Influence of lecithin–lipid composition on physico-chemical properties of nanoliposomes loaded with a hydrophobic molecule

Colloids and Surfaces B: Biointerfaces 115 (2014) 197–204

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Influence of lecithin–lipid composition on physico-chemical properties of nanoliposomes loaded with a hydrophobic molecule Lynda Bouarab a , Behnoush Maherani a , Azadeh Kheirolomoom b , Mahmoud Hasan a , Bahar Aliakbarian c , Michel Linder a , Elmira Arab-Tehrany a,∗ a

Université de Lorraine, Laboratoire d’Ingénierie des Biomolécules, 2, Avenue de la Forêt de Haye, F-54504 Vandoeuvre-Lès-Nancy Cedex, France Department of Biomedical Engineering, University of California, 451 East Health Sciences Drive, Davis, CA 95616, USA c Department of Civil, Chemical and Environmental Engineering (DICCA), University of Genoa, Via Opera Pia 15, 16145 Genova, Italy b

a r t i c l e

i n f o

Article history: Received 21 July 2013 Received in revised form 14 November 2013 Accepted 15 November 2013 Available online 24 November 2013 Keywords: Liposome Cinnamic acid Encapsulation Antioxidant Physico-chemical characterization

a b s t r a c t In this work, we studied the effect of nanoliposome composition based on phospholipids of docosahexaenoic acid (PL-DHA), salmon and soya lecithin, on physico-chemical characterization of vector. Cinnamic acid was encapsulated as a hydrophobic molecule in nanoliposomes made of three different lipid sources. The aim was to evaluate the influence of membrane lipid structure and composition on entrapment efficiency and membrane permeability of cinnamic acid. These properties are important for active molecule delivery. In addition, size, electrophoretic mobility, phase transition temperature, elasticity and membrane fluidity were measured before and after encapsulation. The results showed a correlation between the size of the nanoliposome and the entrapment. The entrapment efficiency of cinnamic acid was found to be the highest in liposomes prepared from salmon lecithin. The nanoliposomes composed of salmon lecithin presented higher capabilities as a carrier for cinnamic acid encapsulation. These vesicles also showed a high stability which in turn increases the membrane rigidity of nanoliposome as evaluated by their elastic properties, membrane fluidity and phase transition temperature. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Among different techniques of encapsulation, nanoliposomes have become very versatile tools in biology, biochemistry and medicine because of their enormous diversity of structure and composition. The main constituents of liposomes are phospholipids, which are amphiphilic molecules containing water soluble, hydrophilic head section and a lipid-soluble, hydrophobic tail section. This property of phospholipids gives liposomes unique properties, such as self-sealing, in aqueous media and make them an ideal carrier system with applications in different fields including food, cosmetics, pharmaceutics, and tissue engineering [1]. Liposomes were first made synthetically in England in 1961 by Alec D. Bangham, who found that phospholipids combined with water form a sphere because of their unique properties. Liposomes are spherical, closed structures, composed of curved lipid bilayers, which enclose part of the surrounding solvent into their interior [2]. Due to their biocompatibility and capability of incorporating

∗ Corresponding author. E-mail address: [email protected] (E. Arab-Tehrany). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.11.034

both hydrophilic and lipophilic drugs, liposomes have been investigated as parenteral drug carrier systems and more recently as transdermal drug delivery systems [2]. The drug delivery properties of liposomes are largely affected by the physico-chemical characteristics of the lipid bilayer, which are determined by factors such as the lipid composition, the particle size and the drug loading [3]. The preparation method of nanoliposomes has some control over the size range (as narrow as possible) and, polydispersity index (as low as possible). By considering these parameters, the extrusion technique was chosen to prepare liposomes. Extrusion is a common method for nanoliposomes production in a laboratory scale and there are numerous reports on liposome preparation with this technique to obtain small particle size [4,5]. Currently, in vitro and animal studies indicate that n-3 PUFAs suppress carcinogenesis. Several studies present a new insight on effectiveness of marine phospholipids for suppression of colon carcinogenesis to investigate growth inhibition and apoptosis inducing effects of n-3 PUFA in the form of marine phosphatidylcholine (PC) on chemically induced (1,2-dimethylhydrazine) colon cancer in rats [6]. Marine lecithin from salmon (Salmosalar) contains a high percentage of polyunsaturated fatty acids (PUFAs), especially

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eicosapentaenoic acid (EPA, C20:5 n-3) and docosahexaenoic acid (DHA, C22:6 n-3), those are critical to several physiological processes [7]. Soya lecithins also consist mainly of contain esterified monoand poly-unsaturated fatty acids such as oleic (C18:1 n-9), linoleic (C18:2 n-6), and linolenic acids (C18:n-3). The linoleic and linolenic acids are categorized as essential fatty acids and are important to human health [8]. The cinnamic acid and its derivatives are well known for their biological and pharmacological properties such as antimicrobial, antioxidant, anti-inflammatory, and antitumoral activity [9]. Some cinnamic acid derivatives represent secondary metabolites in plants and they have been the subjects of a great number of chemical, biological, agricultural and medical studies, the most important category being the hydroxy-cinnamic acids. In fact these biophenols are very effective peroxyl radical scavengers and protect low-density lipoproteins from oxidative modification [10]. Cinnamic (trans-3-phenyl-2-propenoic) acid is widely used in food, cosmetics, and pharmaceuticals fields. Recent studies, devoted to the use of trans-cinnamic acid in cosmetics, revealed its importance as a nonphotosynthetic pigment in photoprotection [11]. Cinnamic acid is an effective anticancer constituent of traditional Chinese herbal medicines. The molecular mechanisms of anticancer effects of this constituent and its target have long been unknown. As a product of a potential tumor suppressor gene, cinnamic acid participates in the regulation of cell growth, proliferation, and cell differentiation [12]. Due to their interesting properties we encapsulated cinnamic acid into liposomal systems as vectors containing the phospholipids to introduce a double functionalization with therapeutic use. They are suitable for low molecular weight drugs, imaging agents, peptides, proteins, and nucleic acids, therefore suitable vectors for the cinnamic acid which has a molecular weight of 148.16 g/mol [5]. Particle size, phase transition temperature and fluidity of membranes are important in the manufacture and application of liposomes. Controlling the above factors is important in using liposomes as drug carrier systems. In addition, entrapment efficiency of liposomes is an important factor in their practical use [5]. In this work, we compared the effect of three different compositions on physico-chemical properties of cinnamic acid encapsulated liposome such as size, electrophoretic mobility, phase transition temperature and fluidity. We used one natural lecithin extracted by enzymatic process and two commercial lecithins. The encapsulation efficiency of cinnamic acid was determined by using HPLC after physical separation of entrapped and non-entrapped cinnamic acid using ultracentrifugation process. Furthermore to characterize the resulting liposomes, a variety of techniques were used to investigate the rheological properties of systems. This study allowed us to explain the correlation between the lipid composition and the physico-chemical properties of nanoliposome and liposomal carrier. In addition we studied the influence of lipid composition on acid cinnamic encapsulation.

2. Materials and methods Trans-cinnamic acid (3-phenylpropenoic acid) (CA) and sodium thiosulfate pentahydrate were purchased from Merck (Germany), Trifluoroacetic acid about 100% from VWR Prolabo (Belgium), methanol for HPLC from group CARLO ERBA reagents (France), BF3 (boron trifluoride)/methanol (purity = 99%) and hexane (purity = 97%) used for gas chromatography (GC) were purchased from Sigma–Aldrich (France) and Fisher (France).

TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5hexatrienep toluenesulfonate) from Invitrogen (City, State). PHOSPHOLIPON 85G (phospholipon with 89.5% soya phosphatidylcholine) from Lipoid PHOSPHOLIPID GmbH (Germany), PL-DHA (phospholipids of docosahexaenoic acid (DHA) from POLARIS (France), and salmon lecithin was extracted by use of a low temperature enzymatic process in the absence of organic solvent [13]. 2.1. Fatty acid composition Fatty acid methyl esters (FAMEs) were prepared as described by Ackman [14]. Separation of FAMEs was carried out on a Shimadzu GC-2010 Plus gas chromatograph, equipped with a flame-ionization detector. A fused silica capillary column was used (60 m × 0.25 mm × 0.25 ␮m film thicknesses SP TM -2380), purchased from Supelco (USA). Injector and detector temperatures were set at 250 ◦ C. A temperature program of column initially set at 120 ◦ C for 2 min, then rising to 220 ◦ C at a rate of 3 ◦ C/min and held at 220 ◦ C for 25 min was used. Standard mixtures (PUFA1 from marine source and PUFA2 from vegetable source; Supelco, Sigma–Aldrich, Bellefonte, PA, USA) were used to identify fatty acids. The results were presented as triplicate analyses. 2.2. Lipid classes The lipid classes of the different fractions were determined by Iatroscan MK-5 TLC-FID (Iatron Laboratories Inc., Tokyo, Japan). Each sample was spotted on ten Chromarod S-III silica coated quartz rods held in a frame. The rods were developed over 20 min in hexane/diethyl ether/formic acid (80:20:0.2, v:v:v), then oven dried for 1 min at 100 ◦ C and finally scanned in the Iatroscan analyzer. The Iatroscan was operated under the following conditions: flow rate of hydrogen, 160 mL min−1 ; flow rate of air, 2 L/min. A second migration using a polar eluent of chloroform, methanol, and ammoniac (65:35:5, v:v:v) made it possible to identify polar lipids [15]. The FID results were expressed as the mean value of ten separate samples. All standards were purchased from Sigma (Sigma–Aldrich Chemie GmbH, Germany). The recording and integration of the peaks were provided by the ChromStar internal software. 2.3. Cinnamic acid solubility To determine the maximum solubility of cinnamic acid in each lipid phase, excess powder of cinnamic acid was added to known volumes of lecithins aqueous solutions ([cinnamic acid]final = 5 mg/mL). The mixtures were then incubated at 37 ◦ C with gentle stirring for more than 48 h. Finally, the solutions were centrifuged at 200,000 × g for 10 min and aliquots of the supernatant saturated solution were diluted and analyzed by HPLC [16]. The supernatant of each sample was pipetted and then 20 ␮l of supernatant was diluted 1000-fold with methanol. Cinnamic acid in methanol with concentration of 20 ␮g/mL was used as standard suspension. HPLC measurements were performed by a HPLC system (Shimadzu, Japan) equipped with a quaternary pump (LC-20AD), an auto-injector (SIL-20AC HT), a UV–vis photodiode array detector (UV–vis PDA, SPD-M20A), a column oven (Zorbax – 15 cm-C18) and Labsolution data software. All suspension was analyzed using isocratic mode of methanol (v/v, 75%) and (H2 O–0.1% trifluoroacetic acid) TFA at pH 3 (v/v, 25%) at a flow rate of 0.4 mL min−1 . The suspensions (20 ␮l) were injected onto a AlltimaTM [HP C18, 3 ␮m (150 × 3 mm i.d.) column (GRACE, Deerfield, IL, USA)] protected by a 2.1 mm × 7.5 mm Alltech guard

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C18 pre-column (GRACE, Deerfield, IL, USA) at 25 ◦ C. Cinnamic acid was detected at 271 nm (Ugazio et al., 2008) and eluted after 10 min. The experiments were performed in triplicates.

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on Netzsch 204 F1 (Netzsch-Geratebau GmbH, Germany) at a scanning rate of 2 ◦ C/min. In addition, Kinexus Pro rheometer (Malvern instruments) was used to confirm the phase transition temperature in the same range of temperature as DSC.

2.4. Preparation of nanoliposomes Large unilamellar vesicles (LUVs) were prepared as described elsewhere [5,15]. Briefly, 2 g of each lecithin was added to 98 mg of distilled water to obtain a solution with 2% (w/w) lecithin. Cinnamic acid was dissolved in lecithin/water mixture at pH 7 to obtain a final cinnamic acid concentration of 0.2 mg/mL before use. The suspension was mixed for 4 h under agitation, and under inert atmosphere (nitrogen). Then, the mixture was subjected to sonication (25 ◦ C) at 40 kHz, and 30% of full power for 30 s (1 s on and 1 s off) to obtain the homogeneous solution (Sonicator Vibra cell 75115, 500 watt, Bioblock Scientific Co.). Finally, the suspension was extruded through a polycarbonate filter (100 nm pore size filter, 11 times) above the phase transition temperature of the vesicles by using an Avanti Mini extruder (Avanti Polar Lipids, Alabaster, USA).

2.8. Rheological study The rheological study was carried out on a Kinexus Pro rheometer (Malvern instruments) equipped with CP4/40: PL 65 geometry (cone and plate). The sample was placed on a flat plate and below a cone. CP4/40: PL65 was 40 mm diameter with 4◦ angle cone over a 65 mm bottom plate diameter. The viscosity variation of liposome suspension against shear rate ramp at 25 ◦ C (between 0.001 and 2 s−1 ) as well as viscosity and membrane elasticity against temperature variation (−10 to 60 ◦ C) at 4 Hz frequency and 0.05% of shear strain were plotted. The plots were made by “Visco-soft” software. Rheological properties measurements were performed directly on liposome suspensions before and after cinnamic acid encapsulation (mean, n = 3).

2.5. Particle size distribution and electrophoretic mobility 2.9. Membrane fluidity Size distribution of liposomes was analyzed by dynamic light scattering technique using a Malvern Zetasizer Nano ZS (Malvern instruments, UK). The software used was DTS Nano, version 6.12 supplied by the manufacturer (Malvern Instruments Ltd., UK). The apparatus was equipped with a 4 mW He/Ne laser emitting 633 nm, measurement cell, photomultiplier and correlator. The samples were diluted in ultra-filtrated water (1:40) and were placed in vertical cylindrical cells (10 mm diameters). The scattering intensity was measured at a scattering angle of 173◦ relative to the source using an avalanche of photodiode detector, and all measurements were carried out at 25 ◦ C [15]. The refractive indices (RI) were fixed at 1.46, 1.49, 1.34 for salmon lecithin, PL-DHA and soya lecithin respectively at 25 ◦ C using refractometer apparatus. The measurements were carried out in five repetitions. Results are presented as an average diameter of the liposome suspension (z-average) with the polydispersity index (PDI). The electrophoretic mobility of nanoliposomes was utilized to evaluate the surface net charge around the lipid droplets. Electrophoretic mobility measurements (␮E) were performed by means of ZetasizerNano ZS. The liposome suspensions were diluted (1:40) to avoid multiple scattering effects and then directly placed into the module; all measurements were carried out at 25 ◦ C and the results are presented as an average electrophoretic mobility of the liposome suspension (z-average). 2.6. Transmission electron microscopy (TEM) Transmission electron microscopy was employed to monitor the microstructure of nanoliposomes with a negative staining method. The nanoliposome samples were diluted 10-folds with distilled water to reduce the concentration of the vesicles. Equal volumes of the diluted sample and a 2% ammonium molybdate solution were combined and left for 3 min at room temperature. A drop of this solution was placed on a Formvar-carbon coated copper grid (200 mesh, 3 mm diameter HF 36) for 5 min. The excess of liquid was drawn off by using filter papers. After drying the grid at room temperature for 5 min, micrographs were made using a Philips CM20 Transmission Electron Microscope operating at 200 kV and recorded using an Olympus TEM CCD camera [17]. 2.7. Phase transition temperature determination Liposome suspension was analyzed with differential scanning calorimetry. Calorimetric scans from −10 to 60 ◦ C were performed

Fluorescence polarization (P) was used to determine membrane fluidity of liposomes by measuring the fluorescent intensity of TMA-DPH, a compound which contains a cationic trimethylammonium substitute that acts as a surface anchor to improve the localization of the fluorescent probe of membrane interiors, DPH. This measurement was carried out according to the conventional method [5]. The solution of TMA-DPH (in ethanol) was added to the liposome suspension to maintain the lipid/probe molar ratio of 250 ([TMA-DPH]final = 8 ␮M). The mixture was then incubated for at least 1 h at room temperature with gentle stirring and then was distributed into a 96-well black microplate at 200 ␮L per well. The fluorescence probe was vertically and horizontally oriented in the lipid bilayer. The fluorescence intensity of samples was measured with Tecan INFINITE 200® PRO (Austria) equipped with fluorescence polarizers. Samples were excited at 360 nm, and emission was recorded at 430 nm under constant stirring at 25 ◦ C. The software used was Magellan 7 from Tecan Group Ltd. (Switzerland). The P value of TMA-DPH was calculated from the following equation: P=

III − GI⊥ III + 2GI⊥

(1)

where III is the intensity of fluorescence parallel to excitation plane, I⊥ is the intensity of fluorescence perpendicular to excitation plane, and G is the factor that accounts for transmission efficiency. The reciprocal value of polarization (1/P) was defined as membrane fluidity. 2.10. Entrapment efficiency determination Cinnamic acid was chosen as a hydrophobic molecule. Percentage of initial cinnamic acid (CA) incorporated into liposomes was measured by using ultracentrifugation of 50,000 × g during 4 h at 25 ◦ C. The non-encapsulated cinnamic acid in the supernatant was detected by HPLC. Total active molecule in the suspensions was also determined [18]. The elution parameters were the same as those used previously in solubility test. Entrapment efficiency was calculated according to the following equation as previously reported [5]. EE (%) =

Ctotal − Cout × 100 Ctotal

(2)

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Ctotal : 20 ␮l of liposome suspension (with concentration of 20 mg/mL) were diluted 100-fold with methanol. Cout : nonencapsulated cinnamic acid diluted 100-fold with methanol.

for PL-DHA, salmon and soya lecithin, respectively. The obtained results also indicated that the presence of polar lipids is predominant in soya lecithin with 93.3 ± 0.1% in comparison to 61.1 ± 0.2% and 45 ± 0.1% in salmon lecithin and PL-DHA, respectively.

2.11. Statistics 3.3. Solubility of cinnamic acid All data are presented as mean ± standard error. Statistical significance was determined by Fischer’s tests with p value lower than 0.05 using stat-graphic software 5.0.

The main fatty acid composition was shown in Table 1. The high proportions of fatty acids found in salmon lecithin contained C22:6 n-3, categorized as polyunsaturated fatty acids (PUFAs), C18:1 n-9, known as monounsaturated fatty acids (MUFAs) and C16:0 as saturated fatty acids (SATs) for salmon lecithin. The largest amount of fatty acid belonged to a polyunsaturated fatty acid, C18:2 n6, with 64.92 ± 0.04% for soya lecithin. The results showed that the presence of C22:6 n-3 and C18:2 n-6 in PL-DHA is more predominant compared to other types of unsaturated fatty acids with 48.07 ± 1.35% and 15.16 ± 1.01%, respectively. Unsaturated fatty acids have a strong effect on the membrane properties. They are known to increase fluidity in a concentrationdependent mode by creating a kinked structure in LC-PUFA [19,20].

Cinnamic acid has a relatively low water-solubility (<0.21 g/L) and a medium oil–water partition coefficient (log P = 1.91) [9]. The amount of a lipophilic material that can be dissolved in an oil phase depends on the molecular characteristics of the oil (e.g., molecular weight, polarity, and interactions) [21,22]. For this reason, we initially measured the maximum amount of cinnamic acid that could be solubilized in different lipid phases at 37 ◦ C. Shorter alkyl chains have more polar groups (oxygen) per unit mass than longer alkyl chains, and consequently more dipole–dipole interactions were observed between polar groups of a lipidic carrier and cinnamic acid molecules [21]. Soya lecithin represented higher solubility of cinnamic acid with 2.75 ± 0.08. This solubility decreased in PL-DHA and salmon lecithin with 2.46 ± 0.15 and 2.11 ± 0.11, respectively. There is no significant difference (p < 0.05) between three solubilities. By considering these experimental results, we used 0.2 mg/mL cinnamic acid concentration in the lipid phase concentrated to 20 mg/mL to prepare the conventional nanoliposomes (lipid/cinnamic acid ratio: 100, w/w), not to saturate the lipid bilayer.

3.2. Lipid classes

3.4. Particle size distribution and electrophoretic mobility

The lipid classes of lecithin were separated by thin-layer chromatography (Iatroscan). The results showed the phosphatidylcholine represented the major class of phospholipids (PL) contained in soya lecithin (89.5% ± 0.1), PL-DHA (44.3 ± 1.7%) and salmon lecithin (33 ± 0.4%). Moreover, the percentage of triacylglycerols (TAG) contained in lecithins was 55 ± 0.2%, 38.9 ± 0.8%, 6.7 ± 0.1%

Determination of vesicle size distribution is a fundamental quality control assay. The mean size and size distribution generally depend on lipid composition and preparation method, e.g. sonication time and the number of cycles in extrusion process. Table 4 presents the results of mean size and electrophoretic mobility of three liposomal formulations. The polydispersity index was the same before and after encapsulation of cinnamic acid. After encapsulation of cinnamic acid the particle size was slightly increased for all three liposomal formulations (Table 2). This modification depends on physico-chemical properties of cinnamic acid such as log P and the size of the molecule. When a hydrophobic molecule partitions into the lipid bilayer of the liposome, the interaction between the acyl chains of bilayer and the active molecule may increase the nanoliposome size. The increase of liposome size can be explained on the basis of the membrane fluidity. The cinnamic acid entrapment in bilayers increased the complex elastic modulus and membrane fluidity. Furthermore, the entrapment of cinnamic acid could increase the liposome size, probably by interacting with lipid acyl chains and altering the acyl chain order. Surface charge of liposomes depends primarily on lipid composition. The results of electrophoretic mobility showed that the nanoliposomes composed of salmon lecithin have a negative charge about −5.68 ± 0.16 ␮mcm/Vs. The electrophoretic mobility of PL-DHA and soya lecithin is lower than salmon lecithin with −4.95 ± 0.15 ␮mcm/Vs and −2.71 ± 0.08 ␮mcm/Vs, respectively. This negative charge is due to the presence of polar phospholipids in composition of three lecithins. As, it mentioned before, soya lecithin was richer in polar lipids with 93.3 ± 0.1% in comparison to salmon lecithin and PL-DHA with 61.1 ± 0.2% and 45.0 ± 0.1%, respectively. Additionally, the presence of negatively charged phospholipids such as phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI) in lipid composition of marine lecithin justified the negatively charge of liposome [23]. After cinnamic acid encapsulation, the electrophoretic mobility decreased. The presence of cinnamic acid in liposomal bilayer

3. Results and discussion 3.1. Fatty acid composition

Table 1 Mean percentage of fatty acid composition of salmon lecithin, soya lecithin and PL-DHA by gas chromatography (area; n = 3 replicate). Fatty acids

Salmon lecithin

Soya lecithin

PL-DHA %

%

SD

%

SD

C14:0 C15:0 C16:0 C17:0 C18:0 C21:0 C22:0 C23:0  SAT

1.72 0.24 18.10 0.49 5.14 1.79 0.75 0.60 28.83

0.11 0.01 0.38 0.02 0.10 0.17 0.03 0.02

– – 13.53 – 3.48 – – – 17.01

– – 0.02 – 0.03 – – –

– 0.28 3.01 – 1.51 0.73 0.25 – 5.78

SD – 0.1 0.06 – 0.03 0.00 0.02 –

C15:1 n-9 C16:1 n-9 C17:1 n-9 C18:1 n-9 C20:1  n-9 MONO

0.54 1.46 1.09 21.67 0.28 25.04

0.02 0.38 0.17 0.12 0.06

– 0.10 – 10.77 – 10.87

– 0.00 – 0.06 –

– 0.18 0.40 13.65 – 14.23

– 0.02 0.04 0.13 –

C18:2 n-6 C20:2 n-6 C20:3 n-6 C20:3 n-3 C20:4 n-6 C22:4 n-6 C18:3 n-3 C20:5 n-3 C22:5 n-3 C22:6  n-3 PUFA

5.28 0.13 0.26 0.16 2.18 2.33 2.37 8.83 2.99 21.60 46.13

0.15 0.07 0.01 0.00 0.19 0.04 0.03 0.15 0.04 0.44

64.92 – – – – – 6.09 – – – 71.01

0.04 – – – – – 0.02 – – –

15.16 0.25 0.19 – 2.36 1.46 0.64 6.96 4.85 48.07 79.94

1.01 0.00 0.04 – 0.07 0.49 0.01 0.15 0.16 1.35

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Table 2 Mean size and electrophoretic mobility of nanoliposomes from three lipid sources before and after encapsulation of cinnamic acid. Nanoliposome composition

Before cinnamic acid encapsulation Size (nm)

Salmon lecithin PL-DHA Soya lecithin

84.9a ± 1.9 94.9b ± 2.6 88.6c ± 0.7

E. mobility (␮mcm/Vs) −5.68a ± 0.16 −4.95b ± 0.15 −2.71c ± 0.08

After cinnamic acid encapsulation PDI

Size (nm)

0.21a ± 0.01 0.20a ± 0.01 0.22a ± 0.02

115.2a ± 2.0 111.8a ± 4.6 93.4b ± 4.5

E. mobility (␮mcm/Vs) −3.54a ± 0.27 −4.02b ± 0.06 −2.34c ± 0.08

PDI 0.16a ± 0.01 0.20a ± 0.01 0.17a ± 0.02

Data were expressed as mean ± SD (n = 3). Significant difference between a, b and c; P < 0.05.

and its interaction with the hydrophobic core may have partially masked the negative charge of nanoliposomes.

3.5. Stability of nanoliposomes Liposome stability is one the most important factors in liposome applications and depends on numerous factors such as size and chemical composition of the vesicles. The stability of the nanoliposomes with and without cinnamic acid was examined at 4 and 25 ◦ C. Stability of the nanoliposomes was evaluated by monitoring the mean particle size, electrophoretic mobility and polydispersity index [24]. Liposome suspensions with small polydispersity index (lower than 0.2) present a narrow droplet size distribution and consequently, higher stability. Nanoliposomes with or without cinnamic acid were found to have stable size distribution during 30 days of storage at 4 and 25 ◦ C. Furthermore no precipitation or loss of encapsulation of cinnamic acid was observed in samples.

3.6. Transmission electron microscopy (TEM) Transmission electron microscope (TEM) method was used to obtain the visual information concerning the morphology and size of liposome. Vesicles in the nanometer size range (after sonication and extrusion through 100 nm pore size filters) were observed by transmission electron microscope. The extrusion step was performed in order to produce nanometric unilamellar vesicles with a homogeneous size distribution. TEM images indicate that the extruded vesicles are in the form of large unilamellar vesicles. The bilayer nature of the vesicles is clearly visible in these micrographs confirming that the prepared lipid vesicles are liposomes (defined as closed continuous bilayer structures) (Fig. 1).

3.7. Phase transition temperature The main phase transition is associated with the melting of the acyl chains in the hydrophobic core of the membrane from the rigid gel phase to the more fluid liquid crystalline phase [25]. Interactions of bioactive compounds with model lipid bilayers could provoke changes in their thermotropic behavior as well as in their conformation properties. These effects were taken into account in the design of liposomal formulations as drug controlled release delivery systems [26,27]. The results reveal that cinnamic acid influences the thermotropic properties of lipidic membranes causing abolition of the pre-transition, as seen in liposome composed of salmon lecithin, and broadening of the phase-transition profile, and decreases the main transition Tc and transition enthalpy H, at increasing concentrations. In the presence of cinnamic acid, a notable decrease in main phase transition temperature Tc and H was observed for both marine and vegetable lecithins (Table 3). Fig. 2 shows DSC thermogram of nanoliposomes made of PLDHA in the presence and absence of cinnamic acid at lipid/CA ratio (lipid/CA: 100, w/w). The DSC trace of nanoliposomes based on PLDHA showed two peaks, one at 2.1 ◦ C which could be corresponding to temperature transition of water and another one at 35.7 ◦ C which is in good relevance by the values obtained for the pure dipalmitoylphosphatidyl choline (DPPC) liposomes at 39.8 ± 1.9 ◦ C [5]. Incorporation of cinnamic acid into liposomes reduced the TC value to 27.33 ± 1.00 ◦ C. The DSC thermogram of nanoliposomes from salmon lecithin presented two peaks corresponding to pre-transition and main phase transition temperatures at 23.26 ± 1.26 ◦ C and 47.86 ± 3.88 ◦ C, respectively. The presence of cinnamic acid in liposome prepared from salmon lecithin caused abolition of the pre-transition and broadened the phase-transition profile and decreased the main transition Tc to 32.8 ± 0.85 ◦ C. Cinnamic acid possesses a hydrophobic portion, with protonated form that makes it able to interact with the hydrophobic part of bilayer and facilitates its permeation into membrane. The decrease in Tc value represents the augmentation of fluidity membrane [28].

Table 3 Phase transition temperature of nanoliposomes from three sources of lecithin before and after cinnamic acid encapsulation. Nanoliposomes composition

Salmon lecithin PL-DHA Soya lecithin Fig. 1. Transmission electron micrograph of nanoliposomes prepared by sonication–extrusion techniques.

Tc (◦ C) Before cinnamic acid encapsulation 23.26 47.86 37.56 32.60 53.65

± ± ± ± ±

1.26 3.88 1.64 3.81 3.74

Data were expressed as mean ± SD (n = 3).

After cinnamic acid encapsulation 32.80 ± 0.85 27.33 ± 1.00 21.20 ± 1.54 53.60 ± 1.44

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Fig. 2. Phase transition temperature of PL-DHA nanoliposomes in the presence and absence of cinnamic acid determined by DSC.

3.8. Rheological study The rheological behavior of nanoliposomes was determined with the apparent viscosity measurement against shear rate at 25 ◦ C [29]. The results showed that the liposomal suspension represents a shear-thinning behavior solution; it means that viscosity of nanoliposome suspension tends to decrease with increasing the shear rate. Nanoliposomes composed of soya lecithin and PL-DHA showed higher complex elastic modulus. This might be due to the flexibility of the fatty acid chains rich in polyunsaturated fatty acids with estimates of 71.01 and 79.94% for soya lecithin and PL-DHA, respectively (Table 4). The complex elastic modulus value of each nanoliposome system was increased after cinnamic acid encapsulation. From a molecular point of view, bioactive substances able to insert themselves or become entrapped in the liposomal bilayer can alter the shape, size distribution and chemical properties of a liposome. Heimburg has theoretically shown that the mechanical properties of phospholipid vesicles are directly coupled with the gel to liquid crystalline transition of a lipid bilayer [30]. Generally, the reduction of elastic modulus directly induces adhesion of liposomes [31]. The phase transition temperature was also determined by rheological measurements. The obtained results of phase transition temperature confirmed the effects of cinnamic acid on membrane

Table 4 Complex elastic modulus of nanoliposome suspension prepared from three sources before and after cinnamic acid encapsulation. Composition

Complex elastic modulus of nanoliposomes (Pa) at 25 ◦ C Before encapsulation

Salmon lecithin PL-DHA Soya lecithin

133.90a ± 1.32 207.60b ± 1.61 246.20c ± 1.51

Data were expressed as mean ± SD (n = 3). Significant difference between a, b and c; p < 0.05.

curvature. The liquid-crystalline (LR) phase of bilayer inverted to hexagonal (HII) phase transition of lipid vesicles. The HII phase is characterized by a high negative curvature. Ligands that either stabilize this curvature or increase the bending elasticity will favor the HII phase and lower Tc [25]. Cinnamic acid promotes formation of the negatively curved bilayer with an inverted hexagonal (HII) phase. Phase transition temperature can also be determined by measuring viscosity and elasticity changes as a function of temperature, using a rheometer. The viscous modulus value decreases and the elastic modulus increases at phase transition temperatures. For the nanoliposomes based on PL-DHA, these temperatures are 2.1 ◦ C and 35.6 ◦ C (Fig. 3a). Membrane viscosity is a characteristic term that describes the ease of movement within the phospholipid bilayer. Recently, membrane viscosity has been investigated as a possible approach to indicate physiological processes within the cell [32]. The phase transition temperature obtained by rheometer was the same value as measured by DSC (Fig. 3b), which confirms that phase transition temperature depends on chemical and physical parameters (such as vesicle size, curvature, and number of lamellae parameter). The thermotropic behavior also provides information on the homogeneity and the lateral organization of the lipid bilayer. Upon heating, a sequence of three endotherms is distinguished: a sub-transition from the crystalline (Lc) to the gel (L␤0) phase, a pre-transition from gel (L␤0) to the rippled gel (P␤0) phase, followed by the main transition into the fluid liquid-crystalline (LR) phase, each observed at a well-defined temperature, Tc , Tp and Tm , respectively [33,34]. The first transition corresponds to a loss of order of the head group lattice, while the latter two values correspond to processes closely connected to acyl chain melting (trans-gauche isomerization) [35].

After encapsulation 542.96a ± 2.46 565.36b ± 11.14 618.83c ± 6.41

3.9. Membrane fluidity The influence of liposome composition on membrane fluidity was investigated by Coderch et al. [36].

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unsaturated acyl chains and increases with decreasing the acyl chain length. The presence of cinnamic acid increased the membrane fluidity of all nanoliposomes due to the alkyl chains rearrangement caused by the cinnamic acid penetration through the lipid bilayer. This phenomenon was observed with the previous measurement of elasticity. The increase of membrane fluidity is more predominant in nanoliposomes composed of salmon lecithin. The effects of cinnamic acid on membrane fluidity are related to its location in lipid bilayer and its interaction with acyl chains. Cinnamic acid decreases the membrane rigidity which is in good agreement with the drug preferential location in the membrane. This behavior can be explained by the fact that in the region where the drug is entrapped would be more disordering or a new rearrangement in lipid bilayer which causes a decrease in packing density than in the other regions of the bilayer. The rotational motions of the probes that result in depolarization of fluorescence are tightly coupled to acyl chain orientational fluctuations and, consequently, reflect the degree of molecular packing (order) in the membrane [39]. 3.10. Entrapment efficiency

Fig. 3. (a) Elastic (G*) and viscous (G ) modulus of PL-DHA nanoliposomes vs. temperature (20 mg/mL), (b) phase transition temperature of nanoliposomes, based on PL-DHA (20 mg/mL) before cinnamic acid encapsulation, by rheometer and DSC measurement.

Calvagno et al. determined a significant difference in the release profiles of drugs by the presence of two factors: (i) the strength of the drug–liposomal lipid interaction and (ii) the fluidity of the bilayer, i.e. by increasing the fluidity of the bilayer, the drug leakage to outer liposomal aqueous compartments was rapidly increased [37]. The presence of saturated FAs increased the packing between phospholipids which expelled the water in the vicinity of the bilayer surface, the evidence of reduced membrane fluidity. On the other hand, unsaturated FAs reduced the packing between phospholipids and preserved the level of hydration, thus maintaining membrane fluidity [38]. The obtained results show that the membrane fluidity depends on lipid composition of nanoliposomes. It also appears that nanoliposomes composed of soya lecithin have higher membrane fluidity than nanoliposomes based on PL-DHA and salmon lecithin (Table 5). Soya lecithin contains a higher proportion of polyunsaturated fatty acids with short chain length compared to other lecithins used. Membrane fluidity is more important in presence of poly

Encapsulating a sufficient amount of a therapeutic agent is one of the most desirable properties of liposomes. However, accommodation of lipophilic compounds in the lipid phase can be problematic, as some drugs can interfere with bilayer formation and stability and thus limit the range and amount of valuable drugs that could be otherwise associated with liposomes. Accommodation of a poorly water-soluble drug in the lipid bilayer of liposomes is often limited in terms of drug to lipid mass ratio [40]. The results proved that the nanoliposomes composed of salmon lecithin show the highest entrapment efficiency estimated about 91.40 ± 1.39%. The entrapment efficiency decreased for PL-DHA and soya lecithin with 76.4 ± 0.98% and 68.63 ± 1.21%, respectively. The interaction between cinnamic acid and lipid bilayers is due to the lack of hydroxyl group. This interaction is enhanced by an acidic pH, where the carboxylic acid is in a protonated form [41]. Cinnamic acid solution gently acidified the lecithin suspension dissolved in distilled water and presented a suspension with pH about 5.5. The cinnamic acid interacted with lipid vesicles at both neutral and acidic pH values. However, the higher entrapment efficiency achieved at pH 5 indicates more favorable interactions between cinnamic acid and the lipid bilayer at acidic pH compared to neutral pH. The pH affects the solubility of cinnamic acid. In an alkaline environment (higher than its pKa ), cinnamic acid is more soluble because it is de-protonated dissociated. The negative charge of nanoliposomes can also play an important role in the attraction and creation of the dipole–dipole bonds interactions between bilayer and cinnamic acid in protonated form.

Table 5 Membrane fluidity of nanoliposomes from three sources before and after encapsulation of cinnamic acid. Lipid composition of nanoliposomes

Membrane fluidity

Before encapsulation Salmon lecithin PL-DHA Soya lecithin

2.24a ± 0.02 2.40a ± 0.02 3.00b ± 0.08

Data were expressed as mean ± SD (n = 3). Significant difference between a and b; p < 0.05.

After encapsulation 2.53a ± 0.01 2.64a ± 0.01 3.02b ± 0.07

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The importance of the negatively charged nanoliposomes is in the following order: salmon lecithin > PL-DHA > soya lecithin. The high entrapment efficiency observed for nanoliposomes based on salmon lecithin can also be proven by difference observed in charge, size, complex elastic modulus and membrane fluidity of these nanoliposomes before and after cinnamic acid encapsulation as compared to other two nanoliposomes. Our findings have shown that liposome size and entrapment efficiency are considerably correlated, as by increasing liposome size, entrapment efficiency is also increased in liposomes with preserved unilamellarity. Consequently, the highest cinnamic acid encapsulation efficiency (91.4%) achieved by liposome which contained the higher content of the neutral zwitterionic PC (26% of total phospholipid content) as compared to other nanoliposomes (5.5–6%). 4. Conclusion The resemblance of liposomes to the model membrane makes them ideal for transportation of biomolecules through human tissues due to their broad bio-distribution and compatibility. Our results showed that cinnamic acid could be able to permeate through nanoliposome membrane. Penetration of a biomolecule into cellular membrane is often the first step to exert its biological activity, followed by the degree of incorporation and the uniform distribution into lipid bilayer. Combination of sonication and extrusion processes often results in formation of nanoliposome with homogeneous size distribution. Physicochemical and structural properties of three different nanoliposomes may be justifying their different capability to interact with hydrophobic molecules such as cinnamic acid. For cinnamic acid, we observed a correlation between the size of the liposome and the entrapment efficiency as by increasing liposome size, entrapment efficiency also increases in liposomes with preserved unilamellarity. The nanoliposomes composed of salmon lecithin presented higher capabilities as a carrier for cinnamic acid encapsulation. Salmon lecithin had the highest entrapment efficiency (about 91.40 ± 1.39%) and an acceptable phase transition temperature for the release of cinnamic acid (32.8 ± 0.85 ◦ C). This temperature is ideal to control the release of active molecule at 37 ◦ C (human body temperature). Fluorescence polarization of TMA-DPH was used to determine the modifications in lipid packing across the bilayer. DSC provides valuable information on the phase transition of lipidic membrane. Both techniques provide information about the biophysical modifications of lipid vesicles, causing by cinnamic acid entrapment in bilayer. Obtained results show that, incorporation of cinnamic acid increased the fluidity membrane and consequently the bilayer permeability. Finally, we can conclude that optimization of liposome formulation can make it possible to deliver the high quantity of bioactive agents (hydrophile or hydrophobe) to target site. In addition, to master the lipid composition allows us to control the bioactive agent’s release.

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