RESEARCH PAPER

a-Tocopherol/chitosan-based nanoparticles:characterization and preliminary investigationsfor emulsion systems application

Antonella Aresta • Cosima Damiana Calvano •

Adriana Trapani • Carlo Giorgio Zambonin •

Elvira De Giglio

Received: 16 October 2013 / Accepted: 22 December 2013 / Published online: 12 January 2014

� Springer Science+Business Media Dordrecht 2014

Abstract The processes of lipids oxidation represent

a great concern for the consumer health because they are

one of the major causes of quality deterioration in fat-

containing products. One of the most effective methods

of delaying lipid oxidation consists in incorporating

antioxidants. The present investigation describes the

formulation of chitosan and novel glycol chitosan

nanoparticles (NPs) loaded with a-Tocopherol (aToc-

NPs). The obtained NPs were characterized by various

techniques, such as particle size (showing mean diam-

eters in the range 335-503 nm) and zeta potential

measurements, X-ray photoelectron spectroscopy and

Fourier transform infrared spectroscopy. The NPs were,

then, added in the preparation of oil-in-water simple

emulsion both to make the lipophilic aToc available in

an aqueous medium and to prevent emulsion oxidation.

For this purpose, a new highly sensitive, simple and

solvent-free method based on a solid phase

microextraction (SPME) coupled to gas chromatogra-

phy mass spectrometry was developed for the determi-

nation of aToc in aqueous medium. All the parameters

influencing SPME, including fiber coating, time and

temperature extraction, pH, ionic strength and desorp-

tion conditions, have been carefully screened. The

method was successfully applied to the determination of

vitamin in the aToc-NPs and its release from NPs-

enriched simple emulsion formulations. SPME pro-

vided high recovery yields and the limits of detection

and of quantification in emulsion were 0.1 and 0.5 lg/

mg, respectively. The precision of the method has been

also estimated. The delay of the lipid oxidation by the

proposed formulations has been evaluated exploiting

the Kreis test on aToc-NPs-enriched emulsions.

Keywords SPME/GC–MS � a-Tocopherol �Chitosan nanoparticles � Emulsions � Lipid

oxidation � Antioxidants

Introduction

The chemical decomposition of fats, oils and other

lipids is one of the major reasons of quality deterio-

ration in fat-containing products, such as food, phar-

maceuticals and cosmetic formulations (Sun et al.

2011; Gibson 2007).

When these processes take place, undesirable odors

and flavors can result and lipid oxidations can

potentially be very harmful to human health due to

A. Aresta (&) � C. D. Calvano � C. G. Zambonin �E. De Giglio

Department of Chemistry, University of Bari, ‘‘Aldo

Moro’’, Via Orabona, 4, 70125 Bari, Italy

e-mail: antonellamaria.aresta@uniba.it

A. Aresta � C. G. Zambonin � E. De Giglio

Interdepartmental Research Center S.M.A.R.T,

Department of Chemistry, University of Bari, ‘‘Aldo

Moro’’, via Orabona, 4, 70125 Bari, Italy

A. Trapani

Department of Pharmacy-Drug Sciences, University of

Bari, ‘‘Aldo Moro’’, Via Orabona, 4, 70125 Bari, Italy

123

J Nanopart Res (2014) 16:2230

DOI 10.1007/s11051-013-2230-0

the formation of undesirable compounds, including

free radicals and reactive aldehydes, sometimes very

toxic (Sun et al. 2011; Esterbauer 1993; Poljsak et al.

2013).

Oil is often present in foods and in pharmaceutical

and cosmetic preparations to form colloidal systems.

The so-called ‘‘biphasic systems’’ refer to water-in-oil

(w/o) and also oil-in-water (o/w) simple emulsion, or

microemulsion or solid–lipid nanoparticles (NPs)

(Ziani et al. 2012). However, lipid oxidation involving

emulsions is generally recognized as being more

complex than in bulk oil systems, since the emulsifi-

cation process leads to the formation of a large

interfacial area, and lipid oxidation is initiated at the

interface between oil and water, where different non-

polar and polar compounds can interact in the system

(Frankel et al. 1994; McClements and Decker 2000;

Cercaci et al. 2007).

One of the most effective approaches to slow down

the lipid oxidation is the incorporation of natural or

synthetic antioxidants.

The natural antioxidants suffer of a short life and,

therefore, for preparations requiring very long shelf

life the use of synthetic antioxidants (i.e., butylated

hydroxyanisole, butylated hydroxytoluene, propyl

gallate, tert-butylhydroquinone, etc.) is preferred.

However, some issues (i.e., toxicity, potential carcin-

ogenicity, etc.) surround the use of synthetic antiox-

idants especially in foods and beverage, so recently the

interest for natural ones has been newly intensified

(Poljsak et al. 2013).

Among natural antioxidants, special attention

should be given to vitamin E, a fat-soluble vitamin

that exists in eight different forms, of which the

a-tocopherol (aToc) is the major radical scavenging

antioxidant in vivo (Traber and Packer 1995; Hoppe

and Krennrich 2000).

Vitamin E efficiently interrupts the chain propaga-

tion of lipid oxidation, thus protecting polyunsaturated

fatty acids and low-density lipoproteins from oxida-

tion. The commercial topical formulations of the type

o/w emulsion often contain vitamin E, exerting both

antioxidant and skin conditioning activities. For this

reason, there is increasing interest in fortifying many

commercial products with vitamin E (Sagalowicz and

Leser 2010). Several studies also suggest that the

bioavailability of vitamin E may be increased when it

is delivered by colloidal formulations rather than in

bulk form (Feng et al. 2009). However, the short life

time of tocopherols induces to develop strategies that

enable to protect and prolong its biological activity. In

this respect, in a previous work (Aresta et al. 2013)

chitosan NPs (CSNPs) loaded with aToc and/or

vitamin C were proposed for the improvement of the

antioxidant(s) shelf life in food packaging.

Starting from the obtained encouraging results, the

present work aimed at extending aToc-loaded NPs as

novel preservatives able to minimize system oxidation

in commercial emulsions applications. In this study,

we describe the formulation and characterization of a

CS derivative, glycol chitosan NPs (GCNPs) loaded

with aToc along with the previous investigated aToc-

loaded CSNPs.

Characterization of the resulting particles was

performed by particle size and zeta potential mea-

surements, Fourier transform infrared spectroscopy

(FT-IR) and X-ray photoelectron spectroscopy (XPS),

whereas for aToc quantification in aqueous medium a

new solid phase microextraction (SPME)–gas chro-

matography (GC)–mass spectrometry (MS) method

was developed. SPME is a quite efficient technique

which can be applied for the extraction of organic

compounds from aqueous solution, thus allowing the

simultaneous extraction and pre-concentration of

analytes from complex matrices (Pawliszyn 1997).

In the literature, the most frequently used methods for

tocopherol determination involve organic solvent

extraction (Abidi 2000; Gruszka and Kruk 2007),

solid phase extraction (Grigoriadou et al. 2007; Mata-

Granados et al. 2009), supercritical fluid extraction

(Fratianni et al. 2002) and pressurized liquid extrac-

tion (Bustamante-Rangel et al. 2007; Delgado-Zamar-

reno et al. 2009). More recently, polymeric materials,

specific towards vitamin E, have been developed to

selectively and accurately bind these analytes from

biological and food samples (Puoci et al. 2007; Liu

et al. 2010).

Herein, a new protocol allows to achieve a selective

recognition of aToc from natural samples in aqueous

mixtures in the absence of organic solvents. Moreover,

SPME represents a vitamin E extraction technique

more advantageous than the conventional ones thus

removing some drawbacks, such as vitamin destruc-

tion when alkaline conditions are used for organic

solvent extraction, or the plugging of the solid phase

cartridges.

Finally, in the present work a simple o/w emulsion

was prepared and, after addition of aToc-loaded NPs,

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the capacity to make the vitamin available in an

aqueous medium has been investigated at 25 and 4 �C

following the new analytical developed method. It was

also demonstrated that the fiber enables a selective

extraction of aToc also from similar commercial

formulations. Overall, the efficacy of the aToc-loaded

NPs in delaying lipid oxidation was also evaluated

following the Kreis test on aToc-NPs-enriched emul-

sions (Narasimhan et al. 1999).

Materials and methods

Materials

The following chemicals were obtained from commer-

cial sources and used as received. Chitosan hydrochlo-

ride (Protasan, UP CL 113, Mw in the

50,000–150,000 g/mol range, deacetylation degree

86 % according to manufacturer instructions) was

purchased from Pronova Biopolymer (Norway). Olive

oil (highly refined, low acidity), KBr, a-Tocopherol

(aToc, C96 % purity), glycerol (over 99.5 % purity),

glycol chitosan (degree of polymerization C400 accord-

ing to supplier instructions), phloroglucinol dihydrate

(97 %), Span 65, Tween 20 were provided by Sigma-

Aldrich, Italy. Sulfobutylether-b-cyclodextrin sodium

salt (SBE7m-b-CD, Mw = 2163 Da, average substitu-

tion degree = 6.40) was purchased from CyDex, Inc.

(USA). SBE7m-b-CD was kept in a desiccator until use.

Ultrapure water (Carlo Erba, Italy) was used throughout

the study. All other chemicals were reagent grade.

NPs preparation

Chitosan NPs in the presence and in the absence of

aToc were prepared according to our previous work

(Aresta et al. 2013). a) Unloaded GCS and CSNPs

Firstly, GCS was dissolved at 0.20 % w/v in acetic

acid solution (0.1 % v/v) whereas CS was dissolved in

double distilled water at the same concentration.

1.5 mL of the resulting solution of each polysaccha-

ride was maintained under magnetic stirring prior to

the addition of 0.5 mL of SBE7m-b-CD aqueous

solution (0.45 % w/v) leading to NPs formation. The

final GCS/SBE7m-b-CD and CS/SBE7m-b-CD mass

ratio was equal to 1.3:1 (w/w). Herein, unloaded NPs

were denoted as ‘‘unloaded GCSNPs’’ and ‘‘unloaded

CSNPs’’. b) aToc-loaded GCS and CSNPs The

preparation of an inclusion complex constituted by

SBE7m-b-CD and aToc was carried out for 24 h at

room temperature and under magnetic stirring. The

reagents were poured in double distilled water in order

to ensure an equimolar ratio between aToc and

SBE7m-b-CD. In particular, SBE7m-b-CD concentra-

tion was set at 4.5 mg/mL in order to provide NPs

formation whereas aToc was set at the concentration

of 1.0 mg/mL. To obtain NPs, 1.5 mL of the above

mentioned polysaccharide solution (0.20 % w/v) was

mixed with 0.5 mL of the SBE7m-b-CD:aToc com-

plex, a process already reported to lead to NPs

formation (Aresta et al. 2013). Finally, for all formu-

lations, the resulting NPs were isolated by centrifuga-

tion (13.200 rpm for 60 min, Eppendorf 5415D

centrifuge, Eppendorf, Germany) and re-suspended

in ultrapure water by manual shaking.

NPs characterization

Particle size and polydispersity index

Particle size and polydispersity index of all tested NPs

were determined in double distilled water by photon

correlation spectroscopy using a Zetasizer NanoZS

(ZEN 3600, Malvern, UK). The determination of the

f-potential was performed using laser Doppler ane-

mometry (Zetasizer NanoZS, ZEN 3600, Malvern,

UK). Particles were centrifuged on a glycerol bed and

the resulting pellet was diluted with KCl 1 mM

following a previously reported procedure (Montene-

gro et al. 2012).

FT-IR spectroscopy

The FT-IR spectroscopy investigation was performed

using a PerkinElmer 1600 FT-IR spectrometer (Perk-

inElmer, Italy). FT-IR spectra were recorded on pure

aToc, unloaded GCS/CSNPs, and aToc-loaded GCS/

CSNPs mixed with an appropriate amount of KBr to

obtain a final concentration of the sample of 1 % w/w.

The range examined was 4,000–400 cm-1 with a

resolution of 1 cm-1.

XPS analysis

XPS analysis was performed with a ThermoVG The-

taprobe spectrometer (Thermo Fisher Scientific, USA)

equipped with a microspot monochromatized Al Ka

J Nanopart Res (2014) 16:2230 Page 3 of 12 2230

123

source. The Al Ka line (1,486.6 eV) was used through-

out this work and the base pressure of the instrument

was 10-9 mbar. The X-ray beam spot was 300 lm. The

analysis was performed by acquiring survey scans

(binding energy [BE] range 0–1,200 eV, FAT mode,

pass energy = 150 eV) and detailed spectra of C1s,

O1s, N1s, S2p, Cl2p, and Na1s regions (FAT mode, pass

energy = 50 eV). Data were analyzed using the

Advantage software package (version 4.75). The sam-

ple charging effects were minimized with a low-energy

flood gun. In any case, charge referencing of all the

experimental BE values was made by setting the

binding energy of C1s hydrocarbon photopeak at

285.0 eV (De Giglio et al. 2011). The examined

samples (nanoparticulate suspensions and standard

vitamin solution) were casted on gold sheets. At least

three replicates for each sample have been analysed.

Stability studies of aToc-CSNPs and emulsions

Freshly prepared aToc-CSNPs were exposed to

different storage temperatures (i.e., 25 and 4 �C) in

order to evaluate their stability under different incu-

bation conditions (Trapani et al. 2013). The systems

were maintained at both temperatures up to 72 h and

particle size of the samples was determined by

Zetasizer NanoZS at scheduled time intervals. Sam-

ples of emulsions (enriched or not with aToc-CSNPs)

were stored at 25 and 4 �C and their stability was also

assessed. The size of emulsion droplets was deter-

mined by direct observation using a light stereomi-

croscope (Leica Galen III) equipped with a Panasonic

(WV CP 230) camera and Leica Qwin v.2.4 software.

The arithmetic mean diameter of the drops was

determined by averaging the individual values of

150 drops taken by each sample.

SPME/GC–MS measurements

Apparatus

GC–MS analysis was performed by a Finnigan TRACE

GC ultra gas chromatograph equipped with a split/

splitless injector and interfaced, by a GC transfer line, to

an ion trap mass spectrometer (Finnigan PolarisQ). The

GC chromatographic column was a Supelco SPB-5 fused

silica capillary column (non-polar, 30 m 9 0.25 lm

inner diameter, 0.25 lm coating thickness, Sigma-

Aldrich). The injector liner i.d. was 1.00 mm.

GC–MS conditions

To optimize the gas chromatographic and MS detec-

tion conditions initial experiments were performed by

direct injection of 0.5 lL of standard compounds

(5 lg/mL of aToc in methyl alcohol). The oven

temperature program was 180 �C (3 min), 25 �C/min

to 300 �C (12 min); helium (99.999 %) was used as

carrier gas at a constant flow of 1 mL/min; inlet and

transfer line temperatures were set at 270 and 300 �C,

respectively. The mass spectrometer was operated in

the electron impact positive (EI?) ion mode with a

source temperature of 270 �C. The electron energy

was 70 eV and the filament current 150 lA. Mass

spectra were acquired scanning from 50 to 500 m/z.

Preparation of solutions for SPME measurements

Firstly, the stock solution (5 mg/mL) was prepared by

dissolving aToc in methyl alcohol and stored in the

dark at -20 �C. The standard solutions for SPME

measurements were prepared in 5 mL amber vials

(Supelco) by diluting the previous stock in 10 %

ethanol in water.

SPME

Two different silica fibers coated with an 85-lm-thick

polyacrylate (PA) and a 100-lm-thick poly-

dimethylsiloxane (PDMS) film, respectively, were

employed for comparative studies. A manual SPME

device (Supelco) was used to hold the fibers. The

SPME device and procedure have been extensively

described elsewhere (Zambonin et al. 2002). The fiber

was inserted directly into the sample solution and

exposed for 30 min under mechanical stirring; then,

the thermal desorption was performed in the injector

of the gas chromatograph for 10 min. The injector was

operated in splitless mode with 5 min sampling time.

To eliminate the carryover effect, after each chro-

matographic run the fiber was subjected to a second

thermal desorption to confirm that no signal from

aToc was detected.

Determination of aToc association efficiency

The amount of aToc loaded in the NPs was calculated

from the difference between the vitamin concentration

in the aqueous medium before (Co) and after (Cs) the

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NP formation. The latter amount was determined

following the separation of aToc-loaded NPs from the

aqueous suspension medium by centrifugation at

13.200 rpm for 60 min. For the determination of

amounts in Co and Cs, each sample was previously

diluted 1:1000 (v/v) in 10 % ethanol in water.

Results were expressed as association efficiency

(A.E.%) calculated as [1-(Cs/Co)] 9 100. Three mea-

surements were made on at least five different batches

of NP formulations.

Emulsion preparation

Oil-in-water (o/w) emulsions (30 % olive oil, v/v)

were prepared by the drop wise addition of oil to 0.1 %

of the surfactant Tween 20 in acetate buffer (0.1 M,

pH 5.4). The emulsions were homogenized with an

Ultraturrax (Janke and Kunkel, Germany) model T25

equipped with an S25 N dispersing tool at 11,400 rpm

for 2 min prior than cooling in an ice bath for 5 min.

The emulsions were studied in the presence and in the

absence of aToc-CSNPs.

In vitro release of vitamin from aToc-CSNPs-

loaded emulsions

For in vitro release studies, aliquots of 5 mg of aToc-

CSNPs-loaded emulsion were re-suspended in 5 mL

of 10 % ethanol in water and left at constant shaking at

room temperature. The aToc content in the NPs for

each aliquot was in the range 37–43 lg, according to

the A.E.% previously determined. Then, the aliquots

were directly subjected to SPME after 0, 3, 24, 48 and

72 h as described above. To evaluate the temperature

effect on the release performance, a parallel test was

carried out at 4 �C. Results, expressed as release

percentages, were obtained calculating the ratio

between each determination and that obtained on a

standard solution set at a concentration of 8.0 lg/mL.

Each experiment was performed in triplicate.

Oil rancidity evaluation test

The oil rancidity test was performed as follows. 10 mL

of bulk oil with and without the addition of aToc

(0.6 mg/g) or 10 mL of emulsion with and without

aToc-CSNPs was stored in the dark at room temper-

ature or exposed to light at 50 �C, to preserve oil or

induce rancidity, respectively. Aliquots of 1 mL were

withdrawn from each sample every 2 days for 2 weeks

and subjected to Kreis test (Narasimhan et al. 1999), a

colorimetric assay where the color produced by the

action of chemical reagents is a qualitative assessment

of fats and/or oils oxidative rancidity. Briefly, to

perform the test, each aliquot was first added with a

similar amount of concentrate hydrochloric acid and

then with 0.1 % (w/v) of phloroglucinol in ether; the

solutions were then mixed for 30 s. The appearance of

a red color indicated the presence of aldehydes and

ketones due to oil rancidity. The reaction tubes were

centrifuged for 2 min at 2,000 rpm, the lower phase

(color red) was collected and transferred into micro-

cuvettes seeds. The resulting spectra were recorded by

a UV/Vis double beam spectrophotometer (Shimadzu

UV-1601 PC, Japan) vs. water as reference solution.

Each assay was carried out in triplicate.

Statistical analysis

Size, zeta potential, SPME/GC–MS data from differ-

ent experimental groups were compared by a one-way

ANOVA and differences at 95 % level of confidence

(p \ 0.05) were considered significant.

Results and discussion

NPs characterization

Physicochemical characterization of NPs

aToc-loaded NPs were formulated according to the

ionic gelation technique which is a simple procedure

in comparison to recent approaches recommended for

polysaccharides based on probe tip sonication (Qui-

nones et al. 2013). Table 1 shows the main physico-

chemical properties of the investigated aToc-loaded

NPs. Concerning size values of unloaded CSNPs and

GCSNPs, the difference in size could be due to the

solvent used for dissolution of the polysaccharide (i.e.,

water for CS versus a slightly acidic solution for GCS).

Nevertheless, when size data referring to aToc-loaded

NPs were considered, for CS-based NPs, no statistical

difference was noted in comparison to the control

unloaded CSNPs (Table 1). On the other hand, for

GCS-based NPs, the introduction of aToc led to

statistically different size values than control, maybe

due to conformation change in the polysaccharide

J Nanopart Res (2014) 16:2230 Page 5 of 12 2230

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network. For all investigated particles, zeta potential

values were always found positive according to the

positive charges arising from chitosan and glycol

chitosan chains. Moreover, the addition of aToc was

seen to cause a deshielding of the external positive

charges for both types of NPs (Tantra et al. 2010). It

could be consistent with the outcomes arising from

XPS analysis (see below), suggesting an external

localization for aToc whose extent depends on the

type of NPs.

Furthermore, all types of particles exhibited high

A.E.% values for aToc (Table 1). However, to

account for the less aToc A.E.% value of GCSNPs,

it seems that presence of the hydrophilic glycolic

branches should reduce the association of the vitamin

to the GCS polysaccharide backbone, acting as steric

hindrances.

FT-IR analysis

FT-IR spectra were acquired for pure aToc and for

both formulations constituted of CSNPs and GCSNPs,

unloaded and aToc-loaded. As far the loaded and

unloaded CSNPs are concerned, our previous obser-

vations (Aresta et al. 2013) are confirmed. Figure 1

reports representative FT-IR spectra

(4,000–800 cm-1) relevant to aToc (A), unloaded

GCSNPs (B), and aToc-GCSNPs (C). For the

unloaded GCSNPs, almost all the bands have been

associated to the stretching vibrations typical of glycol

chitosan and/or SBE7m-b-CD used for their prepara-

tion. The band at around 3,410 cm-1 observed in

Fig. 1b represents the overlapping of the O–H stretch-

ing with N–H stretching; its broad shape could be

ascribed to the formation of hydrogen bonds; this band

overlaps the –OH and –CH3 stretching vibrations

relevant to aToc (Fig. 1a). The bands of 2,924 and

2,875 cm-1, observed for unloaded NPs, represent

aliphatic C–H stretching, and the band at around

1,638 cm-1 represents the stretching of C=O of acetyl

group in amide I.

The absorption peaks at 1,150 cm-1 (anti-symmet-

ric stretching of the C–O–C bridge), 1,080 and

1,030 cm-1 (skeletal vibrations due to C–O stretch-

ing) are due to its saccharide structure (Quinones et al.

2010), while the fingerprint region presents many

intense signals relevant to SBE7m-b-CD. The spectra

of aToc-GCSNPs (Fig. 1c) are dominated by the

broad peaks of glycol chitosan. However, the new

band observed at 1,490 cm-1 (Aresta et al. 2013; Hu

et al. 2012) and the increase in the amide I band at

1,650 cm-1 (Quinones et al. 2012) confirm the

interaction between the CSNPs and GCSNPs and

aToc.

4000 3500 3000 2500 2000 1500 1000

Tra

nsm

itta

nce

(%)

Wavenumber (cm-1)

A

B

C

Fig. 1 FT-IR spectra relevant to: aToc (a), unloaded GCSNPs

(b), alfa-Toc-GCSNPs (c) (range 4,000–800 cm-1)

Table 1 Physicochemical properties of unloaded CS/GCSNPs and vitamin-loaded CS/GCSNPs

Formulation Size (nm) PI f (mV) A.E.%

Unloaded CSNPs 503 ± 64 0.32–0.23 ?33.8 ± 0.2 –

aToc-CSNPs 494 ± 39 0.26–0.21 ?26.8 ± 1.2a 94.7 ± 7.5

Unloaded GCSNPs 335 ± 25 0.34–0.44 ?26.5 ± 1.2 –

aToc-GCSNPs 439 ± 44a 0.45–0.28 ?17.9 ± 2.2a 74.6 ± 8.2

In particular, size, polydispersity index (PI), zeta potential (f) and association efficiency (A.E.%) are reported as mean ± SD, n = 6a Significantly different from the controls, i.e., unloaded CSNPs and unloaded GCSNPs (p \ 0.005)

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XPS analysis

XPS has been employed to investigate the surface

chemical composition of unloaded CSNPs, unloaded

GCSNPs, aToc-CSNPs and aToc-GCSNPs and, in

particular, the vitamin localization on these nanopar-

ticulate systems. Regarding the unloaded CSNPs and

the aToc-CSNPs, XPS data are consistent with the

previous reported observations (Aresta et al. 2013).

Survey scans recorded (data not shown) confirmed the

typical CS or GCS and cyclodextrin components

(carbon, oxygen, and nitrogen) on unloaded CSNPs or

GCSNPs, as expected (De Giglio et al. 2012). On the

other hand, traces of sulfur, sodium and chlorine were

also detected in all examined samples. The presence of

sulfur and sodium was due to the use of SBE7m-b-CD

as cross-linking agent, while the chlorine signal was

relevant to chitosan salt type employed. It can be

underlined that the introduction of aToc in the NPs

preparation solution did not introduce peculiar signals

in the XPS survey spectra, but simply added contri-

butions to carbon and oxygen spectral regions.

At the best of our knowledge, no XPS literature data

were available neither on pure aToc nor on the

investigated vitamin-modified polymeric systems. In

our recent study on vitamin C and/or aToc-loaded

CSNPs, the spectral comparisons with the standard

vitamin(s) carbon regions have been considered to

assess the vitamin(s) surface presence (Aresta et al.

2013). Also in the XPS investigation of unloaded

GCSNPs and aToc-GCNPs, the complexity of the

carbon regions, due to the co-presence of the

contributions arising from glycol chitosan, SBE7m-b-

CD and aToc, did not allow a reliable fit of these

spectral regions. Therefore, as reported in the previous

study (Aresta et al. 2013), a comparison of the

C1s regions relevant to GCSNPs before and after the

addition of the vitamin has been proposed.

In Fig. 2, the comparison of C1s spectra relevant to

unloaded GCNPs/aToc/aToc-GCSNPs is reported.

Unlike what previously observed on the aToc-

CSNPs system, where the surface presence of aToc

was remarkably evident, on the surface of aToc-

GCSNPs sample only a slight presence of aToc can be

detected. Indeed, this finding raised from the small

increase of the aToc-GCSNPs signal at a BE of about

285.0 eV attributable to hydrocarbon/aromatic carbon

photopeak characteristic of aToc.

The XPS investigation allowed us to point out the

difference between the unloaded nanoparticulate sys-

tems and the aToc-modified CSNPs or GCSNPs

samples in terms of surface features. Altogether,

XPS analyses suggested that aToc can be argued on

both NPs surfaces but a marked presence of vitamin

was detected only on aToc-CSNPs samples. These

findings are in agreement with A.E.% data reported in

Table 1.

Stability of NPs and emulsions

A further characterization of aToc-CSNPs concerned

the assessment of their particle size under different

storage temperatures (25 and 4 �C). Results from

stability studies (see Fig. 3) indicated that aToc-

CSNPs kept constant their mean diameter at 25 �C and

this finding may be related to their higher zeta

Fig. 2 Comparison of C1s spectra relevant to unloaded

GCSNPs/aToc/aToc-GCSNPs. (dashed line unloaded

GCSNPs; grey line aToc; black line aToc-GCSNPs)

Fig. 3 Particle size of aToc-loaded CSNPs upon incubation at

different temperatures. Values represent means ± SD (n = 3).

Black bars 4 �C; grey bars 25 �C

J Nanopart Res (2014) 16:2230 Page 7 of 12 2230

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potential values. Indeed, some small changes in

particle size were detected at 4 �C which maybe due

to a different swelling dynamic influenced by low

temperatures (data not shown).

In addition, the stability over the time for the simple

o/w emulsions, both in the presence and in the absence

of aToc-CSNPs, was also evaluated. In these cases,

the size of the emulsion droplets was examined under a

light microscope (up to 72 h), revealing that their

diameters remained unchanged at 4 �C (data not

shown). On the other hand, after 1 day at 25 �C, the

examined samples showed a slight increase in the

average diameter according to Mine et al. (1996).

SPME/GC–MS measurements

An essential step of the work was the optimization of

the SPME conditions for the extraction of aToc.

Therefore, standard solutions at 0.5 lg/mL concen-

tration were obtained daily by progressively diluting

stock solution of 10 % ethanol in water. The vials were

sealed with hole caps and Teflon-faced silicone septa.

Choice of fiber coating material

As well known, the choice of the best stationary phase

is a crucial step, since it directly affects the magnitude

of the partition coefficient between the sample and the

fiber. PA and PDMS are both homogeneous polymer

coatings exhibiting two different polarities, the former

highly polar and the latter non-polar. Since the aToc

features a chromanol ring with a hydroxyl group and a

hydrophobic side chain, the selected fibers could be

both potentially able to extract the aToc forms of

vitamin E.

Extraction time and temperature

Adsorption times ranging from 5 to 120 min were

investigated at 20 �C at constant stirring in order to

optimize the equilibration time for aToc partition

between the aqueous and the polymer phases. The

extraction time profiles were established by plotting

the area counts versus the extraction time. The

equilibrium was considered reached when a further

increase in the extraction time did not produce a

significant increase in the response values. Figure 4

reports the extraction time profiles at 20 �C for both

fibers.

The equilibrium was not reached after 120 min in

both cases, even if PDMS was capable of the most

efficient extraction. It is possible to obtain good

extraction yields and reliable analysis also in non-

equilibrium conditions. Indeed, it is well known that

SPME quantification is feasible even before adsorp-

tion equilibrium is reached since the amount of the

analyte absorbed onto the fiber is proportional to the

initial concentration in the sample matrix, at agitation

conditions and sampling times constant (Gibson

2007). Therefore, both PDMS and an extraction time

of 30 min were selected for the subsequent tests.

Effect of ionic strength and pH

Experiments were performed by increasing progres-

sively the ionic strength of the extraction solutions. As

expected, the response decreased with salt addition

and was in good agreement with the fact that salt

addition improves the recovery almost exclusively in

the case of polar compounds. The same significant

effects were observed by varying the pH of extraction

solutions from 2 to 7.5.

Desorption conditions and ‘‘carryover’’

It has already been reported that the desorption

temperature should be just slightly higher than the

boiling point of a given compound to ensure its

complete desorption from the fiber coating (Arthur

et al. 1992). However, in the present work, desorption

was carried out for 10 min at 270 �C as significant

‘‘carryover’’ effect was observed at lower tempera-

tures or shorter desorption times. Since carryover was

found to be 0.92 % in optimized conditions, the PDMS

Fig. 4 Extraction time profiles obtained with PDMS (filled

diamond) or PA (filled triangle) fibers

2230 Page 8 of 12 J Nanopart Res (2014) 16:2230

123

was subjected to a second thermal desorption after

each chromatographic run.

Recoveries, linear range, detection limits

and precision

For the recovery estimation, the emulsion samples

were added with aToc at 50 and 5 lg/mg, equilibrated

at room temperature, for at least 10 min, and then

5 mg of spiked emulsions re-suspended in 5 mL of

10 % ethanol in water. The samples were left to

equilibrate at room temperature for 10 min before

extraction with the fiber. Then, the recoveries were

calculated as peak area ratio of aToc (standard)/aToc

(spiked emulsion samples); the obtained % recover-

ies ± standard deviation (n = 3) were 92 ± 8 and

remained practically unchanged from 5 to 50 lg/mg

(aToc/emulsion mass ratio).

The linear range of the developed procedure was

evaluated in the concentration interval 0.1–100 lg/mg

using the above spiked emulsions. In the investigated

range, it was found a linear response in the interval

0.3–100 lg/mg with correlation coefficients[0.9997

and an intercept not significantly different from zero at

95 % confidence level.

The limit of detection (LOD) and limit of quantifi-

cation (LOQ) were 0.1 and 0.5 lg/mg, respectively.

LOD was calculated as threefold and LOQ as tenfold the

standard deviation of the calibration curve intercept,

obtained by unweighted linear regression (Long and

Winefordner 1983). The within-day precision was esti-

mated on spiked emulsion samples (50 and 5 lg/mg)

by performing daily three replicates. The same samples

were analyzed three times every day for a period of

5 days for the between-days precision evaluation. The

within-day RSD% and between-days RSD% were 7.2

and 12.5, respectively, and they were found not

concentration dependent. Finally, the potential of the

analytical method was evaluated on a commercial o/w

emulsion for topic use. The successful determination of

aToc in this commercial sample demonstrated that the

fiber also allows a selective extraction of aToc from

complex matrices.

In vitro release of aToc from CSNPs

Due to the higher A.E.% in aToc-CSNPs than aToc-

GCSNPs, only the former were adopted for in vitro

release tests, in view of their application to the emulsion

systems. The release performances were studied over

the time at two different temperatures, i.e., 25 and 4 �C,

according to data arising from NPs and emulsion

stability tests. Release profiles are shown in Fig. 5. As

expected, the low temperature significantly diminishes

the swelling of the CS polysaccharide and, therefore,

lower percentages of aToc were detected in the aqueous

medium. On the other hand, at 25 �C almost 80 % of the

total amount of aToc was available within the first 72 h.

This result suggests a high and fast supply of the vitamin

from the emulsions enriched with aToc-CSNPs when

they are stored at room temperature. These data are in

good agreement with our previous work (Aresta et al.

2013) where it was demonstrated that about 88 % of

active vitamin E was released within 7 days at room

temperature.

Oil rancidity evaluation test

As known, oxidation processes occurring in oils and fats

can originate free radicals which lead to decomposition

of fatty acids into various low molecular weight

compounds as aldehydes and ketones. These processes

are enhanced overall in presence of light, oxygen,

metals and high temperatures (Calvano et al. 2007;

Calvano et al. 2011). So, to limit rancidity, the fats can

be stored at cool temperatures, in the dark and away

from oxygen exposure or, alternatively, could be

enriched with antioxidants. In particular, the type of

added antioxidant is a key factor in controlling these

processes; it is known that lipophilic antioxidants are

Fig. 5 Amount of the aToc release from CSNPs enriching

emulsion systems up to 72 h of monitoring at 4 �C (filled

square) and 25 �C (filled triangle). Data represent mean ± SD

(n = 3)

J Nanopart Res (2014) 16:2230 Page 9 of 12 2230

123

more active in polar systems because they located at the

oil–water interface during oxidation propagation. In this

work, the efficacy of the aToc in slowing down lipid

oxidation was evaluated following the Kreis test on bulk

olive oil, subjected to induced rancidity with and

without the addition of vitamin (see ‘‘Oil rancidity

evaluation test’’ section). Then, the same protocol was

applied to evaluate the oxidation of the emulsion

systems with and without aToc-CSNPs. At the 15th day

of treatment, the test showed the appearance of a clear

red color for all the samples taken as controls (i.e.,

without vitamin supplementation) and no color in those

added with vitamin, confirming the formation of

aldehydes and ketones in control samples. The resulting

absorbance spectra are shown in Fig. 6. Although the

olive oil is little susceptible to rancidity due to the high

content of monounsaturated fatty acids, it was selected

as ‘‘model’’ of the apolar component in the biphasic

simple emulsions which can be destined to different

applications. Further studies will be addressed to gain

insight into the rancidity occurrence of different oils in

the presence of aToc nanoparticulate device.

Conclusions

Herein, aToc-loaded CS and GCSNPs were formu-

lated according to the ionic gelation technique and

characterized as new vitamin delivery systems. FT-

IR and XPS techniques confirmed the aToc

presence on both nanoparticulate systems. A novel

SPME coupled with GC–MS procedure has been

exploited, for the first time, for the determination of

aToc from aqueous matrices using a PDMS fiber.

The method is simple, highly sensitive, fast and

solvent-free. By SPME/GC–MS method the deter-

mination of the vitamin both in NPs investigated

suspensions and in simple emulsion systems was

carried out. CSNPs suspension revealed the higher

A.E.% and both release and storage performances

of this nanosystem suggested the application of this

novel ‘‘nano-preservative’’ for delaying lipids oxi-

dation. Kreis test confirmed the efficacy of the

aToc-loaded CSNPs in stabilizing vitamin-CSNPs-

enriched emulsions. Overall, the obtained detection

limit of aToc allowed the potential application of

the optimized SPME/GC–MS method to commer-

cial emulsions such as food, pharmaceuticals or

cosmetics.

Acknowledgments This project was financed by Universita

degli Studi di Bari (Progetti di Ateneo 2010). Thanks are due to

Public Research Laboratories Networks of Regione Puglia

(Italy)-APQ Research Programme ‘Integrated renewable energy

generation from the regional agro-industrial sector. Project

Code 01’. (Intervento cofinanziato dall’Accordo di Programma

Quadro in materia di Ricerca Scientifica—II Atto Integrativo

Fig. 6 Bulk olive oil (a), o/w simple emulsions (b); solid line samples enriched with aToc or aToc-CSNPs; dotted line sample not

added

2230 Page 10 of 12 J Nanopart Res (2014) 16:2230

123

—PO FESR 2007-2013, Asse I, Linea 1.2-PO FSE 2007-2013

Asse IV ‘‘Investiamo nel vostro futuro’’).

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