Nitric Oxide Delivery using Biocompatible Perfluorocarbon

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Nitric Oxide Delivery using Biocompatible Perfluorocarbon Microemulsion for Antibacterial Effect Moonhyun Choi, Sohyeon Park, Kyungtae Park, Hyejoong Jeong, and Jinkee Hong ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00016 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on February 2, 2019

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ACS Biomaterials Science & Engineering

Nitric Oxide Delivery using Biocompatible Perfluorocarbon Microemulsion for Antibacterial Effect Moonhyun Choi, Sohyeon Park, Kyungtae Park, Hyejoong Jeong and Jinkee Hong* Department of Chemical & Biomolecular Engineering, College of Engineering, Yonsei University 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea *Corresponding author: E-mail address: [email protected], Tel: +82-2-2123-5748

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Abstract Nitric oxide (NO) participates in various physiological and pathophysiological processes for example, as a cell messenger and as an antimicrobial agent of the cell-mediated immune response. The development of NO-releasing materials to carry and deliver NO for biomedical applications has gained immense attention. NO-releasing perfluorooctane (PFO) microemulsion (ME) has been prepared using a simple and time-saving method. Perfluorocarbon (PFC) liquids are halogen-substituted carbon nonpolar oils with enhanced NO gas dissolution capacity. The solubility of NO in PFC liquids is higher than that in water-based fluids. Liquid-gas solubility is governed by Henry’s Law. The cytotoxicity of the NO-unloaded and NO-loaded PFO MEs towards human dermal fibroblast (HDF) was evaluated. The results showed that the NO-loaded PFO ME was highly biocompatible. On the other hand, at high concentrations, the NO-releasing PFO ME accelerated the bacteria (Staphylococcus aureus) death unlike the NO-unloaded PFO ME. Hence, NO-releasing PFO MEs are suitable for biomedical applications such as wound healing and antibacterial agents. Key words: Perfluorocarbon, microemulsion, nitric oxide delivery, biocompatibility, antibacterial effect

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Introduction Nitrogen oxides (NOx) are toxic atmospheric pollutants emitted mainly from industries and automobiles [1]. However, the biological implications of nitric oxide (NO) gas have been considered only over the last 20 years [2]. NO gas is colorless, hydrophilic, and highly reactive (produced by various organisms ranging from bacteria to human) [3–6]. It is an important signaling molecule of a variety of biological functions, and also participates in various biomedical processes depending on its concentration for example, cardiac contractility [7,8], vasodilation [9], antibacterial effects [10], cell proliferation [11,12], and anti-cancer effects [13,14]. It has been reported that lower concentrations of NO exert a direct effect on processes such as cell proliferation and survival, whereas higher concentrations exert an indirect effect through both oxidative and nitrosative stresses [15]. At high concentrations, NO binds covalently to DNA, proteins, and lipids, thereby inhibiting or killing the target pathogens. Therefore, the development of exogenous NO gas delivery systems is imperative to deliver highly sensitive free radical NO gas (a half-life of only a few seconds) in a stable state. Many studies have been carried out to deliver exogenous NO with various in vivo and in vitro effects by using NO-donor functionalized materials (such as amino acids [16], nanoparticles [17, 18], and polymers [19, 20]). Although the release kinetics and loading amounts of NO gas can be controlled using NO-donor functional materials obtained via chemical modification, these processes are challenging because of the high reaction pressure, long time, low efficiency, high sensitivity, unexpected byproducts and reproducibility. On the other hand, there are very few reports on the delivery of exogenous NO by physical interaction. In addition, for practical applications, simple and time-saving methods should be developed for the synthesis of NO carriers. 3



Perfluorocarbon (PFC) liquids, which are immiscible with water, can dissolve large quantities of gases such as O2, CO2, N2, CO, CH4, and NO [21]. PFCs are very stable because of the high strength of the carbon-fluorine bond, one of the strongest bonds in organic chemistry. The electronegativity of fluorine imparts a partial ionic character through partial charges on carbon and fluorine atoms, which shortens and strengthens the bond through favorable covalent interactions [22]. The high solubility of gases in PFCs is attributed to the weak intermolecular interactions (van der Waals interactions) between PFCs, as van der Waals interactions depend on fluctuations in polarity of the electronic cloud. Therefore, PFC liquids are potential gas carriers. Highly nonpolar and biologically inert PFC liquids have been widely studied for O2 delivery as blood substitutes [23–26]. However, NO gas delivery using PFC liquids has not been studied much. Since PFC liquids are hydrophobic in nature, they are generally used in emulsion or particle forms in order to facilitate their dispersion in water for various biomedical applications. In this study, we prepared a nanoscale pluronic F-127 microemulsion (ME) incorporating perfluorooctane (PFO). The PFO ME could be fabricated easily by ultrasonication for just 10 min. NO was incorporated into the PFO ME by carrying out NO gas purging for 2 h. Large amounts of NO gas could be incorporated into the PFC ME. It was found that the interface formed by the emulsion played the most important role in suppressing the contact between NO and O2 gases. Large amounts of NO gas could be released from the PFO ME in 12 h without any controlling system. In addition, we also investigated the effect of NO gas (released from the PFO ME) on the viability of human dermal fibroblast (HDF). Finally, in order evaluate its antibacterial properties, NO gas released from the PFO ME was treated with two types of bacteria: gram-positive Staphylococcus aureus (S. aureus) and gram-negative Pseudomonas aeruginosa (P. aeruginosa). 4


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Experimental section Materials Pluronic F-127, 1,2-Diacyl-sn-glycero-3-phosphocholine (egg yolk phospholipid (EYP)), and PFO were purchased from Sigma Aldrich to prepare the PFO ME. Float-A-lyzer® G2 (MWCO, 12,000-15,000 kDa) was purchased from Spectrum Lab.

Synthesis of PFO ME As the emulsifying agents for PFO, 20 wt% Pluronic F127 and 4 mg/mL EYP solutions were prepared by dissolving them in phosphate buffered saline (PBS, pH ~7.4). The PFO/Pluronic F68 /EYP mixture (6/2/2, v/v/v) was vigorously agitated using a probe-type ultrasonic wave homogenizer (BransonSonifier model 185, USA) to produce nano-sized emulsion droplets. Ultrasonication (30 s ON and 20 s OFF; 10 cycles) was directly applied to the mixture solution placed in a water bath [27]. For removing the excess F-127 and EYP, the as-prepared PFO ME solution was dialyzed using a Float-A-lyzer dialysis membrane.

Characterization of PFO ME The zeta () potentials and average size of the prepared PFO ME droplets were measured by dynamic light scattering (SZ 100, Horiba) at room temperature. The energy filtering transmission electron microscopy (EF TEM) images of the PFO ME were obtained using a LIBER 120 (Carl Zeiss). The PFO ME concentration (number of particles per mL) was measured by Nanosight (LM10, Malvern) 5



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NO loading and release experiment The PFO ME was degassed in high-pressure reactor (custom-made, Hanwoul Engineering Co., Ltd., Republic of Korea) by bubbling pure argon three times (2 min) at room temperature with 1 atm Ar gas. It was then equilibrated with NO gas for 2 h (1 bar, 25 °C). Real-time NO release measurements from PFO ME were performed using a chemiluminescence NO analyzer (NOA, Sievers 280i, GE Analytical instruments, Boulder, CO, USA). This machine is a high sensitivity detector for measuring nitric oxide based on a gas-phase chemiluminescent reaction between nitric oxide and ozone: NO + O3 → NO2* + O2

(1)

NO2* → NO2 +hv

(2)

Emission from electronically exited nitrogen dioxide is in the red and near-infrared region of the spectrum, and is detected by a thermoelectrically cooled, red-sensitive photomultiplier tube.

Cell viability test The cytotoxicity of the PFO ME to HDFs was evaluated using (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT, Cat. No. M2128, Sigma, USA) assays. The cells were cultured in a growth medium composed of the Dulbecco’s modified Eagle’s medium (DMEM, Gibco® Life Technologies, Gaithersburg, USA) and 10% fetal bovine serum (FBS, Gibco® Life Technologies, Gaithersburg, USA), and 1% penicillin/streptomycin antibiotics (Gibco® Life Technologies, Gaithersburg, USA). The medium was maintained at 37 °C in a 5% CO2/95% air humidified atmosphere. The HDFs were seeded in four 24-well plates at 1 mL/well with 6


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densities of 1×104, 0.8×104, 0.6×104, and 0.3×104 cells/mL and incubated for 24 h. After the incubation period, the cultured medium was replaced with a fresh medium, and the PFO ME (30, 10, 5, and 2 μL) was added into each well in each 24-well plate. The wells containing pure medium and the medium with 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, USA) were used as the negative and positive control groups, respectively. The material-treated cells in each well plate were incubated for 1 and 3 days depending on the number of seeded cells. After the incubation, all the cultured media were removed from the well plates, and each well was washed with 1X PBS. Subsequently, the medium containing 10% MTT solution was put into each well and incubated for 2 h. To dissolve the MTT formazan so obtained, the medium was removed and 150 μL DMSO was added to each well. A 100-μL DMSO solution with dissolved formazan was placed in a 96-well plate, and the relative amounts of the viable cells were analyzed by measuring their optical density (OD) at a wavelength of 570 nm.

Antibacterial activity test Two bacteria strains of S. aureus (Gram-positive, S. aureus; 25923) and P. aeruginosa (Gramnegative, P. aeruginosa; 10145) were purchased from American Type Culture collection (ATCC; Manassas, VA, USA). Both the bacteria were cultured in tryptic soy broth media (soybean-casein digest medium; Becton Dickinson and Company (BD), Franklin Lakes, NJ, USA) and maintained in an incubator at 37 °C under aerobic conditions. The PFO ME (100, 30, 10, 5, and 2 μL) was added to 1 mL tryptic soy broth media with S. aureus and P. aeruginosa in a concentration of an optical density (OD)600 of 0.58, and all the groups were incubated for 12 h. Here, the PFO ME-treated and untreated bacterial groups were used as experimental and control groups, respectively. For the evaluation for the antibacterial activity of the PFO ME, 7



both S. aureus and P. aeruginosa were collected from the original well plates where bacteria had been cultured. The harvested bacteria were counted at the OD measured at 600 nm.

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Results and discussion We successfully prepared the PFO ME using the ultrasonication method. For its application as an oxygen carrier, PFO was emulsified using Pluronic F127 and EYP, which are the commonly used emulsifiers for perfluorocarbons [28] to improve their miscibility with water and reduce their density. The size of the resulting MEs is less than 500 nm. Such MEs are generally thermodynamically stable and have a long shelf-life. Figures 1a and 1b show that the size and zeta potential (determined using a size analyzer) of the PFO ME were 317.95±21.25 nm and 22.7±1.01 mV, respectively. From Figure S1, it is clear that the PFO ME could retain its shape even after 1 month and filtration (Figure S1). The high zeta potential of the PFO ME induced repulsion between the ME particles, which resulted in the formation of a stable dispersion in aqueous solutions. If the case of emulsions with very low zeta potential values, there is no force to prevent the aggregation of emulsion particles, which results in the formation of unstable dispersions. The zeta potential value of the PFO ME was sufficiently high for forming stable dispersions.

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Figure 1. Characterization of the PFO ME (a) size distribution, (b) zeta potential, (c) schematic illustration and (d) TEM images. Figure 1c shows the schematic illustration of the PFO ME. The TEM images showed that the PFO ME particles were spherical in shape with a moderately uniform size. The particle size measured from the TEM images can be checked clearly (Figure 1d). The location of the PFO core in PFO MEs is highly critical. In the TEM images, the PFO core (which was in the interior of the PFO ME) could be clearly distinguished from the F-127 and EYP components of the PFO ME. Hence, it can be stated that the PFO ME had a core-shell structure. This is because the PFO region was relatively dark because of the high electron density and electronegativity of the fluorine-containing functional groups, while the regions corresponding to F-127 and EYP were relatively bright because of their low electron density. Hence, it was confirmed that the PFO ME was synthesized successfully.

Figure 2. Incorporation of NO gas into the PFO ME. (a) PFO ME before and after NO gas loading and the release of NO gas from the PFO MEs. (b) Diameter of the PFO ME before (black line) and after (red line) NO gas loading, as obtained by DLS measurements. (c) Kinetics and (d) total amount of NO released from PFO and the PFO ME. 10


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NO is a biatomic free radical (uncharged molecule) containing an unpaired electron [29]. NO can react with oxygen free radicals to form nitrogen oxides, which are involved in protein oxidation reactions under physiological conditions [30]. NO gas remains stable as long as it is not exposed to reactive species. Since the solubility of gases (O2, CO2, N2, NO gas etc.) in liquid PFO is very high, NO gas readily reacts with other soluble gases such as O2 in PFO. As a result, nitrogen oxides (NOx) are easily produced in liquid PFO. Hence, in order to deliver NO gas using PFC liquids, it is necessary to minimize the contact and reaction between NO and O2. In order to achieve this, we made use of the difference between the gas solubilities in aqueous solutions. O2 and NO gases in water show solubilities of 9.09 and 56 mg/L, respectively, in water. The difference in the water and PFO solubilities of NO gas prevents its explosive release and keeps it active. In the PFO ME, an interface was formed between water and PFO. NO gas scarcely reacted with O2 and could be safely stored in the PFO core of the ME (Figure 2a). During the loading of NO gas into the PFO ME, it physically drew in between the PFO molecules. The NO-loaded PFO ME was slightly larger than the PFO ME (Figure 2b). We compared the NO gas release kinetics of PFO and the PFO ME to investigate the air barrier effect of water. Despite the much larger amount of PFO in the liquid state, the release amount of NO from the PFO ME was much larger that from the liquid PFO (Figure 2c). As mentioned above, PFO offers high solubility not only to NO but to other gases as well. The amount of oxygen that can be dissolved in PFO is almost equal to its atmospheric concentration. Liquid PFO facilitates the reaction between NO and O2. Hence, irrespective of the amount of NO purged, NOx gases are produced as soon as PFO is exposed to air. However, NO can be stably stored in PFO, if water blocks its contact O2 in air. As shown in Figure S1 shows that when liquid PFO is in direct contact with air, NOx is produced, which can be confirmed by the brown 11



color of PFO (can be observed with naked eye). On the other hand, no brown color is observed in the case of water-protected PFO, indicating that no NOx is produced in this case. Figure 2d shows that the total amount of NO released from the PFO ME and liquid PFO was 87.4 and 1.094 nmol/L for 12 h, respectively. The amount of NO released from the PFO ME was very high. NO gas can be released by physical gas dissolution phenomenon of PFO that follows Henry’s law. NO gas solubility is directly proportional to the gas’s partial pressure. NO gas can be rapidly and extensively extracted from PFCs when there is difference of NO concentration between PFC and media.

Figure 3. In vitro cytotoxicity test for the PFO ME before/after loading NO gas. HDF cell viability of the PFO ME with and without releasing NO gas at various concentrations (2, 5, 10, 30 µL) for 1 and 3 days. Data are expressed as means ± SD from three experiments as % of control cells. An optimum concentration of NO gas can induce the extracellular signal-regulated kinase phosphorylation process that enhances cell proliferation and cell migration for wound healing [31]. Indeed, it is known that exogenous NO gas can positively regulate cell proliferation and/or the proliferation of neighboring cells. We prepared a variety of concentrations of NO-loaded PFO ME (30 µL-maximum of released NO: 2.622 mol, 10 µL-maximum of released NO: 0.874 mol, 5 µL-maximum of released NO: 0.437 mol, and 2 µL-maximum of released NO: 0.175 mol) for testing cell viability. On day 1, only a slight difference was observed in the cell viabilities of the NO-loaded PFO ME (30 µL) and the PFO ME (10 µL), (Figure 3a). On 12


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day 3, the cell viability of the NO-loaded PFO was significantly higher than that of the PFO ME and control, regardless of the sample concentrations (Figure 3b). In the case of the cell viability of the NO-loaded PFO ME was 20% higher than that of the control sample. It can be stated that the HDF cell growth on days 1 and 3 improved by using 30 and 10 L of the NOloaded PFO ME, and that NO from the PFO ME affects the cell growth and proliferation positively. NO released from PFO MEs plays an important role in the mediation of the cell proliferation and protection effects. Low NO concentrations are required for cyclic guanosine monophosphate (cGMP)-mediated processes. In our NO delivery system, of the amount of NO released was very high initially and decreased gradually in the second half. At the beginning, the NO released from the PFO ME generated nitrosative stresses. However, the level of these stresses was not fatal to eukaryotic cells such as human cells. Hence, in the initial stage, the cells can withstanding the nitrosative stresses and start proliferating gradually because of the slower NO release during the later stages. We found that the cell viability with NO-loaded PFO ME was higher than that obtained with the control and PFO ME samples. Antibacterial activity of NO involves the interaction of NO free radical with reactive oxygen intermediates (hydrogen peroxide and superoxide) to produce various antibacterial molecular species [32]. Furthermore, NO itself can be transformed to peroxynitrite (OONO−), Snitrosothiols (RSNO), nitrogen dioxide (NO2), dinitrogen trioxide (N2O3), and dinitrogen tetroxide (N2O4). These reactive intermediates target DNA, causing deamination and oxidative damages including basic sites, strand breaks, and other DNA alterations [Mutat Res 349:51– 61]. Bacteria make up a large domain of prokaryotic microorganisms. Hence, the weakening or destruction of the cell membrane by NO gas can be fatal. 13



Figure 4. Antibacterial activity test of the PFO ME. (a) Schematic illustration of NO delivery and anti-bacterial mechanism by NO gas from PFC microemulsions. Percent of S. aureus killed by (b) PFO ME and (c) NO-loaded PFO ME in 12 h. We evaluated the antimicrobial property of the NO-loaded PFO ME. Figure 4a displays NO delivery phenomenon (NO moving PFC → media → bacteria cytoplasm). Transfer of NO gas in this system directly depends on partial pressure according to Henry`s law. As shown in Figure 4b, the PFO ME showed a slight antibacterial effect because nanoscale microemulsions can negatively affect the cell membrane of bacteria [33]. In the case of the NO-loaded PFO ME, the antibacterial effect varied significantly with the concentration (Figure 4c). At low concentrations of 2, 5, and 10 µL, the number of S. aureus bacteria killed by the NO-loaded PFO ME was slightly less than that killed by the NO-unloaded PFO ME. This indicates that NO has a positive effect on the growth of bacteria. On the other hand, at high concentrations of 30 and 100 μL, the NO-loaded PFO ME showed good antibacterial efficiency of 52.48 % and 37.89 %, respectively. However, the effect of the NO-loaded PFO ME on the gram14


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negative P. aeruginosa (Figure S3) bacteria was negligible. This is because the cell membrane compositions of S. aureus and P. aeruginosa are different. S. aureus and P. aeruginosa have plasma membrane, periplasmic space, and peptidoglycan. P. aeruginosa has more periplasmic space and its outer membrane is composed of lipopolysaccharide and proteins. This is the reason why P. aeruginosa is more stable than S. aureus.

Conclusion PFO ME was successfully synthesized and could retain its ME form up to 1 month. Unlike the time-consuming chemical methods for the preparation of NO carriers, the method used by us for preparing the PFO ME required only 10 min even when NO gas was purged for 2 h. Large amount of NO could be loaded onto the PFO ME without any chemical reaction and could be released for 12 h. The HDF cell viability studies showed that the PFO ME and NO-loaded PFO ME were nontoxic towards human fibroblasts for three days. The NO-loaded PFO ME showed slightly higher cell viability that the PFO ME. At higher concentrations (100 µL and 30 µL), the NO-loaded PFO ME showed strong antibacterial activity towards S. aureus, indicating that it is a promising material for antibacterial applications. As NO-releasing materials for high biocompatibility, the prevention and dispersal of biofilms, NO-loaded PFO ME with physical interaction would be a great tool to the biomedical and bioanalytical field.

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ASSOCIATED CONTENT Supporting Information. Prevention of the formation of NOx in PFO by the water barrier effect. Stability and antibacterial activity tests (P. aeruginosa) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions All the authors contributed to writing the manuscript. All authors have approved the final version of the manuscript.

ACKNOWLEDGMENTS This research was also supported by the National Research Foundation of Korea (NRF) Grant funded by the Ministry of Science and ICT for First-Mover Program for Accelerating Disruptive Technology Development (NRF-2018M3C1B9066755). This research was also supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2017R1E1A1A01074343).

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Nitric Oxide Delivery using Biocompatible Perfluorocarbon Microemulsion for Antibacterial Effect Moonhyun Choi, Sohyeon Park, Kyungtae Park, Hyejoong Jeong and Jinkee Hong

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Nitric Oxide Delivery using Biocompatible Perfluorocarbon

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