J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 6 0 – 1 2 6 9

.sc iencedi rect .com

Avai lab le at www

journal homepage: www.elsevier .com/ locate / j f f

Lipid nanoparticles based on omega-3 fatty acids as effectivecarriers for lutein delivery. Preparation and in vitrocharacterization studies

Ioana Lacatusua,b, Elena Mitreaa,c, Nicoleta Badeaa,*, Raluca Stana, Ovidiu Opreaa,Aurelia Megheaa,*

aFaculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Polizu Street No. 1, 011061 Bucharest, RomaniabPetroleum-Gas University of Ploiesti, Bucuresti Street No. 39, 100680 Ploiesti, RomaniacBiofarm S.A., Logofat Tautu Street No. 99, 31212 Bucharest, Romania

A R T I C L E I N F O A B S T R A C T

Article history:

Received 9 November 2012

Received in revised form

10 April 2013

Accepted 12 April 2013

Available online 23 May 2013

Keywords:

Lipid nanocarriers

Fish oil

Lutein

Antioxidant activity

In vitro release

1756-4646/$ - see front matter � 2013 Elsevihttp://dx.doi.org/10.1016/j.jff.2013.04.010

* Corresponding author. Tel./fax: +40 0213154E-mail addresses: nicoleta.badea@gmail.c

This study aimed at exploring the behavior of fish oil enriched with x-3 fatty acids in order

to obtain stable lipid nanocarriers (NLCs) with improved characteristics as effective deliv-

ery systems for lutein. The particle size of optimized lutein-NLCs was below 200 nm. The

less ordered arrangement of lipid core revealed by scanning calorimetry and the high

entrapment efficiency of 88.5% clearly indicated the appropriate role of fish oil in obtaining

effective lipid nanocarriers. The evaluation of in vitro antioxidant activity has demonstrated

a significant blocking effect of NLCs, scavenging up to 98% oxygen free radicals. The in vitro

release profile has shown that NLCs are able to ensure a better, in vitro sustained release of

lutein as compared to conventional nanoemulsions.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The remarkable benefits of nanotechnology for the food sec-

tor with a strong potential to influence many aspects of food

systems have been well documented in the past few years.

Even though the applicability of nanotechnology to the food

sector occurred mainly after 2005, it offers interesting manu-

facturing and processing opportunities for the food industry

and promises great market potential. As drug delivery nano-

systems have been widely researched in the pharmaceutical

field, more and more attention has also been paid to the use

of appropriate delivery systems in the food industry. In this

er Ltd. All rights reserved

193.om (N. Badea), a.meghea

context, the nanoencapsulation of bioactive compounds

seems to be an essential requisite for the production of func-

tional foods (Chen, Weiss, & Shahidi, 2006; Duncan, 2011).

The bioactive compounds, usually referred to as ‘‘nutra-

ceuticals’’ are extranutritional constituents that typically oc-

cur in small quantities in foods and exhibit the capacity to

modulate one or more metabolic processes (Ajilla, Anaidu,

Bhat, & Prasada Rao, 2007). Their presence as natural costitu-

ents in foods confers health benefits and provides an alterna-

tive to modern medicine (Biesalski et al., 2009; Madrigal-

Carballo et al., 2010). Many of these bioactive compounds

(e.g. carotenoids, omega-3 fatty acids, phytosterols) are highly

.@gmail.com (A. Meghea).

J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 6 0 – 1 2 6 9 1261

lipophilic and, as a result they have a poor absorption and

limited bioavailability (Barrow, Nolan, & Holub, 2009; Weiss

et al., 2008). Over the last 2 years, some studies have shown

that encapsulation of bioactive compounds could efficiently

overcome the above mentioned issues (Paolino et al., 2011;

Lacatusu, Badea, Oprea, Bojin, & Meghea, 2012; Lacatusu, Ba-

dea, Stan, & Meghea, 2012; Wang et al., 2012). By reducing the

particle size, nanotechnology can contribute to improving the

properties of food compounds (Sauto & Muller, 2006), such as

delivery properties (Chen et al., 2006; Teixeira et al., 2012), sol-

ubility (Patel & San Martin-Gonzales, 2012;Porter, Pouton,

Cuine, & Charman, 2008), and efficient absorption through

cells (Acosta, 2009).

Among the nanoencapsulation systems, the lipid based

nanoparticles are appropriate systems for encapsulation

and delivery of poorly soluble food ingredients. Nanostruc-

tured lipid carriers (NLCs) are the latest generation of nano-

scale encapsulation systems, developed by Muller since 2000

(Muller, Souto, & Radtke, 2000). NLCs are colloidal particles

dispersed in aqueous surfactant solution, with average diam-

eters ranging between 50 and 1000 nm, made of biocompati-

ble and biodegradable lipids (Martins et al., 2012) with well-

established safety profiles and toxicological data (Lim, Baner-

jee, & Onyuksel, 2012; Cushen, Kerry, Morris, Cruz-Romero, &

Cummins, 2012). Up to present, the application of lipid nano-

particles for active food ingredients delivery has been rather

limited, although recent studies have reported the main

advantages of the lipid nanoparticles for oral distribution of

active food nutrients (Fathi, Mozafari, & Mohebbi, 2012;

McClements, Decker, Park, & Weiss, 2009).

Lutein is one of the bioactive food compounds found as

lipophilic pigment in various vegetables (Mitri, Shegokar,

Gohla, Anselmi, & Muller, 2011). Recent studies have shown

a beneficial association between consumption of lutein and

a lower incidence of ocular diseases, including age-related

macular degeneration (Liu & Wu, 2010; Khalil et al., 2012).

Moreover, lutein is able to quench singlet oxygen (Palombo

et al., 2007) and plays an important role in maintaining skin

health by reducing UV-induced inflammation (Stahl & Sies,

2002). Despite these biological activities, lutein is an unstable

molecule which has a very low bioavailability caused by its

poor solubility in the aqueous media. As a result, the nanoen-

capsulation of lutein represents a prerequisite for the produc-

tion of functional foods designed to improve the consumer’s

health.

The production of functional nanostructures with nutra-

ceutical content and improved antioxidant and release prop-

erties is one of the major challenges in the food industry that

will be significantly enhanced by the development of a com-

bined approach of nanotechnology with the specific proper-

ties of natural oils. Starting from this idea, the main

objective of the current research is to investigate and com-

pare the efficacy of x-3 fatty acids in developing new solid

nanocarriers for encapsulation and delivery of poorly soluble

food compounds. The innovative idea of this research was to

combine the advantages of x-3 fatty acids with known biolog-

ical effects (e.g. cardioprotective properties (Harris, Park, & L.

& Isley W., 2003), preventing rheumatoid arthritis and cancer

(Augustina et al., 2011; Nagao & Yanagita, 2005) with physico-

chemical characteristics of the lipid nanomatrix. More pre-

cisely, in this study, the performance of fish oil to obtain a

highly disordered lipid nanomatrix able to protect and accom-

modate the sensitive lutein is explored for the first time. Fur-

thermore, the in vitro antioxidant and release properties of the

lutein loaded into the new nanocarriers were evaluated to

provide some optimal parameters to formulate this nutraceu-

tical into fat-in-water dispersions.

2. Materials and methods

Lutein (20% lutein, w/w, dispersed in corn oil) was purchased

from DSM Special Products (Rotterdam, The Netherlands),

Polyoxyethylenesorbitan monooleate (Tween 80), Dimethyl-

sulphoxide and Hydrogen peroxide were obtained from Merck

(Frankfurt, Germany); Poloxamer 407 (block copolymer of

polyethylene and polypropylene glycol) was supplied by BASF

Chem Trade GmbH (Burgbernheim, Germany), Soybean leci-

thin from Cargill Texturizing Solutions Deutschland GmbH

& Co. (Hamburg, Germany) and Tris[hydroxymethyl] amino-

methane (Luminol) was purchased from Sigma Aldrich Che-

mie GmbH (Seelze, Germany). The fish oil (FO) with omega-3

fatty acids composition of 63% determined starting from tria-

cylglycerols (e.g. 1.33% linolenic acid, C 18:3; 2.00% moroctic

acid, C 18:4; 0.29% eicosanetrienoic acid, C 20:3; <0.05% eico-

sanetetraenoic acid, C 20:4; 32.95% eicosapentaenoic acid

(EPA), C 20:5; 3.81% docosapentaenoic acid (DPA), C 22:5; doco-

sahexaenoic acid (DHA), 22.58% C 22:6) was supplied by Henry

Lamotte Oils Gmbh (Bremen, Germany). Carnauba wax (CW)

was obtained from Kahl Wachsraffinerie (Trittau, Germany)

and glycerol stearate (GS) from Cognis (Monheim, Germany).

2.1. Synthesis of lutein–lipid nanocarriers

The lutein loaded lipid nanocarriers (noted as NLCs-L1–L9)

and unloaded lipid nanocarriers (noted as free NLCs) were

synthesized by using the melting emulsification coupled

with the high shear homogenization technique, previously

reported (Lacatusu et al., 2012; Niculae, Lacatusu, Badea, &

Meghea, 2012). Briefly, in a melted lipid phase composed of

GS, CW and FO, an appropriate amount of lutein–corn oil

dispersion (see Table 1) was added. The lipid mixture was

kept for 5 min before mixing with an aqueous surfactant

phase. The hot emulsion resulted by mixing lipid and aque-

ous phases at 85 �C was submitted to an external mechani-

cal energy by high shear homogenization at 25,000 rpm for

a period of 10 min (Lab rotor–stator High-Shear Homogenizer

SC 250 type, 30,000 rpm, power of 250 W, PRO Scientific (Ox-

ford CT, USA). The resultant pre-emulsion was allowed to

cool down at room temperature, with formation of aqueous

dispersions of lutein lipid nanocarriers. The composition of

each NLC and some physico-chemical characteristics are

presented in Table 1. Finally, the NLCs dispersions were sub-

mitted to a lyophilization process (�55 �C, 72 h), by using an

Alpha 1–2 LD Freeze Dry System equipment (Braunschweigv,

Germany), in order to remove the excess of water and to ob-

tain powders of lutein loaded NLCs and free-NLCs. For the

lutein release study, two reference nanoemulsions (NE) were

prepared by the same experimental protocol and using a

mixture of FO with medium chain triacylglycerols (pur-

chased from Cognis GmbH, Dusseldorf, Germany).

Table 1 – The physico-chemical characterization of prepared lutein-NLCs.

Formulation* Luteinconcentration(%)**

Fish oilcontent (%)***

Average diameters,Zave (nm)

Polydispersityindex (PdI)

Zeta potential f (mV) EntrapmentEfficiency, EE (%)

NLC-L1 0.04 10 387.4 ± 10.24 0.419 ± 0.006 �29.1 ± 0.67 –

NLC-L2 0.04 20 350.2 ± 8.388 0.293 ± 0.001 �30.3 ± 0.97 –

NLC-L3 0.04 30 270.6 ± 5.762 0.217 ± 0.017 �27.4 ± 0.68 –

NLC-L4 0.08 10 366.0 ± 5.543 0.331 ± 0.033 �32.9 ± 1.04 55.8 ± 3.35

NLC-L5 0.08 20 292.5 ± 7.671 0.263 ± 0.010 �34.3 ± 1.76 83.5 ± 4.01

NLC-L6 0.08 30 167.5 ± 0.793 0.172 ± 0.016 �34.2 ± 0.50 88.5 ± 4.21

NLC-L7 0.12 10 375.8 ± 3.242 0.345 ± 0.043 �33.3 ± 0.86 50.1 ± 2.46

NLC-L8 0.12 20 329.2 ± 8.391 0.276 ± 0.008 �32.2 ± 2.33 75.3 ± 4.15

NLC-L9 0.12 30 206.6 ± 1.943 0.221 ± 0.006 �34.5 ± 4.61 76.8 ± 3.89

* All NLCs formulations have been prepared by using 4% surfactant mixture of Tween 80/Lecithin/Block copolymer.

** The lutein concentration in the aqueous dispersion.

*** The composition of fish oil was varied between 10% and 30% from the total lipid content of 10%.

1262 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 6 0 – 1 2 6 9

2.2. Determination of peroxide value and para-anisidinevalue

The experimental protocols applied for the determination of

the peroxide value (PV) and the p-anisidine value (pAV) of fish

oil based NLCs and fish oil references have followed the stan-

dard procedures according to the AOCS (1997) method Cd 8–53

and the International Standard ISO 6885 (2006), respectively.

2.3. Determination of average diameters andpolydispersity index of lutein-NLCs

The particle size parameters of lipid nanoparticles given by the

hydrodynamic diameters, zaverage and polydispersity index, Pdl

of each NLC dispersion were determined by the dynamic light

scattering (DLS) technique (Zetasizer Nano ZS, Malvern Instru-

ments Ltd., Worcestershire, UK), at a scattering angle of 90 and

25 �C. Dispersions were analyzed after appropriate dilution

with deionised water to an adequate scattering intensity prior

to the measurement. The particle size analysis data were eval-

uated using intensity distribution. The average diameters

(based on Stokes–Einstein equation) and polydispersity index

were calculated based on three individual measurements.

2.4. The stability evaluation of lutein-NLCs

The electrophoretic mobility (zeta potential, f) of the lutein-

NLCs and their surface charge have been measured by com-

bining laser Doppler velocimetry and phase analysis light

scattering using a Zetasizer Nano ZS (Malvern, Instruments

Ltd., Worcestershire, UK). Before zeta potential measure-

ments, the aqueous dispersions of NLCs samples were ad-

justed to 50 lS/cm by drop wise addition of 0.9% (w/v) NaCl

solution. The zeta potential results reported are the

mean ± standard deviation of at least three determinations.

2.5. Transmission electron microscopy (TEM) examination

The size and morphological examination of the lipid nanocar-

riers loaded with lutein were observed using a Philips 208 S

electron microscope (Eindoven, Netherlands). The lutein-

NLCs sample was diluted with ultrapure water (in a ratio of

1:50). One drop of the dispersion was deposited on a carbon

film-covered copper grid and kept for 15 min to allow some

of the particles to adhere to the carbon substrate. The sample

was then examined and photographed.

2.6. Determination of lutein entrapment efficiency intolipid nanocarriers

For investigation of the entrapment efficiency (EE%), the free-

lutein concentration (non-encapsulated inside the lipid core)

was detected by standard UV–VIS analysis. The EE% has been

determined using the UV absorption at kmax = 444 nm (the

specific absorption maximum for lutein), according to the

Lambert Beer law. The concentration of free lutein has been

determined on the NLCs loaded with initial concentrations

of 0.08% and 0.12%. In the case of NLCs formulations loaded

with 0.04% lutein, no EE% values are given because the

amount of free lutein in the NLC investigated was below the

detection limit. The percentage of loaded lutein has been cal-

culated from the absorbance by using the calibration curve in

the concentration range of 10.5–79.2 lg/L, with a correlation

coefficient of R = 0.9977 (n = 6).

2.7. Differential scanning calorimetry (DSC)characterization

The changes in the crystalline states of the lipid matrix of

lyophilized free- and lutein loaded NLCs were studied by differ-

ential scanning calorimetry. The DSC analysis was performed

using a Jupiter, STA 449C differential scanning calorimeter

(Netzsch, Selb, Germany). The samples (10 mg) were weighed

into standard alumina pans. An empty pan was used as refer-

ence. The thermal analysis profiles were obtained as the tem-

perature was increased from 30 to 100 �C at a rate of 5 �C/min.

2.8. In vitro determination of antioxidant activity

The in vitro antioxidant activity of pure lutein, free-NLCs and

lutein-NLCs was determined by the chemiluminescence

method (CL) using a Chemiluminometer Turner Design TD

J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 6 0 – 1 2 6 9 1263

20/20, Sunnyvale, CA, USA. A cyclic hydrazide (luminol) has

been used as a light amplifying substance which emits light

when oxidized and is converted into an excited aminophta-

late ion in the presence of oxidizing species. As a generator

system for free radicals, H2O2 has been used in a Tris–HCl buf-

fer solution (pH = 8.4). Reference solutions of lutein with con-

centrations of 60 nM (reference lutein (1), 116 nM (reference

lutein (2) and 176 nM (reference lutein (3) were prepared.

The antioxidant activity (percentage of scavenging of free rad-

icals) was calculated by using the relation:

%AA ¼ I0 � Is

I0� 100

where I0 = the maximum CL for reference at t = 5 s; Is = the

maximum CL for sample at t = 5 s.

2.9. Evaluation of in vitro lutein release from developedlipid nanocarriers

The in vitro lutein release from the prepared lipid nanocarriers

was assessed using the diffusion Franz cells (25 mm in diam-

eter, Hanson Research Corporation, Chatsworth, CA, USA), for

a period of 56 h. The cell consisted in donor and receptor

chambers between which a cellulose membrane (Teknokro-

ma, Spain) was positioned. The experiments were performed

under sink conditions in order to conduct an appropriate lu-

tein dissolution test. The sink conditions involve the use of

a sufficient volume of dissolution medium (receptor fluid),

which should be able to dissolve the total lutein released from

the lipid nanocarriers. For the in vitro experiments, a volume

of 300 lL of lutein-NLC dispersion was placed in the donor

compartment. The receptor fluid (6 mL) consisted in a solu-

tion of 70% phosphate buffered saline (pH = 5.5) and 30% eth-

anol (HPLC purity). The temperature of the receptor chamber

was controlled at 37 �C by a water circulator. At specified

intervals, 300 lL of the receptor medium were withdrawn

and the same volume of fresh phosphate buffer solution

was added. The released lutein concentration was analyzed

by UV–VIS using the same protocol described for the entrap-

ment efficiency.

3. Results and discussion

A critical aspect in order to obtain the desired lipid nanopar-

ticles containing active food ingredients is represented by the

appropriate selection of the oils/lipids used. Starting from this

idea and knowing the poor lutein solubility and stability, the

fish oil enriched in x-3 fatty acid concentrate seems to

meet all those characteristics that make it a suitable candi-

date for the development of bioactive NLCs. Firstly, the fish

oil plays an important role in improving the lutein solubility

and stability. Secondly, it comes with supplementary health

benefits in addition to those of lutein.

3.1. Stability of fish oil based lipid nanocarriers

The chemical stability of fish oil enriched with x-3 fatty acids

is an important issue to be taken into consideration in the

development of appropriate carrier systems for active food

compounds such as lutein or any related compound. When

fish oils undergo oxidation, they produce unstable intermedi-

ary compounds, such as free radicals and hydroperoxides

(known as ‘‘primary oxidation products’’), which are suscepti-

ble to further oxidation reactions, leading to products such as

aldehydes and ketones (secondary oxidation products) (Arab-

Tehrany et al., 2012). The stability and quality of x-3 fish oil

based NLCs were evaluated by assessing the peroxide value

(PV) and the para-anisidine value (pAV) on different lutein-

NLCs as compared to fish oil references. In this context, insig-

nificant changes were observed in the PV of the analyzed

samples. The PV of the reference fish oil was 3.8 ± 0.14 meq/

kg (before processing), while for lutein-NLCs the PV was

4.3 ± 0.23 meq/kg. This minor change observed confirms the

integrity of the fish oil which does not undergo multiple

changes after incorporation into NLC formulations. This

behavior could be assigned to a chemically protective effect

of solid core of the nanoparticles that hinders the oxygen

from reaching the FO and oxidizing it. The use of a ternary

mixture of lipophilic antioxidant (encapsulated lutein) with

glycerol monostearate and carnauba wax could be also

responsible for obtaining an efficient stabilization of FO. Car-

nauba wax is known to manifest antioxidant activity owing to

the rich amount of hydroxyl esters and cinnamic aliphatic

diesters existent in its composition. Moreover, according to

several recent studies (Serfert, Drusch, & Schwarz, 2009; Ave-

rina, Muller, Popov, & Radnaeva, 2011; Aghbashlo, Mobli, Mad-

adlou, & Rafiee, 2012) reported on encapsulation of the

polyunsaturated fatty acids, they can be protected against

light and heat damage, and thus it is possible to suppress or

retard their oxidation.

The first observation was further confirmed by the results

obtained from para-anisidine measurements. A reference FO

sample subjected to the same heat treatment as those used

for NLCs preparation (85 �C during a period of 1/2 h) has a

high para-anisidine value (28.5 ± 0.96) suggesting a rapid oxi-

dation process. All the fish oil based NLCs have shown an

effective protection against oxidation, the pAV varying from

a minimum value of 5.67 ± 0.32 (for NLC-L7) to a maximum

value of 10.1 ± 0.58 (for NLC-L6). By comparing the pAV results

for both lutein-NLCs and reference FO samples (with the FO

amount varying from 10% to 30%), there is a slight increase

in pAV of NLC samples, suggesting the appearance of some

secondary oxidation reactions (Fig. 1). A potential source of

these secondary oxidation products can be caused by the in-

creased temperature (85 �C during a period of 1/2 h) which

rapidly converts the unstable primary oxidation products

(e.g. hydroperoxide, free radicals) into secondary ones (e.g.

carbonyl compounds) detected by the para-anisidine reagent.

However, the pAV values obtained in the present study for the

omega-3 fatty acids based lipid nanoparticles are lower than

the acceptable limit imposed by the World Health Organisa-

tion for human consumption (Max 20). Moreover, as it may

be seen later, the chemiluminescence assay conducted to elu-

cidate the radical scavenging activity of the developed fish oil

based NLCs, supported all these observations. For instance,

the presence of primary oxidation products should be associ-

ated with low antioxidant activity. A decrease of antioxidant

activity has not been confirmed by the in vitro antioxidant as-

say (Fig. 5).

Fig. 1 – The para-anisidine values of the lutein loaded fish oil based NLCs, the reference un-heated fish oil and the fish oil after

employing the ‘‘oxidative stress test’’ (heat treatment at 85 �C, during a period of 1/2 h). n = 3.

1264 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 6 0 – 1 2 6 9

It is worthwhile to mention that the pAV of lutein-NLCs

prepared with a composition of 10%FO (e.g. 6.36 ± 0.52, for

NLC-L4 and 5.67 ± 0.32, for NLC-L7) have been lower than

those prepared with 30%FO (e.g. 10.1 ± 0.63, for NLC-L6 and

10.02 ± 0.58, for NLC-L9, Fig. 1). This difference could be asso-

ciated with the lutein distribution inside the lipid core or in

the outer shell of lipid nanoparticles, as function of FO con-

centration (as will be discussed in further sections). The exis-

tence of lutein adsorbed on the surfactant shell could justify

the retard of the oxidation reactions. In accordance with

these results, it could be presumed that the presence of anti-

oxidant compounds (lutein and carnauba wax) incorporated

into the solid matrix could work sinergistically with FO in or-

der to produce stable lipid nanoparticles.

3.2. Size evaluation of lutein–lipid nanocarriers

The results obtained by the DLS technique have indicated that

the lipid nanocarriers size was significantly influenced by the

fish oil composition and the amount of lutein encapsulated.

The hydrodynamic diameters of lutein-NLCs ranged from

167 to 390 nm. There is a decrease in the average diameters

of NLCs as the fish oil content was increased (e.g. from

375.8 nm for a content of 10% FO up to 206.6 nm for 30% FO

and an initial loading of 0.12% lutein). A reason behind this

decrease in the particle size with increased fish oil loading

could be attributed to differences in the viscosities of the dis-

persed phase and to a better solubility of lipophilic lutein into

the lipid network. Moreover, a proper accommodation of lu-

tein inside the lipid network formed with higher fish oil con-

tent was evidenced by the quantitative analysis of lutein

entrapped into the lipid core, as it will be discussed later.

Fig. 2 – TEM images of optimized lutein-NLCs with different pe

With respect to the influence of the initial lutein concen-

tration on the lipid particles size, the increase in lutein con-

centration from 0.04% to 0.08% has lead to a decrease in the

Z-average values for all the NLCs (Table 1). This behavior

may be explained by considering the previous viscosity char-

acteristics, as a consequence of corn oil influence present in

the lutein suspension. An opposite trend was observed for

NLC formulation that was initially loaded with 0.12% lutein.

An increase in the main diameter (e.g. from 167 nm, for

NLC-L6 to 206 nm, for NLC-L9) could be associated with a pro-

gressive reduction of lutein solubility (from NLC-L7 to NLC-

L9). This means that the lipid core could not accommodate

large amounts of lutein, which would lead to physical adsorp-

tion of lutein on the surface coated with surfactant. As result,

an increase in the diameter size is observed. This assumption

is proved by the results obtained by scanning calorimetry and

UV–VIS spectroscopy.

The particle size estimated by DLS technique was well cor-

related with that found using microscopy analysis. The mor-

phology and sizes of NLCs loaded with different lutein

concentrations can be observed on TEM micrographs

(Fig. 2). According to TEM images, lipid nanocarriers prepared

with 30% FO and loaded with 0.08% and 0.12% lutein, have

spherical shape and diameters less than 180 nm (e.g. NLC-

L6) and 200 nm (e.g. NLC-L9).

3.3. Determination of the electrokinetic potential

The surface particle charge is quantified as zeta potential (f)

which refers to the electrophoretic mobility of the particles

in an electric field (Das & Chaudhury, 2011). The results of

electrokinetic measurements have shown a slight variation

rcent of lutein loading. (a) NLC-L6; (b) NLC-L9; Bar: 500 nm.

Fig. 3 – Evaluation of the lutein loaded NLCs stability by zeta potential distribution.

J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 6 0 – 1 2 6 9 1265

of zeta potential values (Table 1). The combination of alkyl

polyoxy-ethylenesorbitan with soybean lecithin and a block

copolymer has lead to strongly negative surface charges, with

absolute zeta potentials of synthesized bio-active formula-

tions ranging between �29.1 and �34.5 mV. Some representa-

tive zeta potential distributions are presented in Fig. 3. These

close values indicate similar interfacial properties of all devel-

oped NLCs and good physical stability in time, without fur-

ther aggregation. No effect of the lipid core (with 10%, 20%

and 30% fish oil composition) on the surface charge was ob-

served. The only observation to be drawn from these systems

is that the NLCs containing a larger amount of lutein have in-

creased stability as compared to formulations containing

0.04% lutein (Table 1).

3.4. Evaluation of lipid crystallinity before and afterloading with lutein

The scanning calorimetry has been employed in this study to

provide insights on the melting behavior and crystalline

structure of the free and loaded NLCs prepared with fish oil.

The allure of DSC curves reveals the existence of three endo-

thermic effects: one peak located at about 52 �C (specific for

unsaturated x-3 fatty acids and also for glycerol monostea-

rate), one shoulder at �45 �C due to the saturated fatty acids

present in the fish oil composition and one peak at �78 �Ccharacteristic to the lipid component from the carnauba wax.

The perturbation of the lipid network after lutein encapsu-

lation is evident (Fig. 4). This aspect underlines a less ordered

crystalline network of all lutein-NLCs as compared to free-

NLC with significant effect, as it may be seen later, on the

entrapment efficiency of lutein. The most obvious perturba-

tion of the lipid network has been produced by the encapsu-

lation of 0.08% lutein, although the appearance of this effect

was expected for the initial concentration of 0.12% lutein

(e.g. NLC-L5 versus NLC-L8 and NLC-L6 versus NLC-L9,

Figs. 4A and B). This behavior can be correlated with some lit-

erature data which report that lutein has a weak capacity to

be solubilised (Alves-Rodrigues & Shao, 2004). These aspects

support the presumption formulated in the preliminary eval-

uation of lutein-NLCs (pAV measurements and DLS results)

that indicates a possible localization of lutein outside the lipid

Fig. 5 – Enhanced in vitro antioxidant activity by including fish oil enriched in x-3 fatty acids in lipid nanoparticles.

Fig. 4 – Differential scanning calorimetry thermograms of lyophilized free- and loaded lutein-NLCs. (A and B) The influence of

lutein loading (for NLC prepared with 20% and 30% FO); (C) the effect of fish oil composition on the lipid crystallinity.

1266 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 6 0 – 1 2 6 9

Fig. 6 – The release profile of lutein from optimized solid lipid nanocarriers (NLCs) and related nanoemulsions (NE).

J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 6 0 – 1 2 6 9 1267

core. This lutein distribution is investigated in detail in the

last section of this study.

An interesting result was observed by comparing the effect

of FO composition on the crystallization pattern of NLCs.

Fig. 4C exemplifies the depression of the main melting points

and endothermic peaks detected after the increase of FO

amount. Endothermic peaks of NLC-L4, L5 and L6, containing

10%, 20% and 30% FO, respectively, were 53.2/78.4, 52.3/77.5

and 50.9/77 �C, respectively (Fig. 4C). This behavior clearly

indicates the presence of lattice defects inside the lipid core

(many imperfections present in the matrix) which are favor-

able – as it will be seen later – to the encapsulation of a de-

sired amount of lutein and consequently, to obtain good

entrapment efficiencies. Additionally, the shift of the melting

peaks can be explained by the colloidal nature of the samples.

As it was earlier reported (Bunjes, Koch, & Westesen, 2000), a

melting point depression suggests defects in the crystalline li-

pid network and, as a result, smaller particle sizes.

3.5. Lutein entrapment inside the lipid nanocarriers

The determination of the entrapment efficiency (EE%) is an

essential stage in order to predict the suitability of NLCs

based on x-3 fatty acids, as efficient reservoirs for the lyp-

ophil lutein. The entrapment of lutein inside the lipid core

was strongly associated with the lutein and the FO concen-

trations (Table 1). The EE% was improved as the FO content

has been increased to a maximum value of 30% (e.g. from

56% for NLC-L4 prepared with 10% FO to 88.5% for NLC-L6

prepared with 30% FO). These results could be justified by

the high solubility of lutein into the complex lipid core

which contains the liquid fish oil reservoirs. In other words,

a less ordered arrangement of the lipid core is accompanied

by high entrapment efficiency.

Concerning the maximum lutein concentration that can

be encapsulated, it was observed that a higher lutein load-

ing has led to a saturation of the lipid matrix. The EE of

NLC-L9 (with initial concentration of 0.12%, w/v) was lower

than those obtained for a 0.08% lutein loading (Table 1). As

a result, a higher lutein loading led to an increased level of

non-encapsulated lutein (free-lutein localized in the outer

surfactant shell) than the lutein encapsulated inside the li-

pid core. These results are in good agreement with the pre-

vious observations regarding the distribution of lutein in

the lipid core.

3.6. In vitro antioxidant activity of free-lipid nanocarriersand loaded with lutein

The use of fish oil highly concentrated in x-3 fatty acids for

the development of functional nanostructures with antioxi-

dant properties represents an innovative aspect that may

open new opportunities for the food industry. All the synthe-

sized NLC formulations have the potential to develop a high

blocking action of the chain reactions in vitro generated into

the chemiluminescence system (Fig. 5). Despite various

entrapment efficiencies obtained for the developed NLCs, it

was observed that there is no significant difference between

AA% of NLCs loaded with various amounts of lutein. The lipid

nanocarriers containing 60, 116, and 176 nM lutein, manifest

similar capacities to scavenge the free radicals, with values

ranging between 93.5% and 98.2% (Fig. 5). Two relevant

hypothesis could be associated in order to explain this unex-

pected antioxidant behavior: (i) the distribution of lutein in

the lipid core or at the interface does not influence the antiox-

idant capacity; (ii) the existence of a synergistic effect of anti-

oxidant properties of various active compounds contained in

the NLC structure.

Interestingly, by determining the AA% of free-NLCs, the re-

sults demonstrated that the lipid nanocarriers prepared with

10% fish oil do not result in a decrease of AA%, as expected,

considering the previous quantitative results. This could be

due to a size effect (nanostructuration of fish oil), meaning

that whatever the fish oil content, the effect is the same. In

this regard it is worthwhile to mention that beside the liquid

oil that participates in the formation of a highly disordered li-

pid network with many lattice defects able to solubilise an

important lutein concentration, the fish oil is able to develop

new lipid nanocarriers that present themselves antioxidant

properties. Their capacity to scavenge the oxygen free radi-

cals is ranging between 94% and 98%.

3.7. In vitro release study of the lutein-NLCs

The release profiles of lutein from NLCs in phosphate buf-

fered saline media for a period of 56 h of the experiment have

been compared with conventional nanoemulsions (NEs)

(Fig. 6). By comparing the release profile of both kinds of car-

riers, the release of lutein from NLCs was significantly better

than that of related NEs. After 27 h of diffusion experiments,

the lutein release has been almost complete in the case of NEs

1268 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 6 0 – 1 2 6 9

(90% for NE-L6 and 99% for NE-L9), whilst for NLCs the RE%

has reached only 60% for NLC-L6 and 63% for NLC-L9. These

results represent clear proof of the important role played by

fish oil in combination with appropriate solid lipids in

improving the lutein release percentage.

The in vitro release behavior of NLC-L9 loaded with initial

concentration of 0.12% lutein demonstrated the presence of

lutein outside the lipid core, as it was discussed in the previ-

ous sections. A certain amount of lutein was readily released

from the NLC-L9 immediately after the release experiments

(Fig. 6). The lutein release percentage in this case is higher

than that observed for NE-L9 (e.g. 9.9% in NLC-L9 versus

6.5% in NE-L9, during 2 h of release and 13.6% in NLC-L9 ver-

sus 11.8% in NE-L9, after 3 h of release). This percentage ob-

tained during the first 3 h of measurements (for NLC-L9)

corresponds to the free (not encapsulated) lutein (physical ad-

sorbed to the surfactant shell) which manifests a faster re-

lease rate as compared to that of the lutein entrapped

inside the lipid core.

4. Conclusion

Different fish oil based lipid nanocarriers with various lutein

loading were successfully synthesized and their physico-

chemical properties were investigated in detail. The in vitro

characterization of lutein–lipid nanocarriers has shown that

the fish oil plays important roles in improving the antioxidant

capacity. The free- and lutein loaded NLCs have demon-

strated the ability to develop a high blocking effect, with a po-

tential to scavenge up to 98% the oxygen free radicals

generated into the chemiluminescence system. Most impor-

tantly, this comparative study is associated with the main

feature of synthesized fish oil based lipid nanocarriers to ex-

hibit a better in vitro sustained release of lutein as compared

to their related nanoemulsions. It is worthwhile to conclude

that data generated in the current study represent a novel

and effective strategy for lutein delivery, which may be bene-

ficial to future applications in the development of functional

foods with a high applicability spectrum to the food sector.

The formulation of lutein into colloidal nanolipid-in-water

dispersions could serve to successfully incorporate this nutra-

ceutical into water-dispersible food systems.

Acknowledgements

Authors recognize financial support from the European Social

Fund through POSDRU/89/1.5/S/54785 project: ‘‘Postdoctoral

Program for Advanced Research in the field of

nanomaterials’’.

R E F E R E N C E S

Acosta, E. (2009). Bioavailability of nanoparticles in nutrient andnutraceutical delivery. Current Opinion in Colloid and InterfaceScience, 14, 3–15.

Aghbashlo, M., Mobli, H., Madadlou, A., & Rafiee, S. (2012). Thecorrelation of wall material composition with flow

characteristics and encapsulation behavior of fish oilemulsion. Food Research International, 49, 379–388.

Alves-Rodrigues, A., & Shao, A. (2004). The science behind lutein.Toxicology Letters, 150, 57–83.

Ajilla, C. M., Anaidu, K., Bhat, S. G., & Prasada Rao, U. J. S. (2007).Bioactive compounds and antioxidant potential of mango peelextract. Food Chemistry, 105, 982–988.

Averina, E. S., Muller, R. H., Popov, D. V., & Radnaeva, L. D. (2011).Physical and chemical stability of nanostructured lipid drugcarriers (NLC) based on natural lipids from Baikal region(Siberia, Russia). Pharmazia, 66, 348–356.

Arab-Tehrany, E., Jacquot, M., Gaiani, C., Desobry, S., & Linder, M.(2012). Beneficial effects and oxidative stability of omega-3long-chain polyunsaturated fatty acids. Trends in Food Science &Technology, 25, 24–33.

Augustina, M. A., Abeywardena, M. Y., Pattena, G., Heada, R.,Locketta, T., De Lucae, A., & Sanguansri, L. (2011). Effects ofmicroencapsulation on the gastrointestinal transit and tissuedistribution of a bioactive mixture of fish oil, tributyrin andresveratrol. Journal of Functional Foods, 3, 25–37.

Barrow, C. J., Nolan, C., & Holub, B. J. (2009). Bioequivalence ofencapsulated and microencapsulated fish-oilsupplementation. Journal of Functional Foods, 1, 38–43.

Biesalski, H.-K., Dragsted, L. O., Elmadfa, I., Grossklaus, R., Muller,M., Schrenk, D., Walter, P., & Weber, P. (2009). Bioactivecompounds: Definition and assessment of activity. Nutrition,25, 1202–1205.

Bunjes, H., Koch, M. H. J., & Westesen, K. (2000). Effect of particlesize on colloidal solid triglycerides. Langmuir, 16, 5234–5241.

Chen, H., Weiss, J., & Shahidi, F. (2006). Nanotechnology innutraceuticals and functional foods. Food Technology, 60, 30–36.

Cushen, M., Kerry, J., Morris, M., Cruz-Romero, M., & Cummins, E.(2012). Nanotechnologies in the food industry – Recentdevelopments, risks and regulation. Trends in Food Science &Technology, 24, 30–46.

Das, S., & Chaudhury, A. (2011). Recent advances in lipidnanoparticle formulations with solid matrix for oral drugdelivery. AAPS PharmSciTech, 12, 62–76.

Duncan, T. V. (2011). Applications of nanotechnology in foodpackaging and food safety: Barrier materials, antimicrobialsand sensors. Journal of Colloid and Interface Science, 363, 1–24.

Fathi, M., Mozafari, M. R., & Mohebbi, M. (2012).Nanoencapsulation of food ingredients using lipid baseddelivery systems. Trends in Food Science & Technology, 23, 13–24.

Harris, W. S., Park, Y., & Isley, W. L. (2003). Cardiovascular diseaseand long-chain omega-3 fatty acids. Current Opinion inLipidology, 14, 9–14.

Khalil, M., Raila, J., Ali, M., Islam, K. M. S., Schenk, R., Krause, J. P.,Schweigert, F. J., & Rawel, H. (2012). Stability and bioavailabilityof lutein ester supplements from Tagetes flower preparedunder food processing conditions. Journal of Functional Foods,4(2), 602–610.

Lacatusu, I., Badea, N., Oprea, O., Bojin, D., & Meghea, A. (2012).Highly antioxidant carotene-lipid nanocarriers: Synthesis andantibacterial activity. Journal of Nanoparticles Research, 14,902–918.

Lacatusu, I., Badea, N., Stan, R., & Meghea, A. (2012). Novel bio-active lipid nanocarriers for the stabilization and sustainedrelease of sitosterol. Nanotechnology, 23, 455515–455702.

Lim, S. B., Banerjee, A., & Onyuksel, H. (2012). Improvement ofdrug safety by the use of lipid-based nanocarriers. Journal ofControlled Release, 163, 34–45.

Liu, C.-H., & Wu, C.-T. (2010). Optimization of nanostructured lipidcarriers for lutein delivery. Colloid and Surfaces A:Physicochemical and Engineering Aspects, 353, 149–156.

Madrigal-Carballo, S., Lim, S., Rodriguez, G., Vila, A. O., Krueger, C.G., Gunasekaran, S., & Reed, J. D. (2010). Biopolymer coating ofsoybean lecithin liposomes via layer-by-layer self-assembly as

J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 6 0 – 1 2 6 9 1269

novel delivery system for ellagic acid. Journal of FunctionalFoods, 2(2), 99–106.

Martins, S., Costa-Lima, S., Carneiroa, T., Cordeiro-da-Silva, A.,Soutod, E. B., & Ferreira, D. C. B. (2012). Solid lipidnanoparticles as intracellular drug transporters: Aninvestigation of the uptake mechanism and pathway.International Journal of Pharmaceutics, 430, 221–227.

Mitri, K., Shegokar, R., Gohla, S., Anselmi, C., & Muller, R. H. (2011).Lipid nanocarriers for dermal delivery of lutein: Preparation,characterization, stability and performance. InternationalJournal of Pharmaceutics, 414, 267–275.

McClements, D., Decker, E. A., Park, Y., & Weiss, J. (2009).Structural design principles for delivery of bioactivecomponents in nutraceuticals and functional foods. CriticalReviews in Food Science and Nutrition, 49, 577–606.

Muller, R. H., Souto, E. B., & Radtke, M. (2000). PCT application PCT/EP00/04111.

Nagao, K., & Yanagita, T. (2005). Conjugated fatty acids in food andtheir health benefits. Journal of Bioscience and Bioengineering,100, 152–157.

Niculae, G., Lacatusu, I., Badea, N., & Meghea, A. (2012). Lipidnanoparticles based on butyl-methoxydibenzoylmethane: Invitro UVA blocking effect. Nanotechnology, 23, 315704–315714.

Palombo, P., Fabrizi, G., Ruocco, V., Ruocco, E., Fluhr, J., Roberts, R.,& Morganti, P. (2007). Beneficial long-term effects of combinedoral/topical antioxidant treat- ment with the carotenoidslutein and zeaxanthin onhumanskin: A double-blind, placebo-controlled study. Skin Pharmacology and Physiology, 20, 199–210.

Paolino, D., Stancampiano, A. H. S., Cilurzo, F., Cosco, D., Puglisi,G., & Pignatello, R. (2011). Nanostructured lipid carriers (NLC)for the topical delivery of lutein. Drug Delivery Letters, 1, 32–39.

Patel, M. R., & San Martin-Gonzales, F. (2012). Characterization ofergocalciferol loaded lipid nanoparticles. Journal of Food Science,71, N8–N12.

Porter, C. J. H., Pouton, C. W., Cuine, J. F., & Charman, W. N. (2008).Enhancing intestinal drug solubilisation using lipid-baseddelivery system. Advanced Drug Delivery Reviews, 60, 673–691.

Sauto, E. B., & Muller, R. H. (2006). Applications of lipidnanoparticles (SLN and NLC) in food industry. Journal of FoodTechnology, 4, 90–95.

Serfert, Y., Drusch, S., & Schwarz, K. (2009). Chemical stabilisationof oils rich in long-chain polyunsaturated fatty acids duringhomogenisation, microencapsulation and storage. FoodChemistry, 113, 1106–1112.

Stahl, W., & Sies, H. (2002). Canotenoids and protection againstsolar UV radiation. Skin Pharmacology and Applied SkinPhysiology, 15, 291–296.

Teixeira, Z., Dreiss, C. A., Lawrence, M. J., Heenan, R. K., Machado,D., Justo, G. Z., Guterres, S. S., & Duran, N. (2012). Retinylpalmitate polymeric nanocapsules as carriers of bioactives.Journal of Colloid and Interface Science, 382, 36–47.

Wang, Y., Ye, H., Zhou, C., Lv, F., Bi, X., & Lu, Z. (2012). Study on thespray-drying encapsulation of lutein in the porous starch andgelatin mixture. European Food Research and Technology, 234,157–163.

Weiss, J., Decker, E. A., McClements, D. J., Kristbergsson, K.,Helgason, T., & Awad, T. (2008). Solid lipid nanoparticles asdelivery systems for bioactive food components. FoodBiophysics, 3, 146–154.