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: [email protected]; 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]. TheThe AL-CS AL-CS particles particles were were prepared prepared in in three three steps: steps: (i) (i) o/ow/w nanoemulsionnanoemulsion formation formation by by ultrasound ultrasound ofof a amixture mixture of of an an AL AL aqueous aqueous solution, solution, surfactant, surfactant, and and the the organic organic phase phase (oil), (oil), (ii) (ii) ionotropic ionotropic gelation gelation ofof AL AL with with calcium calcium chloride chloride by bystirring stirring for for30 min, 30 min, and and (iii) (iii)CS coating CS coating on AL on particles AL particles by stirring by stirring for 30for min. 30 In min. the Infirst the step first (particle step (particle formation), formation), high-intensity high-intensity ultrasonication ultrasonication was applied was to applied the mixture to the formixture 3 min with for 3 temperature min with temperature controlled controlledwith an ice with bath an (7–10 ice bath°C). The (7–10 ultrasonic◦C). The probe ultrasonic was probecarefully was 1 carefully positioned at the aqueous solution and oil interface. A−1 0.3 g L AL solution was prepared positioned at the aqueous solution and oil interface. A 0.3 g·L AL ·solution− was prepared with distilledwith distilled water and water the and pH thewas pH adjusted was adjusted to 5–5.5. toCS 5–5.5. was dissolved CS was dissolved in 1% acetic in 1%acid acetic aqueous acid solution aqueous andsolution pH was and pHadjusted was adjusted to 4.5–5 to using 4.5–5 usingNaOH NaOH soluti solution.on. The Theexperimental experimental procedure procedure is ispresented presented schematicallyschematically in in Figure Figure 1.1 .

FigureFigure 1. 1. ExperimentalExperimental procedure procedure for for the the formulatio formulationn of ofsodium sodium alginate alginate (AL)-chitosan (AL)-chitosan (CS) (CS) nanoemulsionnanoemulsion encapsulating encapsulating vegetable vegetable oils. oils. In this study, we evaluated various factors that could affect the properties of the AL-CS particles, In this study, we evaluated various factors that could affect the properties of the AL-CS particles, among them, the type of oil and the concentrations of CS and surfactant. For each case, the oil was among them, the type of oil and the concentrations of CS and surfactant. For each case, the oil was added in the first step of the procedure and the volume ratio of AL solution to oil was kept constant at added in the first step of the procedure and the volume ratio of AL solution to oil was kept constant 9 to 1. at 9 to 1. Chemical structures of AL and CS were characterized by Proton nuclear magnetic resonance Chemical structures of AL and CS were characterized by Proton nuclear magnetic resonance (1H-NMR) with a Bruker Ascend 500 MHz spectrometer (Billerica, MA, USA). The molecular weight of (1H-NMR) with a Bruker Ascend 500 MHz spectrometer (Billerica, MA, USA). The molecular weight chitosan was determined by gel permeation chromatography (GPC) from Viscotek (Houston, TX, USA) of chitosan was determined by gel permeation chromatography (GPC) from Viscotek (Houston, TX, with a refractive index detector and pullulan standards. The molecular weight of AL was determined USA) with a refractive index detector and pullulan standards. The molecular weight of AL was by capillary viscometry with an Ubbelohde 1C viscometer (K = 0.02871 mm2 s 2) from Technical Glass determined by capillary viscometry with an Ubbelohde 1C viscometer (K =· −0.02871 mm2·s−2) from Products (St. Dover, NJ, USA). Technical Glass Products (St. Dover, NJ, USA). After the formation of the AL-CS nanoemulsions under different conditions, the characterization of After the formation of the AL-CS nanoemulsions under different conditions, the characterization the particles was carried out measuring their average size, polydispersity index (PDI), and zeta-potential. of the particles was carried out measuring their average size, polydispersity index (PDI), and zeta- potential.2.2.1. Types of Oil

2.2.1. TypesThree of di ffOilerent 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 Three different edible oils were evaluated: sacha inchi, olive,1 and soybean1 oils. All the oil market. The concentrations of AL and CS solutions were 0.3 g L− and 2.0 g L− , respectively. samples were produced by Peruvian companies (Lima, Peru) and· used as they· were obtained from the2.2.2. local Concentration market. The concentrations of CS of AL and CS solutions were 0.3 g·L−1 and 2.0 g·L−1, respectively.

2.2.2. ConcentrationThe effect of the of concentrationCS 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. The effect of the concentration of CS in solution on the particle property was studied.1 Chitosan Different concentrations of CS in solution were used, namely, 0, 0.3, 0.6, 1.5, and 2.0 g L− . 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 1 range of 4000 to 400 cm− . 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 DC Encapsulation efficiency (EE)(%) = initial DC − Free 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 L 1. 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. 1 H-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 (Figure2) 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

Polymers 2019, 11, 1245 6 of 18 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 weightweight (weight (weight average)average) ofof 886886 kDa kDa was was determined determined forfor CSCSwith with a a Malvern Malvern Viscotek Viscotek GPC GPC system system withwith an an aqueous aqueous Novema Novema Max Max analytical analytical linear linear XL XL column column and and a a refractive refractive index index detector. detector. Pullulan Pullulan standardsstandards (PSS(PSS PolymerPolymer StandardsStandards ServiceService GmbH,GmbH, 10–200010–2000 kDa)kDa) werewere usedused asas aa referencereference and and an an HOAcHOAc/NaOAc/NaOAc bu bufferffer (pH (pH= =4.5) 4.5) was was used used as as mobile mobile phase phase [ 29[29].].

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.For This AL, calculation 1H-NMR is morespectroscopy complex wa [30s] used and is to detailed determine in a previousa ratio of work, 1.57 mannuronic/guluronic in which the chemical structureunits. This of calculation this AL sample is more was complex proven to[30] be and adequate is detailed for ionotropic in a previous gelation work, with in which calcium the ions chemical [31]. Astructure molecular of this weight AL sample (viscosity) was ofproven 78 kDa to be was adequate measured for forionotropic AL by gelation capillary with viscosity calcium in ions a 0.1 [31]. M A molecular weight (viscosity) of 78 kDa was measured for AL by capillary viscosity in a 0.1 M NaCl NaCl aqueous solution at 25 ◦C using the Mark–Houwink equation with constants of δ = 0.92 and Kaqueous= 0.0073 solution cm3/g [ 32at ].25 °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 3.1.1. Types of Oil To determine the influence of the oil type on the properties of the nanoemulsion particles’ size, PDI andTo determine zeta potential the influence were measured, of the oil and type the on results the properties are presented of the in nanoemulsion Figure3. The particles’ oil in water size, ALPDI nanoemulsion and zeta potential obtained were inmeasured, the first and stage the of result the processs are presented was compared in Figure with 3. The the oil final in water AL-CS AL nanoemulsion.nanoemulsion ALobtained particles in prepared the first with stage the of di fftheerent process oils showed was compared similar sizes with with the values final around AL-CS 320~340nanoemulsion. nm, and AL an particles increase prepared of particle with size the was differ observedent oils after showed the last similar stage sizes in which with a values coating around with CS320~340 was formed nm, and (Figure an increase3A). Contrary of particle to AL, size CS was is aobserved high molecular after the weight last stage cationic in which polyelectrolyte a coating with in solution;CS was formed therefore, (Figure an increase 3A). Contrary in particle to sizeAL, isCS expected is a high when molecular a layer weight of CS depositscationic overpolyelectrolyte the outer layerin solution; of AL as therefore, a result of an electrostatic increase in attraction particle size [20]. is expected when a layer of CS deposits over the outerIn layer the case of AL of particlesas a result prepared of electrostatic with sacha attraction inchi oil, [20]. the size increase was much higher (30%) than for oliveIn the and case soybean of particles oils, 3% prepared and 1%, respectively. with sacha inchi After oil, storage the size at ~5 increase◦C forseven was much days, thehigher particles (30%) werethan analyzedfor olive again.and soybean The size oils, of particles3% and 1%, prepared respectively. with sacha After inchi storage and oliveat ~5 oils°C for slightly seven increased, days, the showingparticles higher were analyzed stability thanagain. the The particles size of preparedparticles withprepared soybean with oil. sacha The inchi size polydispersityand olive oils slightly of AL particlesincreased, was showing quite low higher ( 0.25) stability and increased than afterthe CSparticles coating prepared (Figure3 B)with in all soybean samples. oil. The size ≤ polydispersityFigure3C shows of AL theparticles changes was of quite the zeta-potential low (≤0.25) and values increased of oil loadedafter CS AL coating and AL-CS (Figure particles. 3B) in all Thesamples. zeta-potential is an important parameter for the electrical characterization of nanoemulsions preparedFigure with 3C polyelectrolytes, shows the changes as theof the adsorption zeta-potential of charged values counterof oil loaded ions canAL and be measured AL-CS particles. by its valueThe zeta-potential and sign [33]. is The an type important of oil used parameter for the preparationfor the electrical of the characterization AL emulsion greatly of nanoemulsions affected the zeta-potential.prepared with As polyelectrolytes, expected, surface as charges the adsorption in the AL of emulsions charged counter prepared ions with can vegetable be measured oils showed by its

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 showednegative negative values because values because of the carboxylate of the carboxylate groups of gr AL,oups a polyanionicof AL, a polyanionic chain [19 chain,34]. The [19,34]. AL emulsion The AL prepared with sacha inchi oil showed the most negative values ( 22 mV), followed by soybean oil emulsion prepared with sacha inchi oil showed the most negative− values (−22 mV), followed by ( 11 mV), and finally olive oil ( 5 mV). After coating with CS, the surface charges of the particles soybean− oil (−11 mV), and finally− olive oil (−5 mV). After coating with CS, the surface charges of the particleschanged changed from negative from negative to positive to aspositive a result as of a re thesult presence of the presence of the protonated of the protonated amino groups amino in groups CS at a inpH CS between at a pH between 4.5 and 5 4.5 [22 and]. Among 5 [22]. Among the three the oils, thr sachaee oils, inchi sacha oil inchi presented oil presented the most the dramatic most dramatic change of its zeta-potential, from 22 to +35 mV, for AL and AL-CS particles, respectively. As mentioned change of its zeta-potential,− from −22 to +35 mV, for AL and AL-CS particles, respectively. As mentionedbefore, the before, zeta-potential the zeta-potential is an indicator is an indicator of not only of not the only surface the surface charge charge of the of particles, the particles, but but also alsotheir their stability stability [34 ].[34]. CS-AL CS-AL particles particles prepared prepared with with sacha sacha inchi inchi oil oil were were expected expected to to be be very very stable stable as astheir their value value of of the the zeta-potential zeta-potential was was inin thethe rangerange of >|>|30|.30|. ThisThis highhigh stability could be be the the result result of of self-assemblyself-assembly of of the the two two polymers polymers around around the the oil oil co corere owing owing to to their their strong strong electrostatic electrostatic attraction attraction fromfrom their their opposite opposite charge charge nature nature [20]. [20].

(A)

(B)

Figure 3. Cont.

Polymers 2019, 11, x FOR PEER REVIEW 8 of 18 Polymers 2019, 11, 1245 8 of 18 Polymers 2019, 11, x FOR PEER REVIEW 8 of 18

(C) (C)

FigureFigure 3. EffectEffect ofof thethe type type of of oil oil on on the the properties properties of AL of nanoemulsion AL nanoemulsion (AL)and (AL) CS and coated CS ALcoated particles AL Figure 3. Effect of the type of oil on the properties of AL nanoemulsion (AL) and CS coated AL particles(AL-CS) on(AL-CS) the particle on the size particle (A), size size polydispersity (A), size polydispersity (B), and zeta-potential (B), and zeta-potential (C), when prepared (C), when and particles (AL-CS) on the particle size (A), size polydispersity (B), and zeta-potential (C), when preparedafter seven and days. after PDI, seven polydispersity days. PDI, polydispersity index. index. prepared and after seven days. PDI, polydispersity index. 3.1.2.3.1.2. Concentration Concentration of of CS CS Solutions Solutions 3.1.2. Concentration of CS Solutions TheThe effect effect of of the the CS CS solution solution concentration concentration on on the the formulation formulation of of AL-CS AL-CS nanoemulsions nanoemulsions was was studiedstudiedThe in ineffect terms terms of of ofthe particles CS solution size, PDI,concentration and zeta-potential. on the formulation A A low concentration of AL-CS nanoemulsions of CS (≤0.6 g·L g wasL−11) ) ≤ · − resultedstudiedresulted inin in termsno no change change of particles in in size size,nor zeta-potentialPDI, and zeta-potential. of the AL-CSAL-CS A low nanoemulsions.nanoemulsions. concentration The Theof CSzeta-potential zeta-potential (≤0.6 g·L−1) remainedresultedremained in with with no achange a negative negative in sign,size sign, norsuggesting suggesting zeta-potential that that the the of positively positively the AL-CS charged charged nanoemulsions. amino amino groups groups The of ofzeta-potential CS CS were were not not enoughremainedenough to to withneutralize neutralize a negative the the negatively negatively sign, suggesting charged charged that carbox carboxylate the positivelyylate groups groups charged in in AL AL amino[35]. [35]. When When groups CS CS of concentration concentration CS were not wasenoughwas increased increased to neutralize to to 1.5 1.5 g·L gtheL−1, negatively1the, the size size increased increasedcharged and carbox and the the ylatezeta-potential zeta-potential groups in became AL became [35]. positive, When positive, CSas can asconcentration can be seen be seen in · − Figurewasin Figure increased 4. This4. This observation to 1.5 observation g·L−1, hasthe hasbeensize beenincreased previously previously and report the reported zeta-potentialed by Natrajan by Natrajan became et al. (2015) et positive, al. (2015)and Choias and can et Choibe al. seen (2011) et al.in [22,33].Figure(2011) [4.However,22 This,33]. observation However, when the when has concentration been the concentrationpreviously of CS report was of higher CSed wasby thanNatrajan higher 1.5 thang·L et −al.1, 1.5 the (2015) g sizeL 1and and, the Choi PDI size ofet and al.particles PDI(2011) of · − decreased[22,33].particles However, decreased and the when zeta-potential and the the concentration zeta-potential was also of was reduced.CS alsowas reduced.higher Wang than and Wang 1.5 his g·L and colleagues−1, his the colleagues size andalso PDI observed also of observedparticles this phenomenondecreasedthis phenomenon and with the withhigher zeta-potential higher concentration concentration was alsoof CS reduced. of solutions, CS solutions, Wang and reported andand reportedhis thatcolleagues thatit may it maybealso the beobserved result the result of thisthe of + saturationphenomenonthe saturation of ion with of logarithmic ion higher logarithmic concentration formed formed by ofelec by CStrostatic electrostatic solutions, attraction and attraction reported forces forces betweenthat betweenit may the be –NH the the –NH 3result+ of 3CS ofof and the CS + thesaturationand –COO the –COO− of AL,ion− of aslogarithmic AL, well as as well an formed increase as an increase by viscosity elec viscositytrostatic of the attraction ofCS the solution, CS solution,forces making between making the reaction the the –NH reaction difficult3 of diCSffi with andcult thethewith AL–COO the surface AL− of surface AL, [35]. as well [35]. 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. Figure(A) Size 4./ PDIEffect and of (CSB) zeta-potential.concentration on the properties of sacha inchi oil loaded CS-AL nanoemulsion. (FigureA) Size/PDI 4. Effect and of ( BCS) zeta-potential. concentration on the properties of sacha inchi oil loaded CS-AL nanoemulsion. (A) Size/PDI and (B) zeta-potential.

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3.1.3.3.1.3. Concentration Concentration of of Surfactant Surfactant SurfactantsSurfactants areare frequently frequently used used in the in preparation the prepar of nanoparticlesation of nanoparticles to optimize theirto optimize physiochemical their physiochemicalproperties and stability.properties Di ffanderent stability. concentrations Different of theconcentrations non-ionic surfactant, of the non-ionic poloxamer surfactant, 407, were poloxamerused to encapsulate 407, were sachaused to inchi encapsulate oil into the sacha AL-CS inchi particles. oil into Thethe eAL-CSffect on particles. the particle The size,effect PDI, on andthe particlezeta-potential size, PDI, is evaluated and zeta-potential as shown is in evaluated Figure5. Poloxameras shown in 407 Figure has a 5. triblock Poloxamer structure 407 has containing a triblock a structurecentral hydrophobic containing a block central of polyoxypropylene hydrophobic block (POP) of polyoxypropylene and two identical (POP) lateral and hydrophilic two identical chains lateralof polyoxyethylene hydrophilic chains (POE) of [polyoxyethylene36]. Poloxamers (POE) have an[36]. amphiphilic Poloxamers character have an amphiphilic with both association character withand adsorptionboth association properties and adsorption and improve properties solubilization and improve and stabilization solubilization of compounds and stabilization as a result of compoundsof their notable as a result physiological of their notable properties physiological and low properties toxicity [37 and]. Poloxamers low toxicity and[37]. poloxaminesPoloxamers and are poloxaminesnon-ionic surfactants are non-ionic that have surfactants diverse applicationsthat have diverse in various applications biomedical in fieldsvarious such biomedical as drug delivery, fields suchmedical as drug imaging, delivery, as well medical as cancer imaging, therapy as [well38]. Inas acancer previous therapy work, [38]. it was In founda previous that anwork, increase it was of foundconcentration that an increase of poloxamer of concentration 407 produces of a poloxamer reduction of 407 both produces particle a size reduction and PDI of [ 17both]. Similar particle results size andwere PDI observed [17]. Similar in AL-CS results nanoemulsion were observed encapsulating in AL-CS nanoemulsion sacha inchi oil. encapsulating According tosacha the literature,inchi oil. Accordingnon-ionic surfactantsto the literature, like poloxamer non-ionic dosurfactants not significantly like poloxamer affect the do zeta-potential not significantly [39,40 affect]. However, the zeta- in potentialour study, [39,40]. a variation However, of the zeta-potentialin our study, wasa variation observed of with the poloxamer zeta-potential concentration was observed (Figure with5B). poloxamerThe zeta-potential concentration of sacha (Figure inchi oil5B). encapsulated The zeta-pot inential AL-CS of particlessacha inchi decreased oil encapsulated when the poloxamerin AL-CS particlesconcentration decreased changed when from the poloxamer 0.1% to 0.3% concentrationw/v and increased changed when from higher 0.1% to a concentration0.3% w/v and increased was used when(0.5% higher and 1.0%, a concentrationw/v). 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 Figureinchi oil 5. loadedEffect of AL-CS poloxamer nanoemulsion. 407 concentration on (A) size and PDI and (B) zeta-potential of sacha inchi oil loaded AL-CS nanoemulsion. 3.2. Emulsion Stability 3.2. EmulsionThe phase Stability separation of the AL-CS nanoemulsions for a long time (six months) was visually comparedThe phase as an separation evaluation of of the their AL-CS physical nanoemulsions stability (Figure for a6 ).long Storage time time,(six months) temperature, was visually type of comparedstabilizer, andas an type evaluation of oil phase of their often physical affect phase stabil separationity (Figure and6). Storage show e fftime,ects liketemperature, creaming type [41– 43of]. stabilizer,The phase and separation type of oil of phase the serum often fromaffect anphase emulsion separation often and arises show because effects of like the creaming flocculation [41–43]. and Themigration phase ofseparation oil molecules of the from serum smaller from droplets an emulsi to largeron often ones arises through because the aqueousof the flocculation phase, which and is migrationknown as of the oil Ostwald molecules ripening from esmallerffect [41 droplets,44]. In AL-CS to larger nanoemulsions, ones through the the concentration aqueous phase, of surfactant which is knownand the as type the of Ostwald oil affected ripening the physical effect stability, [41,44].producing In AL-CS di nanoemulsions,fferent levels of serumthe concentration separation. of surfactantIn AL-CS and the nanoemulsions, type of oil affected poloxamer the physical concentrations stability, of producing 0.1% and different 0.5% and levels the type of serum of oil separation.determined the phase separation of the system. When higher concentrations of poloxamer were used, betterIn physicalAL-CS nanoemulsions, stability of AL-CS poloxamer emulsion concentrations was observed, of showing 0.1% and less 0.5% phase and separation the type than of oil for determinedlower poloxamer the phase concentrations. separation of Notable the system. phase When separation higher of concentrations serum with high of poloxamer turbidity was were visually used, betterobserved physical in samples stability prepared of AL-CS with emulsion 0.1% of was poloxamer, observed, showing showing a high less ratephase of separation creaming (height than for of lowerserum poloxamer (Hs) compared concentrations. with the height Notable of phase cream separation (Hc)) [43,44 of]. serum In comparison with high with turbidity soybean was and visually olive observedoils, AL-CS in nanoemulsionssamples prepared encapsulating with 0.1% of sacha poloxamer, inchi oil showedshowing higher a high physical rate of creaming stability with (height a more of 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 stablemore stable emulsion. emulsion. This observation This observation is in accordance is in accordance with the with results the fromresults Figure from3. Figure The zeta-potential, 3. The zeta- thepotential, direct indicatorthe direct of indicator particle stability,of particle was stability, the highest was ( +the35 mV)highest in AL-CS (+35 mV) nanoparticles in AL-CS encapsulatingnanoparticles sachaencapsulating inchi oil sacha and remained inchi oil afterand re onemained week after of storage. one week of storage.

(A) (B)

Figure 6. Phase separation images of AL-CS nanoemulsions (A: soybean oil, B: sacha inchi oil, C: olive oil)Figure prepared 6. Phase with separation different images surfactant of AL concentrations,-CS nanoemulsions that is, (A: (A) soybean 0.1% and oil, (B B:) 0.5%. sacha The inchi samples oil, C: olive were

storedoil) prepared for six monthswith different at 5 ◦C. 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. FT-IRThe encapsulationAnalysis of sacha inchi oil in AL-CS nanoemulsions was verified by FTIR-ATR spectroscopyThe encapsulation (presented inof Figure sacha7 ).inchi Spectra oil ofin fats AL-CS and oilnanoemulsions samples show peakswas verified and shoulders by FTIR-ATR that are attributedspectroscopy to their(presented specific in functional Figure 7). groupsSpectra [45of ].fats Guti andérrez oil samples et al. and show Guill peaksén et al.and have shoulders previously that reportedare attributed the physicochemical to their specific characterization functional groups of sacha [45]. inchiGutiérrez oil using et al. FTIR and spectroscopyGuillén et al. [46 have,47], 1 whichpreviously coincides reported with the the physicochemical spectra of our sample. characterization The 3010 of cm sacha− band inchi is oil attributed using FTIR to the spectroscopy stretching vibration[46,47], which of cis coincidesolefinic C–H with in the carbon–carbon spectra of our double sample. bonds The [46 3010]. The cm two−1 band absorption is attributed bands atto 2910 the 1 andstretching 2854 cmvibration− correspond of cis olefinic to the methyleneC–H in carbon–carbon asymmetric anddouble symmetric bonds [46]. stretching The two vibration absorption [47]. 1 Thebands characteristic at 2910 and band 2854 atcm 1743−1 correspond cm− corresponds to the methylene to the stretching asymmetric vibration and symmetric of the C=O stretching group of 1 triacylglycerols.vibration [47]. The The characteristic small band band detected at 1743 at cm 1650−1 corresponds cm− is assigned to theto stretching the disubstituted vibration cisof theC= C=OC of 1 thegroup unsaturated of triacylglycerols. acyl groups The and small the band band detected at 1460 cmat 1650− is cm attributed−1 is assigned to the to bending the disubstituted vibrations cis of 1 theC=C CH of 2theand unsaturated CH3 aliphatic acyl groupsgroups [and46,47 the]. Theband two at 1460 bands cm at−1 1238is attributed and 1163 to cm the− bendingcorrespond vibrations to the stretchingof the CH2 vibrationand CH3 aliphatic of the C–O groups ester [46,47]. groups The and two to thebands bending at 1238 vibration and 1163 of cm the−1 correspond CH2 groups to that the 1 arestretching commonly vibration found of in the many C–O typesester groups of vegetable and to oils the [ 47bending,48]. The vibration band that of the appears CH2 groups at 730 that cm− areis attributablecommonly found to the overlapin many of thetypes methylene of vegetable (CH2 )oils rocking [47,48]. vibrations The band and tothat the appears out of plane at 730 vibrations cm−1 is ofattributablecis-disubstituted to the olefinsoverlap [48 of]. Thesethe methylene characteristic (CH2 transmittance) rocking vibrations bands of and sacha to inchithe out oil wereof plane also detectedvibrations in of AL-CS cis-disubstituted nanoemulsions olefins prepared [48]. These with characteristic sacha inchi oil, transmittance which is evidence bands of sacha the successful inchi oil encapsulationwere also detected of the in oil AL-CS into the nanoemulsions nanoemulsion. prepared with sacha inchi oil, which is evidence of the successfulBesides encapsulation the characteristic of th bandse oil into of sachathe nanoemulsion. inchi oil in the nanoemulsion, the interaction between the amineBesides groups the of chitosancharacteristic and the bands carboxylate of sacha groups inchi oil of alginatein the nanoemulsion, can also be evidenced. the interaction The shift between of the 1 peakthe amine at 1660 groups to 1650 of cmchitosan− is attributed and the carboxylate to C=O stretching groups vibration of alginate and can the also disappearance be evidenced. of theThe –NH shift2 1 bendingof the peak peak at 1660 at 1602 to 1650 cm− cmin− chitosan1 is attributed was observed to C=O stretching after the finalvibration stage and of the the AL-CS disappearance nanoemulsion of the 1 formation–NH2 bending [49]. peak The broad at 1602 shoulder cm−1 in near chitosan 3400 cm was− indicatesobserved theafter presence the final of hydroxylstage of andthe AL-CS amino groupsnanoemulsion from AL formation and CS polymers. [49]. The broad shoulder near 3400 cm−1 indicates the presence of hydroxyl and amino groups from AL and CS polymers.

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FigureFigureFigure 7.7. Fourier-transform7.Fourier-transform Fourier-transform infraredinfrared infrared attenuated attenuated attenuated total total total reflectance re flectancereflectance (FTIR-ATR) (FTIR-ATR) (FTIR-ATR) of sachaof ofsacha sacha inchi inchi oil,inchi oil, AL, oil, AL, CS, AL, andCS,CS, and a sampleand a samplea sample of AL-CS of ofAL-CS AL-CS nanoparticles nanopa nanoparticlesrticles with with sacha with sacha inchisacha inchi oil. inchi oil. oil.

3.4. Oil Encapsulation E ciency 3.4.3.4. Oil Oil Encapsulation Encapsulation Efficiencyffi Efficiency NileNileNile red red is is isa a lipophilica lipophilic lipophilic dye dyedye and and has has has been been been used used used to tost to ainstain stain lipids, lipids, lipids, cells, cells, cells, and and oils and oils [50–53]. oils[50–53]. [50 In– 53In this]. this Instudy, study, this study,NileNile red Nilered was was red employed wasemployed employed to tostain stain to vegetable stain vegetable vegetable oils oils to toev oils evaluate toaluate evaluate oil oil encapsulation encapsulation oil encapsulation efficiency. efficiency. efficiency. Figure Figure Figure8 shows8 shows8 showsfluorescencefluorescence fluorescence microscopy microscopy microscopy images images images (Leica, (Leica, (Leica, Wetzlar, Wetzlar, Wetzlar, Germany) Germany) Germany) of of ofa asacha a sacha inchi inchiinchi oil oiloil loaded loaded loaded AL-CSAL-CS AL-CS nanoemulsion,nanoemulsion,nanoemulsion, whichwhich which isis evidenceisevidence evidence ofof ofthethe the successfulsuccessful successful loadingloading loading ofof ofthethe the oil.oil. oil.

FigureFigureFigure 8.8. Microscopy8. Microscopy images images (x100) (x100) of of AL-CS AL-CS nanoemulsion nanoemulsion preparedprepared prepared withwi with thsachasacha sacha inchi:inchi: inchi: fluorescencefluorescence fluorescence filterfilterfilter TX2:BPTX2:BP TX2:BP 560560/40 560/40/40 (red(red (red colorcolor color correspondscorresponds corresponds toto sachatosacha sacha inchiinchi inchi oiloil oil stainedstained stainedwith with withNile Nile Nilered). red). red).

TheTheThe encapsulation encapsulation eefficiencyffi efficiencyciency waswas was alsoalso also determineddetermined determined andan and isisd presentedispresented presented inin inFigureFigure Figure9 9.. The 9.The The unloaded unloaded unloaded oil oil oil fromfromfrom AL-CSAL-CS AL-CS nanoemulsionsnanoemulsions nanoemulsions waswas was separatedseparated separated byby by centrifugationcentri centrifugationfugation usingusing using ethanolethanol ethanol andand and thethe the supernatantsupernatant supernatant waswas was analyzedanalyzedanalyzed with with UV–Vis UV–Vis spectroscopyspectroscopy spectroscopy atat at553553 553 nm.nm. nm. SachaSacha Sacha inchiinchi inchi oiloil oil showedshowed showed thethe the highesthighest highest valuevalue value ofof of loadingloading loading eefficiencyffiefficiencyciency comparedcompared compared with with with olive olive olive and and and soybean soybean soybean oils, oils, oils, although although although the the total the total encapsulatedtotal encapsulated encapsulated oil quantityoil oil quantity quantity was was less was thanlessless than 40% than 40% in 40% the in nanoemulsion. inthe the nanoemulsion. nanoemulsion. Encapsulation Encapsulation Encapsulation of olive of ofolive and olive soybeanand and soybean soybean oil in oil AL-CS oil in inAL-CS AL-CS nanoemulsions nanoemulsions nanoemulsions was lower,waswas lower, aroundlower, around around 30~35%. 30%~35%. 30%~35%. This di ffThiserence This difference difference might be might because might be be ofbecause oilbecause composition of ofoil oil composition composition and physical and and properties physical physical suchpropertiesproperties as density such such andas as density viscosity. density and and viscosity. viscosity.

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Figure 9. The oil encapsulation efficiency of AL-CS nanoemulsions prepared with different types of oil: Figure 9. The oil encapsulation efficiency of AL-CS nanoemulsions prepared with different types of sacha inchi, olive, and soybean (solution to oil ratio was fixed to 9:1). 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 importantMost roleof the in protectionunrefined againstvegetable oxidation, oils naturall both iny oilcontain stability antioxidant and as nutritional compounds benefits that [ 7play]. Sacha an importantinchi seeds role have in recently protection become against a popular oxidation, nutrient both source in oil containing stability highand levelsas nutritional of omega benefits 3 fatty acids [7]. Sachain the inchi form ofseedsα-linolenic have recently fatty acid become ( 45%), a popular various nu typestrient of tocopherolssource containing (like α ,highβ γ, levels and δ-tocopherol), of omega 3 ∼ fattyphytosteroids, acids in the and form phenolic of α-linolenic compounds fatty thatacid are(∼45%), known various antioxidant types of substances tocopherols [5, 7(like–9]. Amongα, β γ, and the δmany-tocopherol), important phytosteroids, desired bioactivities and phenolic for medical compou andnds pharmaceutical that are known applications, antioxidant antioxidant substances activity [5,7– 9].is consideredAmong the as many one of important the principal desired requirements. bioactivities for medical and pharmaceutical applications, antioxidantIn this activity work, we is evaluatedconsidered the as antioxidantone of the principal activity of requirements. the three types of vegetable oil (from sacha inchi,In olives, this work, and we soybeans) evaluated and the the antioxidant AL-CS nanoemulsions activity of the encapsulatingthree types of vegetable the oils. The oil (from antioxidant sacha inchi,activity olives, of samples and soybeans) was measured and the for AL-CS 60 minutes, nanoem andulsions the cumulativeencapsulating percentage the oils. ofThe inhibition antioxidant (%) activityis presented of samples in Figure was 10measuredA,B. Among for 60 the minutes, three typesand the of cumulative oil, sacha inchi percentage oil presented of inhibition the highest (%) is presentedand fastest in capacityFigure 10 for (A) radical and (B). scavenging. Among the Inthree 15 min,types the of oil, reduction sacha inchi of free oil presented radicals reached the highest near and90%. fastest On the capacity other for hand, radical olive scavenging. and soybean In 15 oil mi showedn, the reduction 35% and of 25%, free respectively, radicals reached of free near radical 90%. Onscavenging the other capacity hand, inolive 15 min and and soybean reached oil up showed to 40% and35% 30%, and respectively, 25%, respectively, of cumulative of free inhibition. radical scavengingEven though capacity olive in oil 15 is min well and known reached to up contain to 40% antioxidant and 30%, respectively, components of like cumulative omega fattyinhibition. acids, Eventocopherols, though phenolicolive oil compounds,is well known as to well contain as carotenoids antioxidant similar components to sacha like inchi omega [54,55 fatty], the acids, main tocopherols,difference between phenolic them compounds, is the type as of thewell major as carotenoids tocopherols. similar Olive to oil sacha contains inchi mostly [54,55],α-tocopherol, the main differencewhile the between main tocopherols them is the in type sacha of the inchi major oil aretocopherols.γ- and δ -tocopherols,Olive oil contains which mostly are moreα-tocopherol, effective whileantioxidants the main than tocopherolsα-tocopherol in [56sacha,57]. inchi This explainsoil are γ that- and the δ radical-tocopherols, scavenging which capacity are more of sacha effective inchi antioxidantsoil was higher than than α-tocopherol that of olive [56,57]. oil. Moreover, This explains compared that with the αradical-tocopherol, scavengingγ-tocopherol capacity from of sacha sacha inchiinchi oil is consideredwas higher to than be highlythat of potentolive oil. and Moreover, very effective compared to trap with reactive α-tocopherol, nitrogen oxides γ- tocopherol considered from as sachatoxic toinchi the bodyis considered [57]. The to antioxidant be highly activity potent ofand encapsulated very effective vegetable to trap oils reactive in AL-CS nitrogen nanoparticles oxides consideredwas also evaluated. as toxic to Chitosan the body is [57]. considered The antioxidan as a naturalt activity antioxidant of encapsulated polymer and vegetable its scavenging oils in activityAL-CS nanoparticleshas been proven was against also evaluated. 1,1-diphenyl-2-picrylhydrazyl Chitosan is considered radicals as a (DPPHnatural ),antioxidant hydroxyl radical polymer ( OH), and andits • • scavengingsuperoxide radicalactivity ( hasO – )[been58– 60proven]. The radicalagainst scavenging 1,1-diphenyl-2-picrylhydrazyl capacity of CS without radicals oil incorporation (DPPH•), • 2 hydroxylreached up radical to 30% (•OH), in 60 and min. superoxide radical (•O2–) [58–60]. The radical scavenging capacity of CS withoutEncapsulated oil incorporation vegetable reached oils in up AL-CS to 30% nanoparticles in 60 min. presented similar radical scavenging behaviors comparedEncapsulated with their vegetable free oil samples oils in (Figure AL-CS 10 nanoparticlesB). Sacha inchi presented oil encapsulated similar in AL-CSradical nanoparticlesscavenging behaviorsshowed the compared highest radicalwith their reduction free oil rate samples in a short (Figure period, 10B). 95% Sacha in 15inchi min. oil As encapsulated previously presentedin AL-CS nanoparticlesfor the encapsulation showed ethefficiency highest of vegetableradical reduction oils in AL-CSrate in nanoemulsions a short period, (Figure 95% 9in), 15 sacha min. inchi As previouslyoil showed presented the highest for value the ofencapsulation encapsulation effici rateency in AL-CS of vegetable nanoemulsions oils in AL-CS and also nanoemulsions possesses the (Figurestrongest 9), antioxidantsacha inchi capacity.oil showed The the antioxidant highest value activities of encapsulation of olive and rate soybean in AL-CS oils encapsulatednanoemulsions in andAL-CS also nanoparticles possesses the gradually strongest increasedantioxidant with capacity time and. The reached antioxidant 90% andactivities 70% ofof radicalolive and scavenging, soybean oilsrespectively, encapsulated in 60 in min. AL-CS As the nanoparticles oils were encapsulated gradually increased in stable particles, with time the and release reached profile 90% of and oils from70% ofthem radical might scavenging, affect the graduallyrespectively, increasing in 60 min. antioxidant As the oils activity. were encapsulated in stable particles, the release profile of oils from them might affect the gradually increasing antioxidant activity.

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(A) (B)

(A) (B) Figure 10. Evaluation of the antioxidant activity of (A) free vegetable oils (from sacha inchi, olive, and soybean)Figure 10. andEvaluation (B) oil encapsulated of the antioxidant in AL-CS activity nanoemulsions. of (A) free vegetable *NP: Nanoparticles. oils (from sacha inchi, olive, and Figuresoybean) 10. and Evaluation (B) oil encapsulated of the antioxidant in AL-CS activity nanoemulsions. of (A) free vegetable *NP: Nanoparticles. oils (from sacha inchi, olive, and 3.6. Proteinsoybean) Loading and ( BEfficiency) oil encapsulated in AL-CS nanoemulsions. *NP: Nanoparticles. 3.6. Protein Loading Efficiency The efficiency of protein loading in nanoparticles varies depending on the pH, formulation 3.6. Protein Loading Efficiency methodThe (mechanical efficiency ofenergy protein applied loading in the in nanoparticlesformulation), and varies ratio depending of polymers on theand pH,protein. formulation AL-CS nanoemulsionsmethodThe (mechanicalefficiency are positivelyof energy protein applied charged loading in because thein nanoparticles formulation), of the polycationic varies and ratio depending ofcharacter polymers on of the andchitosan pH, protein. formulation in AL-CSacidic media,methodnanoemulsions which (mechanical is arelocated positively energy on the applied charged outer in becauselayer the offormulat ofemulsion the polycationicion), particles. and ratio character Chitosan of polymers of nanoparticles chitosan and protein. in acidic are AL-CS media,often usedwhichnanoemulsions as iscarrier located materials are on positively the outerto incorporate layercharged of emulsion negativelybecause particles.of charged the polycationic Chitosanmacromolecules nanoparticlescharacter [61]. of In chitosan arethis often study, in used acidicBSA as wasmedia,carrier used materialswhich to study is tolocated the incorporate loading on the efficiency negativelyouter layer of chargedsacha of emulsion inchi macromolecules oil particles. loaded AL-CS Chitosan [61]. Innanoemulsions, this nanoparticles study, BSA because wasare often used it hasusedto studybeen as carrier thebroadly loading materials studied efficiency to asincorporate a of model sacha negatively inchiof protein oil loaded charged [62–65]. AL-CS macromolecules It nanoemulsions,has been reported [61]. because In thisthat study, itahas higher beenBSA concentrationwasbroadly used studied to study of protein as the a model loading produces of efficiency protein larger [ 62particles of– 65sacha]. It andinchi has loading been oil loaded reported efficiency, AL-CS that becausenanoemulsions, a higher more concentration interactions because ofit betweenproteinhas been producesthe broadly polymer larger studied and particles protein as a and causemodel loading an of increase effiproteinciency, in [62–65]. becausethe particle moreIt has size, interactions been which reported results between inthat theimproving a polymer higher loadingconcentrationand protein efficiency cause of protein[17,64]. an increase producesIn our in study, the larger particle the particles size size, of whichAL-CS and loading results particles in efficiency, improvingdid not notably because loading change, more efficiency interactions remaining [17,64]. inbetweenIn a our range study, the from polymer the 410 size to of and430 AL-CS nmprotein for particles allcause concentrations did an notincrease notably ofin BSAthe change, particle (Figure remaining size,11A). which The in a loading rangeresults from inefficiency improving 410 to 430of BSAloadingnm forincreased all efficiency concentrations with [17,64]. an increase of In BSA our in (Figurestudy, its concentration the11A). size The of loadingAL-CS (Figure particles e ffi11B).ciency At did the of not BSA lowest notably increased concentration change, with anremaining increaseof BSA 1 (0.01in itsa rangeg·L concentration−1), fromthe protein 410 (Figure to 430was nm11 notB). for Atentrapped all the concentrations lowest into concentration the ofAL BSA-CS particles. of(Figure BSA (0.0111A). However, g TheL− ),loading theat proteinconcentrations efficiency was not of · 1 BSAentrapped increased into with the−1 AL-CSan increase particles. in its However,concentration at concentrations (Figure 11B). At higher the lowest than 0.5concentration g L , the loading of BSA higher than 0.5 g·L , the loading efficiency of the protein increased substantially to· close− to values near(0.01efficiency 80%, g·L− 1 ofwhich), the protein proteinremained increased was with not more substantiallyentrapped BSA. Ininto to sacha close the AL toinchi values-CS oil particles. nearloaded 80%, AL-CSHowever, which nanoemulsions, remained at concentrations with morethe proteinhigherBSA. In mightthan sacha 0.5 be inchi g·L positioned− oil1, the loaded loading between AL-CS efficiency nanoemulsions,the two of oppo the proteinsitely the proteincharged increased might polyelectrolytes, substantially be positioned toAL between close and toCS, thevalues and two thusnearoppositely the 80%, particle which charged size remained polyelectrolytes,did not change,with more because AL BSA. and BSA CS,In sacha andis a small thus inchi thepolymer oil particle loaded (60 size kDa)AL-CS did compared notnanoemulsions, change, with because CS theor AL.proteinBSA is amight small be polymer positioned (60 kDa) between compared the two with oppo CSsitely or AL. 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(A concentration) on (A) the particle size and PDI and( B) loading efficiency of Figure 11. Effect of protein concentration on (A) the particle size and PDI and (B) loading efficiency AL-CS nanoemulsion prepared with sacha inchi oil. BSA, bovine serum albumin. 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

3.7. In Vitro Release After incubation of AL-CS nanoemulsion in various testing medium, the amount of BSA released from thethe particles particles was was evaluated evaluated and and is presented is present in Figureed in Figure12. In vitro 12. proteinIn vitro releasing protein wasreleasing monitored was andmonitored a very lowand amount a very oflow protein amount was of released protein during was released incubation. during A slightly incubation. higher amountA slightly of proteinhigher wasamount released of protein in the was digestive released enzyme in the containingdigestive enzyme buffer comparedcontaining withbuffer the compared non-digestive with the enzyme non- containingdigestive enzyme buffer, containing showing the buffer, positive showing effect ofthe enzymes positive oneffect the of protein enzymes release on the from protein the particles. release However,from the particles. the maximum However, percentage the maximum of released percenta proteinge onlyof released reached protein less than only 12% reached (gastric less bu thanffer with12% pepsin(gastric enzyme) buffer with in 72 pepsin h. enzyme) in 72 h. In polymeric nanoparticles, the drug release rate depends on several factors: (i) drug solubility; (ii)(ii) desorptiondesorption ofof thethe surface-boundsurface-bound or adsorbedadsorbed drug; (iii) drugdrug didiffusionffusion throughthrough thethe nanoparticlenanoparticle matrix; (iv) nanoparticle matrix erosion or degradation; and (v) the combination of erosion and didiffusionffusion processes [[66].66]. Especially when the nanoparticle is coated by polymer, the release of the drug can be controlled by (iii) drugdrug didiffusionffusion fromfrom thethe polymericpolymeric membrane.membrane. As the CS coating was performed over AL nanoparticles in the second process,process, the protein didiffusionffusion through CS membrane was the decisive factor onon the releaserelease profileprofile inin AL-CSAL-CS nanoparticles.nanoparticles. The combination of AL and CS forms stablestable membranes membranes as as a result a result of entanglements of entanglements and ionic and interactions ionic interactions between between the two polymers, the two whichpolymers, makes which it hard makes for it BSA hard to for diff BSAuse toto thediffuse testing to the medium. testing Besidesmedium. that, Besides the interactionthat, the interaction between BSAbetween and BSA each and of theeach components of the components in the particles in the part mighticles alsomight contribute also contribute to its lowto its release low release of BSA. of AsBSA. many As many proteins, proteins, BSA possesses BSA poss amideesses groupsamide groups and other and polar other functional polar functional groups. groups. They can They interact can withinteract CS, with AL, orCS, other AL, compounds,or other compounds, either by either ionic interactions,by ionic interactions, hydrogen hydrogen bonds, or bonds, other van or other der Walls van forcesder Walls [66, 67forces]. During [66,67]. the During chain interactionthe chain interaction in aqueous in medium, aqueous BSAmedium, and CS BSA form and the CS non-covalently form the non- linkedcovalently complexes linked withoutcomplexes changing without the changing conformation the conformation of BSA molecules. of BSA Themolecules. BSA-CS The complexes BSA-CS arecomplexes potentially are morepotentially functional more and functional stable than and the stable single polymerthan the owingsingle to polymer the combination owing to of the attributescombination of proteinof the attributes and CS polymer of protein [68 and]. 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 accomplished.The ultrasound-assisted Full characterization encapsulation of AL-CS of vegetable particles oils was in AL-CS carried nanoparticles out to optimize was thesuccessfully physical propertiesaccomplished. and Full phase characterization stability of particles. of AL-CS In theparticles formulation was carried of AL-CS out nanoemulsions,to optimize the thephysical types ofproperties oils, concentrations and phase stability of CS, and of particles. surfactant In played the formulation decisive roles of AL-CS on the nanoemulsions, particle characteristics the types like of size,oils, concentrations polydispersity, of and CS, surface and surfactant charge. The played encapsulation decisive roles of bio-active on the particle oils into characteristics AL-CS particles like size, was 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.

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