray ionization mass spectrometry. After self-assembly based on π-π stacking between PpIX and C3N4, PCCN was further prepared. The mechanism of C3N4 induced water splitting was illustrated in Figure 2a. C3N4 has a relatively small band gap (ca. 2.7 eV), and it can be activated under blue light (~420 nm). Then, C3N4 transmits the energy to water and induces the water splitting reaction (requiring 1.23 eV theoretically).20 In this paper, carbon dots was doped to decrease the band gap of C3N4, and red light could be used to trigger the water splitting. As shown in Figure 2b, 5
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C3N4 nanoparticles had obvious absorption at around 340 nm, but weak absorption at red light region. After the doping of carbon dots, nanoparticles displayed an enhanced visible absorption over the entire wavelength range. Indicated by the dynamic light scattering analysis, PCCN showed a hydrodynamic size of 180 nm (PDI = 0.19) and transmission electron microscope (TEM) images also suggested the formation of nanocomposite (Figure S1). X-ray diffraction (XRD) analysis demonstrated that C3N4 atomic structure was largely retained after various modifications (Figure S2). X-ray photoelectron spectra (XPS) was characterized with monochromated Al Ka X-ray source. High oxygen atomic ratio and appearance of amido bond in PCCN indicated the successful modification of polymer (Figure 2d and S3). Thermogravimetry analysis also indicated the polymer incorporating as shown in Figure S4. Also, the PpIX loading capacity of PCCN was calculated to be 9.6%. The interaction between PpIX-PEG-RGD and CCN was further studied and PCCN was added into sodium dodecyl sulphate, Tween 20, urea, NaCl and EDTA solutions, respectively. After 2 h co-incubation, supernatants were collected and UV-Vis spectrum was performed. About 80% of PpIX-PEG-RGD was released after Tween 20 and sodium dodecyl sulphate treatment. This result indicated that hydrophobic competition triggered the self-assembly between PpIX-PEG-RGD and CCN. In contrast, treatments of urea (eliminating hydrogen bonds), NaCl (shielding electrostatic force) and EDTA (eliminating coordination bond) were ineffective for PCCN disassembly.21
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In Vitro Study of Water Splitting and PDT Enhancement. To further prove the water splitting ability of PCCN, a dissolved oxygen meter was used to measure the O2 generation in PBS upon the irradiation of 630 nm laser. After sodium thiosulfate treatment, O2 in PBS was consumed to obtain deoxygenated PBS. Notably, a liquid paraffin seal was also used to isolate deoxygenated PBS from air. As shown in Figure 2e, instantly after the light irradiation, increased O2 concentration could be observed in PCCN and CCN dispersed solutions. After 15-min light irradiation, CCN dispersed solution showed an oxygen concentration of 1.86 times higher than that in C3N4 dispersed solution. The enhanced water splitting effect under 630 nm laser was attributed to the carbon dots doping. PpIX would consumed part of O2 produced by CCN, thus, compared with CCN, reduced O2 generation of PCCN was observed. However, the water splitting triggered O2 generation of PCCN was still faster than its PDT induced O2 consumption. Polymer modified carbon nitride (PCN) without carbon dots decoration showed no O2 generation under 630 nm laser. Based on the experiments, a constant O2 level was found without laser irradiation. Then, the ROS sensor, 2′,7′-Dichlorofluorescin (DCFH) was used to measure the in vitro ROS generation ability of materials in both hypoxic and normoxic PBS.22 As shown in Figure 2f, in the normoxic environment, upon the 15-min laser irradiation for PpIX, PCN and PCCN, similar amount of ROS was produced. After 15 minutes of irradiation in hypoxic environment, the ROS production of PpIX and PCN was extremely weak. Interestingly, in the hypoxic environment, remarkable fluorescence of 7
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DCFH was detected in the PCCN group, verifying the unaffected ROS generation ability under light irradiation. This result indicated that PCCN may overcome hypoxia in cancer cells and improve the PDT efficacy. After confirming light driven water splitting and PDT enhancement of PCCN in PBS, its intracellular O2 generation and ROS production were studied in 4T1 cells. ROS-ID™ is a fluorogenic probe for intracellular hypoxia detection. In the normoxic environment, ROS-ID™ hypoxia detection probe is a weakly fluorescent compound with a nitro group. However, in a hypoxic intracellular environment, nitro group is reduced to hydroxylamine and amino groups, inducing the red fluorescence recovery.23 As shown in Figure 3a, in the normoxic environment, PpIX and PCN treatment displayed red fluorescence. Whereas, in the hypoxic environment, remarkably enhanced red fluorescence was observed in PpIX and PCN treated 4T1 cells after light irradiation. In contrast, weak fluorescence was observed in PCCN treated cells after irradiation in both normoxic and hypoxic environments, indicating PCCN could overcome PDT induced hypoxia. The overcoming of hypoxia also enhanced the ROS generation ability of PDT. Here, 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was used as an intracellular ROS sensor, because DCFH-DA with nonfluorescence can be rapidly hydrolyzed by esterase and oxidized with ROS to produce fluorescent 2′,7′-Dichlorofluorescin (DCF). Confocal laser scanning microscopy (CLSM) images confirmed that in both hypoxic and normoxic environment, PCCN displayed a satisfactory ROS generation ability 8
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after light irradiation. Analyzed by Image J software, the quantitative mean fluorescence intensity study showed that the hypoxia only reduced 3% of DCF fluorescence as compared with normoxic environment. Whilst, 4T1 cells treated with PpIX and PCN had negligible fluorescence in the hypoxic environment, and the quantitative study demonstrated that hypoxia decreased 52% of DCF fluorescence. Cellular Selectivity and Cytotoxicity Studies. In order to achieve a tumor-triggered targeting, RGD sequence was introduced to PCCN. It is well known that most of cancer cells express high level of integrin αvβ3, thus PCCN could efficiently target cancer cells via RGD sequence.24 Here, αvβ3 positive 4T1 cells and αvβ3 negative MCF-7 cells were used to study the cellular selectivity of PCCN,25-27 and the western blot analysis of αvβ3 level in 4T1 cells and MCF-7 cells was shown in Figure S5. After 4-h co-incubation with PCCN, a larger area of fluorescence was observed in 4T1 cells (Figure S6). In contrast, red fluorescence was negligible in MCF-7 cells. Flow cytometry further indicated the addition of free RGD could inhibit the αvβ3 mediated uptake of PCCN in 4T1 cells (Figure S7). After illustrating the targeting ability of PCCN, the efficiency of on-demand PDT was studied. Since C3N4 is able to catalyze NADH regeneration, cell proliferation and cytotoxicity assays based on intracellular redox status would be affected.28 Herein, sulforhodamine B colorimetric assay based on cellular protein content was used, and the cytotoxicity of both PpIX and PCCN in hypoxic and normoxic environments was investigated.29 Without laser irradiation, no significant cytotoxicity of PCCN in 4T1 9
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cells was observed (Figure S8). As presented in Figure 3c, 3d and 3e after laser irradiation, PpIX and PCN showed a significant cytotoxicity in normoxic environment (21% O2), while limited cytotoxicity in hypoxic incubator (
