Controlled release of resveratrol from lipid nanoparticles improves antioxidant effect

10th IFAC Symposium on Biological and Medical Systems 10th IFAC Symposium on Biological and Medical Systems São Paulo, Brazil, September 3-5, 2018 10th IFAC Symposium on Biological and Medical Systems São Paulo, Brazil, September 3-5, 2018 Available online at www.sciencedirect.com 10th IFAC Symposium on Biological and Medical Systems São Brazil, September 3-5, 2018 10th Paulo, IFAC Symposium on Biological and Medical Systems São Paulo, Brazil, September 3-5, 2018 São Paulo, Brazil, September 3-5, 2018

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IFAC PapersOnLine 51-27 (2018) 16–21

Controlled release of resveratrol from lipid nanoparticles improves antioxidant effect Controlled release of resveratrol from lipid nanoparticles improves antioxidant effect Controlled release of resveratrol from lipid nanoparticles improves antioxidant effect a a a b Controlled resveratrol nanoparticles improves antioxidant effect Pedro P. Soldatirelease , Hudson of C. Polonini , Camilafrom Q. Paeslipid , Jelver A. S. Restrepobb, Tânia B. Creczynksi-Pasa , Maria aa a a b Controlled resveratrol nanoparticles improves antioxidant effect a aa b a a b, Tânia B. Creczynksi-Pasa a,* Pedro P. Soldatirelease , Hudson of C. Polonini , Camilafrom Q. Paeslipid , Jelver A. S. Restrepo , Maria

A. F. Brandão Frederico , Nádia R. B. Raposoa,* a A. M. Chavesa, Marcos a b b a Pedro P. SoldatiG. C. Polonini Q. Paesaa,aa,,Jelver A. S.Pittella Restrepo a a b, Tânia B. Creczynksi-Pasa a, Hudson a, Camila b, Maria G. A. M. Chaves , Marcos A. F. Brandão Frederico Pittella , Nádia R. B. B. Creczynksi-Pasa Raposoa,* Pedro P. Soldati C. Polonini Q. Paes A. S. Restrepo a, Hudson a, Camila a,a Jelver b, Tânia b, Maria a a a,* Pedro P. SoldatiG., Hudson C. Polonini , Camila Q. Paes , Jelver A. S. Restrepo , Tânia B. Creczynksi-Pasa , Maria A. M. Chaves , Marcos A. F. Brandão , Frederico Pittella , Nádia R. B. Raposo a a a a,* G. A. M. Chaves a, Marcos A. F. Brandão a, Frederico Pittellaa, Nádia R. B. Raposo a,* G. A. M. Chaves , Marcos A. F. Brandão , Frederico Pittella , Nádia R. B. Raposo a  Núcleo de Pesquisa e Inovação em Ciências da Saúde (NUPICS), Federal University of Juiz de Fora, Juiz de Fora, MG, aa  Núcleo de Pesquisa e Inovação em Ciências da Saúde (NUPICS), Federal University of Juiz de Fora, Juiz de Fora, MG,  36036-900, Brazil. a  (NUPICS), Federal University of Juiz de Fora, Juiz de Fora, MG, ab Núcleo de Pesquisa e Inovação em Ciências da Saúde 36036-900, Brazil. de Pesquisa e Inovação em CiênciasFederal da Saúde (NUPICS), Federal University of Juiz MG, a Núcleo Departamento de Ciências Farmacêuticas, University of Santa Catarina, Florianópolis, SC, 88040-900, Brazil. (NUPICS), Federal University of Juiz Juiz de de Fora, Fora, Juiz de de Fora, Fora, MG, b Núcleo de Pesquisa e Inovação em Ciências da Saúde 36036-900, Brazil. b Departamento de Ciências Farmacêuticas, Federal University of Santa Catarina, Florianópolis, SC, 88040-900, Brazil. 36036-900, Brazil. * Address correspondence to: Núcleo de Pesquisa e Inovação em Brazil. Ciências Saúde (NUPICS), Federal University ofBrazil. Juiz de b 36036-900, de Ciências Farmacêuticas, Federal University of Santa da Catarina, Florianópolis, SC, 88040-900, b Departamento *Fora, Address correspondence to: Núcleo de Pesquisa e Inovação em Ciências da Saúde (NUPICS), Federal University of Juiz de de Farmacêuticas, Federal University of Catarina, Florianópolis, SC, Brazil. b Departamento Ruacorrespondence José Lourenço Kelmer, s/n,de Campus Universitário, JuizCiências deSanta Fora,da MG, 36036-900, Brazil. Tel.:88040-900, +55 32 2102 3809; Departamento de Ciências Ciências Farmacêuticas, Federal University of Santa Catarina, Florianópolis, SC, 88040-900, *Fora, Address to: Núcleo Pesquisa e Inovação em Saúde (NUPICS), Federal University ofBrazil. Juiz de Rua José Lourenço Kelmer, s/n, Campus Universitário, Juiz de Fora, MG, 36036-900, Brazil. Tel.: +55 32 2102 3809; ** Address correspondence to: de3809. Pesquisa ee Inovação Ciências da Saúde (NUPICS), Federal University of de fax: +55 32 2102 E-mail address:em [email protected] (N.R.B. Raposo). Address to: Núcleo Núcleo Pesquisa Inovação Ciências Saúde (NUPICS), Federal of Juiz Juiz de Fora, Ruacorrespondence José Lourenço s/n,de Campus Universitário, Juiz de Fora,da MG, 36036-900, Tel.:University +55 32 2102 3809; fax: Kelmer, +55 32 2102 3809. E-mail address:em [email protected] (N.R.B.Brazil. Raposo). Fora, Rua José Lourenço Kelmer, s/n, Campus Universitário, Juiz de Fora, MG, 36036-900, Brazil. Tel.: +55 32 2102 3809; Fora, Rua José Lourenço s/n, Campus Universitário, de Fora, MG, 36036-900, Tel.: +55 32 2102 3809; fax: Kelmer, +55 32 2102 3809. E-mail address:Juiz [email protected] (N.R.B.Brazil. Raposo). fax: fax: +55 +55 32 32 2102 2102 3809. 3809. E-mail E-mail address: address: [email protected] [email protected] (N.R.B. (N.R.B. Raposo). Raposo). Abstract: Solid lipid nanoparticles (SLN) based on natural seed butter Theobroma grandiflorum were Abstract: Solid lipid nanoparticles (SLN) based on natural seed butter Theobroma grandiflorum were prepared for the topical release of resveratrol. Nanoparticles small particle size and the surface Abstract: Solid lipid nanoparticles (SLN) based on naturalpresented seed butter Theobroma grandiflorum were prepared for the topical release of resveratrol. Nanoparticles presented small particle size and the surface Abstract: Solid lipid (SLN) based on natural seed Theobroma grandiflorum were charge, size and polydispersity index. The controlled revealed a burst size release followed by Abstract: Solid lipid nanoparticles nanoparticles (SLN) based on release naturalkinetics seed butter butter Theobroma grandiflorum were prepared for the topical release of resveratrol. Nanoparticles presented small particle and the surface charge, size and polydispersity index. The controlled release kinetics revealed a burst release followed by prepared for the topical release of resveratrol. Nanoparticles presented small particle size and the surface acharge, sustained drug release from SLNs containing resveratrol (R-SLN). R-SLN showed an increased preparedsize for and the polydispersity topical release index. of resveratrol. Nanoparticles presented small particle size andfollowed the surface The controlled release kinetics revealed a burst release by acharge, sustained drug release from SLNs containing resveratrol (R-SLN). R-SLN showed an increased size and polydispersity index. The controlled release kinetics revealed a burst release followed by antioxidant in 20%from and increased permeation and kinetics retention of resveratrol in the an human skin, size activity and polydispersity index. The controlled release revealed a burst release followed by acharge, sustained drug release SLNs containing resveratrol (R-SLN). R-SLN showed increased antioxidant activity in 20% and increased permeation and retention of resveratrol in the human skin, araising sustained drug release from SLNs containing resveratrol (R-SLN). R-SLN showed an increased the amount ofinresveratrol than 2-fold in resveratrol stratum (SC) compared to araising sustained drug release SLNs containing (R-SLN). R-SLN showed increased antioxidant activity 20%from and over increased permeation and corneum retention of resveratrol in RES. the an human skin, the amount of resveratrol over than 2-fold in stratum corneum (SC) compared to RES. antioxidant activity in 20% and increased permeation and retention of resveratrol in the skin, antioxidant activity 20% and over increased permeation and retention of resveratrol innanoparticles the human human skin, raising amount ofinresveratrol than 2-fold stratum corneum compared to RES. © 2018,the IFAC (International Federation of Automatic Control) Hosting by(SC) Elsevier All rights reserved. Keywords: Antioxidants, resveratrol, natural seed in butter, controlled release, solidLtd. lipid raising the amount of resveratrol over than 2-fold in stratum corneum (SC) compared to RES. raising the amount of resveratrol overnatural than 2-fold stratum corneum (SC) compared RES. Keywords: Antioxidants, resveratrol, seed in butter, controlled release, solid lipidtonanoparticles Keywords: Antioxidants, resveratrol, natural seed butter, controlled release, solid lipid nanoparticles  Keywords: controlled release, solid lipid Keywords: Antioxidants, Antioxidants, resveratrol, resveratrol, natural natural seed seed butter, butter, controlled solid lipid nanoparticles nanoparticles  preferred typerelease, of lipids to improve transdermal drug delivery,  1. INTRODUCTION preferred type of lipids to improve transdermal delivery,  once they have the advantage of beingdrug endogenous 1. INTRODUCTION  preferred typehave of lipids toadvantage improve transdermal drug delivery, once they the of being endogenous preferred type of lipids to improve transdermal drug delivery, 1. INTRODUCTION of human while increasing thedrug skin fluidity, Normal cellular metabolism and processes triggered by components preferred type of lipids toadvantage improve transdermal delivery, once they have theskin of being endogenous 1. INTRODUCTION components of human skin while increasing the skin fluidity, 1. INTRODUCTION once they have the advantage of being endogenous Normal cellular metabolism and processes triggered by which allow the penetration of delivery nanoparticles to external aggressions usually generates reactive oxygen once they have the advantage of being endogenous components ofthe human skin while increasing the skin fluidity, Normal cellular metabolism and processes triggered by which allow penetration of delivery nanoparticles to components human skin while increasing the skin fluidity, external aggressions usually generates reactive oxygen specific sitesof inthe thepenetration skin (Mandawgade and Patravale, 2008). Normal cellular metabolism and processes triggered by species (ROS). The excess of ROS damage cellular structures components of human skin while increasing the skin fluidity, which allow of delivery nanoparticles to Normal cellular metabolism and processes triggered by specific external aggressions usually generates reactive oxygen sites in the skin (Mandawgade and Patravale, 2008). which the of delivery nanoparticles to species (ROS). The excess ofnucleic ROS damage cellular structures On the allow contrary ofpenetration synthetic lipids, natural lipids, including external aggressions usually generates reactive oxygen such as lipids, proteins and acids and is involved in which allow the penetration of delivery nanoparticles to specific sites in the skin (Mandawgade and Patravale, 2008). external(ROS). aggressions usually generates reactive oxygen species The excess ofnucleic ROS damage cellular structures On the contrary of synthetic lipids, natural lipids, including and is involved in specific sites in the skin (Mandawgade and Patravale, 2008). such as lipids, proteins and acids the butters extracted from seeds, present an alternative for the species (ROS). The excess of ROS damage cellular structures worsening a number of human pathological conditions specific sites in the skin (Mandawgade and Patravale, 2008). On the contrary of synthetic lipids, natural lipids, including species The excess ofnucleic ROS damage cellular structures such as (ROS). lipids, proteins and acids and is involved in the butters extracted from seeds, present an alternative for the On the of lipids, natural lipids, including worsening a number of human pathological conditions production of “green” SLN, which is usually considered as such proteins and nucleic acids and is in including cutaneous pathologies (Packer and 2007). On butters the contrary contrary of synthetic synthetic lipids, natural lipids, including the extracted from seeds, present an alternative for the such as as lipids, lipids, proteins and nucleic acids andCadenas, is involved involved in production worsening a number of human pathological conditions of “green” SLN, which is usually considered as including cutaneous pathologies (Packer and Cadenas, 2007). the butters extracted from seeds, present an alternative for the biodegradable and safe for humans and the environment worsening a number of human pathological conditions To enhance efficiency of(Packer this defense mechanism, the butters extracted from seeds, presentusually an alternative for the production of “green” as worsening a the number of human pathological conditions including cutaneous pathologies and Cadenas, 2007). biodegradable and safeSLN, for which humansis and the considered environment To enhance the efficiency of(Packer this defense mechanism, of “green” SLN, which is usually considered as (Soddu et al., including cutaneous pathologies and Cadenas, 2007). exogenous antioxidants are commonly acquired from the diet production production of2014). “green” SLN, which is and usually considered as biodegradable and safe for humans the environment including cutaneous pathologies (Packer and Cadenas, 2007). To enhance the efficiency of this defense mechanism, (Soddu et al., 2014). exogenous antioxidants are commonly acquired from the diet biodegradable and safe for humans and the environment To enhance the efficiency of defense mechanism, or skin absorption (Natarajan al., 2014). biodegradable and safe for humans and the environment (Soddu et al., 2014). Tothrough enhance the efficiency of this thisetacquired defense mechanism, exogenous antioxidants are commonly from the diet In the present work, we developed novel SLNs containing (Soddu et 2014). or through skin absorption (Natarajan etacquired al., 2014). exogenous antioxidants are commonly from the (Soddu et al., al.,butter 2014). the present work, we developed novel SLNstowards containing exogenous antioxidants are(Natarajan commonly from the diet diet In or through skin absorption etacquired al., 2014). natural seed to nanoencapsulate resveratrol the Resveratrol is a non-flavonoid polyphenolic compound In the present work, we developed novel SLNstowards containing or through skin absorption (Natarajan et al., 2014). natural seed butter to nanoencapsulate resveratrol the or throughinskin (Natarajan et al., 2014). In the present work, we developed novel SLNs containing Resveratrol is absorption a peanuts non-flavonoid polyphenolic compound skin penetration, controlled release and increased efficiency abundant grapes, and other foods consumed as part In the present work, we developed novel SLNs containing natural seed butter to nanoencapsulate resveratrol towards the Resveratrol is a non-flavonoid polyphenolic compound skin penetration, controlled release and increased efficiency natural seed butter to nanoencapsulate resveratrol the abundant indiet, grapes, peanuts and other foods consumed as part of the antioxidant. For thisrelease purpose, we used towards the butter Resveratrol is a non-flavonoid polyphenolic compound of human thus considered an exogenous antioxidant natural seed butter to nanoencapsulate resveratrol towards the skin penetration, controlled and increased efficiency Resveratrol is a peanuts non-flavonoid polyphenolic compound abundant indiet, grapes, and other foods consumed as part of the antioxidant. Forof this purpose, we used the butter skin penetration, controlled release and increased efficiency of human thus considered an exogenous antioxidant obtained from seeds the fruits of an Amazon tree, abundant in grapes, peanuts and other foods consumed as part (Amri et al., 2012). It is a lipophilic (partition coefficient of skin penetration, controlled release and increased efficiency of the antioxidant. For this purpose, we used the butter abundant in grapes, peanuts and other foods consumed as part obtained of human thus an (partition exogenous antioxidant from seedsForof this theaspurpose, fruits ofweanused Amazon tree, of butter (Amri et al.,diet, 2012). It considered is a lipophilic coefficient of Theobroma grandiflorum, the natural lipidthe of diet, thus considered an exogenous antioxidant 3.40) and unstable that a broad range of of the the antioxidant. antioxidant. the source. butter obtained from seedsForof this theaspurpose, fruits ofweanused Amazon tree, of human human thusItmolecule considered an has exogenous antioxidant (Amri et al.,diet, 2012). is a lipophilic (partition coefficient of Theobroma grandiflorum, the natural lipid source. 3.40) and unstable molecule that has a broad range of obtained from seeds of the fruits of an tree, Resveratrol containing SLN (R-SLN) andAmazon its source. control (Amri et al., 2012). It is aa lipophilic (partition coefficient of applications such as antioxidant, anti-inflammatory and obtained from seeds of the fruits of an Amazon tree, Theobroma grandiflorum, as the natural lipid (Amri et al., 2012). It is lipophilic (partition coefficient of 3.40) and unstable that has a broad rangeand Resveratrol containing SLN (R-SLN) andprepared its source. control applications such activities asmolecule antioxidant, anti-inflammatory Theobroma grandiflorum, as the natural lipid containing no loading (C-SLN) were and 3.40) and unstable molecule that has a broad range of cardio-protective (Abdel-Latif et al., 2015). Theobroma grandiflorum, as (R-SLN) the natural lipid source. Resveratrol containing SLN and its control 3.40) and unstable molecule that has a broad range of applications such as antioxidant, anti-inflammatory and containing no loading (C-SLN) wereandprepared and cardio-protective activities (Abdel-Latif etandal.,loses 2015). Resveratrol containing SLN (R-SLN) its control characterized in terms of mean hydrodynamic size (Z-ave), applications such as antioxidant, anti-inflammatory and However, resveratrol molecule is unstable its Resveratrol no containing SLN (R-SLN) its (Z-ave), control loading (C-SLN) wereandprepared and applications such activities as antioxidant, anti-inflammatory and containing cardio-protective (Abdel-Latif etandal.,loses 2015). in terms of(PdI), mean hydrodynamic size containing no loading (C-SLN) prepared and However, resveratrol molecule is unstable its characterized polydispersity zeta were potential cardio-protective activities al., 2015). antioxidant properties after (Abdel-Latif short light et exposure, thus containing noin index loading (C-SLN) were prepared and characterized terms of(PdI), mean hydrodynamic size(ζ), (Z-ave), cardio-protective activities (Abdel-Latif et al.,loses 2015). However, resveratrol molecule is unstable and its polydispersity index zeta potential (ζ), and characterized in terms of mean hydrodynamic size (Z-ave), antioxidant properties after short light exposure, thus morphology. The drug release kinetics, antioxidant activity, However, resveratrol molecule is unstable and loses its requesting vehicles that could protect the molecule while characterized in terms of mean hydrodynamic size (Z-ave), polydispersity index (PdI), zeta potential (ζ), and However, resveratrol molecule is unstable and losesthus its morphology. antioxidant properties after short light exposure, The drug release kinetics, antioxidant activity, polydispersity index (PdI), zeta potential (ζ), and requesting vehicles that couldetshort protect the molecule while and penetration in human skin were evaluated to predict the antioxidant properties after light exposure, thus increasing the efficacy (Müller al., 2000). polydispersity index (PdI), zeta potential (ζ), and morphology. The drug release kinetics, antioxidant activity, antioxidant vehicles properties after light exposure, while thus and requesting that couldetshort protect the molecule penetration in drug human skin were evaluated to predict the morphology. The release kinetics, antioxidant activity, increasing the efficacy (Müller al., 2000). efficiency and safety of this nanocarrier. requesting vehicles that could protect the molecule while morphology. Thein drug release kinetics, antioxidant activity, and penetration human skin were evaluated to predict the requesting vehicles that could protect the molecule while increasing the efficacy (Müller et al., 2000). efficiency and safety of this nanocarrier. penetration in human skin were evaluated to predict the Due to their unique physicochemical and biological and increasing the efficacy (Müller et al., 2000). and penetration in human skin were evaluated to predict the efficiency and safety of this nanocarrier. increasing the efficacy (Müller etgood al., 2000). Due to their unique physicochemical and biological efficiency and safety of this nanocarrier. properties, like small size and biocompatibility, SLNs Due to their unique physicochemical and biological efficiency and safety of this nanocarrier. properties, like small size physicochemical and good biocompatibility, SLNs Due to their unique and are able protect incorporated drug against degradation and Due to to unique and biological biological properties, like small size physicochemical and good biocompatibility, SLNs 2. MATERIAL AND METHODS are able totheir protect incorporated drugefficiency against degradation and properties, like small size and good biocompatibility, SLNs modify its release rate for improved (Battaglia and 2. MATERIAL AND METHODS properties, like small size and good biocompatibility, SLNs are ableits to release protect rate incorporated drugefficiency against degradation and modify for improved (Battaglia and 2. MATERIAL METHODS are to protect incorporated drug and Gallarate, Wang et al., 2015). materials AND are able ableits to2012; protect incorporated drug against against degradation degradation 2. AND modify release rate for improved (Battaglia and 2.1 Reagents and Gallarate, 2012; Wang et al., 2015). efficiency 2. MATERIAL MATERIAL AND METHODS METHODS modify its release rate for improved efficiency (Battaglia and 2.1 Reagents and materials modify release ratethe for improved Gallarate, 2012; Wang et al., 2015). efficiency Reagents and(ACN) materials In orderitsto produce imperfect solid core,(Battaglia a coupleand of 2.1 Gallarate, 2012; Wang et al., 2015). The acetonitrile used in the preparation of the mobile 2.1 Reagents and materials Gallarate, 2012; Wang et al., 2015). to In order lipids to produce the imperfect solid core,thea SLNs, coupleand of The 2.1 Reagents and(ACN) materials acetonitrile used in the preparation of thecalcium mobile synthetic are normally chosen prepare phase was HPLC grade, and sodium chloride (NaCl), In order to produce the imperfect solid core, a couple of The acetonitrile (ACN) used in the preparation of the mobile synthetic lipids are normally chosen to prepare the SLNs, and phase was HPLC grade, and sodium chloride (NaCl), calcium In order to the imperfect solid core, couple of it includes triglycerides (e.g., tripalmitin), partial The acetonitrile used in the of mobile (CaCl magnesium sulfate (MgSO4),(NaCl), and potassium 2),(ACN) In commonly order lipids to produce produce the imperfect solid core,theaa SLNs, couple of chloride synthetic are normally chosen (e.g., to prepare and The acetonitrile (ACN) in the preparation preparation of the the mobile phase was HPLC grade, used and sodium chloride calcium it commonly includes triglycerides tripalmitin), partial chloride (CaCl ), magnesium sulfate (MgSO ), and potassium synthetic lipids are normally chosen to prepare the SLNs, and 2 4 2 grade, 4 glycosides (e.g., glyceryl monostearate), fatty acids (e.g., phase was HPLC and sodium chloride (NaCl), calcium dihydrogen phosphate (KH PO ) were of analytical grade, all 2 sodium 4 synthetic lipids are normally chosen (e.g., to prepare the SLNs, and chloride it commonly includes triglycerides tripalmitin), partial phase was HPLC grade, and chloride (NaCl), calcium (CaCl ), magnesium sulfate (MgSO ), and potassium 2 4 glycosides (e.g., glyceryl monostearate), fatty acids (e.g., dihydrogen phosphate (KH22PO grade, all it commonly includes triglycerides (e.g., tripalmitin), partial 4 ) were of analytical 4 stearic acid), sterols (e.g., cholesterol) and waxes (e.g., cetyl chloride (CaCl ), magnesium sulfate (MgSO ), and potassium 2 4 from Sigma-Aldrich (USA). Ultrapure water (H O) obtained 2 it commonly(e.g., includes triglycerides (e.g., tripalmitin), partial glycosides glyceryl monostearate), fatty acids (e.g., chloride (CaCl sulfate (MgSO dihydrogen phosphate (KH2PO of analytical grade, all 2), magnesium 4), and potassium 4) were stearic acid), sterols (e.g., cholesterol) and waxes (e.g., cetyl from Sigma-Aldrich glycosides (e.g., glyceryl monostearate), fatty acids (e.g., 2 O) obtained (USA). Ultrapure water (H 2 palmitate) (Dal Pizzol al., 2014).and Fatty are(e.g., the dihydrogen phosphate (KH2PO4) were of analytical grade, all glycosides (e.g., glyceryl monostearate), fattyacids acids stearic acid), sterols (e.g.,et cholesterol) waxes (e.g., cetyl dihydrogen phosphate (KH analytical grade, all from Sigma-Aldrich (USA). Ultrapure water (H O) obtained 2PO 4) were of 2 palmitate) (Dal Pizzol et al., 2014). Fatty acids are the stearic acid), sterols (e.g., cholesterol) and waxes (e.g., cetyl from Sigma-Aldrich (USA). Ultrapure water (H O) obtained 2 stearic acid), sterols (e.g., cholesterol) and waxes (e.g., cetyl palmitate) (Dal Pizzol et al., 2014). Fatty acids are the from Sigma-Aldrich (USA). Ultrapure water (H2O) obtained palmitate) (Dal Pizzol et Fatty acids the Copyright © 2018, 2018 2405-8963 IFAC (International Federation Hosting by Elsevier Ltd. All rights reserved. palmitate)© (Dal IFAC Pizzol et al., al., 2014). 2014). Fattyof Automatic acids are areControl) the 16 Copyright © 2018 IFAC 16 Copyright 2018 responsibility IFAC 16 Control. Peer review©under of International Federation of Automatic Copyright © 2018 IFAC 16 10.1016/j.ifacol.2018.11.600 Copyright © 2018 IFAC 16 Copyright © 2018 IFAC 16

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in a Direct-Q® 3 UV System (Millipore, Germany) (18.2 MΩ cm resistivity at 25 °C and < 10 ppb total organic carbon) was used throughout analysis. Resveratrol 98% was acquired from JiAherb (China). The butter was commercially available in Mercado Ver o Peso in Belem, Para (Brazil). Pluronic ® F127 was purchased from Sigma-Aldrich (USA).

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The encapsulation efficiency (EE) of the R-SLN formulation was determined indirectly by calculating the amount of free resveratrol (i.e., non-encapsulated) present in the aqueous phase of dispersions. The drug loading (DL) capacity was the ratio of incorporated drug to lipid (w/ w). Afterwards, the free resveratrol was separated by filter centrifugation (3500 rpm for 30min), using an Amicon® Ultra Centrifugal filter device (MWCO 10,000, Millipore, Germany). The flowthrough was collected for quantification by HPLC.

2.2 Resveratrol quantification by HPLC Resveratrol quantification was achieved through standard curves prepared immediately before sample analyses. The runs were determined using a high-performance liquid chromatography (HPLC) method. The HPLC system consisted of a Shimadzu SPD-10AV UV/VIS detector. Separation was achieved in an ACE® 5 C18 analytical column (150 x 4.6 mm, 5 µm), at room temperature. The mobile phase was a mixture of acetonitrile and water (55:45, v/ v) with a flow-rate of 1.2 mL min-1, the injection volume of samples was 25 µL and the UV detection was carried out at 307 nm. The retention time of resveratrol was 1.29 min. The limit of quantification of the method was 1.88 µg mL-1. The method was validated in accordance to Food and Drug Administration (FDA) (Food and Drug Administration, 2001) (data not shown).

The EE and DL of resveratrol in SLN (n=3) were calculated according to the following equations (Equation 1 and 2, respectively) (Souto and Müller, 2006):

Where Wtotal, Wfree were the total of weighted drug and total of unentrapped resveratrol in the system and Wlipid was the total amount of excipients added into the system. 2.7 Controlled release kinetics

2.3 Solid lipid nanoparticles(SLN) preparation

In vitro release tests were performed in 7-mL static vertical Franz diffusion cells with a diffusion area of 1.86 cm2 (Microette Plus®, Hanson Research, USA). The receptor medium was composed of an aqueous solution of artificial human sweat which mimics skin conditions, added with 10% ethanol, to ensure sink conditions and guarantee that the receptor media could not act as a barrier to percutaneous absorption as recommended by FDA guideline (Food and Drug Administration, 1997). The donor compartment contained 200 µL of RES or R-SLNs (equivalent to 90 µg of resveratrol), and the receptor compartment was filled with the respective receptor medium. Hydrophilic polysulfone membrane disc filters (Tuffryn®, 25 mm, 0.45 µm, Pall Corporation, USA) were positioned between the cell compartments.

The SLNs were prepared by modified high shear homogenization technique (Neves et al., 2013). Briefly, the natural seed butter (1% w/v) and resveratrol (0.05% w/v) were used as the organic phase and a suspension containing Pluronic® F-127 (0.7% w/v) as the aqueous phase. Both phases were heated 5 – 10 °C above the lipid’s melting point, separately, and the molten lipid was then dispersed in the aqueous phase by high-speed stirring in an Ultra-Turrax (SilentCrusher M, Heidolph, Germany) for 1 min at 12,000 rpm, followed by 10 min of 35% intensity sonication (Vibra Cell, Sonics, USA). The nanodispersions were cooled at room temperature. 2.4 Physicochemical characterization of SLN The SLNs were characterized by their Z-ave, PdI and ζ, using Zetatrac (Microtrac, USA). The Z-ave and PdI were performed using dynamic light scattering (DLS) and were determined as a measure of the width of the particle size distribution. The ζ was determined by measurement of the electrophoretic mobility. Prior to the measurements, all samples were diluted (1:400, v/v) using ultrapure water to yield a suitable scattering intensity. The analyzed samples were obtained by calculating the average of five runs, in triplicate at room temperature.

The R-SLNs were then carefully applied to achieve complete uniform coverage, with the compartments held together using a clamp. Stirring rate and temperature were kept at 600 rpm and 32 ± 2 ºC, respectively. Aliquots (1 mL) were withdrawn at regular time intervals (0.5 – 24 hours), collected into HPLC vials, and immediately replaced with the receptor medium at the same temperature. The resveratrol concentrations were correspondingly corrected for the replenishments. Withdrawn samples were submitted to HPLC analysis, as described in topic 2.2.2. Data were expressed as the cumulative amount of resveratrol permeated through the membrane, considering the total amount of drug applied.

2.5 Transmission Electron Microscopy (TEM) The SLNs dispersions were set on a copper metal substrate coated with carbon (CF 200-Cu, 300 square mesh cupper, EMS, USA). The material was dried with nitrogen flow and left at room temperature for 12h. The particles were visualized at 100,000 times magnification in a JEM-1011 TEM model (JEOL, USA).

The percentage of diffused drug release (Qt) in the time t was calculated using Equation 3:

where Cmeasured,t is the concentration measured at time t, Vr is the volume of diffusion cell, Va is the aliquot volume, n

2.6 Encapsulation efficiency and drug loading 17

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3M, Brazil) (Andrade et al., 2014). The RS was cut into small pieces. Tape strips with SC and RS were placed in individual tubes and immersed in 5 mL of mobile phase, homogenized with a vortex mixer for 2 min and left in ultrasound bath for 20 min at room temperature. The tubes containing SC and RS were then filtered and analyzed by HPLC.

is the number of the sampling at time t, and Ca is the concentration of the aliquot. Mathematical models were applied to determine the diffusion kinetics: cumulative percentage of drug diffusion was plotted against time (h) and the square root of time (√h) for zeroorder kinetics and Higuchi model, respectively; log of the cumulative percentage of drug diffusion was plotted against time (h) for first-order kinetics. The coefficient of determination (R2) was calculated, and those with value > 0.99 were considered linear. Steady-state diffusion flux (Js) was then determined from the linear slope of the cumulative percentage of resveratrol versus time curves. Here lag time represented the time required to achieve a steady-state flux.

2.10 Statystical analysis All data are reported as mean ± standard error of media (SEM) of at least three replicates/ treatment. For SLN characterization, statistical significance was determined by Student’s t-test. All other experiments were analyzed by Analysis of Variance (ANOVA), and Tukey’s test was used as post hoc pairwise comparisons between treatment groups and control, using the software PRISM 5.0 (GraphPad, USA). Statistical significance is represented as * for p < 0.05.

2.8 Antioxidant activity The scavenging activity of RE, R-SLN and C-SLN was measured according to the 1,1-diphenyl-2-picrylhydrazil free radical (DPPH) method, as described previously (Qin et al., 2014) with minor modification. Briefly, the samples (100 µL) of different antioxidant concentrations (10 to 100 µM resveratrol equivalents) were mixed with 100 µL of 100 µM DPPH that was dissolved in 95% ethanol. The mixture was then shaken vigorously and kept for 30 min in the dark at room temperature. The absorbance of the resulting solution was recorded at 517 nm. The scavenging activity was calculated using the following equation 4:

3. RESULTS AND DISCUSSION 3.1 SLN characterization The DLS measurements presented in Table 1 indicates that the method used for nanoparticle assembling was successful. An illustration of the SLN is shown in Figure 1 A. C-SLN exhibited mono dispersed characteristic with a Z-ave and PdI of 201.81 ± 9.43 nm and 0.26 ± 0.11, respectively. The ζ revealed a negative surface charge equal to -17.21 ± 1.54 mV. On the basis of R-SLN, resveratrol encapsulation did not affect SLNs characteristics (p > 0.05), since it exhibits homogenous size distribution with particle diameter equal to 195.30 nm ± 12.19 nm and PdI of 0.16 ± 0.09 while the ζ revealed negative surface charge (-19.54 ± 1.89 mV). The values of ζ and PdI also indicate the presence of stable and homogenous dispersions. When lipid nanoparticles exhibit a particle size equal or less than 200 nm, they can be able to form a hydrophobic monolayer on the skin, which leads to the hydration of this tissue and enhance drug penetration (Schäfer-Korting et al., 2007).

where A DPPH sample is the value for the sample solution mixed with the DPPH solution; A Sample control is the value for the sample solution mixed with 95% ethanol; and DPPH blank is the value for the 95% ethanol mixed with the DPPH solution. The results are expressed as the ED50 (50% effective dose), given as amount (µM) of antioxidant required to consume half of the free radicals present (µM DPPH). 2.9 Human skin permeation Excised human skin samples were obtained from abdominoplasty surgery from 1 patient (female with 37 years old). The excess of adipose layer was sectioned off from the received tissue with surgical scissors. The skin was then wrapped in aluminum foil and stored at - 80 ºC until use. This protocol followed The Code of Ethics of the World Medical Association (Declaration of Helsinki) and was approved by the Ethics Committee of Federal University of Juiz de Fora (approbation no. 151.275).

Table 1. Physicochemical characterization of blank solid lipid nanoparticles (C-SLN) and RES-loaded solid lipid nanoparticles (R-SLN). C-SLN R-SLN Z-ave (nm) 201.81 ± 5.44 195.30 ± 7.03 PdI 0.26 ± 0.06 0.16 ± 0.05 ζ (mV) -17.21 ± 0.89 -19.54 ± 1.09 Notes: All values represent the mean ± SEM (n=3). No statistically significant differences were observed between any of nanoparticle formulation. Mean particle size (Z-ave), polydispersity index (PdI), and zeta potential (ζ).

The percutaneous absorption was carried out as described in controlled release kinetics (Topic 2.4), with a few modifications. The excised human skin was used in substitution of the polysulfone membrane, while the time intervals for collecting the samples ranged from 0.5 to 12h. An aliquot of 200 µL of RES or R-SLNs was applied on the diffusional surface of the skin in the donor compartment.

The morphology of the SLNs determined by TEM is shown in Figure 1 B and C. The nanoparticles were revealed to have a relatively homogenous spherical shape, while the average size ranged from 150 nm to 200 nm. These results are in accordance to the size found in DLS measurements.

Afterwards, the skin was removed from the diffusion cell and stratum corneum (SC) was separated from the remaining skin (RS) by applying 15 tape strips with adhesive tapes (Durex ®, 18

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treatments with the same concentration. Values represent means ± SEM (n=3). A dose-response curve was obtained for both R-SLN and resveratrol using the DPPH method, and it shows a significant antioxidant activity in a dose-dependent manner. The ED50 value obtained for R-SLN was 0.41 ± 0.03 (µM resveratrol/ µM DPPH), which was lower and statistically different from the value of 0.64 ± 0.05 (µM resveratrol/ µM DPPH) obtained for RES. Thus, it takes almost 40% less resveratrol when it is nanoencapsulated to obtain the same antioxidant effect of an ethanolic solution. This may occur due to a synergistic antioxidant effect of the resveratrol along with SLN, whose composition includes lipids that exert mild antioxidant effect (Cho et al., 2010).

Figure 1. Schematic illustrative nanoparticle and components are shown in Figure A. Representative transmission electron microscopy images of blank-solid lipid nanoparticle (C-SLN) (Figure B) and resveratrol-loaded solid lipid nanoparticles (R-SLN) (Figure C).

3.4 Controlled release kinetics The in vitro release kinetics of resveratrol from RES and RSLNs was investigated for 24h. As shown in Figure 3, RES diffused freely and crossed the polysulfone membrane rapidly. Almost all RES crossed the membrane within 5-10h, whereas R-SLN presented a slow and sustained release of resveratrol.

3.2 Encapsulation efficiency and drug loading Drug encapsulation can be evaluated through two main parameters, drug loading (DL) and encapsulation efficiency (EE), where the former is the ratio between drug and lipid in nanoparticles and the latter is the percentage of drug incorporated into the lipid nanoparticles relative to the total drug added (Battaglia, Gallarate, 2012). The DL and EE were calculated as 3.36 ± 0.11% and 74.12 ± 2.17%, respectively. Since resveratrol has a lipophilic nature, it is suggested that its partition into the lipid matrix is preferred than remaining in the aqueous media. Thus, a high percentage of DL and EE was found in this study, confirming that the drug was well dispersed within the lipid matrix. 3.3 Antioxidant activity Figure 2 shows the DPPH radical scavenging capacity of RSLN compared to RES and C-SLN. R-SLN showed a higher and statistically significant antioxidant activity when compared to all the other treatments. At the concentration of 100 µM, R-SLN presented DPPH radical scavenging activity of 85.70 ± 2.15% compared to RES (68.38 ± 1.13%) and to C-SLN (19.62 ± 0.77%), at the same dilution.

Figure 3. In vitro resveratrol release profile from resveratrol ethanolic solution (RES) or resveratrol-loaded solid lipid nanoparticle (R-SLN). Drug release study was performed at 32 ± 2 °C under stirring (600 rpm) using Franz Diffusion cells containing artificial human sweat + ethanol 10% (v/ v) as receptor media, to ensure sink conditions. Values represent means ± SEM (n=6). The release profile of entrapped resveratrol from R-SLN indicated a biphasic pattern with a burst release phase at the first 4h with a mean of 34.66 ± 5.82% resveratrol released followed by a sustained release over 24h, reaching 80.48 ± 12.20% resveratrol. The initial fast drug release during the first hours could be ascribed to those drugs located on or near the surface of nanoparticles. Besides, the surfactant present in the aqueous phase of the dispersion enhances the solubility of resveratrol and thus enables its faster releasing (Nie et al., 2011). Despite its predominant lipophilic behavior, resveratrol has three hydroxyl groups with a tendency to localize at the interface in the hydrophilic area. Consequently, the physical-chemical natures of SLN and resveratrol both favor localization of resveratrol near the SLN shell, enabling ready release from the nanoparticles (Teskac,

Figure 2. DPPH radical scavenging activity of resveratrolloaded solid lipid nanoparticle (R-SLN), resveratrol ethanolic solution (RES), and blank solid lipid nanoparticle (C-SLN). *All entries showed significant difference (p < 0.05) among

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Kristl, 2010). The slow and sustained release could be attributed to the diffusion of drug molecules through the lipid matrix of SLNs. From a therapeutic standpoint, a burst release profile can be considered advantageous, as sufficient amount of resveratrol is rapidly released from the R-SLN to the skin to exert an initial therapeutic effect. In addition, a subsequent sustained release of the remaining resveratrol from the R-SLN is beneficial in order to keep the therapeutic dosage without the need of repeated application (Bunjes, 2011).

favors its accumulation in the skin (Cevc, 2004; Kyadarkunte et al., 2014). Interestingly, the highest amount of resveratrol in SC may be related to the initial burst release of R-SLN, found in the first 5h (Figure 3). Although the calculated release flux showed 3.88 µg cm-2 h-1 (Table 4), after 12h it was possible to observe the retention of 13.97 µg cm-2 of the active in the skin. According to the results presented in Figure 4, around 60% of the resveratrol is still to be released after 12h, which may account for the overall difference between calculated release flux and amount retained in skin.

The prolonged release of resveratrol was well fitted to Higuchi’s square root model (y = 3.8842 – 0.2732, R2 = 0.9903), as has been reported for drug-loaded SLN systems (Kashanian et al., 2011). The kinetic evaluation showed a sustained release trend, with a flux of 3.88 µg cm-2 h-1, and a lag time of 0.29 min. Linear fits were obtained, indicating that the release profile of resveratrol from the lipid core is diffusion-controlled, which describes a passive release process from vehicle to receptor media proportional to √t (Prausnitz et al., 2004).

On the other hand, there was a difference between the concentration of resveratrol in R-SLN and RES in the skin, suggesting that either RES could not penetrate efficiently in the skin or it has permeated straight away throughout the skin. To elucidate this difference, the concentration of resveratrol in the flow-through solution was submitted to quantification. However, it was found that the amount of active that passed through the skin and reached the receptor media did not reach the limit of quantification for both RES and R-SLN, indicating negligible permeated amount for both treatments (data not shown). This is of particular interest in the dermatological products field, since it is not desirable for topically administered nanocarriers to permeate deeply, reach the blood circulation and cause systemic effects. Thus the result indicates the safety of developed SLN in this regard, while suggesting that the RES was ineffective in penetrating the skin compared to R-SLN.

3.5 Human skin permeation In our current investigation, excised human skin uptake of resveratrol, after delivery from R-SLN to the skin layers (SC and RS), was measured in vitro and the results are presented in Figure 4.

4. CONCLUSION In the present research, SLNs containing T. grandiflorum natural butter as the lipid core were constructed for the delivery of resveratrol to the skin. Through the application of this system, we were able to control the release of the active, while enhancing the antioxidant activity. The balanced lipid constitution of the natural seed butter allowed improved permeation and retention of the nanoparticles and active in the upper layers of human skin. These results demonstrated the great potential of the SLN prepared with natural seed butter to be used in dermatological formulations towards the controlled delivery of hydrophobic substances to the skin.

Figure 4. Human skin uptake of resveratrol after 12h of permeation experiments in Franz diffusion cells. Fullthickness abdominoplasty skin model; receptor media comprised of artificial human sweat + ethanol 10% (v/ v) to ensure sink conditions. Values represent mean ± SEM (n=6), * p < 0.05.

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