Medical

Communication

Biosci. Biotech. Res. Comm. 10(1): 151-160 (2017)

Preparation methods of nanoliposomes containing

Zataria

multi ora

essential oil: A comparative study

Mohammad Hosein Arabi

, Azam Mirzapour

, Hora Chabok

, Mehdi Sha ee

Ardestani

and Mostafa Saffari

Department of Biochemistry, School of Medicine, Kashan Uiversity of Medical Sciences, Kashan, Iran

Department of Radiopharmacy, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Herbal Medicine Research Center & Department of Pharmaceutics, School of Pharmacy, Branch of

Pharmaceutical Sciences. Islamic Azad Universty, Tehran, Iran

ABSTRACT

Nowadays there is a substantial interest on the biological activities of essential oils. Zataria multi ora essential oil

has a wide spectrum of pharmacological activities. However, essential oils are unstable and susceptible to degradation

by oxygen, light and temperature and also low penetration in transdermal administration. Therefore, nanoliposo-

mal systems can be used as promising strategies to overcome these limitations due to their unique set of properties:

Nanoliposomes were prepared via two different methods based on thin  lm hydration method, including extrusion

and sonication methods. The physical properties of nanoliposomes such as particle size, polydispersity index, zeta

potential, encapsulation ef ciency and stability were also determined. In comparison of two methods, sonicated

nanoliposomes had smaller mean particle size and better dispersity while mean encapsulation ef ciency in extruded

ones were higher. It can be deducted by consideration of signi cance level; physicochemical properties of the vesicles

were strongly in uenced by essential oil and cholesterol concentration besides the preparation method.

KEY WORDS: NANOLIPOSOME; ZATARIAMULTIFLORA; ESSENTIAL OIL; THIN FILM HYDRATION; SONICATION

151

ARTICLE INFORMATION:

*Corresponding Author: mostafa.saffary@gmail.com

Received 19

Jan, 2017

Accepted after revision 22

March, 2017

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Online Contents Available at: http//www.bbrc.in/

INTRODUCTION

Zataria multi ora is one of the lamiacea family herbs

with remarkable pharmacological properties. It natively

grows in Iran, Pakistan, and Afghanistan (Saedi Dezaki

et al., 2016). It has various therapeutic effects such as

antiseptic, antispasmodic, carminative, expectorant, anti-

in ammatory, antiparasitic, spasmolytic, antiviral, anti-

bacterial, antifungal and antioxidant properties (Sunar et

al., 2009).

Most of these properties are related to the main

152 PREPARATION METHODS OF NANOLIPOSOMES CONTAINING

ZATARIA MULTIFLORA BOISS

ESSENTIAL OIL BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Mohammad Hosein Arabi et al.

constituents of its essential oils, which are thymol and

carvacrol and signi cant quantities of phenolic monoter-

penes (Misaghi & Basti., 2007; Ali et al., 2000). Essential

oils are con rmed to possess wide spectrum of pharma-

cological properties, besides their classical roles as natu-

ral food additives, such as the antioxidant, antibacterial,

antifungal and anti-in ammatory activities (Shahsavari

et al., 2008; Edris, 2007). Unfortunately, essential oils are

biologically unstable, poorly soluble in water and are

very sensitive to environment (Martin et al., 2007). All

these obstacles restrict the application of essential oils as

candidates for pharmatherapeutic treatments. Currently,

nanoencapsulation of these oils in drug delivery systems,

among which is the use of liposomal encapsulation,

have

been proposed to improve solubility, stability and ef cacy

of essential oil-based formulations (Belay et al., 2011; Saf-

fari

et al., 2016).

Liposomes represent versatile and progressive nano

delivery system for extensive range of biologically active

compounds (Hofheinz et al., 2005). Liposome is a lipoidal

vesicle composed of a bilayer membrane that have been

developed to deliver drug to speci c site in the body for

more than four decades (Chetanachan et al., 2008); El-

Samaligy et al., 2006). Because of biodegradation, non-

toxicity, biocompatibility, non-immunogenicity,

as well

as superior ef cacy, they are excellent carrier systems for

a variety of applications, and in particular for essential

oils (Sinico et al., 2005; Liolios et al., 2009; Saffari

et al.,

2016). Encapsulation in liposome protect the essential oil

from light, air and humidity, increases the solubility of

the oil , enhancing the bioavailability of this drug, drug-

targeting and makes the oil easier to handle (Wen et al.,

2010; Valenti et al., 2001). The aim of this study was to

prepare phosphatidyl choline based nanoliposomes via

optimization the concentrations of essential oil, choles-

terol and lipid composition through different methods for

encapsulating zataria multi ora essential oil. Furthermore,

to compare these methods via physicochemical properties

of nanoliposomes including size, PDI index, zeta potential,

encapsulation ef ciency and drug release pro le.

MATERIALS AND METHODS

Lecithin, Egg PC, DOTAP were purchased from LIPOID

(Germany). Cholesterol was from Sigma-Aldrich Co.

The solvents (chloroform, methanol and ethanol) were

purchased from commercial source and were in analyti-

cal grade. The essential oils were obtained from Barij

essence Co. in Kashan, Iran.

PREPARATION OF LIPOSOME

Liposomes can be formulated and processed to vary in

size, composition and charge (Akbarzadeh et al., 2013).

For production of different types of liposomes, many

methods are available (Meure et al., 2008). All Prepa-

ration methods can be simpli ed as to involve three

basic steps: 1) dissolve the lipids in aqueous media in

order to forming liposomes. 2) Puri cation of prepared

liposomes. 3) Analysis of  nal product. Liposomes in our

study prepared with three different methods as describe

here:

1. Thin  lm hydration

The most conventional and classical method, known

by hydration of dried phospholipid  lms (Bangham et

al., 1965). Multilamellar vesicles (MLVs) were prepared

based on the thin  lm hydration method. Brie y the

precise amount of lecithin, cholesterol and essential oil,

according to several ratios of essential oil to total lipids,

molar ratio of cholesterol to lecithin and lipid compo-

sition (DOTAP), were dissolved in chloform-methanol

(2:1, v/v) in a 500 ml round-bottomed  ask. The organic

solvent was eliminated by rotary evaporator (heidolph

Hei-VAP Germany) under reduced pressure and high

vacuum for 2 hour until a thin  lm was formed on the

walls (above the lipid phase transition temperature, Tc).

Then the obtained lipid  lm was dispersed in Phosphate

Buffer Saline solution (PBS, pH=7.4). This suspension

was allowed to hydrate for 1.5 hour. Finally milky white

suspension is formed. During the process, the conditions

such as speed (120 rpm) and temperature (above the Tc

of lecithin) for conventional liposomes were maintained

constant.

a. Concentration of essential oil

The total lipid concentration to prepare liposomes was

50 mM. Different ratios of essential oil to total lipid (1/2,

1/3, 1/4) with a constant molar ratio of cholesterol to

phosphatidyl choline (1: 1) were prepared.

b. Concentration of cholesterol

After  nding optimum concentration of essential oil

to total lipid, cholesterol concentration was optimized

by various concentrations as 7.1, 6.4, 4.83 mg/ml on a

weight basis and 1:1, 1:2, 1:3 (molar ratio of Cholesterol:

PC) in the overall formulation.

c. Charge-inducing lipids

EO-loaded cationic liposomes were formulated with pc,

1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),

and cholesterol at a 1:1:1 molar ratio of E80: DOTAP:

CH.

2. Sonication method

Sonication is a simple method for reducing the size of

liposomes to the nanoscale and manufacture of nanoli-

posomes. Liposomal suspension of MLVs transferred

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS PREPARATION METHODS OF NANOLIPOSOMES CONTAINING

ZATARIA MULTIFLORA BOISS

ESSENTIAL OIL 153

Mohammad Hosein Arabi et al.

to a tube and placed in ice bath. Then the suspension

was sonicated using prob sonicator (Hielscher UP400S,

Germany).Vertically, the probe of a sonicator was fully

immersed in the middle of the volume. The liposomal

suspension was exposed to ultrasonic irradiation with

an output of 70 Watt amplitude and duration of continu-

ous 20 minute (6 times for 5 min). The energy input into

liposomal dispersion is very high in this method.

3. Extrusion method

To obtain large unilamellar vesicles, the liposomal dis-

persion up to 3 mL was extruded three times through

a polycarbonate membrane (100, 200 nm pore size)

at above the lipid transition temperature by using an

extruder to give a translucent solution.

Different formulation are listed in table 1 based on

their method of preparation and their components and

composition.

Puri cation of Liposomes

Entrapped vesicles were separated from non-entrapped

vesicles using centrifugation method (Shashi et al.,

2012). Vesicular dispersions were centrifuged at 10000

rpm for 10 minutes (laboratory centrifuge Hettich Uni-

versal 320 R). The supernatant was removed and the

liposomes were reconstituted with PBS buffer. Concen-

tration of essential oil in both fractions was determined.

PHYSICOCHEMICAL CHARACTERIZATION OF

LIPOSOMES

Particle Size distribution

Mean vesicle size and polydispersity index (PDI) was

determined by using Zetasizer (nanoZs Malvern ZEN

3600), which the principle is based on the Brownian

motion of particles in medium. Dynamic light scattering

is a simple and rapid method to determine the particle

size and size distribution of liposomes.

Zeta potential () determination

Zeta potential is another important factor that is respon-

sible for a description of the liposome surface charge and

predicts the stability. Charge on loaded vesicles surface

and average zeta potential was determined using Zeta-

sizer ZEN 360 Malvern Instruments (Bhatia et al., 2004).

Imaging of liposomes by Scanning Electron Microscopy

(SEM)

The shape and morphology of drug-loaded liposomal

formulation were observed using a KYKY-EM3200 scan-

ning electron microscopy (SEM, KYKY Instruments,

China). Sample was dispersed on glass slide and gold

paste used as  lament and then viewed using an acceler-

ating voltage of 20 kilovolt at different magni cations.

Images of liposomes exhibited the diameter and

size of vesicles and this is in agreement with the

results of Zetasizer

(Figure 4).

Evaluation of the Encapsulation ef ciency

In case of puri ed liposomal suspension, after the cen-

trifugation, by the sediment, the quantity of essential

oil was measured using a UV Visible Spectrophotometer

at  = 270 nm. The encapsulation ef ciency (EE %) of

essential oil was calculated using the following formula:

S3/ L3

Encapsulation Ef ciency = ---------- × 100

S2/ L2

Which in this equation; S3= EO amount in sediment &

L3= phospholipid amount in sediment,

S2= EO amount before centrifuged & L2= phospho-

lipid amount before centrifuged

In vitro essential oil release studies

Release studies were performed using dialysis membrane

method. In vitro release was done for selected formula-

Table 1. formulation based on method of preparation and composition

Method Composition EO /Total lipid CH:PC

Thin  lm hydration

PC+CH+EO

0.5, 0.33,0.25

0.33

0.5, 0.33, 0.25

0.33

0.5, 0.33, 0.25

0.33

1:1

1:2,1:3

1:1

1:2,1:3

1:1

1:2,1:3

Sonication

PC+CH+EO

PC+CHO+EO

Extrusion

PC+CH+EO

PC: phosphatidyl choline; CH: cholesterol; EO: essential oil

Mohammad Hosein Arabi et al.

154 PREPARATION METHODS OF NANOLIPOSOMES CONTAINING

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ESSENTIAL OIL BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 1. A is effect of essential oil concentration on Size of nanoliposomes and B is effect of cholesterol content

on Size of nanoliposomes

FIGURE 2. A is effect of essential oil concentration on PDI index and B is effect of cholesterol content on PDI

index of nanoliposomes

tion. In brief, 1000 μL of the 7.1 mg/mL essential oil

encapsulated liposome suspension was added in a dialy-

sis bag (MWCO 12kDa, Thermo Fisher Scienti c). The

dialysis system was suspended in a release volume of 100

mL PBS at 25°C and rotated at 100 rpm (1:100 dilution

relation between donor and acceptor compartments). At

scheduled intervals, 1 ml of the release medium was col-

lected for the UV- spectrophotometric assay. The same

volume of fresh PBS buffer at the same temperature was

added immediately to maintain constant release volume.

The length of the dialysis tubing was kept consistent

for all methods to ensure that the surface area available

for dialysis remained constant. To ensure that dilution

between the donor and acceptor compartments provided

sink conditions, a 1:100 dilution study was conducted

and release volume was set at 100 mL PBS pH 6.5.

STATISTICAL ANALYSIS

Result has been reported as mean ± standard deviation

of three times repetition assays for each method. The

mean values compared by T-Test and one-way analy-

sis of variance (ANOVA), and statistical signi cance

declared at P<0.05.

RESULTS & DISCUSSION

MEAN PARTICLE SIZE

The results showed that the mean size of the vesicles

was strongly affected by EO and CH concentration,

besides of preparation method. Since the mean size

of particles prepared by thin  lm method was around

Mohammad Hosein Arabi et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS PREPARATION METHODS OF NANOLIPOSOMES CONTAINING

ZATARIA MULTIFLORA BOISS

ESSENTIAL OIL 155

a few microns, 1426.3± 50 nm, particle size reduc-

tion in nano scale by two methods was quite impres-

sive. According to the table 2, the mean size soni-

cated oil loaded vesicles ranged from about 97.8 to

about 210 nm, while extruded liposomes were larger

(256–489 nm). It’s clearly seen that as the amount of

the essential oil increases, size of vesicles increases. Also

with decrease of cholesterol content, size of liposomes

reduced (Figure 1).

Polydispersity index

All MLV liposomes prepared by thin  lm method had

high PDI index (>0.4), which re ects the heterogeneity

and dissimilarity of their colloidal system. While the size

distribution of sonicated and extruded nanoliposomes

was relatively narrow. Nanoliposomes prepared by the

sonication method were most homogenous in size dis-

tribution than liposomes prepared by two other methods

(Table 2).

As seen in  gure 2 (A) by increasing the concentration

of essential oil in two methods PDI was increased. In the

case of cholesterol,  gure 2 (B), in extruded method PDI

decreases with increased cholesterol levels, this trend is

more imperceptibly in sonication method.

Zeta potential

The zeta potential measurement of liposomal sam-

ple containing DOTAP was +14.7 mV. Other liposomes

showed neutral charge that was owing to lack of charge

ingredients.

Encapsulation Ef ciency

The encapsulation ef ciency greatly depends on lipo-

somal content, lipid concentration, method of prepara-

Table 2. Effect of preparation parameters on Zataria multi ora essential oil Loaded

nanoliposomes in two different preparation methods

Essential oil

concentration mg/ml

Size (nm) PDI EE%

Extrusion method

14.2 489.1±21 0.38±0.15 53.66±0.55

9.46 405.66±67.5 0.31±0.03 56.26±1.99

7.1 296.5±127.49 0.27±0.09 83.03±7.42

Sonication method

14.2 208.333±7.63 0.27±0.006 11.91±0.17

9.46 167.333±2.08 0.18±0.007 14.13±0.36

7.1 97.866±0.80 0.21±0.01 16.67±0.30

All data are mean value ± standard deviation

Table 3. Effect of cholesterol concentrations on properties of Zataria multi ora essential oil

loaded nanoliposomes in two different preparation methods

Cholesterol: lecithin Size(nm) PDI EE%

Extrusion method

Sonication method

1:1 (7.1mg/ml)

1:2 (6.4mg/ml)

1:3 (4.83mg/ml)

405.66±67.50

302.66±62.54

285.033±15.59

0.313±0.03

0.412±0.03

0.424±0.12

56.26±1.99

62.39±4.50

70.56±2.58

1:1

1:2

1:3

167.33±2.08

133.66±0.57

138±1

0.183±0.01

0.172±0.001

0.174±0.0005

14.16±0.31

18.59±0.01

18.96±0.05

All data are mean value ± standard deviation

Mohammad Hosein Arabi et al.

156 PREPARATION METHODS OF NANOLIPOSOMES CONTAINING

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ESSENTIAL OIL BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 3. A is effect of essential oil concentration on encapsulation ef ciency and B is effect of cholesterol

content on encapsulation ef ciency of nanoliposomes

FIGURE 4. SEM image of Zatariamulti oraessential oil loaded liposomes by sonication (right image) and

extrusion (left image) method.

tion and the drug which is used. The results show that

both two methods have acceptable incorporation ef -

ciency. However sonication method gave a lower con-

tent in incorporated essential oil than extrude method

(11-18%). Extruded vesicles showed high encapsulation

ef ciency (53-83%) of the essential oil. The highest

mean entrapment ef ciency was found at 0.25 of EO/

total lipid (Table 2). Moreover, in the case of choles-

terol effect, it was also observed that liposomal formula-

tion PC/Chol (3:1) load the highest amount of the active

ingredient in each method (70.56±2.58 % for extruded

vesicles and 18.96±0.05 % for sonication method) (Table

3). From  gure 3(A), we observed a decrease of incorpo-

rated oil with increase of essential oil concentration in

each method. Also, it can be appreciated that decrease

content of cholesterol lead to increase loading of the

essential oil ( gure 3 B). Results were summarized in

Table 2 and 3. On the whole, ANOVA test showed that oil

concentration in uence on physicochemical properties

of each method signi cantly (p <0.05).

From  gure 3(A), we observed a decrease of incorpo-

rated oil with increase of essential oil concentration in

each method. Also it can be appreciated that decrease

content of cholesterol lead to increase loading of the

essential oil ( gure 3B).

Image of selected nanoliposome

SEM image of prepared nanoliposomes has been shown

in Figure 4. As it can be seen all nanoliposomal parti-

cles have spherical structure and system is homogenous

in both methods. Both formulations in which used EO:

PC: CH 1:4:1. Liposomes prepared by sonication method

Mohammad Hosein Arabi et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS PREPARATION METHODS OF NANOLIPOSOMES CONTAINING

ZATARIA MULTIFLORA BOISS

ESSENTIAL OIL 157

(right image) were smaller than liposomes treated via

extrusion method (left image). It can be due to higher

content of essential oil that  uidize liposomal bilayers

and increase their susceptivity for aggregation.

In vitro essential oil release studies

As showed in Figure 5, the release of EO within 24 hours

from nanoliposomes was very inconsiderable and negli-

gible. In the other words, it can be said that EO leakage

from liposomes does not occur. The selected formulation

was EO: PC: CH 1:4:3 nanoliposomes containing Zataria

essential oil.

Liposome stability

The physical and chemical stability of soni-

cated formula were evaluated at 5°C, 25°C and

37°C for two months. The sonicated formula (1/4) loaded

with Zataria essential oil were stable for at least 8 weeks

at room and refrigerator temperatures as the particle size

and the EE% of the liposomes did not change signi -

cantly during this period (data not shown).

Effect of essential oil concentration on liposomes

characteristics

Different methods presented different results in the for-

mation of the nano-sized liposomes. Due to high energy

used in probe sonicator, liposomes prepared by this

method have, smaller mean size and PDI index com-

pared to extruded ones. Sonication generally produces

SUV liposomes with lower size and lower incorporated

essential oil than MLVs that are according to Sinico et

al  ndings (Sinico et al., 2005). Comparison the aver-

age size of nanoliposomes in different concentration of

essential oil in two methods showed signi cant differ-

ence (p<0.05). It has been showed that nanoliposomes

size could be affected by EO amount incorporated.

Also entrapment of lipophilic compounds in liposome

membrane depends on size of liposome (Schwendener

& Schott, 2017). Varona et al, also showed an essential

oil amount impact on the liposome size (Varona et al.,

2011). According to our results, by increasing the con-

centration of essential oil to total lipid with the same

ratio of PC: CH, average size of the nanoparticles have

increased. Probably because of higher EO concentration

required greater mechanical force in the reduction size

process of vesicles. In study of Detoni and colleagues, it

was not observed any changes in the size of the nano-

particles containing different ratios of Zanthoxylum tin-

goassubia oil to the phospholipid, which is inconsistent

with our study (Detoni et al., 2009).

PDI (poly dispersity index) usually is considered to

be as an indicator of colloidal particle size distribution.

The smaller index indicates the particle distribution is

more limited and so the system will be homogeneous

and more uniform in size (Ruozi et al., 2005). Two meth-

ods had low value PDI index although the mean PDI

index in sonication method is lower than the extrusion

method. In both methods, the mean PDI index changes

made no signi cant difference, in different concentra-

tion of EO (0.05<P). The liposomes encapsulated atrac-

tylodes macrocephala essential oil that formed by the

modi ed RESS had uniform and narrow size distribution

(Wen et al., 2010). The same results have been obtained

by Valenti et al and Sinico et al which is consist with our

study (Sinico et al., 2005; Valenti et al., 2001). On the

contrary, Yoshida et al reported PDI index higher than

0.7 for their MLV dispersions using Eugenia uni ora EO

(Yoshida et al., 2010). According to the above  gure (3),

it can be observed that, in both two methods, the incor-

poration ef ciency decreased when the EO amount was

increased. Ortan et al, showed a slight decrease in the

encapsulation ef ciency of anethum graveolens essential

FIGURE 5. In vitro essential oil release curve over 24 hours

Mohammad Hosein Arabi et al.

158 PREPARATION METHODS OF NANOLIPOSOMES CONTAINING

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ESSENTIAL OIL BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

oil with the increase of its concentration which is consist

with present study (Ortan et al., 2009). Lavandin and

his colleagues obtain varying amounts of encapsulation

ef ciency by loading different ratio of essential oil to

phospholipid (Varona et al., 2011). With increasing of

essential oil concentration the encapsulation ef ciency

increases because of more EO can be encapsulated into

liposomes that is consistent by the studies of Fang et al

and also Varona et al which in contrast with present study

(Varona et al., 2011; Fang et al., 2001). The difference in

the encapsulation ef ciency was explained by Detoni et

al. According to their study, the physical and chemical

properties of essential oils, or changes may be created

in the techniques of liposomes preparation such as con-

centration of phospholipids, cholesterol and the ratio of

EO to total lipid can affect this property (Detoni et al.,

2012). In our study, Signi cance differences in incorpo-

ration ef ciency related to the preparation method was

observed (p<0.05). It has been reported that produced

liposomes by sonication method, may be have lower

encapsulation ef ciency due to the degradation possibil-

ity of phospholipids and encapsulated compounds (Riaz,

1996) . In a similar study, Zataria multi ora essential oil

into the nanoliposomes was encapsulated. The essential

oil encapsulation within liposomes were 18 to 22 % by

providing multiple ways (Khatibi et al., 2015). Celia et

al, have incorporated bergamot essential oil by using

extrude method with an encapsulation ef ciency of 75%

(Ceilia et al., 2015). The result obtained in present study

recon rms approximately their  ndings.

Effect of cholesterol content on liposomes

characteristics

By maintaining the ratio of EO to total lipid (1/3) in

the formulations, the effect of different cholesterol

concentration on liposome features was also deter-

mined. Cholesterol as a usual

composition plays an

important role in the structure of the liposome. Cho-

lesterol due to its amphiphilic properties, perch into

liposome so that a hydroxyl group to the core of water

oriented and the hydrophobic tail to the phospholipid

bilayer of the liposomes. In previous studies it has been

widely reported that the formation and stability of the

liposomes is highly dependent on the ratio of cholesterol

to phospholipid, so have great impact on the behavior

of the carrier in vitro and in vivo conditions (Haeri et

al., 2014). Other effects of cholesterol are control and

reduce membrane permeability, making of hardness to

membrane and layer structural stability (Chan et al.,

2004). Results shown in  gure 1 part B demonstrated

that liposome size increased with the increasing amount

of cholesterol in both two methods. Varona and col-

leagues observed that with lower cholesterol levels, the

size of the liposomes reduced which it’s in line with the

results of this study (Varona et al., 2011). It is reported

that the insertion of cholesterol in liposomal membrane

causes liposomes more rigid and resistant to size reduc-

tion (Briuglia et al., 2015). Comparison the mean size

of two methods, sonication method showed a signi -

cant size reduction (p <0.05). Also the mean PDI index

in different molar concentrations of cholesterol in two

methods showed that changes are signi cant (p<0.05).

Sonication method gave smaller and more homogene-

ous nanoliposomes than extrude method. Lipophilic

molecules, competes with cholesterol molecules for the

lipophilic space in the lipid bilayer (Jaafar-Maalej et al.,

2010) so, cholesterol might distaste the incorporation of

hydrophobic molecules into the liposome bilayer mem-

brane (Fathi Moghaddam et al., 2008; Rezaee et al, 2015;

Saffari

et al, 2013 . Ortan et al in their study showed

that by increasing the amount of Cholesterol, amount

of encapsulated EO (EE%) in liposomes reduced (Ortan

et al., 2009). This result was con rmed by Varona et

al via essential oil of lavandi (Varona et al., 2011). The

same result was obtained in present study with both two

methods and encapsulation ef ciency in both methods

have signi cantly decreased (p < 0.05).

In summary, this study contributes to the understand-

ing effect of preparation method & the different con-

centration of EO, Cholesteol content on Zataria essen-

tial oil loading into liposomes. The study presented here

suggested that the physicoch emical features of loaded

nanoliposomes clearly in uenced by EO concentration,

cholesterol content and liposome preparation method. It

can be deduced by consideration of signi cance level,

sonication method gave smaller nanoliposomes than the

extrude method, while higher EO incorporation obtained

by extruded method.

ACKNOWLEDGEMENTS

The authors thank Ms Zahra Abbasian & Ms Leila Asta-

rakifor their valuable technical assistance. This research

was performed with support from Kashan University of

Medical Sciences.

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