Article pubs.acs.org/Langmuir
Ionic Conductivity as a Tool To Study Biocidal Activity of Sulfobetaine Micelles against Saccharomyces cerevisiae Model Cells Matteo Tiecco,†,‡ Luca Roscini,† Laura Corte,† Claudia Colabella,† Raimondo Germani,*,‡ and Gianluigi Cardinali†,‡ †
Department of Pharmaceutical SciencesMicrobiology, University of Perugia, Borgo XX Giugno 74, I-06121 Perugia, Italy CEMIN, Centre of Excellence on Nanostructured Innovative Materials, Department of Chemistry, Biology and Biotechnology, University of Perugia, via Elce di Sotto 8, I-06123 Perugia, Italy
‡
S Supporting Information *
ABSTRACT: Zwitterionic sulfobetaine surfactants are used in pharmaceutical or biomedical applications for the solubilization and delivery of hydrophobic molecules in aqueous medium or in biological environments. In a screening on the biocidal activity of synthetic surfactants on microbial cells, remarkable results have emerged with sulfobetaine amphiphiles. The interaction between eight zwitterionic sulfobetaine amphiphiles and Saccharomyces cerevisiae model cells was therefore analyzed. S. cerevisiae yeast cells were chosen, as they are a widely used unicellular eukaryotic model organism in cell biology. Conductivity measurements were used to investigate the interaction between surfactant solution and cells. Viable counts measurements were performed, and the mortality data correlated with the conductivity profiles very well, in terms of the inflection points (IPs) observed in the curves and in terms of supramolecular properties of the aggregates. A Fourier transform infrared (FTIR)-based bioassay was then performed to determine the metabolomic stress-response of the cells subjected to the action of zwitterionic surfactants. The surfactants showed nodal concentration (IPs) with all the techniques in their activities, corresponding to the critical micellar concentrations of the amphiphiles. This is due to the pseudocationic behavior of sulfobetaine micelles, because of their charge distribution and charge densities. This behavior permits the interaction of the micellar aggregates with the cells, and the structure of the surfactant monomers has impact on the mortality and the metabolomic response data observed. On the other hand, the concentrations that are necessary to provoke a biocidal activity do not promote these amphiphiles as potential antimicrobial agents. In fact, they are much higher than the ones of cationic surfactants.
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INTRODUCTION Surfactants are molecules that find applications in a wide range of topics thanks to their amphipathic structure.1,2 This feature permits these compounds to interact, in fact, with different kinds of molecules and macromolecules, as well as with biological ones. The examples of the applications of surfactants are numerous, a nonexhaustive list of which is represented by DNA interaction for gene delivery, 3 vectors for the solubilization and transport of hydrophobic solutes,4 and use as components of novel reaction media or solvents.5−7 In all of these topics, small modulations in the molecular structures of the amphiphiles can lead to important changes in the results observed, so a fundamental knowledge of their properties is needed for productive applications. © 2016 American Chemical Society
The interaction of surfactants with microorganisms represents one of the most relevant topics regarding the use of these molecules,8,9 and it also represents one of the most important examples in which differences in the molecular structures of amphiphiles can lead to drastically different results. In fact, amphiphiles can either act as biocides or be innocuous for the cells, depending on their structures. Cationic surfactants can act as biocides against microorganisms,10,11 whereas nonionic and zwitterionic ones are considered innocuous so far, so they are widely used in drug-delivery processes.12,13 Received: November 5, 2015 Revised: January 11, 2016 Published: January 11, 2016 1101
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Table 1. Structures, Acronyms, and Critical Micellar Concentrations (cmc) of the Compounds Investigated in This Worka
a
The cmc values were obtained by surface tension measurements at 25.0 ± 0.1 °C.
In our studies on the biocidal activity of differently structured synthetic surfactants on two yeasts and two bacterial species,27 peculiar and interesting results emerged about the effects of sulfobetaine zwitterionic surfactants. This led us to examine in depth the interaction effects of this class of compounds against microbial cells, due to the importance of these molecules in the drug-delivery topic.28
The mechanisms of interaction of cationic surfactants with microorganisms are not yet defined clearly. Most of what is currently known about their action is derived from the study of the mode of action of antimicrobial cationic peptides, and only very few of these have been well characterized mechanistically.14 However, a large number of papers report about the interaction of these molecules on the membranes or on the cell walls, driven by electrostatic forces between the cationic headgroup of the monomeric surfactants and the anionic charge density of the membrane, as well as by hydrophobic interactions between the tails and the membrane internal parts.14−17 The constant need for novel antibacterial compounds is a clever reason for the increasing use of surfactants in this field, because of the wide range of molecules with different biological or biocidal activity that can be easily synthesized and because microbial cells can develop resistance to commonly used antimycotic and antibacterial drugs.18,19 Nonionic and zwitterionic surfactants are widely used in pharmaceutical or biomedical applications whenever nontoxicity of the molecules is needed.20,21 On the contrary to cationic, the nonionicity of zwitterionic surfactants could lead to weak or absent interactions with charged cell membranes or walls. These compounds are used in the solubilization and transport of hydrophobic drugs.4,22−24 The zwitterionic aggregates permit important hydrophobic molecules to solubilize and transport in water or in biological environments. In the literature are reported patents regarding the use of these micellar aggregates to transport biocidal cationic amphiphiles with comicellization processes.25,26 This process was made to increase the drug-delivery efficiency into biological systems and to decrease their toxicity.
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MATERIALS AND METHODS
Surfactants. The structures of all the compounds employed in this work are reported in Table 1. The acronyms of the molecules used in this work are reported as SBRm-n: SB means sulfobetaine; R refers to the headgroup, specifically to the alkyl group bound at the ammonium (none = methyl, E = ethyl, Pr = propyl); m is the number of methylene groups of the spacer between the ammonium and the sulfate groups; and n is the number of carbon atoms of the hydrophobic chain. Surfactants 3-(N,N-dimethyldodecylammonio)propane-1-sulfonate (SB3-12) and 3-(N,Ndimethyltetradecylammonio)propane-1-sulfonate (SB3-14) were purchased from Sigma-Aldrich and were recrystallized twice in methanol− acetone mixture before use. The other compounds, 3-(N,Ndimethybutylammonio)propane-1-sulfonate (SB3-4), 3-(N,Ndiethyltetradecylammonio)propane-1-sulfonate (SBE3-14), 3-(N,Ndipropyltetradecylammonio)propane-1-sulfonate (SBPr3-14), 4(N,N-dimethyltetradecylammonio)butane-1-sulfonate (SB4-14), 4(N,N-diethyltetradecylammonio)butane-1-sulfonate (SBE4-14), 4(N,N-dipropyltetradecylammonio)butane-1-sulfonate (SBPr4-14), and 5-(N,N-dimethyltetradecylammonio)pentane-1-sulfonate (SB514), were synthesized and purified following procedures reported in previous papers.29−35 Molecular structures were confirmed by means of 1H NMR spectra; the purity of all surfactants and the critical micellar concentrations (cmc) were evaluated through surface tension measurements. 1102
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Langmuir cmc determination with surface tension method. The cmc values were determined using a KSV Sigma 701 tensiometer, equipped with a Du Noüy ring, at 25.0 ± 0.1 °C. Surface tension of 10 mL of Milli-Q water in a vessel was measured to be 72 mN/m. Surface tensions of the samples were measured after addition of the appropriate amount of aqueous surfactant solution. After each addition, the solution in the vessel was stirred for 30 s and then left for 3 min before reading the value. Each measure was repeated at least three times. The cmc value was detected by plotting the surface tension values vs −log [surfactant]. Cultures and growth conditions. The yeast strain Saccharomyces cerevisiae CMC 520 was employed as target. It was obtained from the internal collection of the Microbial Genetics and Phylogenetics Laboratory of the Department of Pharmaceutical Sciences (University of Perugia), and it is also deposited in the collection of the Centraalbureau voor Schimmelcultures (CBS) as CBS 13873. The D1/D2 domain of the LSU (26S) gene showed that this strain is 99.8% similar to the species type strain, thus allowing us to identify it as an authentic S. cerevisiae strain36 (data not shown). Preculture was inoculated at OD600 = 0.2 in 200 mL of YEPD + chloramphenicol medium (yeast extract 1%, peptone 1%, dextrose 2%, chloramphenicol 0.5 g L−1Biolife Italiana S.r.l., Milan, Italy) and grown 18 h at 25 °C, under shaking at 150 rpm. Conductivity measurements. Conductivity was measured on a CRISON GLP-31 conductivity meter at 25.0 ± 0.1 °C (Pharmacia Biotech Multitemp III thermostat). The error of conductometric measurements ranged from 0.08 to 0.52 μS/cm. The conductivity meter was connected to a computer via RS-232. A Watson-Marlow 323 peristaltic pump (with Watson-Marlow tubes) was used for the surfactant solutions and cell suspension additions. A JASCO V-530 spectrophotometer was used to determine cell suspension concentration. The cell suspension was centrifuged at 4 143g (4 500 rpm) for 3 min and then washed twice in distilled sterile water. The cells suspension was finally calibrated at OD600 = 5.00. The OD600 was maintained constant with addition of cell suspension at OD600 = 10.00 with the same rate of addition of the surfactant. Surfactant solution and cell suspension were added at 0.0143 g s−1 to constantly thermostated and stirred water with the peristaltic pump, following the procedure published elsewhere.16 The conductivity values were acquired by the conductivity meter at 1 s−1 and were transferred to a computer running SerialNGAdvDemo software. Biocidal activity. Cells grown as described above were washed twice with distilled sterile water and pelleted in 15 mL polypropylene tube. Surfactant solutions (5 mL) were added directly to the test tubes, and the cells were resuspended. The control (0 mM) was obtained by resuspending the cells directly in distilled sterile water. The tubes were incubated for 1 h at 25.0 °C in a shaking incubator set at 50 rpm. After the incubation, each sample was diluted to determine the viable cell counting of tests and control suspension, in triplicate, on YEPDA plates (yeast extract 1%, peptone 1%, dextrose 2%, agar 1.7%chloramphenicol 0.5 g L−1Biolife Italiana S.r.l., Milan, Italy). The viability (V) was calculated compared to the control as V = [Cv/Ct] × 100, where Cv is the number of viable cells in the tested sample and Ct is the number of viable cells in the control suspension. The biocidal effect of the compounds was tested as cell mortality (M), calculated as M = 100 − V. FTIR-based bioassay. The FTIR bioassay was carried out in parallel to the biocidal activity tests to compare the metabolomic damages to the loss of viability. A 105 μL volume of each cell suspension prepared for the viability analysis was sampled for three independent FTIR readings (35 μL each, according to the technique suggested by Manfait and co-workers37). The FTIR experiments were carried out with a TENSOR 27 FTIR spectrometer, equipped with HTS-XT accessory for rapid automation of the analysis (BRUKER Optics GmbH, Ettlingen, Germany). FTIR measurements were performed in transmission mode. All spectra were recorded in the range between 4000 and 400 cm−1. Spectral resolution was set at 4 cm−1, sampling 256 scans per sample. The software OPUS version 6.5 (BRUKER Optics GmbH, Ettlingen,
Germany) was used to carry out the quality test, baseline correction, vector normalization, and calculation of the first and second derivatives of spectral values. Spectra statistical analyses. The script MSA (metabolomic spectral analysis) employed in this study was developed in “R” language to carry out the following operations on the matrices of spectral data exported as ASCII text from OPUS 6.5. The analytical procedure consisted of calculating the distance between the spectrum of the cells under test and that of the cells without the stressing agent. This procedure was extended to five different spectral regions in order to differentiate the stress response among the different classes of molecules. The procedure could be outlined as follows: (1) Each single spectrum was normalized in order to have the range spanning from 0 to 1 in a way already suggested by Goodacre and colleagues.38 Average spectra from the three repetitions were calculated. (2) Response spectra (hereinafter reported as RS) were calculated as the difference between each average spectrum and the average spectrum of the same cells maintained in water39 (defined as control RS). Response spectra of each agent were plotted with the exclusion of the control RS, which is by definition a straight line with RS = 0. (3) Synthetic stress indexes (hereinafter reported as SIs) of metabolomic stress response were calculated as Euclidean distances of the RS under stress and the control RS. SIs of the whole spectrum and of the five different spectral regions individuated by Kümmerle et al.40 were calculated. The five regions were defined as follows: fatty acids (W1) from 3000 to 2800 cm−1, amides (W2) from 1800 to 1500 cm−1, mixed region (W3) from 1500 to 1200 cm−1, carbohydrates (W4) from 1200 to 900 cm−1, and typing region (W5) from 900 to 700 cm−1. Since the five spectral regions differ in length, their SIs were scaled to the length of the whole spectrum, in order to make the different SIs comparable on the same scale.
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RESULTS AND DISCUSSION Eight sulfobetaine amphiphilic compounds were examined in this work: two of them are commercially available and six of them are synthetic analogues (Table 1). These surfactants are structurally diversified in terms of their hydrophobic chain length, length of the polymethylene spacer between the ammonium and the sulfonate, and on their headgroup dimensions. An additional sulfobetaine compound was studied to determine the role of the amphiphilicity of these molecules: SB3-4 compound has the same headgroup of the amphiphiles but has a much shorter hydrophobic tail, preventing its capability of aggregation in water. The effects of the zwitterionic amphiphiles were evaluated using the yeast Saccharomyces cerevisiae as target cells, a widely used unicellular eukaryotic model organisms in cell biology.41 S. cerevisiae cells have dimensions of 5−10 μm, and they have a negatively charged lipid bilayer due to the substantial presence of phosphomannan.42 Conductivity and viable counts measurements were performed. The mortality data correlated with the conductivity profiles very well, in terms of the inflection points observed in the curves. A FTIR-based bioassay was then performed to determine the metabolomic stress-response of the cells subjected to the action of the sulfobetaine surfactants.39 Also this metabolomic response of the cells correlated well with the data observed with the other techniques. Conductivity measurements. Conductivity profiles have been studied recently on cell suspensions subjected to the action of cationic surfactants. They showed interesting results that correlated well with microbiological and FTIR data.10,16,17,27 The conductivity curves observed with sulfobetaine surfactants are interesting because they permit one to observe conductivity values that are dependent only on the cell 1103
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Figure 1. Conductivity profile of Saccharomyces cerevisiae cells suspension in the presence of SBE3-14 surfactant. Temperature = 25.0 ± 0.1 °C; S. cerevisiae cells suspension OD600 = 5; average SD result of 1.15 corresponding to a variation coefficient of 0.0047.
contributions. Zwitterionic nonamphoteric amphiphiles do not contribute to the overall conductivity for their neutral overall charge. The conductometric profile of S. cerevisiae cells suspension in the presence of SBE3-14 surfactant is reported in Figure 1. It was obtained with a semiautomated method that permitted us to maintain cell concentration OD600 at the constant value of 5, and it was obtained from 560 reading points.16 As can be easily observed in the curve, the overall conductivity remains constant until a specific value of surfactant concentration, hereinafter referred to as inflection point (IP), where it increases rapidly. This IP occurs at a concentration value close to the critical micellar concentration of the sulfobetaine surfactant in pure water. The same behavior was observed in all the conductometric profiles of other surfactants employed, except for the nonamphiphilic molecule SB3-4 (all the profiles are reported in Figure S1 in Supporting Information). The IP concentration values, calculated as the intercept of the two regression curves before and after the discontinuity, and the tensiometric cmc values of the surfactants are reported in Table 2. The increase of the conductivity values observed could be due to ionic species (such as inorganic salts or small charged organic molecules) released by the cells because of the interaction with the zwitterionic micellar aggregates. The ionic species, enclosed inside the cells, can contribute a little to the overall conductivity, because of their low mobility due to the large dimensions of the cells compared to the single molecules. Their release in solution provokes an increase in the observed conductivity for their increased mobility. It is the opposite case of what was observed in conductivity cmc determination of charged surfactants: in that case the micelle formation provokes a decrease in monomer mobility and therefore a decrease of the slope of the conductometric curves.1 In the conductometric studies of cationic surfactants in the presence of cell suspensions, the relevant inflection points observed were at amphiphile concentrations lower than cmc, and the microbiological and FTIR studies also confirmed the action of the surfactant monomers on the cells.10,17 In the case
Table 2. Tensiometric cmc Values of the Sulfobetaines and Inflection Points (IPs) in the Conductometric Curves Observed in the Presence of Saccharomyces cerevisiae Cellsa compound
(IP), mM
cmc, mM
SB3-4 SB3-12 SB3-14 SBE3-14 SBPr3-14 SB4-14 SBE4-14 SBPr4-14 SB5-14
n.a. 2.63 ± 0.12 0.255 ± 0.011 0.343 ± 0.017 0.290 ± 0.017 0.345 ± 0.010 0.348 ± 0.016 0.324 ± 0.017 0.261 ± 0.013
n.a. 2.16 0.288 0.250 0.190 0.370 0.354 0.310 0.277
a
IP values observed in conductometric curves in the presence of Saccharomyces cerevisiae cells at OD600 = 5 concentration, reported with the errors calculated via replicas.
of sulfobetaine surfactants, the inflection points are only observed at micelle formation. Zwitterionic micelles have different and peculiar properties compared to the ones of charged micelles, particularly on their ionic structure and on their ion-uptake peculiar capability.43−45 In the recent literature the authors postulated many different models to interpret these peculiarities. Souza and co-workers treated the theme of specific ion association on zwitterionic micelles, reported as “chameleon-like” behavior.46−48 In previous works regarding micellar effects on reactivity and on micellar structure of zwitterionic surfactants, it has emerged that micelles of zwitterionic sulfobetaines have a “cationic-like” behavior30,49−51 that is due to the charge distribution in the micellar structures. In these works the micellar aggregates are described as a spherical capacitor. A zwitterionic sulfobetaine surfactant micelle has, in fact, an overall neutral charge, but the positive charges are in an inner smaller sphere and the negative sulfates are in an outer larger one. In other recent works, it has emerged that even zwitterionic micellar aggregates with an inner negative charge and an outer positive one acted as cationic micelles.52 So the pseudocationic behavior of a sulfobetaine micellar aggregate 1104
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Figure 2. Conductivity profile of SB3-14 surfactant in water (a); conductivity vs time profile of S. cerevisiae cells water suspension (b); conductivity profile of S. cerevisiae cell suspension in the presence of SB3-14 surfactant (c). Temperature = 25.0 ± 0.1 °C; S. cerevisiae cells suspension OD600 = 5; average SD result of 1.15 corresponding to a variation coefficient of 0.0047. In (a) and (c) are reported conductivity vs surfactant concentration profiles; in (b) is reported a time course of the conductivity of the cell suspension without surfactant, acquired with the same times of (a) and (c) experiments.
Figure 3. Mortality/conductivity vs concentration profile for SB4-14 surfactant with S. cerevisiae cells. Mortality profile (gray) of SB4-14 surfactant compared with conductivity profile (black). T = 25.0 ± 0.1 °C; cell OD600 = 5.
could depend on many factors. The first factor is the charge distribution, and it leads to comparing the micellar aggregates to capacitors. However, the different charge densities and hydration degrees are responsible for the cationic behavior of other zwitterionic systems with opposite charge distributions. In zwitterionic molecules the positive charge, in fact, relies on a single atom (an ammonium) for most of the structures, while the negative charge is delocalized in different oxygen atoms (such as the cases of sulfate, sulfonate, phosphate, and carboxylate groups). As a consequence the negative charge density is lower; therefore, the aggregates act as cationic ones.
For these peculiar properties, the zwitterionic micelles can act as cationic aggregates. Cells are sensitive to cationic molecules such as cationic surfactants,14,53,54 so the interaction of the zwitterionic sulfobetaine micelles could occur due to the pseudocationic behavior of these aggregates. The interaction of the micellar aggregates with the cells could subsequently cause the opening of channels or holes in the membrane,14 and this could cause the release of the substances that make the conductivity values increase in the cell suspensions. 1105
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Langmuir Table 3. Mortality Data (%) of S. Cerevisiae Cells Subjected to the Action of Sulfobetaine Compounds 104 [SB3-14], mM
mortality, %
104 [SBE3-14], mM
mortality, %
104 [SBPr3-14], mM
mortality, %
0.05 0.15 0.25 0.35 0.45 0.55 104 [SB4-14], mM
0.00 9.50 7.75 55.66 90.05 97.59 mortality, %
0.07 0.17 0.27 0.37 0.47 0.57 104 [SBE4-14], mM
0.00 7.55 9.12 70.75 81.76 87.42 mortality, %
0.01 0.05 0.10 0.40 0.50 0.60 104 [SBPr4-14], mM
1.69 0.00 0.00 32.63 36.72 46.33 mortality, %
0.05 0.15 0.25 0.37 0.50 0.60 104 [SB3-12], mM
0.00 3.33 5.33 34.00 46.00 68.33 mortality, %
0.05 0.15 0.25 0.40 0.50 0.60 104 [SB5-14], mM
0.00 14.75 13.57 58.11 76.99 80.83 mortality, %
0.05 0.15 0.25 0.40 0.51 0.61 104 [SB3-4], M
0.00 2.89 0.00 16.76 15.99 25.24 mortality, %
0.67 1.32 1.99 3.00 3.67 4.59
2.59 18.45 11.65 57.93 51.94 56.31
0.07 0.13 0.20 0.27 0.33 0.40
0.00 0.00 0.00 18.45 14.00 11.97
1.13 2.25 3.37 4.50 5.62 6.75
0.00 0.00 7.44 0.00 6.80 3.00
Biocidal activity. The studies of the mortality of S. cerevisiae model cells, subjected to the action of sulfobetaine surfactants, were performed via viable plate counts. These measurements were made on six points of concentration for each surfactant: three at concentrations lower than the conductometric IP value and three at higher concentrations. In this manner an evaluation of the meaning of these critical conductometric phenomena could be achieved. In Figure 3 is reported a graph of mortality/conductivity vs concentration of SB4-14 surfactant in S. cerevisiae suspension. In Figure S3 the profiles of all the surfactants are reported, and in Table 3 are reported all the acquired data. From these measurements it is clear that the increases of conductivity observed at the inflection points correspond to a decrease in cell viability. In all the surfactants examined, in fact, the three points at concentrations higher than the cmc showed a mortality increase whereas the data observed at lower concentrations did not show any mortality increase. This phenomenon did not occur with SB3-4 compound, which did not show this behavior in all the examined concentrations. This could occur because it is not an amphiphilic molecule, so it cannot form micellar aggregates that can interact with the cells. The increases observed in conductometric curves could be due to a release of ionic species from the cells, which is caused by the interaction of the amphiphiles that provoke cell death. The cationic behavior of the micelles permits the interaction of the surfactants with the cells, and the surfactant structure has impact on the mortality data observed. Bigger mortality increases were observed, in fact, for higher cationic densities on the ammonium group in the surfactants. In SBR3-14 series is observed a mortality decrease from 97% (SB3-14) to 87% (SBE3-14) then to 46% (SBPr3-14) as the length of the head alkyl residues increases. This could be due to the polarization of the headgroup that increases from methyl to ethyl and propyl groups, which leads to lower cationic charge density of the ammonium. The SBR4-14 compounds showed an analogous behavior but with some differences because of an effect of charge neutralization. In fact, a mortality of 68% for SB4-14 is observed, as well as a mortality of 81% for SBE4-14 and 25%
The conductometric curve observed with SB3-4 molecule (Figure S1) is a confirmation of this phenomenon. The absence of a hydrophobic tail in the molecular structure, therefore the absence of amphiphilicity, determines the impossibility of formation of micellar aggregates. In this case no inflection points were observed in the whole concentration profile, so the phenomenon has to be ascribed to the micellar aggregates and not to the headgroup of the amphiphiles. The small increases of the conductivity values observed in the baseline of the curves are due to the cells: the microbial species in water suspension could provoke an increase of the conductometric values in time. This is due to the release of conducting materials from the cells in these conditions. In Figure 2 are reported the conductivity profile of SB3-14 molecule (panel a), a time course of the conductivity of S. cerevisiae cell suspension at OD600 = 5 (panel b), and the conductivity profile of the cell suspension in same surfactant (panel c). From this figure it is clear that no inflection points could be detected in the conductometric curves of the surfactants alone and on the S. cerevisiae cell suspensions alone, except for the slight increases observed in the cell suspensions time courses. In Figure S2 (Supporting Information) a longer time conductivity profile of S. cerevisiae cells in water is reported; it was acquired in 2 h of timing (the time of all the conductivity experiments in this work is ∼10 min). The surfactant concentration values observed in the inflection points are slightly different from the surface tension cmc (Table 2), but the values are often dependent on the technique used to determine them.55,56 The change of the slope of the curves at higher surfactant concentration than cmc, observed in all the profiles, could not be linked to other critical phenomena, because the values observed were not reproducible in subsequent experiments, while on the contrary the inflection points were; they cannot be treated in the same manner of the IP. Mortality measurements and FTIR-based bioassay were then performed to verify the microbiological meaning of the increase of conductivity values observed in the curves. 1106
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Figure 4. Stress index (SI) histograms and mortality of S. cerevisiae cells subjected to the action of SBPr3-14 zwitterionic surfactant. Dashed red line = mortality; black = global stress index (GSI); red = W1 fatty acids; green = W2 amides; purple = W3 mixed region; light blue = W4 carbohydrates; orange = W5 typing region.
FTIR-based bioassay. FTIR spectroscopy has been used in metabolomic stress-response assays developed to evaluate the stress induced by synthetic surfactants10,17,27 or by novel green solvents7,58 on model cells. This technique was used to evaluate the metabolomic response of S. cerevisiae cells subjected to the action of sulfobetaine surfactants. In this manner, a brief evaluation of the cell compartments involved in the interaction with the surfactants was also achieved. Due to the high number of studied conditions, the whole FTIR spectra of the cells could not be analyzed, so the stress indexes (SIs) were studied. These synthetic indexes are determined as Euclidean distance between the response spectrum (RS) of the cells under test and that of the cells without the stressing agent (see Materials and Methods section). This procedure was extended to five different spectral regions in order to differentiate the stress response among the different cell compartments.59,60 Another index, the global stress index (GSI), was added to these just because it rapidly and efficaciously considers the global stress of the cells; in fact, it represents the SI of the global spectrum in only one index. In Figure 4 are reported the SIs, reported as histograms of each concentration point, of the S. cerevisiae cells subjected to the action of the SBPr3-14 amphiphile. The SIs of the cells subjected to the action of all the surfactants employed in this work are reported in Figure S4 in Supporting Information section. These measures were performed at the same concentration points used in viable count measurements, and the corresponding mortality data are reported in the same graphs. The metabolomic responses of the cells can be analyzed considering the same subclasses of surfactants, in the same manner of viable counts experiments. Even in these measures, concentration-dependent results were observed, with nodal points corresponding to the IP values. In the SBR3-14 series the SIs can be ordered in three main groups corresponding to different surfactants concentrations: two pre-IPs, two next to the IPs, and two after the IPs. As can be easily observed, considering all the histograms and the GSIs, the highest metabolomic response was observed at the two concentrations
for SBPr4-14. In this case the length of the spacer could provoke a partial neutralization of the positive charge made by sulfate groups that can get closer to the ammonium due to a folding of the headgroup. This can reduce the cationic behavior of the monomers, and therefore, it impacts on the mortality data observed in the series. In this case the bulkiness of the head alkyl residues have an impact on the polarization of the headgroup and also on this phenomenon, leading to weaker neutralizations due to the steric hindrance. This neutralization phenomenon was also observed in the literature with other zwitterionic carboxybetaine surfactants with NMR studies.57 The same phenomenon lead to the mortality data observed for SB5-14 surfactant. In this case the higher length of the spacer showed an even stronger impact on the biocidal activity for its higher capability of folding and, therefore, of charge neutralization. In this case a very slight increase of mortality is observed at concentrations higher than the IP (∼15%). The hydrophobic chain length has an impact on the biocidal activity of these compounds: longer chains on the monomers showed higher biocidal activities (∼56% of mortality for SB3-12 and ∼98% for SB3-14). This behavior was already observed in other studies on the toxicity of cationic synthetic surfactants.10,17 As these mortality data can be interpreted with structure− activity relationships, it is possible to choose a specific surfactant for a specific role. Longer alkyl residues on the headgroup and longer spacers between the ammonium and the sulfate can provoke a decrease of charge density, and therefore a lower biocidal activity. This makes these compounds suitable for application where a low toxicity is needed, for instance, drug delivery. Even if the concentrations needed to provoke a biocidal effect are quite high compared to the ones observed for cationic surfactants, whenever an increase of biocidal activity is needed, a specific sulfobetaine surfactant could be chosen. It should have a spacer that can prevent a headgroup folding (for example, a rigid one), a smaller headgroup that could provoke a higher cationic charge density of the molecules, and a long hydrophobic chain. 1107
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next to the inflection point, while a weaker response in all the spectral regions was observed before and after the cmc value of the surfactants. The same metabolomic behavior was observed in the SBR4-14 series, but with some differences. Even in this class an increase of metabolomic response was observed next to the inflection points, but the responses after them were higher than the ones observed in the SBR3-14 series. Even the observed mortality data were a little lower than the ones of the SBR3-14 class, indicating a more effective response of the cells to the stressing agent. SB4-14 surfactant provoked a slightly different metabolomic response of the cells at low concentrations: the metabolomic responses increase with the concentration with a little decrease in the last sample. As already showed with the other experiments, SB3-12, SB514, and SB3-4 compounds showed peculiar results. SB3-12 surfactant induced a high metabolomic response in the cells at low concentrations, while inducing very weak ones at concentrations higher than the IP. The same behavior was observed with SB5-14 amphiphile, which showed a high metabolomic response of the cells at low surfactant concentrations, while it was very low after the IP. SB3-4 compound did not induce any significant mortality of the cells, and the metabolomic responses were very weak in all the samples analyzed, even if the concentrations were much higher than the ones studied with the other compounds. The concentrations of the surfactants, and the conductometric IPs observed, had an impact also on the detected FTIR responses. The employed FTIR bioassay showed that the cells have a strong response next to the cmc of the compounds but also at lower concentrations. This could mean that the FTIR could detect phenomena that occur even before the IPs that do not determine the cells’ death, but can activate their metabolomic response. The SIs calculated in the five spectral regions were then used to briefly evaluate the cell compartments involved in the interaction with the surfactants.40,60 The action of SB3-14 surfactant involved three main cell compartments: primarily carbohydrates (W4), then mixed region (W3) and amides (W2). The same spectral regions were observed in the whole SBR3-14 series, with different intensities: the highest response was observed first for SBE3-14 surfactant and then for SBPr314 and for SB3-14. The absorption bands of the carbohydrates present within the cell wall dominate the carbohydrates region (W4). The mixed region (W3) is a spectral region containing information from phosphate-carrying compounds, proteins, and fatty acids. The amides region (W2) is dominated by the amide I and amide II bands of proteins and peptides; the fatty acids spectral region (W1) is dominated by the stretching vibrations of the functional groups usually present in the fatty acid components of the various membrane amphiphiles. The increase of the spacer length (SBR4-14 series) provoked the same metabolomic response trend, but with weaker intensities, corresponding to lower mortalities of the cells. SB3-12 surfactant showed an interesting metabolomic response profile with mixed region (W3), amides (W2), and fatty acids (W1) region involved. The same cell compartments were involved in the action of SB5-14 amphiphile. Even SB3-4 compound provoked a response in the same cell compartments, even if weaker. For these three compounds a small biocidal activity is detected and the same spectral regions were involved, different from the SBR3-14 and SBR4-14 series with higher biocidal capability.
Article
CONCLUSIONS
The developed conductometric method, thanks to its speed, permitted us to obtain relevant and accurate data on interaction between zwitterionic surfactant and living cells. Sulfobetaine amphiphiles can interact with eukaryotic model cells Saccharomyces cerevisiae at concentrations higher than their critical micellar concentration values. This was due to the particular structure of the zwitterionic micellar aggregates that can act as pseudocationic aggregates because of their charge distribution and charge densities. This behavior of the micelles permits the interaction of the surfactants with the cells, and the structure of the surfactant monomers has an impact on the mortality and on the metabolomic response data observed. These phenomena, even if they are still to be defined in their exact mechanisms, involved specific parts of the cells, as the FTIR data showed: carbohydrates, mixed region, and amides of the cells. The release in solution of conductive material coincided with significative mortality increases, as respectively showed in conductivity and in viable plate counts experiments. These conductivity experiments are interesting because with simple and rapid experiments they can give relevant information on the nodal concentration values (IPs) that provoke cell mortality increases. Relevant metabolomic responses of the cells are also observed corresponding to these concentrations as showed with SIs with FTIR spectroscopy bioassay. The amphiphile structures showed important structure− biocidal activity relationships that can be useful in the synthesis of specific sulfobetaine surfactants for specific roles. Small changes in the molecular structures led, in fact, to strong differences in the biocidal activity of the compounds. The absence of biocidal capability of the SB3-4 compound showed that the activity of these molecules must be ascribed to their amphiphilic nature and not only to their sulfobetaine moiety. The cationic charge densities in the monomers have a significant importance on the biocidal activity of these compounds. The increase of the dimensions of the headgroup (methyl-, ethyl- and propylammonium) led to a decrease of the biocidal activity; this is due to the polarization of the headgroup that leads to lower cationic charge density of the ammonium. The decrease in mortality observed for SB5-14 micelles has to be ascribed to the folding of the sulfonate on the ammonium and its neutralization. The hydrocarbon chain length also has relevance on the biocidal activity, leading to ∼50% of mortality observed for 12C SB3-12 compound up to ∼90% for 14C SB314. These data could be useful in the synthesis and use of synthetic sulfobetaine surfactants, because structural characteristics can strongly impact on their biocidal activity; this is useful when a low toxicity is needed, such as in drug-delivery topic. The synthesis of sulfobetaine surfactants with specific structural characteristics can lead to an increased biocidal activity of these molecules. In fact, rigid spacers between the ammonium and the sulfonate, long hydrophobic chains, and the absence of any electron-releasing groups next to the ammonium can lead to an increase of their biocidal capability. However, the concentrations needed to provoke these biocidal phenomena are quite high, and much higher than the ones observed for cationic amphiphiles, so these molecules could not be considered good biocides or antimicrobial agents. 1108
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b04077. Conductivity profiles of all the compounds studied in S. cerevisiae cell suspensions (Figure S1), time-course conductivity profile of Saccharomyces cerevisiae cells in water suspensions (Figure S2), mortality/conductivity vs concentration profiles for sulfobetaine surfactants with S. cerevisiae cells (Figure S3), stress index histograms and mortality of S. cerevisiae cells subjected to the action of zwitterionic surfactants (Figure S4), mortality data of S. cerevisiae cells subjected to the action of sulfobetaine compounds (Table 1S) (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: + 39 075 585 5538. Fax: +39 075 585 5560. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the Ministero per l’Università e la Ricerca Scientifica e Tecnologica, MIUR (Rome, Italy) [PRIN “Programmi di Ricerca di Interesse Nazionale” 2010−2011, no. 2010FM738P], and Regione Umbria (POR FSE 2007− 2013, Risorse CIPE, Perugia, Italy) for funding. The authors gratefully thank Antonella Megale for the technical help.
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