Preparation and optimization of quercetin-loaded liposomes for wound healing, using response surface methodology

Abstract

The basic objective of this study was to prepare quercetin-loaded liposomes by the thin film hydration method. The liposomal formulation was optimized using response surface methodology (RSM). A rotation speed of 75 rpm and a water bath temperature of 46°C were finalized as the best for optimized drug-loaded liposomal formulation. In vitro characterization of the quercetin-loaded liposomal formulation was done through some parameters including the entrapment efficiency (EE), drug release (DR), and mean particle size; the resulting values of 86.5 ± 0.42%, 76.5%, and146 nm were found to be standard characterized values respectively. It is concluded that quercetin-loaded liposomal formulations achieve sustained release of drug in wound areas.

Keywords::

• liposome
• quercetin
• response surface methodology
• temperature

Introduction

A wound is a common occurrence in which the skin is torn by various means (mechanical, physical, or chemical), which can cause serious pathological conditions (Clark 1996, Rakhimov et al. 2000). Wound healing involves a complex process. Healing occurs in a cascade of changes, including: 1) hemostasis, 2) inflammation, 3) granulation, 4) fibrogenesis, 5) neo-vascularization, 6) wound contraction, and 7) epithelialization (Gupta et al. 2006, Hurler and Skalko-Basnet 2012). Quercetin is a bioflavonoid polyphenolic phytoconstituent having potential anti-inflammatory and anti-oxidant properties. It directly inhibits the various proinflammatory agents and has also been reported for its potential immunomodulatory, gastro-protective, anti-tumor, cardio-protective, and bacteriostatic effects (Fan et al. 2003, Rice-Evans et al. 2007). Quercetin has been widely used as a therapeutic agent in different disease conditions. Apart from its potential therapeutic effects, it suffers from some limitations like low aqueous solubility, and low bioavailability. The low bioavailability of this drug requires its administration at a high concentration to produce therapeutic effects (Park et al. 2013). Therefore, some novel carriers are required to overcome the problems mentioned above. Vesicular carriers can play a vital role in enhancing the solubility and bioavailability of the active moiety; with these advantages offered by vesicular carriers, high therapeutic potential can be achieved. In addition, vesicular carriers assist in providing sustained release of the active moiety (Rahman et al. 2010). At present, vesicular carriers are being widely explored for use in drug delivery in topical applications, due to their unique phospholipid complex. Liposomes are colloidal carriers containing phospholipids in the composition of their bilayered structure, which resembles the lipid cell membrane of the human body (Shaji and Iyer 2012). Additionally, they can load hydrophilic as well as lipophilic drugs in their core, and exhibit biocompatibility with low toxicity. The optimization of liposomes plays a major role in designing the appropriate formulations for novel vesicular systems. The response surface method (RSM) is commonly used for the optimization of novel vesicular formulations using various kinds of drugs. In the present research, we focused on the enhancement of poor aqueous solubility and improvement in the bioavailability of drug (Varde et al. 2013). In this work, quercetin-loaded liposomes were prepared by the thin film hydration method using a vacuum rotary evaporator. The optimization of the liposomal formulation was done using RSM combined with miscellaneous designs. The variables selected were the temperature of the water bath (X₁), and the rotation speed of the rotatory evaporator (X₂); the response variables were the drug release (DR) as Y₁, mean particle size diameter (MD) as Y₂, and the entrapment efficiency (EE) as Y_3, of the liposome. The levels for these variables were determined from the preliminary trials. Furthermore, the selected formulation had maximum entrapment of drug and minimum in vitro release in 24 h.

Materials and methods

Materials

Quercetin, phosphatidylcholine, and cholesterol were purchased from HiMedia Chemicals (Mumbai, India). HPLC grade solvents were purchased from Merck (Mumbai, India). All other materials and solvents used were of analytical grade.

Preparation of quercetin-loaded liposomes

Liposomes were prepared using the thin-film hydration method (Ruozi et al. 2005). Phospholipid, cholesterol and quercetin, were dissolved in 25 mL of a chloroform–methanol mixture (4:1) using a fixed molar ratio. The mixtures were evaporated in a rotary evaporator (IKA RV10 Digital Rotary Evaporator, IKA Pvt. Ltd. Bangalore, Karnataka, India) at 15 min, at a speed of 70 rpm and a temperature of 46°C, to remove traces of solvent and also to form a film. The film was hydrated with phosphate buffer (pH 7.4) for 1 h at room temperature, which was above the lipid transition temperature. The vesicle dispersion was then homogenized using a probe sonicator (FS-500, Frontline), passed through a 0.45 μm filter (Minisart CA 26 mm), and stored until use.

Optimization of formulation parameters for liposomes

As a preliminary study, the formulation technique was optimized by studying various process parameters, such as the variable rotational speed of the rotary evaporator and the temperature of the water bath during processing. It was found that thickness and uniformity of the lipid film varies depending upon the rotational speed of the evaporating flask. The optimum speed was found to be 75 rpm. The film obtained after rotary evaporation was kept overnight under vacuum, to be dried and removed. Further, it was observed that liposomes prepared using a phosphate buffer of pH 7.4 as the hydration medium, showed better % DE compared to those prepared with water as the hydration medium (Zhang et al. 2010).

The above two observations were further corroborated by the values for entrapment efficiency (EE) of the formulation. The optimized formulation parameters were then used to formulate further batches. Suitable batches were then prepared to study in detail the interactions of quercetin with the lipids, and their effects on the entrapment and the particle size of the final formulation, using experimental designing techniques.

Experimental design

A 3-level factorial-response surface methodology (3LF-RSM) was used to study the effect of different variables dependent on the properties of the formulation, like mean particle size, percentage drug release (% DR), and entrapment efficiency (% EE) of the prepared liposomes, and independent variables including temperature (X₁) and rotation speed (X₂) (Table I). The best fitted model for statistical analysis was considered significant when the P value was less than 0.05. The predicted R² value and ANOVA were pursued to confirm the best-fit of the model. Three-dimensional (3D) surface plots were used to establish the relationship between independent variables and dependent variables (response).The desirability functions of particle size and DR were at the minimum level, while that of EE was at the maximum level, which was used for optimization of formulations (Rawat et al. 2007).

Table I. Independent variables along with their coded level, actual level, and respective response values of different batches of quercetin-loaded liposomes.

	Coded level		Actual level		Responses			Polydispersity Index (PDI)	Zeta Potential (mV)
Form code	X1	X2	X1	X2	Drug Release (%)	Particle Size (nm)	Entrapment Efficiency (%)
Q1	− 1	− 1	40	50	75.09	591.9	69.4	0.372 ± 0.032	− 16.3±(− 1.43)
Q2	0	− 1	45	50	60.32	213.6	76.3	0.289 ± 0.214	− 17.7±(− 2.36)
Q3	1	− 1	50	50	71.51	425.7	78.5	0.248 ± 0.024	− 14.2±(− 5.47)
Q4	− 1	0	40	75	69.55	418.1	71.8	0.265 ± 0.321	− 14.1±(− 4.61)
Q5	0	0	45	75	58.55	158.2	78.3	0.145 ± 0.025	− 14.2±(− 1.32)
Q6	1	0	50	75	66.51	334.3	86.5	0.067 ± 0.326	− 10.6±(− 1.25)
Q7	− 1	1	40	100	66.23	395.1	54.6	0.128 ± 0.245	− 13.9±(− 2.87)
Q8	0	1	45	100	57.15	146.8	59.2	0.250 ± 0.347	− 12.5±(− 6.02)
Q9	1	1	50	100	64.45	303.9	70.3	0.345 ± 0.478	− 11.6±(− 6.24)

Differential Scanning Calorimetry analysis

Differential scanning calorimetry (DSC) analysis was performed to ascertain the absence of potential interactions between the components of the liposomal formulation and quercetin, to confirm the formation of liposomes. The possibility of any interaction between the phospholipid, quercetin, and liposomes, during preparation, were assessed by thermal analysis of the liposome samples. A model DSC (Perkin Elmer Jade, California, USA, Department of Pharmaceutical Sciences, Dibrugarh University, Assam, India) was used to determine melting point and enthalpy for the liposomal formulation. A sample equivalent to approximately 5 mg was placed in an aluminum pan and DSC analysis was carried out at a nitrogen flow rate of 20 mL/min and a heating rate of 5°C/min, from 50°C to 305°C. An empty aluminum pan was placed on the reference platform. The thermal analysis of sample parameters in the DSC thermogram are the onset temperature (T₀), the peak temperature or the gel to liquid-crystalline transition^™, the end-set temperatures (Te and T₀), and enthalpy change of the transition (Begum et al. 2012, Epstein et al. 2008).

Transmissions electron microscopy

The morphology of the vesicles in the optimized batch was determined by a negative stain electron microscopy method using a transmission electron microscope (Hitachi J 500, Japan. North East Hill University Shillong, India). The surface morphology and size of the optimized liposomes were analyzed by transmission electron microscopy (TEM). The optimized aqueous dispersion of the quercetin-loaded liposomes was placed on copper grids coated with 1% aqueous phosphotungstic acid, and dried at room temperature for observation. After drying, the specimen was viewed under the microscope at 10–100 fold enlargement. The magnification for the TEM images was 150,000 x (Liu and Wu 2010).

Determination of mean particle size, PDI and zeta potential of the quercetin-loaded liposomes

The mean particle size (z-average) of the liposomes, and the poly dispersity index (PDI) as a measure of the width of particle size distribution, were calculated using photon correlation spectroscopy (PCS) using a Zetasizer (Nano ZS 90, Malvern Instruments, UK) at a temperature of 25°C and a 90⁰ scattering angle. The liposomal formulation was diluted with double distilled water, to weaken opalescence before measurements. The surface charge was assessed by measuring the zeta potential of liposomes, based on the Smoluchowski equation, using the same equipment at 25°C with an electric field strength of 23 V/cm. Three independent measurements were performed for each sample. The samples were analyzed 24 h after preparation (Jin et al. 2006).

Determination of percentage of entrapment efficiency of quercetin-loaded liposomes

The % EE was evaluated by determining the amount of free quercetin in the aqueous medium, which was separated by using the cooling micro centrifuge (5430 R, Eppendorf India Ltd.) (Jin et al. 2006). The aqueous dispersion of the quercetin-loaded liposomes was placed in the cooling centrifuge tubes and the speed of the centrifuge was kept at 12,000 rpm for 20 min at 4°C. The concentration of quercetin in the aqueous phase was determined using the drug content in both supernatants after centrifugation, and was measured by the HPLC method developed. For the HPLC analysis, a mobile phase system comprising of methanol/water (50/50% v/v) was utilized. The solvents were mixed, filtered through a membrane filter of 0.45 micron pore, and degassed before use. The chromatography system comprised of a LC-10AT VP liquid chromatogram pump equipped with a SDP-10A VP UV-VIS detector and an injector with a 20-microliter loop. Samples were injected into a RP-18 column (4.6 × 250 mm). The flow rate in the mobile phase was 1mL/min. Quercetin was analyzed at a wavelength of 372 nm. The %EE was calculated by the following equation:

$$\%\text{Entrapment}\text{efficiency}=\frac{\text{weight}\text{of}\text{quercetin}\text{used}-\text{weight}\text{of}\text{free}\text{quercetin}}{\text{weight}\text{of}\text{quercetin}\text{used}}\times 100$$

In vitro drug release from quercetin liposomes

In vitro release of quercetin from optimized quercetin liposomes was determined by the diffusion cell apparatus (EMFD-08 Orchid scientific & Innovative India Pvt. Ltd. Nasik, Maharashtra, India) using a dialysis membrane (molecular weight cutoff 10,000 Da). The dialysis membrane was kept in double distilled water for 24 h before being utilized in the diffusion cell apparatus. The aqueous dispersion of quercetin liposomes (2 mL) was placed in the donor compartment, the receptor compartment was filled with the dissolution medium (phosphate buffer of pH 7.4), and the temperature was maintained at 35 ± 0.5^∘C by continuous stirring at 100 rpm. Aliquots of 2 mL were withdrawn at intervals of 0, 1, 2, 3, 4, 5, 6, 12, 16 and 24 h. They were filtered after withdrawal and the apparatus was immediately replenished with 2 mL of the fresh buffer medium. The aliquots withdrawn were diluted sufficiently and 20 μl solution was injected into the HPLC system for analysis, at a wavelength of 372 nm (Maitani et al. 1990).

Storage stability studies

The storage stability studies were carried out with the optimized quercetin-loaded liposomes. A 10 mL sample of quercetin- liposome dispersion with 2 mg/mL drug concentration was taken into glass vials and stored at 4°C and 25°C for 3 months. The stability test was analyzed on the basis of particle size, zeta potential, and % EE determined in the dispersion, with a sampling frequency of 1 month (Epstein et al. 2008).

Results and discussion

Preparation of quercetin-loaded liposomes

Several batches of liposomes were prepared to study the effect of the rotation speed of the rotary evaporator and the temperature of the water bath, by using the thin film hydration method, which is an easy method that can be utilized in the laboratory production of liposomes. For homogeneous distribution of quercetin inside the lipid phase, 25 mL chloroform/methanol mixture (4/1) was incorporated. The homogenization speed and sonication time were optimized at 15,000 rpm for 10 and 5 min at 50 W, respectively.

Analysis of optimization data for the quercetin-loaded liposomes

The observed responses of nine formulations were fitted to various models by using Design-Expert software trial version 9.0.1. It was seen that the quadratic models were the best-fit for the responses studied, that is, mean particle size, % EE, and % DR. The quadratic equations generated for the responses are given as follows:

$$\begin{array}{l}\text{Particle}\text{size}=+144.44-56.87{\text{X}}_1-64.23{\text{X}}_2+18.75{\text{X}}_1{\text{X}}_2+238.63{\text{X}}_1{}^1+42.63{\text{X}}_2{}^2\\ \%\text{EE}=+78.48+6.58{\text{X}}_1-6.88{\text{X}}_2+1.65{\text{X}}_1{\text{X}}_2+0.58{\text{X}}_1{}^1-10.82{\text{X}}_2{}^2\\ \%\text{DR}=+58.06-1.40{\text{X}}_1-3.18{\text{X}}_2+0.45{\text{X}}_1{\text{X}}_2+10.22{\text{X}}_1{}^1+0.92{\text{X}}_2{}^2\end{array}$$

Where X₁ and X₂ represent the coded values of the temperature of the water bath and the rotation per minute of the rotary evaporator, respectively. The positive value of a factor in the above equations points out the enhancement of that response, and vice versa. All values of the correlation coefficient (R²), SD, percentage coefficient of variation, and results of ANOVA are shown in Table II. A value of R² and results of ANOVA for the dependent variables confirmed that the model was significant for the response variables observed.

Table II. Summary of results of regression analysis of responses and analysis of variance for drug release, particle size, and EE.

Parameters	DF	SS	MS	F	P value	R^2	SD	Coeff. of variance (%)
Drug Release (%)
Model	5	283.77	56.75	20.68	0.0157 Significant	0.9718	1.66	2.53
Residual	3	8.23	2.74
Total	8	292.00
Mean Particle Size
Model	5	1.631E + 005	32618.32	24.77	0.0121 Significant	0.9764	36.29	10.93
Residual	3	3949.82	1316.61
Total	8	1.670E + 005
% Entrapment Efficiency
Model	5	773.61	154.72	37.77	0.0065 Significant	0.9844	2.02	2.82
Residual	3	12.29	4.10
Total	8	785.90

Experimental design

Based on the preliminary experiments and our previous studies, two factors (temperature of the water bath and rotation speed of the rotary evaporator) were identified as key factors responsible for % EE, % DR, and mean particle size of the liposome. The temperature of the water bath was chosen because the temperature reaches the phase transition temperature of the water bath system, the water molecules penetrate into the lattice between the phospholipid molecules which spontaneously form a multi-bilayered structure, and the rotation speed of the rotary evaporator affects the lipid film formation. Contact of the film with hot water is increased by lowering the speed, and with a higher speed, the film is not formed properly because of a lack of time for the lipid to form a film, and the proper liquid to gel transitions of the lipid do not occur. Both of these may have caused the uneven distribution of heat, leading to the formation of an uneven film.

Predicted optimum ranges of the independent variables are listed in Table III. The results that fit point out that the optimized liposome formulation with high % EE, low % DR, and small mean diameter of particles, was obtained at the rotation speed of 75 rpm and a water bath temperature of 46°C, respectively. Table III shows that the observed values of the batch prepared with the optimized formula are very close to the predicted values, with low percentage bias, suggesting that the optimized formulation was trustworthy and rational.

Table III. Comparison of the observed and predicted values for the liposomes prepared under predicted optimum conditions.

		Predicted optimum range
S. No.	Responses variable	X1	X2	Predicted value	Observed value	Bias %
1	% Drug Release	46	75	58.43	54.31	7.05
2	% EE	46	75	80.32	86.5	− 7.69
3	Mean particle size (nm)	46	75	146.8	143.6	2.179

The relationship between the dependent and independent variables is further elucidated by constructing the response-surface plot. The three dimensional (3D) response-surface graphs generated by the Design-Expert software (trial version 9.0.1) for the most statistically significant variables on the evaluated parameters are presented in Figure 1. The 3D response-surface curves are used for studying the interaction patterns. On the basis of the 3D response-surface graphs, it can be said that the lipid and drug concentrations and the rotation speed of rotary evaporator produce a significant effect on mean particle size, % EE, and %DR. The graphs show that with increasing the concentration of lipid in formulation, mean particle size and % EE increased, but % DR decreased, and vice versa. In case of the second factor (rotation speed), it was responsible for minimum mean particle size and higher %EE and low % DR.

Figure 1. Surface plots showing the effect of variables on (A) particle size, and (B) % Entrapment Efficiency, and (C) % Drug Loading.

Differential scanning calorimetry

The thermal behavior of quercetin, phosphatidylcholine, cholesterol and the physical mixture were studied using DSC. The DSC thermogram of quercetin showed an endotherm at 119.24°C and 145.33°C. For cholesterol, the melting process took place with the maximum peak at 149.08°C. The thermogram of the physical mixture was almost the overlap of each individual component, except for some slight differences. The DSC thermogram of the physical mixture showed the peak of cholesterol at ∼147.5°C, and the integrated peak of quercetin at ∼172.38°C. The DSC spectrum of the complex reveals the characteristic absence of the melting peak of quercetin at 295.23°C, as shown in Figure 2A'C. The area under the curve for the quercetin thermogram was less as compared to that of quercetin alone. This may be due to the melting of the lipid components and their interactions with quercetin. Partial incorporation of quercetin in the melted lipid is likely. The complete disappearance of the drug's endothermal peak was instead observed for systems obtained by freeze-drying. This phenomenon can be assumed as proof of interactions between the components of the respective binary systems. This can be considered as indicative of drug amorphization and/or inclusion complex formation.

Figure 2. DSC thermograms of quercetin, cholesterol and physical mixture.

Transmission electron microscopy

In order to investigate the morphology and size of the optimal quercetin-loaded liposome, TEM was used. The TEM photomicrograph of the quercetin-loaded liposome is shown in Figure 3. These liposomes seem to be pear-like and small. The optimized quercetin-loaded liposomes in the formulation showed a nonspherical shape with a particle size of about 100 nm, which is almost the same as the results determined using the Zetasizer, and the formulation consists of a mixed population of unilamellar and small multilamellar vesicles (around 100 nm in diameter).

Figure 3. TEM image of optimized quercetin-loaded liposome.

Particle size, PDI, and zeta potential of the quercetin-loaded liposomes

Optimized preparations, as predicted by the experimental designing, were successfully prepared. The particle size, PDI, and zeta potential of the quercetin-loaded liposomes are depicted in Table I. The mean particle sizes, PDI values, and zeta potentials of the nine formulations in total were obtained, and seen to be in the range of 146–591 nm, 0.067–0.372, and − 10.6 to − 17.7 mV, respectively.

Percentage entrapment efficiency

The values for % EE of the quercetin-loaded liposomes are depicted in Table I. It can be seen that the linear effect of phosphatidylcholine and quercetin concentration was significant (0.0065). The effect of independent variables on quercetin-loaded liposomes is that at higher quercetin concentration, EE was increased, due to which more quercetin was encapsulated into the vesicles. Besides, increased ratio of phosphatidylcholine increased the EE. The values for % EE of all the nine formulations were obtained, and seen to be in the range of 78.14 ± 0.56%–86.5% ± 1.21%, respectively. The nature of the drug plays a significant role in the determining the EE, because the drug is encapsulated in the lipid phase. Quercetin is a lipophilic drug, and its solubility is also higher in methanol (conclusion drawn from the study of partition coefficient), therefore the %EE was found to be noticeably higher.

In vitro release studies

The in vitro release curve of the optimal quercetin-loaded liposomal suspension in a phosphate buffer of pH 7.4 at 35 ± 0.5°C, is shown in Figure 4. The cumulative % DR of the optimized quercetin-loaded liposomal suspension was 75.09% in 24 h. The in vitro release curve showed the initial burst release with about 40% of the drug released during the first two hours; after that, the release was sustained from the optimized quercetin-loaded liposomes. The burst release occurred due to the presence of the free quercetin in the external phase and on the surface of the liposome. The lipophilic nature of the quercetin could be the reason for the sustained release of the drug from the internal lipid phase after the initial burst release.

Figure 4. Release profile of the optimized quercetin-loaded liposome in phosphate buffer of pH 7.4, at 37°C.

Storage stability studies

Storage stability studies were conducted on optimized liposomes using the particle size, zeta potential, and EE as the prime parameters. There was a negligible or slight increase in the particle size during the three-month storage at 4°C and 25°C, from 146.8 ± 1.65 nm to 150.61 ± 1.68 nm and 140.25 ± 1.61 nm, respectively. In case of the zeta potential, similar results were seen for three-month storage at 4°C and 25°C, from a value of − 10.6± (− 1.25) mV to − 17.7± (− 2.36) mV and − 14.2± (− 1.32) mV, respectively. The % EE of the optimized batch was initially found to be 86.5% ± 1.21%, while that after three-month storage at 4°C and 25°C was found to be78.14% ± 0.56% and 78.5% ± 0.48%, respectively, indicating that the drug can be retained within the liposomes for a sufficient period of time. On storage of the liposomes, there was no significant change occurring in the size, zeta potential, and % EE of the liposomes. Hence, they were found to be stable under the storage conditions tested (at 4°C and 25°C) for a total period of 3 months.

Conclusions

The effect of the temperature of the water bath and the rotation speed of the rotary evaporator in preparing quercetin-loaded liposomes was studied. The quercetin-loaded liposomes were optimized using the miscellaneous design-response surface methodology, by fitting a second order model to the response data. Second-order polynomial models were obtained for predicting particle size and encapsulation efficiency. It was observed that increasing the temperature of the water bath increased the particle size and entrapment efficiency. The effect of the two variables, i.e. temperature of the water bath (X₁) and the speed of rotation (X₂), with their interactions, were evaluated and modeled. The best maximum of entrapment efficiency (86.5% ± 1.21%) and minimum particle size (146.8 nm ± 1.65 nm) were found at a water bath temperature of 46°C and rotation speed of 75 rpm. The release profile of the liposomes produced was investigated in a phosphate buffer media, and it showed prolonged release during 24 h, with up to 75.09% release. The drug release behavior of the liposomes exhibited a biphasic pattern, with the burst release at the initial stage and sustained release subsequently. These results indicated that the liposomes obtained in this study could potentially be exploited as a carrier with an initial dose and prolonged release when therapeutically desired. The quercetin-loaded liposomes showed an acceptable stability.

Acknowledgments

The authors are thankful to the Director, University Institute of Pharmacy, Pt. Ravi Shankar Shukla University, Raipur, Chhattisgarh, India, for providing necessary infrastructural facilities.

Declaration of interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

The authors would like to thank and acknowledge UGC-MRP-41-748-2012, UGC-RA-70371/2012, DST-FIST and UGC-SAP for providing financial support for this work.