Polyhexamethyleneguanidine phosphate induces cytotoxicity through disruption of membrane integrity

Accepted Manuscript Title: Polyhexamethyleneguanidine phosphate induces cytotoxicity through disruption of membrane integrity Authors: Jeongah Song, Kyung Jin Jung, Seok-joo Yoon, Kyuhong Lee, Bumseok Kim PII: DOI: Reference:

S0300-483X(19)30002-2 https://doi.org/10.1016/j.tox.2019.01.001 TOX 52145

To appear in:

Toxicology

Received date: Revised date: Accepted date:

4 July 2018 17 December 2018 3 January 2019

Please cite this article as: Song J, Jung KJ, Yoon S-joo, Lee K, Kim B, Polyhexamethyleneguanidine phosphate induces cytotoxicity through disruption of membrane integrity, Toxicology (2019), https://doi.org/10.1016/j.tox.2019.01.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Polyhexamethyleneguanidine phosphate induces cytotoxicity through disruption of membrane integrity

Jeongah Song1, Kyung Jin Jung2, Seok-joo Yoon3, Kyuhong Lee4,5*, Bumseok Kim6*

Animal Disease Research center, Korea Institute of Toxicology, Jeonbuk 56212, Korea

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Analytical Research center, Korea Institute of Toxicology, Daejeon 34114, Korea

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Systems Toxicology Center, Predictive Toxicology Department, Korea Institute of Toxicology, Daejeon 34114,

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Korea

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Inhalation Toxicology Research Center, Korea Institute of Toxicology, Jeonbuk 56212, Korea

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Human and Environment Toxicology, University of Science and Technology, Daejeon 34114, Korea

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Biosafety Research Institute and Laboratory of Pathology (BK21 Plus Program), College of Veterinary Medicine,

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Chonbuk National University, Iksan, 54596, Korea

Bumseok Kim, Ph.D.

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* Corresponding author

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Biosafety Research Institute and Laboratory of Pathology (BK21 Plus Program),

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College of Veterinary Medicine, Chonbuk National University, 79, Gobong-ro, Iksan 54596, Korea Tel.: +82-63-850-0953

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E-mail: [email protected]

Kyuhong Lee, Ph.D. Inhalation Toxicology Research Center, Korea Institute of Toxicology, Human and Environment Toxicology, University of Science and Technology,

30, Baekhak 1-Gil, Jeongeup-si, Jeollabuk-do 56212, Korea Tel.: +82-63-570-8740 Fax: +82-63-570-8797 E-mail: [email protected], [email protected]

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Abstract Polyhexamethyleneguanidine phosphate (PHMG-P) is a polymeric biocide with a guanidine group. It has multiple

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positive charges in physiological conditions due to nitrogen atom in the guanidine and this cationic property contributes antimicrobial effect by disrupting cell membranes. To determine whether the cationic nature of PHMG-P results in cytotoxicity in human cell lines, anionic compounds were treated with PHMG-P. The cytotoxic

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effect was evaluated with ROS production and HMGB1 release into media. To verify the protection effect of anion against PHMG-P-induced cell death in vivo, a zebrafish assay was adopted. In addition, membrane disruption by

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PHMG-P was evaluated using fluorescein diacetate and propidium iodine staining. As a result, anionic substances

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such as DNA and poly-L-glutamic acids, decreased PHMG-P induced cell death in a dose-dependent manner.

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While HMGB1 and ROS production increased with PHMG-P concentration, the addition of anionic compounds with PHMG-P reduced the ROS production and HMGB1 release. The mortality of the zebrafish increased with

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PHMG-P concentration and co-treatment of anionic compounds with PHMG-P decreased mortality in a dosedependent manner. In addition, FDA and PI staining confirmed that PHMG-P disrupts plasma membrane. Taken

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toxicity.

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together, a cationic property is considered to be one of the main causes of PHMG-P-induced mammalian cell

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Keyword: Polyhexamethyleneguanidine phosphate; cation; membrane disruption

1. Introduction The guanidine family of antiseptics has been widely used for many years in medicine and the food industry, as well as in plastics, fabric softeners, paints, swimming pools and paper (NICNAS, 2012). It is known to be harmless and less toxic than other antiseptics (Oule et al., 2012). However, a tragic outbreak of pulmonary disease in Korea in 2011 was reported to have originated from humidifier disinfectants containing the guanidine family of

antiseptics (polyhexamethyleneguanidine phosphate (PHMG-P) or oligo(2-(2-ethoxy)ethoxyethyl guanidium chloride (PGH)) (Korea Centers for Disease Control and Prevention, 2011). Some victims died from rapid development of lung fibrosis. Until now, over 179 people have died and at least 280 people have suffered from lung damage associated with humidifier disinfectants (Press release of Ministry of Environment, 2018). Guanidine has multiple positive charges in physiological conditions and this cationic property contributes an

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antimicrobial effect by disrupting the bacterial cell membrane through electrostatic interactions with negative charges of acidic phospholipid (Maillard 2002; McDonnell and Russell 1999). Oule et al. showed that PHMG hydrochloride disrupts the cell wall and inner membrane and precipitates the proteins and nucleic acids inside the

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cells (Oule et al., 2012). However, in addition to the bactericidal effect, cationic agents including guanidine are cytotoxic to immortalized cell lines and erythrocytes (Creppy et al., 2014; Fischer et al., 2003; Ranaldi et al., 2002). Polycationic materials such as polyhexamethylene biguanidine (PHMB), polyethylenimine, chitosan, and

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poly-L-lysines perturb tight junction and change the membrane permeability (Elferink 1991; Fischer et al., 2003;

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Ranaldi et al., 2002; Shen and Ryser 1978). These detrimental effects are considered to be influenced by molecular

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weight, charge density, and structure of cationic polymers (Fischer et al., 2003; Ranaldi et al., 2002; Schulz et al., 2012). In addition, Hoet et al. previously reported that other polycationic paint components such as polyuria,

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polyamide, and polyamine salt exhibit cytotoxicity in various cell lines and primary cells (Hoet et al., 2001; Hoet

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et al., 1999).

Dying cells release intracellular and cell-surface molecules extracellularly. These endogenous cellular components can act as danger signals which include adenosine triphosphate (ATP) (Mariathasan et al., 2006), uric acid crystals

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(Shi et al., 2003), hyaluronan and heparin sulfate (Feng et al., 2012; Yamasaki et al., 2009), amyloid-β (Halle et al., 2008), and high-mobility group box 1 (HMGB1) (Sims et al., 2010) and lead to inflammation and T cell

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responses (Sims et al., 2010). HMGB1 is a DNA-binding nuclear protein and is tightly bound to chromatin when cells remain intact. However, it is released into the extracellular milieu when the cells die. Released HMGB1 can induce inflammation through activating Toll-like receptors or inflammasomes (Chi et al., 2015) and promote the

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proliferation of fibroblast via NF-κB signaling pathway. It is therefore involved in fibrotic diseases including cystic fibrosis, liver/renal fibrosis, and pulmonary fibrosis (Li et al., 2015) as well as inflammatory diseases such as sepsis and rheumatoid arthritis (Andersson and Erlandsson‐Harris 2004; Huang et al., 2010). Zebrafish is emerging as an alternative vertebrate model in biological research with unique advantages compared to the rodent model (Dooley and Zon 2000; Hill et al., 2005; Sukardi et al., 2011). Zebrafish larvae are small enough to put in 96-well plates and embryo is transparent, revealing the phenotype under microscope. One female

fish lays 200-300 eggs per week, enabling a large screening size. The Wellcome Trust’s Sanger Institute sequenced the genome of the zebrafish and found that about 70% of human genes are similar to those of the zebrafish (Howe et al., 2013; Sukardi et al., 2011). Zebrafish have thus become attractive in environmental and biomedical research. Some biological knowledge obtained from cell culture studies of various cell types is not easy to apply to an in vivo model, such as, delivering agonists or antagonist/inhibitors to a target organ or target cell is difficult in living

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organism. In this regard, more diverse models including zebrafish are needed. In the current study, we attempt to verify whether PHMG-P, a major ingredient of humidifier disinfectant, induces cytotoxicity with its cationic nature in various cell lines. And we implemented various experimental methods

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which we expect to be effective screening tools for assessing pulmonary toxicity.

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2. Materials and methods

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2.1. Materials

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PHMG-P was obtained from BOC Sciences (NY, USA) and DNA and Poly-L-glutamic acids (molecular weight

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of 3000-15000, 15000-50000, and 50000-15000) were purchased from Sigma Aldrich.

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2.2. Cell culture

A549, a human lung epithelial cell, was purchased from American Type Culture Collection (Rockville, MD, USA). MRC-5, a human lung fibroblast, and THP-1, a monocyte cell, were obtained from Korean cell bank. A549 cell

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and MRC-5 were suspended in RPMI 1640 and EMEM, respectively, both supplemented with 10% fetal bovine

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serum (FBS) (Thermo Scientic) and penicillin/streptomycin. THP-1 cells were grown in RPMI 1640 supplemented with 10% FBS and penicillin/streptomycin and differentiated into macrophage-like cells with 100 nM phorbol myristate acetate (PMA) (Sigma-Aldrich) for 24 hrs. After differentiation, THP-1 cells were washed

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with PBS, refilled with fresh medium, and then treated with test materials.

2.3. Cell viability A549 and MRC-5 were plated at a density of 5 X 103 cells per well. THP-1 cells were plated at a density of 1 X 105 per well and differentiated with 100 nM PMA overnight. After 24 hrs, the cells were treated with 10 μl of 0.25~20 μg/mL of PHMG-P and the cell cytotoxicity was measured after 24, 48, and 72 hrs using the cell counting kit (CCK)-8 (Dojindo, Japan) according to the manufacturer’s protocol. Briefly, 10 μL of CCK-8 was added to

each well and incubated at 37℃ for 2 hrs. Cell viability was evaluated by measuring the optical density (O.D.) at 450 nm absorbance using spectrophotometric analysis with SpectraMax M3 microplate reader from Molecular Devices (Sunnyvale, USA). Cell viability was expressed as a percentage of the vehicle control. All experiment was performed 3-5 times independently.

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2.4. Effect of polyanionic compounds on cell viability in PHMG-P-treated cells To evaluate the effect of polyanionic compounds on cytotoxicity, DNA or poly-L-glutamic acids were treated with

PHMG-P by modification of a protocol described previously (Hoet et al., 2001). A549 and MRC-5 were plated at

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a density of 0.75 X 104 cells per well. THP-1 cells were plated at a density of 1 X 105 per well and differentiated

as described above. After 24 hrs, DNA at concentrations ranging from 12.5 μg/mL to 100 μg/mL was added into each well and then PHMG-P was added. At each time points, cell viability was measured as described above.

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Poly-L-glutamic acids (molecular weight of 3000-15000, 15000-50000, and 50000-15000) at concentration

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2.5. Cytokine measurement and western blot analysis

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ranging from 2 μg/mL to 100 or 200 μg/mL were treated as the same method to DNA.

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A549 (1.2 X 105 per well), MRC-5 (1.2 X 105 per well), and THP-1 (2 X 106 per well) were seeded in 6-well cell culture plates. THP-1 cells were differentiated as described above. Twenty four hours after plating the cells, they

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were rinsed with new medium and 25-100 μg/mL of DNA and 10 μg/mL or 20 μg/mL of PHMG-P were added to the wells. Following 24 hr incubation, cell supernatants were collected and centrifuged at 15000 rpm for 15 min

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to remove debris. The supernatants were used for HMGB1 and IL-1β analysis. Equal quantities of each sample were resolved using SDS-PAGE and the samples were then transferred to nitrocellulose membranes (Biorad,

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USA). The membranes were incubated with 5% non-fat milk in Tris-buffered saline with 0.05% Tween 20 (TBST). Anti-rabbit HMGB1 antibody (Abcam, Cambridge, UK) was diluted in skim milk in TBST and incubated overnight at 4°C while shaking. The membranes were washed 3 times with 0.05% TBST and incubated at room

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temperature for 1 h with anti-rabbit horseradish peroxidases-conjugated secondary antibodies. After washing 4 times, the membranes were visualized using chemiluminescent western blotting detection reagents (Thermo, Rockford, USA). All the bands were obtained using Image Lab software (Biorad, USA). IL-1β was quantified using IL-1β ELISA kit (R&D systems, Minneapolis, USA) according to the manufacture’s protocol. The experiment was performed in duplicate.

2.6. Detection of ROS generation mediated by PHMG-P A549, MRC-5, and THP-1 cells were seeded at a density of 1 X 105, 1 X 105, and 1 X 106 per well, respectively. Twenty-four hours after plating the cells, they were rinsed with new medium. Ten μg/mL of PHMG-P with or without 25- 50 μg/mL of DNA were added to the wells. Following 24 hr incubation, each well was washed with phosphate-buffered saline (PBS) and the dichlrohihydrofluoresceine diacetate (DCF-DA, 25 μM) was loaded for

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30 min. The cells were lysed by adding 300 μL 0.1 N NaOH and 200 μL lysate was used for measurement of fluorescence intensity using spectrophotometric analysis with SpectraMax M3 microplate reader (Molecular

Devices, Sunnyvale, USA) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The

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lysates were collected and total protein concentration was determined using the BCA protein assay (Sigma

Aldrich). The fluorescence intensity was adjusted according to the protein concentration. The experiment was

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performed in duplicate.

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2.7. FDA and PI staining and flow cytometric analysis

The suspended, undifferentiated THP-1 cells were seeded at a density of 1 X 106 per tube. Fluorescein diacetate

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(FDA; Sigma) and proidium iodide (PI; Sigma) staining were performed by modification of a previously described

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protocol (Hong et al., 2004). For FDA staining, 25 μg/mL FDA/acetone solution was added and incubated for 1 hr in the dark. After washing with PBS, PHMG-P (2.5 ~ 20 μg/mL) and DNA (100 μg/mL) was added and

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incubated for another 1 hr. For PI staining, PHMG-P and DNA were added and incubated for 1 hr in the dark. And then 1 μg/mL of PI was added and incubated for 1 hr. After staining, each FDA or PI-stained sample was washed

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with PBS and the cell pellets were resuspended in 1 mL PBS. The fluorescence signal of the individual cells was acquired using the BD FACS Caliber (BD Biosciences, CA, USA). Data were analyzed for 1 × 10 4 cells/sample

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using the BD CELLQuest software (BD Biosciences, CA, USA).

2.8. Zebrafish assay

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Wild type AB zebrafish (Danio rerio) were maintained on a 14:10 hour light:dark cycle at 28°C. Healthy fertilized embryos were collected following natural spawning and were reared in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) at 28.5 °C. From the previous zebrafish embryo acute toxicity test for 96 hr, PHMG-P concentration was set at 1.25 μg/mL (data not shown). For DNA treatment, the eggs of ~1 day post fertilization (dpf) were dechorionated mechanically with forceps, and 10-12 larvae were then placed into a 12well plate. DNA was treated and 1.25 μg/mL PHMG-P were then added to each well. Coagulation of embryos,

lack of somite formation, non-detachment of tail, and lack of heartbeat were considered as lethality (OECD guideline, 2013). Mortality was observed at various times. During the test, the E3 medium with fresh chemicals solutions was replenished every day. Each experiment was repeated on at least 3 clutches. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Korea Institute of Toxicology.

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2.9. Zeta potential measurement The surface charge (zeta potential) of PHMG-P, DNA, and mixture of PHMG-P and DNA were characterized using the ZetaSizer Nano ZS (Malvern Instruments Inc., UK), utilizing electrophoretic light scattering (ELS) or

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Laser Doppler velocimetry (LDV). The zeta potential of the particle is calculated from the measured electrophoretic mobility using the Smoluchowski equation. PHMG-P and DNA was diluted with PBS (pH 7.4).

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2.10. Data analysis

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One-way analysis of variance (ANOVA) with Tukey’s or Dunnett’s T3 post-hoc test was used to determine

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differences among groups (SPSS Ver15.0.0, SPSS Inc., USA). A p value less than 0.05 was considered statistically significant. All data are presented as mean ± standard error (SE). The number of samples in each group is indicated

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3. Results

3.1. Cell viability

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in the figure legends.

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To investigate the toxic effect of PHMG-P on various types of cells, a cell viability assay was carried out in A549, MCR-5, and THP-1 cells. These cells were chosen to investigate the adverse effect of PHMG-P on lung-related cells. A549 cells are one of lung epithelial cells and differentiated THP-1 cells, macrophage-like cells, play an important role in immune systems. And MRC-5 cells, lung fibroblast cells, provide structural integrity. PHMG-P

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decreased the cell viability in a dose- and time-dependent manner in all three cell lines (Fig. 1). Interestingly, THP-1 cell was more resistant to cell death than A549 or MRC-5 (Supplementary Fig 1.). Exposure of A549 and MRC-5 to 5 μg/mL PHMG-P reduced cell viability by 44.7% and 64.9% at 24 hr, 19% and 25.8% at 48 hr, and 11.2 % and 12.7 % at 72 hr, respectively, whereas 5 μg/mL PHMG-P exposure decreased cell viability by 86.9% at 24 hr, 73.8 % at 48 hr, and 69.4 %at 72 hr in THP-1 cell.

3.2. Protective effect of polyanionic compounds on PHMG-induced cytotoxicity Polyanionic compounds, DNA and various poly-L-glutamic acids, prevented cytotoxic effect of PHMG-P in cells. Incubation with DNA ranging from 12.5 to 100 μg/mL or various molecular weight of poly-L-glutamic acid ranging from 2 to 200 μg/mL did not induce any cytotoxicity in A549 cell (data not shown). Cell viability increased in all three cell lines in a DNA dose-dependent manner. In A549 lung epithelial cell, MRC-5 lung

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fibroblast cell, and THP-1 cell, 13.7%-71.3%, 14.4%-66.8%, and 35.2%-78.6% of cell viability at 5 μg/mL-20 μg/mL PHMG-P increased to 29.4%-98.4%, 16.7%-96.2%, and 61.9%-99.8% in a DNA dose-dependent manner, respectively (Fig. 2). The protective effect of DNA on polycation-induced cytotoxicity was then measured in a

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time-dependent manner. Surprisingly, the addition of DNA effectively prevented PHMG-P mediated cytotoxicity

for 72 hr in A549 cell (Fig. 2D). Although the cell viability decreased at low concentration of DNA in a timedependent manner, the treatment of 100 μg/mL DNA protected PHMG-P-induced cell death for 72 hr. This

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protective effect was not reproduced in MRC-5 and THP-1 cell (Fig. 2E and F). Even the 100 μg/mL DNA

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treatment resulted in a decrease of cell viability from 91.4% at 48 hr to 40.0% at 72 hr in MRC-5 and 82.5% at 24

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hr to 44.6% at 72 hr in THP-1.

Poly-L-glutamic acids, another polyanionic compound, also reduced the cytotoxicity mediated by PHMG-P. The

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cell viability by poly-L-glutamic acids treatment with PHMG-P showed a similar pattern to that of DNA treatment

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in all three cell lines (Figs. 3).

3.3 Measurement of HMGB1, IL-1β, and ROS

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HMGB1 is a biomarker for cell necrosis. In order to confirm the PHMG-P induced cell death, the release of HMGB1 was evaluated. Exposure of A549 cell or MRC-5 or THP-1 cell to PHMG-P resulted in the secretion of

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HMGB1 in the cell supernatant in a dose-dependent manner. The addition of DNA to A549, MRC-5, and THP-1 cell followed by PHMG-P treatment decreased HMGB1 secretion into the media as shown in Fig. 4A. Proinflammatory cytokine IL-1β was also increased with PHMG treatment in a dose-dependent manner and

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decreased with the co-treatment of DNA and PHMG (Fig. 4B) in PMA-differentiated THP-1. IL-1β was not detected in A549 and MRC-5 cell (data not shown). A549 and MRC-5 cells are not immune cells, therefore, they do not seem to produce as much as IL-1β as differentiated THP-1 or enough IL-1β to be detected in the ELISA kit that we used. PHMG-P is known to generate ROS in A549 or raw cells (Jung et al., 2014; Kim et al., 2015a; Kim et al., 2015b). The neutralization of the positive charge of PHMG with the negative charge of DNA would affect ROS generation.

DCF fluorescence, the intracellular ROS marker, was measured in A549 cells, MRC-5, and differentiated THP-1 cells. DCF fluorescence was markedly increased with 10 μg/mL PHMG-P compared to control cells of A549 or MRC-5. Twenty four hr incubation with 12.5, 25, and 50 μg/mL DNA reduced ROS generation by 37.0%-49.1% and 22.2%-25.9% compared to the 10 μg/mL PHMG-P-treated A549 and MRC-5 cells, respectively (Fig. 4C).

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Increase of ROS was not detected in THP-1 cells (data not shown).

3.4. PI and FDA staining and flow cytometric analysis

To determine the cell membrane disturbance by PHMG-P, FDA and PI staining were adopted. FDA is a non-

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fluorescent nonpolar compound and readily enters the intact cells. It is then hydrolyzed by endogenous esterase, resulting in free fluorescein, which is polar and is not able to escape the intact membrane. On the other hand, PI

does not enter the intact membranes and readily enters the cells with damaged membranes ((Ross et al., 1989;

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Umebayashi et al., 2003). Therefore, FDA and PI are markers for intact and damaged membrane, respectively. As

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shown in Fig 5A, FDA-positive cells were markedly decreased with PHMG-P treatment (76.0%, 32.7%, 26.6%

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and 2.2% at 2.5, 5, 10, and 20 μg/mL PHMG-P, respectively) in a dose-dependent manner compared to the control (93.7%). The addition of DNA to PHMG-P dramatically increased the percentages of the FDA-positive cells to

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the level of control at all concentrations of PHMG-P. Unlike FDA, the PI-positive cells slightly increased in the

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PHMG-P-treated cell in a dose-dependent manner, and when DNA was added into PHMG-P, the percentages of PI-positive cells decreased to the control level.

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3.5. Protective effect of polyanionic compounds on cytotoxicity of PHMG in zebrafish Next, the protective effect of polyanionic compounds on PHMG-P mediated cell death was observed in an in vivo

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zebrafish model. The mortality of zebrafish induced by 1.25 μg/mL PHMG-P was markedly increased in a timedependent manner, whereas the treatment of DNA reduced mortality in a DNA concentration-dependent manner

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as shown in Fig 5. No mortality was observed with DNA over 4 μg/mL.

3.6. Zeta potential measurement To verify whether PHMG-P and DNA interact with each other, the zeta potential was measured. The zeta potential is an electrokinetic potential existing between the particle surface and the dispersing solution (Fedele et al., 2011). Zeta potential can be altered by adsorbing of oppositely charged ions which are sourced from different electrolytes, cationic or anionic surfactants present in the dispersing solution (Fairhurst, 2013). The surface charges of PHMG-

P and DNA were positive and negative, respectively (Table 1). However, the addition of DNA in the PHMG-P solution showed a shift of zeta potential from positive to negative charge, as expected.

4. Discussion and conclusions

Cationic antiseptics or disinfectants have been widely used and are still promising candidates for the development

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of biocides because they have excellent microbicidal activity with low toxicity (Carmona-Ribeiro and de Melo Carrasco 2013). The bactericidal mechanism of cationic compounds has been well studied. PHMB, one of these

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cationic compounds, causes bactericidal effect by being attracted toward the negatively charged cell surface with

the adsorption to phosphate-containing compounds. This causes impairment of outer membrane and PHMG binds to phospholipids in the inner membrane, following complete loss of membrane function (McDonnell and Russell

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1999). However, polycationic materials have recently been shown to induce a cytotoxic effect in immortalized cell lines, primary cells or red blood cells (Fischer et al., 2003; Hoet et al., 2001). The mechanism of cytotoxicity

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induced by polycation is not yet fully understood yet. Creppy et al. showed that PHMB induces a significant

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increase in LDH leakage and cytokine activity in human cell lines, Caco-2 cells, Neuro-2A, and HepG2 cells.

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However, no increase has been observed in programmed cell death-related gene or protein expression at 24 hr treatment (Creppy et al., 2014). On the contrary, Jung et al. reported that PHMG induced cytotoxicity in a dose-

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dependent manner, and that apoptosis related genes and protein such as GADD45B, BAX, FAS, and JUN were upregulated in A549 cells at 24 hr treatment (Jung et al., 2014). Ranaldi et al. and Kim et al. reported that

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polycationic materials enhance the membrane permeability by affecting tight junctions and morphological changes in the F-actin cytoskeleton in Caco-2 cells (Ranaldi et al., 2002) and by decreasing transepithelial

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electrical resistance and increasing paracellular flux in Calu3, THP-1, and HMC-1 cocultures (Kim et al., 2016). Hong et al. showed that polyamidoamine dendrimer with amine-terminated surface forms holes in supported lipid bilayers and KB and Rat2 cell lines (Hong et al., 2004). On the other hand, cationic polymers including chitosan,

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thpolylysine, and polyethylenimine have been extensively studied and widely used as a tool for gene delivery because they bind to negatively charged nucleic acid following ionic interaction with the negative charge on the cell membrane and release of DNA into cytoplasm (Jin et al., 2014; Prevette et al., 2010). Taken together, it is assumed that the target of PHMG-P is a cell membrane. PHMG-P decreased the cell viability in a dose- and time-dependent manner in all three cell lines (lung epithelial cells, monocytes, and lung fibroblast). The differentiated THP-1 cell seems more resistant to PHMG-P. Jung et al.

showed that 4 different cell lines have also different cell viability on exposure to PHMG-HCl (Jung et al., 2014). This might be because each cell has different cell surface charge. Cell surface charge is affected by phospholipid composition, the levels of charged components on the plasma membranes, and the activities of ion channel on the plasma membrane (Chen et al., 2016; Goldenberg and Steinberg, 2010). If the cells have more anionic lipid head groups within the membrane or positively charged glycoprotein on the membrane, PHMG-P can bind to the

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anionic moiety and kill the cells effectively. To verify whether PHMG-P kills cells with its positive charges, negative charged materials were added to cells

with PHMG-P. Polyanionic materials such as DNA and different molecular weights of poly-L-glutamic acids

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effectively protected against PHMG-P induced cell death. The viability patterns of both materials seem quite similar, which means that the DNA and different molecular weights of poly-L-glutamic acids act via the same mechanism; in other words, other anionic materials also have a protective effect against PHMG-P induced cell

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death. At 20 μg/mL PHMG-P, the polyanionic substances reduced cell death from over 80% to less than 20%.

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This protective effect was sustained upto 72 hr in A549 cell in a DNA concentration-dependent manner, which

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firmly supports the assumption that PHMG-P leads to cell death due to its polycationic nature. In the case of MRC-5 and PMA-differentiated THP-1, this protective effect was not sustained up to 72 hr. This might be because

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endocytic or phagocytic ability may enhance cytotoxicity. MRC-5 can ingest materials with clathrin-mediated

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endocytosis or phagocytosis (Ng et al., 2015; Couzinet et al., 2000). Couzinet et al. reported that phagocytic ability of MRC-5 is higher than A549 cells and much lower than macrophage (Couzinet et al., 2000). Differentiated macrophages are known to have higher phagocytic activity than monocytes (Schwende et al., 1996; Lunove et al.,

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2011) (Supplementary data Fig. 2). Endocytosed or phagocytosed DNA might lead to the depletion of DNA in the

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medium, which might enhance cytotoxicity. In addition, PHMG-P enters into the cytoplasm through damaged membrane and then interacts with the membranes of organelles such as endoplasmic reticulum or mitochondria. This would cause the dysfunction of organelles such as disruption of energy supply and protein folding, production of free radicals, and cell death. In addition, 17%-32% cell death derived from PHMG-P and DNA cotreatment at

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24 hr might evoke inflammatory response or cellular damage, leading to high cell death even in the presence of DNA at 48 hr and 72 hr. As shown in Fig. 4, proinflammatory cytokine IL-1β was detected in THP-1 cells. Various proinflammatory cytokines produced by macrophage-like THP-1 can influence the viability of THP-1 cells. While cation-mediated cytotoxicity can easily be performed in an in vitro experiment, it appears not to be easy to carry out in in vivo, rodents study. Therefore, a zebrafish assay was adopted in this study. Zebrafish has many advantages including transparent appearance at larva stage, rapid and ex utero development, availability of many

transgenic lines with fluorescing organs or tissues, and known full genome data (Howe et al., 2013; Sukardi et al., 2011). In this study, we used 1 day old dechorionated zebrafish larvae because they have no physical barriers such as scale. Although the route of administration is oral in the zebrafish experiment, the exposed environment seems to be similar to that of a lung in which various lung cells interact directly with PHMG-P in the lining fluid. Treatment of PHMG-P in 24 hour old zebrafish larvae showed significant mortality in a time-dependent manner

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and the addition of polyanionic DNA effectively increased the survival of zebrafish larvae in a dose-dependent manner. The hypothesis that PHMG-P-mediated cytotoxicity is due to the cationic nature is clearly proven in our in vivo model. Additionally, zebrafish is a good in vivo model for connecting the in vitro test models.

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The HMGB1 level was measured to confirm the cell death induced by PHMG-P. HMGB1 is a DNA-binding

nuclear protein that acts as a transcriptional regulator and stabilizes nucleosomes in the nucleus (Park et al., 2003). However, it is translocated to cytosol and released into the extracellular milieu when cells are damaged. Therefore,

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it can usually be used as a necrotic marker (Sims et al., 2010). In addition, it can be actively secreted from

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macrophage, natural killer cells, dendritic cells, and endothelial cells (Harris et al., 2012). When it is released, it

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provokes inflammatory response by inducing cytokine release and acting as a damage-associated molecular pattern (DAMP) molecule. It is associated with inflammatory diseases such as sepsis, rheumatoid arthritis, and

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systemic lupus erythematosus (Andersson and Erlandsson‐Harris 2004; Huang et al., 2010; Lu et al., 2015). In

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addition, elevated levels of HMGB1 were detected in patients with idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease (Hamada et al., 2008; Ko et al., 2014). HMGB1 is known to regulate the NLRP3 activation and IL-1β maturation in a NF-κB-dependent manner (Chi et al., 2015). In our previous study, we showed

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that PHMG-P activates NALP3 inflammasome in mice (Song et al., 2018); it is thus possible that DAMP such as HMGB1 released from necrotic cells is responsible for the prolonged inflammation and subsequent fibrotic

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changes. Increased HMGB1 was detected in the supernatant of the PHMG-P-treated A549, MRC-5, and THP-1 cells and treatment of DNA reduced HMGB1 level, which also confirms that the cation of PHMG-P induces cell death.

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Kim et al. and Jung et al. reported that ROS play an important role in PHMG-P-related pathogenesis (Jung et al., 2014; Kim et al., 2016). ROS are generated intracellularly in plasma membranes, mitochondria, and endoplasmic reticulum through multiple stimuli such as hypoxia, TNF-α, growth factors, cytokines, and integrin signaling (Holmstrom and Finkel 2014; Novo and Parola 2008). The major sources of ROS within the cell are mitochondria and membrane-bound NADPH oxidase (Holmstrom and Finkel 2014). ROS can be cytotoxic and can lead to inflammation and aging (Nathan and Cunningham-Bussel 2013). The DNA treatment reduced ROS production in

A549 and MRC-5 cells. The decreased ROS production might be partly due to the intact membrane integrity. Positive-charged PHMG-P can pass freely through the compromised plasma membrane, enter into the cytosol, then bind to and disrupt the mitochondria membrane and lead to the production of enormous amounts of ROS. However, the neutralization of a positive charge on PHMG-P might result in an intact plasma membrane, less access to mitochondria, and reduced ROS production.

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To verify that the target of PHMG-P is the cell membrane, we stained PHMG-P treated cells with FDA and PI. FDA is usually used as a marker for live cells with intact membranes, as it can readily enter a cell with intact membrane but cannot transverse cells with an intact membrane. On the contrary, PI can enter cells that have

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damaged membranes, and is thus used as a marker for damaged or dead cells. FDA and PI staining results exhibited

that PHMG-P disturbed the cell membrane integrity. The cotreatment of anionic DNA and PHMG-P led to increased FDA positive cells and decreased PI positive cells. These results suggest that PHMG-P seems to mediate

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membrane damage through ionic interaction. We verified this with measurement of zeta potential. As expected,

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the surface charge of DNA and PHMG-P are negative and positive, respectively. However, the surface charge of

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the PHMG-P and DNA mixture converted from positive to negative with the increase of added anionic DNA concentration. Zeta potential data showed indirectly that PHMG-P interacts with DNA.

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The disruption of membrane integrity or membrane hole formation induced by the cationic nature of PHMG-P does not seem to be entirely responsible for the cell death because cells possess several membrane repair

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mechanisms. The repair of mechanically damaged plasma membrane or closure of small wounds (< 1 μm) occurs within seconds to minutes (Andrews et al., 2014; Draeger et al., 2011; Mellgren 2011). Cell death is likely to occur

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when membrane damage exceeds the repair capacity of the cells. At the same time, the intracellular uptake of PHMG-P through endocytosis and/or damaged membrane and downstream events such as ROS generation or

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further interaction with negatively charged proteins in the cytosol, are also considered to be responsible for cell death.

In this study, we showed that PHMG-P induced cytotoxicity in a dose- and time-dependent manner in various

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cells. The positive charge of PHMG-P can bind phospholipid or negatively charged cell surface components (heparin sulfate proteoglycans or integrins) or proteins in the membrane, which leads to the disruption of membrane integrity, alteration of metabolism, cellular transport, cell migration, and cell survival (Bishop et al., 2007; Frohlich 2012; Khalil et al., 2006). PHMG-P is also considered to act on organelle membranes such as mitochondria, ER, lysosome in addition to the plasma membrane, and to contribute to the production of ROS, apoptosis, and cytotoxicity. These toxic effects of PHMG-P were protected by neutralizing the positive charges of

PHMG-P with negative charges, which was confirmed with the reduction of HMGB1, cytokine IL-1β expression, and ROS production. In conclusion, our current and previous in vitro and in vivo data showed that PHMG-P mediated inflammatory and fibrotic changes in the lung and other tissues (Song et al., 2014; Kim et al., 2016; Kim et al., 2013) are initiated in part by cell membrane damage. This study implies that other polycationic materials that can be inhalable or

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inhalant drug candidates can cause the same pulmonary toxicity as PHMG-P, although the dose is the key point for the manifestation of toxicity. In vitro assays performed in this study, a neutralization test using anionic

compounds and FDA/PI staining, are considered to be useful and simple tools for screening whether a chemical

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can induce cytotoxicity via polycation-mediated effects.

Funding

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This study was supported by Basic Science Research Program through the National Research Foundation of Korea

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(NRF) funded by the Ministry of Education (No. 2017R1D1A1B04032833).

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Figure legend

Fig. 1 Cytotoxicity of A549, MRC-5, and THP-1 cells exposed to PHMG-P. A549 (A), MRC-5 (B), and THP-1 cells (C) were incubated with PHMG-P (0.25-20 μg/mL) for 24 hr, 48 hr, and 72 hr. Cytotoxicity was measured using CCK-8. Cell viability was expressed as a percentage of the vehicle control.

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Data are expressed as mean ± SE of three to four separate experiments; * p < 0.05, **p < 0.01 indicates significant

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difference compared to the vehicle control.

Fig. 2 Protective effect of DNA on PHMG-induced cytotoxicity. A549 (A, D), MRC-5 (B, E), and THP-1 cells (C, F) were co-incubated with DNA (12.5-100 μg/mL) and PHMGP (5, 10, 20 μg/mL in A-C, 20 μg/mL in D and 15 μg/mL in E) for 24 hr (A-C) and up to 72 hr (D and F). Cytotoxicity was measured using CCK-8 kit. Cell viability was expressed as a percentage of the vehicle control. Data are expressed as mean ± SE of three to four separate experiments; * p < 0.05, **p < 0.01 indicates significant

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difference compared to the vehicle control.

Fig. 3 Protective effect of poly-L-glutamic acids on PHMG-induced cytotoxicity. A549 (A), MRC-5 (B), and THP-1 cells (C) were co-incubated with 3 different molecular weight (molecular weight of 3000-15000, 15000-50000, and 50000-15000) of poly-L-glutamic acids (2-200 μg/mL) and PHMG-P (20 μg/mL) for 24 hr. Cytotoxicity was measured using CCK-8. Cell viability was expressed as a percentage of the vehicle control. Data are expressed as mean ± SE of three to five separate experiments; * p < 0.05, **p < 0.01

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indicates significant difference compared to the vehicle control.

Fig. 4. Detection of HMBG1, IL-1β, and ROS production in A549, MRC-5, and THP-1 cells exposed to PHMG-P and DNA. A549, MRC-5, and THP-1 cells were incubated with PHMG-P (10 and 20 μg/mL) without or with DNA (12.5100 μg/mL). After 24 hr incubation, cell supernatants were collected and used for detection of HMBG1 (A) and IL-1β (B, THP-1). HMBG1 was detected by western blot and IL-1β was quantified by ELISA. For ROS detection

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(C), A549 cells were washed with PBS and added to DCF-DA. After 30 minutes, cells were lysed with NaOH and lysates were used for measurement of fluorescence intensity. Values are expressed as mean ± SE. Values are

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significant compared to control: * p < 0.05, **p < 0.01 and from PHMG-P only: # p<0.05, ## p<0.01.

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Fig 5. Flow cytometric measurement of THP-1 cells using FDA (A) and PI (B) staining. THP-1 cells were treated with PHMG-P (2.5-20 μg/mL) only or DNA (100 μg/mL) only or both PHMG-P and DNA before PI

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staining and after FDA staining. Control sample was treated with distilled water.

Fig 6. Protective effect of DNA on PHMG-induced mortality in zebrafish. Zebrafish larvae (~ 24 hr post fertilization) were dechorionated with forceps and incubated with 1.25 μg/mL PHMG-P only or with DNA (0.02-10 μg/mL). The mortality was observed at designated time. Data are expressed

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as mean ± SE (n = 10–12); * p < 0.05, **p < 0.01 indicates significant difference compared to the PHMG-P group.

Table legend

Table 1. Zeta potential measurements of PHMG-P, DNA, and PHMG-P and DNA mixture. Each sample was measured three times. Average and standard deviations of each sample are shown.

measured three times. Average and standard deviations of each sample are shown.

Mean value

Standard deviation

PHMG-P

(mV)

(mV)

1

3.81

0.99

2.5

16.13

1.40

5

12.97

1.00

10

13.77

2.58

20

13.27

1.10

100

11.20

1.70

12.5

-24.00

1.55

25

-18.17

2.45

50

-20.13

75

-18.73

100

-17.70

200

-18.43

0.40

7.32

0.25

-6.03

0.44

-20.57

1.03

1.45 3.15

12.5

20

25

20

50

20

75

-21.97

0.50

20 20

100 200

-23.23 -21.13

1.64 1.29

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20

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1.63

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DNA

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Concentration (μg/mL)

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Table 1. Zeta potential measurements of PHMG-P, DNA, and PHMG-P and DNA mixture. Each sample was