Accepted Manuscript

Design of dipalmitoyl lecithin liposomes loaded with quercetin and rutin and their
release kinetics from carboxymethyl cellulose edible films

A. Silva-Weiss, M. Quilaqueo, O. Venegas, M. Ahumada, W. Silva, F. Osorio, B.

Giménez

PII: S0260-8774(18)30001-3
DOI: 10.1016/j.jfoodeng.2018.01.001
Reference: JFOE 9134

To appear in: Journal of Food Engineering

Received Date: 8 November 2017

Revised Date: 30 December 2017
Accepted Date: 1 January 2018

Please cite this article as: Silva-Weiss, A., Quilaqueo, M., Venegas, O., Ahumada, M., Silva, W., Osorio,
F., Giménez, B., Design of dipalmitoyl lecithin liposomes loaded with quercetin and rutin and their
release kinetics from carboxymethyl cellulose edible films, Journal of Food Engineering (2018), doi:
10.1016/j.jfoodeng.2018.01.001.

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 ACCEPTED MANUSCRIPT
1 Design of dipalmitoyl lecithin liposomes loaded with quercetin and rutin and their

2 release kinetics from carboxymethyl cellulose edible films

4 A. Silva-Weiss1*, M. Quilaqueo2, O. Venegas 1, M. Ahumada3, W. Silva1, F. Osorio1, B.

5 Giménez1.

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7 Department of Food Science and Technology, Universidad de Santiago de Chile,

8 USACH. Avenida Ecuador 3769, Santiago, Chile.

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9 Chemical Engineering Department, Universidad de La Frontera, PO Box 54-D,

10 Temuco, Chile

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Bio-nanomaterials Chemistry and Engineering Laboratory, University of Ottawa Heart
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12 Institute, 40 Ruskin Street, Ottawa, Canada.
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14 *Corresponding author: Dra. Andrea Silva-Weiss. Department of Food Science and

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15 Technology, Technological Faculty, Universidad de Santiago de Chile. E-mail address:

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16 andrea.silva@usach.cl Tel.: +56 2 27180556.

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25 Abstract

26 Quercetin and rutin were encapsulated in liposomes based on dipalmitoyl lecithin. The

27 effect of liposomal formulation stage for flavonol incorporation and the size-reducing

28 method on encapsulation efficiency (EE) of flavonols and physical properties of

29 liposomes were evaluated. In addition, the release mechanism and kinetics of

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30 polyphenols from carboxymethyl cellulose edible films were studied through modeling

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31 and simulation equations. When flavonols were incorporated during the phospholipid

32 film formation stage, low polydispersity index (0.32 and 0.20) and high EE (88.9 and

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33 74.1%) of quercetin and rutin, respectively, were obtained. Sonication gave liposomes

with higher zeta potential (36.9 – 42.4 mV) than extrusion (13.3 – 17.1 mV), and

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34 AN
35 quercetin-loaded liposomes were the most stable during 21 days of storage. In CMC

36 films, diffusion coefficients of flavonols were higher for non-encapsulated flavonols

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37 than encapsulated flavonols. The release of non-encapsulated quercetin and rutin from

38 CMC films was 25% and 24% higher than in the case of encapsulated quercetin and
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39 rutin at day 21. The released mechanism agreed with Fickian diffusion for encapsulated
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40 and non-encapsulated quercetin, whereas the release mechanism of encapsulated and

41 non-encapsulated rutin agreed with non-Fickian diffusion. These results highlight the
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42 relevance of using liposomes as encapsulation technology, able to preserve polyphenols

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43 and control their release in the design of edible films with antioxidant activity for
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44 improving food shelf life.

45 Keywords: Controlled release, encapsulation, edible film, flavonol, physical stability.

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50 1. Introduction

51 In recent years, the food industry has shown increasing interest in incorporating ingredients

52 with functional and health-promoting properties such as polyphenols. Flavonols as quercetin

53 (3,3′,4′,5,7-pentahydroxyflavone) and their glycoside rutin (3,3′,4′,5,7-

54 pentahydroxyflavone-3-rhamnoglucoside) are polyphenols extensively studied and used

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55 as model flavonol for their strong antioxidant activity and their abundance in plants and

foods (Munin & Edwards-Levy, 2011). However, potential health benefits of these

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56

57 antioxidants and their application in functional foods as ingredients have been limited

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58 due to their reduced stability under processing conditions and/or during storage (pH,

59 temperature, light, oxygen), low water solubility and poor bioavailability (Emami et al.,

60
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2016). Such limitations can be overcome by the use of delivery systems for
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61 encapsulation, protection and controlled release of flavonoids.
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62 Among the encapsulation technologies used for polyphenols compounds (Fang &

63 Bhandari, 2010), encapsulation in liposomes allows improving their bioactivity and

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64 bioavailability (Emami et al., 2016; Munin & Edwards-Levy, 2011). Liposomes are
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65 self-assembled spherical vesicles made with biodegradable and non-toxic phospholipids

66 with hydrophilic head and hydrophobic fatty acid tail. They contain an internal aqueous
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67 core entirely enclosed by one (unilamellar) or multiple concentric lipid bilayers

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68 (multilamellar). These liposomal systems can transport and control the release of several
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69 hydrophilic, lipophilic and amphiphilic bioactive compounds, including food

70 ingredients and nutraceuticals that may be incorporated into the formulation of

71 functional foods (Emami et al., 2016, Pinilla, Noreña & Brandelli, 2017; Rashidinejad

72 et al., 2014). In order to increase the stability of liposomes by delaying oxidation and

73 provide controlled release due to the formation of a gel phase below their transition

74 temperature, saturated lipids such as dipalmitoyl phosphatidylcholine (DPPC) can be

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75 used, which have higher gel to crystalline liquid transition temperature (Tm = 41 °C)

76 compared to unsaturated phospholipids.

77 The location of polyphenols in the liposomal structure (aqueous core, lipid bilayer or

78 aqueous-lipid interface) may affect the release of these bioactive compounds from

79 liposomes. This location will depend on the physicochemical characteristics of both

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80 polyphenols and lipids that form the bilayer (Cadena et al., 2013; Park et al., 2013;

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81 Pawlikowska-Pawlęga et al., 201; Ulrih et al., 2015). Furthermore, the stage of the

82 polyphenol incorporation during the liposome formulation can also affect the location of

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83 these compounds in the liposomal system. However, to the best of our knowledge, the

effect of the stage of polyphenol incorporation on the stability properties of liposomes

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85 has not been studied.

86 Edible films and coatings are among the active packaging technologies for minimally
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87 processed foods, offering additional protection for primary packaging to maintain or

88 improve the overall quality of foods and prolong their shelf life (Bonilla et al., 2012).
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89 Plant extracts rich in polyphenols and phenolic compounds have been shown to increase
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90 the antioxidant activity, UV light and oxygen barrier ability of edible films, improving
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91 food preservation (Bonilla et al., 2012; Silva-Weiss et al., 2013b). However,

92 polyphenols can interact with the biopolymeric matrix (Silva-Weiss et al., 2013a), and
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93 as a consequence, physical and functional properties of the matrix may be modified

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94 (Silva-Weiss et al., 2013b; Siripatrawan & Harte, 2010) and bioactivity of polyphenols

95 reduced. The combined use of edible films and coatings with encapsulation of

96 antioxidant polyphenols is an alternative technology that has not been studied in depth.

97 This combination could protect the active compounds from environmental factors,

98 processing and storage conditions (Quirós-Sauceda et al., 2014), allowing them to

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99 maintain their bioactive properties and control their release to extend the shelf life of

100 foods.

101 The aim of this research was to study the physical properties of dipalmitoyl lecithin

102 based liposomes with quercetin or rutin incorporated in three different stages of

103 preparation. Furthermore, one stage for flavonols incorporation was selected to evaluate

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104 the effect of sonication and extrusion as size-reducing methods. Additionally, the

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105 liposomal formulations were incorporated into edible films, and the release of the

106 flavonols from the matrix film was evaluated.

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107 2. Materials and methods

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108 2.1. Materials
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109 Liposome: dipalmitoyl lecithin (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, DPPC,

110 16:0, C40H80NO8P, M.W.:734.05 g/mol, purity ≥ 99%, Avanti Polar Lipids, USA),
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111 ultra-pure cholesterol (CH, Calbiochem, Japan), quercetin (Q, purity ≥ 95%) and rutin
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112 (R, purity ≥ 94%) (Sigma-Aldrich, Chile), and potassium phosphate buffer (PPB) 10
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113 mM and pH 7.4. Edible films: sodium carboxymethyl cellulose (CMC) substitution

114 degree 0.87%, sodium content 8.7% and M.W. 95 kDa (DOW, Switzerland) and
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115 glycerol (>99.0%; Sigma-Aldrich, Chile). Folin-Ciocalteu reagent (Merck, Chile).

116 Ethanol (>99.98%) and chloroform (> 99.98%) were supply by J.T. Baker (USA), and
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117 methanol by Sigma-Aldrich (Chile). High quality water purified in a Milli-Q system
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118 was used in all the experiments.

119 2.2. Evaluation of flavonol incorporation stage in liposome formulation

120 2.2.1. Flavonol suspension preparation

121 The flavonol suspensions (0.3 mg of Q or R/mL) were prepared from stock suspensions

122 of Q or R in ethanol (0.5 mg/mL) by diluting with PPB 10 mM at pH 7.4. According to

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123 preliminary studies, both flavonol suspensions showed higher stability at pH 7.4 than at

124 pH 5.8 or pH 6.7, with transparent yellow color and without precipitate formation at

125 least until 50 min.

126 2.2.2. Formulation of liposomal suspension (LS)

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127 The LS were formulated according to the thin-film hydration method (Bangham,

128 Standish & Watkins, 1965). Composition of LS was DPPC (1 mg/mL), CH (0.20

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129 mg/mL) and flavonol (Q or R) (0.3 mg/mL). DPPC:CH (5:1 w/w) were dissolved in

chloroform:methanol (3:1 v/v), and the mixture was dried in a rotavapor (Büchi V-100,

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130

131 India) at 65 °C for 15 min, obtaining a thin and homogeneous film. The traces of

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132 solvent in the film were removed using a gaseous nitrogen stream at 101 kPa for 5 min.
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133 To obtain the multilamellar LS, the phospholipid film was rehydrated with PPB 10 mM

134 pH 7.4 and stirred for 5 min. Then, the lamellarity of liposomes was reduced by
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135 applying 10 cycles of freezing and thawing at -180 and 60 °C, respectively.
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136 2.2.3. Liposome size-reducing by extrusion

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137 After the lamellarity reduction, the LSs were transferred into a nitrogen pressurized

138 homemade extruder and extruded 10 times through polycarbonate filters (400 nm
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139 nominal pore size, Nucleopore, Corning Costar) slightly above the DPPC transition

140 temperature (44 ± 2 °C) with a nitrogen stream at 2017 kPa. This procedure maximizes
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141 the formation of unilamellar liposomes.

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142 2.2.4. Incorporation of flavonols in LS and characterization of liposomes

143 Q and R were added in different stages of the LS formulation (ratio DPPC:flavonol 3:1

144 w/w; Mignet et al., 2013) for the flavonols to be located at different zones of the

145 liposome structure and/or different depths inside the phospholipid membrane: i) during

146 the phospholipid film formation (PFF) stage, where flavonols are dispersed in the
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147 organic phase of phospholipids dispersion, prior to solvent evaporation; ii) during the

148 phospholipid film hydration (PFH) stage; iii) during the liposome resuspension (LRS)

149 stage, where the flavonol suspension was incorporated after the size-reducing process.

150 In the latter, empty LS was centrifuged at 9000 rpm for 15 min, the supernatant was

151 discarded and the pellet (liposomes) was resuspended with the flavonol suspension. All

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152 LS were stored at 4 °C until analysis (maximum 6 days).

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153 2.2.4.1. Physical characteristics

The average hydrodynamic diameter (DH), polydispersity index (PI), and zeta potential

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154

155 in absolute value (ZP) of PFF, PFH and LRS liposomal systems were assessed by

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156 dynamic light scattering and measurement of electrophoretic mobility in a Zetasizer
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157 Nano-ZS (Malvern Instruments, Malvern, UK). The measurements were performed at

158 25 °C, with a refractive index of 1.334, a wavelength of 630 nm and a detection angle of
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159 173°. The samples of LS were diluted with PPB 10 mM pH 7.4 in a ratio 1:100. All the

160 measurements were performed in triplicate.

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161 2.2.4.2. Encapsulation efficiency (EE)

162 The LS were centrifuged for 1 h at 15000 rpm and 25 °C, and the concentration of the
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163 non-encapsulated flavonols in the supernatant was determined using a

164 spectrophotometer (Aquamate 8000, Orion, USA) at 367 and 358 nm for Q and R,
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165 respectively. The EE was calculated according to equation:

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= − / × 100 Eq. 1

166 where Ctotal is the total concentration of flavonol in the liposomal suspension (30

167 mg/100 mL LS), and CNE is the concentration of the non-encapsulated flavonol in the

168 external liquid phase.

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169 Based on the physical characteristics and encapsulation efficiency of liposomes, one

170 stage for flavonols incorporation was selected to evaluate the effect of sonication and

171 extrusion as size-reducing method.

172 2.3. Evaluation of the method for reducing liposome size

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173 In order to reduce the size of the liposomes, two methods were compared: extrusion

174 (detailed in the section 2.2.3.) and sonication. Liposome size-reducing by sonication

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175 was performed by immersing the LS in an ultrasonic bath (Bransonic, Branson, USA),

where 10 cycles of 3.5 min were applied, at intervals of 1 min. These size-reducing

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176

177 methods were applied on LS with Q (LSQ), LS with R (LSR) and LS without flavonol

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178 or empty LS (LSE). These
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179 LS were prepared according to section 2.2.2., and the flavonols were incorporated

180 during the stage selected in section 2.2.4. Physical characteristics of LS and
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181 encapsulation efficiency of flavonols were determined according to section 2.2.4.1. and
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182 2.2.4.2., respectively, and one of the size-reducing processes was selected to evaluate
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183 the morphology by transmission electron microscopy (TEM) and the stability of the

184 liposomal formulations during storage at 4ºC.

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185 2.3.1. Transmission electron microscopy (TEM)

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186 The morphology of the LSQ, LSR and LSE systems obtained under the selected size-
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187 reducing process was evaluated by TEM (Tecnai 12, Philips, The Netherlands) at 80kV,

188 using the negative-stain method. The LS were diluted with PPB at ratio 1:10, and an

189 aliquot of 15 µL was placed on a formvar-carbon coated copper grid (300 mesh, 3 mm

190 diameter HF 36), and stained with uranyl 2% during 1 min. Then, the samples were

191 dried in an oven (LDO-150F model, LabTech, Korea) at 25 °C for 10 min.

192 2.3.2. Stability of liposomes during storage

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193 The physical stability of the LSE, LSQ and LSR systems was evaluated at day 1 and 21

194 of storage at 4 °C by measuring the average hydrodynamic diameter (DH),

195 polydispersity index (PI), and zeta potential as described in section 2.2.4.1.

196 2.4. Incorporation and release of flavonols from edible film

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197 CMC-based edible films with LSQ (EF-LSQ) and LSR (EF-LSR) were prepared by

198 casting. CMC was hydrated in distilled water (2% w/v) at 24000 rpm (IKA T18,

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199 Germany) and left in ultrasonic bath for 10 min to remove the bubbles. Glycerol was

added to the CMC solution (35% w/w of CMC) and shaken for 5 min. LSQ or LSR,

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201 with 0.3 mg/mL of Q or R, were added to the coating suspensions (50% v/v) and shaken

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202 for 10 min. In addition, edible films with free Q (EF-SQ) or R (EF-SR) were prepared
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203 by adding the flavonol solutions into the coating suspensions. The final theoretical

204 concentration of flavonols in all the coating suspensions was 0.15 mg/mL. The pH of
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205 the coating suspension was 7.3 in average. An amount of 25 g of the coating

206 suspensions was poured into a polystyrene plate of 144 cm2 and dried at 25 °C for 24 h.
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207 All the films were conditioned at 25 °C for 3 days in a desiccator at 58% RH by using a
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208 sodium bromide saturated solution. The thickness of the films varied in the range of 35-
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209 45 µm.

210 2.4.1. In vitro release of polyphenols from edible films

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211 The release of polyphenols, both encapsulated (LSQ and LSR) and non-encapsulated
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212 (SQ and SR), from film to fatty food simulant was estimated through the quantification

213 of total phenolic content (TPC) for 7 days and at day 21 at 25 °C. A mixture of

214 water:ethanol 50:50 (v/v) was used as fatty food simulant, according to the European

215 Union Regulation 10/2011 on plastic materials intended to come into contact with food

216 (EU, 2011). A piece of film (6 cm2) was weighed and placed in a tube with 9 mL of
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217 water:ethanol 50:50 (v/v). All the tubes were shaken at 100 rpm and maintained at 25

218 °C in a thermoregulated bath (D3006, GFL, Germany). Three independent samples

219 were analyzed for each time of release. The TPC was determined by the Folin-Ciocalteu

220 method (Singleton & Rossi, 1965), and the results were expressed as µg gallic acid

221 equivalent (GAE)/mL.

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222 The release data were fitted to Korsmeyer–Peppas kinetic model (Equation 2) to

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223 determine the release mechanism of flavonols from CMC edible films.

Mt
= KR · tnR

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Eq.2
M∞

224 Where Mt / M∞ is fraction of TPC released , Mt is amount of TPC in food simulant at

225

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time t (s), M∞ is the equilibrium amount of TPC in the food simulant (50% ethanol) that
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226 corresponds to the initial TPC present in the film, KR is the release rate constant and

227 indicates the release mechanism of the active agent from films (Ritger & Peppas, 1987).
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228
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229 A derived analytical equation from Fick’s second law of diffusion, valid for low valid
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230 for low release fractions (0 ≤ Mt / M∞ ≤ 0.67) (Crank, 1975; Equation 3), was applied to

231 obtain the effective diffusion coefficient (D, m2/s) of flavonols from the film.
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1
Mt 4  D ⋅t  2
= ⋅  Eq.3
M ∞ 2d p  π 
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232 Where is the half thickness of the film (m) and t is the time (s)
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233 Finally, the fraction of TPC released into the food simulant over the time was simulated

234 using an analytical diffusional equation for planar sheet to non-steady state (Crank,

235 1975; Equation 4), valid for the entire range of release fractions, taking into count

236 unidirectional diffusion and isotropic structure. Thus, microstructural effects like
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237 porosity, tortuosity, and homogeneous distribution inside the film are related in

238 calculation of the effective diffusion coefficient.

 ( 2 n + 1) 2 π 2  Eq.4
∞ − ⋅ D ⋅t 
Mt 8
= 1− ∑
2
 
⋅e
4d p

n = 0 ( 2 n + 1) ⋅ π
2 2
M∞

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239 For this simulation n = 1000 was used for ensuring convergence, is the half thickness

240 of the film (m), t is the time (s) and diffusion coefficients were obtained from the slope

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241 of the plot of Mt/M∞ as a function of t1/2 (Figure 3, Eq.3). The mathematical processing

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242 of data and the simulation were carried out in MATLAB (MathWorks, Inc) software.

243 The error between the experimental and theoretical release fraction was determined

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244 through the equation 5.
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n
M t,theor M t,exp Eq.5
∑
i=1 M∞
-
M∞ i
Error =
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2.7. Statistical analyses

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245
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246 The reported results correspond to the mean value of three batches ± standard deviation.

247 Statistical analysis was performed by one-way analysis of variance (ANOVA) followed
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248 by Tukey's test to determine the significant differences among groups (Statgraphics

249 Centurion XVI version 16.1.03, StatPoint Technologies, Inc., Warrenton, VA, USA).
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250 Statistically significant differences were considered at p≤0.05.

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251 3. Results and discussion

252 3.1. Characteristics of liposomes with flavonols added in different stages of the

253 liposome formulation

254 The EE and physical properties of LS with flavonols incorporated during different

255 stages of the liposomal formulation are shown in Table 1. The EE of Q was
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256 significantly higher (p≤0.05) than that of R in all the liposomal systems (Table 1). The

257 stage of flavonol incorporation during the liposomal formulation significantly affected

258 the EE values both in Q-loaded (LSQ) and R-loaded (LSR) liposomes (p≤0.05). When

259 the flavonols were placed in the organic phase for the phospholipid film formation

260 (PFF), they were expected to be mainly entrapped in the phospholipidic bilayer. The

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261 high EE values of both flavonols in the PFF systems (88.9% for LSQ and 74.1% for

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262 LSR) may be related to the hydrophobic character of these compounds (Mignet et al.,

263 2013), allowing them to locate at the phospholipidic bilayer. However, R showed lower

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264 EE than Q in the PFF systems, as the relative hydrophobicity of the flavonols decreases

265 as the level of hydroxylation increases. Furthermore, in contrast with R, Q has a planar

266
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configuration which allows it to easily intercalate into the phospholipid bilayer structure
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267 (van Dijk, Driessen, & Recourt, 2000). High EE values of R (71%) and Q (86%) have
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268 been also reported for egg phosphatidylcholine (EPC) based liposomes, with 9:2 as

269 molar ratio EPC:flavonol at pH 5.6 (Goniotaki et al. 2004). Additionally, Cadena et al.
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270 (2013) obtained EE values for Q of 97.3% in liposomes of soy

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271 phosphatidylcholine:cholesterol 3.5:1 when it was entrapped in the phospholipidic

272 bilayer.
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273 The lowest EE values were obtained when the flavonols were incorporated during the
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274 stage of phospholipid film hydration (PFH) (69.8% for LSQ and 31.8% for LSR). In
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275 this case, they are expected to be mainly in the aqueous inner cavity, where they can

276 interact with the lipid headgroups by hydrogen or electrostatic bonds (adsorption) at the

277 water-lipid interface, and penetrate into the lipid bilayer (absorption) in the liquid-

278 crystalline state (Sirk et al., 2008, Ulrih et al., 2015). Q (pKa = 6.74) is partially ionized

279 at physiological pH (pH 7.3), and this flavonol has been reported to be located at the

280 water-lipid interface, affecting only the polar region of the bilayer but not the
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281 hydrophobic core (Pawlikowska-Pawlega et al., 2003). The EE values of R were

282 significantly lower than those of Q (p≤0.05), which may be attributed to the lower lipid-

283 aqueous partition coefficient of R (log P 0.76) compared to Q (log P 1.82; Rothwell et

284 al., 2005). Nii & Ishii (2005) reported a correlation between EE of several drugs and

285 their partition coefficients, and compounds with higher log P showed higher EE values.

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286 Compared to PFF systems, the lower values of EE obtained in PFH systems may be

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287 attributed to the higher solubility of flavonols in the phospholipids as well as to the

288 lamellarity and size-reducing processes, where a leakage of the liposomal content may

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289 occur. In other study, EE values of resveratrol and quercetin in liposomes were also

290 higher when these polyphenols were placed in the phospholipidic bilayer than when

291
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they were placed in the inner aqueous cavity (Cadena et al., 2013).
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292 When R and Q were incorporated during the stage of liposome resuspension (LRS),
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293 they tended to mainly locate at the surface of the liposomes, where they can be adsorbed

294 onto the lipid bilayer by hydrogen and electrostatic bonds, since flavonols are not likely
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295 to intercalate into the lipid bilayer when DPPC is in the gel state (25 °C; Ulrih et al.,
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296 2015). Significant differences (p≤0.05) were found in EE values between LSQ-LRS

297 (92.7%) and LSR-LRS (55.4%) that may be related to the different molecular weight
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298 (Mw) of flavonols (Mw Q = 302 g/mol; Mw R = 610 g/mol), since flavonols with lower
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299 molecular weight tend to be easily adsorbed on the surface of liposomes (Kerdudo et al.,
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300 2014).

301 The physical properties of LS were also affected by the stage of flavonol incorporation

302 (Table 1). All the liposomal formulations showed an average DH in the range of 170 −

303 425 nm, and therefore, they can be classified as large unilamellar vesicles (LUV)

304 (Emami et al., 2016). LSR showed lower sizes than LSQ, except when the flavonols

305 were incorporated during LRS stage. This may be because glycosylated flavonols, such
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306 as R, have more hydroxyl groups than Q in their structure and may form a larger

307 number of hydrogen-bonds with the polar headgroups of the phospholipids in the lipid

308 bilayer, leading to a higher packing density of the phospholipid membrane (Olilla et al.,

309 2002; Ulrih et al., 2015). Moreover, the interactions among the polyphenol molecules

310 may reduce the size of the liposome central core, and consecuently reduce the liposome

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311 size (Hidalgo et al., 2010). In the case of Q-loaded liposomes, PFF and PFH liposomes

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312 showed similar DH values (p>0.05) and higher (p≤0.05) than those obtained for LRS

313 (PFH ≈ PFF > LRS). For the R-loaded liposomes, PFH system showed the highest DH

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314 values, followed by LRS and PFF (PFH˃LRS˃PFF; p≤0.05).

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315 The PI values for LSQ and LSR varied between 0.20 and 0.60. The PI values can vary
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316 between 0 and 1, where 0 indicates completely monodisperse systems and 1 indicates

317 polydispersed system. A liposomal suspension with PI values below 0.4 can be
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318 considered as a homogenous and monodisperse distribution of particles (Chessa et al.,

319 2011). LSQ showed lower PI values than LSR, except when the flavonols were
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320 incorporated during the PFF stage. As in the case of DH values, the stage of flavonol
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321 incorporation influenced significantly the polydispersity of the liposomal formulations

322 (p ≤ 0.05). The PI values followed the same trend both in LSQ and LSR, and the highest
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323 polydispersity was obtained for PFH liposomes, followed by LRS and PFF (PFH > LRS
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324 > PFF). Thus, the incorporation of Q and R during the PFF stage gave liposomal
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325 suspensions with the lowest PI values (0.20 and 0.32 for LSR-PFF and LSQ-PFF,

326 respectively).

327 The zeta potential influences the stability of particles during the storage, and liposomal

328 systems with absolute ZP value greater than ∼ 30 mV are considered stable due to the

329 relatively high repulsive forces, minimizing aggregation or sedimentation of their

330 particles. Zeta potential values were always negative. The stage of flavonol
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331 incorporation affected significantly the ZP values. When compared with ZP values of

332 empty DPPC liposomes (13.2 mV, Table 2), the entrapment of Q and R in the

333 phospholipid bilayer led to the lowest change in ZP values (16.5 mV for LSQ-PFF and

334 17.1 mV for LSR-PFF). However, the incorporation of flavonols in the inner aqueous

335 cavity (during the PFH stage) or adsorbed on the liposome surface (during the LRS

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336 stage) gave rise to noticeable increases in ZP values. Interactions between DPPC and

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337 the encapsulated flavonols may induce changes in the surface charge of liposomes,

338 through changes of orientation of the choline and phosphate groups and compression of

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339 the diffuse part of electric double layer (Chibowski & Szczés, 2016). In general, the ZP

340 was higher in Q-loaded liposomes (LSQ; Table 1). As ZP values of liposomes mainly

341
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depends on the phospholipid composition, the encapsulated compound and the
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342 dispersion medium, a wide range of ZP values can be found for Q or R-loaded

liposomal systems in the literature (Babazadeh, Ghanbarzadeh & Hamishehkar, 2017;

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343

344 Cadena et al., 2013; Goniotaki et al., 2004; Guo et al., 2016).
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345 Considering the high EE values and the low PI values found in liposomal systems with
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346 flavonols incorporated during the PFF stage (LSQ-PFF and LSR-PFF), these systems

347 were selected to continue with the following experiments.

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348 3.2. Evaluation of the size-reducing method

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349 Table 2 shows the EE and physical characteristics of liposomal suspensions with Q or R
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350 incorporated during the PFF stage and subjected to extrusion (LSQ-E, LSR-E) or

351 sonication (LSQ-S, LSR-S) as size-reducing method. All liposomes showed were large

352 unilamellar vesicles (LUV >100 nm), characterized by a large aqueous phase to lipid

353 ratio and high efficiency at entrapping large volume of hydrophilic material (Emami et

354 al. 2016).

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355 The EE values of Q were higher than those of R for both size-reducing methods,

356 especially in the case of sonicated liposomes. The EE values of polyphenols in

357 liposomal systems may be affected by several factors such as structural and

358 physicochemical properties of the polyphenol, type of phospholipid forming the bilayer

359 and method used for liposome preparation (Emami et al., 2016). When LS are subjected

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360 to extrusion as size-reducing method, the forced passage through the capillaries of the

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361 membrane or filter exerts high shear forces, leading to a rupture of the membranes and

362 the leakage of the entrapped compounds (Taylor et al., 2005). However, EE values of Q

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363 and R in extruded and sonicated liposomes, respectively, were close and no statistical

364 differences were obtained (p>0.05), denoting that liposome preparation either by

365
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sonication or extrusion led to similar levels of EE of Q and R, respectively. This may be
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366 related to the stage of the polyphenol incorporation during the liposome formulation. As
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367 the flavonols were incorporated during the PFF stage, they are expected to be mainly

368 entrapped in the phospholipidic bilayer instead of in the inner aqueous cavity.
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369 As mentioned above, the relative planar structure adopted by quercetin it confers a
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370 markedly higher affinity for membranes (van Dijk et al., 2010), leading to a higher EE

371 (Goniotaki et al., 2004). In the case of R, the bulky substituent at the carbon-3 (rutinosyl
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372 group), forces the B-ring out of the plane of the AC-ring, inducing a dihedral angle of
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373 38° between the plane of the AC-ring and that of the B-ring (Moalin et al., 2011), giving
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374 a weaker interaction with the bilayer, and therefore lower EE values (Goniotaki et al.,

375 2004). Goniotaki et al. (2004) encapsulated Q and R in the phospholipid bilayer of EPC

376 liposomes (EPC:flavonol 9:2 molar ratio) by the thin-film hydration method, using

377 sonication as size-reducing method. In contrast with this study, the hydration of the lipid

378 film was performed with water at pH 5.6. These authors also reported higher EE values

379 of Q (86%) than R (71%), attributed to the mentioned difference in the structure
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380 features between both polyphenols. In contrast, using a probe sonicator, R loaded in

381 ceramide-CH-oleic acid-EPC liposome at pH 7.4 showed higher EE than Q (Park et al.,

382 2013).

383 The encapsulation of Q or R gave a significant decrease (p≤0.05) in the size of

384 liposomes obtained by both extrusion and sonication methods, and therefore, LSR and

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385 LSQ showed lower liposomal size (DH) than LSE. This result is in contrast with other

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386 studies where the encapsulation of Q or R led to an increase of the liposome size

387 (Babazadeh et al., 2012; Goniotaki et al., 2004). The lower DH of the flavonol-loaded

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388 liposomes may be attributed to flavonol-phospholipid and flavonol-flavonol interactions

that increase the packing of the lipid molecules and reduce the size of the central core of

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389 AN
390 liposome (Olilla et al., 2002, Hidalgo et al., 2010; Ulrih et al., 2015). The size-reducing

391 method, extrusion or sonication, did not have a net effect on liposome size. In the case
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392 of LSE and LSQ, sonicated liposomes were smaller than extruded liposomes (p≤0.05),

393 whereas the smallest R-loaded liposomes were obtained by extrusion (p≤0.05).
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394 Goniotaki et al. (2004), using sonication as size-reducing method, reported higher sizes
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395 than those obtained in this study for Q-loaded EPC-based liposomes (414 nm vs. 260

396 nm), but similar sizes for R-loaded liposomes (424 nm vs. 391 nm). Regarding
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397 polydispersity, LSQ and LSR obtained by extrusion showed significant lower PI values
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398 (p≤0.05) than those obtained by sonication (Table 2). In the case of extruded samples,
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399 the incorporation of flavonols gave a significant decrease in polydispersity (p≤0.05),

400 whereas there were not significant differences (p ˃ 0.05) in PI values between LSE and

401 flavonol-loaded liposomes obtained by sonication.

402 As shown in Table 2, the LS obtained by sonication showed ZP absolute values in the

403 range 36.9 - 42.4 mV, and therefore significantly higher than those obtained by

404 extrusion that ranged from 13.2 mV to 17.1 mV (p≤0.05). This involves that liposomal
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405 suspensions obtained by sonication may have higher stability to destabilization

406 phenomena such as aggregation and fusion over time, sometimes causing a leakage of

407 the encapsulated molecules (Alavi et al., 2017). Furthermore, the incorporation of Q or

408 R in LS obtained by extrusion led to a slight but significant decrease in ZP values, and

409 LSQ-E and LSR-E showed slightly lower ZP values than LSE-E (Table 2). However, all

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410 the liposomal systems obtained by sonication showed similar ZP values, in the range

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411 36.9 - 42.4 mV (Table 2; p>0.05).

412 Considering the high zeta potential values together with the high EE of flavonols found

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413 in LS obtained by sonication, this size-reducing method was selected to be evaluated by

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414 the morphology by TEM together with the stability of the liposomal formulations
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415 during storage at 4 ºC.

416 3.3. Morphology and stability of flavonol-loaded liposomes obtained by sonication

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417 Figure 1 shows the TEM micrographs of liposomal suspensions with and without Q or
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418 R incorporated during the PFF stage and obtained by sonication as size-reducing
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419 process. All the LS had spherical shape and they were unilamellar. The presence of

420 agglomerations of liposomes was more noticeable in LSE than in LSQ and LSR, where
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421 dispersed liposomes were mainly observed. The real diameter of liposomes calculated

422 from the image analysis of TEM photographs showed that LSE had the highest size
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423 (range of 100-600 nm), followed by LSR (range of 70-180 nm) and LSQ (range of 20-
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424 100 nm). In all LS, two size populations were identified.

425 The LS were stored at 4 °C during 21 days to evaluate their physical stability over time.

426 Figure 2 shows the physical characteristics of LSE, LSQ and LSR at day 1 and 21 of

427 storage. As shown by DH values, LSQ had the smallest size at day 1 of storage, with

428 values of 260 nm, whereas LSE and LSR showed similar sizes (p˃0.05), in the range
 ACCEPTED MANUSCRIPT
429 391 - 436 nm. The liposome size did not increase significantly (p˃0.05) during the

430 storage in LSE and LSQ. However, a significant 27% increase in DH values was found

431 in the case of LSR at day 21 (p≤0.05). In other studies, the increase in liposome size

432 during storage has been related to hydrolysis of phosphatidylcholine from their ester

433 bonds in aqueous environments, causing particle aggregation (Hou et al., 2013). All the

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434 liposomal systems showed PI values in the range 0.57 – 0.59 at the beginning of the

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435 storage, and no significant differences (p˃0.05) were found among them. Furthermore,

436 the PI values were not affected by the storage at 4 ºC in any of the liposomal systems,

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437 and similar values were obtained both at day 1 and 21 (p˃0.05). Regarding the zeta

438 potential, all the systems showed similar ZP values and higher than 30 mV (p˃0.05) at

439
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the beginning of the storage. Similar ZP values were found at day 1 and 21 of storage
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440 for LSE. However, ZP values decreased significantly in those liposomal formulations
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441 with flavonols (LSQ and LSR) during the storage, especially in the case of LSR that

442 showed ZP values below 20 mV at day 21. At the end of the storage (day 21) only LSE
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443 and LSQ samples showed ZP values over 30 mV, suggesting that these systems were
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444 stable during the storage in terms of zeta potential. These results denote the high

445 stability of LS formulations during the storage period studied, especially in the case of
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446 LSE and LSQ. Although the liposomal formulations with flavonols showed significant

changes in size (LSQ) and zeta potential (LSQ and LSR) during the storage at 4 ºC, the
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447
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448 most noticeable changes were observed in LSR, which showed the lowest ZP value at

449 day 21. According to Goniotaki et al. (2004), EPC-based liposomes containing Q or R

450 were retained their stability during 2 months of storage at 4 ºC, and no significant

451 differences were found in liposome size or zeta potential between the beginning and the

452 end of the storage period for any of the liposomal formulations.

453 3.4. In vitro release of polyphenols from edible films

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454 Figure 3 shows the release profile of Q and R from CMC-based films fitted to a derived

455 equation from Fick’s second law of diffusion (Eq. 3). Non-encapsulated flavonols

456 showed higher diffusion coefficients (D = 5.34 × 10-17 m2/s, R2 = 0.992 for EF-SQ and D

457 = 6.20 × 10-17 m2/s, R2 = 0.992 for EF-SR) from the films than liposome-encapsulated

458 flavonols (D = 1.76 × 10-17 m2/s, R2 = 0.847 for EF-SLQ and D= 1.36 × 10-17 m2/s, R2 =

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459 0.982 for EF-SLR). Pravilović et al. (2015) studied the diffusion of thyme extract

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460 polyphenols, both non-encapsulated and encapsulated in several liposome formulations,

461 from cellulose acetate membranes. In accordance with our results, all the encapsulated

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462 polyphenols (D = 1.69 – 2.31 × 10-9 m2/s) diffused less that polyphenols directly

incorporated into the membrane (D = 8.77 x 10-9 m2/s), allowing a controlled release

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463

over time. In other study, Zhang & Zhao (2014), using the same model to evaluate
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464

465 diffusion coefficient of non-encapsulated flavonoids from low-density polyethylene

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466 films into aqueous food simulant (30% ethanol), reported a higher release of flavonoids

467 than that obtained in this study, with D values of 9.20 x 10-13 m2/s.
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Figure 4 shows the release profile of Q and R from the CMC-based films (EF-SQ, EF-
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468

469 LSQ, EF-SR, EF-LSR) to food simulant, where experimental and theoretical values
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470 (calculated with Eq. 4) are shown. The experimental results fit theoretical data with

471 error values (Eq. 5) of 0.012 for EF-SQ, 0.063 for EF-LSQ, 0.023 for EF-SR and 0.015
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472 for EF-LSR. According to Figure 4, the fraction of flavonol released increased with
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473 time in all the samples, but a significant effect of the form of flavonol incorporation into

474 the films was found. A higher release of Q and R was observed in films with Q or R

475 added as suspensions (SQ and SR) than in films where these flavonols were

476 incorporated as LS throughout the study, suggesting that polyphenol-polymer

477 interactions are weaker than polyphenol-lipid interactions. Thus, the fraction of Q

478 released from the matrix films was 10% and 24% higher in EF-SQ than in EF-LSQ at
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479 days 7 and 21, respectively. In contrast, the fraction of R released from the CMC-based

480 films was 15% and 25% higher in EF-SR than in EF-LSR, respectively, at the same

481 days of storage. Moreover, noticeable differences were found among the release of

482 liposome-encapsulated Q and R from the matrix films. thus, EF-LSQ showed higher

483 release than EF-LSR (5 and 10% higher at days 7 and 21 days, respectively). This result

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484 is in agreement with other studies, where quercetin had a faster diffusion rate than rutin

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485 due to its higher membrane permeability (Park et al., 2013). Rutin, due to its higher

486 number of hydroxyl groups, may form a larger number of hydrogen bonds with the

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487 polar headgroups of the phospholipids in the lipid bilayer, leading to a higher packing

488 density of the phospholipid bilayer and, therefore, a lower permeability of the liposomal

489 membrane.
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490 The analytical Korsmeyer–Peppas model was applied to explain the flavonols release
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491 mechanism from CMC films. Table 3 shows the kinetic parameters obtained from

492 release curves for non-encapsulated flavonols (SQ and SR) and liposome-encapsulated
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493 flavonols (LSQ and LSR). The release kinetics showed a good correlation with
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494 mathematical model applied (R2 ≥ 0.898 for all the systems, see Table 3). Fickian

diffusion is defined by equal to 0.50 and non-Fickian by greater than 0.50 (Ritger
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495

496 & Peppas, 1987). According to values (0.440 for EF-SQ and 0.374 for EF-LSQ),
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497 Fickian diffusion was the principal release mechanism of Q encapsulated and non-
AC

498 encapsulated from EF. However, in the case of EF with R encapsulated and non-

499 encapsulated, values (0.510 for EF-SR and 0.548 for EF-SLR) indicated that R

500 release mechanism agreed with non-Fickian or anomalous diffusion where different

501 mechanisms such as diffusion and relaxation of the polymer chains may occur

502 simultaneously.

503 4. Conclusions
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504 A 2-step delivery system was developed in this study by incorporating flavonol-loaded

505 liposomes into CMC edible films in order to protect Q and R and control their release.

506 The incorporation of Q and R during the PFF stage of liposomal formulation results in

507 higher encapsulation efficiency of both flavonols, quercetin and rutin, since these

508 polyphenols have higher solubility in the phospholipid dispersion. Sonication gave rise

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509 to liposomes with higher zeta potential values than extrusion, and Q-loaded liposomes

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510 obtained by sonication showed higher stability than those loaded with R during storage

511 at 4 ºC. The encapsulation efficiency was significantly higher for Q than for R,

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512 regardless the stage of flavonol incorporation and the size-reducing method used,

513 showing the influence of the flavonol structural features. A controlled release over time

514
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of both Q and R was observed when flavonol-loaded liposomes were incorporated into
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515 CMC edible films. The results obtained in this study highlight the relevance of using an
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516 encapsulation technology such as liposomes, able to preserve polyphenols and control

517 their release, in the design of edible films with antioxidant activity for improving food
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518 shelf life.

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519 Acknowledgements
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520 This work was financially supported by The National Fund for Scientific and

521 Technological Development, FONDECYT 11140509 (CONICYT – Chile) and DICYT-

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522 USACH.
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523 Conflict of interest

524 This article represents the authors own work and we are not aware of any conflict of

525 interest.

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656 Figure Captions

657 Figure 1. TEM micrographs of LS obtained by sonication: a) without addition of

658 polyphenol (LSE), b) with Q (LSQ) and c) with R (LSR) incorporated during the PFF

659 stage.

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660 Figure 2. Physical characteristics of liposomes obtained by sonication, without

661 polyphenols (LSE, white bars) and with Q (LSQ, black bars) or R (LSR, grey bars)

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662 incorporated during the PFF stage, at days 1 and 21 of storage at 4 °C. Means ±

standard deviation (n=3). Small letters indicate significant differences (p ≤ 0.05) among

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663

664 samples for each sampling day.

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665 Figure 3. Release profile of Q and R from CMC edible films fitted to Eq.3 (EF) with
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666 encapsulated flavonols (LSQ, ▲ and LSR, ●) and non-encapsulated (SQ, and SR, ○)

667 flavonols in simulant food medium at 25 °C.

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668 Figure 4. Release profile of flavonols from CMC edible films simulated with Eq. 4
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669 (lines) and experimental data. a) Encapsulated Q (LSQ, ▲) and non-encapsulated Q

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670 (SQ, ). b) Encapsulated R (LSR, ●) and non-encapsulated R (SR, ○) in simulant food

671 medium at 25 °C.

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638 Table 1. Physical characteristics and encapsulation efficiency of liposomes with

639 flavonols incorporated in different formulation stage

Sample Stage Average DH PI (-) ZP (mV) EE (%)

(nm)
LSQ PFF 424 ± 13 bB 0.32 ± 0.04 bA -16.5 ± 0.5 aB 88.9 ± 5.1 bB
425 ± 22 bB 0.46 ± 0.01 aC -45.8 ± 4.3 aA 69.8 ± 7.2 bA

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PFH
LRS 254 ± 5.2 aA 0.39 ± 0.03 aB -43.9 ± 4.1 aA 92.7 ± 10.5 bB
LSR PFF 170 ± 16 aA 0.20 ± 0.01 aA -17.1 ± 0.6 aC 74.1 ± 11.4 aC

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PFH 367 ± 43 aC 0.60 ± 0.03 bC -37.2 ± 1.2 bA 31.8 ± 10.1 aA
LRS 288 ± 19 bB 0.49 ± 0.01 bB -22.3 ± 0.9 bB 55.4 ± 9.7 aB

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640 PFF: stage of phospholipid film formation; PFH: stage of phospholipid film hydration;

641 LRS: stage of liposome resuspension. Mean ± standard deviation (n=3). Small letters

642
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643 the same stage. Capital letters indicate significant differences among formulations with
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644 the same flavonol incorporated during different stages of the liposomal formulation.
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645 Table 2. Physical characteristics and encapsulation efficiency of liposomes with Q or R

646 incorporated during the stage of phospholipid film formation (PFF) using extrusion (E)

647 or sonication (S) as size-reducing methods.

Sample Average DH PI (-) ZP (mV) EE (%)

(nm)

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LSE-E 941 ± 132 bC 0.67 ± 0.17 aB -13.2 ± 0.5 bB -
LSQ-E 424 ± 13 bB 0.32 ± 0.04 aA -16.5 ± 0.5 bA 88.9 ± 5.1 aB

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LSR-E 170 ± 16 aA 0.20 ± 0.01 aA -17.1 ± 0.6 bA 74.1 ± 11.4 aA
LSE-S 436 ± 54 aB 0.57 ± 0.09 aA -37.6 ± 1.9 aA -

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LSQ-S 260 ± 13 aA 0.57 ± 0.12 bA -42.4 ± 3.2 aA 97.4 ± 7.4 bB
LSR-S 391 ± 62 bB 0.59 ± 0.10 bA -36.9 ± 2.1 aA 72.0 ± 6.6 aA
648 E: extrusion, S: sonication, LSQ: liposomal suspension with quercetin, LSR: liposomal

649
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suspension with rutin, LSE: liposomal suspension empty. Mean ± standard deviation
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650 (n=3). Small letters indicate significant differences between the same sample and
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651 different size-reducing process; Capital letters indicate significant differences among

652 samples obtained with the same size-reducing process.

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653 Table 3. Kinetics parameters obtained from release curve of flavonol from CMC edible

654 films using Korsmeyer–Peppas model.

Sample KR R2 Release mechanism

EF-SQ 0.126 0.440 0.995 Fickian diffusion
EF-SR 0.111 0.510 0.988 Non-Fickian diffusion

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EF-LSQ 0.107 0.374 0.898 Fickian diffusion
EF-LSR 0.051 0.548 0.982 Non-Fickian diffusion
655

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Figure 1.

a)

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1 µm

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b)

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1 µm
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c)
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2 µm
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LS-E
800 LS-R

Average diameter (nm)

aA LS-Q
aA
600
bA
bA
aB
400
aA

200

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0
1 21

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Storage day

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LS-E
0.8 LS-R
aA
aA aA bA
Polydispersity index

LS-Q

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aA aA
0.6
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0.4

0.2
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0.0
1 21
D

Storage day
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0
EP

-10
Zeta potential (mV)

-20 cB
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-30
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bB
-40 LS-E
aA aA aA LS-R
LS-Q
aA
-50
1 21
Storage day
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Figure 3.

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Figure 4.

a)

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b)
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Highlights
• Liposomal suspensions of dipalmitoyl lecithin containing quercetin (LSQ) or rutin

(LSR) were designed.

• Encapsulation efficiency (EE) was affected by the stage of flavonol incorporation

• The higher EE of Q suggested the influence of the flavonol structural features

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• LSQ obtained by sonication showed higher stability than LSR during storage at 4 ºC

• Dipalmitoyl lecithin liposome controlled the release of Q and R from CMC films

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