ROS-Responsive Nanoparticles for Suppressing the

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ROS-responsive nanoparticles for suppressing the cytotoxicity and immunogenicity caused by PM2.5 particulates Yixian Zhang, Haolan Zhang, Zhengwei Mao, and Changyou Gao Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00174 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 19, 2019

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Biomacromolecules

ROS-responsive nanoparticles for suppressing the cytotoxicity and immunogenicity caused by PM2.5 particulates Yixian Zhang1, Haolan Zhang1, Zhengwei Mao1*, Changyou Gao1,2* 1. MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China 2. Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Zhejiang University, Hangzhou 310058, China *Corresponding authors. Email: [email protected] (Z. Mao), [email protected] (C. Gao).

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Abstract Although the negative impacts of particulate matter (PM2.5) on human health have been well recognized, very a few efforts have been paid to find new strategies to suppress the toxicity of PM2.5 both in vitro and in vivo. In this study, reactive oxygen species (ROS)-responsive nanoparticles made of poly(1,4-phenleneacetonedimethylene thioketal) (PPADT) were used to load immunosuppressant drug tacrolimus (FK506) with a drug loading efficiency of around 44%. The PPADT particles showed very good ROS-responsiveness, and were degraded in an oxidation environment. By exhausting intracellular ROS, they could effectively suppress the toxicity of A549 lung epithelial cells and RAW264.7 macrophages induced by the PM2.5 particulates collected from three different regions in China. Moreover, the inflammatory response of PM2.5 could also be significantly suppressed, showing much better performance than the free FK506 drugs both in vitro and in vivo. This concept-proving research demonstrates the promising application for the ROS-sensitive drug release particles in dispelling the toxicity and suppressing the inflammation of PM2.5 pollutes, shedding a new light in the design and applications of stimuli-responsive systems in the bionanotechnology and healthcare fields. Key words: Poly(thioketal), PM2.5, ROS-responsive, particles, FK506, inflammation.

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1. Introduction The air pollution is becoming one of the biggest challenges to human health. The particulate matter (PM) especially the particles with a median aerodynamic diameter less than 2.5 m (PM2.5) can retain in the air for a long time, and can be delivered to other regions because of their small size. There are a lot of hazardous substances in the compositions of PM2.5, such as heavy metal ions, organic compounds, and microorganisms and so on.1-3 These ambient air particles can break through the nasal passage, interfere with the bronchi and alveoli, and intrude into the bloodstream, aggravating respiratory and cardiovascular diseases.4 Moreover, the components in PM2.5 smaller than 100 nm are more liberate with the ability to spread to elsewhere in human body, leading to atherosclerosis, heart attack and other diseases.5-8 According to statistics and estimation, about 800 thousand of premature deaths are related to air pollution every year worldwide.9,10 The underlying relationship between healthy issues and PM2.5 has not been totally elaborated, especially the mechanisms at cellular levels. Nonetheless, the inhalation or instillation of PM2.5 can definitely increase intracellular reactive oxygen species (ROS) to a great extent, resulting in a reduction in cell viability, cell apoptosis and even genotoxicity.11-14 Besides, PM2.5 is believed in association with the inflammatory response. For example, Chio et al15 demonstrated that the instillation of PM extract could exacerbate the neutrophils recruitment and cause lung inflammation and injury. The inhaled PM could promote cytokine release, such as tumor necrosis factor alpha (TNF-), interleukin 6 (IL-6) and transforming growth factor beta (TGF-), as well as increase of NF-B-related inflammatory and gene expression.16-18 Thus, it is urgent to find solutions dealing with the cytotoxicity and inflammatory response induced by PM2.5. As the released heavy metal ions and the aroused intracellular ROS are the leading causes for cytotoxicity and tissue damage, chelating the

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heavy metal ions and reducing the ROS level may be the key point for eliminating the toxicity induced by PM2.5. In our previous work, amino acids-modified chitosan nanoparticles (NPs) are used to chelate the released Cu2+ and thus significantly suppress the cytotoxicity of CuO NPs.19 Besides, the involvement of antioxidants such as polyphenols, vitamin C and mannitol can effectively decrease intracellular ROS level and thereby reduce the cytotoxicity of nanomaterials.20,21 For example, Zhang et al reported that bovine serum albumin (BSA) particles encapsulated with curcumin can efficiently scavenge intracellular ROS and reduce the cytotoxicity of CuO nanoparticles.22 However, the adverse effects of these antioxidants such as toxicity at high concentration limit their application. Stimuli-responsive drug delivery systems have attracted great attention over the past decades, as the drug can be released on demand with a fewer side effects. These materials can be sensitive to external stimuli such as light,23,24 temperature,25 magnetic field26 or some intracorporal environments such as enzyme,27 pH,28 glutathione,29 and ROS30-35. In particular, ROS-responsive materials have been popularly used as smart gene and drug delivery vehicles because a higher ROS level is involved in tissue injury and many chronic diseases, such as atherosis, diabetes and rheumatism.36-38 The ROS in normal serum is about 1~810-6 mol/L, which can be activated to 10~1000 times higher in predilecting sites.39 Xia et al40 prepared a poly(amino thioketal) (PATK) for DNA delivery, and found that the PATK/DNA complexes are able to achieve efficient cell transfection in prostate cancer cells. Martin et al41 demonstrated that the ROS-responsive polyurethane can be degraded at the damaged tissue with a varied degree of degradation according to the tissue repairment. Lee and his colleagues 42-44 prepared a series aromatic oxalate-based polymer particles and verified their ROS-responsiveness in the application of oxidation-sensitive prodrug release. In this study, ROS-responsive poly-(1,4-phenleneacetonedimethylene thioketal) (PPADT) NPs loaded

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with tacrolimus (FK506) are prepared for suppressing the toxicity and inflammation caused by PM2.5. PPADT is predicted as a ROS scavenger because of its ROS-responsiveness,45 which can be degraded by intracellular ROS and thus consume exorbitant ROS (Scheme 1). The inhibiting effects of cytotoxicity and secretion of inflammatory factors induced by PM2.5 collected from three different regions in China are studied by simultaneously co-incubating the PM2.5 with A549 lung epithelial cells and RAW264.7 macrophages in the existence of PPADT NPs or PPADT/FK506 NPs, respectively. The in vivo detoxifying effects are also investigated with intratracheal instillation to rats. To the best of our knowledge, this is the first study to suppress the cytotoxicity of PM2.5 with a ROS consumer polymer NPs and to inhibit simultaneously the inflammation with an immunosuppressant, which shows stimuli-responsive delivery under ROS induced by PM2.5.

2. Materials and methods 2.1 Materials 1,4-Benzenedimethanethiol (BDT), 2,2-dimethoxypropane (DMP), poly(vinyl alcohol) 1788 (alcoholysis degree=87~89%) and p-toluenesulfonic acid (PTSA) were purchased from Aladdin Chemistry Co. Ltd., China. Anhydrous toluene, anhydrous ethyl acetate, n-hexane, Nile red (NR), sodium chloride (NaCl) and ethylene diamine tetraacetate acid disodium (EDTA) were purchased from

Sinopharm

Chemical

Reagent

Co.,

Ltd.,

China.

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich. Dulbecco’s modified eagle medium (DMEM), trypsin and other cell culture reagents were obtained from Life technology, USA. RIPA Lysis Buffer was purchased from Beyotime Biotechnology. Micro BCA Protein Assay Kit was purchased from Thermo Fisher Scientific Co., Ltd.

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Enzyme linked immunosorbent assay (ELISA) kits for TNF-α and IL-6 were bought from Boster, China. All chemicals were of analytical grade and used as received if not specifically described. Milli-Q water was used throughout the experiments.

2.2 Preparation and characterization of PPADT NPs 2.2.1 Polymer synthesis and characterization Poly(1,4-phenleneacetonedimethylene thioketal) (PPADT) was synthesized via a condensation polymerization.46 Briefly, two-necked flask was charged with distilled toluene, BDT (0.68 g, 4 mmol) and DMP (492 μL, 4.0 mmol), and then equipped with a metering funnel and distillation head for removal of the methanol by-product. The mixture was stirred continuously and heated to 95 oC before a catalytic amount of re-crystallized PTSA (2.29 mg, 0.012 mmol) in 2 mL distilled ethyl acetate was added to start the reaction. After 1 h, a solution of DMP (492 μL, 4.0 mmol) in 10 mL anhydrous toluene was added to the metering funnel, and the funnel stopcock was set so that DMP was added drop-wise at the rate of 30 μL/min for 12 h. The reaction was magnetically stirred for additional 12 h at 95 oC to achieve sufficient reaction. The resulting polymers were isolated by precipitation twice in cold n-hexane and dried at 60 oC under vacuum to yield a brown solid. The polymers were vacuum-dried. Their structure was confirmed by

1H

nuclear magnetic resonance (1HNMR)

spectroscopy (Bruker DMX500 equipment operated at 500 MHz using CDCl3 as solvent and tetramethylsilane as reference): δ (ppm), 7.28 (4H), 3.85 (4H), and 1.60 (9H). Molecular weight and molecular weight distribution was measured on a Waters 1515 gel permeation chromatography setup using poly(methyl methacrylate) standards for calibration. Tetrahydrofuran was used as an eluent at a flow rate of 0.5 mL/min at 40 °C.

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The polymers were incubated in 5 mM H2O2 solution at 37 oC for different time, and the variation of their molecular weight was monitored by GPC. It is worth mentioning that the PPADT was degraded too fast or too slow in other concentrations of H2O2. 2.2.2 Preparation and characterization of PPADT NPs The PPADT NPs loaded with FK506 (PPADT@FK506) were prepared via an oil-in-water (O/W) emulsification and solvent evaporation method.47 Briefly, FK506 (10 mg) and PPADT (25 mg) were dissolved in 1 mL chloroform to form the oil phase, which was filtered through a 0.22 μm filter to remove the undissolved substances. This organic solution was then added to 10 mL of 1% (w/v) PVA aqueous solution, which was then vortexed thoroughly. The suspension was ultrasonicated using a probe sonicator with 40% amplitude (output power 13 W, Sonicator 4000, Misonix, USA, equipped with a probe with a tip diameter of 3.2 mm) in an ice bath for 5 min. The organic solvent in the suspension was evaporated in vacuum. The nanoparticles (NPs) were finally recovered by centrifuging the remaining mixture at 10,000 g for 10 min. The non-encapsulated FK506 and the dispersants were removed by washing 6 times with sterilized water. Nile red-loaded NPs (PPADT@NR) were prepared with the same method except that 500 μg of Nile red was dissolved in 0.5 mL chloroform and added to a solution of PPADT (25 mg) in chloroform (1 mL). Unloaded PPADT NPs were made following the same procedures without addition of FK506 or Nile red. All the particles were stored at 4 oC before use. The morphology of PPADT and PPADT@FK506 NPs was measured by transmission electron microscopy (TEM, Philips TECNAL-10). The sample for TEM was prepared by adding a drop of the particles suspension in water onto a copper grid with a carbon membrane and dried at ambient condition overnight. The size and surface charge of the particles were determined using Beckman

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Delsa TM Nano (Beckman Coulter) with a He–Ne laser operating at a wavelength of 677 nm and temperature of 25 oC. The particles were dispersed in water and DMEM containing 10% fetal bovine serum (FBS, Sijiqing Co. Ltd., Hangzhou, China), respectively. To measure the amount of FK506 loaded within the NPs, 1 mL of the sample was lyophilized and redissolved in 1 mL of chloroform in a bath type sonicator for 1 h to dissolve the polymers and extract FK506. After filtration through a 0.45 μm filter, the amount of FK506 in the filtrate was analyzed using reverse phase high-performance liquid chromatography (HPLC). A calibration curve was obtained using a series of FK506 solutions of different concentrations. PPADT NPs were incubated in 5 mM H2O2 solution at 37 oC for different time, and the hydrodynamic diameter and morphology variation of the NPs were characterized by DLS and TEM, respectively. 2.2.3 Intracellular stimulus release To study the effect of PM2.5-stimulated enhancement of intracellular ROS on drug release behavior of PPADT@FK506 NPs, the PPADT@NR NPs were chosen as a model to investigate the ROS-responsiveness of PPADT particles. In brief, A549 and RAW264.7 cells were seeded on 24-well plates at a density of 4.0×104 and 6.4×104 cells per well, and were allowed to attach for 16 h, respectively. Then the culture medium was replaced with fresh one containing 100 µg/mL PPADT@NR NPs or 100 µg/mL PM2.5 collected from Beijing and 100 µg/mL PPADT@NR NPs for different time, respectively. At pre-determined time intervals, the cells were washed three times with PBS and detached by trypsinization. The uptake amount of NPs was determined by flow cytometry (FACS Calibur, BD). Confocal laser scanning microscopy (CLSM; LSM 510, Carl Zeiss) was used to evaluate the NR release from the PPADT@NR NPs intuitively through fluorescent staining of the cell nucleus. Briefly,

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after the cells were co-incubated with 100 µg/mL PPADT@NR NPs or 100 µg/mL PM2.5-BJ and 100 µg/mL PPADT@NR NPs for 3 h, 12 h and 24 h, they were carefully washed three times with PBS, and fixed with 4% paraformaldehyde solution for 30 min at room temperature. After further treatment with 0.5% Triton/PBS solution at 4 oC for 10 min and washing three times with PBS, they were dispersed in 1% BSA/PBS solution to block nonspecific adsorption for 2 h. The cell nuclei were finally stained with DAPI (100 ng/mL) at 37 oC for 1 h. After washing three times with PBS, the cells were observed under CLSM.

2.3 Collection and cytotoxicity of PM2.5 2.3.1 Collection of PM2.5 PM2.5 was collected using Libra Plus 20 (A.P. BUCK, USA) from three typical areas of China with different air quality: (1) in front of the Department of Polymer Science and Engineering in the campus of Zhejiang University, Hangzhou, Zhejiang province (PM2.5-HZ); (2) nearby Jiyuan steel mill in Jiyuan, Henan province (PM2.5-JY); (3) Haidian District in Beijing (PM2.5-BJ). The particulates were collected on SiO2 fiber filters (Munktell, Sweden) and weighed before use. The filter with certain amount of PM2.5 was sterilized under UV light for 15 min, cut into pieces and immersed into sterile water. The particles were released with the assistance of ultrasonication (30 min). The solution containing PM2.5 was centrifuged at 10,000 g for 10 min. The supernatant was carefully collected and was sterilized by passing through a 220 nm filter, which was recognized as PM2.5 leaching medium. Meanwhile, the substance at the bottom after centrifugation was redispersed into water by ultrasonication, filtered with an eight-layer gauze for three times and centrifuged at 10,000 g for 10 min to collect the particulate component in PM2.5. Finally, the solid constituent was mixed with

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PM2.5 leaching medium to get the PM2.5 suspension. 2.3.2 Characterization of PM2.5 The morphology of the insoluble substance in PM2.5 was observed by a scanning electron microscope (SEM, FEI SIRION-100). The SEM samples were prepared by adding a drop of the particulate component of PM2.5 in water onto a small piece of glass and dried at ambient condition. The size and surface charge of the PM2.5 were determined using a Delsa Nano instrument (Beckman Coulter) with a He–Ne laser operating at a wavelength of 677 nm and at 25 oC. The suspension was dissolved in aqua regia (HCl: HNO3=3:1, v/v), and the metal contents in the mixture were quantified using inductively coupled plasma-mass spectrometry (ICP-MS, XSENIES, USA). 2.3.3 Cytotoxicity of PM2.5 A549 cells and RAW264.7 cells were purchased from Cell Bank of Typical Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cells were maintained in a regular growth medium consisting of high glucose DMEM (Gibco, USA), supplemented with 10% fetal bovine serum (FBS, Sijiqin Inc., China), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 oC in a 5% CO2 humidified incubator. A549 and RAW264.7 cells were plated at a density of 1.0×104 and 1.6×104 cells per well in a 96-well plate and cultured overnight, respectively. The medium was replaced with fresh one containing different concentrations of PM2.5 suspension derived from different areas. After being incubated with the PM2.5 for 24 h, the cells were further incubated with a medium containing MTT (0.5 mg/mL) for 3 h. The dark blue formazan crystals generated by mitochondrial dehydrogenase in living cells were dissolved in dimethylsulfoxide. The absorbance at 570 nm was measured by a microplate reader (Biorad model 680). The data were normalized to that of untreated control cells (100%).

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2.4 Toxicity suppression of PPADT NPs and PPADT@FK506 NPs The cytotoxicity of PPADT NPs and PPADT@FK506 NPs with various concentrations was evaluated by MTT assay. To appraise the suppression effect of PPADT NPs to the cytotoxicity induced by PM2.5, the A549 and RAW264.7 cells were co-cultured with 100 µg/mL PPADT NPs or PPADT@FK506 NPs and 100 µg/mL PM2.5-HZ, PM2.5-JY and PM2.5-BJ for 24 h, respectively. The cell viability was measured by the MTT assay, and the data were normalized to that of the untreated control cells (100%). The oxidation-sensitive probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma-Aldrich) was employed to determine the intracellular ROS level. DCFH-DA is an amphiphilic nonfluorescent molecule that readily crosses the cell membrane, and is deacetylated by esterases and then oxidized to the highly fluorescent 2’,7’-dichlorfluorescein (DCF) in the presence of intracellular ROS.48,49 In this study, A549 and RAW264.7 cells were seeded on 24-well plates at a density of 4.0×104 and 6.4×104 cells per well, and were allowed to attach for 16 h, respectively. The cells were then incubated with 100 µg/mL PM2.5 suspension from three regions or the mixture of 100 µg/mL PM2.5 and 100 µg/mL of PPADT NPs or PPADT@FK506 NPs for 24 h, respectively. The intracellular ROS generation was studied by flow cytometry (FACS Calibur, BD) according to the method described previously.50 Cells treated with 10 mM H2O2 for 10 min and untreated cells were used as positive and negative controls, respectively.

2.5 Inflammatory modulation of PPADT@FK506 NPs The RAW264.7 cells were seeded on a 96-well plate at a density of 1.6×104 cells per well and cultured

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overnight to allow cell attachment. They were then incubated with 100 µg/mL PM2.5 suspension from three regions or the mixture of 100 µg/mL PM2.5 and 100 µg/mL of PPADT NPs or PPADT@FK506 NPs for 24 h, respectively. Secretion of TNF-α and IL-6 was measured in the supernatant of the cultured RAW264.7 cells by ELISA kits according to the user manual. The remained cells were lysed with RIPA lysis buffer, and the protein content that is proportional to the cell number was measured by a micro BCA kit. Finally, the relative secretion of TNF-α and IL-6 was normalized to the protein content. Untreated cells were used as a negative control.

2.6 Instillation and inflammation assessment in vivo Male SD rats, approximately 220 g, were used for the in vivo assessment. The procedures were performed in accordance with the ‘‘Guidelines for Animal Experimentation” by the Institutional Animal Care and Use Committee, Zhejiang University. The animals were anesthetized with halothane, cannulated with a laryngoscope to expose the trachea, and 200 µL NPs suspensions in saline were instilled into the lungs every 3 d for 3 times, respectively: 1) 0.9% NaCl (control), 2) 1 mg/mL PM2.5, 3) 1 mg/mL PM2.5 + 1 mg/mL PPADT NPs, 4）1 mg/mL PM2.5 + 1 mg/mL PPADT@FK506 NPs and 5) 1 mg/mL PM2.5 + 360 µg/mL free FK506 (the same drug amount as 1 mg/mL PPADT@FK506 NPs). Animals were further raised for 3 d after the last instillation, and then sacrificed with a single intraperitoneal injection of 2 mL sodium pentobarbitone (200 mg/mL). The trachea was cannulated with a luer port cannula, which was then tied in place. The trachea and lungs were harvested by dissection for H&E staining, and the lungs were lavaged in situ with a single 8 mL volume of sterile saline for analysis of released lactate dehydrogenase (LDH). Besides, part of the lungs was grinded with a slurry pipe and centrifuged at 3000 rpm for 15 min, and the TNF-α and IL-6

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contents in the supernatant were analyzed with the ELISA kits, respectively. The results were normalized to the particle-free control.

2.7 Statistical analysis The data are expressed as mean ± standard deviation with 5 and 8 samples in parallel for cell and rat experiments, respectively. The statistical significant difference between groups is determined by one-way analysis of variance (ANOVA) in the Origin software. The Tukey Means Comparison method is performed, and the statistical significance is set as p

ROS-Responsive Nanoparticles for Suppressing the

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