polymers Article Ultrasound-Assisted Encapsulation of Sacha Inchi (Plukenetia volubilis Linneo.) Oil in Alginate-Chitosan Nanoparticles Mariela Elgegren 1, Suyeon Kim 2,*, Diego Cordova 1, Carla Silva 3, Jennifer Noro 3, Artur Cavaco-Paulo 3 and Javier Nakamatsu 1 1 Department of Science, Chemistry Division, Pontificia Universidad Católica del Perú PUCP, Av. Universitaria 1801, Lima 32, Peru 2 Department of Engineering, Pontificia Universidad Católica del Perú PUCP, Av. Universitaria 1801, Lima 32, Peru 3 Centre of Biological Engineering, University of Minho, Campus De Gualtar, 4710-057 Braga, Portugal * Correspondence: skim@pucp.pe; Tel.: +511-626-2000  Received: 24 June 2019; Accepted: 18 July 2019; Published: 27 July 2019  Abstract: Sacha inchi oil is rich in essential and non-essential fatty acids and other types of bioactive agents like tocopherols and polyphenolic compounds, which are very well-known antioxidants. In this study, the encapsulation of sacha inchi oil in alginate (AL) and chitosan (CS) nanoparticles was achieved with the assistance of high-intensity ultrasound. Nanoemulsion is the most effective delivery and high stability system for lipophilic bioactive agents. Chitosan and surfactant concentrations were varied to study their effect on particle formulations. Size, zeta-potential, polydispersity, and stability of particles were determined in time to optimize the preparation conditions. Sacha inchi oil encapsulated in AL-CS nanoparticles showed a higher loading efficiency and stability for short and long periods compared with other vegetable oils such as olive and soybean. Also, because of the types of tocopherols present in sacha inchi oil (γ- and δ-tocopherols), a much higher antioxidant activity (95% of radical reduction in 15 min) was found in comparison with nanocapsules with olive oil, which contain α-tocopherols. The particles showed high efficiency of protein loading at high concentration of bovine serum albumin (BSA) and a low rate of leaching profiles in various testing media like simulated gastric and intestinal fluids with/without enzymes, that is, pepsin 0.1% (w/v) and pancreatin 0.1% (w/v), respectively. Keywords: sacha inchi; nanoemulsion; tocopherols; antioxidant; Nile red; protein loading 1. Introduction Sacha inchi (Plukenetia volubilis L.), also known as Inca peanut, is a native plant found in the Amazon rainforest of Peru, Ecuador, Brazil and other parts of South America [1–5]. The seeds of sacha inchi are characterized as rich in oil and proteins with aminoacids such as cysteine, tyrosine, threonine, and tryptophan [1,2,6]. Sacha inchi oil contains important macronutrients; bioactive compounds; heat-labile substances; and an unusually high content of essential fatty acids, that is, C18:3 omega-3 (α-Ln, cis, cis,cis-9,12,15-Octadecatrienoic acid; α-linolenic) and C18:2 omega-6 (L, cis,cis-9,12-octadecadienoic acid; α-linoleic) fatty acids, representing about 82% of the total oil content [5,6]. Besides the essential fatty acids, tocopherols, carotenes, phytosterols, and polyphenols are also present [5–9], these compounds are responsible for the health benefits and bioactivities attributed to sacha inchi. The beneficial effects of essential fatty acids on human health have been evaluated for treating diseases like arthritis, cancer, coronary heart disease, diabetes, hypertension, and inflammatory skin diseases [10–12]. Tocopherols and polyphenols are representative antioxidant agents commonly Polymers 2019, 11, 1245; doi:10.3390/polym11081245 www.mdpi.com/journal/polymers Polymers 2019, 11, 1245 2 of 18 found in natural sources [11,12]. In the medical and pharmaceutical field, antioxidant activity, together with antimicrobial activity, is one of the most searched for bioactivities. Reactive oxygen species (ROS) are generally produced in the organism cells by oxidative stress and are closely associated with several diseases [13]. When ROS are formed during the pathogenesis of wounds and injuries, undesirable oxidative damage to proteins, nucleic acid, and lipids, as well as depletion of mitochondrial DNA from human skin, can occur [14]. Antioxidants prevent the damage caused by ROS through radical scavenging or prevention of the generation of these reactive species by iron binding [14], and thus they are important substances to protect the organism from the damage caused by the oxidative stress. Considering the nutrient composition of sacha inchi oil, it is an optimal candidate to become a natural antioxidant agent for medical and pharmaceutical applications. High-intensity ultrasound has been extensively used over many years to synthesize nanostructured materials. Ultrasonic waves can produce high-energy chemical reactions in liquids in a brief time through acoustic cavitation, which provides a unique interaction of energy and matter [15,16]. Among many applications, the formulation of nanoemulsions (w/o or o/w) has been successfully carried out using ultrasound resulting in low polydispersity (homogeneous), size reduction, high stability, and no aggregation of particles [16–18]. In the present work, we employed ultrasound to form oil-in-water (o/w) nanoemulsions using sodium alginate (AL) and chitosan (CS) polymers to achieve stable nanostructured vehicles for medical and food applications. The sacha inchi essential oil was encapsulated to improve the bioactivity of AL-CS nanoparticles like antioxidant activity. Herein, we explore the encapsulation of other types of vegetable oils like soybean and olive oils, and their effects on the formulation and product properties of AL-CS nanoemulsions were studied and compared to sacha inchi oil. Emulsification is a very suitable system for encapsulating and delivering lipophilic components in aqueous media [19–22]. Among many polymeric nanocarriers for drug delivery system, CS and AL are the most broadly studied because of their biocompatibility, antimicrobial activity, non-toxicity, biodegradability, mucoadhesiveness, and longer in vivo circulation time, among others [21,22]. AL and CS are obtained from naturally occurring polysaccharides that form polyelectrolytes of opposite charges in solution. AL forms an anionic chain owing to the carboxylate groups, while CS forms a positively charged polymer chain owing to its protonated amine groups. Together, they can form stable polyelectrolyte complexes. Several factors must be considered for AL-CS polyelectrolyte complex formation, such as pH of the solution, molecular weight of polymers, and the ratio of CS to AL [22]. The effect of the type of oil and the chitosan and surfactant concentrations on the particle´s size, polydispersity, zeta-potential, and stability were evaluated. Encapsulation of sacha inchi in AL-CS nanoparticles was verified using fluorescence microscopy and Fourier-transformed infrared attenuated total reflectance spectroscopy (FTIR-ATR). Antioxidant activity of AL-CS nanoemulsions containing sacha inchi oil was also evaluated by measuring its free radical scavenging capacity. 2. Experimental 2.1. Materials Commercial chitosan (CS) and sodium alginate (AL) obtained from Sigma-Aldrich (St. Louis, MO and Milwaukee, WI, USA, respectively), were used for the formulation of nanoparticles. Three different commercial vegetable oils were evaluated for encapsulation: sacha-inchi, olive, and soybean oils; all were produced in Peru and purchased from local suppliers (Inca-inchi, El Olivar, and Primor, respectively. Lima, Peru). Non-ionic surfactant poloxamer 407 (pluronic f127), lyophilized bovine serum albumin (BSA) with molecular weight 66 kDa, Nile red (Nile blue oxazone) dye, and other chemicals were purchased from Sigma-Aldrich and used without any further treatments. Polymers 2019, 11, 1245 3 of 18 2.2. Preparation of AL-CS Particles Sacha inchi oil loaded AL-CS nanoemulsions were prepared according to methods reported by Polymers 2019, 11, x FOR PEER REVIEW 3 of 18 Lertsutthiwong et al (2009) and Natrajan et al. (2015), with some modifications [21,22]. TTheh eAAL-LC-CS Spapratritcilcelse swwereer eprperpeparaerded inin thtrheree estsetpeps:s (:i)( io)/ow/w nannaoneomemuluslisoino nfofromrmataiotino nbyb yulutrltarsaosuonudn d ofo af ammixitxutruer eofo afna nAAL Laqauqeuoeuosu ssosloultuiotino,n s,usrufrafcatcatnatn, ta,nadn dthteh eorogragnainci cphpahsaes e(o(iol)i,l )(,ii()i ii)oinoontortorpoipci cgeglealtaiotino n ofo Af AL Lwwithit hcaclcailucimum chclohrliodreid beyb sytirsrtiinrrgin fogrf 3o0r m30inm, iann,da n(idii)( CiiiS) cCoSatcionagt oinng AoLn pAaLrtipcalersti bclye sstbiryrisntgir froinr g 30fo mr i3n0. Imn itnh.e Ifnirstth setefipr s(tpastretipcl(ep faorrtmicaletiofonr)m, haitgiohn-i)n,thenigshit-yin utletnrassitoyniuclattriaosno nwiacas taiopnplwiedas toa pthpeli medixttourteh e fomr i3x mtuirne wfoirth3 tmeminpweriathtutreem cponertarotullreedc wonitthro alnle idcew biaththa ◦ (n7–ic1e0 b°aCt)h. T(7h–e1 0ultCra)s.oTnhiec purltorbaes ownaics pcarorebfeuwllya s pocasriteifounlelyd paots itthioe naeqduaetotuhse saoqluuetioouns asnodlu toioiln inantedrfoaiclei.n Ate r0fa.3c eg. ·LA−10 .A3Lg ·L −1 soluAtioLns owluatsi opnrewpaasrepdre wpaitrhed dwistiitlhleddi swtiallteedr awndat tehrea pnHd twhaesp aHdjwusatseda dtoju 5s–t5e.d5.t CoS5 –w5a.5s. dCisSsowlvaesdd iinss 1o%lv aecdeitnic 1a%cida caeqtuiceoaucisd saoqluuteioonu s ansodl uptiHon wanasd padHjuwstaesda dtoju s4t.e5d–5t ou4s.i5n–g5 uNsainOgHN asOolHutsioonlu. tiTohne. Tehxepeerxipmeerinmtaeln tparlopcreodcuerdeu ries ipsrperseesnetnetde d scshcehmemataitciaclalyll yinin FiFgiugurer e1.1 . FiFgiugruer e1.1 .ExEpxerpiemriemnteanl taplropcreodcuedreu rfeorf otrheth eforfomrumlautliaotnio nof ofsosdoiduimum alagligniantae te(A(AL)L-c)-hcihtoitsoasna n(C(SC)S ) nannaoneomemuluslisoino nenecnacpaspuslualtaintign gvevgeegteatbalbel eoiolsil.s . InI nthtihsi sstsutduyd,y w, we eeveavlauluataetded vavrairoiuous sfafcatcotrosr sthtahta ctocuoludl dafafeffcetc tthteh eprporpoepretrietise os fo tfhteh Ae AL-LC-SC Spapratirctlieclse, s, amamonogn gthtehmem, t,hteh etytpyep eofo foioli lanandd ththe ecoconncecnentrtartaitoinosn sofo fCCS Sanandd susurfrafcatcatnant.t F. oFro reaecahc hcacsaes,e t,hteh eoiol iwl wasa s adadddeded inin ththe efifirsrts tsstetepp oof fththee pprroocceedduurree aanndd tthhee vvoolluummee rraattiioo ooff AALL ssoolluuttiioonn ttoo ooiillw waassk keeppttc coonnsstatannt ta t at9 9t oto1 1. . CChehmemiciacla sl tsrturcutcutruerse sofo fAALL anandd CCS Swwereer echcharaarcatcetreirziezded bby yPProrototonn nnuuclcelaera1 rmmagagnneteitci crerseosonnanancec e (1(HH-N-NMMRR) w) witihth a aBBrurukkeer rAAsscceenndd 550000 MMHHzz ssppeeccttrroommeetteerr ((BBiilllleerriiccaa,, MMAA,, UUSSAA)).. TThheem mooleleccuulalarrw weeigighht to f ofc hchitiotsoasnanw wasads edteetremrminiendedby bgye gl eple prmeremateiaotniocnh rcohmroamtoagtroagprhayph(GyP (CG)PfCro) mfroVmis cVoitsecko(tHeko u(Hstoouns, tToXn,, UTXSA, ) UwSAith) awriethfr aac trievferainctdivexe dinedteecxt odreatnedctopru lalunlda npsutlalnudlaanr dsst.anTdhaermdso.l eTchuela mr woleeicgh 2ultao 2r fwAeLigwhat sodf eAteLrm winaes d − dbetyercmapiinlleadr ybvyi sccaopmilelatrryy wviisthcoamneUtrbyb ewloithhd aen1 CUvbibsecloomhdetee r1(CK v=is0c.o0m28e7t1erm (mK =·s 0.0)2fr8o7m1 mTemch2·nsi−c2)a lfrGolmas s TPecrhondiuccatls G(Slat.ssD Porvoedr,uNctJs, (USSt.A D)o. ver, NJ, USA). AAftfetre rththe efoformrmaatitoionn oof fththee AALL--CCSS nnaannooeemmuullssiioonnss uunnddeerr ddiiffffeerreenntt ccoonnddiittiioonnss,,t thheec chhaarraaccteterrizizaatitoionno f oft htehep apratirctliecslews awsacas rcraierdrieodu tomuet amsueraisnugrtihnegi rthaveierr aagveersaizgee, psiozley,d pisopleyrdsiistypeinrsdietyx (iPnDdeI)x, a(PndDzI)e,t aa-npdo tzeenttai-al. po2.t2e.n1t.iaTly. pes of Oil 2.2.1. TTyhpreees odfi ffOeirle nt edible oils were evaluated: sacha inchi, olive, and soybean oils. All the oil samples were produced by Peruvian companies (Lima, Peru) and used as they were obtained from the local marTkherte. eT hdeifcfoernecnetn terdaitbiolen sooilfsA wLearne deCvaSlusoaltuedti:o nsascwhear ein0c.h3ig, oLl−iv1 ea,n adn2d.0 sgoyb−e1an oils. All the oil · ·L , respectively. samples were produced by Peruvian companies (Lima, Peru) and used as they were obtained from th2e.2 l.o2c.aCl omnacreknettr.a Ttihoen coofnCceSntrations of AL and CS solutions were 0.3 g·L−1 and 2.0 g·L−1, respectively. 2.2.2. CTohneceeffnetrcattoiofnth oef cCoSn centration of CS in solution on the particle property was studied. Chitosan was dissolved in a 1% (v/v) acetic acid solution and the pH was adjusted to 5 using NaOH solution. DiffTehreen etfcfoecntc oenf ttrhaet icoonnscoefnCtrSatiinons oolfu CtioSn inw seorleuutisoend ,onna tmhee lpy,a0rt,i0cl.3e ,p0r.6o,p1e.r5t,ya wndas2 .s0tugdLie−d1· .. Chitosan was dissolved in a 1% (v/v) acetic acid solution and the pH was adjusted to 5 using NaOH solution. Different concentrations of CS in solution were used, namely, 0, 0.3, 0.6, 1.5, and 2.0 g·L−1. Polymers 2019, 11, 1245 4 of 18 2.2.3. Concentration of Surfactant The surfactant plays a very important role in the formation of emulsion particles. Poloxamer 407 was added to the AL aqueous solution so that concentrations of 0.1, 0.2, 0.3, 0.5, and 1% (w/v) were obtained. 2.3. Emulsion Stability (Phase Separation) AL-CS nanoemulsions prepared with the different oils and different concentration of surfactant were stored for six months, and their physical stability was evaluated by observing if phase separation occurred (creaming phenomenon). Creaming index was obtained from the ratio between the height of the cream layer (Hc) and the total height of cream and serum layers (Hs + Hc = He, the total height of emulsion) according to Equation (1). Hc and Hs values were measured directly from the vials without shaking them. Hc Creaming index (CI)(%) = × 100 (1) Hs + Hc 2.4. Size, PDI, and Zeta-Potential A Zetasizer Nano ZS (Malvern Instruments Inc., Worcester, UK) was used to obtain the values of average size, PDI, and zeta potential. Samples were diluted with ultrapure water before measurements in a ratio of 95:5 (water/sample). All measurements were performed with at least three samples. 2.5. Fourier-Transform Infrared Attenuated Total Reflectance (FTIR-ATR) Spectroscopy Analysis The encapsulation of sacha inchi oil in the AL-CS nanoemulsion was verified by FTIR-ATR spectroscopy using an FTIR/NIR (near infrared) frontier spectrophotometer (Perkin Elmer, Waltham, MA, USA). FTIR analysis is a simple and fast technique to determine the characteristic functional groups of samples. Besides that, it is not necessary to use any type of solvent to prepare samples for analysis. In the analysis of vegetable oils, a few drops of each oil sample were positioned in contact with ATR on a multi-bounce plate of crystal at controlled ambient temperature (25 ◦C). The lyophilized AL-CS nanoemulsion samples were also analyzed in the same manner. The ATR crystal was carefully and thoroughly cleaned using pure acetone after each run. IR spectra were recorded in the wavelength range of 4000 to 400 cm−1. The background spectrum of the empty ATR crystal was collected before proceeding to scan the sample. 2.6. Oil Encapsulation Efficiency Test Using Nile Red Dye The oil was stained with Nile red, a lipophilic dye, before the preparation of AL-CS nanoemulsions. Then, 30 mL of AL-CS nanoemulsions prepared with stained vegetable oils were lyophilized. After lyophilization, the free unencapsulated oil was separated using ethanol. The lyophilized AL-CS nanoemulsions prepared with stained oil were washed with 20 mL of ethanol with stirring in a vortex for 1 min and then centrifuged. The supernatant ethanolic solution was used to determine the efficiency of oil encapsulation in the AL-CS nanoemulsions using UV-visible spectrophotometry at 553 nm, with maximum absorbance of Nile red dye. Dye concentration (DC) in the alcoholic solution was determined from a calibration curve at the same wavelength and was used to calculate the oil content in AL-CS nanoemulsion samples. The encapsulation efficiency was obtained from Equation (2). All measurements were performed using at least triplicate samples and the average value was determined. DC Encapsulation efficiency (EE) (%) = initial DC −DCFree DC × 100 (2) DCinitial 2.7. Evaluation of Antioxidant Activity The antioxidant activity was evaluated according to the 2,2’-azino-bis(3-ethylbenzothiazoline- 6-sulphonic acid (ABTS) radical cation decoloration assay [23,24]. ABTS•+ free radical cation was Polymers 2019, 11, 1245 5 of 18 obtained by the reaction between 7 mM ABTS solution and 2.45 mM potassium persulfate. The solution was stored for 12 h in the dark, at room temperature before use. A total of 1 mL of the AL-CS emulsion was lyophilized before antioxidant evaluation. The ABTS•+ free radical solution was diluted with ethanol to reach an absorbance of 0.700 ± 0.025 at 734 nm and the absorbance of 3 mL of this solution was set as a control (Acontrol). The lyophilized sample was added to 3 mL of the diluted ABTS•+ free radical solution and the absorbance was measured during one hour (Asample). All measurements were performed using at least triplicate samples and the sample’s free radical scavenging capacity was calculated with the following Equation (3): Acontrol −Asample Radical scavenging effect (%) = × 100 (3) Acontrol 2.8. Protein Loading Efficiency BSA was studied as a model protein molecule to evaluate the loading efficiency and releasing behaviors of AL-CS particles. Various concentrations of 0.01, 0.03, 0.1, 0.5, 0.8, and 1.0 g −1 ·L . BSA were dissolved in the solutions containing 0.3 g L−1 · of AL and 0.5% (w/v) of surfactant. After adding BSA, the mixture was subjected to ultrasonication, ionotropic gelation, and CS coating. The BSA loading capacity of AL-CS particles was quantitatively evaluated by measuring the unloaded free BSA after emulsion formation. The separation of unloaded BSA from emulsion was acquired using a Vivaspin 20 filters (cut-off 100 kDa) by centrifugation (Beckman Allegra X155, Indianapolis, IN, USA) at 3000 rpm for 30 min at 25 ◦C. The BioRed DC protein assay was used to measure the concentration of protein in the filtered solution separated from particles. The loading efficiency was obtained using the same Equation (2) from the oil encapsulation efficiency calculation. The BSA loaded AL-CS nanoemulsions were further tested. All measurements were performed using at least triplicate samples and the average value was determined. 2.9. In Vitro Release Behavior The release response of BSA loaded AL-CS nanoemulsions was studied with various testing medium, that is, simulated gastric and intestinal fluids with/without enzymes, that is, pepsin 0.1% (w/v) and pancreatin 0.1% (w/v), respectively, were prepared as described according to the United States Pharmacopeia (USP) guidelines [25]. A total of 5.05 mL of the AL-CS nanoemulsions was taken and mixed with the same volume of testing medium and incubated at the physiological temperature of 37 ◦C under gentle agitation [26]. After a certain time, like 1, 4, 8, 24, and 72 h, 10 mL of incubated samples was taken and placed in a Vivaspin 20 (cut-off 100 kDa), and then centrifuged at 3000 rpm for 30 min to separate the released BSA from the nanoemulsion [27]. The concentration of released BSA from particles was measured using the BioRed DC protein assay. 3. Results and Discussion As an ecological, environmentally benign, non-toxic, and cost-effective method for the synthesis of nanoparticles, Kumar and his colleagues have studied the potential use of the leaf extract or seed oil of sacha inchi to produce silver or gold nanoparticles, and nanocatalyst [1–4]. They used sacha inchi extracts or oils as a reducing agent for silver or gold compounds and obtained small size particles (<100 nm). In the present study, we applied sacha inchi oil to obtain bioactive nanoemulsions formulated with two natural polyelectrolytes, AL and CS, and the effects on the particle properties were compared with olive and soybean oils. 1H-NMR analysis of CS was obtained at 70 ◦C in DCl/D2O, with a Bruker Ascend 500 MHz spectrometer, with an AvanceIII HD console (Bruker, Billerica, MA, USA). A degree of deacetylation of 80.59% was calculated from the spectrum (Figure 2) based on the peaks for hydrogens in C1 (H1A, δ 4.54–4.58 ppm) and the acetyl group (H7A, δ 2.01 ppm) of the acetylated units and hydrogens in C1 (H1D, δ 4.84–4.86 ppm) and C2 (H2D, δ 3.15–3.17 ppm) of the deacetylated units [28]. A molecular Polymers 2019, 11, x FOR PEER REVIEW 6 of 18 Pδol y4m.5er4s–240.1598, 1p1,p1m24)5 and the acetyl group (H7A, δ 2.01 ppm) of the acetylated units and hydrogens6 ionf 1C81 (H1D, δ 4.84–4.86 ppm) and C2 (H2D, δ 3.15–3.17 ppm) of the deacetylated units [28]. A molecular wweeigighht t( w(weeigighhtta avveeraraggee))o off8 8866k kDDaaw waassd deetetermrminineeddf oforrC CSSw witihtha aM MaalvlveernrnV VisicsocotetekkG GPPCCs ysystsetemm wwitihtha anna aqquueeoouussN NoovveemmaaM Maaxxa annaalylytitciacal ll ilnineeaarrX XLLc ocolulummnna anndda ar erferfaractcitviveei ninddexexd deteetcetcotor.r.P Puulllululalann sstatannddaarrddss( P(PSSSP PoolylymmeerrS StatannddaarrddssS SeerrvviciceeG GmmbbHH,,1 100––22000000 kkDDaa)) wweerreeu usseedda assa ar erefefererenncceea anndda ann HHOOAAc/cN/NaaOOAAccb buufffefrer( p(pHH= =4 4.5.5))w waassu usseedda assm moobbilielep phhaasese[ 2[299].]. Figure 2. Proton nuclear magnetic resonance (1H–NMR) spectrum of chitosan (CS) solution in DCl/D2O, Figure 2. Proton nuclear magnetic resonance (1H–NMR) spectrum of chitosan (CS) solution in at 70 ◦C (500 MHz). DCl/D2O, at 70 °C (500 MHz). For AL, 1H-NMR spectroscopy was used to determine a ratio of 1.57 mannuronic/guluronic units.FTohri sAcLa,l c1uHla-tNioMn Ris mspoercetrcoosmcoppleyx w[3a0s] uanseddi stod edtaeitleerdmiinnea par eravtiioou sofw 1o.5rk7, minawnnhuicrhonthice/gchuelumriocnailc sturnuicttsu. rTehoisf cthalicsuAlaLtisoanm isp lmeowrea scopmropvleenx t[o30b]e aandde iqsu daetteafiolerdi oinn oat rporpevicioguesla wtioonrkw, iinth wchailcchiu tmhei ochnesm[3i1c]a.l Astmruoclteucruel oafr twhiesi gAhLt s(avmispcoles iwtya)so pfro78veknD toa bwea asdmeqeuaastuer feodr fioonr oAtrLopbiyc gcaeplaitliloanry wviitshc coaslictyiuimn iaon0s.1 [3M1]. NAa Cmloalqecuueloaur swseoilguhtti o(vnisacto2s5ity◦C) ouf s7i8n gkDthae wMasa rmk–eHasouurewdi nfokr eAqLu abtyio cnapwililtahryc ovnissctaonsittsyo ifn δa =0.10 .M92 NanadCl Kaq=u0e.o0u07s3 soclmu3ti/ogn[ 3a2t ]2.5 °C using the Mark–Houwink equation with constants of δ = 0.92 and K = 0.0073 cm3/g [32]. 3.1. Characterization of AL-CS Nanoemulsions 3.1. Characterization of AL-CS Nanoemulsions 3.1.1. Types of Oil 3.1.1T.o Tdyepteesr mofi nOeilt he influence of the oil type on the properties of the nanoemulsion particles’ size, PDI anTdo dzetaerpmoitneen thiael iwnfelrueenmcea osfu trhede ,oailn tdypthe eorne tshuelt ps raorpeeprtrieesse onft etdhei naFnigouemreu3l.sioTnh epaoriltiicnlews’ astiezre, APLDnI anode zmetual spiotnenotbiatal iwnedre imnetahseurfierdst, asntadg teheo fretshueltps raorcee pssrewseanstecdo minp Fairgeudrew 3i.t Thhteh eoilfi inna wl AatLer-C ASL nnaannooeemmuulslisoionn. AoLbtpaainrteidcl eisnp rtehpea rfeirdstw sitahgteh eodf ifftheere nptrocilesssh owwase dcsoimpilaresdiz ewsiwthi ththvea lfuineasla rAouLn-CdS 3n20a~n3o4e0mnumls,ioan.d AaLn pinacrtriecaleses pofreppaarrteicdle wsitzhe twhea sdoifbfeserervnet doialsf tsehrotwheelda stimstialagre siinzewsh wicihtha vcaoluateisn garwouitnhd C3S20w~a3s40fo nrme, dan(dFi agnu rienc3rAea).sCe onf tprartyictloe AsiLze, CwSaiss oabhseigrhvemd oalfetceur ltahrew laesitg shttacgaet iionn wichpicohly ae lceocatrtoinlygt we inth soClSu twioans; ftohremreefdor (eF, iagnurine c3rAea)s. eCionnptraarrtyic lteo sAizLe, iCs Sex ips eac theidghw mheonleaculalayre rwoefigChSt dceaptiosniitcs opvoelyretlheectoruoltyetre lainy esrooluf tAioLna; sthaeresfuorlte,o af nel einctcroesatsaet iicna pttarartcitciloen s[i2z0e] .is expected when a layer of CS deposits over the outeIrn ltahyeecr aosfe AofLp aasr tai crlesupltr eopf aerleedctwroisthtastiacc haattrinacthiiooni l[,2t0h]e. s ize increase was much higher (30%) than for oliIvne tahned csaosyeb oefa npaoritlisc,l3e%s parnedpa1r%ed, r ewsipthec stiavcehlya. iAncftheir ositlo, rtahgee saizt e~ 5in◦cCrefaosres wevaesn mdauycsh, thhigehpearr t(i3c0le%s) wtheraena fnoarl yozlievde agnadin s.oTyhbeasniz oeiolsf, p3a%rt iacnleds 1p%re, praersepdecwtiivthelysa. cAhfateinr cshtoi raangde oalti v~e5 o°iCls fsolrig shetvlyenin dcareyass, etdh,e shpoarwtiicnlgesh wigehrer asntablyilzietyd tahgaaninth. Tehpea rstiizcele osfp praerptaicrleeds wpriethpasroeydb weaintho sila.cThha einscizhei panodly odliisvpee rosiiltsy solifgAhtLly pianrctricelaessewd,a ssqhuowiteinlogw h(i≤g0h.e2r5 )satnadbiilnitcyr etahsaend atfht er CpSarctoicalteisn gp(rFeipgaurred3 Bw) intha lslosyambepalne s.oil. The size polyFdigisupreers3iCtys ohfo AwLs tphaertcihclaensg wesaso fqtuhiteez leotwa- p(≤o0t.e2n5t)i alnvda ilnucerseoasfeodil alfotaedr eCdS AcoLaatinndg A(FLig-CuSrep 3aBr)t iicnle asl.l Tshaemzpelteas-.p otential is an important parameter for the electrical characterization of nanoemulsions prepaFreigduwrei t3hCp sohloywelse ctthreo lcyhtaens,gaes tohf ethaed zsoetrap-tpiontenotfiaclh vaarglueeds coofu onilt eloraiodneds cAaLn abnedm AeLa-sCuSre pdabrtyiciltess. vTahluee zaentda-psiogtnen[3ti3a]l. iTsh aent yimpepofrtoainl tu speadrafmorethere pforre pthaera etiloenctroifcathl echAaLraecmteurilzsaiotinong roeaf tnlyanaoffemcteudlsitohnes zpetrae-paortedn twiali.thA psoelxypeelectcetdro, lsyutrefsa, caesc hthaer gaedssionrtphteioAnL oef mchualsrigoends cporeupnatererd iownist hcavne gbeeta mbleaosiulsresdh obwye idts Polymers 2019, 11, x FOR PEER REVIEW 7 of 18 Polymers 2019, 11, 1245 7 of 18 value and sign [33]. The type of oil used for the preparation of the AL emulsion greatly affected the zeta-potential. As expected, surface charges in the AL emulsions prepared with vegetable oils snheogwaetidv enevgaalutievseb veacaluuesse boefctahuescea orbf othxey lcaatrebgorxoyulpasteo gfrAoLu,pas poof lAyaLn, iao pnoiclycahnaiinon[1ic9 c,3h4a]i.nT [h1e9,A34L].e Tmhuel sAioLn epmreuplsairoend pwreitphasreadch wa iitnhc hsiacohilas hinocwhie doitlh sehmowosetdn tehgea tmivoesvt anleugeasti(v−e2 2vamluVe)s, f(o−l2lo2 wmeVd)b, yfosloloywbeeadn boyi l s(o−y1b1eamnV o)i,l a(−n1d1 fimnVal)l,y aonldiv feinoaillly(− o5livmeV o)i.l A(−f5te mr cVo)a. tAinftgerw ciothatCinSg, wthieths uCrSf,a tcheec shuarrfgaecse ocfhathrgeepsa orft itchlees pcahratincgleesd cfhroanmgnedeg farotimve nteogpaotisvitei vtoe apsoasirtievseu latso af rtehseupltr eosfe tnhcee porfetsheencper ootfo tnhaet epdroatmoninaotegdr aomupinsoin gCroSuaptsa inp HCSb aett wa epeHn b4e.5twaneedn5 4[.252 a]n. dA 5m [o22n]g. Athme othnrge ethoei ltsh,rseaec ohialsi,n scahcihoai ilnpcrheis oeinl tperdesthene tmedo stht ed rmamosat tdicracmhaantigce cohfanitgsez eotfa -iptso tzeenttai-apl,oftreonmtia−l,2 f2rotom+ −3252m toV , +fo35r AmLVa, nfdorA ALL-C aSnpda rAtiLcl-eCsS, rpesaprteicctleivse, lrye.sApescmtiveenltyi.o nAesd mbeenfotiroen, etdhe bezfeotrae-,p tohtee nzteitaal-pisotaenntiinadl iisc aatno rinodficnaototro onfl ynotth oenslyu rtfhaec esucrhfaacreg echoafrgthe eofp tahret ipclaerst,icbleust, baulsto atlhsoe itrhsetiarb sitlaitbyil[i3ty4 ][.3C4]S. -CASL-ApLa rptiacrlteisclpesre ppraerpeadrewdi twhisthac shaachinac ihnichoiil owil ewreereex pexepcteecdtetdo tboe bvee vryersyt asbtalbelaes atsh tehierivr avlauleueo fotfh theez ezteata-p-pootetenntitaial lw waassi inn tthhee rraannggee ooff >>||3300||. . TThhiiss hhiigghh ssttaabbiilliittyy ccoouulldd bbee tthhee rreessuultlt oof f sseelfl-fa-asssseemmbblyly oof fththee twtwoo ppoolylymmeersr saarorouunndd ththee ooili lccoorree oowwiningg ttoo tthheeirir ssttrroonngg eelleeccttrroossttaatticic aattttrraacctitoionn frfroomm ththeeirir ooppppoossititee cchhaarrggee nnaattuurree [[2200]]. . (A) (B) Figure 3. Cont. Polymers 2019, 11, x FOR PEER REVIEW 8 of 18 PPoollyymmeerrss 22001199,, 1111,, x1 2F4O5R PEER REVIEW 8 8ooff 1188 (C) (C) FFiigguurree 33.. EEffffeecctt oofft htheet ytpyepeo foofi looinl othne tphreo pperrotpieesrotifeAs Lofn aAnLo enmanuolseimonu(lAsiLo)na (nAdLC) Sacnoda tCedS AcoLapteadrt iAclLes pFaigrtuicrlee s3 .( AEfLfe-CctS )o fo tnh et htey ppea rotfi coleil soinze t h(Ae )p, rsoipzee rtpieosly odfi sApLer snianoemulsion (AL) and CS coated AL (AL-CS) on the particle size (A), size polydispersity (B), and ztyet a(-Bp)o, taenndti azle(tCa)-,pwotheenntiaplr e(pCa),r ewdhaennd ppraerptiacrleeds a(AnL-CS) on the particle size (A), size polydispersity (B), and zeta-potential (C), when after seven dda ayfst.erP sDeIv,epno dlyadyiss.p PeDrsIi,t ypoinlyddeixs.persity index. prepared and after seven days. PDI, polydispersity index. 33.1.1.2.2. .CCoonncceenntrtraatitoionn ooff CCSS SSooluluttioionnss 3.1.2. Concentration of CS Solutions TThhee eeffffeecctt off tthee CS solluttiion conccenttrraattiioonn oon tthee fforrmullaattiioonn off AL--CSS nanoeemuullssiioonnss waass ssttuuddieiTedhd ei inne ftfteeercrmt sos f otffh pe aCrtSi csloesl ustiizoen, PcoDnIc, eanntdra tzieotna -opno ttehnet ifaol.r mAu llaotwio nco onfc eAnLtr-aCtSio nn aonfo CemS u(≤ls0io.6n sg ·wL−−a11)s ) rsretesusudulilteteeddd iininn tneorom csh aonf gpeea irinti csliezse snizoer, zPeDtaI-, paontden zteiatal -opfo tthene tAiaLl. -CAS l onwan cooenmcuenlstiroantiso. n Tohhfee CzSet ta(a≤--0p.oo6t teegnn·tLtiia−a1l)l rrreeesmuaalitineneded di nw initthoh aca hnnaeengggaaetti ivivnee sssiiigzgnen, ,nssuougrg ggzeeeststatini-npggo ttthehanattt itathhle eo pfp ootshsieitti ivAveelLyly- CcchSha anrrgagenedod eaammuinilnsoio oggnrrsoo.u uTpphss eo ofzf CeCtSaS -pwoeetrreeen ntniooatlt erenenmoouuagignhhe tdtoo wnneietuuht traraa lnliizezege atththieve enn eseigggaantti,i vsveuelglyyg cechshatairnrgggee dtdh cacaatr rtbbhooex xpyylolaastitete igvgreroloyuu pcphss aiinrng eAdL a[[m3355i]n].. oW ghrheoenun pCsS So cfco oCnnScce ewnntetrrraeat tiniooonnt ewnaaoss uiingnchcr reteoaa snseeeddu tttoro a1l1.i5.z5 eg g·tL·hL−e1−, n1t,hetgeh asetiizsveiez ilenyic ncrhecraaesragesdeed ad ncaadnr btdhoetxh yzeelatzatee-tp ago-rptoeountpetisna tlii nba leAcbLaem c[a3em5 p].eo Wspiothisveineti, v CaesS, cacasonnc abcneen sbtereeasntei oiennn FwiingauFs irigenu c4rr. eeTa4hs.iesdT o htbois e1or.5bv saget·iLrov−n1a, thtihoaesn bshiezaeesn ib npecrerenevaiposreuedsv layion rudes ptlhyoerrt ezpedto abr-ytpe NodtaebtnyrtaiNjaalna bt eretac jaaalmn. (e2e 0tp1ao5ls.)i ta(i2nv0de1, C5a)sh aocnai dnet bC aehl .s o(e2ie0en1t 1ian)l . [F(22i2g0,1u313r)e]. [ 4H2.2 To,3wh3ies].v oeHbrso, ewrvheavetneiro t,nhw eh hcaeosn bcteehenent crpoartneicovenino outrfs aCltyiSo r wnepaoosfr hCteiSgdhw beayrs tNhaaigtnrh a1ej.ar5n tgh e·Lat n−a1l,1 .t .(h52e0g s1Li5z)−· e 1a a,nntdhd eC PshDiozIie oeaftn apdla. rP(t2Di0clI1e1os)f d[p2ea2cr,rt3ei3ca]ls.e eHsddo aewcnerdev aetsrhe,e dw zahenetnda -ttphhoeet cezonentiaca-elpn owtrtaaesnti toainalsl oowf rCaesSd awulscaoesd rh.e idgWuhacenerd gth. aaWnnad 1n .5hg igas· nLcd−o1,lh lteihsaegc souilzelese aaagnlusdoe P soDablIs ooefro vpbeasdret ritcvhleeisds pdtheiecsnrepoahmseeendno omanne dwn oittnhhe wh zigtehtaeh-rip gcohotenerncectinoantlr cawetniaotsrn a aotlifso oCn Sro efsdoCulSuctesidol.n usWt,i oaannsdg, raaennpddo rehtepidso rtcthoealdlte tiahtg mautaeitys mbaleas yoth beoe brtesheseurvlrtee dsouf lttthhoiesf sptahhteuenrsaotmiuorenan tooiofn ni owonif tlhioo ghnaiglroihtgeharm rciiotchn fmcoerincmtfreoadrtmi obnyed oelfbe CyctSer losesoctltaurtotiicso tnatsti,rc aaanctdtiro ranec ptfiooorrncteefdso rbtcheeatswt bietee tmnw ateyhe enb e–t NhtheHe– 3rN+e osHfu 3Cl+t Soo faf tnChdeS tsahanetd u–rCtahOteioO–nC− ooOffO Aio−Lno, alfosAg wLare,ilatlhs amws aiecnl lf ioansrcmareneadisn ebc vyrei seaclseoecsvtirtiyosc sootfas ttiihtcye a oCtftSrt ahscoetliCuoStnio sfnool,r umcteiasok nbi,nemtgw atehkeeinn rg etahthcete i–orNnea Hdcit3f+i fooincfu dCltiSffi w acinuthdlt ttwhheiet Ah–CLthO seuOAr−f Laocfs eAu [rL3fa,5 ac].es w[3e5l]l. as an increase viscosity of the CS solution, making the reaction difficult with the AL surface [35]. (A) (B) (A) (B) Figure 4. Effect of CS concentration on the properties of sacha inchi oil loaded CS-AL nanoemulsion. F(iAg)uSreiz 4e./ PEDffIecatn odf (CBS) zceotnac-epnottreanttiioanl. on the properties of sacha inchi oil loaded CS-AL nanoemulsion. (FAig) uSirzee 4/P. EDfIf eacntd o f( BC)S z ceotan-cpeontternattiiaoln. on the properties of sacha inchi oil loaded CS-AL nanoemulsion. (A) Size/PDI and (B) zeta-potential. Polymers 2019, 11, 1245 9 of 18 Polymers 2019, 11, x FOR PEER REVIEW 9 of 18 33.1.1.3.3. .CCoonncceenntrtraatitoionn ooff SSuurrffaaccttaanntt SSuurrffaaccttaannttss aareref refqrueqenutelnytulys eduisnedth eipnr etphaer atpiorenpoafrnaatinoonp aortfi clneasntoopoaprttimicliezse thtoe iropphtyimsioizceh emthieciarl pphryospieorcthieesmaincadl sptarboiplietryt.ieDs iffaenrde nsttcaobnilciteyn.t rDatiifofenrseonft thcoenncoennt-rioantiiocnssu roffa ctthane t,npoonl-oioxnamic ersu4r0f7a,ctwanert,e puosleodxatomeenr c4a0p7s, uwlaetrees uascehda tino cehnicoaipl sinutloatteh seaAchLa- CinScphai rotiilc lienst.oT thhee eAffLe-cCt So npathrteicpleasr.t iTclhees eizffee,cPt DoIn, athned pzaerttai-cpleo tseizneti, aPlDisI,e avnadlu zaetetad-pasotsehnotwianl iisn eFviagluuraete5d. aPso slohxoawmne rin4 F07ighuarse a5.t rPiobllooxckamsterru 4ct0u7r ehacso na ttariinbilnogcka scterunctrtualreh ycdornotpaihnoinbgic ab loceckntoraf lp holyydorxoypphroobpiyc lebnloec(kP OofP )paonlydotxwyporiodpeynlteincael (lPatOerPa)l ahnydd rtowpoh iliidcecnhtaicianls loatfepraoll yhoyxdyreotphhyilleicn ceh(aPinOsE o)f[ p36o]l.yoPxoyleotxhaymleenres (hPaOvEe) a[n36a].m Ppohloipxahmiliecrsc hhaarvaec taenr awmitphhibpohtihlica scshoacriaacttioern wanitdh abdostohr pastisooncipatrioopne ratnieds aadnsdorimptpiorno vperosopleurbtiielisz aatniodn imanpdrosvtaeb siloizluabtiiolinzaotifocno manpdo usntadbsilaizsaatiorens uolft coofmthpeoiurnndost aabs lae rpeshuylst ioofl othgeicira lnoptraobpleer ptiheyssaionldogliocwal ptorxoipceitrytie[3s 7a]n. dP loowlo xtoamxiceirtsy a[3n7d]. pPoollooxxaammienress anarde pnoolno-xiaomniicnseusr faarcet annotns -tihoantich asvuerfdaicvtearnstes atphpalti chaatvioen sdiivnevraseri oaupspbliicoamtioednisc ainl fivealdrisosuusc bhiaosmderduigcadl efliievledrsy , smucehd iacsa ldirmuagg dinegli,vaesryw, emlleadsiccaaln icmeratghienrga,p ays [w38e]l.l Ians acapnrecevri otuhserwapoyrk [,3i8t ]w. Ians fao purnedvitohuats awnoirnkc,r eita sweaosf fcoounncde ntthraatt iaonn ionfcpreoalosxe aomf ecron40c7enptrroadtiuocne sofa preodlouxcatimonero 4f0b7o tphropdaurtcicelse as irzeedauncdtioPnD Iof[ 1b7o].thS ipmairltairclree ssuizltes awnedr ePDobI s[e1r7v].e dSiminilAarL -rCesSunltas nwoeerme uolbsisoenrveendc ainp sAuLla-CtinS gnsaancoheamiunlcshioino iel.ncAapccsourldatiinngg tsoacthhea liintecrhai touirle. , Ancocno-riodninicg stuor tfhacet alintetsralitkuerep, onloonxa-imoneircd sournfoatctsaignntsi filickaen tplyolaoffxeacmt ethr edzoe ntao-tp soitgennitfiiacla[n3t9ly,4 0a]f.feHcto twheev zeert,ai-n pooutrensttuiadl y[,3a9v,4a0r]i.a tHioonwoefvtehre, zinet ao-upro testnutdiayl,w aa svoabrisaetriovned owf itthhep ozleotxa-apmoeternctoianlc ewnatrsa toiobnse(rFviegdu rwe 5itBh) . pTohleoxzaemtae-pr octoenncteianltoraftsioacnh (aFiingcuhrie o5iBl e).n Tcahpes zuelatate-pdoitnenAtLia-lC oSf psaarcthical eisncdheic roeial seendcawphseunlattheed pinol oAxLa-mCeSr pcaornticcelnestr daeticorneacsheadn wgehdenfr tohme p0o.1l%oxatom0e.r3 %conwc/evnatnradtiionnc rcehaasnegdewd hfreonmh 0ig.1h%er tao c0o.3n%ce wnt/vra atniodn inwcarseausseedd w(0h.5e%n haingdhe1r.0 a% c,own/cve)n. tration was used (0.5% and 1.0%, w/v). (A) (B) Figure 5. Effect of poloxamer 407 concentration on (A) size and PDI and (B) zeta-potential of sacha Fiingcuhrieo 5il. lEofafdeectd oAf Lp-oCloSxnaamneore m40u7l scioonc.entration on (A) size and PDI and (B) zeta-potential of sacha inchi oil loaded AL-CS nanoemulsion. 3.2. Emulsion Stability 3.2. EmThuelsipohna Ssteabsieliptya ration of the AL-CS nanoemulsions for a long time (six months) was visually comTpharee dphaassae nseepvaarluataitoino noof fththe eAirLp-hCySs incaanl osetambuillistyio(nFsi gfourr ea6 l)o. nSgt otriamgee (tsimix em, toenmthpse)r awtuasre v, itsyupaelloyf csotmabpilaizreedr, aans danty epveaolufaotiilopnh oafs ethoefitre pnhayffseiccat lp shtaasbeilsiteyp a(Friagtiuorne a6n).d Sstohroawgee fftiemctes, ltiekmepcreeraamtuirneg, t[y4p1e– 4o3f] . sTtahbeilpizhears, eansdep tayrpaet iofn ooilf pthaessee oruftmen farfofmecta pnheamseu slespioanraotifotenn anardi ssehsobwe ceaffuescetso lfikteh ecrfleaomcciunlga t[i4o1n–4a3n]d. Tmhieg rpahtiaosne osefpoailramtioolnec oufl etshfer osmerusm aflrloermd raonp elemtsutlosiloanr goefrteonn easritsherso ubgechatuhsee aoqfu tehoeu fslopchcauslea,tiwonh icahndis mknigorwatnioans othf eoOil smtwoaleldcurliepse fnrionmg esffmeactll[e4r1 d,4r4o]p. lIentsA tLo- lCaSrgnearn ooneems uthlsriooungs,ht htheec oanqcueenoturast piohnaosef ,s wurhfaicchta inst kannodwtnh eatsy pteheo f Ooisltawffaelcdt edriptheenipnhgy seifcfaelcts ta[4b1il,i4t4y],.p rInod AucLin-CgSd inffaenreonetmleuvlseilosnosf, stehrue mcosnecpeanrtartaiotino.n of surfacItnanAt La-nCdS tnhaen toyepme uolfs ioinl sa, fpfeocltoexda mtheer pchoynsciecanlt rsattaiboinlistyo, fp0r.o1d%ucaindg d0.i5ff%ereanntd letvheelst yopf eseorfuomil sdeeptaerramtiionne.d the phase separation of the system. When higher concentrations of poloxamer were used, betteIrn pAhLy-sCicSa lnsatanboielimtyuolsfioAnLs,- CpSoleomxaumlseior ncwonacseonbtrsaetriovneds ,osfh o0w.1%in galneds s0p.5h%as eansedp athrae titoynpeth oafn ofoilr dloewteremr pinoelodx tahme eprhcaosnec seenptararatitoionns. oNf tohtea bslyestpehmas. eWshepena rhaitgiohnero cfosnecruenmtrwatiitohnhs iogfh ptoulrobxiadmityerw waesrvei ususeadll,y boebttseerr vpehdyisnicasal mstpableilsitpyr eopf aAreLd-CwSi tehm0u.1ls%ioonf wpoaslo oxbasmerevr,esdh, oswhoinwginagh liegshs rpahteasoef scerepaamraitniogn( htheaignh ftoorf lsoewruerm p(oHlosx)acmomerp caornecdewntirtahtitohnesh. Neiogthatbolef pcrheasme s(eHpca)r)a[t4io3,n4 4o]f. sIenrucomm wpiathri shoignhw tuitrhbsidoiytbye wanasa vnidsuoallilvye oobilsse,rAveLd-C inS nsaamnopelmesu plsrieopnasreendc awpistuhl a0t.1in%g osaf cphoaloinxcahmi oeril, sshoweindgh aig hiegrhp hraytsei coafl csrtaebamilitinygw (ihtheiaghmt oorfe serum (Hs) compared with the height of cream (Hc)) [43,44]. In comparison with soybean and olive oils, AL-CS nanoemulsions encapsulating sacha inchi oil showed higher physical stability with a Polymers 2019, 11, 1245 10 of 18 Polymers 2019, 11, x FOR PEER REVIEW 10 of 18 smtaobrlee setmabulels ieomn.uTlshioisno. bTsheirsv oatbisoenrvisatinioanc icso rind aanccceorwdiathncteh ewrietshu tlthsef rroemsulFtsig furroem3 .FTighuerzee 3ta. -Tphoete znetitaal-, tphoetednirteiaclt, itnhdei cdaitroercot finpdairctiactloers otafb pilaitryt,icwlea ssttahbeilhitiyg,h wesats( +th35e mhiVgh) ienstA (L+3-C5 SmnVan) oinp aArtLic-CleSs ennacnaoppsaurltaitcilnegs seancchaapsinuclahtiinogil saancdhare imncahini eodil aafntedr roenmeawineeedk aofftesrt oornaeg ew. eek of storage. (A) (B) Figure 6. Phase separation images of AL-CS nanoemulsions (A: soybean oil, B: sacha inchi oil, C: olive oFiilg)uprree p6.a Prehdaswei sthepdairffaetrieonnt ismuarfgaecst aonf tAcLon-CceSn ntraantoioenms,utlhsiaotniss, ((A:) s0o.y1%beaann doi(lB, B) :0 s.5a%ch.aT ihnechsai moipl,l Ces: owleivree sotiol)r epdrefporarseixd mwoitnht hdsifafte5re◦nCt .surfactant concentrations, that is, (A) 0.1% and (B) 0.5%. The samples were stored for six months at 5 °C. 3.3. FT-IR Analysis 3.3. FTTh-IeR eAnncaalpyssuisl ation of sacha inchi oil in AL-CS nanoemulsions was verified by FTIR-ATR spectTrohsec oepnyca( prseusleanttieodn inofF isgaucrhea7 )i.nScphei cotrial oinf faAtsLa-CndS onilasnaomemplueslssihonows wpeaask svaenridfieshdo ublyd eFrTs ItRh-aAt aTrRe aspttercibtruotsecdoptoy t(hperierssepnetceidfi cinf uFnigcutiroen 7a)l. gSrpoeucptrsa[ 4o5f ]f.aGtsu atniédr roeizl seatmalp. laens dshGouwil lpéenaekts aaln. dh asvheouplrdeverios uthslayt raerpe oarttterdibtuhteedp htoy sitchoecirh esmpeiccaiflicc hfaurnaccttieornizaal tigornouopfss a[4ch5]a. iGncuhtiiéorrilezu seitn galF. TaInRd spGeucitlrloénsc oept yal[.4 6h,a4v7]e, wprheivcihoucosliyn criedpeosrtwedit hthteh ephspyseicctorachoefmoiucrals cahmapralec.teTrihzeat3i0o1n0 ocfm sa−c1hbaa indchiis oaitlt urisbiuntge FdTtIoRt hspeescttrreotscchoinpgy v[4ib6,r4a7t]io, nwohficchis cooleinficniidceCs –wHitihn tchaer bsopne–cctararb ofn oduoru sbalembpolen.d Tsh[4e6 3].0T10h ecmtw−1o baabnsodr pist iaotntrbibauntdesda tto2 9th10e astnrdetc2h8i5n4gc vmib−r1actioornre ospf ocnisd otloeftinhiec mCe–tHhy ilne ncearabsoynm–mcaertbroicn adnodubsylem bmoentdrisc [s4t6r]e.t cThhien gtwvoib raabtsiornp[t4io7n]. Tbahnedcsh art a2c9te1r0i satincdb a2n85d4 actm17−14 3cocrmre−s1poconrdr etsop othned smteoththyleenster eatscyhminmg evtirbicr aatinodn soyfmthmeeCtr=icO stgrreotcuhpinogf tvriibarcaytligolny c[4e7ro].l Ts.hTe hcheasrmacatlelrbisatnicd bdanedte actte 1d74a3t c1m65−01 ccomrr−e1spisonasdssi gton ethdet sotrtehtechdiinsgu bvsibtirtauttieodn coifs tChe= C=oOf tghreouupn soaft utrriatceydlgalycycelrgorlos.u Tphsea nsmdatlhl ebbanandd daette1c4te6d0 camt 1−615i0s camttr−1i bisu taesdsitgonethde tob etnhde idngisuvbibstriattuitoends coisf tCh=eCC oHf 2thaen udnCsaHtu3 raaltiepdh atciyclg grroouuppss[ a4n6,d4 7th].eT bhaendtw aot 1b4a6n0d csmat−11 i2s 3a8ttarinbdut1e1d6 3toc tmhe− 1bceonrdriensgp ovnibdratotiothnes sotfr ethtceh CinHg2 vainbdra CtiHon3 aolfipthaetiCc –gOroeusptes r[4g6r,o4u7p]. sTahned twtoo tbhaenbdesn adt i1n2g38v iabnrdat 1io1n63o cfmth−1e cCoHrre2sgproonudp stot hthaet astrreetccohminmg ovniblyraftoioun dofi nthme Can–yOt eyspters gorfovuepgse taanbdle to itlhse[ 4b7e,n4d8]i.ngT hveibbratnidont hoaf tthaep pCeHar2 sgarot u7p30s tchma−t 1aries acottmribmuotanblyle ftouthned oivne rmlaapnoyf tthyepems eothf yvl enget(aCbHle2 )oriolsc k[i4n7g,4v8i]b. raTthioen bsaand ttohatht eaopupteoafrsp latn e73v0ib cramti−o1 niss oatftcriisb-udtiasbulbes ttiotu ttehde oolvefiernlasp[4 8o]f. Tthhee sme ecthhayrlaecnter i(sCtiHc 2t)r arnoscmkiinttga nvciebbratniodnsso fasnadc htao inthceh ioouilt woef replaalnsoe dviebteracttieodnisn oAf cLi-sC-dSisnuabnsotietmutuedls ioolnefsinpsr e[p48a]r.e Tdhwesiteh cshaacrhaactienrcishtiico tirl,awnshmicihttaisnecve ibdaendcse off sthacehsau icnccehssi fouill ewnecraep aslusloa tdioenteoctfetdh eino iAl Lin-tCoSt hneanoaenmoeumlsuiolsniso np.repared with sacha inchi oil, which is evidence of the succeBsessfuidle esntchaepcshualraaticotenr iosft itchbea onidl sinotfos tahceh anai ncoheimoiullisniotnhe. nanoemulsion, the interaction between the aminBeegsriodueps sthoef cchiatorasactnerainsdtict hbeancadrsb oxf yslaactheag irnocuhpis ooifl ainlg tihnea tneacnaoneamlsuolsbieonev, itdhen icnetder. aTchtieosnh bifettowfethene ptheea kamatin16e6 g0rtoou1p6s5 o0fc cmh−it1oissanat tarnibdu ttheed ctaorCbo=xOylsattre tgcrhoinugpsv iobfr altgioinaatne dcathne adlsiosa bpep evairdanenceceodf.t Thehe– NshHif2t boef nthdein pgeapke akt 1a6t6106 0to2 1c6m5−01 cimn−c1 hisit aotstarnibwutaesdo tbos Cer=vOe dstarefttecrhtihneg fivnibarlasttiaogne aonfdt htheeA dLis-aCpSpneanraonemceu olfs itohne f–oNrmH2a tbioend[4in9g]. pTehaekb raot a1d60sh2 ocumld−e1 rin ecahri3to4s0a0nc mw−a1s ionbdsiecravtesdt haefteprr etsheen cfeinoafl hsytadgreo xoyfl athned AamL-iCnoS gnraonuopesmfurolsmioAn LfoarnmdatCioSnp [o4l9y]m. Tehrse. broad shoulder near 3400 cm−1 indicates the presence of hydroxyl and amino groups from AL and CS polymers. Polymers 2019, 11, 1245 11 of 18 PolPyomlyemrse 2rs0 1290,1 191, ,1 x1 ,F xO FRO PRE PEERE RE RVEIEVWIE W 11 1o1f o1f8 18 FiigFuigreu r77e.. F7o.o uFuroriuieerr-i-tetrra-antnrsasfnfoosrrmfmori mninf rfiarnarferedadrae tadtte tanetunteauntaeutdeadteo tdtoa tltaorlet arfllee frcletafclnteacnetac(neF cT(eFI RT(F-IARTTI-RAR-T)ARoTf)R soa)f c ohsfa csihancach hiani coinhilci, hoAii Llo, ,iAlC, LSA, L, aCnSdC, Sa,n sadan mad p saal emsaopmflAep loLef- CoAfS LAn-CLa-nSC onSpa anraotnipcoaleprstairwctlieictshl ews aiwtchhi tsaha icsnhaccahh iano cinhl.ci hoi lo. il. 33..443... 4OO. iiOll EEilnn Eccnaappcassuupsllaauttliiaootnnio EEnffif fEiccfifieiecnnicecynyc y NNiiNlleei lrreee drde idiss iasa lailp ilpiopopophphilhiilciil cidcy ddeyy aeen aadnn hdda hhsa absse bebenee eunns ueusdes tedod tsott aositnast ilanipi nlipdlisidp, scid,e clsle,sl,cl sae,nl ladsn, odai nlosdi l[s5o 0[i5–l0s5–3[5]5.30 I]–n. 5I tn3h] ti.shI isnst ustthduiysd, y, sNtuiNldeiy lr,ee Ndre iwdle awrsea desm wepmalspoyelomeydpe tdloo tysoet asditnato ivnse vtgaeigntaevbtaleebg loeeti laosbi ltlsoe teoovi leasvltuaoaluteeav toaeli ulo eailnt eecnaocpialspeunsluactaliapotsniou enlfa fetiifcofiinecnieecffinyc.c yFie.i gnFuicgyru.e rF8ei gs8hu sorhewo8sw s sfhlufolowuroessrflecesucnoecrneec semc eimnccrieocrsmocosiccporyop syicm oipamgyaeigsme s(aL g(eeLiscea(iLc, aeW,i cWeat,zeWltazerlta,z rlG,a erG,rGmereamrnmayna) yno)y f) ooaf fasa asscaahccahh aain icinnhccih hioio iloi lillo oalaoddaeedde dAA LLA--CLC-SSC S nnaannaooneeommeuumllsusiiolosnnio,, nww, hhwiicchhhic iihss eiesvv eiiddveiednneccenec ooeff otthfh eteh sseuu csccuceecscssesffsuuslflu lloola aldodaiindnggin oogff otthfh eteh ooeiil lo.. il . FFiiggFuuigrreue r88e.. 8M. iiMccrrioocssrccooospcpoyyp iiym iaamggeaesgs e((xsx 11(0x0010)0) 0oo)ff oAAfL LA--CCLS-SC nnSaa nnoaoneeomeuumllssuiiolosnnio ppnrr epeppraearpreeaddre wdi itwthhi tsshaa ccshhaacah iinanc cihnhici::h flfil:uu foolurreeossrcceeesnnccecene ce fifillttfeeirlrt TeTrXX T22X::BB2PP:B 55P66 050/6/44000/4 ((r0ree d(dre ccdoo lcloorrl cocoro rcrrroeerssrppeosonpndodsnsdt toso s tsaoac cshhaacahi nianc cihnhicio hoiili los stital aisnitnaeeidndew wdi tiwhthiN tNhi liNeleri lreed dr)e.).d ). ThTeh een ecnacpaspulsluattliiaotnio enffif feicfcifieiecnnicecynyc wya awssa aslls saools doee dtteerrtmeriimnneeidnde aadnn dadn iidss ipsrr epessreensnteteneddte iidnn iFFniig gFuuigrreue 9r9e.. 9T.h Teh uen ulonalodaede odii llo il frofrmo mA LA--LC-SC nSa naoneomeumllusiilosniosn ws awsa sse speapratrteadte bdy b cye cnettnriitfruifguagtiiaotnio uns uiinsgin egt hetahnaonll oaln adn tdh teh seu spueprnerantaantatn wt awsa s anaanlyazlyezde dw iwthit Uh VU–VV–iisVs sisspp esecpctetrrcootsrscocoscpoyp aaytt a55t55 3535 nn3m n..m S.a Scahcah iian cinhci hoiii llo silh sohwoewde tdth teh heii ghhigehset svta vllualeu oeff ollfo alodaiidngin g effiffeicfcfiieiecnicecynyc ycoo mcmoppmaarpreaeddrew dwi tiwhthiot lhoivl ioevleiav naedn adsno dsyo bsyeobayenbaeonai lnosi, loasil,l tsah,l otauhltoghuhogtuhge htht oteht atelo etaonltc aealnp ecsnaucplastpueslduatoleaidtle qdoui lao niqlt uiqtayunawtnitatyis tywle sawsa s tlhesalesn sts4h 0tah%na 4nin0 4%t0h %ien n itnah nteho nea mnauonlesomieoumnl.usEilosnnico.a nEp.n sEucnalacpatsipuosnlautoliafotoniol ionvf e oofal ionvldeiv saeon aydnb sdeoa synoboyeibalenian onAi lo Linl- C iAnS LAn-LaCn-SCo nSeam nauonlesomioeumnlsuswilosaniosn s lwoawsea rls,o awloroewur,en rad, rao3ru0o~nu3dn5 d%3 0.3%0T~%h3i~5s3%d5.i% ffT.e hrTeishn icdse idfmfiefirfgehnretcnbec eemb imegchiagtuh sbte eb oebf eobciealcucaosuems eop fo osofi itloi oiclno cmaonpmdopspoihtsiyiotsnioi cna nladpn rdop phpeyhrstyiicseaiscl al spurpcohrpoeaprsteidretesine ssu istcyuhc aahns da dsve dnisescniotsysii ttayyn .adn vdi svcioscsoitsyi.t y . Polymers 2019, 11, 1245 12 of 18 Polymers 2019, 11, x FOR PEER REVIEW 12 of 18 Figure 9. The oil encapsulation efficiency of AL-CS nanoemulsions prepared with different types of oil: Fsiagcuhraei n9.c hTih,eo loivile e,nacnadpssouylabteioann (esfofilcuiteinocnyt oofo AilLra-CtiSo wnaansofiexmeudltsoio9n:s1 )p. repared with different types of oil: sacha inchi, olive, and soybean (solution to oil ratio was fixed to 9:1). 3.5. Evaluation of Antioxidant Activity 3.5. Evaluation of Antioxidant Activity Most of the unrefined vegetable oils naturally contain antioxidant compounds that play an impoMrtoasntt roof lethine purnortefcitnioend avgeaginetsatbolxei doailtsio n,abtuortahlliyn ociolnsttaibni liatyntaionxdidasantu tcroitmiopnoaul nbednse tfihtast[ 7p].laSya cahna iimncphoirsteaendt srhoalev einre pcernottleyctbieocno mageaainpsot pouxliadrantiuotnri,e bnot tsho uirnc eocilo nsttabiniliintyg haingdh laesv enlustorfitoiomneagl ab3enfaetftiytsa c[7id].s Sinacthae ifnocrmhi osefeαd-sli nhoalveen ircefcaetntytlya cbidec(o∼m45e% a) p, voapruiolaurs ntyuptreisenotf tsocuorpche ecroonlsta(ilnikiengα ,hβigγh, laenvdelδs- otofc ompheegrao l3) , fpahttyt oacstiderso iind sth, aen fdorpmh eonf oαl-ilcincolmenpiocu fnatdtys tahcaidt a(r∼e4k5n%o)w, vnaarinotuios xtiydpaenst osfu tbosctoapnhcesro[5ls, 7(–li9k]e. Aα,m βo γn,g anthde δm-taoncyopimheprorlt)a, npthdyetosisrteedrobiidosa,c atinvdit ipehs efonromlice dcoicmalpaonudnpdhs atrhmata acereu tkicnaolwapnp alinctaitoixoindsa,natn stiuobxsidtaancteasc [t5iv,7it–y 9is]. cAonmsoidnegr etdhea smoanneyo ifmthpeoprtraintc idpeaslireqdu birieomacetnivtsit.ies for medical and pharmaceutical applications, antioxInidtahnist awcotirvki,tyw ies ecvoanlsuiadteerdedth aesa onntieo oxfid thane tparcitnicviiptyalo rfetqhueitrhemreentytsp. e s of vegetable oil (from sacha inchIi,no tlhivise ws,oarnkd, wsoey ebveaalnusa)teadn dthteh aenAtiLox-CidSanta nacoteivmituyl soifo tnhsee tnhcraeep styuplaetsi nogf vthegeeotialbs.leT ohiel (afrnotmio xsiadcahnat ianccthivi,i toyliovfes,a amnpdl essoywbaesanmse) aasnudre tdhef oAr L6-0CmS inaunteose,maunldsitohnes ceunmcauplsautilvaetinpge rtcheen toaiglse. oTfhien ahnibtiiotixoinda(n%t) aisctipvrietyse onft esdaminplFeisg wuraes 1m0eAa,sBu.rAedm fonr g60t hmeinthurtees, taynpde sthoef couiml, usalacthivaei npcehrcieonitlapgree osef nintehdibtihtieonh i(g%h)e ist parnedsefnatsetdes itnc Faipgaucriety 10fo (rAr)a adnidca (lBs)c. aAvmenognign tgh.eI tnhr1e5e mtyipne,st hoef orield, suaccthioan inocfhfir oeiel prardesiceanltsedre tahceh hedighnesatr a9n0d% f.asOtenstt hcaepoatchiteyr fhoarn rdad, ioclaivl secanvdensgoiynbge. aIn 1o5i lmsihno, wtheed re3d5u%ctaionnd o2f5 f%re,er reasdpieccatlisv reelya,choefdfr neearra 9d0i%ca.l Oscna vtehneg iontghecra phaacnitdy, inol1iv5em ainda nsdoyrebaecahne douilp sthoo4w0e%d a3n5d%3 0a%n,dr e2sp5%ec,t ivreslpy,eocfticvuemlyu, loaft ivfreeien hriabditiicoanl . sEcvavenentghionugg chapoalcivitey ioni l15is mwine lalnkdn roewacnhetod ucopn ttoa 4in0%an atniodx 3id0%an, trecsopmecptoivneelny,t sofl ickuemoumlaetgivae fianthtyibiatcioidns. , Etovceonp thheoruolgsh, pohlievneo oliicl ciso mwpeollu knndosw, ans two ecllonatsacina raontetniooxiiddsansitm ciolamrptonseancths aliiknec hoim[5e4g,a5 5f]a,ttthy eamcidaisn, tdoicffoeprhenercoelsb,e tpwheennoltihce cmomisptohuentydps,e aosf twhelml asjo cratrooctoepnhoeidros lss.imOilliavre tooi lscaocnhta iinnscmhio [s5t4ly,5α5]-,t otchoep mhearionl , dwifhfeilreenthce bmetawineetnoc tohpehme riso ltshein tyspaec hoaf tihnec hmiaojoilr atorecoγp-haenrodlsδ. -Otolcivoep ohielr coolsn,tawinhsi cmhoasrtelym α-otroecoepffheectriovle, wanhtiiloex tidhea nmtsatihna ntoαc-otpohcoeprohlesr oinl [5sa6c,5h7a] . iTnhchisi eoxipl larine sγt-h atntdh eδr-atodcicoaplhsecarovlesn, gwinhgiccha paarcei tmy ofresa ecfhfaecitnicvhei aoniltiwoxaisdhaingths etrhtahna nα-tthoactoopfhoelrivole [o5i6l.,5M7]o. rTeohvise re,xcpolmaipnasr tehdawt tihthe αra-tdoiccoapl hsecraovle,nγg-tioncgo cpahpearociltfyr oomf ssaacchhaa iinncchii oisilc wonassi hdiegrhedert tohbaen hthigaht loyf poolitveen toailn. dMvoerreyoveffere,c ctoivmeptoarteradp wreitahc tαiv-teoncoitprohgeeronl,o γxi-d toescocponhseirdoelr ferdomas staocxhica tiontchei biso dcyon[5si7d].erTehde taon tbioe xhidigahnltya cptoivteitnyt oafnedn cvaeprsyu leaftfeedctviveeg ettoa btrleapo ilrseainctAivLe- CnSitrnoagneonp aorxtiidcleess cwoanssiadlseoreedv alsu taotxeidc. tCo hthiteo sbaondiys [c5o7n]s. iTdheree adnatisoaxnidaatunrt aalcatinvtiitoyx iodfa enntcpaoplsyumlaetreadn vdeigtestsacbalvee oniglsin ing aAcLti-vCitSy nhaansobpeaerntipclreosv ewnaasg aalisnos te1v,a1l-udaiptehde.n Cylh-2it-opsicarny lihs ycdornasziydlerraedi caasl sa( DnaPtPuHra•l )a, hnytidorxoidxyalnrta pdoiclyalm(•eOr Han)d, a intsd sscuapveernogxiindge raacdtiivciatly( •hOa – 2s )b[e5e8n– 60p]r.oTvhene ragdaicinalsts c1a,v1e-dnigpinhgencyalp-2a-cpiticyryoflhCySdrwazityhlo uratdoiiclailnsc o(rDpPoPraHti•o)n, hreyadcrhoexdylu rpadtoic3a0l %(•OinH6)0, amnidn .superoxide radical (•O2–) [58–60]. The radical scavenging capacity of CS withoEuntc oaipl siunlcaotrepdovreagtieotnab rleeaocihlsedin uApL t-oC S30n%an ionp 6a0r tmicliens. p resented similar radical scavenging behaviors compEanrceadpwsuitlahtethde irvefrgeeetaobilles amoiplsl eisn( FAigLu-rCeS1 0nBa).nSoapcahratiicnlcehs i poirleesnencatepdsu lsaimtedilainr AraLd-iCcSaln asncaovpeanrtgiicnlegs bsehhoawveiodrsth ceohmigphareesdt r waditihca tlhreeidr ufrcetieo onilr astaeminplaess h(Foirgtupreer i1o0dB,)9. 5S%achina 1in5cmhii no.ilA esnpcarpevsuioluatseldy pinr eAseLn-CteSd nfoanr othpearetinccleasp ssuhloawtioend etffihec ihenigchyesotf vraedgiectaalb lreedouilcstiionnA rLa-tCe Sinn aan oshemorut lpsieorniosd(,F i9g5u%re i9n) ,1s5a cmhaini.n Achsi porielvshioouwsleyd pthreeshenigtehde sftovra ltuhee oefnecnapcaspuslautliaotnio neffriactieenincyA oLf- CvSegneatnaboleem ouillssi oin sAaLn-dCaSl snoapnosesmesuslessiotnhse (sFtirgounrgee 9st),a snatcihoxa iidnacnhti coaipl ashciotyw. eTdh tehaen htiiogxhiedsat nvtaaluceti voift ieenscoafposulilvaetiaond rastoey ibne aAnLo-CilsS ennacnaopesmulualtseidonisn aAnLd- CalSson panoosspeasrsteics ltehseg srtardounaglelsyt iannctrieoaxsieddanwt icthaptaimcietya. nTdher eaancthioexdid90a%nt aanctdiv7i0ti%es ooff roaldivicea alnsdca svoeynbgeianng , orielssp eencctiavpeslyu,liante6d0 imn iAn.LA-CsSt hneaoniolspwaretirceleesn cgarpasduulaaltleyd iinncrsetaabsleedp warittihc lteism, teh aenrdel eraesaechperdofi 9l0e%of aonilds f7r0o%m othf eramdimcaigl hsctaavffeencgt itnhge, grreasdpuecatlilvyeilnyc, riena s6i0n gmaint. iAoxsi dthaen toailcst iwvietrye. encapsulated in stable particles, the release profile of oils from them might affect the gradually increasing antioxidant activity. Polymers 2019, 11, x FOR PEER REVIEW 13 of 18 Polymers 2019, 11, 1x2 F4O5 R PEER REVIEW 1133 ooff 18 (A) (B) (A) (B) Figure 10. Evaluation of the antioxidant activity of (A) free vegetable oils (from sacha inchi, olive, and soFyigbueraen1) 0a.nEdv (aBlu) oatiilo enncoafpthsuelaantetido xinid AanLt-CacSt invaitnyooefm(uAl)siforenes.v *eNgePt:a Nblaenooiplsa(rftriocmless. acha inchi, olive, and Fsoigyubreea n1)0.a Envda(lBu)atoiiolne nocf athpesu alnattieodxiidnaAnLt a-CctSivnitayn ooef m(Au)l fsrioeen sv.e*gNetPa:bNlea oniolsp (afrrtoimcle ssa. cha inchi, olive, and 3.6. Prsotyebine aLno)a adnindg ( BE)f foiicli encya psulated in AL-CS nanoemulsions. *NP: Nanoparticles. 3.6. Protein Loading Efficiency 3.6. TPhroet eeinff iLcoiaednicnyg oEff fipcrieontceyin loading in nanoparticles varies depending on the pH, formulation methoTdh e(meeffichciaenniccayl oefneprrgoyte ainpploliaeddi ning tihne nfoarnmopualarttiioclne)s, avnadri ersatdioe poef npdoilnygmoerns tahnedp pHro, tfeoinrm. AuLla-tCioSn nmaneotheTmohdue(l mseifoefnicchsi eaanrcieyc a polofes nipteirrvogetyelyian pc hlpoalairedgdiendign btienhc eanfuaosnremo pouafl ratthtiiceol enps) o,vlayancrdiaetrsiao dtnieoicp oecfnhpdaorinalycgmt eoren ro stfha cneh dpitpHors,oa tfneo irinmn. uAalcaLitd-iCoicnS mmneaedntiohaeo,m dw u(hmlsicieohcn hissa anlroieccapalto eesdnit eiovrnge lyyt haceph paorluigeteeddr iblnae yctheareu sofeof roemfmtuhulealstpiioonnly )cp, aaatnriotdinc lriecastc.ih oCa orhafi ctpoteosralynomf nceahrnist ooapsnaadnr tpiincrloeatsce iiadnri.ce AmoLfet-deCniaS , uwnsaehndioc ahesm icsualrlosrciioearnt esmd aaorteen rpitaholess iottoiuv tieenlryco lacryhpeaorrgaoetfede nmbeegucalastuiivosneel ypo afc rhtthaicrelg epesdo. lCmychaaictriooosnmainco lnceahcnuaorleapsca t[re6tri1 c]ol.e fI snca htrihetiosos fasttneu ndinyu ,s aeBcdSidAaics wmcaaersrd uiiesare, mdw athotie csrhtiua ilds ytl otchianetc eloodra pdoonirn agtth eeefn foeicugitaeetnrivc lyea lyoefc rsh aoacfrh geaem dinumclshaiioc ornoi lmp laooraltediceludels eA.s CL[6h-1Ci]tS.o sInnaantn honiesamnstouupldsayiro,tniBcsSl,eA sb ewacraaeus osuefst eitdn hutaosse sdtbu eadesyn c tahbrreroielaord amldyia ntsegtrueiadffilisec dtioe n aicnsy coaor fpmsoaorcadhteeal ninoecfg hapitirovoietlellyion ac dh[ae6dr2g–Ae6d5L] -m. CIaStc nrhoaamns oeblemeceunl esrsioe [np6so1,r].bt eIednc attuhhsiase ts itauh dahysi,g bBheSeeArn cwbornaocsae dunlstyreadst titouond s ioteufd dpayrso tthaeeimn l opadrdoeildnougfc eepfsrf iolcatireginecry[ 6p o2a–fr 6tsi5ac]cl.ehsaI t aihnnadchs liob oaeideln ilnorgae dpeefofdric tAeiedLn-ctChyaS, t bnaeacnhaoiugeshmee mur lcosoiroenn cisen,nt betrreaacctaituoiosnne osi ft bpheratowst eebienenep ntrh oebd rpuoocaeldyslmlya ergsr teaurndpdiae rpdtri coalteses ina n cmdauloosdaeed lai nnog fin efficprrecoaiteseneicn iy n,[ b6teh2ce–a 6pu5as]e.r tmiIct olerh esaiiszn etbe, erwaecnht iicorhen psroebsreuttewldtse eitnnh atihtm eapp roohlviygimnhger locaoandndicnpegnro tertfeafitinicoicenan uocsfy e p[a1rno7,t6ien4icn]r. epInars ooeduiunr cstethsue dlapyra,g rtethirce lp esaiszrietzi ceol,fe wAs haLin-cCdhS lr oepasaudrlittniscglie nesf idfmiicdpie rnnoocvtyi nn, gobtelaocbaludysi ncehg maenoffigrecei ,ei rnetcmeyraa[i1cnt7ii,on6ng4s] . inbIn eato wruaernesgnteu t dhfryeo, mptho el4y1smi0z etoro a4fn3A0dL np-CmroS tfepoiarn r atcilacl luceosend caiednn nitnroacttrineooanstsae b oilnfy BtchSheAa np (gaFerit,gircuelreme sa1iiz1neAi,n )wg. Thinhiceah l roraeansdguielntfgsr oienmf fiimc4i1ep0nrtcooyv 4ion3fg0 BlnSomAad fiionnrcgra eellaffcsioecdniec nwecniytthr [a 1at7ino, 6ni4ns]c.or IfenaB soSeuA irn (sF tiiutgsdu cyroe, nt1hc1eeAn st)ir.zaTet hiooefn lA o(LaFd-igCinuSgr pee affi1r1tcBici)el.en Asc ydt itodhf enB loSotAw nieonstctar bceolayns eccdhenawntrigatheti,o arnne mionfac iBrneSiaAnsge (0in.0 ia1t srgac·onLng−1ce)e, fnrthtorema tp i4or1no0t( eFtioing 4uw3r0ea sn1 1mnBo )ft.o Aer ntatltrlha cepoplnoecwde enistntrtacoto inothcnees n oAtrfL aB-tiCSoASn po(FfairBgtSuicArlee (s10. 1.0HA1o)g.w ·TLeh−ve1e )rl,o,t ahadet ipncogron etcefefininctiwreanaticsoynn os ft hBeignShtArea ripn tpchreaedna si0ne.dt5o wgt·hiLteh−1 A,a tnLh -eiCn lcSoraepdaisrnetgi ic nlee fisft.isc Hcieononcwcyee novtfer rat,htiaeot npc ro(oFnticegeiunnr teirn a1ct1rioBean).ss Aehdti gtshuebe rlsottawhnaetnisat 0lcl.yo5 ntgoc −1 ·e Lcnltor,saeth ioteon l voaafl dBuiSensAg n(ee0ffia.0rc 1i8 e0gn%·cLy,− 1ow),f hthiceh p prrroeomteteianiinni newdcra ewsa sintehodt mseunobtrsreta apBnpStieAadl.l yIinntot osca ltcohhseae AtionLcv-haCil Suo eipsl anlroetaiacdrlee8sd0.% AH, Lwo-whCieScvh enrae, nmaotae icmnoeundlcsewionintrhsa,m titohoneres phBriSogAthe.ienIrn mtshaiagcnhh at0 .bi5ne cg hp·Lio−os1ii,tl itlohonea edldoe adbdeAitnwLg-e CeeSfnfin ctahieneno tcewym oou fol sptihopeno sps,irttoehtleyeip ncrh oiantercgirneedams piegdohl tysubelbeespcttaornsoitltiyiaotlenlyse, d tAob Lec tlaownsede ntCot Shv, eaaltnuwdeos thnoupeasp rot hs8ei0t %eplay, rwtcihchlaiecr ghsie zdree mpdoiadliy nneeoldetc cwthroiatlnhyg tmees, o,brAeecL aBuaSnsAed .B CISnSA ,s iasnc ahd ast mhinuacslhl tpih oeolipyl maloretaircd (le6d0s ikAzDeLad-C)i dcSo mnoaptnacorheeamdn ugwlesi,tihobn eCscS,a utohsre ApBLrSo.A teiisna msmigahltl bpeo lpyomseitrio(6n0edk Dbeat)wcoemenp tahre dtwoit ohpCpSosoirteAlyL .charged polyelectrolytes, AL and CS, and thus the particle size did not change, because BSA is a small polymer (60 kDa) compared with CS or AL. (A) (B) Figure 11. Effect of protein(Aco)n centration on (A) the particle size and PDI and ((B)) loading efficiency of FAigLu-rCeS 1n1a. nEoffeemctu olsf iponropterienp acroendcewnittrhatsiaocnh oanin (cAh)i tohiel. pBaSrAti,cbleo sviiznee asnedru PmDaI labnudm (iBn.) loading efficiency of AL-CS nanoemulsion prepared with sacha inchi oil. BSA, bovine serum albumin. Figure 11. Effect of protein concentration on (A) the particle size and PDI and (B) loading efficiency of AL-CS nanoemulsion prepared with sacha inchi oil. BSA, bovine serum albumin. Polymers 2019, 11, 1245 14 of 18 Polymers 2019, 11, x FOR PEER REVIEW 14 of 18 33..77.. IInn VViittrroo RReelleeaassee AAfftteerr iinnccuubbaattiioonn ooff AALL--CCSS nnaannooeemmuullssiioonn iinn vvaarriioouuss tteessttiinngg mmeeddiiuumm,, tthhee aammoouunntt ooff BBSSAA rreelleeaasseedd ffrroomm ththeep apratirctlieclsews aws aesv aeluvaatluedataendd aisnpdr eiss epntreedseinntFeidg uinre F12ig. uInrev i1t2ro. pInr ovteitinror epleraostiening wrealsemasoinngit owreads amnodnaitvoereryd loanwda ma ovuenryt olfopwr oatmeinouwnats orfe lpearsoetedind uwriansg rinelceuabsaetdio dnu. rAinsgli ginhctluybhaitgiohner. aAm soluignhttolyf phriogtheienr wamasourenlte aosfe pdrointetihne wdaisg erestlievaeseedn ziny mthee cdoingteasitnivine genbzuyffmere ccoomntpaainreindgw biuthffetrh econmopna-dreigde wstiivthe tehnez nyomne- cdoignetastiinvien genbzuyffmere, cshonotwaiinnigngth beupfofesri,t isvheoewffiencgt othfee npzoysmitievse oenffethcte opfr oenteziynmreelse aosne tfhreo mprtohteeinp arretliecalesse. Hfroomwe tvheer ,ptahretimcleasx.i Hmouwmepveerrc, ethntea mgeaoxifmreulema speedrcpernottaeigne oonf lryelreeaascehde dprloestesitnh oannl1y2 r%ea(cghaesdtr liecsbsu tffhearn w12it%h p(geapsstirnice bnuzfyfmere w) iinth7 p2ehp.sin enzyme) in 72 h. IInn ppoollyymmeerriicc nnaannooppaarrttiicclleess,, tthhee ddrruugg rreelleeaassee rraattee ddeeppeennddss oonn sseevveerraall ffaaccttoorrss:: ((ii)) ddrruugg ssoolluubbiilliittyy;; ((iiii)) ddeessoorrppttiioonn ooff tthhee ssuurrffaaccee--bboouunndd oorr aaddssoorrbbeedd ddrruugg;; ((iiiiii)) ddrruugg ddiiffffuussiioonn tthhrroouugghh tthhee nnaannooppaarrttiiccllee maattrriixx;; ((iivv)) nnaannooppaarrttiiccllee maattrriixx eerroossiioonn oorr ddeeggrraaddaattiioonn;; aanndd ((vv)) tthhee ccoombbiinnaattiioonn ooff eerroossiioonn aanndd ddiiffffuussiioonn pprroocceesssseess [[6666]].. EEssppeecciiaallllyy wwhheenn tthhee nnaannooppaarrttiiccllee iiss ccooaatteedd bbyy ppoollyymeerr,, tthhee rreelleeaassee ooff tthhee ddrruugg ccaann bbee ccoonnttrroolllleedd bbyy ((iiiiii)) ddrruugg ddiiffffuussiioonn ffrroom tthhee ppoollyymeerriicc meembbrraannee.. AAss tthhee CCSS ccooaattiinngg wwaass ppeerrffoorrmeedd oovveerr AALL nnaannooppaarrttiicclleess iinn tthhee sseeccoonndd pprroocceessss,, tthhee pprrootteeiinn ddiiffffuussiioonn tthhrroouugghh CCSS meembbrraannee wwaass tthhee ddeecciissiivvee ffaaccttoorr oonn tthhee rreelleeaassee pprroofifillee iinn AALL--CCSS nnaannooppaarrttiicclleess.. TThhee ccoombbiinnaattiioonn ooff AALL aanndd CCSS ffoorrmss sstatabblelem memembrbarnaenseass aasr eas urletsouflet notaf negnletmanegnltesmaenndtsio nanicdi nitoenraicc tiionntesrbaecttwioenesn btheetwtweeonp othlyem tewrso, wpohliycmh emrsa,k weshiitchh amrdakfeosr iBt ShAartdo fdoirff BuSsAe ttoo tdhieffutesset itnog thmee tdesiutimng. Bmeesdidiuems t.h Baets, itdheesi tnhtaetr,a tchtieo inntbeertawcteioenn BbeStAweaennd BeSaAch aonfd tehaechco omf pthoen ceonmtspionntehnetsp ianr ttihcele psamrtiigclhets amlsioghcto anltsroib cuotnetrtiobuittse ltoow itsr elloewas reeloefaBseS Aof. ABSsAm. aAnsy mpraonteyi npsr,oBteSiAnsp, oBsSsAes speossasemssidese gamroiudpes garnoduposth aenrdp ooltahrefru pnocltaiorn faulngcrtoiounpasl. gTrhoeuypcsa. nThinetye rcaacnt winittehraCcSt, wAiLth, oCrSo, tAheLr, coorm otphoeurn cdosm, epiothuenrdbsy, eioitnhiecri bnyte iroanctiico innst,ehraycdtrioognesn, hbyodnrdosg,eonr boothnedrsv, aonr odtehreWr vaallns fdoerrc eWs a[6ll6s, 6fo7r].ceDsu [r6i6n,6g7t]h. eDcuhraining itnhtee rcahcatiionn inintearaqcuteioonu sinm aeqduieuomu,sB mSAedaiunmd ,C BSSfAor amndth CeSn foonr-mco tvhael ennotnly- lcionvkaeldenctolym lpinlekxeeds wcoimthpoluetxecsh awnigthinogutt hcehacnongifnogrm thatei ocnonoffoBrmSAatimono loecf uBleSsA. TmhoeleBcSuAle-sC. SThceo mBpSAle-xCeSs acoremppolteexnetsi aallrye mpoorteenfutinaclltyio nmaol raen dfusntacbtiloentahla nanthde sstianbgllee pthoalynm tehreo wsiningglet optohleymcoemr boinwaitniogn toof tthhee acottmribbuinteastioofnp orfo ttheein aattnrdibCutSesp oolfy pmroetre[i6n8 a].nd CS polymer [68]. Figure 12. Cumulative release of BSA over 72 h in different mediums and the control sample. Figure 12. Cumulative release of BSA over 72 h in different mediums and the control sample. 4. Conclusions 4. Conclusions The ultrasound-assisted encapsulation of vegetable oils in AL-CS nanoparticles was successfully accomThpeli ushlterdas. oFuunldl-cahssairsatecdte erinzcaatpiosnuloaftioAnL o-Cf vSepgeatratibcllee soiwls ains AcaLr-rCieSd noauntoptoarotipctliems wizaest shueccpehsysfsuiclalyl pacrocopmerptileisshaendd. Fpuhlal scehsatraabcitleitryizoaftiponar toicf leAsL. -ICnSt hpearftoircmlesu lwataios ncaorfrAiedL -CouStn taon ooepmtimuliszieo ntsh,et hpehytyspiceasl opfroopilesr,tcioesn caenndt rpahtiaosnes sotafbCilSit,ya nodf psaurrtfiaccletas.n Itnp tlhaye efdordmeucilsaitvioenr oolfe As oLn-CtSh enpanaroteicmleuclshioarnasc, ttehreis ttyicpselsi koef soiizlse,, cpoonlycednistpraetrisointys, oafn CdSs,u arnfadc seucrhfaarcgtaen. tT phleayeendca dpescuislaivtieo nroolefsb oion- athctei vpearotiilcslein cthoaAraLc-tCerSisptiacrst ilcilkees swizaes, polydispersity, and surface charge. The encapsulation of bio-active oils into AL-CS particles was evidenced by FT-IR and fluorescence microscopy analyses, and the sacha inchi oil showed a higher rate of encapsulation than other vegetable oils like soy and olive oils. Besides the oil encapsulation, Polymers 2019, 11, 1245 15 of 18 evidenced by FT-IR and fluorescence microscopy analyses, and the sacha inchi oil showed a higher rate of encapsulation than other vegetable oils like soy and olive oils. Besides the oil encapsulation, the sacha inchi oil encapsulated AL CS particles presented the best behavior on the phase stability with a long period. Sacha inchi oil and AL-CS particles loading the same oil presented very strong antioxidant activity. Owing to the stable polymeric membrane and interaction with CS or other components, the release rate of BSA from AL-CS was very slow and only reached less than 12% (gastric buffer with pepsin enzyme) in 72 h. Sacha inchi oil encapsulated in AL-CS nanoemulsions displayed possible applications as a drug delivery system with high stability and controlled release properties. Author Contributions: Conceptualization, S.K. and J.N.; methodology, S.K. and J.N.; formal analysis, S.K. and J.N.; investigation, M.E., S.K., D.C., C.S., J.N., A.C.-P. and J.N.; resources, S.K. and J.N.; data curation, S.K. and J.N.; writing—original draft preparation, M.E. and S.K.; writing—review and editing, J.N.; visualization, S.K. and J.N.; supervision, J.N.; project administration, S.K. and J.N.; funding acquisition, S.K. and M.E. Funding: This research was funded by Grant 151-2017-FONDECYT from consejo nacional de ciencia tecnología e innotecnológicavación (CONCYTEC), Peru. Conflicts of Interest: The authors declare no conflict of interest. References 1. Kumar, B.; Smita, K.; Cumbal, L.; Debut, A. Sacha inchi (Plukenetia volubilis L.) oil for one pot synthesis of silver nanocatalyst: An ecofriendly approach. Ind. Crops Prod. 2014, 58, 238–243. [CrossRef] 2. Kumar, B.; Smita, K.; Cumbal, L.; Debut, A. 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