ORIGINAL PAPER

Nanoencapsulation of Alpha-linolenic Acid with ModifiedEmulsion Diffusion Method

Salam M. Habib • Ayed S. Amr • Imad M. Hamadneh

Received: 23 March 2011 / Revised: 7 September 2011 / Accepted: 14 October 2011

� AOCS 2011

Abstract Nanocapsules of alpha-linolenic acid (a-LA)

were prepared by a modified emulsion diffusion technique

with encapsulation efficiency of 93%. Polylactic acid

(PLA) was used as the encapsulating polymer with acetone

and ethyl acetate as organic solvents, and Tween 20, gel-

atin and Pluronic-F68 in water as stabilizers. Two ratios of

organic to aqueous phases were used with each solvent and

stabilizer. Nanocapsule dispersions with a particle size less

than 100 nm and a zeta potential as high as 33 mV were

obtained as verified by scanning electron microscopy and

the dynamic light scattering technique respectively. Both

particle size and zeta potential were influenced by such

preparation conditions as the type of stabilizer, type of

organic solvent and the organic to aqueous phase ratio.

Acetone was superior to ethyl acetate, and Tween 20 was

superior to each of Pluronic-F68 and gelatin in obtaining

smaller, less aggregated nanocapsules. An organic to

aqueous phase ratio of 1:5 was shown to be more suitable

for the formation of smaller nanocapsules, particularly

when acetone was used as the organic solvent.

Keywords Nanoencapsulation � Omega-3 fatty acid �PLA � Particle size � Zeta potential

Introduction

Omega-3 fatty acids have long been recognized for their

health benefits. They are important in the development of

both vision and cognitive functions in infants [1] as they

are believed to induce a group of physiological processes

such as inflammatory responses, vasodilation, pain and

fever [2]. Moreover, these fatty acids are believed to have

several beneficial effects on common diseases such as

cardiovascular diseases, certain types of tumors and neu-

rological disorders such as Alzheimer [3].

Nowadays, a wide variety of food products including

bread and bakery products, milk and its derivatives,

spreadable fats, eggs, juices, soft drinks, meat and poultry

have been enriched with such omega-3 fatty acids as a-LA

(18:3, 9,12,15-octadecatrienoic acid), EPA (20:5, eicosa-

pentaenoic acid), and DHA (22:6, docosahexanoic acid)

[4]. Nevertheless, the family of omega-3 fatty acids is

known to be highly susceptible to oxidation and degrada-

tion by heat, which limits their nutritive value and

decreases the shelf life of foods enriched with them [5].

Nanotechnology can be used in the design, character-

ization, production and application of structures, devices

and systems on the nano-scale [6]. Nanoencapsulation is a

viable, non-traditional technique that can be employed

for enhancing some of the characteristics of bioactive

materials, such as water solubility, storage and thermal

stability and various sensory attributes. Tachaprutinun

et al. [7] have recently improved the thermal stability of

astaxanthin, an important industrial carotenoid pigment, by

polymeric nanoencapsulation. Sane and Limtrakul (2009)

[8] have successfully employed nanoencapsulation to pro-

tect retinyl palmitate from photodegradation induced by

UV radiation. Moreover, as the naturally occurring pungent

odor of capsaicin impedes its beneficial utilization, using

This work is patent pending.

S. M. Habib � A. S. Amr (&)

Department of Nutrition and Food Technology,

Faculty of Agriculture, University of Jordan,

Amman 11942, Jordan

e-mail: ayedamr@ju.edu.jo

I. M. Hamadneh

Department of Chemistry, Faculty of Science,

University of Jordan, Amman 11942, Jordan

123

J Am Oil Chem Soc

DOI 10.1007/s11746-011-1960-3

nanoencapsulation of capsaicin has effectively hidden its

pungent odor [9]. Nanoencapsulation also found use in the

pharmaceutical and nutraceutical industries as it has been

used to effect targeted delivery, minimized toxicity,

enhanced bio-distribution and higher cell uptake of some

drugs and nutraceuticals [10, 11].

Therefore, nanoencapsulation of omega-3-fatty acids is

considered a novel method for protecting them from vari-

ous deleterious environmental conditions; hence, there is a

need to study the conditions that influence the character-

istics of the nanocapsules of omega-3-fatty acids in order to

use them as functional ingredients in foods. The objective

of the current study is to prepare nanocapsules of a-LA

using poly-lactic acid (PLA) as encapsulating material

following a modified emulsion-diffusion technique, and to

study the variables that influence their characteristics.

Materials and Methods

Materials

Alpha-Linolenic acid (a-LA) ([99% purity), poly(D, L-lac-

tide) with inherent viscosity of 0.55–0.75 dL/g (Fig. 1),

Tween 20�1 and Pluronic-F68� were purchased from

Sigma-Aldrich. All other chemicals were of analytical or

HPLC grade.

Preparation of the Nanocapsules

Alpha-LA nanocapsules were prepared as water disper-

sions by a modified emulsion-diffusion method [12] as

follows: approximately 120 mg of PLA was dissolved in

6 mL of the organic solvent (ethyl acetate or acetone),

heated to facilitate dissolution and 60 mg of a-LA were

dissolved separately in 6 mL of the same organic solvent

without heating. The two solutions were mixed to obtain

the organic phase which was added drop-wise to twice and

five times its volume of the aqueous phase (1% Tween 20,

Pluronic-F68 or gelatin in water) with high shear mixing

using a rotor–stator device Ingenieurburo CAT 9120

(10,000 rpm/min) for 5 min. Thus, 12 mixtures were

obtained (two organic phases 9 three aqueous pha-

ses 9 two ratios of organic: aqueous phase). To form the

nanocapsule dispersions, each of the 12 mixtures was

diluted up to 300 mL with distilled water and stirred

manually for a few minutes. The organic solvent was

removed under a vacuum using a rotary evaporator (Lab-

orota 4001) at 35 �C. Alpha-LA nanocapsule dispersions

were kept in a refrigerator (4 �C) till analysis. Freshly

prepared oil-in-water non-encapsulated emulsions with the

same composition obtained by mixing a-LA with distilled

water using Tween 20 as the stabilizer were used as a the

control in the study.

Characterization of the Nanocapsules

Particle Size Determination

Particle size as Mean Intensity Diameter was measured by

the dynamic light scattering technique, using the Zetatrac

particle size and zeta potential analyzer model NPA152

with a measuring range of 0.8 nm–6.5 lm, distilled water

(Refractive index = 1.33) was used as a reference and the

nanocapsules were considered as light absorbing object.

The particle concentration in the sample was diluted to five

times its original volume with distilled water during anal-

ysis in order to avoid multiple-scattering effect. All mea-

surements were conducted at ambient temperature. The

final particle diameter was calculated as the average of at

least three readings. The system was run by Microtrac�

FLEX Windows Software V 10.5.4.

Zeta Potential Determination

Zeta potential was estimated based on electrophoretic

mobility under an electric field using the zeta potential

particle size analyzer mentioned earlier. The relation

between zeta potential and mobility is given by the

Smoluchowski equation:

f ¼ lg=e ð1Þ

Where f = zeta potential, l = mobility, g = viscosity,

e = dielectric constant. The zeta potential of the

nanocapsule dispersions was measured under a 5 V/cm

Fig. 1 The chemical structure of polylactic acid (PLA) (a), and

alpha-linolenic acid (a-LA) (b)

1 Any mentioning of a trademark does not imply endorsement by the

authors.

J Am Oil Chem Soc

123

electric field and a dielectric constant of 79 (of water) at

31 ± 1 �C.

Scanning Electron Microscopy

The appearance of the nanocapsule populations was visu-

alized by scanning electron microscopy (SEM). A drop of

the nanocapsule dispersion was deposited on an aluminum

stub, coated with adhesive carbon, left to dry at ambient

temperature, then sputtered for 3 min with platinum using a

coating machine model K550 X. Samples were analyzed

with a scanning electron microscope model InspectTM

F 50

at an accelerating voltage of 3–5 kV and magnification of

about 1059 using Everhardt Thornley Secondary Electron

Detector (ETD). At least, five different spots of each sample

were visualized. The system was equipped with a model

400-NAV-CAM camera and XT Microscope server

software.

Extraction of a-LA

Total (both nanoencapsulated and the free) a-LA from the

dispersions was extracted by taking 2 mL of a-LA dis-

persion and mixing it with double its volume of 1:1 ethyl

acetate:petroleum ether solution in a 20-mL vial in a vortex

mixer at high speed (2,400 rpm/min) for 1 min. The upper

organic layer containing most of the a-LA was transferred

to a small beaker by the dropper. To get the residual a-LA,

the dispersions were double extracted with petroleum ether

followed by adding a volume of 10% sodium chloride

solution then centrifugation at 2,500 rpm/min for 5 min.

The upper layer containing the residual a-LA was com-

bined with the first extract and the organic solvents were

removed under a stream of nitrogen. The extracted a-LA

was dissolved in 2 mL of methanol for HPLC analysis.

The free not encapsulated portion of a-LA was extracted

by taking a 2-mL volume of a-LA nanocapsule dispersion,

shaking it gently with double its volume of petroleum ether

[13] in a 20-mL vial for 5 min. The upper organic layer

containing the free a-LA and the organic solvent was

transferred to a small beaker (by a dropper) from which the

organic solvent was removed under a stream of nitrogen,

and the remaining a-LA was reconstituted in 2 mL of

methanol for HPLC analysis.

HPLC Analysis

A Shimadzu HPLC system was used. It consisted of an LC-

10AT pump, an SPD-10A ultraviolet–visible light detector

and a CR8A Chromatopac digital integrator. A beta-basic-

C18 column (250 mm long, 4.6 mm internal diameter,

5 lm particle diameter) from Thermoelectron was used for

a-LA quantification.

Alpha-LA was eluted isocratically at a flow rate of

0.8 mL/min using methanol and 10 mM acetate buffer pH

7.8 (88:12, v/v) as mobile phase after filtering through a

0.45-lm nylon membrane filter and ultrasonicated to

remove dissolved gases. The injection volume was 5 ll and

the retention time of a-LA was 5.1 min. The detector

wavelength was set to 210 nm. A standard curve was

constructed using a stock solution of pure a-LA in meth-

anol with a concentration range of 0.02–400 ppm and an

R-squared value of 0.998. All runs were carried out at

ambient room temperature.

Determination of Encapsulation Efficiency

The percentage of a-LA incorporated during nanoparticle

preparation was determined by estimating both the encap-

sulated and the free (non-encapsulated) a-LA by a vali-

dated HPLC method described below. Encapsulation

efficiency (EE) of the technique were calculated according

to the following equations [14]:

Encapsulated a-LA ¼ Total a-LA � Free a-LA

in the dispersion ð2Þ

Encapsulation efficiency ¼ Encapsulated a-LA

Total a-LA� 100%

ð3Þ

Statistical Analyses

An ANOVA/Mixed General Linear Model procedure was

performed on the data, following Randomized Complete

Block Design with factorial arrangement, whereas a Pro-

tected LSD test was used for mean separation at the 5%

level of probability (The SAS System software package,

version 9.2, SAS Institute, Cary, NC). All experiments

were performed in duplicate.

Results and Discussion

Alpha-LA Nanocapsule Formation

The formation of a-LA nanocapsules involved the addition

of the organic phase composed of the PLA and a-LA to the

aqueous phase (continuous phase) containing the stabilizer.

Nanoparticles were spontaneously formed in the continu-

ous phase when the organic solution containing PLA was

added, thus resulting in a transparent dispersion. Nano-

particles are formed due to differences in surface tension

between the aqueous and organic phases, which cause

interfacial turbulence in the system leading to the contin-

uous solvent flow away from regions of low surface tension

and the aggregation of polymer on the hydrophobic surface

J Am Oil Chem Soc

123

[15]. The use of the stabilizer is important for nanoparticle

formation by this method. Stabilizer plays the role of dis-

persing nanoparticles and preventing them from coagula-

tion and precipitation in the dispersion system. Hence, it is

suggested that the shells of the nanocapsules consist of an

adsorbed surfactant layer (stabilizer/emulsifier) while the

inner spheres consist of a-LA and the polymer [16]. The

theoretical a-LA concentration in these dispersions was

about 250 mg/L while the actual concentration was

190 ± 10 mg/L as confirmed by HPLC analysis with an

average efficiency of 76%. Kolanowski et al. [17] reported

an average extraction efficiency of about 98% for the

extraction of fish oil from microencapsulated with modified

cellulose using Soxhlet extraction with hexane.

Particle Size of a-LA Nanocapsules

The SEM images (Fig. 2) show that a-LA capsules have

spherical shapes with a particle size as small as 50 nm

regardless of the type of stabilizer used, although samples

prepared with gelatin gave larger size particles due to

aggregation. However, the mean size of the capsules as

observed by the particle size analyzer ranged between 75

and 5,000 nm (Fig. 3). Polymeric particles are considered

to be of nano-size if they are below 500 nm in size [15].

The rather large size particles obtained in some prepara-

tions is due to aggregation between particles as they are

visualized as one large mass by the particle size analyzer.

Monomodal distributions were the predominant pattern

observed, while bimodal patterns were seen in some cases

(Fig. 3). Most of the bimodal distribution patterns were

observed when ethyl acetate was used as the solvent

(Fig. 3b). Sane and Limtrakul (2009) [8] reported bimodal

and trimodal distribution patterns of retinyl palmitate

nanoparticles with PLA prepared using a supercritical fluid

CO2 technique without organic solvents. They attributed

these patterns to the degree of saturation of the active and

the encapsulating material. These findings indicate that

more than just one factor contributes to this phenomenon.

Effect of Solvent on the Size of Nanocapsules

As indicated in (Fig. 4), the type of solvent plays a sig-

nificant role in determining the size of nanocapsules aside

from the role of the stabilizer used in their preparation.

Moreover, statistical analysis indicates that the solvent

used has a general highly significant (P B 0.01) effect on

the size of the nanoparticles (Table 1). Nanocapsules

Fig. 2 SEM images of a-LA nanocapsules prepared with acetone as

the organic solvent and Pluronic-F68 (a), Tween 20 (b) and gelatin

(c) as the stabilizer and organic:aqueous phase ratios of 1:5

c

J Am Oil Chem Soc

123

prepared with acetone as the solvent were significantly

(P B 0.05) smaller, in size, than those prepared with ethyl

acetate (Table 2). This could be attributed to the lower

boiling point of acetone than ethyl acetate, which makes its

evaporation more efficient and rapid. In addition, acetone is

considered a fully water-soluble solvent, whereas ethyl

acetate is partially water-soluble. The rate of solvent dis-

solution in water and evaporation out of the system seems

to play a critical role in determining the size of the

nanocapsules.

Smaller nanoparticles of poorly water-soluble drugs

were obtained when acetone was used as the organic sol-

vent as compared to other such solvents such as tetrahy-

drofuran and N,N-dimethylacetamide [18, 19]. However,

Song et al. [20] observed that the use of a non-ionic sta-

bilizer, such as Pluronic-F68, results in reducing the effect

of the organic solvent on the mean particle sizes of poly(D,

L-lactide-co-glycolide) nanoparticles. This was attributed

to repulsion forces between particles, where the steric

Fig. 3 Particle size distribution

of a-LA nanocapsules prepared

with acetone (a), and ethyl

acetate (b) and different

emulsifiers at organic:aqueous

phase ratio of 1:5

Fig. 4 The effect of the type of solvent on the size of nanocapsules

prepared with different types of stabilizers

J Am Oil Chem Soc

123

hindrance of the non-ionic stabilizer was not enough to

show the significant differences in the effect of the type of

solvent on the particle size [21].

The Effect of the Type of Stabilizer on the Size

of Nanocapsules

In general, the type of stabilizer had a highly significant

effect on the particle size of the nanocapsule dispersions

obtained (Table 1). Particles prepared with Tween 20 as the

stabilizer were significantly smaller (P B 0.05) than those

prepared with Pluronic-F68 or gelatin (Fig. 3, Table 2).

Results show that nanocapsule dispersions prepared with

gelatin as the stabilizer had the largest mean particle sizes

compared to those prepared with other stabilizers regardless

of the solvent used (Fig. 4). This might be due to that gelatin

has a variable large molecular weight depending on the

source and method of extraction as it consists of a mixture of

single or multi-stranded polypeptides with the molecular

weight of each above 100 9 103. In addition, gelatin chains

in general, have the tendency to coalesce and permanently

form large units, which may result in coalescence of the

nanocapsules prepared with it, resulting in lump-like struc-

tures, as shown in the SEM image (Fig. 2c). This effect was

compounded by the fact that gelatin used in this work had a

relative high bloom/gel strength (275 g/cm2) which makes it

more prone to coalesce than other grade gelatins.

Using Pluronic-F68 as the stabilizer in this study gave

nanocapsule dispersions with particle sizes falling between

those obtained with Tween 20 and gelatin (Fig 3). Plu-

ronic-F68 has a molecular weight (8,350 Da) that falls

between those of Tween 20 (1,226.5 Da) and gelatin

(80–40 kDa). The results imply that in general, the smaller

the molecular weight of the stabilizer the smaller the size

of the nanodispersions when all other factors are held

constant. This is expected as the smaller the size of the

components of the dispersion, the smaller its size would be

despite the fact that considerable amount of compacting

takes place in the process of nanoparticle formation.

It is suggested that the behavior of the stabilizer in the

process of encapsulation is influenced by its molecular

geometry, expressed as Critical Packing Parameter (CPP)

Table 1 ANOVA table for the effect of the type of solvent, stabilizer, and organic:aqueous phase ratio on the size and zeta potential of

nanocapsules

Source of variation Particle size (nm) Zeta potential (mV)

df Mean square F df Mean square F

Solvent 1 2.248 9 107 161.61** 1 1764.22 268.20**

Organic:aqueous phase ratio 1 412,152.3 2.96 1 129.13 19.63**

Stabilizer 2 1.158 9 107 83.25** 2 670.67 101.96**

Solvent*organic:aqueous ratio 1 62,577.1 0.45 1 473.22 71.94**

Solvent*stabilizer 2 5,075,646.4 36.49** 2 181.63 27.61**

Organic:aqueous ratio*stabilizer 2 350,787.9 2.52 2 79.84 12.14**

Solvent*organic:aqueous ratio*stabilizer 2 1,524,250.1 10.96** 2 3.02 0.459

** Significant at the 1% level of probability

R Squared = 0.975 (Adjusted R Squared = 0.948)

Table 2 The main effects of the type of solvent, organic:aqueous phase ratio and stabilizer on mean particle size and mean zeta potential of

different nanocapsule preparations

Factor Mean Particle size (nm) Mean zeta potential (mV)

Solvent Ethyl acetate 2,411a -4.46a

Acetone 475b -21.61b

Aqueous:organic phase ratio 1:5 1,312a -10.71a

1:2 1,574a -15.35b

Stabilizer Pluronic-F68 1,309b -9.03a

Tween 20 313c -23.51b

Gelatin 2,708a -6.56a

Means within the same column and same factor group having the same superscripts are not significantly different (P B 0.05) according to a

protected-LSD test

J Am Oil Chem Soc

123

[22] which is defined as the ratio of the volume of the

hydrophobic part of the stabilizer (tail) to the minimum

interfacial area occupied by the hydrophilic part (head

group) [23]. Stabilizer molecules with high CPP take a

minimum geometrical orientation, which makes them take

a vesicular or circular shape, thus decreasing the size of the

produced capsules. On the other hand, stabilizers with low

CPP take larger geometrical orientation such as a laminar

shape thus increasing the size of the capsules prepared with

them. The relatively small molecular size of Tween 20, and

the high degree of hydrophobicity of Pluronic-F68 help

confer a high CPP on them [24, 25] and consequently

smaller capsule sizes are obtained when they are used as

stabilizers. On the other hand, gelatin which has a low CPP,

due to the presence of several helical conformations in its

molecular structure and chain penetration between its

several strands, gave larger capsules.

The Effect of the Organic:Aqueous Phase Ratio

on the Size of the Nanocapsules

Table 1 shows that the organic:aqueous phase ratio has no

significant effect on the size of the nanocapsules regardless

of the solvent and type of stabilizer used. However, when

the effect of organic:aqueous phase ratios on the mean

particle size was evaluated within individual solvents

(Table 3), it was observed that nanodispersions prepared

with a 1:5 ratio had significantly (P B 0.05) lower mean

particle sizes than those prepared with a 1:2 ratio only

when acetone was used as the solvent. This finding con-

firms an earlier report by Aliabadi et al. [19] to the effect

that the average nanocapsule diameter of cyclosporine, a

hydrophobic drug, increased by decreasing the acetone to

water ratio. This probably is due to the higher tendency for

aggregation of the nanocapsules prepared using ethyl ace-

tate compared to acetone, which makes it difficult to see

the difference in the size, if present, between the 1:2 and

1:5 organic:aqueous phase ratio of nanocapsules prepared

using ethyl acetate as the solvent.

Zeta Potential of a-LA Nanocapsules

The zeta potential is an indicator of the degree of aggre-

gation of colloidal particles due to electrostatic attraction

caused by charges on their surfaces [26]. Similar charges

cause repulsion of the particles from each others while

opposite charges cause their attraction. Particles with a

high zeta potential show higher repulsion, among others,

than those with a lower potential value, therefore, this

translates into less aggregation in the suspension.

Effect of the Type of Solvent on the Zeta Potential

of Nanocapsules

As shown by statistical analysis (Table 1) the type of sol-

vent has a highly significant effect (P B 0.01) on the zeta

Table 3 The effect of second level interactions between the type of solvent, stabilizer and organic:aqueous phase ratio on particle size

distribution and zeta potential of different nanocapsule preparations

Factors Particle size (nm) Zeta potential (mV)

Solvent*stabilizer Ethyl acetate*Pluronic-F68 2,390b 5.00a

Ethyl acetate*Tween 20 434cd -18.24d

Ethyl acetate*Gelatin 4,410a -0.14b

Acetone*Pluronic-F68 228d -23.07e

Acetone*Tween 20 192d -28.77f

Acetone*Gelatin 1,006c -12.98c

Solvent*aqueous:organic phase ratio Ethyl acetate*1:5 2,331a 2.30a

Ethyl acetate*1:2 2,491a -11.22b

Acetone*1:5 293c -23.73d

Acetone*1:2 658b -19.49c

Aqueous:organic phase ratio*stabilizer 1:5*Pluronic-F68 951c -6.05b

1:5*Tween 20 224d -24.63d

1:5*Gelatin 2,762a -1.47a

1:2*Pluronic-F68 1,667b -12.02c

1:2*Tween 20 402cd -22.39d

1:2*Gelatin 2,654a -11.65c

* Means within the same column and same factor group having the same superscripts are not significantly (P B 0.05) different according to the

protected-LSD test

J Am Oil Chem Soc

123

potential of the nanocapsules. Table 2 shows that a-LA

nanocapsules prepared with acetone had absolutely sig-

nificantly (P B 0.05) higher zeta potential values than

those prepared with ethyl acetate, which is probably due to

their higher tendency to aggregate than those prepared with

acetone. This result is in line with those observed earlier in

the case of the effect of solvent on particle size referred to

above. The better water dissolution and lower boiling point

of acetone seem to influence the zeta potential in the same

manner as they did with particle size.

Effect of the Type of Stabilizer on Zeta Potential

of Nanocapsules

In all cases, when Tween 20 was used as the stabilizer, the

produced nanocapsules had higher absolute zeta potential

values (P B 0.05) than those produced using Pluronic-F-68

or gelatin (Table 2). On the other hand, no significant

difference was observed between the zeta potentials of

nanocapsules prepared with Pluronic-F68 and gelatin as

stabilizers. Nonetheless, as shown by first order interaction,

the produced nanocapsules using Pluronic-F68 had higher

absolute zeta potential values than those prepared with

gelatin as the stabilizer in all cases except when 1:2

organic:aqueous phase ratio was used (Table 3). Again,

this is in conformity with the results obtained in the case of

the effect of type of stabilizer on the size of nanocapsules.

It is worth indicating that even Tween 20 and Pluronic-F68

are nonionic molecules, nanocapsules prepared with them

as stabilizers exhibit a mild negative surface charge. A

similar observation was reported by a number of workers

and was attributed in part to the adsorption of OH- species

from the aqueous phase [27, 28]. Nevertheless, for the

nanoparticle suspensions to be stable and have enough

repulsion forces to prevent aggregation, their absolute zeta

potential values should be at least in the range of

15–30 mV or higher [29]. All nanocapsules prepared with

Tween 20 as the stabilizer (Table 2) had mean zeta

potential values within this range. While those prepared

with Pluronic-F68 gave similar results only when they were

used with acetone as the solvent. All samples prepared with

gelatin gave mean zeta potentials outside the recommended

range regardless of the other preparation factors.

The Effect of the Organic:Aqueous Phase Ratio

on the Zeta Potential of Nanocapsules

As shown in Table 3, when acetone was used as solvent,

nanocapsules prepared with an organic:aqueous phase ratio

of 1:5 had significantly (P B 0.05) higher absolute mean

zeta potential values than when the ratio was 1:2, and both

were within the recommended range. Samples prepared

with ethyl acetate gave mean zeta potential values outside

the recommended range whether the organic:aqueous

phase ratio was 1:2 or 1:5. Although, those dispersions

prepared with a 1:2 ratio showed higher zeta potential

values than those prepared with a 1:5 ratio (Table 3).

Encapsulation Efficiency of a-LA

Encapsulation efficiency (EE) was calculated for the

nanocapsule dispersions prepared with acetone as the

organic solvent, Tween 20 as the stabilizer and 1:5

organic:aqueous phase ratio as these conditions gave the

best results in the case of particle size and zeta potential.

EE was 93% with a CV of 1.2. Other researchers have

obtained EE between 39 and 77% for encapsulation of

ascorbyl palmitate in chitosan nanoparticles and reported

that it was a function of the ascorbyl palmitate concen-

tration used in the preparation [30]. Wu et al. [31] obtained

EE of more than 99% for quercetin by a nanoprecipitation

technique with Eudragit� E polymer and polyvinyl alcohol

as the stabilizer. It is expected that EE is highly associated

with the affinity between the core and the encapsulating

material. Affinity between these two substances seems to

be high enough to give this EE. It was also observed that

increasing the concentration of the stabilizer would vastly

improve the EE of the system [16]. More work is needed to

investigate the effect of preparation parameters on the EE

of a-LA nanocapsules.

Conclusion

In this study, a-LA was successfully nanoencapsulated

with polylactic acid using a modified emulsion-diffusion

technique with an efficiency of 93%. The sizes of the

nanoparticles obtained as well as their zeta potentials were

influenced significantly by the type of organic solvent

used, the stabilizer and the ratio of organic to aqueous

phase. Larger capsules were obtained with ethyl acetate

than with acetone. The latter organic solvent also gave the

smallest capsules especially when used in an organic to

aqueous phase ratio of 1:5. Tween 20 was more effective

than the other two stabilizers in producing smaller cap-

sules and preventing aggregation of capsules as observed

by SEM and a particle size analyzer. Monomodal patterns

of particle size distribution were obtained in most of the

cases, yet some bimodal patterns were obtained especially

when ethyl acetate was used as the solvent. In some cases

where gelatin was used as the stabilizer, aggregation was a

problem, this was overcome by using stabilizers with

lower molecular weight such as Tween 20 and Pluronic-

F68.

J Am Oil Chem Soc

123

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