preparation and optimization of quercetin-loaded liposomes for wound healing, using response surface...

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Artifi cial Cells, Nanomedicine, and Biotechnology, 2014; Early Online: 1–7Copyright © 2014 Informa Healthcare USA, Inc.ISSN: 2169-1401 print / 2169-141X onlineDOI: 10.3109/21691401.2014.975238

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

Rajendra Jangde & Deependra Singh

University Institute of Pharmacy, Pt. Ravi Shankar Shukla University, Raipur, Chhattisgarh, India

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) infl ammation, 3) granulation, 4) fi brogenesis, 5) neo-vascularization, 6) wound contrac-tion, and 7) epithelialization (Gupta et al. 2006, Hurler and Skalko-Basnet 2012). Quercetin is a biofl avonoid polyphe-nolic phytoconstituent having potential anti-infl ammatory and anti-oxidant properties. It directly inhibits the various proinfl ammatory agents and has also been reported for its potential immunomodulatory, gastro-protective, anti-tumor, cardio-protective, and bacteriostatic eff ects (Fan et al. 2003, Rice-Evans et al. 2007). Quercetin has been widely used as a therapeutic agent in diff erent disease conditions. Apart from its potential therapeutic eff ects, it suff ers from some limita-tions like low aqueous solubility, and low bioavailability. Th e low bioavailability of this drug requires its administration at a high concentration to produce therapeutic eff ects (Park

et al. 2013). Th erefore, some novel carriers are required to overcome the problems mentioned above. Vesicular carriers can play a vital role in enhancing the solubility and bioavail-ability of the active moiety; with these advantages off ered 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 bilay-ered 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. Th e optimization of liposomes plays a major role in designing the appropriate formulations for novel vesicular systems. Th e response sur-face 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 bioavail-ability of drug (Varde et al. 2013). In this work, quercetin-loaded liposomes were prepared by the thin fi lm hydration method using a vacuum rotary evaporator. Th e optimization of the liposomal formulation was done using RSM combined with miscellaneous designs. Th e variables selected were the temperature of the water bath (X 1 ), and the rotation speed of the rotatory evaporator (X 2 ); the response variables were the drug release (DR) as Y 1 , mean particle size diameter (MD) as Y 2 , and the entrapment effi ciency (EE) as Y 3, of the liposome. Th e levels for these variables were determined from the preliminary trials. Furthermore, the selected for-mulation had maximum entrapment of drug and minimum in vitro release in 24 h.

Materials and methods

Materials Quercetin, phosphatidylcholine, and cholesterol were pur-chased from HiMedia Chemicals (Mumbai, India). HPLC grade solvents were purchased from Merck (Mumbai,

Correspondence: Dr. Deependra Singh, Assistant Professor, University Institute of Pharmacy, Pt. Ravi Shankar Shukla University, Amanaka G.E. Road, Raipur, Chhattisgarh 492010, India. Mobile: 09302910443. Offi ce: 07712262832. E-mail: [email protected]

(Received 26 September 2014 ; accepted 7 October 2014 )

Abstract The basic objective of this study was to prepare quercetin-loaded liposomes by the thin fi lm 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 fi nalized 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 effi ciency (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

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2 R. Jangde & D. Singh

India). All other materials and solvents used were of analytical grade.

Preparation of quercetin-loaded liposomes Liposomes were prepared using the thin-fi lm hydration method (Ruozi et al. 2005). Phospholipid, cholesterol and quercetin, were dissolved in 25 mL of a chloroform – metha-nol mixture (4:1) using a fi xed molar ratio. Th e 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 fi lm. Th e fi lm was hydrated with phosphate buff er (pH 7.4) for 1 h at room tem-perature, which was above the lipid transition temperature. Th e vesicle dispersion was then homogenized using a probe sonicator (FS-500, Frontline), passed through a 0.45 μ m fi l-ter (Minisart CA 26 mm), and stored until use.

Optimization of formulation parameters for liposomes As a preliminary study, the formulation technique was opti-mized 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 fi lm varies depend-ing upon the rotational speed of the evaporating fl ask. Th e optimum speed was found to be 75 rpm. Th e fi lm obtained after rotary evaporation was kept overnight under vacuum, to be dried and removed. Further, it was observed that liposomes prepared using a phosphate buff er 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).

Th e above two observations were further corroborated by the values for entrapment effi ciency (EE) of the formulation. Th e optimized formulation parameters were then used to formulate further batches. Suitable batches were then pre-pared to study in detail the interactions of quercetin with the lipids, and their eff ects on the entrapment and the particle size of the fi nal formulation, using experimental designing techniques.

Experimental design A 3-level factorial-response surface methodology (3LF-RSM) was used to study the eff ect of diff erent variables dependent on the properties of the formulation, like mean particle size, percentage drug release (% DR), and entrapment effi ciency (% EE) of the prepared liposomes, and independent variables

including temperature (X 1 ) and rotation speed (X 2 ) (Table I). Th e best fi tted model for statistical analysis was considered signifi cant when the P value was less than 0.05. Th e predicted R 2 value and ANOVA were pursued to confi rm the best-fi t of the model. Th ree-dimensional (3D) surface plots were used to establish the relationship between independent variables and dependent variables (response).Th e desirability func-tions 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).

Diff erential Scanning Calorimetry analysis Diff erential scanning calorimetry (DSC) analysis was per-formed to ascertain the absence of potential interactions between the components of the liposomal formulation and quercetin, to confi rm the formation of liposomes. Th e pos-sibility of any interaction between the phospholipid, quer-cetin, and liposomes, during preparation, were assessed by thermal analysis of the liposome samples. A model DSC (Perkin Elmer Jade, California, USA, Department of Pharma-ceutical Sciences, Dibrugarh University, Assam, India) was used to determine melting point and enthalpy for the lipo-somal formulation. A sample equivalent to approximately 5 mg was placed in an aluminum pan and DSC analysis was carried out at a nitrogen fl ow rate of 20 mL/min and a heat-ing rate of 5 ° C/min, from 50 ° C to 305 ° C. An empty alumi-num pan was placed on the reference platform. Th e thermal analysis of sample parameters in the DSC thermogram are the onset temperature (T 0 ), the peak temperature or the gel to liquid-crystalline transition ™ , the end-set temperatures (Te and T 0 ), and enthalpy change of the transition (Begum et al. 2012, Epstein et al. 2008).

Transmissions electron microscopy Th e 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). Th e sur-face morphology and size of the optimized liposomes were analyzed by transmission electron microscopy (TEM). Th e optimized aqueous dispersion of the quercetin-loaded lipo-somes 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. Th e magnifi ca-tion for the TEM images was 150,000 x (Liu and Wu 2010).

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

Coded level

X1 X 2

Actual level

X 1 X 2

Responses

Polydispersity

Index (PDI)

Zeta Potential

(mV) Form code Drug Release

(%) Particle Size

(nm) Entrapment

Effi ciency (%)

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)

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Optimization of quercetin-loaded liposomes 3

Determination of mean particle size, PDI and zeta potential of the quercetin-loaded liposomes Th e 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 cor-relation spectroscopy (PCS) using a Zetasizer (Nano ZS 90, Malvern Instruments, UK) at a temperature of 25 ° C and a 90 0 scattering angle. Th e liposomal formulation was diluted with double distilled water, to weaken opalescence before mea-surements. Th e 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 fi eld strength of 23 V/cm. Th ree independent measurements were performed for each sample. Th e samples were analyzed 24 h after preparation (Jin et al. 2006).

Determination of percentage of entrapment effi ciency of quercetin-loaded liposomes Th e % 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). Th e aqueous dispersion of the quer-cetin-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. Th e concentration of quercetin in the aque-ous 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. Th e solvents were mixed, fi ltered through a membrane fi lter of 0.45 micron pore, and degassed before use. Th e 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). Th e fl ow rate in the mobile phase was 1mL/min. Quercetin was analyzed at a wavelength of 372 nm. Th e %EE was calcu-lated by the following equation:

% Entrapment effi ciency �

weight of quercetin used�weight of free quercetin

� 100weight of quercetin used

In vitro drug release from quercetin liposomes In vitro release of quercetin from optimized quercetin liposomes was determined by the diff usion cell apparatus (EMFD-08 Orchid scientifi c & Innovative India Pvt. Ltd. Nasik, Maharashtra, India) using a dialysis membrane (molecular weight cutoff 10,000 Da). Th e dialysis mem-brane was kept in double distilled water for 24 h before being utilized in the diff usion cell apparatus. Th e aqueous dispersion of quercetin liposomes (2 mL) was placed in the donor compartment, the receptor compartment was fi lled with the dissolution medium (phosphate buff er 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 with-drawn at intervals of 0, 1, 2, 3, 4, 5, 6, 12, 16 and 24 h. Th ey were fi ltered after withdrawal and the apparatus was imme-diately replenished with 2 mL of the fresh buff er medium.

Th e aliquots withdrawn were diluted suffi ciently and 20 μ l solution was injected into the HPLC system for analysis, at a wavelength of 372 nm (Maitani et al. 1990).

Storage stability studies Th e storage stability studies were carried out with the opti-mized quercetin-loaded liposomes. A 10 mL sample of quercetin- liposome dispersion with 2 mg/mL drug concen-tration was taken into glass vials and stored at 4 ° C and 25 ° C for 3 months. Th e 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 eff ect of the rotation speed of the rotary evaporator and the temperature of the water bath, by using the thin fi lm hydra-tion method, which is an easy method that can be utilized in the laboratory production of liposomes. For homoge-neous distribution of quercetin inside the lipid phase, 25 mL chloroform/methanol mixture (4/1) was incorporated. Th e 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 Th e observed responses of nine formulations were fi tted to various models by using Design-Expert software trial ver-sion 9.0.1. It was seen that the quadratic models were the best-fi t for the responses studied, that is, mean particle size, % EE, and % DR. Th e quadratic equations generated for the responses are given as follows:

Particle size � � 144.44 – 56.87 X 1 � 64.23 X 2 � 18.75 X 1 X 2 � 238.63 X 1 1 � 42.63 X 2 2

% EE � � 78.48 � 6.58 X 1 � 6.68 X 2 � 1.65 X 1 X 2 � 0.58 X 1 1 � 10.82 X 2 2

% DR � � 58.06 – 1.40 X 1 -3.18 X 2 � 0.45 X 1 X 2 � 10.22 X 1 1 � 0.92 X 2 2

Where X 1 and X 2 represent the coded values of the tem-perature of the water bath and the rotation per minute of the rotary evaporator, respectively. Th e positive value of a factor in the above equations points out the enhancement of that response, and vice versa. All values of the correlation coeffi -cient ( R 2 ), SD, percentage coeffi cient of variation, and results of ANOVA are shown in Table II. A value of R 2 and results of ANOVA for the dependent variables confi rmed that the model was signifi cant for the response variables observed.

Experimental design Based on the preliminary experiments and our previous studies, two factors (temperature of the water bath and rota-tion speed of the rotary evaporator) were identifi ed as key factors responsible for % EE, % DR, and mean particle size of the liposome. Th e temperature of the water bath was cho-sen because the temperature reaches the phase transition

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factor (rotation speed), it was responsible for minimum mean particle size and higher %EE and low % DR.

Diff erential scanning calorimetry Th e thermal behavior of quercetin, phosphatidylcholine, cholesterol and the physical mixture were studied using DSC. Th e DSC thermogram of quercetin showed an endo-therm at 119.24 ° C and 145.33 ° C. For cholesterol, the melting process took place with the maximum peak at 149.08 ° C. Th e thermogram of the physical mixture was almost the overlap of each individual component, except for some slight diff er-ences. Th e 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. Th e DSC spectrum of the com-plex reveals the characteristic absence of the melting peak of quercetin at 295.23 ° C, as shown in Figure 2A – C. Th e area under the curve for the quercetin thermogram was less as compared to that of quercetin alone. Th is 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. Th e complete disappearance of the drug ’ s endo-thermal peak was instead observed for systems obtained by freeze-drying. Th is phenomenon can be assumed as proof of interactions between the components of the respective binary systems. Th is can be considered as indicative of drug amorphization and/or inclusion complex formation.

Transmission electron microscopy In order to investigate the morphology and size of the optimal quercetin-loaded liposome, TEM was used. Th e TEM photomicrograph of the quercetin-loaded liposome is shown in Figure 3. Th ese liposomes seem to be pear-like and small. Th e 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

temperature of the water bath system, the water molecules penetrate into the lattice between the phospholipid mol-ecules which spontaneously form a multi-bilayered struc-ture, and the rotation speed of the rotary evaporator aff ects the lipid fi lm formation. Contact of the fi lm with hot water is increased by lowering the speed, and with a higher speed, the fi lm is not formed properly because of a lack of time for the lipid to form a fi lm, and the proper liquid to gel transi-tions of the lipid do not occur. Both of these may have caused the uneven distribution of heat, leading to the formation of an uneven fi lm.

Predicted optimum ranges of the independent variables are listed in Table III. Th e results that fi t 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, sug-gesting that the optimized formulation was trustworthy and rational.

Th e relationship between the dependent and independent variables is further elucidated by constructing the response-surface plot. Th e three dimensional (3D) response-surface graphs generated by the Design-Expert software (trial ver-sion 9.0.1) for the most statistically signifi cant variables on the evaluated parameters are presented in Figure 1. Th e 3D response-surface curves are used for studying the inter-action patterns. On the basis of the 3D response-surface graphs, it can be said that the lipid and drug concentra-tions and the rotation speed of rotary evaporator produce a signifi cant eff ect on mean particle size, % EE, and %DR. Th e 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

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

Predicted optimum range

S. No. Responses variable X 1 X 2 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

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

Signifi cant0.9718 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

Signifi cant0.9764 36.29 10.93

Residual 3 3949.82 1316.61 Total 8 1.670E � 005

% Entrapment Effi ciency Model 5 773.61 154.72 37.77 0.0065

Signifi cant0.9844 2.02 2.82

Residual 3 12.29 4.10 Total 8 785.90

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determined using the Zetasizer, and the formulation consists of a mixed population of unilamellar and small multilamel-lar vesicles (around 100 nm in diameter).

Particle size, PDI, and zeta potential of the quercetin-loaded liposomes Optimized preparations, as predicted by the experimental designing, were successfully prepared. Th e particle size, PDI, and zeta potential of the quercetin-loaded liposomes

are depicted in Table I. Th e mean particle sizes, PDI val-ues, 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 effi ciency Th e values for % EE of the quercetin-loaded liposomes are depicted in Table I. It can be seen that the linear eff ect of phos-phatidylcholine and quercetin concentration was signifi cant

Design-Expert® SoftwareFactor Coding: Actualdrue release (%)

Design points above predicted valueDesign points below predicted value75.09

57.15

X1 = A: temp X2 = B: rotation speed

–1 –0.5

00.5

1

–1 –0.5

0 0.5

1

55

60

65

70

75

80 (A)

(B)

(C)

drue

rele

ase

(%)

A: temp (0C)B: rotation speed (rpm)

58.43458.434

Design-Expert® SoftwareFactor Coding: Actualparticle size (nm)


146.8


–1–0.5

00.5

1

–1 –0.5

0 0.5

1

100

200

300

400

500

600

parti

cle

size

(nm

)


146.8146.8

Design-Expert® SoftwareFactor Coding: ActualEE (%)


54.6


–1

–0.5

0

0.5

1

–1

–0.5

0

0.5

1

50

60

70

80

90

EE (%

)


80.32780.327

Figure 1. Surface plots showing the eff ect of variables on (A) particle size, and (B) % Entrapment Effi ciency, and (C) % Drug Loading.

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is a lipophilic drug, and its solubility is also higher in metha-nol (conclusion drawn from the study of partition coeffi cient), therefore the %EE was found to be noticeably higher.

In vitro release studies Th e in vitro release curve of the optimal quercetin-loaded liposomal suspension in a phosphate buff er of pH 7.4 at 35 � 0.5 ° C, is shown in Figure 4. Th e cumulative % DR of the optimized quercetin-loaded liposomal suspension was

(0.0065). Th e eff ect of independent variables on quercetin-loaded liposomes is that at higher quercetin concentration, EE was increased, due to which more quercetin was encapsu-lated into the vesicles. Besides, increased ratio of phosphati-dylcholine increased the EE. Th e 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. Th e nature of the drug plays a signifi cant role in the determining the EE, because the drug is encapsulated in the lipid phase. Quercetin

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

0102030405060708090

0 5 10 15 20 25 30

Dru

g R

elease %

Time in hrs

quercetin liposomes

Figure 4. Release profi le of the optimized quercetin-loaded liposome in phosphate buff er of pH 7.4, at 37 ° C. Figure 3. TEM image of optimized quercetin-loaded liposome.

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Acknowledgments

Th e 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

Th e authors declare that there are no confl icts of interest regarding the publication of this paper.

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

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75.09% in 24 h. Th e in vitro release curve showed the initial burst release with about 40% of the drug released during the fi rst two hours; after that, the release was sustained from the optimized quercetin-loaded liposomes. Th e burst release occurred due to the presence of the free quercetin in the external phase and on the surface of the liposome. Th e 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.

Storage stability studies Storage stability studies were conducted on optimized lipo-somes using the particle size, zeta potential, and EE as the prime parameters. Th ere 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. Th e % 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 suffi cient period of time. On storage of the liposomes, there was no signifi cant 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

Th e eff ect of the temperature of the water bath and the rotation speed of the rotary evaporator in preparing quercetin-loaded liposomes was studied. Th e quercetin-loaded liposomes were optimized using the miscellaneous design-response surface methodology, by fi tting a second order model to the response data. Second-order polyno-mial models were obtained for predicting particle size and encapsulation effi ciency. It was observed that increas-ing the temperature of the water bath increased the par-ticle size and entrapment effi ciency. Th e eff ect of the two variables, i.e. temperature of the water bath (X 1 ) and the speed of rotation (X 2 ), with their interactions, were evalu-ated and modeled. Th e best maximum of entrapment effi -ciency (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. Th e release profi le of the liposomes produced was investigated in a phosphate buff er media, and it showed prolonged release during 24 h, with up to 75.09% release. Th e drug release behavior of the liposomes exhibited a biphasic pattern, with the burst release at the initial stage and sustained release subsequently. Th ese 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. Th e quercetin-loaded liposomes showed an acceptable stability.

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preparation and optimization of quercetin-loaded liposomes for wound healing, using response surface...

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