Molecular Organization of Dipalmitoylphosphatidylcholine Bilayers

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Molecular Organization of Dipalmitoylphosphatidylcholine Bilayers Containing Bioactive Compounds 4‑(5-Heptyl-1,3,4-thiadiazol-2-yl) Benzene-1,3-diol and 4‑(5-Methyl-1,3,4-thiadiazol-2-yl) Benzene-1,3diols Dariusz Kluczyk,†,∇ Arkadiusz Matwijczuk,*,‡,∇ Andrzej Górecki,⊥ Monika M. Karpińska,# Mariusz Szymanek,§ Andrzej Niewiadomy,∥,# and Mariusz Gagoś*,† †

Department of Cell Biology, Institute of Biology and Biochemistry, Maria Curie−Skłodowska University, 20-033 Lublin, Poland Department of Biophysics, §Department of Agricultural Machines Theory, and ∥Department of Chemistry, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland ⊥ Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland # Institute of Industrial Organic Chemistry, Annopol 6, 03-236 Warsaw, Poland ‡

S Supporting Information *

ABSTRACT: This article presents the results of spectroscopic studies of two compounds from the 1,3,4-thiadiazole group, that is, 4-(5-methyl1,3,4-thiadiazole-2-yl)benzene-1,3-diol (C1) and 4-(5-heptyl-1,3,4-thiadiazole-2-yl)benzene-1,3-diol (C7), present at different molar concentrations in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) liposome systems. In the case of both investigated compounds, fluorescence measurements revealed the presence of several emission bands, whose appearance is related to the molecular organization induced by changes in the phase transition in DPPC. On the basis of the interpretation of Fourier transform infrared spectra, we determined the molecular organization of the analyzed compounds in multilayers formed from DPPC and the 1,3,4-thiadiazoles. It was found that the compound with a longer alkyl substituent both occupied the lipid polar head region in the lipid multilayer and interacted with lipid hydrocarbon chains. In turn, the compound with a shorter alkyl substituent interacted more strongly with the membrane polar region. On the basis of the knowledge from previous investigations conducted using different solvents, the fluorescence effects observed were related to the phenomenon of molecular aggregation. The effects were strongly influenced by the structure of the compound and, primarily, by the type of the alkyl substituent used in the molecule. The substantial shortening of fluorescence lifetimes associated with the effect of long-wave emission (with a maximum at 505 nm) decay also confirms the model of aggregation effects in the analyzed systems. Similar effects can be very easily distinguished and associated with respective forms of the compounds in biologically relevant samples.



dihydroxyphenyl system.2 1,3,4-Thiadiazoles3 with a resorcyl fragment substituent are a very promising group of novel pharmaceuticals with neuroprotective and anticancer activity.4−6 Representatives of this compound group have been applied in medicine and science in multifarious ways, for example, as metal-complexing compounds,7,8 oxidation inhibitors, and different types of dyes.9 The 1,3,4-thiadiazoles analyzed are compounds with mostly anticancer,10,11 neuroprotective,12 antifungal,13 antibacterial,14 anti-inflammatory,15 antioxidant,16 and antihypertensive17 activity.

INTRODUCTION Neoplastic and neurodegenerative diseases are the major causes of lethality as well as long-term or permanent disability in the populations of highly developed countries. Still, one of the biggest clinical problems related to the application of new or already available pharmaceuticals is their very high toxicity. This problem is caused by the extremely narrow scope of action of the newly designed and existing compounds. Modern medicine dealing with neurodegenerative diseases assigns an especially difficult and challenging task to scientists searching for new compounds with specific molecular characteristics and multidirectional activity. Great hope in the fight against these diseases is placed on compounds from the group of 2-amino1,3,4-thiadiazoles or 1,3,4-thiadiazoles1 with a substituted 2,4© 2016 American Chemical Society

Received: September 16, 2016 Revised: October 26, 2016 Published: October 31, 2016 12047

DOI: 10.1021/acs.jpcb.6b09371 J. Phys. Chem. B 2016, 120, 12047−12063

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molecular CT states,31 often accompanied by twisting of a part of the molecule, that is, the so-called twisted intramolecular charge transfer states.32 Additionally, dual fluorescence can be evoked by molecule tautomerism in excited states, that is, the so-called photoautomers. Other determinants of the appearance of dual fluorescence are excimer emissions appearing with an increasing concentration of the solution (ct.). To end the overview of the theoretical attempts at the elucidation of the occurrence of several fluorescence bands, it is worth mentioning that the excited-state intramolecular proton transfer is an equally interesting and popular explanation of this process.33,34 Moreover, the very interesting mechanism involved in dual fluorescence, that is, the anti-Kasha mechanism, reported by Brancato et al. in 2015 should be mentioned.35 However, molecular aggregation combined with the structure of the alkyl substituent may have the greatest impact on the fluorescence effects observed in the lipid environment in the case of the 1,3,4-thiadiazoles investigated in this study.36,37 The analyses of the 1,3,4-thiadiazole analogues carried out with stationary and excited-state fluorescence spectroscopy demonstrated three fluorescence forms (as well as the dual fluorescence effect in some temperature and concentration ranges) induced by changes in the concentration of the compounds and temperature in the DPPC liposome medium. Determination of the details of the organization of 1,3,4-thiadiazole molecules in the lipid medium is important for recognition of their pharmacological applications and for further model research.

Given their confirmed high therapeutic and neuroprotective properties, 4-(5-heptyl-1,3,4-thiadiazole-2-yl)benzene-1,3-diol (C7, Scheme 1A) and 4-(5-methyl-1,3,4-thiadiazole-2-yl)Scheme 1. Chemical Structure of the C1 Molecule (Panel A) and the C7 Molecule (Panel B)

benzene-1,3-diol (C1, Scheme 1B) were chosen for the spectroscopic studies of the mechanism of molecular interactions in liposomal membranes. It should be emphasized that, in addition to their pharmacological activity, the 1,3,4thiadiazole compounds chosen for the analyses exhibit interesting spectroscopic effects, for example, the keto/enol tautomerism effect induced by changes in environment polarizability,18,19 crystal polymorphism and solvatomorphism effects,20,21 and interactions in lipid membranes.22 Most importantly, however, the investigated 1,3,4-thiadiazole compounds exhibit dual fluorescence effects induced by changes in the pH, concentration, and temperature of the environment.23,24 Additionally, 1,3,4-thiadiazoles are interesting ligands, exhibiting a capability of complexation with ions of d-block metals, which has already been confirmed and published.25 The attempt to link the spectroscopic and structural effects presented in the previous studies cited above may be especially important for elucidation of the pharmacological performance of the analogues and the entire group of 2amino-1,3,4-thiadiazoles. The aim of these investigations was to conduct spectroscopic studies of the molecular organization of two 1,3,4-thiadiazole analogues, that is, C1 and C7, in 1,2-dipalmitoyl-sn-glycero-3phosphatidylcholine (DPPC) liposome systems and to elucidate the fluorescence effects in comparison with results presented in other previous studies. The spectroscopic techniques used, that is, optical absorption and fluorescence spectroscopy, analysis of fluorescence lifetimes (time-correlated single photon counting (TCSPC)), differential scanning calorimetry (DSC) measurements, circular dichroism (CD) methods, and Fourier transform infrared (FTIR) spectroscopy measurements, have shown the complexity of the physical processes involved in the presence of three fluorescence emission bands in C7 and C1 in the lipid medium. The FTIR spectroscopy measurements have revealed the most probable regions of interactions of the analyzed compounds with the DPPC lipid. The effects of dual fluorescence26 or several fluorescence bands27 are associated with emergence of two/several clearly separated emission spectra due to electronic excitation.28 They can be induced by changes in the environment, for example, solvent polarity, pH of the medium,29 temperature fluctuations,30 and changes in the concentration of the compound. The most popular attempts at the elucidation of these fluorescence effects are based on the appearance of intra-



MATERIAL AND METHODS Materials. 4-(5-Methyl-1,3,4-thiadiazol-2-yl)benzene-1,3diol (C1) and 4-(5-heptyl-1,3,4-thiadiazol-2-yl)benzene-1,3diol (C7) (see Scheme 1A,B) were synthesized in the Department of Chemistry of the University of Life Sciences in Lublin; details of the procedure are described elsewhere.38 The purification procedure of the 1,3,4-thiadiazole compounds is described in detail in refs 20, 38. Lipids. Synthetic lipid DPPC was purchased from Sigma Chem. Co. Water. Water required for the preparation of buffers was first deionized in an HLP10 deionizer (Hydrolab). Buffers. Phosphate-buffered saline (PBS) was used for the formation of lecithin liposomes (Sigma-Aldrich). Formation of Liposomes. Liposome formation (24) with C1 and C7 (1, 3, 5, 10, 15, 20, 25, and 30 mol % relative to the lipid) was carried out by mixing measured volumes of a chloroform solution of the lipid (DPPC) with a C1/C7 methanol solution in glass tubes. The mixture was evaporated under a nitrogen stream and additionally dried under ca. 10−5 bar vacuum. PBS buffer with pH 7.4 was added to the dry sample to obtain a lipid concentration of 0.3 mg DPPC/1 mL buffer. By 3 × 3 s sonication at a temperature of 45 °C (Techpan ultrasonic disintegrator UD−20) with a frequency of 22 kHz and an amplitude of 1−8 μm, unilamellar liposomes were formed with DPPC and C1/C7 with a diameter from 90 to 110 nm (ca. 80% of the liposome population measured with the elastic light-scattering technique). Methods. Measurements of Electronic Absorption and Fluorescence Spectra. Electronic absorption spectra of C1 and C7 were recorded on a Cary 60 UV−vis spectrophotometer (Agilent) equipped with a thermostatted cuvette holder with a Peltier block. The temperature was controlled with a 12048

DOI: 10.1021/acs.jpcb.6b09371 J. Phys. Chem. B 2016, 120, 12047−12063

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Figure 1. (A−C) Electronic absorption spectra with changes in the temperature for C7 in the DPPC medium at the selected mol % concentrations. The proportion of the components was as follows: C7 3%, DPPC 97% (A), C7 15%, DPPC 85% (B), and C7 25%, DPPC 75% (C). Panels D−F show fluorescence emission spectra corresponding to the samples from Panels A−C. The emission spectra for all analyzed samples were obtained at an excitation wavelength in the absorption band maximum (Em(Ex327)). For clarity of the results, only the spectra obtained at temperature of 22, 35, 41.5, 51, and 60 °C are presented.

thermocouple probe (Cary Series II from Varian) placed directly in the sample. Fluorescence excitation and emission spectra were collected using an F7000 (Hitachi) fluorescence spectrophotometer with a 0.2 nm resolution and corrected for the lamp and photomultiplier spectral characteristics. Excitation and emission slits were set to 5 nm. Synchronous spectra were recorded using a Cary Eclipse spectrofluorometer (Varian). All spectra were recorded at 22 °C (unless stated otherwise). The excitation and emission monochromators of the spectrofluorimeter were scanned synchronously (0.0 nm interval between excitation and emission wavelengths), and the slits were set to achieve a spectral resolution of 5 nm. The spectral

analysis was performed with the use of Grams/AI 8.0 software (Thermo Electron Corporation). FTIR Measurements. Infrared absorption spectra were recorded on a Vertex 70 FTIR (Bruker) spectrometer. The attenuated total reflection (ATR) configuration was used with 20 internal reflections of the ZnSe crystal plate (45° cut). Typically, 16 scans were collected, Fourier-transformed, and averaged for each measurement. Absorption spectra at a resolution of one data point per 1 cm−1 were obtained in the region between 4000 and 600 cm−1. The instrument was continuously purged with N2 for 40 min before and during measurements. The ZnSe crystal plate was cleaned with ultrapure organic solvents from Sigma-Aldrich. The spectral 12049

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Figure 2. (A−C) Electronic absorption spectra with changes in the temperature for C1 in the DPPC medium at the selected mol % concentrations. The proportion of the components was as follows: C1 3%, DPPC 97% (A), C1 15%, DPPC 85% (B), and C1 25%, DPPC 75% (C). Panels D−F show fluorescence emission spectra corresponding to the samples from Panels A−C. The emission spectra for all analyzed samples were obtained at an excitation wavelength in the absorption band maximum (Em(Ex327)). For clarity of the results, only spectra obtained at temperature values of 22, 35, 41.5, 51, and 60 °C are presented.

analysis was performed with the Grams/AI software from ThermoGalactic Industries. Dry DPPC/C1−C7 films were prepared by evaporating the mixture under a stream of argon. The spectra of C1/C7 in DPPC in different molar ratios were obtained in dry purified air (relative humidity, RH < 5%, 31 °C). They were recorded in the first heating cycle from 25 to 90 °C with 0.5−3 °C intervals. TCSPC Measurements. TCSPC measurements were performed using a FluoroCube fluorimeter (Horiba, France). The samples were excited using a pulsed NanoLED diode at 372 nm (pulse duration of 150 ps) operated with 1 MHz repetition. To avoid pulse pileup, the power of the pulses was adjusted to an appropriate level using a neutral gradient filter. Fluorescence emission was recorded using a picosecond detector TBX-04

(IBH, JobinYvon, UK). The DataStation and the DAS6 software (JobinYvon (IBH, UK)) were used for data acquisition and signal analysis. All fluorescence decays were measured in a 10 × 10 mm2 quartz cuvette, using an emitter cutoff filter with transmittance for wavelengths longer than 408 nm. The excitation profiles required for the deconvolution analysis were measured without the emitter filters on a light-scattering cuvette. All measurements were performed in water at 20 °C and various pH or in pH 7 and increasing (or decreasing) temperature. Each case of fluorescence decay was analyzed with a multiexponential model described by the equation It =

∑ αi exp( −t τi ) i

12050

(1) DOI: 10.1021/acs.jpcb.6b09371 J. Phys. Chem. B 2016, 120, 12047−12063

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The Journal of Physical Chemistry B where αi and τi are the pre-exponential factor and the decay time of component i, respectively. Best-fit parameters were obtained by minimization of the reduced χ2 value as well as residual distribution of the experimental data. The average lifetime of fluorescence decay was calculated according to the following equation ⟨τ ⟩ =

maximum at 377 nm (as in Panel D) and at 421 nm (Panel E). However, Panel F, which shows the high 25% molar concentration of the C7 compound in DPPC, presents a clear dual fluorescence effect with a maximum at 434 nm and a distinct long-wave fluorescence effect with a maximum at 505 nm. After the main lipid phase transition, the long-wave fluorescence emission rapidly decays and an emission band at 434 nm can only be observed. Next, a distinct slope on the short-wave side, which is characteristic of emission with a maximum at approximately 372/7 nm, is visible. Noteworthy, in the case of the high C7 concentrations in DPPC, there are three fluorescence forms, depending on the amount of the compound used. As indicated in previous studies, these effects in the investigated molecules should be associated with the phenomenon of molecular aggregation, which occurs in model biological liposome systems. To elucidate and understand the reported effect, C1 (Scheme 1B), that is, a 1,3,4-thiadiazole analogue of C7, was chosen for the analyses performed at different DPPC concentrations. The compound differs from C7 only in the structure of the alkyl substituent: C7 contains a 7-hydrocarbon chain, whereas C1 has only one carbon atom in the substituent group. The same fluorescence measurements as in the case of C7 were performed for C1. Similar to Figure 1, Figure 2 presents absorption and electronic fluorescence spectra for C1 at different molar concentrations in DPPC. Panels A−C present electronic absorption spectra for C1 at different molar concentrations in DPPC, depending on the changes in the medium temperature (3 mol % C1 in DPPC in Panel A, 15 mol % C1 in DPPC in Panel B, and 25 mol % C1 in Panel C). In all absorption spectra, there is a broad band (as in Figure 1) between ca. 300 and 340 nm with a maximum at 320 nm (319, 318 nm), which is characteristic of the π → π* electron transition in the analyzed molecule. In contrast to C7, it exhibits thermochromic shifts accompanying the changes in the temperature. In the case of C1, there is distinct underpinning of the maximum absorption band at a wavelength of 345 nm in all presented spectra. The position of the band on the long-wave (345−348 nm) side indicates a possibility of the existence of forms of the compound other than the monomeric forms in the investigated samples (J-dimers or larger N-aggregates). In turn, Panels D−F present (as in Figure 1) fluorescence emission spectra for C1 at different molar concentrations in DPPC (3 mol % C1 in DPPC in Panel D, 15 mol % C1 in DPPC in Panel E, and 25 mol % C1 in Panel F). The excitation wavelength for all the samples corresponds to the maximum of the absorption spectra, that is, ca. 320 nm. There are several spectral forms of the fluorescence emission spectra in this case as well: a very clear form with a maximum at 437/8 nm and a much less intensive form at 380 nm. The increase in the medium temperature is accompanied by a slight solvatochromic shift of the main maximum emission band (437 nm); in addition, the band with a maximum at 380 nm is present only at the lower concentrations of the compound in the lipid but is almost invisible at the higher concentrations. Noteworthily, in the case of C1, there is no long-wave emission with a maximum at 505 nm, likewise in C7 at the higher concentrations. It should be emphasized that, first, the differences in the structure of the alkyl substituent in the analyzed 1,3,4-thiadiazoles exert a clear effect on the number of fluorescence emission bands of the compound, and, second, they also have an impact on the mode of molecular aggregation of the investigated compounds. The longer 7-hydrocarbon chain in C7 may promote formation of

∑i αiτi2 ∑i αiτi

(2)

CD Measurements. CD measurements were carried out on a J-710 CD spectrometer (JASCO, Japan) using a quartz cell of 1 cm and 1 mm path length. Spectra were recorded at 23 °C from 240 to 500 nm at a resolution of 0.2 nm with a scan rate of 20 nm/min. DSC Measurements. DSC measurements were carried out with the use of a DSC1 (Mettler Toledo) calorimeter and standard aluminum 40 μL crucibles. One crucible was filled with 30 μL of the sample, whereas another one was filled with an equal (weight) amount of PBS buffer as a reference. The samples and their concentrations are described in the Formation of Liposomes section. Thermograms were recorded between 20 and 60 °C with a scan rate of 1 °C/min and under a nitrogen flow of 50 mL/min.



RESULTS AND DISCUSSION Panels A−C in Figure 1 present electronic absorption spectra for C7 at different molar concentrations in DPPC, depending on the temperature changes in the analyzed medium (for 3 mol % C7 in DPPC in Panel A, 15 mol % C7 in DPPC in Panel B, and 25 mol % C7 in Panel C). Depending on the fluctuations in the temperature of the solution, slight changes in the shape of the recorded spectra can be observed. In all of the absorption spectra, there is a broad band extending from ca. 300 to 350 nm, with a maximum at 327/8 nm, which is characteristic of the π → π* electron transition in the analyzed molecule and does not exhibit solvatochromic shifts accompanying changes in the temperature. Slight underpinning of the maximum absorption band at a wavelength of 345/6 nm can be clearly noted for all of the spectra. The position of this band on the long-wave side (345/6 nm) and the substantially lower intensity evidently indicate the probable existence of forms of the compound other than the monomeric forms in the analyzed samples (dimers or N-aggregates). Particularly noteworthy is the effect presented in Panels D−F, which show fluorescence emission spectra corresponding to the respective electronic absorption spectra from Panels A−C. The excitation wavelength for all of the samples corresponds to the maximum of the absorption spectra at 327 nm. As can be seen in Panels D−F, there are different spectral bands, depending on the compound (C7) concentration in the liposome system. Panel D, showing the fluorescence emission spectra for 3 mol % C7 in DPPC, presents a single fluorescence band with a maximum at 372 nm, which is shifted toward a shorter wavelength of 368 nm, in particular after the main phase transition in DPPC. In the initial gel phase, slight broadening of the fluorescence emission band at 450 nm can be noted. Next, the increase in the concentration of C7 to 15% in the DPPC liposome system is accompanied by the appearance of new fluorescence bands (Panel E). Before the phase transition, a dual fluorescence effect with a maximum at ∼400 and 505 nm is clearly visible. With the temperature increase and lipid transition from phase Lβ′ to Lα, the long-wave fluorescence (505 nm) form decays and there are forms with a 12051

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Figure 3. Temperature dependence of the position of the main absorption maximum for C7 (Panels A−C) and for C1 (Panels D−F) at different molar concentrations in the DPPC liposomes. Panels A and D present dependences for 3% C7/C1 in DPPC, Panels B and E for 10% C7/C1 in DPPC, and Panels C and F for 20% C7/C1 in DPPC, respectively. The dependences are shown as a ratio of the wavenumber and temperature.

aggregates, and their exciton interactions may evoke fluorescence emission with a maximum at 505 nm. It should also be noted that similar effects were observed in solvents and media characterized by different concentrations of hydrogen ions for C1 and C7. The proposed model of the aggregation effect and its impact on the number of spectral forms can be clearly seen in the model biological DPPC systems. To illustrate the effect of the aggregation process in the analyzed compounds in DPPC liposomes on changes in the phase transition, Figure 3 presents temperature determinants of the position of the maximum of the main band in the electronic absorption spectrum for C7 (Panels A−C) and C1 (Panels D− F) at different molar concentrations of both compounds in DPPC. The position of the main absorption maximum for the analyzed compounds has different values in the temperature range before and after the main Lβ′ → Lα phase transition in DPPC. In the case of the different molar concentrations of C7 in DPPC, only a slight effect of the compound on the main phase transition is observed, in particular at the lower concentrations (