1. Introduction
Liposomes can act as a suitable vesicle to enhance the solubility of hydrophobic bioactive compounds by encapsulating the compounds within the membrane bilayers. An appropriate composition of the lipid phase, along with formulation protocol and preparation techniques, will lead to the successful design of a liposomal system. Flavonoid quercetin (QU) (3,3′,4′,5,7-pentahydroxyflavone) is a semi-lipophilic molecule which is of great interest to researchers due to its great nutritional value. This compound has been shown, in vitro, to be a strong antioxidant and is one of the most powerful scavengers of reactive oxygen species, such as O
2−, NO and ONOO- [
1]. The free radical-scavenging effect of QU is based on its ability to donate proton. As reported by Boots and co-workers [
2], QU can reduce inflammatory pain via the inhibition of oxidative stress and cytokine production. In addition, QU is able to interact and permeate lipid bilayer and such capacity is very important because there is a positive correlation between the ability to incorporate into membranes and antioxidant activity [
3,
4]. Once incorporated into membranes, QU changes the biophysical parameters of the membrane such as its fluidity, cooperativity and the temperature of phase transition. However, despite these beneficial properties, QU has a low absorption rate in the gastrointestinal tract, instability in physiological media and under the Biopharmaceutics Classification System, it is classified as a class IV compound (low solubility, low permeability) [
5]. It is believed that low bioavailability of crystalline QU as a pure substance (as used in our study) is the result of its low solubility in the digestive tract [
6]. The encapsulation method allowed overcoming the problems related to crystalline QU solubilization. Since sufficient QU solubility is only achieved in amphiphilic systems [
7], one alternative to enhance QU bioavailability is by encapsulating it in amphiphilic nanocarriers, such as in mixed soy lecithin (ML)-based liposome systems. The fatty layer on the outskirts of the liposome confines and protects the enclosed compound material until the liposome travels to the site, adheres to the outer membrane of the target cell and delivers the payload [
8]. The process avoids many of the transitional stages of a conventional delivery options. Therefore, development of novel nanovehicles that are capable of solubilizing QU to exert its bioactivity in inhibiting ultraviolet B-induced cutaneous oxidative stress and inflammation is of great significance. In accordance with this, correctly loading QU within the ML-based liposomes containing different membrane components is critically important for improvement of QU’s in vitro bio-accessibility because the lipid bilayers are affected by membrane components and exhibit a specific particle curvature and shape, which affect the in vitro digestion and dispersion capacities as well. In addition, the size and thickness as well as the entrapment efficacy and vesicular stability have been shown to be related to the composition of liposomal wall materials that can further affect its digestion behavior [
9,
10]. The use of soy lecithin as non-synthetic mixed phospholipids for producing liposomes does not raise any food legislation concerns and provides additional nutritional value owing to its high polyunsaturated fatty acids (PUFA) composition [
11]. Nevertheless, the predominance of highly unsaturated fatty acids typically results in more permeable and less stable bilayers [
12], and may eventually generate hydroperoxides and secondary oxidation products as a result of environmental stresses (mainly ultraviolet (UV) light), and whose accumulation in food/ nutraceutical systems could give rise to rancidity and a concomitant reduction in shelf life. Therefore, as a powerful antioxidant, QU is a good choice to be used as an efficient inhibitor against the degradation of ML-based liposomes as a result of light-induced oxidation.
It is of equal importance to produce stress-resistant liposomes via modulation of the lipid bilayers’ composition to protect the bioactivity of the encapsulated substances in the membrane, as well as to maintain the vesicles’ entirety. Like cholesterol, phytosterols (PSs) are also able to modulate a number of different membranes functions, such as acyl chain order [
13], elasticity [
14] and lateral organization [
15]. Previous studies have investigated the addition of PSs to phospholipid vesicles, and demonstrated that their presence modifies the physical properties of the outer membrane [
16]. However, despite the literatures, our understanding of the molecular basis of and specificity of the interactions with different PSs types and with mixed membrane phospholipids remain limited. In this regard, two types of PSs, namely β-sitosterol (βS) (C
29H
50O) and stigmasterol (ST) (C
29H
48O), were added in the liposomal formulations with the aim of improving the packing of the ML membranes. Moreover, incorporation of several biomaterials in an encapsulation system may enhance the bioactivity of individual components [
17]. PSs have been shown to decrease the serum cholesterol levels and the ratio of the low-density lipoprotein (LDL) to high-density lipoprotein (HDL) bound cholesterol in serum [
18]. In addition, PSs have been found to increase the oxidation stability of lipids [
19].
Bilayer membranes are mosaic of areas maintained by the cytoskeleton network, whereby the lipid nature of the membranes is believed to play an important role in the formation of these areas. Squalene (C
30H
50), a natural isoprenoid compound, could be conjugated to phospholipids in the formation of liposomes with the intention of improving the membrane lipophilicity and consequently, the affinity towards the environment of lipid bilayers. As reported by Richens et al. [
20], oily natural substances such as squalene can modify the membrane dipole potential. The adhesion capability of squalene could be affected by its lipophilic character and its tendency to integrate with the ML membrane bilayer. The “fit” between the two imperfect adjoining chains (unsaturated fatty acids) could adhere well after bringing squalene into lipid bilayers containing mono- and polyunsaturated lipid chains. In fact, aliphatic organic molecules have stronger interplays than aromatic compounds, because branches on a carbon chain will reduce the hydrophobic effect of that molecule, and a linear carbon chain can form the largest hydrophobic interaction producing steric hindrance by carbon branches. This dynamic is consistent with the findings of Ott et al. [
21], who used squalene for dual delivery of hydrophilic and lipophilic actives. They declared that the steric hindrances of squalene could deliver postponed release functionality for nanostructured lipid carriers. Moreover, the addition of non-ionic surfactants such as Tween 80 was deemed to affect the hydrophilicity, particle size, fluidity and integrity of mixed liposomes [
22]. The major difference between surfactant-incorporated liposomes, flexible liposomes and surfactant-free liposomes is the high and stress-dependent adaptability of such surfactant conjugated vesicles. This difference is demonstrated in the motive force given by the osmotic gradient between the outer and inner leaflets of the membrane of the liposomes. Edge activators with a high radius of curvature could definitely increase the fluidity and flexibility, and boost the deformability of the bilayers [
23]. Apparently, such elastic membranes are suited for maintaining the integrity of the lamellar vesicles, breeding effective durability of the free radical scavenging system against UV-light-induced degeneration. Therefore, the use of various active ingredients for the fabrication of liposomes via an extrusion technology is expected to confer triple benefits; improved formation of stable liposomes for effective nutraceutical/ food-grade delivery applications, enhanced nutritional value through naturally-occurring bioactive compounds addition, and reduced photodegradation rate via the use of appropriate ratios of liposome ingredients.
Taking into account these notions, the aim of this study was to develop, optimize and characterize the liposomal formulation intended for antioxidant therapy by means of encapsulating QU with the addition of βS and ST. The main target of the research was to find the optimum formulation to produce QU-nanoliposomes with the highest encapsulation efficiency (EE), and highest stability (i.e., least degradation of the nanoliposomes against UV light as compared to other synthesized composites) using response surface methodology (RSM). Furthermore, this research aimed to provide a better understanding of QU physicochemical properties, stability and bioactivity in response to various factors and consequently focused on the physicochemical stability of the proposed ML nanoliposome system. The extrusion technique for nanosizing the liposomes was employed. The effect of adding the PSs (βS and ST) on photo quality, stability and potency of delivery properties of liposomes were investigated. Cured ML (instead of a pure specific phospholipid), a helper lipid (squalene) and a non-ionic surfactant (Tween 80) were used in the formation of the bilayers. This work also aimed to provide a structural understanding of the effects of the selected PSs on the stabilization, functionalization and encapsulation performances of the liposomes.
2. Materials and Methods
2.1. Materials
ML (composition as shown in
Table 1) used in this research was sourced from Ncalai Tesque, Inc. (Kyoto, Japan). Squalene (99% purity; for synthesis) and polyoxyethylene sorbitan monooleates (Tween 80) were purchased from Merck Inc. (Darmstadt, Germany). Cholesterol (95%), QU (≥95%) and ST (≥95%) were supplied by Acros Organics Inc. (Branchburg, NJ, USA). βS (≥95%) was obtained from Amresco Inc. (Cleveland, OH, USA). All other chemicals and reagents, such as chloroform (CAS n. 67-66-3), and ethanol (CAS n. 64-17-5) (Sigma-Aldrich, Milan, Italy), used were of analytical grade. Deionized water was used in all experiments.
2.2. Liposome Preparation via the Extrusion Method
A pre-liposomal formulation containing 5.8% (
w/
w) of lipid was previously optimized in a separate study by varying the proportions of ML, cholesterol, Tween 80 and squalene containing 5% (
w/
w) ML, 0.3% (
w/
w) cholesterol, 0.75% (
w/
w) Tween 80 and 0.5% (
w/
w) squalene. The mass ratios of the pre-liposomal composition were selected based on our preliminary study and those reported by other studies [
24,
25]. The ML-based liposome was purified to formulate for the present study. In the next step, the optimal pre-liposome (OP
1) was loaded with QU, βS and ST via a film-extrusion method based on established procedures [
26]. Briefly, a thin lipid film, named as organic phase, consisting of the OP
1 composition, with or without QU, βS and ST (see mass ratios in
Table 2), was produced from a mixture of ethanol-chloroform (1:1
v/
v) at a final volume of 10 mL. After the solvent removal by rotary evaporation (Rotary Evaporator, Ultra Lab, Delhi, India), the lipidic thin film was hydrated in phosphate buffered saline (PBS; pH 7.4, 0.05 M) and shaken using a magnetic stirrer (60 rpm, 1 h) above T
m (60 °C). The temperature above the T
m refers to liposome including the components mixture. The obtained liposomes were then homogenized using a bath sonicator (Loba Life, Mumbai, India) at 60 °C for 1 min to obtain a homogeneous suspension for the extrusion process. Finally, the lipid particles were extruded eight times through a 100 nm polycarbonate membrane (Millipore USA) filter by means of an Avanti Mini-Extruder (Avanti Polar Lipids, Inc., Alabaster, Alabama, USA) at above the T
m to achieve uniform particle size distribution. In this manuscript, the concentration of QU is always expressed as %
w/
w of the total formulation weight. Component ratios of lipid formulations are also always expressed in %
w/
w.
2.3. Particle Size, Polydispersity Index (PDI) and Particle Size Stability Measurement
The mean particle size (z-average), uniformity and PDI of the liposomes were assessed by dynamic light scattering (DLS) using a NanoZS90 instrument (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK) at room temperature. All solutions were diluted 10-fold in PBS before measurement. The vesicular stability against UV light (280–320 nm) was analyzed to examine the impact of variable concentrations on physical properties of liposomes. All measurements were carried out 24 h after liposome preparation in order to avoid degradation of the phospholipids and to make sure that lipid vesicles were uniform in dispersion for particle size and PDI measurements. The liposomes were always stored in darkness and at 4 °C. Duplicate analyses were run for each sample.
The particle size value change rate caused by UV-light exposure given as a percentage was calculated using Equation (1):
where the value change rate represents the percentage increase of the particle size of liposomes after one time submission to UV-light irradiation.
2.4. Zeta Potential (ZP)
The stability in the term of surface charge was further investigated by determining ZP 24 h after liposome preparation using the Malvern Zetasizer Nano series (Zetasizer Nano ZS; Malvern Instruments Ltd., Worcestershire, UK). The liposomal preparations were gently dispersed in PBS (10 fold) at room temperature to produce a yellow suspension prior to the measurements. All measurements were performed in triplicates.
2.5. Encapsulation Efficiency of QU (EEQU)
Standard curves were prepared using stock solution of 1 mM QU and dimethyl sulfoxide (DMSO) as a blank. The calibration curves were linear (R2 > 0.996) against the QU concentration (1–10 µg mL−1).
Entrapment efficiency of QU-loaded liposome was determined by assessing the difference between the total amount of QU and the amount of free QU present in the liposome. The amount of QU encapsulated in the QU-ML-based liposomes was identified by an indirect method via centrifugation technique using a centrifuge (Model: 3740, KUBOTA MFG. CORP, Tokyo, Japan) at 5000 rpm for 15 min at 4 °C. The experiment of QU quantification was carried out at 370 nm [
27]. The concentration of QU was measured via UV/V is spectrophotometry (Agilent Technologies, Basel, Switzerland) at 370 nm. The
EEQU was then calculated according to Equation (2):
where
Wtotal is the total
QU weight in liposomes suspension;
Wfree is the weight of free QU.
2.6. Assessment of DPPH (2, 2-diphenyl-1-picrylhydrazyl) Scavenging Rate, and DPPH Scavenging Rate of Loss against UV Light
The percentage of antioxidant activity was evaluated via the DPPH method [
28]. Sample solutions were allowed to react for 30 min with DPPH (100 µM) in the dark, subsequently the decrease in absorbance of the liposomal samples was measured at 517 nm using UV–Vis spectroscopy (Agilent Technologies, Basel, Switzerland). The control mixture was prepared by replacing the sample with 50 µL of ethanol. Lower absorbance of the reaction mixture indicated higher radical-scavenging activity. The percentage inhibition of radical scavenging activity was calculated by Equation (3)
where
As and
Ac represents the absorbance of the sample and the control, respectively.
The percentage of antioxidant activity was expressed in Trolox equivalent antioxidant capacity (
TEAC). Often, Trolox is used as a reference compound and the capacity is expressed as Trolox equivalent [
29].
Trolox (0.250 g) was dissolved in 50 mL of 75 mM phosphate buffer (pH 7.4) to make a 0.02 M stock solution for the preparation of working solutions of Trolox (0, 25, 50, 100, 150 and 200 µM) [
30]. The
TEAC corresponds to the Trolox concentration (µM). The
TEAC was determined using the following Equation:
Equation (4) was obtained from a linear least squares fit (R2 = 0.9809, n = 5) to a plot of Trolox versus %antioxidant activity. Of this, “x” refers to TEAC value and “y” refers to %antioxidant activity value obtained from Equation (3).
The loss/degradation of
TEAC (%) as a result of photodegradation (280–320 nm, 6 h) was then obtained as follows (Equation (5)):
where
TEACb and
TEACa are the percentage of
TEAC before and after UV-light irradiation, respectively.
2.7. Fourier-Transform Infrared Spectroscopy (FTIR) Assay
Infrared absorption spectra of the three added compounds (QU alone, βS alone and ST alone), free liposomes and liposomes with QU, βS and ST addition were recorded using a Fourier-transform infrared absorption spectrometer (FTIR-8400S, Shimadzu, Japan). Absorption spectra at a resolution of one data point every 0.6 cm−1 were obtained in the region between 4000 and 750 cm−1 using a clean crystal as the background. All experiments were performed at 20 °C.
2.8. Thermal Stability
The optimal liposomes (OP1 and OP2) were transferred into boiling tubes with screw caps and immersed in water bath set at different temperatures (30–110 °C) for 30 min, then immediately cooled to room temperature under running water. Samples were then stored overnight at room temperature prior to particle size and uniformity measurements.
2.9. Transmission Electron Microscopy (TEM)
Liposomes with QU, βS and ST and the control liposome were visualized via negative stain electron microscopy. A drop of the liposome suspension (~0.1 mg/mL) was deposited onto on a carbon film-coated copper grid. After 60 s, excess solution was removed by tapping the edge of grid with filter paper. A drop of 1% uranyl acetate solution was then applied to the same grid for 60 s. The grid was again tapped dry and further dried in the desiccator overnight. Images were taken on a transmission electron microscope (Hitachi H-7100, Tokyo, Japan).
2.10. Experimental Design and Statistical Analysis
RSM with a three-factor, two-level Box-Behnken Design (BBD) was used to optimize the composition concentrations of the liposomal formulations. Three independent factors were studied, specifically QU dosage (%
w/
w), βS dosage (%
w/
w) and ST dosage (%
w/
w), at 3 different levels for each (
Table 2). Preliminary experiments were carried out to obtain the ranges of the studied parameters. The response measured were the liposomal particle size (Y
1), ZP (Y
2), EE (Y
3), TEAC (Y
4), liposomal particle size value change rate (Y
5) and percentage loss of TEAC (Y
6), as shown in
Table 3 and
Table 4. The experimental plan was designed and the results obtained were analyzed using Minitab 16 (Minitab Inc., State College, PA, USA). Each term of the model was tested statistically, and
F-ratio significance was confirmed at a
p-value of 0.05, as determined by Tukey’s test. Verification of model validity was confirmed by comparing the experimental data with the predicted results from the optimized model.