On the effect of serum on the transport of reactive oxygen species across phospholipid membranes Endre J. Szili, Sung-Ha Hong, and Robert D. Short Citation: Biointerphases 10, 029511 (2015); doi: 10.1116/1.4918765 View online: http://dx.doi.org/10.1116/1.4918765 View Table of Contents: http://scitation.aip.org/content/avs/journal/bip/10/2?ver=pdfcov Published by the AVS: Science & Technology of Materials, Interfaces, and Processing Articles you may be interested in Ionic size effects to molecular solvation energy and to ion current across a channel resulted from the nonuniform size-modified PNP equations J. Chem. Phys. 140, 174102 (2014); 10.1063/1.4872330 Fabrication of long poly(dimethyl siloxane) nanochannels by replicating protein deposit from confined solution evaporation Biomicrofluidics 6, 026504 (2012); 10.1063/1.4730371 Effect of interactions on molecular fluxes and fluctuations in the transport across membrane channels J. Chem. Phys. 128, 085101 (2008); 10.1063/1.2831801 Membrane Fusion of Giant Liposomes of Neutral Phospholipid Membranes Induced by La3+ and Gd3+ AIP Conf. Proc. 708, 316 (2004); 10.1063/1.1764153 Nonequilibrium molecular dynamics simulations of transport and separation of supercritical fluid mixtures in nanoporous membranes. I. Results for a single carbon nanopore J. Chem. Phys. 119, 6810 (2003); 10.1063/1.1605373
On the effect of serum on the transport of reactive oxygen species across phospholipid membranes Endre J. Szili,a) Sung-Ha Hong, and Robert D. Shortb) Mawson Institute, University of South Australia, Adelaide SA 5095, Australia and Wound Management Innovation Cooperative Research Centre, Australia
(Received 11 February 2015; accepted 10 April 2015; published 24 April 2015) The transport of plasma generated reactive oxygen species (ROS) across a simple phospholipid membrane mimic of a (real) cell was investigated. Experiments were performed in cell culture media (Dulbecco’s modified Eagle’s medium, DMEM), with and without 10% serum. A (broad spectrum) ROS reporter dye, 2,7-dichlorodihydrofluorescein (DCFH), was used to detect the generation of ROS by a helium (He) plasma jet in DMEM using free DCFH and with DCFH encapsulated inside phospholipid membrane vesicles dispersed in DMEM. The authors focus on the concentration and on the relative rates (arbitrary units) for oxidation of DCFH [or the appearance of the oxidized product 2,7-dichlorofluorescein (DCF)] both in solution and within vesicles. In the first 1 h following plasma exposure, the concentration of free DCF in DMEM was 15 greater in the presence of serum (cf. to the serum-free DMEM control). The DCF in vesicles was 2 greater in DMEM containing serum compared to the serum-free DMEM control. These data show that serum enhances plasma ROS generation in DMEM. As expected, the role of the phospholipid membrane was to reduce the rate of oxidation of the encapsulated DCFH (with and without serum). And the efficiency of ROS transport into vesicles was lower in DMEM containing serum (at 4% efficiency) when compared to serum-free DMEM (at 32% efficiency). After 1 h, the rate of DCFH oxidation was found to have significantly reduced. Based upon a synthesis of these data with results from the open literature, the authors speculate on how the components of biological fluid and cellular membranes might affect the kinetics of consumption of plasma generated C 2015 American Vacuum Society. [http://dx.doi.org/10.1116/1.4918765] ROS. V
I. INTRODUCTION Nonthermal plasma sources typically used in plasma medicine are highly reactive gases that readily activate the ambient air generating a rich gaseous mixture of reactive oxygen species (ROS) and reactive nitrogen species (RNS), or collectively RONS. When solution is exposed to plasma, some of these RONS dissolve from the gas phase into solution. Further, the plasma or the reactive plasma effluent can directly activate new RONS in the liquid and “downstream” other new RONS may be formed from subsequent reactions in solution.1–3 Many of the RONS generated by plasma (directly or indirectly) in solution are also generated naturally in vivo and within cells and are known to regulate physiological and signaling processes.4 There is a rapidly growing literature that links RONS to the therapeutic outcomes afforded by plasma.5 The potential role of RONS in the regulation of cellular processes has been demonstrated.4,6,7 A number of explanations have been put forward to describe the mechanisms by which plasma generated RONS regulate cellular and physiological processes. The general consensus is that RONS, either directly/indirectly generated by plasma in solution, activate/modify the constituents of the biological milieu or cell media or the external cell membrane, resulting in the oxidation of proteins, lipids, and other macromolecules.8,9 These oxidized/modified products then a)
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b)
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interact with cell membrane receptors or directly with the internal cell components (if they diffuse into the cell), resulting in the activation/deactivation of signaling processes and changes in cellular activity. The role of plasma generated RONS has been linked to biological outcomes including stimulation of cell proliferation, migration, and angiogenesis;10–13 regulation of stem cell differentiation;14 deactivation of bacteria and biofilms;15–22 destruction of tumors;23–25 enhancement of wound healing;26–31 and tissue regeneration.32,33 However, it is not clear if the RONS act by modifying components outside of the cell or inside of the cell. This becomes even less clear considering that plasma can have effects deep into biological material such as biofilms34 and solid tumors24 which can be several micrometers to millimeters in thickness; this is despite many of the plasma generated RONS having short lifetimes and diffusion lengths.8 Earlier studies in the field of plasma interactions with organic surfaces would indicate the effects of plasma should be localized to no more than a few nanometers of the target.35–37 This is clearly not the case in plasma tissue treatment: in the context of cancer treatment, Barekzi and Laroussi argue that the plasma generated RONS stimulate cell reactions or signaling mechanisms that may be transmitted deep into the tissue.38 Supporting this hypothesis, in a recent review, Graves proposes an “oxy-nitroso shielding burst model” whereby surface layer cells communicate to cells deeper within the tissue, similar to other manifestations of cell stress.8
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C 2015 American Vacuum Society V
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029511-2 Szili, Hong, and Short: On the effect of serum on the transport of ROS
In addition to the deactivation of tumors and biofilms at tissue surfaces, our previous research suggests that plasma can be used to deliver RONS to cells that are embedded deep within the affected tissues.39–42 Our data suggest the direct plasma transport of RONS occurs to at least 150 lm into “soft” tissues. But other (undefined) processes further transport RONS deeper, to depths of at least 3.5 mm.43 At these depths, we speculate that the original primary (and more reactive) RONS are likely to have been “consumed” by the surrounding tissue macromolecules. Our findings are of interest in the context of the work of Svarnas et al.44 and Kim et al.45 They show using synthetic cell and live cell models that plasma generated RONS directly interact with, and modify, cell membrane structures, which will alter the subsequent permeability of these membranes to further RONS. Cell membranes are equipped with various channels that allow the passive diffusion or active transport of RONS into and out of the cell. For example, superoxide anion (O2•–), which is formed in liquids by atmospheric-pressure plasmas,46 can initiate intracellular signaling by penetration through cell membrane anion channels, whereas redox signaling from hydrogen peroxide (H2O2) is catalyzed by NADPH oxidase (Nox 2).47 H2O2 signaling is crucial to many physiological functions such as the promotion of the respiratory burst in phagocytes (to protect the host from chronic infection).47 Consequently, in normal cells, the transport of key signaling RONS across cellular membranes is not a random process.48 It follows that the direct delivery of RONS across cell membranes by plasma exposure (or the modification of cell membranes by plasma generated RONS) could be an important strategy in the treatment of diseases. For example, in cancers (such as leukemia) the modulation of H2O2 transport through specific aquaporins affects redox signaling, which is linked to cell proliferation.49 The plasma assisted transport of RONS across cell membranes (or modification of membranes) could potentially and significantly perturb the normal regulation of cellular signaling processes.50 In this study, we expose phospholipid membrane vesicles suspended in DMEM (Ref. 39) (as mimics of a cell membrane) to a helium (He) plasma jet to investigate the transport of plasma generated ROS across the phospholipid membrane. The vesicles encapsulate a (broad spectrum) ROS reporter dye, 2,7-dichlorodihydrofluorescein (DCFH); the dye is oxidized mainly by ROS (although some RNS may also oxidize the dye), converting the nonfluorescent dye to fluorescent 2,7-dichlorofluorescein (DCF).51,52 The fluorescence intensity (FI) of DCF is related to the concentration of ROS inside the solution or vesicles.39 The rate of DCFH oxidation induced by plasma generated ROS was compared between DCFH in cell culture media (free DCFH) and DCFH encapsulated within vesicles, and with and without the addition of serum. We focus specifically on the concentration and on the rate of appearance of DCF (that is oxidized product appearance) given by d[DCF]/dt, both in solution and encapsulated within vesicles. Biointerphases, Vol. 10, No. 2, June 2015
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SCHEME 1. Conceptual overview of the experimental approach to explore the effect of serum on the transport of ROS across phospholipid membranes.
Our aim in this study is to explore RONS transport across a phospholipid membrane and how intercellular components, such as macromolecules, might perturb this process, and its kinetics (Scheme 1). II. EXPERIMENT A. Preparation of 2,7-dichlorodihydrofluorescein
DCFH (Sigma-Aldrich) was prepared using a similar protocol to the one described by Low et al.53 A 1 mg ml1 stock of DCFH diacetate was prepared in ethanol and stored at 20 C until use. Ester hydrolysis was induced by adding 500 ll of stock DCFH to 2 ml of 10 mM NaOH and incubating for 30 min at 25 C in the dark. The solution was then neutralized by adding 10 ml of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer (for HEPES recipe, see Ref. 54). The dye was mixed at a ratio of 50:50 in Dulbecco’s Modified Eagle’s medium (DMEM, SigmaAldrich) with and without 10% (v/v) fetal bovine serum (FBS, Sigma-Aldrich) (herein referred to as serum). (In the case of vesicle studies, serum was added post vesicle formation.) The volume used in experiments was 100 ll per well of a 96 multiwell plate. Fluorescence measurements were recorded at a kexcitation of 485 nm and a kemission of 520 nm for DCF using a BMG Labtech Fluostar Omega microplate reader. The issue of the variability in the FI of DCFH (when converted to DCF) between experiments55–57 was overcome by preparing fresh DCFH stocks, keeping the DCFH/DCF solution in the dark when possible and normalizing the data to an internal control (nonoxidized DCFH) for each of the experiments. B. Preparation of synthetic phospholipid membrane cell model
A synthetic cell model, consisting of phospholipid membrane vesicles encapsulating DCFH, was used to monitor the
029511-3 Szili, Hong, and Short: On the effect of serum on the transport of ROS
flux of plasma generated ROS across the phospholipid membrane. Phospholipids and all other chemicals were obtained from Sigma-Aldrich and Sephadex G25 columns supplied by GE Healthcare. The phospholipid membrane comprised 1,2distearoyl-sn-glycero-3-phospho-(10 -rac-glycerol) (sodium salt) (DSPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol. The phospholipid vesicles mimic the natural cell membrane, i.e., the vesicle membrane comprises a multilipid composition and 20% cholesterol.58 The vesicles were synthesized using the procedure reported elsewhere.54 A 100 ll stock solution was added to 200 ll of chloroform, followed by drying with nitrogen gas. Then, the dry film of lipid was rehydrated in 5 ml of 5 mM deacetylated DCFH (Sec. II A), heated to 65 C, and freeze-thawed three times before being extruded through 100 nm polycarbonate membranes in a LiposoFast basic hand-held extruder. The solution was then purified using a Sephadex G25 column to remove nonencapsulated dye. The vesicles were mixed at a ratio of 50:50 in DMEM with and without serum (serum was added post vesicle formation) to a total volume of 100 ll per well of a 96 multiwell plate. This gave a final vesicle concentration of 5 1011 vesicles ml1. Fluorescence measurements were recorded as described in Sec. II A. Using a protocol outlined in our previous investigation,39 we confirmed that the plasma operating conditions employed herein [directly below] do not rupture vesicles that are freely suspended in solution.
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relative rate at which plasma oxidizes nonfluorescent DCFH to fluorescent DCF is compared for free DCFH in DMEM and DCFH encapsulated within phospholipid membrane vesicles. The rate of DCF appearance (arbitrary units) is taken from d½DCF dt ¼ Slope of FI DCF vs time:
Rate of product apperance ¼
Figures 1(a) and 1(b) show the FI of DCF as function of incubation time after plasma exposure. Figure 1(a) shows that in serum-free media at the 1 h (3600 s) time point (after plasma exposure), the FI of DCF is 4 greater for the free DCFH than the vesicle-encapsulated DCFH. This indicates that the phospholipid membrane presents a barrier to the transport of plasma generated ROS (from solution) into vesicles. When DMEM is supplemented with serum, comparing the FI values in Fig. 1(b) with those in Fig. 1(a), reveals that there is a profound and positive (15) increase in the ROS concentration in solution. This result was initially somewhat surprising—and we comment upon it below. In serum, the elevated ROS concentration is most evident in solution but can be seen even within the vesicles. (At 1 h, the
C. Plasma jet exposure
The plasma source was of a “jet” configuration. It comprised a glass pipette with an inner diameter of 1 mm, surrounded by two external hollow electrodes and separated at a distance of 10 mm. The end of the tube protruded 1 mm out from the bottom electrode. The operating parameters of the plasma were 1 l min1 of He; applied voltage of 5.5 kVpeak-peak; and frequency of 10 kHz. Analysis of the current and voltage waveforms confirmed that the discharge ignites a few times per AC cycle with emission coinciding with distinct current peaks on the driving current waveform (supplementary material).59 The plasma jet was operated into the ambient air, and the exposure time was 1 min. The top of the 96-well plate, containing the test cell solutions, was positioned 25 mm underneath the end of the glass pipette using a laboratory scissor jack covered with an insulating sheet of polytetrafluoroethylene (PTFE). The flow rate of the He gas was controlled with a mass flow controller and power was supplied with a custom-built unit as described elsewhere.60 III. RESULTS AND DISCUSSION We employed DCFH as a (broad spectrum) ROS reporter; DCFH is a well-characterized dye for the detection of ROS in biological systems.57 When nonfluorescent DCFH is oxidized by ROS, it is converted to fluorescent DCF. The intensity of DCF fluorescence (FI) can be correlated to the concentration of ROS in the solution or in cells.52 The Biointerphases, Vol. 10, No. 2, June 2015
FIG. 1. Concentration of ROS is raised by the addition of serum and the efficiency of ROS transport across the phospholipid membrane is significantly reduced by the addition of serum. The FI for DCF for free dye in DMEM and DCF encapsulated within vesicles without serum (a) or with serum (b). The rate of DCFH oxidation (DCF appearance) is determined from the slope at 1 h. The graph inset in (a) has a lower y-axis scale range to illustrate the difference in the slope of the trend lines. The error bars for each datum point are not shown for the sake of clarity. The error in the slope of the curves are in (a) 62.8 107 for DCFH encapsulated within vesicles and 63.5 107 for DCFH in solution and (b) 63.7 107 for DCFH encapsulated within vesicles and 68.8 106 for DCFH in solution (also see Table I).
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TABLE I. Rate of appearance of free DCF (in solution) and encapsulated DCF without and with the addition of serum. Rate of DCF appearance was measured at 1 h for the 0–1 h time period and at 15 h for the 10–15 h time period (post plasma exposure). Rate of DCF appearance: d[DCF]/dt Cell media type
Time-period postplasma exposure (h)
Free dye in solution
Dye in vesicles
0–1 10–15 0–1 10–15
1.16 105 (63.52 107) 7.38 106 (61.04 107)a 2.1 104 (68.8 106) 8.44 106 (61.39 107)a
3.76 106 (62.83 107) 5.06 108 (63.20 107) 7.5 106 (63.69 107) 2.52 106 (65.63 107)a
Serum-free DMEM DMEM þ serum
a
Negative values indicate the oxidation of DCFH has stopped and a decay in the converted fluorophore.
level of FI in vesicles with serum is 2 greater than in vesicles without serum.) Initially, in solution with the addition of serum, the ROS concentration is raised by over an order of magnitude. And, in the presence of serum, the vesicle phospholipid membrane provides an effective barrier to ROS ingress. The DCF FI at 1 h is now 22 greater for free DCFH than for the vesicle-encapsulated DCFH. Table I summarizes the rate of DCF appearance in DMEM with and without serum. We acknowledge that in Fig. 1(b), that for the dye in solution, d[DCF]/dt is not a constant over the first hour; however, for the purpose of following analysis, this approximation is acceptable. The time points chosen were 1 and 15 h after the initial plasma exposure. In the first 1 h following plasma exposure, the rate of DCF appearance is greater in DMEM with serum compared to serum-free DMEM. However, the efficiency of ROS transport from solution into the vesicles, calculated from rate of appearance of DCF within vesicles/rate of appearance of free DCF 100%, is reduced from 32% for serum-free DMEM to 4% for DMEM with serum. At 10–15 h following plasma exposure, the rate of DCF appearance for free dye in solution and within vesicles, decreases in DMEM both with and without serum (Table I). The negative values in Table I most probably indicate the decay in fluorescence in DCF (after the oxidation of DCFH had stopped).
We had initially anticipated that the rate of DCF appearance would be reduced in DMEM containing serum: Tian and Kushner have shown through mathematical modeling that plasma generated ROS are largely consumed by alkane hydrocarbons,3 and further, serum contains amino acids and antioxidant enzymes that will react with ROS.61,62 Grzelak et al. have shown that the light-dependent formation of ROS in DMEM is significantly reduced in the presence of 10% serum.63 As a control, we established that serum does not affect the FI of DCF (i.e., fully oxidized DCFH) (data not shown). In an additional experiment, we measured the rate of DCFH oxidation with a single ROS (H2O2). The experiment was performed in DMEM and in a buffer of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) with and without serum. The experiment involved mixing free DCFH into DMEM or HEPES with and without serum. Then, 50 lM of H2O2 was added and the FI (of DCF) was measured. Figure 2(a) shows that the oxidation of DCFH by H2O2 in HEPES or DMEM is enhanced by serum. For comparison, we performed the same experiment but replaced DCFH with 5,6-carboxyfluorescein (CF). The excitation and emission wavelengths of CF are very similar to DCF. However, the FI of CF is not expected to be sensitive to ROS. Figure 2(b) shows that the FI of CF in HEPES or DMEM is not affected
FIG. 2. Serum enhances the oxidation of DCFH in buffer (HEPES) and cell media (DMEM). (a) The fluorescence intensity (FI) of DCF was compared in HEPES and DMEM with and without addition of serum. The experiment involved mixing free DCFH (0.00005 M) in HEPES, HEPES þ FBS, DMEM, and DMEM þ FBS and then adding H2O2 (50 lM) before measuring the FI. The FI of all solutions without DCFH and H2O2 were also measured and were not fluorescent. (b) The FI of CF, under the same conditions, was compared. CF was not expected to be sensitive to ROS. The error bars are calculated from the standard deviation of the mean from three replicate samples. Biointerphases, Vol. 10, No. 2, June 2015
029511-5 Szili, Hong, and Short: On the effect of serum on the transport of ROS
by serum in the presence H2O2. This experimental control increases our confidence that the addition of serum enhances the rate of oxidation of DCFH in the presence of ROS. We do not have a full explanation as to why serum enhances ROS generation in our study, but reduced ROS generation in the previous study by Grzelak et al.63 We speculate the contradicting results might be attributed to the different mechanisms of ROS generation by plasma and ROS generation under a standard fluorescent laboratory light, as employed by Grzelak et al.63 Serum may reduce the lightdependent generation of ROS in solution; however, ROS generated in the plasma effluent will still solvate from the gas phase into the solution.2,3 In the presence of serum, we speculate that the ROS react with serum macromolecules leading to the generation of alkyl radicals.3 A major component of serum is albumin. Alkyl radicals in turn can react with molecular oxygen to give alkylperoxyl radicals (ROO•).64 Setsukinai et al. observed that after excluding the highly reactive but short-lived hydroxyl radical (•OH) and peroxynitrite (ONOO), the ROO• radical is the most reactive radical with DCFH.65 These “secondary” macromolecular ROS arising from the oxidation of serum will be substantially larger than the initial ROS species. We therefore think it is highly unlikely these secondary ROS can traverse the phospholipid membrane. More likely, decomposition products of these ROS (under ambient laboratory conditions) are responsible for the formation of DCF in vesicles suspended in DMEM containing serum. The ingress of ROS into cells will strongly influence the behavior and ultimately the fate of any cell. Although we are cognizant of the limitation of our synthetic phospholipid vesicles cf. real cells, below we discuss a few ideas on how plasma and plasma generated ROS might broadly interact with real cells. These ideas are put forward in the spirit of trying to improve our understanding of plasma induced effects within cells and tissue. Based upon a synthesis of our data and what is known from the open literature, we suggest that: (1) within 1 h following plasma exposure, the solution (or extracellular matrix in the case of “real” cells in tissue) concentration of ROS would be (initially) much higher than the vesicle (intracellular) ROS concentration; (2) short-lived ROS react quickly with macromolecules in the solution, leading to the generation of larger (perhaps less cell-impermeable) but more stable ROS; (3) other effects during plasma exposure such as electric fields, sheer stress, and pressure may increase (at least temporarily) the permeability of the phospholipid membrane to ROS;66 longer term effects probably include a change in pH, modifications of the intercellular components and oxidation of the phospholipid membrane; (4) the ROS concentration gradient described in (1) and any changes in the permeability of the phospholipid membrane described in (3) will lead to an ingress of ROS into cells; (5) a (real) cell (as opposed to our synthetic vesicle) will initially respond to the influx of ROS by up-regulation of its antioxidant defenses; (6) if the flux of ROS is too high, this cell may undergo apoptosis/necrosis; (7) after about 1 h post plasma, Biointerphases, Vol. 10, No. 2, June 2015
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TABLE II. Membrane surface area to volume ratio (SA:VOL) of the vesicles and prokaryotic and eukaryotic cells. The SA:VOL were based on an average diameter for each cell type and assuming that the vesicles and cells are spherical.
Membrane description 100 nm diameter vesicles 2000 nm diameter prokaryotic cells 10 000 nm diameter eukaryotic cells
Surface area (SA) of membrane: 4pr2 (nm2)
Volume (VOL) of vesicle or cell: 4/3pr3 (nm3)
SA:VOL (3/r)
3.14 104
5.24 105
3/50
1.26 107
4.19 109
3/1000
3.14 108
5.24 1011
3/5000
the extracellular and intracellular ROS concentration begin to equilibrate. This time period will presumably be dependent on the exact ROS dose—we can see in Fig. 1(b) at the 1 h point in solution a negative change in d[DCF]/dt; (8) if the cell survives plasma exposure, in the longer term (depending on the flux of ROS into the cell) the cell may return to its state prior to plasma treatment or it may be irreversibly changed (e.g., phenotypically). Additionally, to be considered is that plasma generated ROS may oxidize cell membranes and therefore also increase permeability to further ROS over a longer period than the initial plasma exposure.67,68 And it is because of point (5), and later point (6), that it is difficult to make direct measurements of ROS ingress into viable cells. The ability of a cell to cope with the burst of ROS may be affected by the cell membrane surface area to cell volume ratio. In Table II, we compare the (theoretically calculated) surface area:volume ratio (SA:VOL) of our vesicles to eukaryotic and prokaryotic cells. We appreciate that the SA:VOL of the phospholipid vesicles is much larger than real cells. Therefore, we readily acknowledge the possibility that ROS may be transported faster into our vesicles, cf. real cells. In general, the SA:VOL is five times higher for prokaryotic cells than eukaryotic cells. This ratio might result in a much faster transportation of ROS into bacterial cells (cf. mammalian cells). A consequence of this faster ingress of ROS into bacterial cells might be that bacterial cells are more vulnerable to damage from ROS. At least in part, this may explain why plasma can be used to effectively decontaminate wounds without detrimentally affecting the surrounding healthy tissue. (This does not take into account any differences there may be in the relative permeability of different cell membranes.) In a previous paper, we argue that plasma is a superior method for the effective delivery of ROS into cells and tissue, cf. the topical application of ROS from solution.41 In our current study, our approach is restricted in that we cannot probe what happens during the 1 min of plasma exposure, but our data certainly support the concept that plasma is an effective method to deliver ROS into cells and tissues. This process is enhanced by the presence of serum in the treated fluid.
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IV. SUMMARY AND CONCLUSIONS The key conclusions drawn from this study are that plasma generated ROS can be transported across a model phospholipid membrane, and that in the context of real cells, this indicates that phospholipid membranes will inhibit (but not prevent) ROS ingress. Plasma generated ROS react with media components (such as serum) and this has a profound and positive impact on the fluid ROS concentration. We speculate that these “additional” ROS species are likely to be alkyl peroxyl radicals. These ROS are substantially larger radicals and will not be so readily transported across cell membranes. In context of plasma treatment of real tissues, we should anticipate the substantial generation of “secondary” macromolecular ROS within tissue and tissue fluids, which will “bath” embedded cells. It may be that the ROS that traverse the phospholipid membrane are small “tertiary” ROS derived from the “secondary” ROS under ambient conditions. Establishing a better insight into the nature and physical transport mechanisms of ROS or RNS into cells and tissue is important: not only does it helps strengthen our scientific understanding for the use of plasma in medicine and biology but also provides a rational basis for determining optimal and/or safe plasma doses. ACKNOWLEDGMENT The authors thank the Wound Management Innovation CRC for partially funding this work through project RP 2.11. 1
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P. Y. Kim, Y.-S. Kim, I. G. Koo, J. C. Jung, G. J. Kim, M. Y. Choi, Z. Yu, and G. J. Collins, PLoS One 6, e24104 (2011). 22 X. Pei, X. Lu, J. Liu, D. Liu, Y. Yang, K. Ostrikov, P. K. Chu, and Y. Pan, J. Phys. D: Appl. Phys. 45, 165205 (2012). 23 M. Keidar, A. Shashurin, O. Volotskova, M. A. Stepp, P. Srinivasan, A. Sandler, and B. Trink, Phys. Plasmas 20, 057101 (2013). 24 M. Keidar, R. Walk, A. Shashurin, P. Srinivasan, A. Sandler, S. Dasgupta, R. Ravi, R. Guerrero-Preston, and B. Trink, Br. J. Cancer 105, 1295 (2011). 25 J. Y. Kim, Y. Wei, J. Li, P. Foy, T. Hawkins, J. Ballato, and S.-O. Kim, Small 7, 2291 (2011). 26 T. von Woedtke, S. Reuter, K. Masur, and K. D. Weltmann, Phys. Rep. 530, 291 (2013). 27 G. Lloyd, G. Friedman, S. Jafri, G. Schultz, A. Fridman, and K. Harding, Plasma Processes Polym. 7, 194 (2010). 28 Nasruddin et al., Clin. Plasma Med. 2, 28 (2014). 29 C. H. Park et al., J. Phys. D: Appl. Phys. 47, 435402 (2014). 30 G. Isbary et al., Clin. Plasma Med. 1, 25 (2013). 31 A. V. Nastuta, I. Topala, C. Grigoras, V. Pohoata, and G. Popa, J. Phys. D: Appl. Phys. 44, 105204 (2011). 32 M. J. Steinbeck, N. Chernets, J. Zhang, D. S. Kurpad, G. Fridman, A. Fridman, and T. A. Freeman, PLoS One 8, e82143 (2013). 33 M. Miletic´ et al., J. Phys. D: Appl. Phys. 46, 345401 (2013). 34 Z. Xiong, T. Du, X. Lu, Y. Cao, and Y. Pan, Appl. Phys. Lett. 98, 221503 (2011). 35 R. M. France and R. D. Short, J. Chem. Soc., Faraday Trans. 93, 3173 (1997). 36 G. Desmet, A. Michelmore, E. J. Szili, S.-J. Park, J. G. Eden, R. D. Short, and S. A. Al-Bataineh, RSC Adv. 3, 13437 (2013). 37 P. M. Bryant, E. J. Szili, T. Whittle, S.-J. Park, J. G. Eden, S. Al-Bataineh, D. A. Steele, R. D. Short, and J. W. Bradley, Surf. Coat. Technol. 204, 2279 (2010). 38 N. Barekzi and M. Laroussi, Plasma Processes Polym. 10, 1039 (2013). 39 S.-H. Hong, E. J. Szili, A. T. A. Jenkins, and R. D. Short, J. Phys. D: Appl. Phys. 47, 362001 (2014). 40 S. E. Marshall, A. T. A. Jenkins, S. A. Al-Bataineh, R. D. Short, S.-H. Hong, N. T. Thet, J.-S. Oh, J. W. Bradley, and E. J. Szili, J. Phys. D: Appl. Phys. 46, 185401 (2013). 41 E. J. Szili, J. W. Bradley, and R. D. Short, J. Phys. D: Appl. Phys. 47, 152002 (2014). 42 S.-H. Hong, E. J. Szili, A. T. A. Jenkins, and R. D. Short, J. Phys. D: Appl. Phys. 48, 029501 (2015). 43 E. J. Szili, J.-S. Oh, S.-H. Hong, A. Hatta, and R. D. Short, J. Phys. D: Appl. Phys. 48, 202001 (2015). 44 P. Svarnas, S. H. Matrali, K. Gazeli, S. Aleiferis, F. Clement, and S. G. Antimisiaris, Appl. Phys. Lett. 101, 264103 (2012). 45 K. Kim, H. Jun Ahn, J.-H. Lee, J.-H. Kim, S. Sik Yang, and J.-S. Lee, Appl. Phys. Lett. 104, 013701 (2014). 46 A. Tani, S. Fukui, S. Ikawa, and K. Kitano, Jpn. J. Appl. Phys., Part 1 54, 01AF01 (2015). 47 A. B. Fisher, Antioxid. Redox Signaling 11, 1349 (2009). 48 G. P. Bienert, A. L. B. Møller, K. A. Kristiansen, A. Schulz, I. M. Møller, J. K. Schjoerring, and T. P. Jahn, J. Biol. Chem. 282, 1183 (2007). 49 F. Vieceli Dalla Sega, L. Zambonin, D. Fiorentini, B. Rizzo, C. Caliceti, L. Landi, S. Hrelia, and C. Prata, Biochim. Biophys. Acta, Mol. Cell Res. 1843, 806 (2014). 50 G. P. Bienert, J. K. Schjoerring, and T. P. Jahn, Biochim. Biophys. Acta, Biomembr. 1758, 994 (2006). 51 A. Gomes, E. Fernandes, and J. F. C. Lima, J. Fluoresc. 16, 119 (2006). 52 A. Gomes, E. Fernandes, and J. L. F. C. Lima, J. Biochem. Biophys. Methods 65, 45 (2005). 53 S. P. Low, K. A. Williams, L. T. Canham, and N. H. Voelcker, J. Biomed. Mater. Res. Part A 93A, 1124 (2010). 54 S. E. Marshall, S.-H. Hong, N. T. Thet, and A. T. A. Jenkins, Langmuir 29, 6989 (2013). 55 P. Bilski, A. G. Belanger, and C. F. Chignell, Free Radical Biol. Med. 33, 938 (2002). 56 M. G. Bonini, C. Rota, A. Tomasi, and R. P. Mason, Free Radical Biol. Med. 40, 968 (2006). 57 X. Chen, Z. Zhong, Z. Xu, L. Chen, and Y. Wang, Free Radical Res. 44, 587 (2010). 58 G. van Meer, D. R. Voelker, and G. W. Feigenson, Nat. Rev. Mol. Cell Biol. 9, 112 (2008).
029511-7 Szili, Hong, and Short: On the effect of serum on the transport of ROS 59
See supplementary material at http://dx.doi.org/10.1116/1.4918765 for applied voltage and current waveforms of the helium plasma jet. 60 S. A. Al-Bataineh, E. J. Szili, A. Mishra, S.-J. Park, J. G. Eden, H. J. Griesser, N. H. Voelcker, R. D. Short, and D. A. Steele, Plasma Processes Polym. 8, 695 (2011). 61 B. Halliwell, FEBS Lett. 540, 3 (2003). 62 E. Takai, T. Kitamura, J. Kuwabara, S. Ikawa, S. Yoshizawa, K. Shiraki, H. Kawasaki, R. Arakawa, and K. Kitano, J. Phys. D: Appl. Phys. 47, 285403 (2014). 63 A. Grzelak, B. Rychlik, and G. Bartosz, Free Radical Biol. Med. 30, 1418 (2001).
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E. Damiani, B. Kalinska, A. Canapa, S. Canestrari, M. Wozniak, E. Olmo, and L. Greci, Free Radical Biol. Med. 28, 1257 (2000). 65 K.-i. Setsukinai, Y. Urano, K. Kakinuma, H. J. Majima, and T. Nagano, J. Biol. Chem. 278, 3170 (2003). 66 J. M. Pouvesle, The Second International Workshop on Plasma for Cancer Treatment, Nagoya, Japan, 16–17 March 2015. 67 M. Leduc, D. Guay, R. L. Leask, and S. Coulombe, New J. Phys. 11, 115021 (2009). 68 S. Yonson, S. Coulombe, V. Leveille, and R. L. Leask, J. Phys. D: Appl. Phys. 39, 3508 (2006).
On the effect of serum on the transport of reactive oxygen species across phospholipid membranes Endre J. Szili,a) Sung-Ha Hong, and Robert D. Shortb) Mawson Institute, University of South Australia, Adelaide SA 5095, Australia and Wound Management Innovation Cooperative Research Centre, Australia
(Received 11 February 2015; accepted 10 April 2015; published 24 April 2015) The transport of plasma generated reactive oxygen species (ROS) across a simple phospholipid membrane mimic of a (real) cell was investigated. Experiments were performed in cell culture media (Dulbecco’s modified Eagle’s medium, DMEM), with and without 10% serum. A (broad spectrum) ROS reporter dye, 2,7-dichlorodihydrofluorescein (DCFH), was used to detect the generation of ROS by a helium (He) plasma jet in DMEM using free DCFH and with DCFH encapsulated inside phospholipid membrane vesicles dispersed in DMEM. The authors focus on the concentration and on the relative rates (arbitrary units) for oxidation of DCFH [or the appearance of the oxidized product 2,7-dichlorofluorescein (DCF)] both in solution and within vesicles. In the first 1 h following plasma exposure, the concentration of free DCF in DMEM was 15 greater in the presence of serum (cf. to the serum-free DMEM control). The DCF in vesicles was 2 greater in DMEM containing serum compared to the serum-free DMEM control. These data show that serum enhances plasma ROS generation in DMEM. As expected, the role of the phospholipid membrane was to reduce the rate of oxidation of the encapsulated DCFH (with and without serum). And the efficiency of ROS transport into vesicles was lower in DMEM containing serum (at 4% efficiency) when compared to serum-free DMEM (at 32% efficiency). After 1 h, the rate of DCFH oxidation was found to have significantly reduced. Based upon a synthesis of these data with results from the open literature, the authors speculate on how the components of biological fluid and cellular membranes might affect the kinetics of consumption of plasma generated C 2015 American Vacuum Society. [http://dx.doi.org/10.1116/1.4918765] ROS. V
I. INTRODUCTION Nonthermal plasma sources typically used in plasma medicine are highly reactive gases that readily activate the ambient air generating a rich gaseous mixture of reactive oxygen species (ROS) and reactive nitrogen species (RNS), or collectively RONS. When solution is exposed to plasma, some of these RONS dissolve from the gas phase into solution. Further, the plasma or the reactive plasma effluent can directly activate new RONS in the liquid and “downstream” other new RONS may be formed from subsequent reactions in solution.1–3 Many of the RONS generated by plasma (directly or indirectly) in solution are also generated naturally in vivo and within cells and are known to regulate physiological and signaling processes.4 There is a rapidly growing literature that links RONS to the therapeutic outcomes afforded by plasma.5 The potential role of RONS in the regulation of cellular processes has been demonstrated.4,6,7 A number of explanations have been put forward to describe the mechanisms by which plasma generated RONS regulate cellular and physiological processes. The general consensus is that RONS, either directly/indirectly generated by plasma in solution, activate/modify the constituents of the biological milieu or cell media or the external cell membrane, resulting in the oxidation of proteins, lipids, and other macromolecules.8,9 These oxidized/modified products then a)
Electronic mail: [email protected] Electronic mail: [email protected]
b)
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interact with cell membrane receptors or directly with the internal cell components (if they diffuse into the cell), resulting in the activation/deactivation of signaling processes and changes in cellular activity. The role of plasma generated RONS has been linked to biological outcomes including stimulation of cell proliferation, migration, and angiogenesis;10–13 regulation of stem cell differentiation;14 deactivation of bacteria and biofilms;15–22 destruction of tumors;23–25 enhancement of wound healing;26–31 and tissue regeneration.32,33 However, it is not clear if the RONS act by modifying components outside of the cell or inside of the cell. This becomes even less clear considering that plasma can have effects deep into biological material such as biofilms34 and solid tumors24 which can be several micrometers to millimeters in thickness; this is despite many of the plasma generated RONS having short lifetimes and diffusion lengths.8 Earlier studies in the field of plasma interactions with organic surfaces would indicate the effects of plasma should be localized to no more than a few nanometers of the target.35–37 This is clearly not the case in plasma tissue treatment: in the context of cancer treatment, Barekzi and Laroussi argue that the plasma generated RONS stimulate cell reactions or signaling mechanisms that may be transmitted deep into the tissue.38 Supporting this hypothesis, in a recent review, Graves proposes an “oxy-nitroso shielding burst model” whereby surface layer cells communicate to cells deeper within the tissue, similar to other manifestations of cell stress.8
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C 2015 American Vacuum Society V
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029511-2 Szili, Hong, and Short: On the effect of serum on the transport of ROS
In addition to the deactivation of tumors and biofilms at tissue surfaces, our previous research suggests that plasma can be used to deliver RONS to cells that are embedded deep within the affected tissues.39–42 Our data suggest the direct plasma transport of RONS occurs to at least 150 lm into “soft” tissues. But other (undefined) processes further transport RONS deeper, to depths of at least 3.5 mm.43 At these depths, we speculate that the original primary (and more reactive) RONS are likely to have been “consumed” by the surrounding tissue macromolecules. Our findings are of interest in the context of the work of Svarnas et al.44 and Kim et al.45 They show using synthetic cell and live cell models that plasma generated RONS directly interact with, and modify, cell membrane structures, which will alter the subsequent permeability of these membranes to further RONS. Cell membranes are equipped with various channels that allow the passive diffusion or active transport of RONS into and out of the cell. For example, superoxide anion (O2•–), which is formed in liquids by atmospheric-pressure plasmas,46 can initiate intracellular signaling by penetration through cell membrane anion channels, whereas redox signaling from hydrogen peroxide (H2O2) is catalyzed by NADPH oxidase (Nox 2).47 H2O2 signaling is crucial to many physiological functions such as the promotion of the respiratory burst in phagocytes (to protect the host from chronic infection).47 Consequently, in normal cells, the transport of key signaling RONS across cellular membranes is not a random process.48 It follows that the direct delivery of RONS across cell membranes by plasma exposure (or the modification of cell membranes by plasma generated RONS) could be an important strategy in the treatment of diseases. For example, in cancers (such as leukemia) the modulation of H2O2 transport through specific aquaporins affects redox signaling, which is linked to cell proliferation.49 The plasma assisted transport of RONS across cell membranes (or modification of membranes) could potentially and significantly perturb the normal regulation of cellular signaling processes.50 In this study, we expose phospholipid membrane vesicles suspended in DMEM (Ref. 39) (as mimics of a cell membrane) to a helium (He) plasma jet to investigate the transport of plasma generated ROS across the phospholipid membrane. The vesicles encapsulate a (broad spectrum) ROS reporter dye, 2,7-dichlorodihydrofluorescein (DCFH); the dye is oxidized mainly by ROS (although some RNS may also oxidize the dye), converting the nonfluorescent dye to fluorescent 2,7-dichlorofluorescein (DCF).51,52 The fluorescence intensity (FI) of DCF is related to the concentration of ROS inside the solution or vesicles.39 The rate of DCFH oxidation induced by plasma generated ROS was compared between DCFH in cell culture media (free DCFH) and DCFH encapsulated within vesicles, and with and without the addition of serum. We focus specifically on the concentration and on the rate of appearance of DCF (that is oxidized product appearance) given by d[DCF]/dt, both in solution and encapsulated within vesicles. Biointerphases, Vol. 10, No. 2, June 2015
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SCHEME 1. Conceptual overview of the experimental approach to explore the effect of serum on the transport of ROS across phospholipid membranes.
Our aim in this study is to explore RONS transport across a phospholipid membrane and how intercellular components, such as macromolecules, might perturb this process, and its kinetics (Scheme 1). II. EXPERIMENT A. Preparation of 2,7-dichlorodihydrofluorescein
DCFH (Sigma-Aldrich) was prepared using a similar protocol to the one described by Low et al.53 A 1 mg ml1 stock of DCFH diacetate was prepared in ethanol and stored at 20 C until use. Ester hydrolysis was induced by adding 500 ll of stock DCFH to 2 ml of 10 mM NaOH and incubating for 30 min at 25 C in the dark. The solution was then neutralized by adding 10 ml of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer (for HEPES recipe, see Ref. 54). The dye was mixed at a ratio of 50:50 in Dulbecco’s Modified Eagle’s medium (DMEM, SigmaAldrich) with and without 10% (v/v) fetal bovine serum (FBS, Sigma-Aldrich) (herein referred to as serum). (In the case of vesicle studies, serum was added post vesicle formation.) The volume used in experiments was 100 ll per well of a 96 multiwell plate. Fluorescence measurements were recorded at a kexcitation of 485 nm and a kemission of 520 nm for DCF using a BMG Labtech Fluostar Omega microplate reader. The issue of the variability in the FI of DCFH (when converted to DCF) between experiments55–57 was overcome by preparing fresh DCFH stocks, keeping the DCFH/DCF solution in the dark when possible and normalizing the data to an internal control (nonoxidized DCFH) for each of the experiments. B. Preparation of synthetic phospholipid membrane cell model
A synthetic cell model, consisting of phospholipid membrane vesicles encapsulating DCFH, was used to monitor the
029511-3 Szili, Hong, and Short: On the effect of serum on the transport of ROS
flux of plasma generated ROS across the phospholipid membrane. Phospholipids and all other chemicals were obtained from Sigma-Aldrich and Sephadex G25 columns supplied by GE Healthcare. The phospholipid membrane comprised 1,2distearoyl-sn-glycero-3-phospho-(10 -rac-glycerol) (sodium salt) (DSPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol. The phospholipid vesicles mimic the natural cell membrane, i.e., the vesicle membrane comprises a multilipid composition and 20% cholesterol.58 The vesicles were synthesized using the procedure reported elsewhere.54 A 100 ll stock solution was added to 200 ll of chloroform, followed by drying with nitrogen gas. Then, the dry film of lipid was rehydrated in 5 ml of 5 mM deacetylated DCFH (Sec. II A), heated to 65 C, and freeze-thawed three times before being extruded through 100 nm polycarbonate membranes in a LiposoFast basic hand-held extruder. The solution was then purified using a Sephadex G25 column to remove nonencapsulated dye. The vesicles were mixed at a ratio of 50:50 in DMEM with and without serum (serum was added post vesicle formation) to a total volume of 100 ll per well of a 96 multiwell plate. This gave a final vesicle concentration of 5 1011 vesicles ml1. Fluorescence measurements were recorded as described in Sec. II A. Using a protocol outlined in our previous investigation,39 we confirmed that the plasma operating conditions employed herein [directly below] do not rupture vesicles that are freely suspended in solution.
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relative rate at which plasma oxidizes nonfluorescent DCFH to fluorescent DCF is compared for free DCFH in DMEM and DCFH encapsulated within phospholipid membrane vesicles. The rate of DCF appearance (arbitrary units) is taken from d½DCF dt ¼ Slope of FI DCF vs time:
Rate of product apperance ¼
Figures 1(a) and 1(b) show the FI of DCF as function of incubation time after plasma exposure. Figure 1(a) shows that in serum-free media at the 1 h (3600 s) time point (after plasma exposure), the FI of DCF is 4 greater for the free DCFH than the vesicle-encapsulated DCFH. This indicates that the phospholipid membrane presents a barrier to the transport of plasma generated ROS (from solution) into vesicles. When DMEM is supplemented with serum, comparing the FI values in Fig. 1(b) with those in Fig. 1(a), reveals that there is a profound and positive (15) increase in the ROS concentration in solution. This result was initially somewhat surprising—and we comment upon it below. In serum, the elevated ROS concentration is most evident in solution but can be seen even within the vesicles. (At 1 h, the
C. Plasma jet exposure
The plasma source was of a “jet” configuration. It comprised a glass pipette with an inner diameter of 1 mm, surrounded by two external hollow electrodes and separated at a distance of 10 mm. The end of the tube protruded 1 mm out from the bottom electrode. The operating parameters of the plasma were 1 l min1 of He; applied voltage of 5.5 kVpeak-peak; and frequency of 10 kHz. Analysis of the current and voltage waveforms confirmed that the discharge ignites a few times per AC cycle with emission coinciding with distinct current peaks on the driving current waveform (supplementary material).59 The plasma jet was operated into the ambient air, and the exposure time was 1 min. The top of the 96-well plate, containing the test cell solutions, was positioned 25 mm underneath the end of the glass pipette using a laboratory scissor jack covered with an insulating sheet of polytetrafluoroethylene (PTFE). The flow rate of the He gas was controlled with a mass flow controller and power was supplied with a custom-built unit as described elsewhere.60 III. RESULTS AND DISCUSSION We employed DCFH as a (broad spectrum) ROS reporter; DCFH is a well-characterized dye for the detection of ROS in biological systems.57 When nonfluorescent DCFH is oxidized by ROS, it is converted to fluorescent DCF. The intensity of DCF fluorescence (FI) can be correlated to the concentration of ROS in the solution or in cells.52 The Biointerphases, Vol. 10, No. 2, June 2015
FIG. 1. Concentration of ROS is raised by the addition of serum and the efficiency of ROS transport across the phospholipid membrane is significantly reduced by the addition of serum. The FI for DCF for free dye in DMEM and DCF encapsulated within vesicles without serum (a) or with serum (b). The rate of DCFH oxidation (DCF appearance) is determined from the slope at 1 h. The graph inset in (a) has a lower y-axis scale range to illustrate the difference in the slope of the trend lines. The error bars for each datum point are not shown for the sake of clarity. The error in the slope of the curves are in (a) 62.8 107 for DCFH encapsulated within vesicles and 63.5 107 for DCFH in solution and (b) 63.7 107 for DCFH encapsulated within vesicles and 68.8 106 for DCFH in solution (also see Table I).
029511-4 Szili, Hong, and Short: On the effect of serum on the transport of ROS
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TABLE I. Rate of appearance of free DCF (in solution) and encapsulated DCF without and with the addition of serum. Rate of DCF appearance was measured at 1 h for the 0–1 h time period and at 15 h for the 10–15 h time period (post plasma exposure). Rate of DCF appearance: d[DCF]/dt Cell media type
Time-period postplasma exposure (h)
Free dye in solution
Dye in vesicles
0–1 10–15 0–1 10–15
1.16 105 (63.52 107) 7.38 106 (61.04 107)a 2.1 104 (68.8 106) 8.44 106 (61.39 107)a
3.76 106 (62.83 107) 5.06 108 (63.20 107) 7.5 106 (63.69 107) 2.52 106 (65.63 107)a
Serum-free DMEM DMEM þ serum
a
Negative values indicate the oxidation of DCFH has stopped and a decay in the converted fluorophore.
level of FI in vesicles with serum is 2 greater than in vesicles without serum.) Initially, in solution with the addition of serum, the ROS concentration is raised by over an order of magnitude. And, in the presence of serum, the vesicle phospholipid membrane provides an effective barrier to ROS ingress. The DCF FI at 1 h is now 22 greater for free DCFH than for the vesicle-encapsulated DCFH. Table I summarizes the rate of DCF appearance in DMEM with and without serum. We acknowledge that in Fig. 1(b), that for the dye in solution, d[DCF]/dt is not a constant over the first hour; however, for the purpose of following analysis, this approximation is acceptable. The time points chosen were 1 and 15 h after the initial plasma exposure. In the first 1 h following plasma exposure, the rate of DCF appearance is greater in DMEM with serum compared to serum-free DMEM. However, the efficiency of ROS transport from solution into the vesicles, calculated from rate of appearance of DCF within vesicles/rate of appearance of free DCF 100%, is reduced from 32% for serum-free DMEM to 4% for DMEM with serum. At 10–15 h following plasma exposure, the rate of DCF appearance for free dye in solution and within vesicles, decreases in DMEM both with and without serum (Table I). The negative values in Table I most probably indicate the decay in fluorescence in DCF (after the oxidation of DCFH had stopped).
We had initially anticipated that the rate of DCF appearance would be reduced in DMEM containing serum: Tian and Kushner have shown through mathematical modeling that plasma generated ROS are largely consumed by alkane hydrocarbons,3 and further, serum contains amino acids and antioxidant enzymes that will react with ROS.61,62 Grzelak et al. have shown that the light-dependent formation of ROS in DMEM is significantly reduced in the presence of 10% serum.63 As a control, we established that serum does not affect the FI of DCF (i.e., fully oxidized DCFH) (data not shown). In an additional experiment, we measured the rate of DCFH oxidation with a single ROS (H2O2). The experiment was performed in DMEM and in a buffer of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) with and without serum. The experiment involved mixing free DCFH into DMEM or HEPES with and without serum. Then, 50 lM of H2O2 was added and the FI (of DCF) was measured. Figure 2(a) shows that the oxidation of DCFH by H2O2 in HEPES or DMEM is enhanced by serum. For comparison, we performed the same experiment but replaced DCFH with 5,6-carboxyfluorescein (CF). The excitation and emission wavelengths of CF are very similar to DCF. However, the FI of CF is not expected to be sensitive to ROS. Figure 2(b) shows that the FI of CF in HEPES or DMEM is not affected
FIG. 2. Serum enhances the oxidation of DCFH in buffer (HEPES) and cell media (DMEM). (a) The fluorescence intensity (FI) of DCF was compared in HEPES and DMEM with and without addition of serum. The experiment involved mixing free DCFH (0.00005 M) in HEPES, HEPES þ FBS, DMEM, and DMEM þ FBS and then adding H2O2 (50 lM) before measuring the FI. The FI of all solutions without DCFH and H2O2 were also measured and were not fluorescent. (b) The FI of CF, under the same conditions, was compared. CF was not expected to be sensitive to ROS. The error bars are calculated from the standard deviation of the mean from three replicate samples. Biointerphases, Vol. 10, No. 2, June 2015
029511-5 Szili, Hong, and Short: On the effect of serum on the transport of ROS
by serum in the presence H2O2. This experimental control increases our confidence that the addition of serum enhances the rate of oxidation of DCFH in the presence of ROS. We do not have a full explanation as to why serum enhances ROS generation in our study, but reduced ROS generation in the previous study by Grzelak et al.63 We speculate the contradicting results might be attributed to the different mechanisms of ROS generation by plasma and ROS generation under a standard fluorescent laboratory light, as employed by Grzelak et al.63 Serum may reduce the lightdependent generation of ROS in solution; however, ROS generated in the plasma effluent will still solvate from the gas phase into the solution.2,3 In the presence of serum, we speculate that the ROS react with serum macromolecules leading to the generation of alkyl radicals.3 A major component of serum is albumin. Alkyl radicals in turn can react with molecular oxygen to give alkylperoxyl radicals (ROO•).64 Setsukinai et al. observed that after excluding the highly reactive but short-lived hydroxyl radical (•OH) and peroxynitrite (ONOO), the ROO• radical is the most reactive radical with DCFH.65 These “secondary” macromolecular ROS arising from the oxidation of serum will be substantially larger than the initial ROS species. We therefore think it is highly unlikely these secondary ROS can traverse the phospholipid membrane. More likely, decomposition products of these ROS (under ambient laboratory conditions) are responsible for the formation of DCF in vesicles suspended in DMEM containing serum. The ingress of ROS into cells will strongly influence the behavior and ultimately the fate of any cell. Although we are cognizant of the limitation of our synthetic phospholipid vesicles cf. real cells, below we discuss a few ideas on how plasma and plasma generated ROS might broadly interact with real cells. These ideas are put forward in the spirit of trying to improve our understanding of plasma induced effects within cells and tissue. Based upon a synthesis of our data and what is known from the open literature, we suggest that: (1) within 1 h following plasma exposure, the solution (or extracellular matrix in the case of “real” cells in tissue) concentration of ROS would be (initially) much higher than the vesicle (intracellular) ROS concentration; (2) short-lived ROS react quickly with macromolecules in the solution, leading to the generation of larger (perhaps less cell-impermeable) but more stable ROS; (3) other effects during plasma exposure such as electric fields, sheer stress, and pressure may increase (at least temporarily) the permeability of the phospholipid membrane to ROS;66 longer term effects probably include a change in pH, modifications of the intercellular components and oxidation of the phospholipid membrane; (4) the ROS concentration gradient described in (1) and any changes in the permeability of the phospholipid membrane described in (3) will lead to an ingress of ROS into cells; (5) a (real) cell (as opposed to our synthetic vesicle) will initially respond to the influx of ROS by up-regulation of its antioxidant defenses; (6) if the flux of ROS is too high, this cell may undergo apoptosis/necrosis; (7) after about 1 h post plasma, Biointerphases, Vol. 10, No. 2, June 2015
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TABLE II. Membrane surface area to volume ratio (SA:VOL) of the vesicles and prokaryotic and eukaryotic cells. The SA:VOL were based on an average diameter for each cell type and assuming that the vesicles and cells are spherical.
Membrane description 100 nm diameter vesicles 2000 nm diameter prokaryotic cells 10 000 nm diameter eukaryotic cells
Surface area (SA) of membrane: 4pr2 (nm2)
Volume (VOL) of vesicle or cell: 4/3pr3 (nm3)
SA:VOL (3/r)
3.14 104
5.24 105
3/50
1.26 107
4.19 109
3/1000
3.14 108
5.24 1011
3/5000
the extracellular and intracellular ROS concentration begin to equilibrate. This time period will presumably be dependent on the exact ROS dose—we can see in Fig. 1(b) at the 1 h point in solution a negative change in d[DCF]/dt; (8) if the cell survives plasma exposure, in the longer term (depending on the flux of ROS into the cell) the cell may return to its state prior to plasma treatment or it may be irreversibly changed (e.g., phenotypically). Additionally, to be considered is that plasma generated ROS may oxidize cell membranes and therefore also increase permeability to further ROS over a longer period than the initial plasma exposure.67,68 And it is because of point (5), and later point (6), that it is difficult to make direct measurements of ROS ingress into viable cells. The ability of a cell to cope with the burst of ROS may be affected by the cell membrane surface area to cell volume ratio. In Table II, we compare the (theoretically calculated) surface area:volume ratio (SA:VOL) of our vesicles to eukaryotic and prokaryotic cells. We appreciate that the SA:VOL of the phospholipid vesicles is much larger than real cells. Therefore, we readily acknowledge the possibility that ROS may be transported faster into our vesicles, cf. real cells. In general, the SA:VOL is five times higher for prokaryotic cells than eukaryotic cells. This ratio might result in a much faster transportation of ROS into bacterial cells (cf. mammalian cells). A consequence of this faster ingress of ROS into bacterial cells might be that bacterial cells are more vulnerable to damage from ROS. At least in part, this may explain why plasma can be used to effectively decontaminate wounds without detrimentally affecting the surrounding healthy tissue. (This does not take into account any differences there may be in the relative permeability of different cell membranes.) In a previous paper, we argue that plasma is a superior method for the effective delivery of ROS into cells and tissue, cf. the topical application of ROS from solution.41 In our current study, our approach is restricted in that we cannot probe what happens during the 1 min of plasma exposure, but our data certainly support the concept that plasma is an effective method to deliver ROS into cells and tissues. This process is enhanced by the presence of serum in the treated fluid.
029511-6 Szili, Hong, and Short: On the effect of serum on the transport of ROS
IV. SUMMARY AND CONCLUSIONS The key conclusions drawn from this study are that plasma generated ROS can be transported across a model phospholipid membrane, and that in the context of real cells, this indicates that phospholipid membranes will inhibit (but not prevent) ROS ingress. Plasma generated ROS react with media components (such as serum) and this has a profound and positive impact on the fluid ROS concentration. We speculate that these “additional” ROS species are likely to be alkyl peroxyl radicals. These ROS are substantially larger radicals and will not be so readily transported across cell membranes. In context of plasma treatment of real tissues, we should anticipate the substantial generation of “secondary” macromolecular ROS within tissue and tissue fluids, which will “bath” embedded cells. It may be that the ROS that traverse the phospholipid membrane are small “tertiary” ROS derived from the “secondary” ROS under ambient conditions. Establishing a better insight into the nature and physical transport mechanisms of ROS or RNS into cells and tissue is important: not only does it helps strengthen our scientific understanding for the use of plasma in medicine and biology but also provides a rational basis for determining optimal and/or safe plasma doses. ACKNOWLEDGMENT The authors thank the Wound Management Innovation CRC for partially funding this work through project RP 2.11. 1
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