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Feb 28, 2015 - Abstract Despite the extended use and well-documented information, there are insufficient reports concerning the ef- fects of propranolol on the ...
J Membrane Biol (2015) 248:683–693 DOI 10.1007/s00232-015-9780-2

Morphological Effects Induced In Vitro by Propranolol on Human Erythrocytes Mario Suwalsky • Pablo Zambrano • Fernando Villena • Marcela Manrique-Moreno Marı´a Jose´ Gallardo • Malgorzata Jemiola-Rzeminska • Kazimierz Strzalka • Ana Marı´a Edwards • Sigrid Mennickent • Nathan Dukes



Received: 28 November 2014 / Accepted: 29 January 2015 / Published online: 28 February 2015 Ó Springer Science+Business Media New York 2015

Abstract Despite the extended use and well-documented information, there are insufficient reports concerning the effects of propranolol on the structure and functions of cell membranes, particularly those of human erythrocytes. Aimed to better understand the molecular mechanisms of its interactions with cell membranes, human erythrocyte and molecular models of the red cell membrane were utilized. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of propranolol to perturb the multibilayer structures of DMPC and DMPE was evaluated by X-ray diffraction. Moreover, we took advantage of the capability of differential scanning calorimetry to detect the changes in the thermotropic phase behavior of lipid bilayers resulting

from propranolol interaction with DMPC and DMPE multilamellar vesicles. In an attempt to further elucidate their effects on cell membranes, the present work also examined their influence on the morphology of intact human erythrocytes by means of defocusing and scanning electron microscopy. Results indicated that propranolol induced morphological changes to human erythrocytes and interacted in a concentrationdependent manner with phospholipid bilayer.

M. Suwalsky (&)  P. Zambrano Faculty of Chemical Sciences, University of Concepcio´n, Concepcio´n, Chile e-mail: [email protected]

M. Jemiola-Rzeminska  K. Strzalka Malopolska Centre of Biotechnology, Jagiellonian University, Krako´w, Poland

F. Villena Faculty of Biological Sciences, University of Concepcio´n, Concepcio´n, Chile M. Manrique-Moreno Faculty of Exact and Natural Sciences, University of Antioquia, Medellı´n, Colombia M. J. Gallardo Center for Optics and Photonics, University of Concepcio´n, Concepcio´n, Chile

Keywords Propranolol  Beta blocker  Erythrocyte membrane  Phospholipid bilayer Abbreviations RBC Red blood cell suspension SEM Scanning electron microscopy DMPC Dimyristoylphosphatidylcholine

A. M. Edwards Faculty of Chemistry, Pontifical Catholic University of Chile, Santiago, Chile S. Mennickent Faculty of Pharmacy, University of Concepcio´n, Concepcio´n, Chile N. Dukes Faculty of Medicine, Pontifical Catholic University of Chile, Santiago, Chile

M. Jemiola-Rzeminska  K. Strzalka Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krako´w, Poland

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DMPE DSC MLV SEM DM

M. Suwalsky et al.: Morphological Effects on Human Erythrocytes

Dimyristoylphosphatidylcholine Differential scanning calorimetry Multilamellar vesicles Scanning electron microscopy Defocusing microscopy

Introduction Beta blockers are a class of drugs that target the beta receptors, which are found on cells of the heart muscle, smooth muscles, airways, arteries, kidneys, and other tissues that are part of the sympathetic nervous system. They lead to stress responses, especially when they are stimulated by adrenaline. Beta blockers interfere with the binding to the receptor of adrenaline and other stress hormones. These blockers are particularly used for the management of cardiac arrhythmias, protecting the heart from a second heart attack (secondary prevention) and hypertension (Freemantle et al. 1999; Cruickshank 2010). In 1962, Sir James W. Black found the first clinically significant beta blockers, propranolol and pronethalol; the discovery revolutionized the medical management of angina pectoris (Van der Vring 1999) and is considered by many to be one of the most important contributions to clinical medicine and pharmacology of the 20th century (Stapleton 1997). Propranolol (Fig. 1) is a sympatholytic non-selective beta blocker. It is on the World Health Organization’s List of Essential Medicines, a list of the most important medication needed in a basic health system (WHO 2013). It has been reported that 80 % of the administered propanolol binds specifically to membrane lipids (Albertini et al. 1990). In fact, most cardiac drugs are lipid soluble molecules that readily partition into the membrane bilayer (Suwalsky et al. 1994; Herbette et al. 1988). The interaction of drugs with membranes play an essential role in their biological activity, since drugs must interact with membranes through their entire in vivo course, and their therapeutic target is often embedded within membranes.

Fig. 1 Schematic formula of propranolol

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Therefore, the study of the influence of propranolol on membrane organization could shed light on cardioprotective properties that are not related with beta adrenoceptor antagonism (Pereira-Leite et al. 2013). Aimed to better understand the molecular mechanisms of the interaction of propranolol with cell membranes, we have utilized human erythrocytes and molecular models of its membrane. Human erythrocytes were chosen because of their only one membrane and no internal organelles constitute an ideal cell system for studying interactions of chemical compounds with cell membranes (Chen and Huestis 1997). On the other hand, although less specialized than many other cell membranes they carry on enough functions in common with them such as active and passive transport, and the production of ionic and electric gradients to be considered representative of the plasma membrane in general. The molecular models of the erythrocyte membrane consisted in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of cell membranes, particularly of the human erythrocyte, respectively (Boon and Smith 2000; Devaux and Zachowsky 1994). The capacity of propranolol to perturb the bilayer structures of DMPC and DMPE was evaluated by X-ray diffraction; intact human erythrocytes were observed by defocusing (DM) and scanning electron microscopy (SEM). These systems and techniques have been used in our laboratories to determine the interaction with and the membrane-perturbing effects of other therapeutic compounds, including antiarrhythmic drugs (Suwalsky et al. 1994, 2009, 2011, 2013).

Materials and Methods X-ray Diffraction Studies of DMPC and DMPE Multilayers The capacity of propranolol to perturb the structures of DMPC and DMPE multilayers was evaluated by X-ray diffraction. Synthetic DMPC (lot 140PC-251, MW 677.9) and DMPE (lot 140PE-59, MW 635.9) from Avanti Polar Lipids (AL, USA), and propranolol hydrochloride (MW 295.8, 99 %) from Aldrich (Milwaukee, WI) were used without further purification. About 2 mg of each phospholipid were introduced into Eppendorf tubes which were then filled with 200 ll of (a) distilled water (control), and (b) an aqueous solutions of propanolol a range of concentrations (4–40 lM for DMPC and 1–7 mM for DMPE experiments). The specimens were shaken, incubated for 30 min at 30 and 60 °C with DMPC and DMPE, respectively, and centrifuged for 15 min at 2,500 rpm. Samples

M. Suwalsky et al.: Morphological Effects on Human Erythrocytes

were then transferred into 1.5 mm diameter special glass capillaries (Glas-Technik & Konstruktion, Berlin, Germany) and X-ray diffracted utilizing Ni-filtered CuKa radiation from a Bruker Kristalloflex 760 (Karlsruhe, Germany) X-ray system. Specimen-to-film distances were 8 and 14 cm, standardized by sprinkling calcite powder on the capillary surface. The relative reflection intensities and interplanar spacings were obtained from an MBraun PSD50 M linear position-sensitive detector system (Garching, Germany) and ASA software; no correction factors were applied. Data analyses were performed by means of Origin 3.0 software (Origin Lab Corp., USA). The experiments were performed at 18 ± 1 °C, which is below the main phase transition temperature of both DMPC and DMPE. Higher temperatures would have induced transitions onto fluid phases making the detection of structural changes harder. Each experiment was performed in triplicate. Differential Scanning Calorimetry (DSC) Studies on DMPC and DMPE Liposomes Appropriate amounts of DMPC or DMPE dissolved in chloroform were gently evaporated to dryness under a stream of gaseous nitrogen until a thin film on the wall of the glass test tube was formed. To remove the remnants of moisture, the samples were subsequently exposed to vacuum for 1 h, and then dry lipid films were suspended in distilled water. Propranolol was added in the concentration range of 0.10–0.75 mM. The multilamellar liposomes (MLV) were prepared by vortexing the samples at the temperature above gel-to-liquid crystalline phase transition of the pure lipid (about 30 °C for DMPC and 60 °C for DMPE). DSC experiments were performed using a NANO DSC Series III System with Platinum Capillary Cell (TA Instruments, USA). The calorimeter was equipped with the original data acquisition and analysis software. In order to avoid bubble formation during heating mode, the samples were degassed prior to being loaded by pulling a vacuum of 0.3–0.5 atm on the solution for a period of 10–15 min. Then, the sample cell was filled with about 400 ll of MLV suspension and an equal volume of buffer was used as a reference. The cells were sealed and thermally equilibrated for about 10 min below starting temperature of the run. All measurements were made on samples under three-bar pressure. The data were collected in the range of 0–40 °C (DMPC) and 30–70 °C (DMPE) at the scan rate 1 °C min-1 both for heating and cooling. Scans of buffer as a sample and a reference were also performed to collect the apparatus baseline. For the check of the reproducibility, each sample was prepared and recorded at least three times. Each data set was analyzed for thermodynamic parameters with the software package supplied by TA Instruments.

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Scanning Electron Microscopy (SEM) Studies on Human Erythrocytes One blood drop from a human healthy donor not receiving any pharmacological treatment was obtained by puncturing a previously disinfected finger and received in an Eppendorff tube containing 1,000 ll of phosphate buffer saline (PBS), pH 7.4 and 1 mg/ml of bovine serum albumin (BSA). Red blood cells (RBC) were centrifuged (1,000 rpm 9 10 min), washed three times in PBS and BSA, and then distributed in several Eppendorf tubes that were centrifuged; the supernatants were replaced by 250 ll of propanolol dissolved in PBS and BSA in a range of concentrations, and then incubated at 37 °C for 1 h, period in line with the larger effects induced by compounds on red cell shape (Zimmermann and Soumpasis 1985; Malheiros et al. 2000). Controls were cells resuspended in PBS and BSA without propanolol. Samples were incubated at 37 °C for 1 h and centrifuged at 1,000 rpm for 10 min, the supernatant was replaced by 500 ll of 2.5 % glutaraldehyde and left to rest for 24 h at 4 °C. Samples were washed three times in distilled water and centrifuged (1,000 rpm 9 10 min.); about 10 ll of each sample was placed on siliconized Al glass-covered stubs, air-dried at room temperature, gold coated for 3 min at 13.3 Pa in a sputter device (Edwards S 150, Sussex, England), and examined in a scanning electron microscope (JEOL JSM6380LV, Japan). Defocusing Microscopy (DM) Studies of Human Erythrocytes RBC was obtained from a healthy donor under no pharmacological treatment and were received in PBS 19 pH 7.4 with 1 mg ml-1 of BSA. RBC solution was prepared diluting the washed blood 20 times in a solution of PBS and BSA. Propranolol solution was prepared in the same preparation of PBS and BSA. In order to carry out the analysis, 1.7 ml of RBC diluted solution was placed in an acrylic cuvette and visualized at the optical microscope. After that, a morphologically normal erythrocyte was visualized and selected (Etcheverry et al. 2012) and the concentration of propranolol was increased until 140 lM (propanolol administration). To incorporate the drug, 200 ll of the erythrocyte sample was extracted and 200 ll of a solution of propranolol was added with the required concentration of the drug. The ‘‘recovery process’’ was performed removing 200 ll of the liquid and adding 200 ll of PBS 19 pH 7.4 and BSA. This procedure was performed in order to dilute the sample and decrease the concentration of propranolol reaching almost zero. To make three-dimensional reconstructions, two images were captured in the defocus positions ?1 and -1 lm to obtain three-

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Results

propanolol. As reported elsewhere, water did not significantly affect the bilayer structure of DMPE (Suwalsky 1996). Figure 2b shows that increasing concentrations of propanolol did not cause any significant effect on DMPE reflection intensities, all of which still remained practically unchanged even with 7 mM propanolol.

X-ray Diffraction Studies of DMPC and DMPE Multilayers

Differential Scanning Calorimetry (DSC) Studies on DMPC and DMPE Liposomes

Figure 2a exhibits results obtained by incubating DMPC with water and propanolol. As expected, water altered the structure of DMPC as its bilayer repeat (phospholipid bilayer width plus the layer of water) increased from about ˚ in its dry crystalline form to 65 A ˚ when immersed in 55 A water, and its small-angle reflections, which correspond to DMPC polar terminal groups, were reduced to only the first two orders of the bilayer width. On the other hand, only ˚ showed up in the wide-angle one strong reflection of 4.2 A region which corresponds to the average distance between fully extended acyl chains organized with rotational disorder in hexagonal packing (Suwalsky 1996). These results were indicative of the less ordered state reached by DMPC bilayers. Figure 2a discloses that after being exposed to 4 lM propanolol concentration there was a considerable weakening of the small- and wide-angle reflection intensities (indicated as SA and WA in the figure, respectively) which with 40 lM concentration practically disappeared. From these results, it can be concluded that propanolol produced a significant structural perturbation of DMPC bilayers. Figure 2b shows the results of the X-ray diffraction analysis of DMPE bilayers incubated with water and

The representative high-sensitivity DSC heating thermograms obtained for pure DMPC multibilayer vesicles and binary mixtures of DMPC and propranolol at 0.1–0.75 mM content are shown in Fig. 3a. In the thermal range of 0–30 °C, fully hydrated DMPC bilayers in the absence of any additives underwent a strong and sharp main-transition at 24.03 °C, with an enthalpy change (DH) of

dimensional shape reconstruction and in the defocus position ?4 lm to obtain the amplitude of thermal fluctuations (Etcheverry et al. 2012; Mesquita et al. 2006).

Fig. 2 Microdensitograms from X-ray diffraction patterns of a dimyristoylphosphatidylcholine (DMPC) and b dimyristoylphosphatidylethanolamine in water and incubated with propranolol; (SA) small-angle and (WA) wide-angle reflections

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Fig. 3 a Representative DSC curves obtained for multilamellar DMPC liposomes containing different propranolol concentrations. Scans were obtained at a heating rate of 1 °C min-1; b a plot of phase transition temperature of DMPC multilamellar liposomes determined for cooling and heating scans as a function of propranolol content

M. Suwalsky et al.: Morphological Effects on Human Erythrocytes

17.04 kJ/mol, which corresponds to the conversion of the rippled gel phase (Pb0 ) to the lamellar liquid–crystal La phase. On the other hand, at 15.26 °C with a DH of 2.62 kJ/mol the smaller transition (Lb0 ? Pb0 phase transition), so called pretransition took place. Here, the transition temperatures correspond to the transition peak at the maximal peak heat and the transition enthalpies correspond to the integrated area under the peak divided by the lipid concentration. The results for the thermodynamic data of the pure DMPC are in agreement with previous reports (for reviews see Marsh 1991; Koynova and Caffrey 1998). As it can be clearly seen, the presence of propranolol induced a rather mild perturbation of the thermotropic behavior of DMPC vesicles. While the main phase transition temperature was shifted to lower values by not more than 1.13 °C for the highest examined concentration, the pretransition underwent a concentration-dependent shift (DTmax = 5.45) (Fig. 3b) Moreover, the latter was gradually diminished as for 0.5 mM propranolol, it was hardly observed in heating curves and completely lost under cooling. Figure 4a show representative high-sensitive DSC heating thermograms obtained for DMPE and DMPE liposomes containing propranolol. In the thermal range of 30–70 °C, the pure DMPE bilayers exhibited a strong and sharp main-transition at 50.43 °C, with an enthalpy change (DH) of 24.73 kJ/mol, arising from the conversion of gel-to-liquid–crystal phase. The transition was reversible and the shape of the peak is roughly symmetrical. Thermodynamical parameters found for DMPE are concurrent with the literature data (Lewis and McElhaney 1993). When propranolol was added to DMPE vesicles, only slight effect on the thermotropic phase behavior was observed upon both heating (Fig. 4b) and cooling (not shown). Although transition temperature decrease did not exceed 1 °C (Fig. 4b), there is an evidence of asymmetry in the heating endotherm which may reflect the onset of phase separation. The effectiveness in perturbations of DMPE thermotropic phase transition exerted by propranolol was further analysed in terms of thermodynamic parameters. Tables 1 and 2 present values of temperature, enthalpy and entropy for DMPC and DMPE systems, respectively, determined on the basis of heating and cooling scans. Scanning Electron Microscopy (SEM) Studies of Human Erythrocytes The effects of the interaction of propranolol with human erythrocytes were evaluated in vitro by SEM. The resulting micrographs (Fig. 5) show that the drug induced notorious changes in the morphology of the RBC. The normal resting shape of the human red blood cell is a flat biconcave disk (discocyte) *8 lm diameter which can be observed in

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Fig. 4 a Representative DSC curves obtained for multilamellar DMPE liposomes containing different propranolol concentrations. Scans were obtained at a heating rate of 1 °C min-1; b a plot of phase transition temperature of DMPE multilamellar liposomes determined for heating and cooling scans as a function of propranolol content

Fig. 5a, corresponding to the erythrocytes incubated with PBS 1X (pH 7.4) (control). On the other hand, morphological analysis of the results revealed that propranolol changed the normal shape of the RBC in a dose-dependent manner. Figure 5b (50 lM) clearly shows that discocytes underwent a partial transformation into stomatocytes (erythrocytes with cup-like shapes), 100 lM propranolol (Fig. 5c) increased the formation of stomatocytes; some echinocytes (erythrocytes with crenated shapes) and knizocytes (triconcave redcell shape) were also observed, and with 200 lM (Fig. 5d) the large majority of cells were stomatocytes. Defocusing Microscopy (DM) Studies of Human Erythrocytes SEM studies showed an increase in the stomatocyte population when the concentration of propranolol was

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Table 1 Thermodynamic parameters of the pretransition and main phase transition of pure, fully hydrated DMPC multilamellar liposomes and DMPC/propranolol mixtures determined from heating and cooling scans collected at a heating (cooling) rate of 1 °C min-1 Compound

DMPC ? propranolol

Conc (mM)

Pretransition heating

Main-transition heating

Pretransition cooling

Main-transition cooling

DH (kJ/ mol)

DH (kJ/ mol)

DH (kJ/ mol K)

DH (kJ/ mol)

DS (J/ mol K)

Tm (°C)

DS (J/ mol K)

Tm (°C)

DS (J/ mol K)

Tm (°C)

DS (J/ mol K)

Tm (°C)

0.00

2.62

0.92

15.26

17.04

5.73

24.03

1.02

0.36

9.45

18.56

6.26

23.27

0.10

3.58

1.25

13.48

21.15

7.12

23.86

0.62

0.22

8.80

22.63

7.64

23.00

0.25

2.80

0.98

12.45

20.77

7.00

23.59

0.11

0.04

7.91

23.41

7.91

22.72

0.50

2.55

0.90

11.14

20.62

6.95

23.25

0.10

0.03

8.77

24.19

8.19

22.34

0.75

1.74

0.61

9.81

20.62

6.96

22.90







25.14

8.52

22.03

Concentration of DMPC was 1 mM, concentration of propranolol is given in the table The accuracy for the main phase transition temperature and enthalpy was ±0.01 °C and ±0.8 kJ/mol, respectively

Table 2 Thermodynamic parameters of the phase transition of pure, fully hydrated DMPE multilamellar liposomes and DMPE/propranolol mixtures determined from heating and cooling scans collected at a heating (cooling) rate of 1 °C min-1 Compound

DMPE ? propranolol

Conc (mM)

Heating

Cooling

DH (kJ/mol)

DS (J/mol K)

Tm (°C)

DH (kJ/mol)

DS (J/mol K)

0.00

24.73

7.64

50.43

28.35

8.82

48.37

0.10

19.94

6.16

50.4

22.20

6.90

48.81

0.25

21.58

6.67

50.16

24.62

7.66

48.26

0.50

24.86

7.70

49.89

28.40

8.84

48.06

0.75

28.18

8.73

49.61

33.12

10.33

47.41

Tm (°C)

Concentration of DMPE was 1 mM, concentration of propranolol is given in the table The accuracy for the main phase transition temperature and enthalpy was ±0.01 °C and ±0.8 kJ/mol, respectively

increased. In order to observe the change of RBC morphology in real time, optical microscopy was used. RBCs were visualized and then one single RBC was selected to study the whole processes, which involved two steps. First an increase of propranolol concentration until 140 lM, called ‘‘propranolol administration’’, and a second step called the ‘‘recovery process’’ when the concentration of propranolol was decreased until reaching almost zero. Figure 6 shows the morphological changes experienced by the red cells by increasing concentrations of propranolol; a complete discocyte-stomatocyte transformation was observed with 125 lM propranolol (Fig. 6c). It was observed that when the propranolol concentration decreased the RBC recovered its normal discocyte shape (Fig. 6e). It was also observed that lower concentrations of the drug, *50 lM (Fig. 6b, d) induced a distortion in the RBC shape characterized by a hollow in the central part of the cell (blue color indicates lower height area). To investigate what takes place in the RBC membrane during drug administration and in the recovery process, the amplitude of thermal fluctuations was measured at each propranolol concentration during the ‘‘propranolol administration’’ and in the ‘‘recovery process’’. Figure 7 (red dots) shows that the increase of propranolol concentration caused an

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increase in the amplitude of thermal fluctuations in the concentration range 20–140 lM. Fluidity changes were expressed in an increase in average fluctuations of the membrane, and became maximal with 125 lM propranolol. This result suggests that the compound was interacting with the red cell membrane intercalating into the membrane lipid bilayer. In the process of cell recovery (Fig. 7; blue dots), the amplitudes of the fluctuations began to decrease to values slightly higher than those of the initial conditions.

Discussion Interactions with biological membranes are of utmost importance for drug pharmacokinetics (molecular pathway to specific receptor site) and pharmacodynamics (specific interactions with high affinity receptor sites) actions. It is not only dependent on their partitioning into lipid bilayer and drug permeability but also their therapeutic target are often anchored in membranes. Moreover, it is believed that numerous cardiovascular and other diseases are related to modifications of membrane lipid composition and structure. Our studies here represent an attempt to analyse the

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Fig. 5 Effects of propranolol on the morphology of human erythrocytes. SEM images of a untreated erythrocytes (control), and incubated with b 50 lM, c 100 lM, and d 200 lM propranolol

molecular basis of membrane structural perturbations induced by propranolol using the human erythrocyte membrane as a model membrane system. In order to understand the location and interaction of propanolol with the erythrocyte membrane lipid bilayer, molecular models constituted by DMPC and DMPE bilayers were used. They are classes of lipids preferentially located in the outer and inner monolayers of the human erythrocyte membrane, respectively (Boon and Smith 2000; Devaux and Zachowsky 1994). Results by X-ray diffraction on the interaction of propranolol with DMPC showed that the drug produced a significant structural perturbation of the lipid bilayer, whereas no effects were observed in DMPE, even at very high propranolol concentrations (Fig. 2). DMPC and DMPE differ only in their terminal amino groups, these being ?N(CH3)3 in DMPC and ?NH3 in DMPE. DMPE molecules pack tighter than those of DMPC due to their smaller polar groups and higher effective charge, resulting in a very stable bilayer system held by electrostatic interactions and hydrogen bonds. However, the hydration of

DMPC results in water filling the highly polar interbilayer spaces with the resulting increase of their width (Suwalsky 1996). This phenomenon might allow the incorporation of propranolol into DMPC bilayers and their consequent interaction. In fact, it was observed that propranolol in a concentration as low as 4 lM induced a considerable weakening of the small- and wide-angle reflection intensities (indicated as SA and WA in the figure, respectively) which with 40 lM all reflections practically disappeared. This result implies that both the polar and hydrophobic regions of DMPC were perturbed by the insertion of the drug in the lipid bilayer. Given the amphipathic nature of propranolol, it is very likely that its naphthalene moiety locates into DMPC hydrocarbon core and its positively charged amine moiety interacts with the polar region negatively charged phosphate groups (Fig. 8); the latter interactions would lead to a disruption of the electrostatic attractions that maintain DMPC molecules in their bilayer arrangement. Herbette et al. (1983, 1985, 1986) arrived to similar conclusions, although their structural studies were mainly performed by neutron diffraction at or above the

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Fig. 8 Schematic drawing of the interaction of DMPC with propranolol Fig. 6 Defocused images and three-dimensional reconstruction of: a RBC under normal conditions (control); b, c administration of propranolol; d, e recovery process. Propranolol concentrations a 0 lM, b 50 lM, c 125 lM, d 53 lM, e 0 lM

Fig. 7 Average fluctuations amplitude eurms in the processes of propranolol administration (red points) and recovery (blue points)

phase transition temperature of DMPC and using higher propranolol concentrations. On the other hand, an X-ray scattering study on the interaction of propranolol with

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dipalmitoylphosphatidylcholine (DPPC) in the gel (Pb0 ), ripple gel (Pb0 ), and fluid (L) phases concluded that propranolol interacted with DPPC polar headgroup region (Pereira-Leite et al. 2013). Another study, performed by Fo¨rst et al. (2014) on palmitoyl-oleoyl phosphatidylcholine (POPC) by fluorescence spectroscopy and molecular dynamics simulations, concluded that propranolol preferentially resides in the head group region of POPC. DSC is a method of choice in the study of thermotropic behavior of lipids and monitor the extent of perturbation induced by the presence of bioactive compound in the system. Among the key parameters that affect the extent of absorption, distribution, metabolism, and excretion are drug lipophilicity, charge and size. According to the Mannhold classification based on octanol/water system, propranolol is considered as highly lipophilic (logP 3.00 ± 0.07) (Mannhold 2005). Consequently, its molecules should be located within the hydrophobic part of the lipid bilayer with a considerable potential to disturb the order of the acyl chain regions. Since such penetration reduces the interfacial tension and affects the lateral

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interactions between apolar chains, as a result the transition cooperativity should be decreased and the calorimetric peaks broaden. Our DSC data, in agreement with previously published calorimetric (Albertini et al. 1990; Herbette et al. 1985, 1986) and fluorescence anisotropy studies (Pereira-Leite et al. 2006) confirmed that propranolol shows ability to lower DMPC Tm and broaden the melting endotherm (Fig. 3). However, our data reveal that much more pronounced drug effect was observed on Lb0 ? Pb0 phase transition that was diminished in dose-dependent way and almost lost in the presence of the highest propranolol concentration. The abolishing of DMPC pretransition is probably associated with the fact that propranolol molecules lie between the lipid chains thus filling the potential void space in the hydrocarbon region created by the bulky DMPC head groups that normally induces the acylchain tilt. As a result, reduction of the overall head group/ acyl chain mismatch is observed and declining of acyl chain tilt in the gel phase (Lb0 ). This in turn could imply that propranolol perturbs the chain packing in a manner that reduces the structural differences between lamellar gel phase and rippled phase leading to lower enthalpy and entropy values of the pretransition experimentally observed (Table 1). This concept is in agreement with data published by Ru¨ppel et al. (1982) that glycophorin at the concentration as low as 0.0004 % is sufficient to stabilize the ripple form at lower temperatures, and clearly suggests that direct hydrophobic lipid-drug interactions do not explain the disappearance of the pretransition. Hence, the effect of propranolol could be considered as being mainly mechanical, i.e., governed predominantly by the sterical restriction and hindrance of the ordered lipid matrix. Consistently, neutron diffraction data (Herbette et al. 1985) obtained for propranolol deutered in the naphthalene moiety proved that a rigid and planar naphthalene group is located within the hydrocarbon core region of DMPC bilayer and can act as a spacer between phospholipids. Moreover, such location of the naphthalene moiety would position the charged amine group of propranolol within the water layer that hydrates the phospholipid headgroups and promote interactions with deprotonated phosphate group of DMPC. This concept is supported by the fact that propranolol pKa of 9.49 (Ishihama et al. 2002) gives rise to the prediction that more than 99 % of its molecules are in the cationic form at pH 7.4. Thus, it is justified to consider the liposome/buffer partition coefficient (Pereira-Leite et al. 2013) that takes into account not only hydrophobic but also electrostatic interactions and reflects behavior of both ionic and neutral form of drug molecules. Such approach offers a good explanation for the strongest influence of propranolol on Lb0 ? Pb0 phase transition found in our DSC studies and it is in line with the findings of fluorescence measurements by Pereira-Leite et al. (2006). They showed stronger

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lowering effect on Tm when TMA-DPH located closer to the membrane surface was used as a fluorophore to measure fluorescence anisotropy as well as the gradual increase of the membrane surface potential dependent on b-blocker concentration (Pereira-Leite et al. 2013). Based on our calorimetric studies performed using model membranes formed from DMPE, we can conclude that fluidizing effect of propranolol at examined concentration range is not solely restricted to phosphatidylcholine bilayer. In DMPE/ propranolol system, a decrease in phase transition cooperativity along with the dose-dependent shift of Tm was observed (Fig. 4). Moreover, inspection of the shape of heating thermograms for the higher propranolol content suggest that ideal solution are not formed in either the liquid crystal or the gel phase. However, in contrast to arsenic compounds (Jemiola-Rzeminska et al. 2007) that displayed greater affinity toward the surface of ethanolamine- than choline-containing liposomes, propranolol molecules are unable to take advantage of increased polarity of DMPE surface, consequent to the unshielded positive charge on the nitrogen atom. As a result, the extent of perturbations induced by the presence of the drug was comparable for both lipid species. Both SEM and DM observations showed that propranolol induced morphological alterations to the red cells from the normal discoid shape to cup-shaped stomatocytes. According to the bilayer couple hypothesis (Sheetz and Singer 1974; Lim and Wortis 2002) shape changes induced in erythrocytes by foreign molecules are due to differential expansion of the two monolayers of the red cell membrane. Thus, stomatocytes are formed when the compound inserts into the inner monolayer, whereas spiculated-shaped echinocytes are produced when it locates into the outer moiety. The finding that propranolol induced the formation of stomatocytes indicates that it was inserted in the inner leaflet of the erythrocyte membrane. This conclusion differs from that obtained from X-ray experiments carried out in DMPC and DMPE multilayers. In fact, our results showed that propranolol only interacted with DMPC, kind of lipid which preferentially locates in the outer monolayer of the human erythrocyte membrane, whereas DMPE does it in the inner one. On the other hand, DSC results on MLV showed that propranolol induced comparable perturbing effects on DMPE and DMPC. This discrepancy might be explained by the fact that liposomes, which resemble more to cell membranes, are more fluid than the multilayers used in the X-ray experiments thus allowing better interactions with the drug. However, interactions of propranolol with other components located in the inner moiety of the human erythrocyte membrane cannot be disregarded. Amazingly, studies on the interaction of propranolol with human erythrocytes are scanty. In two of them, Surewicz et al. (1981, 1982) suggested that although both membrane lipids and

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proteins are involved in propranolol-membrane interactions, the crucial role is played by proteins located in the polar environment of the membrane surface. On the other hand, Godin et al. (1976) postulated that proteins and particularly phospholipid components are involved in propranolol-induced erythrocyte membrane perturbation; they concluded that the drug-induced perturbation in protein amino or sulfhydryl groups correlated well with propranolol-induced phospholipid alterations, suggesting interdependence of proteins and lipids structural interactions. On the other hand, it has been reported that propranolol also interacted with acidic phospholipids (Surewiz and Leiko 1981), lipids mostly located in the inner monolayer of the erythrocyte membrane (Virtanen et al. 1998). Therefore, it can be concluded that in human erythrocyte, the drug preferentially interacts with both proteins and lipids located in its membrane inner moiety. The membrane-fluidizing properties of b-blockers are considered to be related with beneficial effects of these drugs including cardioprotective action (Pereira-Leite et al. 2013). This involves not only the blockage of b-adrenoceptors, but also drug antioxidant activity (Kramer et al. 2012; Gomes et al. 2006). This property could be facilitated due to the fact that in more disordered membranes antioxidants can interact with free radicals more efficiently (Pereira-Leite et al. 2013). Propranolol concentrations used in this work were higher than the about 1 lM reported therapeutic concentration in plasma (Wong et al. 1979), although some of our results were observed below the approximately 15 lM toxic concentration (Macvey and Corke 1991; Joviv-Stosic et al. 2011). The structure of cell membranes is much more complex than the simple systems used in this work and, therefore it is difficult to predict the situation in biological membranes. On the other hand, biophysical studies normally require high concentrations in order to detect effects induced by drugs. As a matter of fact, whereas the propranolol concentrations used in this work were in the 4–200 lM range, similar studies were performed using mM concentrations (Fo¨rst et al. 2014; Surewicz et al. 1981, 1982; Godin et al. 1976; Surewicz and Leyko 1981). Taking into account these considerations, our results might be of pharmacological interest in order to understand the structural effects of propranolol in cell membranes. Acknowledgments To FONDECYT (Project 1130043). This work was partially supported from PIA-CONICYT PFB0824 and from FONDECYT 3140167. Calorimetric measurements were carried out using the instrument purchased thanks to financial support of European Regional Development Fund (contract No. POIG.02.01.00-12167/08, Project Malopolska Centre of Biotechnology).

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