Accepted Manuscript Title: Hybrid Encapsulation Structures based on -carotene-Loaded Nanoliposomes within Electrospun Fibers Author: Rafael Henrique de Freitas Zˆompero Amparo L´opez-Rubio Samantha Cristina de Pinho Jos´e Mar´ıa Lagaron Lucimara Gaziola de la Torre PII: DOI: Reference:
S0927-7765(15)00149-6 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.03.015 COLSUB 6955
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
16-10-2014 12-2-2015 5-3-2015
Please cite this article as: R.H.F. Zˆompero, A. L´opez-Rubio, S.C. Pinho, J.M. Lagaron, L.G. Torre, Hybrid Encapsulation Structures based on rmbeta-carotene-Loaded Nanoliposomes within Electrospun Fibers, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.03.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Hybrid Encapsulation Structures based on β-carotene-Loaded
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Nanoliposomes within Electrospun Fibers
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Rafael Henrique de Freitas Zômpero1, Amparo López-Rubio2, Samantha Cristina de Pinho3, José María Lagaron, Lucimara Gaziola de la Torre1*
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University of Campinas, UNICAMP, School of Chemical Engineering, 13083-970, Campinas, São Paulo, Brazil. Novel Materials and Nanotechnology Group, Institute of Agrochemistry and Food Technology, IATA, 46980, Paterna, Valencia, Spain.
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Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga, SP, Brazil
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*Corresponding author, [email protected], phone: +55 19 35210397
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Abstract
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Hybrid
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incorporated within the polymeric ultrathin fibers produced through electrospinning were
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developed to improve the photostability of the antioxidant. These novel materials were
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intended to incorporate β-carotene into water-based food formulations, overcoming the
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existing limitations associated with its hydrophobic character. Initially, both empty and
24
antioxidant-loaded nanoliposomes were developed and incorporated into polyvinyl
25
alcohol (PVOH) and polyethylene oxide (PEO) solutions. The changes in the solution
26
properties were evaluated to determine their effects on the electrospinning processing.
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The mixed polymer solutions were subsequently electrospun to produce hybrid
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nanoliposome-loaded ultrathin fibers. FTIR analysis confirmed the presence of
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phospholipid molecules inside the electrospun fibers. These ultrathin fibers were
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evaluated regarding their morphology, diameter, internal β-carotene distribution and
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stability against UV irradiation. Liposomal release studies from the electrospun fibers
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were also undertaken, confirming the presence of the liposomal structures after
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dissolving the electrospun fibers in water.
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1. Introduction
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Carotenoids are often used as food additives due to their properties as vitamins,
6
colorants and antioxidants. β-carotene is the most widely studied naturally occurring
7
carotenoid commonly found in fruits and vegetables (Paiva & Russell, 1999). This
8
carotenoid might have anti-cancer and anti-cardiovascular disease properties (Dulinska
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et al., 2005; Vainio, 2000; D’odorico, 2000). However, carotenoids (including β-
10
carotene) are sensitive molecules, being susceptible to oxidation by light, heat and
11
oxygen exposure. In addition, they are hydrophobic compounds, hindering direct
12
applications to aqueous-based food formulations. Micro- and nanoencapsulation
13
techniques are plausible options for overcoming the drawbacks related to adding
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carotenoids to foods.
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Previously, microencapsulation has been used to protect sensitive substances against
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the influences of adverse environments. The term “microencapsulation” designates a
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technology that involves wrapping solids, liquids or gases in small capsules, which can
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release their contents under specific conditions (Champagne & Fustier, 2007). This
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process emerged from the micro/nanotechnology field, finding success in many
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industries by conferring remarkable properties to products, such as pharmaceuticals,
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foods, cosmetics, textiles and many other products used during daily life (Risch, 1995).
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Encapsulation techniques can be used to solubilize the hydrophobic compounds in
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water-based foods, stabilizing reactive compounds and protecting sensitive molecules
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from oxidation, moisture, light exposure, temperature and other extreme conditions.
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These protecting abilities are of outmost importance for
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functional food products (Gibbs et al., 1999). Another important characteristic of
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encapsulation technologies for food applications is their ability to mask undesirable
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odors and flavors, improving the product acceptance by the consumer. Several types of
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nanoparticles are studied for these applications, being liposomes amongst the most
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the development of novel
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promising ones due to their wide range of possible diameters and surface charges, as
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well as their nutraceutical properties and encapsulation abilities for both hydrophilic and
3
hydrophobic compounds (Lasic, 1993). A more recently explored method for
4
microencapsulation of compounds in food is the electrohydrodynamic processing
5
comprised of electrospinning and electrospraying processes (Bhardwaj & Kundu, 2010;
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Fernandez et al., 2009). Electrospinning is a simple and highly versatile method for
7
producing fibers, which are in the submicron range and have a large surface to volume
8
ratio, through the action of an external electric field applied between two electrodes and
9
imposed on a polymer solution or melt. Electrohydrodynamic processing for food
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applications is still in its early stages of development, but has demonstrated a great
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potential for developing innovative products with specific new properties. Another
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advantage of this technique is its low cost and high productivity, making it ideal for
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industrial purposes (Ramakrishna et al., 2005). This technique has recently
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demonstrated tremendous potential in food science through the development of novel
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functional ingredients and more efficient food packaging structures (Fabra et al., 2013;
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López-Rubio et al., 2009; López-Rubio et al., 2012; Torres-Giner et al., 2008; Torres-
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Giner & Lagaron, 2009). Combining electrospinning with other technologies from the
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micro/nano encapsulation field can broaden the possibilities for new products with
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improved functionalities. One interesting and promising combination of technologies
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involves trapping liposomal nanostructures within electrospun fibers to stabilize
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compounds (Mickova et al., 2012). Therefore, the aim of the present work was to
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develop novel hybrid materials comprising the two above-mentioned promising
23
technologies for the encapsulation and protection of β-carotene. Consequently, β-
24
carotene-loaded nanoliposomes were incorporated within polyvinyl alcohol (PVOH) and
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polyethylene oxide (PEO) electrospun fibers because both water soluble polymers are
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of great interest for the food and pharmaceutical industries (Peresin et al., 2009;
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Supaphol & Chuangchote, 2007; Baba et al., 2010; Deitzel et al., 2001).
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2. Experimental Part
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2.1. Materials PVOH was kindly supplied by Plásticos Hidrosolubles S.L. (Valencia, Spain) as a thin
5
film form, and the PEO with a Mw of 200 kDa was supplied by Sigma-Aldrich in powder
6
form (Madrid, Spain). The hydrogenated soybean phosphatidylcholine (HSPC)
7
PHOSPHOLIPON 80H from Lipoid (Ludwisghafen, Germany) and ethanol 96% (v/v)
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were supplied by Panreac (Barcelona, Spain) and used to produce the nanoliposomes.
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Synthetic β-carotene (minimum purity 97%) was purchased from Fluka Analytical
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(Madrid, Spain). n-Hexane (98% purity) from Fluka Analytical (Madrid, Spain) was used
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to quantify the β-carotene. The β-carotene was kept frozen and in darkness until use.
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All of the samples obtained from the performed experiments were protected from light
13
until analysis.
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2.2. Nanoliposomes production The nanoliposomes were prepared using ethanol injection method. The lipids (or lipids
17
and -carotene) were dispersed in ethanol (above the main phase transition
18
temperature) and injected at 30 mL/min into a jacketed reactor containing water (100
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mL) while controlling the temperature (60°C) and stirring speed (1336 rpm using a
20
Cowles type impeller) over 5 min. This heating process is necessary due to high lipid
21
transition temperature (approximately 51°C). The final nanoliposome dispersion
22
contains 10% (v/v) ethanol/water and 20 mM lipid. To produce nanoliposomes
23
containing encapsulated β-carotene, the alcoholic phase was prepared differently,
24
adding a 0.5% molar ratio of β-carotene/Phospholipid. The percentage of the β-carotene
25
added to the nanoliposomes was established based on preliminary studies (results not
26
shown). The average hydrodynamic diameter, polydispersity and zeta potential of the
27
nanoliposomes and -carotene-loaded nanoliposomes were measured using Malvern
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Zeta-Sizer Nano-ZS equipment. Since the aim of this study was β-carotene protection,
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hydrogenated lipids were used to avoid peroxidation processes (Huang & Chung, 1998
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and Krinsky & Deneke , 1982).
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2.3. Nanoliposome/Polymer Formulations The solutions used for the electrospinning process were composed of a polymer (PVOH
7
or PEO) and nanoliposomes produced through the ethanol injection method. The
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polymer/water mass ratio was 20%, and this value remained constant for all the
9
formulations. Three different lipid/polymer mass ratios were studied for empty
10
nanoliposomes and β-carotene loaded nanoliposomes: 2.5%, 5% and 7.5%. The
11
polymeric solutions without addition of nanoliposomes were also electrospun to
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evaluate the effect of the empty nanoliposomes or β-carotene loaded nanoliposomes on
13
the properties of the polymeric solutions and in the produced fibers.
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2.4. Electrospinning process The electrospinning apparatuswas a FluidNatek® instrument, trademark of BioInicia
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S.L. (Valencia, Spain), equipped with a variable high voltage 0–30 kV power supply was
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used. The anode was attached to a stainless steel needle with a 0.9 mm internal
18
diameter, which was connected to the polymer/nanoliposome solutions kept in a 5 ml
19
plastic syringe through a PTFE wire. The syringe was placed on a digitally controlled
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syringe pump with the needle directed toward the collector. The electrospun structures
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were collected on a stainless steel plate attached to the copper grid collector. The
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electrospinning conditions were maintained at 24°C and 60% RH by enclosing the
23
equipment in a specific chamber with temperature and humidity control. The collector
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was placed 10 cm from the capillary tip. The syringe pump delivered the PVOH and
25
PEO polymer solutions at 0.1 ml/h, and the voltage was maintained at 10 kV. The
26
conditions were the same for all of the electrospinning processes. The collected
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materials were stored in a desiccator at 0% RH and protected from light.
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2.5. Characterization of the polymeric solutions The rheological properties of the various polymeric solutions were evaluated with a
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Thermo Haake RS1 controlled stress rheometer (Thermo Scientific, United States,
4
Waltham, MA) in the cone-plate configuration, both of which were made of titanium. The
5
angle between the surface of the cone and the plate was of 1 degree, and a distance of
6
1 mm from the plate was kept during the measurements. The temperature during the
7
measurements was 20 ± 1°C and maintained using a Phoenix P1 Circulator device
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(Thermo Haake). Different frequencies and shear rates were applied to the samples,
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and the behavior of the samples toward the determined conditions was evaluated using
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Rheowin Pro Software v.3.61 Haake. Before applying the stress, the samples remained
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in contact for 10 min with the cone-plate system to achieve thermal equilibrium. The
12
surface
13
nanoliposomes was measured using the Wilhemy plate method in an EasyDyne K20
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tensiometer (Krüss GmbH, Hamburg, Germany). All measurements were conducted in
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duplicate.
the
polymer
solutions
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2.6. Fourier transform infrared spectroscopy The ATR-FTIR spectra of the different electrospun materials obtained (pure polymeric
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fibers and fibers containing empty or β-carotene-loaded nanoliposomes) were collected
20
in a controlled chamber at 24°C and 60% RH by coupling the ATR accessory Golden
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Gate of Specac Ltd. (Orpington, UK) to a Bruker (Rheinstetten, Germany) FTIR Tensor
22
37 equipment. All of the spectra were collected by averaging 20 scans at a 4 cm-1
23
resolution. The spectral data were compared using the Opus Viewer software
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(Rheinstetten, Germany).
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2.7. Scanning Electron Microscopy (SEM) The morphologies of the electrospun fibers were examined using a Hitachi S-4100
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Scanning Electron Microscope. The different samples were sputtered with a gold–
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palladium mixture under vacuum. All of the SEM experiments were carried out at 30 kV.
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The diameters of the electrospun fibers were measured by averaging the diameters of
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at least 100 randomly chosen electrospun fibers from the SEM micrographs in their
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original magnification through the ImageJ software (Maryland, USA).
3
2.8. Raman microspectroscopy The Raman spectra were collected with a Jasco NRS-3100 Confocal Micro-Raman
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spectrophotometer (Jasco Inc., Easton, MD) using a 100x objective to evaluate the β-
7
carotene distribution within the developed hybrid electrospun fibers. The Raman
8
chemical images were collected in point-by-point mode by plotting the area of the β-
9
carotene band at 1500 cm-1 and were constructed by collecting 15x15 spectra spaced
10
equally along the selected sample area. The β-carotene generates very large Raman
11
scattering patterns and, thus, very short acquisition times were needed to record
12
intense spectra showing only the signal for β-carotene within the electrospun fibers.
13
This signal intensity was used to construct the Raman images.
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2.9. Nanoliposomes release from electrospun fiber mats PEO and PVOH nanofibers containing 7.5% (m/m) nanoliposomes and β-carotene-
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loaded nanoliposomes (0.01 g of nanofibers mat) were hydrated in water (5 mL) with
18
gentle stirring at 25ºC. After dissolution was complete, the aqueous phase was
19
observed through Transmission Electron Microscopy. The obtained solution was
20
carefully applied to a 400 mesh cooper grid, rested for 5 min and dried using filter
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paper. Uranyl acetate (1% v/v) was then added to the sample on the cooper grid for
22
coloring, resting for 1 min before being dried with clean filter paper. The prepared
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samples were placed inside the transmission electron microscope sample port for
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image acquisition using ZEISS CLM 902 and CCD Proscan systems.
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2.10. Stability tests by UV-vis irradiation The PEO and PVOH electrospun fiber mats with the maximum nanoliposome content
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(7.5% m/m in relation to polymer) and, consequently, the highest β-carotene content
29
were evaluated relative to their ability to protect the photosensitive antioxidant molecule.
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For this purpose, the antioxidant degradation rate was compared with that of β-carotene
2
encapsulated in nanoliposomes (aqueous solution) and free β-carotene crystals
3
dissolved in hexane. Samples from each system were placed in glass flasks and
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exposed to an Osram Ultra-Vitalux 300W UV lamp over 6 h. The distance between the
5
lamp and the flasks was 10 cm. Samples were collected every hour and the intact β-
6
carotene concentration was determined using Agilent 8453 spectrophotometer (Santa
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Clara, USA) at 450 nm. The β-carotene concentration of the control samples containing
8
β-carotene crystals dissolved in hexane was determined directly by spectrophotometry.
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For the aqueous solutions of the β-carotene-loaded nanoliposomes, ethanol was added
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to disrupt the nanoliposomes (releasing the entrapped β-carotene) before hexane was
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added to extract the β-carotene, generating a 2-phase system. The organic (upper)
12
phase was separated, and the β-carotene concentration was determined by
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spectrophotometry. For the PEO and PVOH electrospun fibers containing the
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antioxidant-loaded nanoliposomes, the fibers were initially placed in water until
15
completely dissolved before adding the ethanol and hexane for the liposomal disruption
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and β-carotene extraction, respectively.
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A statistical analysis was performed to evaluate whether significant differences in the
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relative β-carotene concentration over time under UV light exposure for the different
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protective systems. For this evaluation, a student’s t-test was performed using the
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Statistica 7® software. The confidence interval used in the analysis was 80% due to
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inherent variations in the process.
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3. Results and discussion
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3.1. Surface tension and rheological properties of the polymeric solutions containing “empty” nanoliposomes
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The solution surface tension is an important parameter that directly influences the
9
electrospinning process (Ramakrishna et al., 2005) because the intensity of the
10
electrical field must overcome the surface tension of the solution, expelling an electrified
11
jet from the Taylor’s cone formed on the needle tip (Doshi & Reneker, 1995; Taylor,
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1969). Therefore, the surface tension of the PEO and PVOH solutions containing
13
different amounts of “empty” nanoliposomes were evaluated and subsequently related
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to the fiber morphology (see Supplementary material Figure 1S).
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It was observed that the effect of adding empty nanoliposomes on the surface tension
16
was different between the PEO and PVOH solutions. While a gradual decrease in
17
surface tension was observed when increasing the liposomal content in the PEO
18
solution (from 59.5 mN/m to 53 mN/m), the surface tension of the PVOH solutions
19
remained constant and close to 45 mN/m. The PVOH solutions already had a low
20
surface tension and therefore remained unaffected by the added nanoliposomes.
21
Polymeric
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carotene/phospholipid molar ratio) at the same lipid/polymer mass ratios were also
23
subjected to surface tension measurements and no significant differences were
24
observed with respect to the surface tension of the solutions with “empty”
25
nanoliposomes.
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Surface tension is an important parameter during electrospinning, but alone it cannot
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completely explain the suitability of a solution for the development of electrospun
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structures. Adding nanoliposomes could also affect other important parameters involved
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in electrospinning, such as viscosity and viscoelasticity. These parameters, together
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solutions
containing
β-carotene-loaded
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(0.5%
β-
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with the electrical conductivity of the solutions, are the major factors that determine the
2
suitability of the polymeric solutions for electrospinning and the morphology of the
3
obtained structures. Three typical structures, which are primarily beads, beaded fibers
4
and bead-free fibers, can form depending on the solution properties in combination with
5
several process parameters (such as the applied voltage or the distance to the
6
collector) (Ramakrishna et al., 2005). To understand the morphological features of the
7
obtained hybrid structures, the rheological behavior of the PVOH and PEO-based
8
solutions was analyzed. Figures 1A and 1B show the relationship between the shear
9
rate and the applied stress for the PVOH and PEO solutions, respectively, that contain
10
different lipid/polymer mass ratios. In addition, Figures 1C and 1D show the correlation
11
between the shear rate and apparent solution viscosity.
12
Figures 1A and 1C reveal that the pure PVOH solution (without phospholipid addition)
13
presents Newtonian characteristics; the viscosity remained independent from the
14
applied shear rate. Increasing the phospholipid content increased the viscosity and
15
shifted the rheological behavior toward a pseudoplastic character, reducing its viscosity
16
when increasing the shear rate. The increased viscosity when increasing the
17
lipid/polymer mass ratio likely occurred through the interactions between the hydroxyl
18
groups along the polymer chain and the phospholipid head groups (Antunes et al.,
19
2009; Boggs et al., 1986).
20
For PEO, adding phospholipids caused a slight change on the rheological properties of
21
the solution, mainly in the range from 0 to 100 s-1. Figures 1B and 1D show that the
22
PEO solutions, even without phospholipids, exhibited a pseudoplastic behavior that was
23
retained after adding the nanoliposomes. This pseudoplastic behavior indicates that
24
when high shear rates were applied, the molecular structure of the entangled polymer
25
chains acquired a preferential orientation in the flow direction, decreasing the viscosity
26
of the solution (shear thinning flow behavior). For pure PVOH (Newtonian), this
27
phenomenon was not observed.
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PVOH
PEO B
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Figure 1 - Rheological behavior of PVOH and PEO solutions containing different phospholipid mass ratios: (A,B) stress-shear rate curves; (C,D) apparent viscosity-shear rate curves. All the measurements were performed at 20°C in independent duplicates. Experimental error: 10%.
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Viscoelastic materials, when subjected to shear forces, simultaneously undergo viscous
2
and elastic deformations (Chronakis & Kasapis, 1995). The viscoelasticity is important
3
during electrospinning and, thus, in addition to characterizing the viscosity of the various
4
sample solutions, the viscoelastic behavior in terms of the elastic modulus (G’) and
5
viscous modulus (G’’) were also analyzed and the corresponding graphs can be seen in
6
the supplementary material (Figure 2S).
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For samples in gel state, the storage modulus (G’) prevails over the loss modulus (G’’).
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The obtained data revealed that only PVOH solutions with the greatest nanoliposomes
10
loading exhibited this gel state behavior at certain frequencies, which corresponded to
11
the greater viscosity values (Chronakis & Kasapis, 1995). Concentrated, but not gelled
12
solutions, display slightly higher G’’ than G’. For the PEO polymer solution, adding
13
nanoliposomes did not significantly change its viscoelastic behavior; the G’ and G’’
14
curves remained virtually the same. However, for the PVOH polymer solutions, adding
15
nanoliposomes significantly affected the G’ and G’’ moduli, increasing the storage
16
modulus relative to the loss modulus. In other words, adding nanoliposomes to the
17
PVOH formulations increased the storage and restitution capacities, increasing the
18
rigidity of the polymeric networks and the entanglement of the polymer chains
19
(Chronakis & Kasapis, 1995; Ambrosio et al., 1999). In fact, for the PVOH solution
20
containing the greatest nanoliposome loading, a crossover of G’ and G’’ was observed,
21
pointing out to the gelling point of this specific solution at a certain frequency, which was
22
related to its viscosity. As previously mentioned, the hydroxyl groups from PVOH can
23
interact with the phospholipid head groups from the nanoliposomes and, the crossover
24
between G’ and G’’ indicates the viscosity for their critical entanglement giving rise to a
25
gel. From Figure 2S, it can also be observed that in the case of the PVOH solution with
26
5% nanoliposome content, G’ and G’’ curves approach at very low frequencies
27
corresponding to greater viscosity values.
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3.2. Characterization of the encapsulation structures
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3.2.1. Morphological characterization
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The morphology of the different structures obtained with and without nanoliposomes
5
was studied using Scanning Electron Microscopy (SEM). The SEM images from the
6
electrospun fibers containing empty nanoliposomes or nanoliposomes loaded with β-
7
carotene are shown in Figure 2.
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Figure 2 - SEM images showing the electrospun fibers: (A) neat PEO fibers; (B) neat PVOH fibers; (C) PEO fibers containing 7.5% (m/m – lipid/polymer) empty nanoliposomes; (D) PVOH fibers containing 7.5% (m/m – lipid/polymer) empty nanoliposomes; (E) PEO fibers containing 7.5% (m/m – lipid/polymer) nanoliposomes loaded with 0.5% mol β-carotene; (F) PVOH fibers containing 7.5% (m/m – lipid/polymer) nanoliposomes loaded with 0.5% mol β-carotene. The scale bars correspond to 5 μm. The red arrows point to beads on nanofibers.
7
Figure 2 shows that ultrathin electrospun fibers were obtained for all of the
8
compositions. The average diameter of the fibers was calculated by analyzing 100
9
random electrospun structures. The largest and smallest diameters were also
10
determined. Table 1S (Supplementary Material) summarizes the morphological analysis
11
of each lipid/polymer mass ratio studied for the PVOH and PEO electrospun fibers.
12
The
13
nanoliposomes generated essentially the same results. Increasing the nanoliposome
14
content in the fibers increased the mean, minimum and maximum diameters of the
15
electrospun fibers (Table 1S, Supplementary Material). When comparing the
16
electrospun fibers containing empty nanoliposomes with those containing β-carotene-
17
loaded nanoliposomes, the obtained mean diameter was the same. Specifically,
18
incorporating β-carotene into the nanoliposomes did not influence the diameters of the
19
electrospun fibers. Incorporating 7.5% (m/m) nanoliposomes on PEO formulations
20
increased the mean diameter by approximately 32%, while adding the same amount of
21
nanoliposomes to the PVOH formulations increased mean diameters of the fibers by
22
109% (Table 1S, Supplementary Material). This difference could be explained by the
23
rheological properties of the electrospun formulations, which directly interfered with the
24
electrospinning process dynamics and, consequently, on fibers diameter. When
for
the
electrospun
fibers
containing
β-carotene-loaded
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comparing the rheological behavior of both polymer types (cf. Figures 1 and 2S), adding
2
nanoliposomes changed viscosity and viscoelasticity characteristics of the PVOH
3
solutions more than those of PEO solutions, resulting in higher PVOH solution
4
viscosities, which are related with greater fiber diameters (Uyar & Besenbacher, 2009).
5
Moreover, the morphologies of the PVOH and PEO electrospun fibers were slightly
6
different. The fibers produced from PVOH solutions were smoother and, in general, only
7
a few beads were observed. In contrast, more beaded fibers were observed for PEO,
8
which could not be related to the presence of nanoliposomes as neat PEO electrospun
9
fibers exhibited the same structure. Uyar and coworkers (2009) incorporated
10
cyclodextrins into PEO nanofibers and observed that, when increasing the viscosity of
11
the solution, bead-free fibers were obtained. Therefore, increasing the solution viscosity
12
decreases the likelihood of beads appearing along the fibers. The molar mass of the
13
polymer is also known to influence the morphology of the electrospun fibers. In this
14
study, the PEO molecular weight used was 200 kDa, enabling the use of low
15
polymer/water mass ratios.
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3.2.2. FTIR analysis of the encapsulation structures
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Fourier transform infrared spectroscopy was used to corroborate that the lipidic
20
molecules were effectively incorporated within the electrospun fibers. Figure 3A shows
21
the spectra for the pure PVOH fibers and the hybrid fibers containing 7.5%
22
nanoliposomes. The bottom spectrum corresponds to the pure lipid and was used as
23
reference for evaluating the presence of nanoliposomes within the hybrid fibers.
24
Similarly, Figure 3B shows the spectra corresponding for the PEO materials. An
25
enlarged view of the CH stretching region is also included in both figures so that the
26
bands associated with the nanoliposomes can be discerned (cf. to arrows).
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A)
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Figure 3- FTIR spectra of A) PVOH-based structures and B) PEO-based structures. From bottom to top: HSPC lipid; pure electrospun fibers without nanoliposomes; and electrospun fibers with 7.5% (m/m) added nanoliposomes. The arrows point to the spectral bands related to the presence of nanoliposomes.
Page 16 of 27
Hybrid fibers containing β-carotene-loaded nanoliposomes were also analyzed by
2
Fourier transform infrared spectroscopy, but the obtained spectra were identical to the
3
ones containing only empty nanoliposomes. Consequently, this technique could not
4
detect the presence of the antioxidant. The presence of nanoliposomes within the
5
electrospun fibers was confirmed through the CH stretching bands at 2920 cm-1 and
6
2960 cm-1, which are characteristic of the pure lipid. For PEO, only the band at 2920
7
cm-1 was apparent as the other band was overlapped with the spectral band from the
8
neat polymer.
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3.3. Characterization of the hybrid encapsulation structures with the β-caroteneloaded nanoliposomes
12 13
3.3.1. Distribution of β-carotene
14
The β-carotene distribution within the electrospun fibers was analyzed by Confocal
15
Raman Imaging spectroscopy. This technique is very useful because β-carotene
16
exhibits strong Raman scattering. Figure 3S from the supplementary material shows a
17
typical Raman spectrum for β-carotene. The three major vibrational bands are in
18
accordance with those reported in previous studies (Fernandez et al., 2009) and were
19
used to evaluate the distribution and stability of the carotenoid in selected portions of
20
the electrospun fiber mats. The PVOH and PEO polymers showed no signal over the
21
tracked Raman shift, avoiding any interference. To obtain the Raman images, the area
22
of the band at 1500 cm-1 was integrated over the studied 15x15 µm area. For each of
23
the Raman images, the individual spectrum in different areas with various signal
24
intensities were compared, to assess whether the antioxidant was degraded within the
25
fibers. Figures 4SA and 4SB (Supplementary Material) show the typical Raman images
26
obtained for the PVOH and PEO fiber mats, respectively, with the corresponding
27
individual spectra.
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1 2
Fernandez et al. (2009) studied the changes in the β-carotene Raman spectra upon UV
4
irradiation, observing significant differences in the non-oxidized carotenoid spectrum.
5
Comparing the obtained spectra in Figure 4S with the ones obtained by Fernandez et
6
al. after the UV treatment, it can be stated that the β-carotene molecules inside the
7
obtained electrospun fibers were not degraded, as no peaks related to oxidation were
8
observed. Therefore, the electrospinning process did not affect the stability of the
9
bioactive during encapsulation. Figures 4SA and 4SB show that the distribution of β-
10
carotene inside the nanofiber mats can be compared between the encapsulating
11
polymers. The materials containing encapsulated β-carotene molecules were white (not
12
the characteristic orange color of the β-carotene compound), which may be interesting
13
for certain applications. Figures 4SA and 4SB show that the distribution of β-carotene
14
inside the electrospun fibers was not homogeneous; β-carotene clusters were scattered
15
throughout the image. β-carotene was better distributed within the PEO materials,
16
according to Figure 4SB. The presence of small amounts of β-carotene was detected,
17
even in the regions with the weakest Raman signal. The changes in the β-carotene
18
distributions observed between both electrospun polymers are most likely due to the
19
different molecular interactions. The rheological results displayed in Figure 1 reveal that
20
PVOH had a stronger interaction with the incorporated phospholipids, imparting a
21
greater variation in the apparent viscosity of the solution. This greater interaction most
22
likely occurred between the polar head groups of the phospholipids and the hydroxyl
23
groups present along the PVOH molecules (Boggs et al., 1986). These stronger
24
intermolecular interactions, associated to the electrospinning process, might change
25
bilayer conformation, leading to bilayer fractures, vesicle aggregation or fusion, phase
26
transition and phase separation, and loss of entrapped β-carotene may also occur
27
(Crowe et al 1985), possibly explaining the low β-carotene homogeneity along PVOH
28
electrospun fibers.
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3.3.2. Stability tests via UV-Vis irradiation The abilities of the two polymeric matrices used to protect β-carotene-loaded
4
nanoliposomes were evaluated upon exposure to ultraviolet light. The double
5
encapsulation layer (nanoliposome + polymer fiber) was expected to improve the
6
stability of β-carotene compared to the free carotenoid molecules or those encapsulated
7
using just one of the techniques. The degradation of the β-carotene-loaded
8
nanoliposomes in aqueous phase and the free β-carotene in hexane were also
9
evaluated after ultraviolet light exposure. The β-carotene decay profiles for each system
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11 12 13 14 15 16 17 18 19
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Figure 4 - Relative β-carotene concentrations in the different systems during UV light exposure. For the hybrid systems, a 7.5% lipid/polymer ratio was used. The lines are just to guide the eye. The SD are from three different and independent samples. A student’s t-test (p-value
S0927-7765(15)00149-6 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.03.015 COLSUB 6955
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
16-10-2014 12-2-2015 5-3-2015
Please cite this article as: R.H.F. Zˆompero, A. L´opez-Rubio, S.C. Pinho, J.M. Lagaron, L.G. Torre, Hybrid Encapsulation Structures based on rmbeta-carotene-Loaded Nanoliposomes within Electrospun Fibers, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.03.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Hybrid Encapsulation Structures based on β-carotene-Loaded
2
Nanoliposomes within Electrospun Fibers
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Rafael Henrique de Freitas Zômpero1, Amparo López-Rubio2, Samantha Cristina de Pinho3, José María Lagaron, Lucimara Gaziola de la Torre1*
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University of Campinas, UNICAMP, School of Chemical Engineering, 13083-970, Campinas, São Paulo, Brazil. Novel Materials and Nanotechnology Group, Institute of Agrochemistry and Food Technology, IATA, 46980, Paterna, Valencia, Spain.
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Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of São Paulo (USP), Pirassununga, SP, Brazil
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*Corresponding author, [email protected], phone: +55 19 35210397
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Abstract
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Hybrid
20
incorporated within the polymeric ultrathin fibers produced through electrospinning were
21
developed to improve the photostability of the antioxidant. These novel materials were
22
intended to incorporate β-carotene into water-based food formulations, overcoming the
23
existing limitations associated with its hydrophobic character. Initially, both empty and
24
antioxidant-loaded nanoliposomes were developed and incorporated into polyvinyl
25
alcohol (PVOH) and polyethylene oxide (PEO) solutions. The changes in the solution
26
properties were evaluated to determine their effects on the electrospinning processing.
27
The mixed polymer solutions were subsequently electrospun to produce hybrid
28
nanoliposome-loaded ultrathin fibers. FTIR analysis confirmed the presence of
29
phospholipid molecules inside the electrospun fibers. These ultrathin fibers were
30
evaluated regarding their morphology, diameter, internal β-carotene distribution and
31
stability against UV irradiation. Liposomal release studies from the electrospun fibers
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based
on
β-carotene-loaded
nanoliposomes
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were also undertaken, confirming the presence of the liposomal structures after
2
dissolving the electrospun fibers in water.
3 4
1. Introduction
5
Carotenoids are often used as food additives due to their properties as vitamins,
6
colorants and antioxidants. β-carotene is the most widely studied naturally occurring
7
carotenoid commonly found in fruits and vegetables (Paiva & Russell, 1999). This
8
carotenoid might have anti-cancer and anti-cardiovascular disease properties (Dulinska
9
et al., 2005; Vainio, 2000; D’odorico, 2000). However, carotenoids (including β-
10
carotene) are sensitive molecules, being susceptible to oxidation by light, heat and
11
oxygen exposure. In addition, they are hydrophobic compounds, hindering direct
12
applications to aqueous-based food formulations. Micro- and nanoencapsulation
13
techniques are plausible options for overcoming the drawbacks related to adding
14
carotenoids to foods.
15
Previously, microencapsulation has been used to protect sensitive substances against
16
the influences of adverse environments. The term “microencapsulation” designates a
17
technology that involves wrapping solids, liquids or gases in small capsules, which can
18
release their contents under specific conditions (Champagne & Fustier, 2007). This
19
process emerged from the micro/nanotechnology field, finding success in many
20
industries by conferring remarkable properties to products, such as pharmaceuticals,
21
foods, cosmetics, textiles and many other products used during daily life (Risch, 1995).
22
Encapsulation techniques can be used to solubilize the hydrophobic compounds in
23
water-based foods, stabilizing reactive compounds and protecting sensitive molecules
24
from oxidation, moisture, light exposure, temperature and other extreme conditions.
25
These protecting abilities are of outmost importance for
26
functional food products (Gibbs et al., 1999). Another important characteristic of
27
encapsulation technologies for food applications is their ability to mask undesirable
28
odors and flavors, improving the product acceptance by the consumer. Several types of
29
nanoparticles are studied for these applications, being liposomes amongst the most
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the development of novel
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promising ones due to their wide range of possible diameters and surface charges, as
2
well as their nutraceutical properties and encapsulation abilities for both hydrophilic and
3
hydrophobic compounds (Lasic, 1993). A more recently explored method for
4
microencapsulation of compounds in food is the electrohydrodynamic processing
5
comprised of electrospinning and electrospraying processes (Bhardwaj & Kundu, 2010;
6
Fernandez et al., 2009). Electrospinning is a simple and highly versatile method for
7
producing fibers, which are in the submicron range and have a large surface to volume
8
ratio, through the action of an external electric field applied between two electrodes and
9
imposed on a polymer solution or melt. Electrohydrodynamic processing for food
10
applications is still in its early stages of development, but has demonstrated a great
11
potential for developing innovative products with specific new properties. Another
12
advantage of this technique is its low cost and high productivity, making it ideal for
13
industrial purposes (Ramakrishna et al., 2005). This technique has recently
14
demonstrated tremendous potential in food science through the development of novel
15
functional ingredients and more efficient food packaging structures (Fabra et al., 2013;
16
López-Rubio et al., 2009; López-Rubio et al., 2012; Torres-Giner et al., 2008; Torres-
17
Giner & Lagaron, 2009). Combining electrospinning with other technologies from the
18
micro/nano encapsulation field can broaden the possibilities for new products with
19
improved functionalities. One interesting and promising combination of technologies
20
involves trapping liposomal nanostructures within electrospun fibers to stabilize
21
compounds (Mickova et al., 2012). Therefore, the aim of the present work was to
22
develop novel hybrid materials comprising the two above-mentioned promising
23
technologies for the encapsulation and protection of β-carotene. Consequently, β-
24
carotene-loaded nanoliposomes were incorporated within polyvinyl alcohol (PVOH) and
25
polyethylene oxide (PEO) electrospun fibers because both water soluble polymers are
26
of great interest for the food and pharmaceutical industries (Peresin et al., 2009;
27
Supaphol & Chuangchote, 2007; Baba et al., 2010; Deitzel et al., 2001).
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1
2. Experimental Part
3 4
2.1. Materials PVOH was kindly supplied by Plásticos Hidrosolubles S.L. (Valencia, Spain) as a thin
5
film form, and the PEO with a Mw of 200 kDa was supplied by Sigma-Aldrich in powder
6
form (Madrid, Spain). The hydrogenated soybean phosphatidylcholine (HSPC)
7
PHOSPHOLIPON 80H from Lipoid (Ludwisghafen, Germany) and ethanol 96% (v/v)
8
were supplied by Panreac (Barcelona, Spain) and used to produce the nanoliposomes.
9
Synthetic β-carotene (minimum purity 97%) was purchased from Fluka Analytical
10
(Madrid, Spain). n-Hexane (98% purity) from Fluka Analytical (Madrid, Spain) was used
11
to quantify the β-carotene. The β-carotene was kept frozen and in darkness until use.
12
All of the samples obtained from the performed experiments were protected from light
13
until analysis.
15 16
2.2. Nanoliposomes production The nanoliposomes were prepared using ethanol injection method. The lipids (or lipids
17
and -carotene) were dispersed in ethanol (above the main phase transition
18
temperature) and injected at 30 mL/min into a jacketed reactor containing water (100
19
mL) while controlling the temperature (60°C) and stirring speed (1336 rpm using a
20
Cowles type impeller) over 5 min. This heating process is necessary due to high lipid
21
transition temperature (approximately 51°C). The final nanoliposome dispersion
22
contains 10% (v/v) ethanol/water and 20 mM lipid. To produce nanoliposomes
23
containing encapsulated β-carotene, the alcoholic phase was prepared differently,
24
adding a 0.5% molar ratio of β-carotene/Phospholipid. The percentage of the β-carotene
25
added to the nanoliposomes was established based on preliminary studies (results not
26
shown). The average hydrodynamic diameter, polydispersity and zeta potential of the
27
nanoliposomes and -carotene-loaded nanoliposomes were measured using Malvern
28
Zeta-Sizer Nano-ZS equipment. Since the aim of this study was β-carotene protection,
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hydrogenated lipids were used to avoid peroxidation processes (Huang & Chung, 1998
2
and Krinsky & Deneke , 1982).
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2.3. Nanoliposome/Polymer Formulations The solutions used for the electrospinning process were composed of a polymer (PVOH
7
or PEO) and nanoliposomes produced through the ethanol injection method. The
8
polymer/water mass ratio was 20%, and this value remained constant for all the
9
formulations. Three different lipid/polymer mass ratios were studied for empty
10
nanoliposomes and β-carotene loaded nanoliposomes: 2.5%, 5% and 7.5%. The
11
polymeric solutions without addition of nanoliposomes were also electrospun to
12
evaluate the effect of the empty nanoliposomes or β-carotene loaded nanoliposomes on
13
the properties of the polymeric solutions and in the produced fibers.
14 15
2.4. Electrospinning process The electrospinning apparatuswas a FluidNatek® instrument, trademark of BioInicia
16
S.L. (Valencia, Spain), equipped with a variable high voltage 0–30 kV power supply was
17
used. The anode was attached to a stainless steel needle with a 0.9 mm internal
18
diameter, which was connected to the polymer/nanoliposome solutions kept in a 5 ml
19
plastic syringe through a PTFE wire. The syringe was placed on a digitally controlled
20
syringe pump with the needle directed toward the collector. The electrospun structures
21
were collected on a stainless steel plate attached to the copper grid collector. The
22
electrospinning conditions were maintained at 24°C and 60% RH by enclosing the
23
equipment in a specific chamber with temperature and humidity control. The collector
24
was placed 10 cm from the capillary tip. The syringe pump delivered the PVOH and
25
PEO polymer solutions at 0.1 ml/h, and the voltage was maintained at 10 kV. The
26
conditions were the same for all of the electrospinning processes. The collected
27
materials were stored in a desiccator at 0% RH and protected from light.
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2.5. Characterization of the polymeric solutions The rheological properties of the various polymeric solutions were evaluated with a
3
Thermo Haake RS1 controlled stress rheometer (Thermo Scientific, United States,
4
Waltham, MA) in the cone-plate configuration, both of which were made of titanium. The
5
angle between the surface of the cone and the plate was of 1 degree, and a distance of
6
1 mm from the plate was kept during the measurements. The temperature during the
7
measurements was 20 ± 1°C and maintained using a Phoenix P1 Circulator device
8
(Thermo Haake). Different frequencies and shear rates were applied to the samples,
9
and the behavior of the samples toward the determined conditions was evaluated using
10
Rheowin Pro Software v.3.61 Haake. Before applying the stress, the samples remained
11
in contact for 10 min with the cone-plate system to achieve thermal equilibrium. The
12
surface
13
nanoliposomes was measured using the Wilhemy plate method in an EasyDyne K20
14
tensiometer (Krüss GmbH, Hamburg, Germany). All measurements were conducted in
15
duplicate.
the
polymer
solutions
containing
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quantities
of
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2.6. Fourier transform infrared spectroscopy The ATR-FTIR spectra of the different electrospun materials obtained (pure polymeric
19
fibers and fibers containing empty or β-carotene-loaded nanoliposomes) were collected
20
in a controlled chamber at 24°C and 60% RH by coupling the ATR accessory Golden
21
Gate of Specac Ltd. (Orpington, UK) to a Bruker (Rheinstetten, Germany) FTIR Tensor
22
37 equipment. All of the spectra were collected by averaging 20 scans at a 4 cm-1
23
resolution. The spectral data were compared using the Opus Viewer software
24
(Rheinstetten, Germany).
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2.7. Scanning Electron Microscopy (SEM) The morphologies of the electrospun fibers were examined using a Hitachi S-4100
28
Scanning Electron Microscope. The different samples were sputtered with a gold–
29
palladium mixture under vacuum. All of the SEM experiments were carried out at 30 kV.
30
The diameters of the electrospun fibers were measured by averaging the diameters of
Page 6 of 27
1
at least 100 randomly chosen electrospun fibers from the SEM micrographs in their
2
original magnification through the ImageJ software (Maryland, USA).
3
2.8. Raman microspectroscopy The Raman spectra were collected with a Jasco NRS-3100 Confocal Micro-Raman
6
spectrophotometer (Jasco Inc., Easton, MD) using a 100x objective to evaluate the β-
7
carotene distribution within the developed hybrid electrospun fibers. The Raman
8
chemical images were collected in point-by-point mode by plotting the area of the β-
9
carotene band at 1500 cm-1 and were constructed by collecting 15x15 spectra spaced
10
equally along the selected sample area. The β-carotene generates very large Raman
11
scattering patterns and, thus, very short acquisition times were needed to record
12
intense spectra showing only the signal for β-carotene within the electrospun fibers.
13
This signal intensity was used to construct the Raman images.
15 16
2.9. Nanoliposomes release from electrospun fiber mats PEO and PVOH nanofibers containing 7.5% (m/m) nanoliposomes and β-carotene-
17
loaded nanoliposomes (0.01 g of nanofibers mat) were hydrated in water (5 mL) with
18
gentle stirring at 25ºC. After dissolution was complete, the aqueous phase was
19
observed through Transmission Electron Microscopy. The obtained solution was
20
carefully applied to a 400 mesh cooper grid, rested for 5 min and dried using filter
21
paper. Uranyl acetate (1% v/v) was then added to the sample on the cooper grid for
22
coloring, resting for 1 min before being dried with clean filter paper. The prepared
23
samples were placed inside the transmission electron microscope sample port for
24
image acquisition using ZEISS CLM 902 and CCD Proscan systems.
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2.10. Stability tests by UV-vis irradiation The PEO and PVOH electrospun fiber mats with the maximum nanoliposome content
28
(7.5% m/m in relation to polymer) and, consequently, the highest β-carotene content
29
were evaluated relative to their ability to protect the photosensitive antioxidant molecule.
Page 7 of 27
For this purpose, the antioxidant degradation rate was compared with that of β-carotene
2
encapsulated in nanoliposomes (aqueous solution) and free β-carotene crystals
3
dissolved in hexane. Samples from each system were placed in glass flasks and
4
exposed to an Osram Ultra-Vitalux 300W UV lamp over 6 h. The distance between the
5
lamp and the flasks was 10 cm. Samples were collected every hour and the intact β-
6
carotene concentration was determined using Agilent 8453 spectrophotometer (Santa
7
Clara, USA) at 450 nm. The β-carotene concentration of the control samples containing
8
β-carotene crystals dissolved in hexane was determined directly by spectrophotometry.
9
For the aqueous solutions of the β-carotene-loaded nanoliposomes, ethanol was added
10
to disrupt the nanoliposomes (releasing the entrapped β-carotene) before hexane was
11
added to extract the β-carotene, generating a 2-phase system. The organic (upper)
12
phase was separated, and the β-carotene concentration was determined by
13
spectrophotometry. For the PEO and PVOH electrospun fibers containing the
14
antioxidant-loaded nanoliposomes, the fibers were initially placed in water until
15
completely dissolved before adding the ethanol and hexane for the liposomal disruption
16
and β-carotene extraction, respectively.
17
A statistical analysis was performed to evaluate whether significant differences in the
18
relative β-carotene concentration over time under UV light exposure for the different
19
protective systems. For this evaluation, a student’s t-test was performed using the
20
Statistica 7® software. The confidence interval used in the analysis was 80% due to
21
inherent variations in the process.
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3. Results and discussion
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3.1. Surface tension and rheological properties of the polymeric solutions containing “empty” nanoliposomes
8
The solution surface tension is an important parameter that directly influences the
9
electrospinning process (Ramakrishna et al., 2005) because the intensity of the
10
electrical field must overcome the surface tension of the solution, expelling an electrified
11
jet from the Taylor’s cone formed on the needle tip (Doshi & Reneker, 1995; Taylor,
12
1969). Therefore, the surface tension of the PEO and PVOH solutions containing
13
different amounts of “empty” nanoliposomes were evaluated and subsequently related
14
to the fiber morphology (see Supplementary material Figure 1S).
15
It was observed that the effect of adding empty nanoliposomes on the surface tension
16
was different between the PEO and PVOH solutions. While a gradual decrease in
17
surface tension was observed when increasing the liposomal content in the PEO
18
solution (from 59.5 mN/m to 53 mN/m), the surface tension of the PVOH solutions
19
remained constant and close to 45 mN/m. The PVOH solutions already had a low
20
surface tension and therefore remained unaffected by the added nanoliposomes.
21
Polymeric
22
carotene/phospholipid molar ratio) at the same lipid/polymer mass ratios were also
23
subjected to surface tension measurements and no significant differences were
24
observed with respect to the surface tension of the solutions with “empty”
25
nanoliposomes.
26
Surface tension is an important parameter during electrospinning, but alone it cannot
27
completely explain the suitability of a solution for the development of electrospun
28
structures. Adding nanoliposomes could also affect other important parameters involved
29
in electrospinning, such as viscosity and viscoelasticity. These parameters, together
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solutions
containing
β-carotene-loaded
nanoliposomes
(0.5%
β-
Page 9 of 27
with the electrical conductivity of the solutions, are the major factors that determine the
2
suitability of the polymeric solutions for electrospinning and the morphology of the
3
obtained structures. Three typical structures, which are primarily beads, beaded fibers
4
and bead-free fibers, can form depending on the solution properties in combination with
5
several process parameters (such as the applied voltage or the distance to the
6
collector) (Ramakrishna et al., 2005). To understand the morphological features of the
7
obtained hybrid structures, the rheological behavior of the PVOH and PEO-based
8
solutions was analyzed. Figures 1A and 1B show the relationship between the shear
9
rate and the applied stress for the PVOH and PEO solutions, respectively, that contain
10
different lipid/polymer mass ratios. In addition, Figures 1C and 1D show the correlation
11
between the shear rate and apparent solution viscosity.
12
Figures 1A and 1C reveal that the pure PVOH solution (without phospholipid addition)
13
presents Newtonian characteristics; the viscosity remained independent from the
14
applied shear rate. Increasing the phospholipid content increased the viscosity and
15
shifted the rheological behavior toward a pseudoplastic character, reducing its viscosity
16
when increasing the shear rate. The increased viscosity when increasing the
17
lipid/polymer mass ratio likely occurred through the interactions between the hydroxyl
18
groups along the polymer chain and the phospholipid head groups (Antunes et al.,
19
2009; Boggs et al., 1986).
20
For PEO, adding phospholipids caused a slight change on the rheological properties of
21
the solution, mainly in the range from 0 to 100 s-1. Figures 1B and 1D show that the
22
PEO solutions, even without phospholipids, exhibited a pseudoplastic behavior that was
23
retained after adding the nanoliposomes. This pseudoplastic behavior indicates that
24
when high shear rates were applied, the molecular structure of the entangled polymer
25
chains acquired a preferential orientation in the flow direction, decreasing the viscosity
26
of the solution (shear thinning flow behavior). For pure PVOH (Newtonian), this
27
phenomenon was not observed.
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PVOH
PEO B
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Figure 1 - Rheological behavior of PVOH and PEO solutions containing different phospholipid mass ratios: (A,B) stress-shear rate curves; (C,D) apparent viscosity-shear rate curves. All the measurements were performed at 20°C in independent duplicates. Experimental error: 10%.
4 5
Page 11 of 27
Viscoelastic materials, when subjected to shear forces, simultaneously undergo viscous
2
and elastic deformations (Chronakis & Kasapis, 1995). The viscoelasticity is important
3
during electrospinning and, thus, in addition to characterizing the viscosity of the various
4
sample solutions, the viscoelastic behavior in terms of the elastic modulus (G’) and
5
viscous modulus (G’’) were also analyzed and the corresponding graphs can be seen in
6
the supplementary material (Figure 2S).
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For samples in gel state, the storage modulus (G’) prevails over the loss modulus (G’’).
9
The obtained data revealed that only PVOH solutions with the greatest nanoliposomes
10
loading exhibited this gel state behavior at certain frequencies, which corresponded to
11
the greater viscosity values (Chronakis & Kasapis, 1995). Concentrated, but not gelled
12
solutions, display slightly higher G’’ than G’. For the PEO polymer solution, adding
13
nanoliposomes did not significantly change its viscoelastic behavior; the G’ and G’’
14
curves remained virtually the same. However, for the PVOH polymer solutions, adding
15
nanoliposomes significantly affected the G’ and G’’ moduli, increasing the storage
16
modulus relative to the loss modulus. In other words, adding nanoliposomes to the
17
PVOH formulations increased the storage and restitution capacities, increasing the
18
rigidity of the polymeric networks and the entanglement of the polymer chains
19
(Chronakis & Kasapis, 1995; Ambrosio et al., 1999). In fact, for the PVOH solution
20
containing the greatest nanoliposome loading, a crossover of G’ and G’’ was observed,
21
pointing out to the gelling point of this specific solution at a certain frequency, which was
22
related to its viscosity. As previously mentioned, the hydroxyl groups from PVOH can
23
interact with the phospholipid head groups from the nanoliposomes and, the crossover
24
between G’ and G’’ indicates the viscosity for their critical entanglement giving rise to a
25
gel. From Figure 2S, it can also be observed that in the case of the PVOH solution with
26
5% nanoliposome content, G’ and G’’ curves approach at very low frequencies
27
corresponding to greater viscosity values.
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3.2. Characterization of the encapsulation structures
2 3
3.2.1. Morphological characterization
4
The morphology of the different structures obtained with and without nanoliposomes
5
was studied using Scanning Electron Microscopy (SEM). The SEM images from the
6
electrospun fibers containing empty nanoliposomes or nanoliposomes loaded with β-
7
carotene are shown in Figure 2.
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Figure 2 - SEM images showing the electrospun fibers: (A) neat PEO fibers; (B) neat PVOH fibers; (C) PEO fibers containing 7.5% (m/m – lipid/polymer) empty nanoliposomes; (D) PVOH fibers containing 7.5% (m/m – lipid/polymer) empty nanoliposomes; (E) PEO fibers containing 7.5% (m/m – lipid/polymer) nanoliposomes loaded with 0.5% mol β-carotene; (F) PVOH fibers containing 7.5% (m/m – lipid/polymer) nanoliposomes loaded with 0.5% mol β-carotene. The scale bars correspond to 5 μm. The red arrows point to beads on nanofibers.
7
Figure 2 shows that ultrathin electrospun fibers were obtained for all of the
8
compositions. The average diameter of the fibers was calculated by analyzing 100
9
random electrospun structures. The largest and smallest diameters were also
10
determined. Table 1S (Supplementary Material) summarizes the morphological analysis
11
of each lipid/polymer mass ratio studied for the PVOH and PEO electrospun fibers.
12
The
13
nanoliposomes generated essentially the same results. Increasing the nanoliposome
14
content in the fibers increased the mean, minimum and maximum diameters of the
15
electrospun fibers (Table 1S, Supplementary Material). When comparing the
16
electrospun fibers containing empty nanoliposomes with those containing β-carotene-
17
loaded nanoliposomes, the obtained mean diameter was the same. Specifically,
18
incorporating β-carotene into the nanoliposomes did not influence the diameters of the
19
electrospun fibers. Incorporating 7.5% (m/m) nanoliposomes on PEO formulations
20
increased the mean diameter by approximately 32%, while adding the same amount of
21
nanoliposomes to the PVOH formulations increased mean diameters of the fibers by
22
109% (Table 1S, Supplementary Material). This difference could be explained by the
23
rheological properties of the electrospun formulations, which directly interfered with the
24
electrospinning process dynamics and, consequently, on fibers diameter. When
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comparing the rheological behavior of both polymer types (cf. Figures 1 and 2S), adding
2
nanoliposomes changed viscosity and viscoelasticity characteristics of the PVOH
3
solutions more than those of PEO solutions, resulting in higher PVOH solution
4
viscosities, which are related with greater fiber diameters (Uyar & Besenbacher, 2009).
5
Moreover, the morphologies of the PVOH and PEO electrospun fibers were slightly
6
different. The fibers produced from PVOH solutions were smoother and, in general, only
7
a few beads were observed. In contrast, more beaded fibers were observed for PEO,
8
which could not be related to the presence of nanoliposomes as neat PEO electrospun
9
fibers exhibited the same structure. Uyar and coworkers (2009) incorporated
10
cyclodextrins into PEO nanofibers and observed that, when increasing the viscosity of
11
the solution, bead-free fibers were obtained. Therefore, increasing the solution viscosity
12
decreases the likelihood of beads appearing along the fibers. The molar mass of the
13
polymer is also known to influence the morphology of the electrospun fibers. In this
14
study, the PEO molecular weight used was 200 kDa, enabling the use of low
15
polymer/water mass ratios.
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3.2.2. FTIR analysis of the encapsulation structures
19
Fourier transform infrared spectroscopy was used to corroborate that the lipidic
20
molecules were effectively incorporated within the electrospun fibers. Figure 3A shows
21
the spectra for the pure PVOH fibers and the hybrid fibers containing 7.5%
22
nanoliposomes. The bottom spectrum corresponds to the pure lipid and was used as
23
reference for evaluating the presence of nanoliposomes within the hybrid fibers.
24
Similarly, Figure 3B shows the spectra corresponding for the PEO materials. An
25
enlarged view of the CH stretching region is also included in both figures so that the
26
bands associated with the nanoliposomes can be discerned (cf. to arrows).
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Figure 3- FTIR spectra of A) PVOH-based structures and B) PEO-based structures. From bottom to top: HSPC lipid; pure electrospun fibers without nanoliposomes; and electrospun fibers with 7.5% (m/m) added nanoliposomes. The arrows point to the spectral bands related to the presence of nanoliposomes.
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Hybrid fibers containing β-carotene-loaded nanoliposomes were also analyzed by
2
Fourier transform infrared spectroscopy, but the obtained spectra were identical to the
3
ones containing only empty nanoliposomes. Consequently, this technique could not
4
detect the presence of the antioxidant. The presence of nanoliposomes within the
5
electrospun fibers was confirmed through the CH stretching bands at 2920 cm-1 and
6
2960 cm-1, which are characteristic of the pure lipid. For PEO, only the band at 2920
7
cm-1 was apparent as the other band was overlapped with the spectral band from the
8
neat polymer.
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3.3. Characterization of the hybrid encapsulation structures with the β-caroteneloaded nanoliposomes
12 13
3.3.1. Distribution of β-carotene
14
The β-carotene distribution within the electrospun fibers was analyzed by Confocal
15
Raman Imaging spectroscopy. This technique is very useful because β-carotene
16
exhibits strong Raman scattering. Figure 3S from the supplementary material shows a
17
typical Raman spectrum for β-carotene. The three major vibrational bands are in
18
accordance with those reported in previous studies (Fernandez et al., 2009) and were
19
used to evaluate the distribution and stability of the carotenoid in selected portions of
20
the electrospun fiber mats. The PVOH and PEO polymers showed no signal over the
21
tracked Raman shift, avoiding any interference. To obtain the Raman images, the area
22
of the band at 1500 cm-1 was integrated over the studied 15x15 µm area. For each of
23
the Raman images, the individual spectrum in different areas with various signal
24
intensities were compared, to assess whether the antioxidant was degraded within the
25
fibers. Figures 4SA and 4SB (Supplementary Material) show the typical Raman images
26
obtained for the PVOH and PEO fiber mats, respectively, with the corresponding
27
individual spectra.
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1 2
Fernandez et al. (2009) studied the changes in the β-carotene Raman spectra upon UV
4
irradiation, observing significant differences in the non-oxidized carotenoid spectrum.
5
Comparing the obtained spectra in Figure 4S with the ones obtained by Fernandez et
6
al. after the UV treatment, it can be stated that the β-carotene molecules inside the
7
obtained electrospun fibers were not degraded, as no peaks related to oxidation were
8
observed. Therefore, the electrospinning process did not affect the stability of the
9
bioactive during encapsulation. Figures 4SA and 4SB show that the distribution of β-
10
carotene inside the nanofiber mats can be compared between the encapsulating
11
polymers. The materials containing encapsulated β-carotene molecules were white (not
12
the characteristic orange color of the β-carotene compound), which may be interesting
13
for certain applications. Figures 4SA and 4SB show that the distribution of β-carotene
14
inside the electrospun fibers was not homogeneous; β-carotene clusters were scattered
15
throughout the image. β-carotene was better distributed within the PEO materials,
16
according to Figure 4SB. The presence of small amounts of β-carotene was detected,
17
even in the regions with the weakest Raman signal. The changes in the β-carotene
18
distributions observed between both electrospun polymers are most likely due to the
19
different molecular interactions. The rheological results displayed in Figure 1 reveal that
20
PVOH had a stronger interaction with the incorporated phospholipids, imparting a
21
greater variation in the apparent viscosity of the solution. This greater interaction most
22
likely occurred between the polar head groups of the phospholipids and the hydroxyl
23
groups present along the PVOH molecules (Boggs et al., 1986). These stronger
24
intermolecular interactions, associated to the electrospinning process, might change
25
bilayer conformation, leading to bilayer fractures, vesicle aggregation or fusion, phase
26
transition and phase separation, and loss of entrapped β-carotene may also occur
27
(Crowe et al 1985), possibly explaining the low β-carotene homogeneity along PVOH
28
electrospun fibers.
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1
3.3.2. Stability tests via UV-Vis irradiation The abilities of the two polymeric matrices used to protect β-carotene-loaded
4
nanoliposomes were evaluated upon exposure to ultraviolet light. The double
5
encapsulation layer (nanoliposome + polymer fiber) was expected to improve the
6
stability of β-carotene compared to the free carotenoid molecules or those encapsulated
7
using just one of the techniques. The degradation of the β-carotene-loaded
8
nanoliposomes in aqueous phase and the free β-carotene in hexane were also
9
evaluated after ultraviolet light exposure. The β-carotene decay profiles for each system
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Figure 4 - Relative β-carotene concentrations in the different systems during UV light exposure. For the hybrid systems, a 7.5% lipid/polymer ratio was used. The lines are just to guide the eye. The SD are from three different and independent samples. A student’s t-test (p-value
