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Challenges in application of Langmuir Monolayer Studies to Determine the Mechanisms of Bactericide Activity of Ruthenium Complexes Bianca Sandrino, Jessica Fernanda Affonso de Oliveira, Thatyane M. Nobre, Patricia Appelt, Akash Gupta, Marcio Peres de Araujo, Vincent M. Rotello, and Osvaldo Novais Oliveira Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02247 • Publication Date (Web): 20 Nov 2017 Downloaded from http://pubs.acs.org on November 23, 2017
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Challenges in application of Langmuir Monolayer Studies to Determine the Mechanisms of Bactericide Activity of Ruthenium Complexes B. Sandrinoa, J. F. A. de Oliveirab,c, T. M. Nobrea, P. Appeltd, A. Guptae, , M.P. de Araujod, V. M. Rotelloe and O. N. Oliveira Jr.a a
São Carlos Institute of Physics, University of São Paulo, CP 369, 13560-970, São Carlos, SP,
Brazil. b
National Laboratory of Synchrotron Light (LNLS), CP 6192, 13083-970, Campinas, SP, Brazil.
c
Institute of Chemistry, State University of Campinas (Unicamp), CP 6154,13083-970, Campinas,
SP, Brazil. d
Department of Chemistry, Federal University of Paraná, CP 19081, 81531-980, Curitiba, PR,
Brazil. e
Department of Chemistry, University of Massachusetts Amherst, 01003, Amherst, MA, USA.
* Corresponding author: Tel.: + 55 16 33739825 E-mail address:
[email protected] (Bianca Sandrino)
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Abstract The effects induced by antibiotics on the bacteria membrane may be correlated with their bactericidal activity, and such molecular-level interactions can be probed with Langmuir monolayers representing the cell membrane. In this study, we investigated the interaction between [Ru(mcbtz)2(PPh3)2] (RuBTZ, mcbtz= 2-mercaptobenzothiazoline) and [Ru(mctz)2(PPh3)2] (RuCTZ, mctz= 2-mercaptothiazoline) with Langmuir monolayers of a lipid extract of Escherichia coli, an extract of lipopolysaccharides (LPS) and a zwitterionic phospholipid, dioleoylphosphatidyl choline (DOPC). RuBTZ and RuCTZ had little effects on DOPC, which is consistent with their negligible toxicity toward mammalian cells that may be approximated by a zwitterionic monolayer. Also small were their effects on LPS. In contrast, RuBTZ and RuCTZ induced expansion in the surface pressure isotherms and decreased the compressional modulus of the Escherichia coli lipid extract. While the more hydrophobic RuBTZ seemed to affect the hydrophobic tails of the E. coli extract monolayer to a larger extent, according to polarizationmodulated infrared reflection absorption spectroscopy (PM-IRRAS) results, evidence of a stronger RuBTZ interaction could not be confirmed unequivocally. Therefore, interaction with the E. coli cell membrane cannot be directly correlated with the observed higher bactericide activity of RuBTZ, in comparison to RuCTZ. This appears to be a case in which Langmuir monolayer studies do not suffice to determine the mechanisms responsible for the bactericide activity.
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1. Introduction The indiscriminate use of antibiotics has contributed to the emergence and development of drug-resistant microorganisms,1which has brought the need to develop antimicrobials with novel structures for controlling bacterial and microbial infectious diseases.2,3Metal-based drugs, for instance, have been studied in medicinal chemistry,4,5with many transition metal complexes displaying biological activity. Metal complexes are advantageous because they can provide more stereochemical variability due to their range of coordination geometries, thus allowing for introducing different organic molecules that may facilitate interaction and biorecognition. For example, platinum compounds have been used in cancer treatment,6 silver compounds possess antibacterial properties,7,8 and gold compounds can be used for treating rheumatoid arthritis.6 Ruthenium-based compounds, in particular,exhibit significant anticancer9 and antifungidal10 activity, in addition to serving as catalysts in biological11 and non-biological systems.12Ruthenium has unique properties desirable for drug design, including its ability to bind nucleic acids and proteins, and the range of accessible oxidation states4. Furthermore, ruthenium drugs present low toxicity owing to the iron-mimicking propertywhen bound to biomolecules.4,6Some ruthenium complexes also show antibacterial properties, which should be related to their effects on the bacteria membrane, but the molecular mechanisms involved are not known in detail.4 Four mechanisms have been suggested in the literature to govern antibacterial action. In three of those there is participation of membrane proteins, more specifically with inhibition or regulation of enzymes relevant for biosynthesis in the cell wall, protein synthesis, and nucleic acid metabolism and repair13. The fourth mechanism is unique in the sense that it does not involve other membrane components rather than those responsible for the membrane structure. This action mechanism is based on disrupting the membrane structure14, being especially relevant to fight highly resistant bacteria because the latter cannot develop resistance against antibiotics that are capable of destroying or causing leakage in the membrane15. For Gram-negative bacteria this mechanism is rather complex since the antibiotics have to disrupt both the inner, lipidic membrane as well as the outer bacterial wall made of lipopolysaccharides (LPS)15. To cross the LPS barrier,
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antibiotics use porin channels to reach the periplasmic space, where they can bind to their targets and develop their activity. Porins are beta barrel proteins that cross the membrane and are responsible for the passive diffusion of molecules, including the influx of antibiotics (mostly hydrophilic and smaller than 1500 Daltons). Testing the probable action of drugs via the membrane disruption mechanism is thus crucial for drug design. Experiments with whole cell membranes to verify such a mechanism are not possible with present technology, and therefore use is made of cell membrane models such as Langmuir monolayers16 and vesicles17. Even though the Langmuir monolayers mimic only half a membrane, they are convenient owing to the possible control of lateral packing and membrane composition. Furthermore, studies can be made with drugs that are water-soluble or not, as the drug may be injected into the subphase or co-spread with the monolayer-forming material. In this study, we used Langmuir monolayers to simulate the inner plasmatic membrane and the outer membrane ofEscherichia colito investigate the effects from two ruthenium complexes, namely [Ru(mcbtz)2(PPh3)2] complex, mcbtz= 2-mercaptobenzothiazoline, referred to as RuBTZ (Fig. 1A) and [Ru(mctz)2(PPh3)2] complex, mctz= 2-mercaptothiazoline (RuCTZ, Fig. 1B). The aim is to try andcorrelate the effects of these complexes on the cell membrane model with their inhibition of bacterial growth18 and bactericide activity.As we shall show, in spite of a thorough comparative study with the complexes and three types of monolayer, the monolayer studies are not sufficient to establish such correlation.
S
S S
S N
Ph3P
Ru
Ru N
Ph3P
N
Ph3P
S
A)
N
Ph3P S S
B)
S
Figure 1: A) Ruthenium complex with 2-mercaptobenzothiazoline (RuBTZ) and (B) with 2mercaptothiazoline (RuCTZ).
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2. Experimental 2.1. Materials All chemicals used were of reagent grade or comparable purity, and the solvents were purchased from Sigma–Aldrich. ThecomplexesRuBTZ and RuCTZ were synthesized through a well-established route18. The E. coli extract (PE 57.5 wt%, PG 15.1 wt%, CL 9.8 wt% and unknown content 17.6 wt%) and 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) were acquired from Avanti Polar Lipids. The lipopolysaccharides (rough strains) from Escherichia coli J5 (Rc mutant) were purchased fromSigma–Aldrich. All of these compounds were used without further purification.
2.2. Determination of Antimicrobial Activities of RuBTZ and RuCTZ Experiments to determine the minimal inhibitory concentration (MIC) were conducted with CD-2 strain (Escherichia coli). CD-2 was cultured overnight in minimal M9 medium at 37°C and speed of 275 rpm until reaching the stationary phase, and then harvested by centrifugation and washed three times with 0.85% sodium chloride solution. The optical density at 600 nm was used to determine the concentration of resuspended bacterial solution. M9 medium was used to make dilution of bacterial solution to a concentration of 1x106 CFU/mL. Then, a volume of 50 μL of this solution was added into a 96-well plate and mixed with 50 μL of RuBTZ or RuCTZ solutions in M9, yielding a final bacterial concentration of 5x105 CFU/mL.For preparing the initial solutions with the complexes, 2 mg were dissolved in 1 mL DMSO, and then 128 μL of these solutions were mixed with M9 to complete 400 μL to obtain the concentration of 0.64 mg/mL for each complex. From this solution (0.64 mg/mL), a new one with half of concentration 0.32 mg/mL was obtained and then dilutions were made to have concentrations ranging from 160 to 1.25 μg/mL. Neat DMSO (without any complex) was also tested to evaluate its possible bactericidal effect. A growth control group without Ru complexes and a sterile control group with only growth
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medium were used. All the experiments were conducted in triplicate and performed simultaneously.
2.3. Cytotoxicity assays for mammalian cells 3T3 cells were maintained in high glucose Dulbecco's Modified Eagle's Medium (DMEM) (DMEM; ATCC 30-2002) supplemented with 10% fetal bovine serum (Fisher Scientific, SH3007103), 100 units/mL penicillin and 100 µg. mL-1 Streptomycin (Gibco). The cells were grown in 96-well plates at 37 °C with 5% CO2 atmosphere incubation.Approximately 1.5x1043T3 cellsper well were seeded onto 96-well plates. After 24 h the medium was replaced with a fresh one containing the Ru complexes. Incubation for 24 or 48 h followed, and then cell viability was determined using AlamarBlue®assay according to the manufacturer's protocol (Invitrogen Biosource). After washing with a phosphate-buffered saline (PBS) solution three times, cells were treated with 120 μL of 10% alamar blue in serum-containing media and incubated at 37 °C under an atmosphere of 5% CO2 for 3 h. The solution from each well was transferred into a 96-well black microplate. The red fluorescence, resulting from the reduction of Alamar blue solution, was quantified (excitation/emission: 560 nm/590 nm) on a SpectroMax M5 microplate reader (Molecular Device) to determine cell viability. Cells without any complex were considered as 100% viable. All experiments were conducted in triplicate, performed simultaneously and the reported values are averages. The cell viability was expressed as a percentage relative to the control according to Equation 1.
𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) =
Fluorecencesample 𝐹𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒𝐶𝑜𝑛𝑡𝑟𝑜𝑙
× 100
[1]
whereFluorescencesample is the sample fluorescence in the presence of the complex and Fluorescencecontrol is the fluorescence of the control group (untreated cells).
2.4. Langmuir monolayers
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Langmuir monolayers were produced by spreading the solutions of the compounds onto the surface of a Milli-Q water in a Mini KSV Langmuir trough (KSV instrument Ltd, Helsinki, Finland) equipped with a Wilhelmy plate as surface pressure sensor. DOPC, LPS and E. coli extract were dissolved in CHCl3.The solutions for the monolayers incorporating the Ru complexes were obtained by mixing appropriate volumes of stock solutions made of 10 mg mL -1 for the Ru complex solubilized in DMSO and 2 mg mL-1 for E. coli extract (or LPS or DOPC) in CHCl3. The relative concentrations of 8 and 16 % of the Ru complexes mean the amount of material to replace the lipid or LPS in the film, with a final solution concentration of 2 mg/mL.Aliquots of 40 µL of these solutions were then spread onto the water surface with a micrometric syringe. For LPS, PBS at pH 7.4 was used as the subphase since stable LPS films could not be formed on ultrapure water. The solvent was allowed to evaporate for ca. 10 min before monolayer compression. The surface pressure versus area per molecule (π-A) isotherms were recorded with symmetrical compression of monolayers with two barriers at a constant speed of 10 mm min-1 (~5 Å2 molecule-1 min-1). The experiments were performed at 20±1°C, and the isotherms were made in duplicate. The effect of neat DMSO on the membrane model was also evaluated and the results are given in the Supplementary Material. In this experiment, rather than using 16% of CHCl3as in the monolayers with E coli, DOPC and LPS, DMSO was used to verify its effect by mimicking the DMSO volume used for the 16% mixed films.
2.4.1 Polarization Modulation Infrared Reflection Absorption Spectroscopy - PM-IRRAS. PM-IRRAS measurements were taken with a KSV PMI 550 instrument (KSV instrument Ltd, Helsinki, Finland). The experimental setup used was similar to that described by Geraldo et al19. The Langmuir trough is placed in a way that the light beam reaches the monolayer at a fixed incidence angle of 80, at which the intensity is maximum with a low level of noise. The incoming light is continuously modulated between s- and p- polarization at a high frequency (50 kHz), which allows for the simultaneous measurement of the spectra for the two polarizations. The difference between the spectra provides surface-specific information, and the sum gives the
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reference spectrum. The effect from water vapor is reduced with the simultaneous measurements. The mixed films studied had 8% of Ru complexes in Ecoli.
2.4.2 Brewster Angle Microscopy (BAM) The BAM images were obtained using an ellipsometerAccurion EP4 with Ultraobjective, camera SVS-Vistek eco 285, laser at 658 nm and 50 mWwith 100% of power. The angle of observation was 53.1°, polarization at 2° and analyzer at 10°.The principles of this technique are explained in ref.20 The geometric correction in the images was performed with software datastudio, applying in Y-axis a factor of 1.6655 and producing images with 450 x 570 microns. During the measurements, the grey scale was adjusted depending on the reflectivity, since the image of the interfacial film is formed by the contrast of regions with lower film concentration (dark regions - without reflection) and spots where the water surface is covered with high film concentrations (bright regions - reflection).
3. Results and Discussion 3.1 Bactericidal activity of RuBTZ and RuCTZ The data for thebactericidal effect againstE. coli, obtained with the 96-well plate microdilution method, are shown inFigure 2A for RuBTZ, RuCTZ and DMSO at distinct concentrations. Comparison with DMSO is important because the ruthenium complexes are not soluble
in
aqueous
solutions,
similarly
to
hydrophobic
drugs
such
as
AmoxycillinClarithromycinand Rifampicin21.The bactericidal activity for RuBTZ was consistently higher, especially above 40 µg/mL, where the activity of RuCTZwas not statistically significantly different from the blank controldue to precipitation of the complex, which has a limited solubility in water.At these higher concentrations, DMSO itself had a significant bactericide effect, which was even higher than that for RuBTZ at the highest concentration tested. Therefore, the efficient bactericidal activity of RuBTZin Figure 2A could be ascribed to some combination with the effect from DMSO.In summary, RuBTZ displayed significant bactericidal
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activity against E. coliwhereasRuCTZ had only minor effects, even at high concentrations. With regard to the possible cell toxicity of the ruthenium complexes, we performed cell viability assays using 3T3 mammalian cells. The data in Figure 2B show no significant toxic effect from either RuBTZ or RuCTZ, or DMSO, except at very high concentrations.
Figure2.(A) Optical density at 600 nm, which is correlated to the bactericide activity of the Ru complexes and DMSO. The lower density means higher bactericidal effect. The grey bar corresponds to the control sample, for which no bactericide effect exists. The values for RuBTZ (red bars), RuCTZ(blue bars) and DMSO (black bars) at different concentrations refer to bacterial growth inhibition after incubation for 24 h.(B) Cell viability for 3T3 mammalian cells for incubations for 24 and 48 h into solutions containing varied concentrations of RuBTZ (red), RuCTZ (blue) and DMSO (black). The data are compared to the control group with 100% viability. Data shown are the mean for each condition ± SD. Statistical analysis was performed according to the t-test. The letters on top of the bars (a, b and c) indicate mean P
