Abstract: The effects of alkylresorcinols (ARs) obtained from the acetonic extract of whole-wheat grains (Triticum sativum) on the stability, encapsulation efficiency and permeability of liposomes was investigated in this study. This extract could form vesicles alone (AR liposomes) as well as incorporation of the extracted ARs with phosphatidylcholine (PC) (PC:AR liposomes). AR liposomes had higher size stability than PC liposomes as illustrated by the optical densities. Using Patent blue violet (PBV) as a marker, it was observed that AR liposomes had higher encapsulation efficiency than PC liposomes, especially at pH 10.5. In addition, AR liposomes had more permeability than PC liposomes, especially at pH 6.5, while PC:AR liposomes had intermediate permeability. The percentages of PBV released from PC:AR, AR and PC liposomes during the first hour of incubation at pH 10.5 were 45, 55 and 30%, respectively. Further, after 15 days of incubation at room temperature, the oxidation indices for the AR and PC:AR liposomes were lesser (0.29 and 0.33, respectively) than that for PC liposomes (0.41).
INTRODUCTION
Liposomes are colloidal structures formed by self-assembly of amphiphilic lipid molecules in a solution (Imura et al., 2003). Liposomes are widely used as carriers for various molecules in cosmetic and pharmaceutical industries. In addition, food and agricultural industries have extensively investigated the use of liposome encapsulation to develop delivery systems that can entrap unstable compounds such as antimicrobials, flavors, antioxidants and bioactive ingredients and protect their functionality. Liposomes can entrap both hydrophobic and hydrophilic compounds, prevent decomposition of the entrapped compounds and release the entrapped compounds at designated targets (Benech et al., 2002; Shehata et al., 2008).
Liposomes are used as delivery systems for molecules such as drugs (Johnston et al., 2008; Wagner, 2007) and cosmetics and as models of biological membranes (Imura et al., 2003).
Intensive research has been carried out on developing liposome formulations of antibiotics which can improve the pharmacokinetic properties and antimicrobial activity of the antibiotics (Sezer et al., 2004; Pinto-Alphandary et al., 2002) and on the incorporation of different substances such as steroids, carboxyacyl phospholipids and water-soluble polymers in liposomes (Kallinteri et al., 2004; Mozafari, 2005).
Owing to their biodegradability, biocompatibility, low toxicity and ability to entrap both lipophilic and hydrophilic drugs (Johnston et al., 2007) and facilitate site-specific drug delivery to tumor tissues (Hofheinz et al., 2005), liposomes have gained value both as an experimental system and commercially, as a drug-delivery system. Many studies have been conducted on liposomes with the aim of reducing drug toxicity and/or targeting specific cells (Omri et al., 2002; Schiffelers et al., 2001; Stano et al., 2004).
Resorcinolic lipids are commonly present in cereal grains such as rye, wheat, barley and millet (Ross et al., 2003; Linko et al., 2005). These lipids have been reported to have antiparasitic, anticancer, antifungal, antioxidant, (Siddhuraju et al., 2002) and antimicrobial activity (Muroi and Kubo, 1996). They are also potent inhibitors of many enzymes such as prostaglandin synthase (Ha and Kubo, 2005; Hendricha et al., 2002), have several biological activities (Tyman, 2005; Verstraeten et al., 2003), exhibit strong amphiphilic character (Agata et al., 2008) and show high affinity for lipid bilayers as well as for biological membranes (Stasiuk and Kozubek, 2008).
The purpose of this study was to prepare more stable liposomes obtained from crude extract of whole-wheat grains and to form mixed liposomes (PC:AR) from phosphatidycholine and isolated AR from the acetonic extract. The effects of pH on the encapsulation efficiency and permeability of these liposomes in vitro were also studied.
MATERIALS AND METHODS
Phosphatidylcholine preparation: A dry lipid film was prepared by dissolving 5 mg of phosphatidylcholine (PC) (Phospholipid GmbH, Cologne, Germany) in chloroform and then drying it in a vacuum evaporator (RV 05-ST: Janke and Kunkel, IKA, Germany).
Extraction of alkylresorcinols from whole-wheat grains: A whole-wheat grain sample (20 g) was ground in a coffee grinder. The powder was extracted by continuously shaking the powder in acetone for 30 h at Room Temperature (RT). The extract was filtered using filter papers and evaporated to dryness in a rotary evaporator to obtain a dry residue.
Next, the dry residue was dissolved in chloroform (1 mL) and analyzed by performing Thin-Layer Chromatography (TLC). The solvent system used was chloroform-ethyl acetate (85:15, vol/vol). ARs were specifically detected by immersing the plate in a solution of 0.05% fast blue B (Sigma-Aldrich Chemie GmbH, Germany) in 5% acetic acid (Kulawinek et al., 2008). The AR spots (fractions) which stained reddish-violet in color, were removed from the plate, combined and dissolved in chloroform. After filtration, a part of this sample was again analyzed by TLC to check for the purity of AR. Pure AR molecules were used in all experiments (Mejbaum et al., 1978) and olivetol (Sigma-Aldrich Chemie GmbH, USA) was used as alkylresorcinol standards.
Preparation of phosphatidylcholine: resorcinolic lipids mixture: We dissolved 4 mg of PC and 1 mg of AR in chloroform and dried the solution in a rotary evaporator to obtain a dry film.
Liposome preparation: The dry lipid films obtained after sample preparation were hydrated by the addition of 1 mL of a 1 mM solution of Patent Blue Violet (PBV) (Sigma-Aldrich Chemie GmbH, Germany) in borate buffer (30 mM H3BO3, 70 mM KCL). The hydration was performed in buffers at 2 pH levels, 6.5 and 10.5. The hydrated samples were vortexed and liposomes were obtained by ultrasonication in an ultrasonic bath (Clifton, England) until the suspension became clear. Gel filtration to separate the non-capsulated PBV was performed in a Sephadex G-50 (Fluka Chemie AG, Switzerland). Liposomes obtained at different pH levels were used in the encapsulation efficiency and permeability experiments.
For spectroscopic and stability experiments, the dry lipid films were hydrated with borate buffer at pH 6.5, vortexes and finally, ultrasonicated in an ultrsonic bath (Clifton, England) to obtain liposomes.
Optical densities: The absorbance spectrum of the liposome suspensions was measured over a range of wavelengths (350-700 nm) with a UV-visible spectrophotometer (Biotech Engineering Management Co. Ltd., UK). Spectrophotometric means were determined according to the Klenine method and were used to measure the diameters of the liposomes (Trofimove and Nisnevich, 1990).
Oxidative index: To measure the Oxidative Index (OI), the Absorbance (A) of the liposome suspension was measured at 215 and 233 nm after addition of 3.0 mL of absolute ethanol. The OI was calculated according to the following formula: OI = A at 233 nm/A at 215 nm (New, 1990).
Determination of liposome entrapped volume: For measuring the PBV-encapsulation efficiency of liposomes, 0.1 mL of 10% Triton X-100 was added to a sample of the liposome suspension, mixed, centrifuged and the absorbance of the mixture was measured at 635 nm. The results were calculated as percentage (%) of PBV encapsulated (Przeworska et al., 2001).
Determination of liposome permeability: The release of PBV from the liposomes at various time intervals (0-11 h) was determined spectrophotometrically at 635 nm as per a method reported in a previous study (Przeworska et al., 2001). The results were calculated as percentage (%) of PBV released.
Statistical analysis: For all analysis, 3 replicates were used and the average values are reported. Values are represented as mean±SD.
RESULTS AND DISCUSSION
The influence of the hydrophobicity of ARs on the properties of the hydrophobic regions of the bilayer is greater than that on the properties of the hydrophilic regions. The unsaturated hydrocarbon chain in AR molecules causes extensive changes in the order of phospholipids. Many studies have shown that biological activity depends strongly on the structural characteristics of the specific homolog (Kozubek et al., 1988).
The acetonic extracts obtained from wheat (Triticum sativum) seeds were separated using TLC and subsequently, stained with fast blue B, the observed spots (Fractions) had different chromatographic mobilities and specifically detected by a reaction with fast blue B (Data not shown). Staining with fast blue B is a typical characteristic of resorcinolic compounds.
Alkylresorcinolic lipids that were extracted from whole-wheat grains could form vesicular structures. We prepared the following 3 types of liposomes: AR liposomes, PC liposomes, PC:AR liposomes. Figure 1a and b show the absorption spectra of these liposomes at day 0 and day 15 after formation. The absorbance data were used to estimate liposome aggregation and to study the influence of the molecules on the distribution of liposome size. At 400 nm, the turbidity of the AR and PC:AR liposome suspensions was more than that of the PC liposome suspension (control); the absorbance for AR, PC:AR and PC liposomes were, respectively 0.8, 0.7 and 0.52 at 0 day and 1.0, 0.9 and 0.78 at 15 days (Fig. 1a, b).
| Fig. 1(a,b): | The optical densities for PC, AR and PC:AR liposomes at pH 6.5 and at a spectral range of 350-700 nm at room temperature. Spectrum at (a) 0 days and (b)15 days |
| Table 1: | Size (diameter) and oxidation index of PC, AR and PC:AR liposomes measured at day 0 and at day 15 after incubation at room temperature and at pH 6.5 |
AR molecules are thought to have 2 types of effects on liposomes: they can increase the stability and they can change the shape such that the curvature is enhanced and the size is reduced. Table 1 shows the size measurements for liposomes incubated at RT at day 0 and day 15. The measurements taken at day 0 showed that the diameter of PC liposomes (average, 130 nm) was more than that of PC:AR liposomes (average, 102 nm) and AR liposomes (average, 80 nm), suggesting that AR addition can modify the size of the PC bilayer. After 15 days of incubation at RT, it was found that the average sizes were little increased for AR liposomes (average, 105 nm) and PC:AR liposomes (average, 160 nm) compared to PC liposomes (average, 220 nm).
Addition of AR molecules increased the resistance of the liposome lipids to oxidation (Table 1). Table 1 shows that the OIs of the AR, PC:AR and PC liposomes were 0.29, 0.33 and 0.41, respectively at day 15 of incubation; thus, AR liposomes were more resistant to oxidation than PC:AR and PC liposomes. AR molecules play a role in scavenging of free radicals and enhance the rigidity and stability of the bilayer. A previous report suggested that ARs could more efficiently stabilize liposomes in certain drug formulations than cholesterol could (De Maria et al., 2005).
Another report suggested that AR molecules might have these effects because of their strong amphiphilic character and conical shape; these features can lead to an increase in the curvature of a bilayer, decrease in the vesicle size and enhancement of the rigidity and stability of the bilayer (Hendrich and Kozubek, 1991).
| Fig. 2: | Effect of pH on the encapsulation efficiency (percentage) for the marker, Patent Blue Violet (PBV) when the liposomes were prepared at pH 6.5 and pH 10.5 |
The stability of AR and PC:AR liposomes was better than that of PC liposomes which illustrated the change in the molecular shape of the membrane lipids and the hydrogen bonding between the hydroxyl groups of AR molecules and the polar head-groups of the phospholipids (Przeworska et al., 2001).
We observed that the liposomes efficiently entrapped aqueous solutions. Figure 2 shows the PBV-encapsulation efficiency of the PC, AR and PC:AR liposomes prepared at pH 6.5 and 10.5. For liposomes prepared at pH 6.5, the total entrapped PBV volume for AR liposomes was 2.7-fold higher than that for PC liposomes, while PC:AR liposomes had an intermediate total entrapped PBV volume. In addition, the PC, PC:AR and AR liposomes prepared at pH 10.5 had higher total entrapped volumes (45, 65, 92%, respectively) than the ones prepared at pH 6.5 (30, 45, 79%, respectively). The differences between the total entrapped volume for PC liposomes and that for PC:AR liposomes were 15 and 20% when the liposomes where prepared at pH 6.5 and pH 10.5, respectively.
The difference in the composition of the AR and PC:AR liposomes, i.e., presence of AR molecules, lead to a higher percentage of encapsulation. This is because of the amphiphilic character of ARs and the interaction of ARs with the phospholipids which increased the order in the phospholipids and the curvature of the bilayer.
Figure 3a and b show the effect of pH on the release of PBV from PC, AR and PC:AR liposomes prepared at 2 different pH levels. The PC liposomes had a higher permeability than the AR and PC:AR liposomes during incubation at RT for 11 h. During the first hour of incubation, PC, PC:AR and AR liposomes released about 30, 70 and 75%, respectively of the encapsulated PBV at pH 6.5 (Fig. 3a) and about 60, 45 and 55%, respectively at pH 10.5 (Fig. 3b). The PC:AR liposomes completely released the PBV after 9 h of incubation at both the pH values, whereas PC liposomes completely released the PBV after 10 and 11h of incubation at pH 6.5 and 10.5, respectively. This suggests that the presence of AR makes a PC bilayer more permeable. At pH 10.5, increased ionization of the hydroxyl groups increases the zeta potential which leads to a change in the shape of the liposomes via increased ordering and an increase in the stability of the liposome. Therefore, the presence of AR molecules changes liposome properties and tend to increase their stability, increase their curvature, increase their encapsulation efficiency and enhance their permeability.
In conclusion, present results suggest that the use of AR molecules obtained from the crude acetonic extract of Jordanian whole-wheat grains or addition of the isolated AR molecules to PC molecules made changes in the physical and chemical characteristics of the liposomes. AR and PC:AR liposomes are more stable, have higher encapsulation efficiency and more permeable than PC liposomes, especially at pH 10.5 where the ionization of the functional groups is changed.
| Fig. 3(a,b): | Solute retention (percentage of PBV released by the PC, AR and PC:AR liposomes at (a) pH 6.5 and (b) pH 10.5 |
Further studies are needed to identify the different types of ARs and phospholipids present in the acetonic extracts of Jordanian whole-wheat grains and to applicate these liposomes as a carrier system for some drugs.
ACKNOWLEDGMENTS
This work was supported by Mutah University (Scientific Research grants No. 792/14/120).