J Am Oil Chem Soc DOI 10.

1007/s11746-011-1960-3

ORIGINAL PAPER

Nanoencapsulation of Alpha-linolenic Acid with Modied Emulsion 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 modied emulsion diffusion technique with encapsulation efciency of 93%. Polylactic acid (PLA) was used as the encapsulating polymer with acetone and ethyl acetate as organic solvents, and Tween 20, gelatin 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 veried by scanning electron microscopy and the dynamic light scattering technique respectively. Both particle size and zeta potential were inuenced 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 benets. 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 inammatory responses, vasodilation, pain and fever [2]. Moreover, these fatty acids are believed to have several benecial effects on common diseases such as cardiovascular diseases, certain types of tumors and neurological 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, eicosapentaenoic 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 degradation 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, characterization, 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 protect retinyl palmitate from photodegradation induced by UV radiation. Moreover, as the naturally occurring pungent odor of capsaicin impedes its benecial 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

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 various deleterious environmental conditions; hence, there is a need to study the conditions that inuence the characteristics 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 modied emulsion-diffusion technique, and to study the variables that inuence their characteristics.

Fig. 1 The chemical structure of polylactic acid (PLA) (a), and alpha-linolenic acid (a-LA) (b)

Materials and Methods Materials Alpha-Linolenic acid (a-LA) ([99% purity), poly(D, L-lactide) with inherent viscosity of 0.550.75 dL/g (Fig. 1), Tween 201 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 dispersions by a modied 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 ve times its volume of the aqueous phase (1% Tween 20, Pluronic-F68 or gelatin in water) with high shear mixing using a rotorstator device Ingenieurburo CAT 9120 (10,000 rpm/min) for 5 min. Thus, 12 mixtures were obtained (two organic phases 9 three aqueous phases 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 (Laborota 4001) at 35 C. Alpha-LA nanocapsule dispersions were kept in a refrigerator (4 C) till analysis. Freshly
1

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 nm6.5 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 ve times its original volume with distilled water during analysis in order to avoid multiple-scattering effect. All measurements were conducted at ambient temperature. The nal 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 eld using the zeta potential particle size analyzer mentioned earlier. The relation between zeta potential and mobility is given by the Smoluchowski equation: f lg=e 1

Any mentioning of a trademark does not imply endorsement by the authors.

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

123

J Am Oil Chem Soc

electric eld and a dielectric constant of 79 (of water) at 31 1 C. Scanning Electron Microscopy The appearance of the nanocapsule populations was visualized 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 TM with a scanning electron microscope model Inspect F 50 at an accelerating voltage of 35 kV and magnication of about 1059 using Everhardt Thornley Secondary Electron Detector (ETD). At least, ve 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 dispersion 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 combined with the rst 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 LC10AT pump, an SPD-10A ultravioletvisible light detector and a CR8A Chromatopac digital integrator. A beta-basicC18 column (250 mm long, 4.6 mm internal diameter, 5 lm particle diameter) from Thermoelectron was used for a-LA quantication.

Alpha-LA was eluted isocratically at a ow rate of 0.8 mL/min using methanol and 10 mM acetate buffer pH 7.8 (88:12, v/v) as mobile phase after ltering through a 0.45-lm nylon membrane lter 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 methanol with a concentration range of 0.02400 ppm and an R-squared value of 0.998. All runs were carried out at ambient room temperature. Determination of Encapsulation Efciency The percentage of a-LA incorporated during nanoparticle preparation was determined by estimating both the encapsulated and the free (non-encapsulated) a-LA by a validated HPLC method described below. Encapsulation efciency (EE) of the technique were calculated according to the following equations [14]: Encapsulated a-LA Total a-LA Free a-LA in the dispersion Encapsulation efficiency 2

Encapsulated a-LA 100% Total a-LA 3

Statistical Analyses An ANOVA/Mixed General Linear Model procedure was performed on the data, following Randomized Complete Block Design with factorial arrangement, whereas a Protected 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 continuous phase when the organic solution containing PLA was added, thus resulting in a transparent dispersion. Nanoparticles are formed due to differences in surface tension between the aqueous and organic phases, which cause interfacial turbulence in the system leading to the continuous solvent ow away from regions of low surface tension and the aggregation of polymer on the hydrophobic surface

123

J Am Oil Chem Soc Fig. 2 SEM images of a-LA nanocapsules prepared with acetone as c the organic solvent and Pluronic-F68 (a), Tween 20 (b) and gelatin (c) as the stabilizer and organic:aqueous phase ratios of 1:5

[15]. The use of the stabilizer is important for nanoparticle formation by this method. Stabilizer plays the role of dispersing nanoparticles and preventing them from coagulation and precipitation in the dispersion system. Hence, it is suggested that the shells of the nanocapsules consist of an adsorbed surfactant layer (stabilizer/emulsier) 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 conrmed by HPLC analysis with an average efciency of 76%. Kolanowski et al. [17] reported an average extraction efciency of about 98% for the extraction of sh oil from microencapsulated with modied 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 preparations 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 uid CO2 technique without organic solvents. They attributed these patterns to the degree of saturation of the active and the encapsulating material. These ndings 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 signicant 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 signicant (P B 0.01) effect on the size of the nanoparticles (Table 1). Nanocapsules

123

J Am Oil Chem Soc Fig. 3 Particle size distribution of a-LA nanocapsules prepared with acetone (a), and ethyl acetate (b) and different emulsiers 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

prepared with acetone as the solvent were signicantly (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 efcient and rapid. In addition, acetone is considered a fully water-soluble solvent, whereas ethyl acetate is partially water-soluble. The rate of solvent dissolution 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 solvent as compared to other such solvents such as tetrahydrofuran and N,N-dimethylacetamide [18, 19]. However, Song et al. [20] observed that the use of a non-ionic stabilizer, 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

123

J Am Oil Chem Soc 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) df Solvent Organic:aqueous phase ratio Stabilizer Solvent*organic:aqueous ratio Solvent*stabilizer Organic:aqueous ratio*stabilizer Solvent*organic:aqueous ratio*stabilizer ** Signicant at the 1% level of probability R Squared = 0.975 (Adjusted R Squared = 0.948) 1 1 2 1 2 2 2 Mean square 2.248 9 107 412,152.3 1.158 9 107 62,577.1 5,075,646.4 350,787.9 1,524,250.1 F 161.61** 2.96 83.25** 0.45 36.49** 2.52 10.96** Zeta potential (mV) df 1 1 2 1 2 2 2 Mean square 1764.22 129.13 670.67 473.22 181.63 79.84 3.02 F 268.20** 19.63** 101.96** 71.94** 27.61** 12.14** 0.459

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 Solvent Aqueous:organic phase ratio Stabilizer Ethyl acetate Acetone 1:5 1:2 Pluronic-F68 Tween 20 Gelatin Mean Particle size (nm) 2,411a 475
b a a b

Mean zeta potential (mV) -4.46a -21.61b -10.71a -15.35b -9.03a -23.51b -6.56a

1,312 1,574 1,309 313

c

2,708a

Means within the same column and same factor group having the same superscripts are not signicantly different (P B 0.05) according to a protected-LSD test

hindrance of the non-ionic stabilizer was not enough to show the signicant 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 signicant effect on the particle size of the nanocapsule dispersions obtained (Table 1). Particles prepared with Tween 20 as the stabilizer were signicantly 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 structures, 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). Pluronic-F68 has a molecular weight (8,350 Da) that falls between those of Tween 20 (1,226.5 Da) and gelatin (8040 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 inuenced by its molecular geometry, expressed as Critical Packing Parameter (CPP)

123

J Am Oil Chem Soc 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 Solvent*stabilizer Ethyl acetate*Pluronic-F68 Ethyl acetate*Tween 20 Ethyl acetate*Gelatin Acetone*Pluronic-F68 Acetone*Tween 20 Acetone*Gelatin Solvent*aqueous:organic phase ratio Ethyl acetate*1:5 Ethyl acetate*1:2 Acetone*1:5 Acetone*1:2 Aqueous:organic phase ratio*stabilizer 1:5*Pluronic-F68 1:5*Tween 20 1:5*Gelatin 1:2*Pluronic-F68 1:2*Tween 20 1:2*Gelatin Particle size (nm) 2,390b 434 228
cd a

Zeta potential (mV) 5.00a -18.24d -0.14b -23.07e -28.77f

4,410
d d

192

1,006

-12.98c 2.30a -11.22b -23.73d -19.49c -6.05b -24.63d

2,331a 2,491a 293c 658

b c

951

224d 2,762 402

cd a a b

-1.47a -12.02c -22.39d -11.65c

1,667 2,654

* Means within the same column and same factor group having the same superscripts are not signicantly (P B 0.05) different according to the protected-LSD test

[22] which is dened 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 signicant 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 signicantly (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 nding conrms 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 acetate compared to acetone, which makes it difcult 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 aggregation 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 solvent has a highly signicant effect (P B 0.01) on the zeta

123

J Am Oil Chem Soc

potential of the nanocapsules. Table 2 shows that a-LA nanocapsules prepared with acetone had absolutely signicantly (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 inuence 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 signicant difference was observed between the zeta potentials of nanocapsules prepared with Pluronic-F68 and gelatin as stabilizers. Nonetheless, as shown by rst 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 1530 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 signicantly (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 Efciency of a-LA Encapsulation efciency (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 concentration 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 afnity between the core and the encapsulating material. Afnity 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 modied emulsion-diffusion technique with an efciency of 93%. The sizes of the nanoparticles obtained as well as their zeta potentials were inuenced signicantly 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 capsules 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 PluronicF68.

123

J Am Oil Chem Soc

References
1. Jensen CL (2006) Effects of n-3 fatty acids during pregnancy and lactation. Am J Clin Nutr 83:1452S1457S 2. Barcelo-Coblijn G, Murphy EJ (2009) Alpha-linolenic acid and its conversion to longer chain n-3 fatty acids: benets for human health and a role in maintaining tissue n-3 fatty acid levels. Prog Lipid Res 48:355374 3. Riediger ND, Othman RA, Suh M, Moghadasian MH (2009) A systemic review of the roles of n-3 fatty acids in health and disease. J Am Diet Assoc 109:668679 4. Moghadasian MH (2008) Advances in dietary enrichment with n-3 fatty acids. Critic Rev Food Sci Nutr 48:402410 5. Liu QS, Wang JQ, Bu DP, Khas E, Liu KL, Wei HY, Zhou LY, Beitz DC (2010) Inuence of linolenic acid content on the oxidation of milk fat. J Agric Food Chem 58:37413746 6. Ozin G, Arsenault AC, Cademartiri L (2009) Nanochemistry: a chemical approach to nanomaterials. RSC Publishing, Cambridge 7. Tachaprutinun A, Udomsup T, Luadthong C, Wanichwecharungruanga S (2009) Preventing the thermal degradation of astaxanthin through nanoencapsulation. Int J Pharm 374:119124 8. Sane A, Limtrakul J (2009) Formation of retinyl palmitate-loaded poly(L-lactide) nanoparticles using rapid expansion of supercritical solutions into liquid solvents (RESOLV). J Supercrit Fluids 51:230237 9. Wang JC, Chen SH, Xu ZC (2008) Synthesis and properties research on the nanocapsulated capsaicin by simple coacervation method. J Dispers Sci Technol 29:687695 10. Mishra B, Patel BB, Tiwari S (2010) Colloidal nanocarriers: a review on formulation technology, types and applications toward targeted drug delivery. Nanomed: Nanotechnol, Biol Med 6:924 11. Huang Q, Yu H, Ru Q (2010) Bioavailability and delivery of nutraceuticals using nanotechnology. J Food Sci 75:R50R57 12. Quintanar D, Fessi H, Doelker E, Alleman E (2005) Method for preparing vesicular nanocapsules 6884438 13. Helrich K (ed) (1990) Ofcial methods of analysis of the association of ofcial analytical chemists. AOAC Int, Arlington 14. Xing F, Cheng G, Yi K, Ma L (2005) Nanoencapsulation of capsaicin by complex coacervation of gelatin, acacia, and tannins. J Appl Polym Sci 96:22252229 15. Quintanar D, Allemann E, Fessi H, Doelker E (1998) Preparation techniques and mechanisms of formation of biodegradable nanoparticles from preformed polymers. Drug Dev Ind Pharm 24:11131128 16. Khoee S, Yaghoobian M (2009) An investigation into the role of surfactants in controlling particle size of polymeric nanocapsules containing penicillin-G in double emulsion. Eur J Med Chem 44:23922399

17. Kolanowski W, Laufenberg G, Kunz B (2004) Fish oil stabilisation by microencapsulation with modied cellulose. Int J Food Sci Nutr 55:333343 18. Hornig S, Bunjes H, Heinze T (2009) Preparation and characterization of nanoparticles based on dextrandrug conjugates. J Colloid Interface Sci 338:5662 19. Aliabadi HM, Elhasi S, Mahmud A, Gulamhusein R, Mahdipoor P, Lavasanifar A (2007) Encapsulation of hydrophobic drugs in polymeric micelles through co-solvent evaporation: The effect of solvent composition on micellar properties and drug loading. Int J Pharm 329:158165 20. Song KC, Lee HS, Choung IY, Cho KI, Ahn Y, Choi EJ (2006) The effect of type of organic phase solvents on the particle size of poly(D, L-lactide-co-glycolide) nanoparticles. Colloids Surf A: Physicochem Eng Aspects 276:162167 21. Kwon H-Y, Lee J-Y, Choi S-W, Jang Y, Kim J-H (2001) Preparation of PLGA nanoparticles containing estrogen by emulsication-diffusion method. Colloids Surf A: Physicochem Eng Aspects 182:123130 22. Zhu Y, Zhang G, Yang H, Hong X (2005) Inuence of surfactants on the parameters of polylactide nanocapsules containing insulin. J surfactants deterg 8:353358 23. Tadros TF (2005) Applied surfactants: principles and applications. Wiley-VCH, Weinheim 24. Uchegbu IF, Florence AT (1995) Non-ionic surfactant vesicles (niosomes): physical and pharmaceutical chemistry. Adv Colloid Interface Sci 58:155 25. Swarnakar N, Jain V, Dubey V, Mishra D, Jain NK (2007) Enhanced oromucosal delivery of progesterone via hexosomes. Pharm Res 24:22232230 26. Singh R, Lillard JW Jr (2009) Nanoparticle-based targeted drug delivery. Exp Mol Pathol 86:215223 27. Choi M-J, Ruktanonchai U, Min S-G, Chun J-Y, Soottitantawat A (2010) Physical characteristics of sh oil encapsulated by b-cyclodextrin using an aggregation method or polycaprolactone using an emulsion-diffusion method. Food Chem 119:16941703 28. Yin L-J, Chu B-S, Kobayashi I, Nakajima M (2009) Performance of selected emulsiers and their combinations in the preparation of b-carotene nanodispersions. Food Hydrocoll 23:16171622 29. Couvreur P, Barrat G, Fattal E, Legrand P, Vauthier C (2002) Nanocapsule technology: a review. Crit Rev Ther Drug Carrier Syst 19:99134 30. Yoksana R, Jirawutthiwongchai J, Arpo K (2010) Encapsulation of ascorbyl palmitate in chitosan nanoparticles by oil-in-water emulsion and ionic gelation processes. Colloids Surf B Biointerfaces 76:292297 31. Wu T-H, Yen F-L, Lin L-T, Tsai T-R, Lin CC, Chamd T-M (2008) Preparation, physicochemical characterization, and antioxidant effects of quercetin nanoparticles. Int J Pharm 346:160168

123