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Rayanne R. Pereira1,3 Ucuu` ba Fat Characterization and Use Antonio T. A. Gomes2 Matteo Testi3 to Obtain Lipid Nanoparticles by Annalisa Bianchera3,4 High-Pressure Homogenization with Full Roseane M. Ribeiro-Costa2 Cristina Padula3 Factorial Design Jose´ O. C. Silva Ju´ nior1 Fabio Sonvico3,4,* Ucuu`ba fat was characterized and used as the lipid phase for the production of nanostructured lipid carriers (NLCs). The acid, iodine, refraction, and saponifi- cation values as well as the oxidative stability of the fat were determined. The was found to be the main fatty acid present in the fat. NLCs were produced by high-pressure homogenization with tocopheryl polyethylene glycol 1000 succinate (TPGS) or Tween 80 as surfactant. A full factorial experimental 2 This is an open access article under design 2 was applied to evaluate the impact of production parameters on particle the terms of the Creative Commons size and polydispersity index (PDI) of the nanocarrier. The design of experiments Attribution-NonCommercial- NoDerivs License, which permits (DoE) demonstrated that the increase of pressure and number cycle homogeniza- use and distribution in any tion positively contributed to reduce the mean particle size and PDI of NLCs only medium, provided the original work is properly cited, the use is when TPGS was present. non-commercial and no modifications or adaptations are made. Keywords: Full factorial experimental design, High-pressure homogenization, Nanostructured lipid carriers, Ucuu`ba fat Received: September 05, 2020; revised: February 10, 2021; accepted: March 08, 2021 Supporting Information available online DOI: 10.1002/ceat.202000404

1 Introduction a mixture of solid lipids or more commonly a single saturated triglyceride, while the lipid phase of the NLC is a mixture of The continuous search for new materials has always accompa- solid and liquid lipids. The NLC composition circumvents nied the growth of pharmaceutical and cosmetic industries. some of the limitations of SLNs, such as the low encapsulation The Amazon rainforest is the largest tropical in the efficiency and poor stability during storage. Moreover, it is world and shelters a wide variety of with high potential common for SLN lipids to undergo polymorphic transforma- of application in various fields. Indeed, Amazonian oils and tions during storage which promote instability of the colloidal fats have been widely studied as a raw material for cosmetic, system and expulsion of the drug [6–10]. pharmaceutical, chemical, and other industries [1, 2]. Several methods have been applied for NLC production, but surinamensis, popularly known as Ucuu`ba, is a tree native to among others, high-pressure homogenization (HPH) presents the Amazon, distributed in several states of northern . several advantages such as large-scale production, avoidance of Ucuu`ba seeds are rich in fat that is used in cosmetics produc- organic solvents, sterilization during processing, and short tion and in traditional medicine [3]. Besides the popular use, Cordeiro et al. [4] reported on the antibacterial activity of – 1 Ucuu`ba fat against Staphylococcus aureus, while more recently Dr. Rayanne R. Pereira, Dr. Jose´ O. C. Silva Ju´nior our group [5] used Ucuu`ba fat as the lipid phase of the solid Laboratory R&D Pharmaceutical and Cosmetic, Federal University of Para´, Street Augusto Correa 01, 66075110 Bele´m, PA, Brazil. lipid nanoparticles for drug delivery to the nail. 2 Emulsions and creams are widely used for topical adminis- Prof. Antonio T. A. Gomes, Prof. Roseane M. Ribeiro-Costa Laboratory of Pharmaceutical Nanotecnology, Federal University of tration of active ingredients, either in cosmetics or pharma- Para´, Street Augusto Correa 01, 66075110 Bele´m, PA, Brazil. ceuticals. "Nano" drug delivery systems are generally derived 3Dr. Rayanne R. Pereira, Matteo Testi, Dr. Annalisa Bianchera, Dr. Cris- from conventional pharmaceutical systems, and in this sense tina Padula, Prof. Fabio Sonvico nanoemulsions, microemulsions, and lipid nanoparticles can [email protected] be considered offshoots of traditional emulsions. Lipid nano- Department of Food and Drug, University of Parma, Viale delle particles were firstly developed in the 1990s, physically differ- Scienze 27/a, Parma, 43124, Italy. ing from nano- and microemulsions because their lipid phase 4Dr. Annalisa Bianchera, Prof. Fabio Sonvico is solid. Two types of such lipid-based colloidal systems have Biopharmanet–TEC Interdepartmental Center for the Development been proposed: solid lipid nanoparticles (SLNs) and nano- of Health Products, University of Parma, Pad. 33, Science and Tech- structured lipid carriers (NLCs). The lipid phase of the SLN is nology Campus, Parma, 43124, Italy.

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manufacturing time [11, 12]. Moreover, HPH process parame- The oxidative stability index (OSI) was determined on a ters influence NLC characteristics such as particle size distribu- Metrohm Rancimat (Model 743, Herisau, Switzerland) follow- tion, encapsulation efficiency, and physical stability [13]. ing the AOCS official method (AOCS Cd 12b-92) [15]. The Design of experiments (DoE) is becoming commonplace in conditions of the test were the following: 5 g of sample, flow of pharmaceutical technology because it allows the evaluation of oxygen 20 L h–1, temperature 110 C. The induction time was the influence of production variables on quality parameters of determined by the OSI inflection point at a maximum of the the formulation. In addition, it usually obtains the information second derivative of the conductivity curve. with a limited number of experiments, reducing the process development and production costs. There are several types of DoE models among which to select and the choice depends on 2.3 Lipid Fatty Acid (FA) Profile Characterization the researcher’s objectives. For example, a full factorial experi- ment allows the investigator to study the impact of each factor, The fatty acid profile of Ucuu`ba fat was obtained by gas chro- as well as the effects of the interactions between the factors on matography coupled to mass spectrometry (GC-MS). Con- the response variables [5, 13]. version of triglycerides to methyl esters was performed via Knowing the popular use of Ucuu`ba fat and its importance saponification and esterification with potassium hydroxide in to the Amazon region, the aim of this work was to produce methanol (0.1 M) and hydrochloric acid in methanol (0.12 M) NLCs from this fat in view of potential cosmetic or pharmaceu- [16]. Analysis of the fatty acid profile was conducted according tical applications. The work was divided into two parts. In the to the following specifications: column SH-Rtx-5 first part, the physicochemical characteristics, fatty acid profile, 30 m ·0.25 mm; entrainment gas: helium with flow of and oxidative stability of Ucuu`ba fat was examined. In the 1.5 mL min–1; injection volume of 1 mL (split at 1:50 ratio); tem- second phase, the production of Ucuu`ba fat-based NLCs by perature gradient used was a heating ramp starting at 60 C for HPH was investigated using a full factorial 22 to evaluate the 2 min, followed by heating to 200 C at a rate of 10 C min–1, influence of the homogenization process parameters on the heating from 200 C to 240 C at the rate of 2 C min–1 and particle size and the polydispersity index (PDI) of the formula- keeping the sample at the maximum temperature for 24 min. tions. The detector operated at atemperature of 250 C. Fatty acid peaks were identified using the NIST mass spectral database of the QP2010 ULTRA GC-MS (Shimadzu, Kyoto, Japan) equip- 2 Materials and Methods ment used.

2.1 Materials 2.4 Formulation of NLCs Ucuu`ba fat () was provided by Amazon Oil Industry (Ananindeua, Brazil). D-a-Tocopheryl polyethylene Production of NLCs was performed by HPH. Capryol was used glycol 1000 succinate (TPGS) was supplied by ISOCHEM as liquid lipid, Ucuu`ba fat was the solid lipid, TPGS or Tween (Gennevilliers, France). Propylene glycol monocaprylate type II 80 served as surfactant. Two formulations of NLCs were pre- (Capryol90) was acquired from Gattefosse´ (Saint Priest, pared, i.e., NLCTPGS and NLCTW80. NLCTW80 was made using France). Other reagents were of analytical purity grade. Ultra- 5 % w/v Tween 80 and 10 % w/v of lipid phase containing pure water was obtained with a PureLab Pulse water system Ucuu`ba/Capryol at 4:1 w/w ratio. NLCTPGS was prepared using (Elga Veolia, Milan, Italy). 1.25 % w/v TPGS and 1.25 % w/v lipid phase containing a ratio Ucuu`ba/Capryol at 4:1 w/w ratio. The lipid-phase melting point was determined by differential scanning calorimetry in 2.2 Physicochemical Characterization comparison to Ucuu`ba fat. Differential scanning calorimetry was carried out on a DSC-60 plus equipment (Shimadzu, The physicochemical characterization of Ucuu`ba fat was per- Kyoto, Japan). A sample (5 mg) was placed in a closed alumi- formed by means of classical tests, which use acid-base titra- num pan and subjected to a heating rate of 10 C min–1 to a tions in accordance to the Brazilian Pharmacopoeia (6th edi- temperature of 100 C with nitrogen flowing at 50 cm3min–1. tion) [14] and American Oil Chemists’ Society (AOCS) [15]. Ucuu`ba fat-based NLCs were prepared using an emulsifica- The following properties were determined: saponification index tion step, followed by homogenization and a final solidification (AOCS Cd 3-25), refractive index at 40 C (Cc 7-25), and io- phase. The lipid phase was made by melting Ucuu`ba fat (melt- dine index (Cd 1c-85). The acid value of the fat was determined ing point ~ 42 C) at 10 C above the lipid melting point and according to the following procedure: Ucuu`ba fat was solubi- by mixing it with the liquid lipid (Capryol) and the surfactant. lized in a mixture of ethyl alcohol and ethyl ether (1:1), which Then, the aqueous phase, preheated at 70 C, was added gradu- was neutralized with 0.1 M KOH solution. The acidity index ally into the lipid phase under continuous magnetic stirring. was calculated by Eq. (1) [14]: Subsequently, the emulsification step was completed using an 5:61n Ultra-Turrax TP18/10 (IKA Werke GmbH & Co., Staufen, Acid value ¼ (1) m Germany) operated at 10 000 rpm for 1 min. Then, the emul- sion was processed using a laboratory high-pressure homoge- where n is the volume of KOH used to neutralize the solution nizer (Panda Plus 2000, GEA Niro Soavi, Parma, Italy) and m is the mass of the fat. equipped with a ceramic homogenizing valve group and a shell

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and tube heat exchanger (Exergy, Garden City, NY, USA) used dicate a high content of free fatty acids derived from the hy- to control the process temperature at 70 C with a water ther- drolysis of triglycerides and affect the biological and organolep- mostat (ECO Silver E4, Lauda, Assago, Italy). Nanoparticles tic properties, as well as the physical stability of the vegetable were then left to cool to ambient temperature for allowing lipid fat and of the formulations prepared with it [1, 2]. The acid val- phase solidification. ue determined for Ucuu`ba fat was 38.76 mg KOH g–1. The maximum acid value for cold-pressed and unrefined oils and fats is generally set to 4 mg KOH g–1 according to the FAO 2.5 Full Factorial Design 22 Codex Alimentarius [19]. However, the acid value of 38.76 KOHg–1 can be considered normal for Ucuu`ba fat, since –1 –1 The NLCTW80 and NLCTPGS production, performed by HPH, acid values of 33.69 mg KOH g and 32.22 mg KOH g have was studied through a full factorial design with two factors, two been reported in literature [3, 19]. The hypothesis is that li- levels, and three replicates of the central point. The factors in- gnans and neolignans, present in the Ucuu`ba fat, are reacting vestigated were homogenization pressure and number of ho- with KOH during titration, leading to higher acid values not mogenization cycles, while the responses were particle size and related to an effective elevated content in free fatty acids [2, 20]. PDI. The experimental design was developed and analyzed by The refractive, saponification, and iodine values are parame- means of the software Design-Expert (version 7.0.0, Stat-Easc, ters that can be used to ascertain the authenticity of vegetable Inc., Minneapolis, MN, USA). The Pareto chart was generated oils and fats, as their values strongly depend on the composi- using Minitab Statistical Software (ver. 19, MINITAB, State tion in fatty acids of the triglycerides present in the fat. The –1 College, PA, USA). The DoE responses, i.e., particle size and refractive (1.45) and iodine (8.5 g I2 100 g ) values are directly PDI, were determined by dynamic light scattering using a Zeta- related to the triglycerides content in fatty acid chains present- sizer Nano ZS (Malvern Pananalytical, Malvern, UK) applying ing unsaturation and high carbon chains. In particular, the non-invasive back-scattering (scattering angle 173) at a tem- lower the content in unsaturated fatty acids, the lower the value perature of 25 C. The NLCs were diluted (1:100) with ultra- of these parameters [1, 17]. pure water prior to measurements. Average particle size The Ucuu`ba fat showed a saponification value of 230.21 mg (Z-average) and PDI obtained through the cumulants analysis KOH g–1 (Tab. 1). The saponification index is inversely propor- of scattering data by the instrument software were assessed in tional to the molecular weight of the fatty acid chains present triplicate and results were expressed as mean and standard in the triglycerides of the vegetable fat [21]. Generally, vegeta- deviation. ble oils and fats are composed by triglycerides containing predominantly long-chain fatty acids with high molecular weight and typically present saponification indexes between 3 Results and Discussion 180–200 mg KOH g–1 [1, 21]. Oxidative stability is a global physicochemical parameter 3.1 Physicochemical Parameters reflecting the quality of vegetable oils and fats. It is expressed as an induction time, i.e., the time required for the formation Natural fats obtained from the Amazon flora have interesting oil oxidation products when the oil is submitted to extreme physicochemical characteristics for their application in the cos- conditions of temperature and humidity [1, 21]. The oxidative metic and pharmaceutical sector. However, it is pivotal to know stability of a fat is affected by several factors, such as its compo- the specific properties and composition of Amazonian natural sition in fatty acids, the presence of antioxidant substances, the oils and fats for the development of new products, as well as exposure to oxygen, light as well as storage temperature. The establishing control protocols to implement quality standards induction time of Ucuu`ba fat was found to be longer than 20 h, [1, 2, 17, 18]. Several physicochemical parameters of Ucuu`ba a value comparable to induction times reported for other Ama- fat, such as acidity, iodine, refraction, and saponification values, zonian fats, such as the Murumuru fat (> 40 h) and the Buriti were investigated in order to evaluate the quality of the fat in oil (32.7 h) [21]. Since a high quantity of saturated fatty acids use (Tab. 1). in the composition of triglycerides could be the cause for the The acid value is a common parameter for the specification oxidative stability of the Ucuu`ba fat, a complete fatty acid pro- of vegetable oils and fats. In particular the quality of the fat is file characterization of the fat was carried out. often evaluated through the acid value [2]. High acid values in-

Table 1. Physicochemical properties of the Ucuu`ba fat. 3.2 Fatty Acid Profile

Parameters Value Tab. 2 presents the Ucuu`ba fat fatty acid composition deter- mined by GC-MS. –1 Acid value 38.76 ± 1.1 mg KOH g From the collected data it became evident that myristic acid –1 Iodine index 8.50 ± 0.02 g I2 100 g is the main fatty acid (68.23 %) present in the triglycerides composing Ucuu`ba fat, followed by lauric (18.32 %), palmitic Refractive index 1.45 ± 0.01 (5.8 %), and (3.96 %) (Tab. 2). Myristic acid is a Saponification index 230.21 ± 0.02 mg KOH g–1 14-carbon chain saturated fatty acid widely distributed in fats throughout the animal and kingdoms [22]. Topically Oxidative stability index (OSI), 110 C > 20 h applied on the skin, myristic acid proved to have bactericidal

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Table 2. Percentage composition of the main fatty acids in Virola surinamensis fat determined by gas chromatography.

Fatty acid Percentage [%] Fatty acid type Total percentage [%]

Caprylic acid (C8:0) 0.34 ± 0.3401 SFAa) 94.37

Capric acid (C10:0) 0.29 ± 0.2935

Lauric acid (C12:0) 18.32 ± 5.2309

Myristic acid (C14:0) 68.23 ± 5.6124

Palmitic acid (C16:0) 5.86 ± 0.3453

Stearic acid (C18:0) 1.10 ± 0.3883

Arachidic acid (C20:0) 0.89 ± 0.3167

Palmitoleic acid (C16:1, w-7) 0.25 ± 0.07211 MUFAb) 4.21

Oleic acid (C18:1) 3.96 ± 0.2787

Linoleic acid (C18:2) 0.89 ± 0.3167 PUFAc) 0.89 a)SFAs, saturated fatty acids); b)MUFAs, monounsaturated fatty acids; c)PUFAs, polyunsaturated fatty acids. activity against S. epidermidis [23] and wound healing activity liquid lipids used as lipid phase for the production of nano- [24]. structured lipid carriers. is a saturated medium-chain fatty acid (C:12) The lipid mixture selected to prepare the NLCs (Ucuu`ba also present in Babassu, , and Tucuma˜ oil [2, 18]. It is fat/Capryol mixture ratio 4:1 w/w) showed a relatively small well studied with respect to its antimicrobial activity, being reduction in the temperature of the endothermic peak attribut- effective against the Propionibacterium acnes [25], and also ed to the melting of the pure solid lipid (from 42.13 C to just against Staphylococcus aureus, Bacillus cereus, Salmonella typhi- 41.40 C, Fig. 1). Therefore, it can be assumed that the lipid murium, and Escherichia coli at a concentration of 5 % [26]. It mixture is solid at room temperature but with a reduced crys- has also been used in the manufacture of creams, soaps, and tallinity of the solid fat, a point that has been observed previ- other cosmetics, due to its surfactant, emollient, and humectant ously for the mixture of these two lipids [5]. properties [18]. In the pharmaceutical field, it is essential to identify the Palmitic and oleic acids are widely applied in cosmetic and parameters that can affect the quality and effectiveness of pharmaceutical products. is emulsifying and formulations. In this regard, the DoE is the ideal tool to evalu- emollient, and is quite common in products intended for per- ate the influence of different process variables on the quality sonal hygiene [1]. Oleic acid activates lipid metabolism, restor- parameters of formulations [6, 16]. Hence, a full factorial ing the barrier function of the skin and is recog- nized as a drug penetration agent, through the skin and mucous membranes [25]. Hence, the fat of Ucuu`ba can be a source of diverse fatty acids with interesting implications for its use as component in cosmetic, medicinal, and personal care products [1, 18, 27].

3.3 Full Factorial Design 22

In recent years, one of the most interesting applica- tions for fats has been their use as valuable raw materials for drug delivery systems, such as lipid nanoparticles. In this sense, the fat properties along with its melting point, crystallization, and polymor- phic transformations represent the most critical factors contributing to the loading capacity for drug molecules and affecting the long-term stability of lipid nanoparticles [28]. In this regard, initially differential scanning calo- rimetry (DSC) was used to characterize the melting Figure 1. DSC traces of the Ucuu`ba fat and the lipid phase used for NLC manu- point and crystallinity of the mixture of solid and facturing (Ucuu`ba fat/Capryol mixture ratio 4:1 w/w).

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design 22 was used to evaluate the influence of the two main affecting the average particle size and PDI of the nanostruc- process parameters of HPH, i.e., number of cycles and pres- tured lipid carriers produced. The Pareto chart showing the sure, on the average particle size and particle size distribution standardized effects for the process parameters along with the of Ucuu`ba fat-based NLCs. Furthermore, two alternative sur- same result indicates that the model is not significant (Fig. 2). factants, namely, Tween 80 (NLCTw80) or the vitamin E deriva- The NLCTPGS had particle sizes ranging from 46.50 to tive TPGS (NLCTPGS), were employed to stabilize the lipid 67.58 nm, while the PDI ranged from 0.150 to 0.278 (Tab. 3). In nanoparticles gained with the Amazonian fat. The results contrast to results obtained in the case of NLCTw80, both the obtained in the DoE are presented in Tab. 3. PDI and the particle size of NLCTPGS were affected significantly 2 NLCTw80 showed particle sizes ranging from 94.5 to (p < 0.05) by the number of cycles and the pressure. The R 121.2 nm, while the PDI ranged from 0.131 to 0.150. The parti- shows a value of 0.9993, indicating that the linear model cle size and the PDI were analyzed separately and the depen- explained 99.93 % of data. dence of the production parameters was studied by the analysis The effect of the factors on the particle size of NLCTPGS is of variance (ANOVA) test (Tab. S1 in Supporting Information). described by Eq. (2) obtained by linear regression: It was found that in the case of NLCs produced with Tween 80 as stabilizing surfactant the variation in the homoge- Particle size ¼3:82X1 6:50X2 þ 4:04X1X2 þ 53:2 (2) nization cycles and the pressure did not generate statistically significant modification of both mean particle size and PDI. The ANOVA results indicate that both factors exhibited a The ANOVA test performed on DoE results found for negative effect on the particle size due to their coefficients NLCTW80 exhibited a p-value > 0.05, indicating that the mathe- (pressure –3.82; cycle –6.5, see Tab. S2 in Supporting Infor- matical model describing the obtained results is statistically mation). These ANOVA findings demonstrate that both the insignificant. In brief, the variation of the number of homoge- pressure (p = 0.0019) and homogenization cycles (p = 0.0006) nization cycles and the operating pressure are not significantly showed a reduction effect on particle size response (Tab. S2).

2 Table 3. Results of DoE 2 experiments for NLCTw80 and NLCTPGS.

Sample Factors NLCTW NLCTPGS Pressure [bar] Number of homogenization cycles Particle size [nm] PDI Particle size [nm] PDI

1 1000 3 94.7 0.144 51.86 0.209

2 750 6.5 115 0.143 48.38 0.163

3 750 6.5 111.2 0.142 48.29 0.169

4 1000 10 103.8 0.134 46.93 0.152

5 500 3 100.7 0.150 67.58 0.278

6 750 6.5 102.5 0.133 47.77 0.171

7 500 10 121.2 0.131 46.5 0.15

Figure 2. Pareto chart plots by pressure (X1) and homogenization cycles (X2) for particle size (a) and PDI (b) of the NLCTw80.

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The increase in the number of cycles of homogenization con- sure, since linear coefficients present in the two models were tributes to the reduction of the particle size (Fig. 3a). The parti- greater. In fact, the increase in the number of cycles of homoge- cle size decreases up to 21.08 nm when the number of cycles in- nization from 3 to 10 reduces the NLC size from 67.6 to creases from 3 to 10, keeping the pressure at 500 bar (Tab. 3). 46.9 nm and the PDI value from 0.278 to 0.150. The Pareto The increase in pressure also contributes to the decrease in par- charts confirm linear coefficient values, also showing the largest ticle size. The Pareto chart and the coefficient linear show the effect on size and PDI linked to the number of homogenization two factors and the interaction between the factors were statis- cycles (Fig. 3d). tically significant (Fig. 3b). In addition, the largest effect on In the preparation of NLC by HPH, the increase in the num- particle size is the cycle homogenization (Fig. 3c). ber of cycles and in pressure intensifies the mechanical stress The effect of the factors on the PDI of NLCTPGS was and break-up of dispersed phase droplets. When the droplet described by the linear regression in Eq. (3): fragmented, the water-soluble surfactant stabilizes the surface of newly formed droplets. Therefore, the time for adsorption of PDI ¼0:017X1 0:046X2 þ 0:018X1X2 þ 0:20 (3) the emulsifier at the freshly created interface determines the efficiency of the lipid-phase size reduction. Highly efficient The ANOVA test results indicate that both pressure droplet disruption, as increase pressure and number of cycle (p = 0.0151) and homogenization cycle (p = 0.002) contribute homogenization, can be a problem when the surfactant does significantly to decrease the PDI value, with negative coeffi- not stabilize the droplets quickly enough with the result that cients (Tab. S2). The coefficient of determination R2 has a value the smaller droplets can coalesce. As a consequence, it is also of 0.9968, demonstrating a good fitting of the linear model that possible that the droplet size remains constant at a determined explained 99.68 % of data. Overall, the increase of pressure and limit value, as in the case of NLCTW80, for which the process number of homogenization cycles determine a decrease of parameters in the range explored did not significantly modify mean particle size and PDI, i.e., a reduction of the particle size the particle size and PDI [29]. distribution (Figs. 3a and 3b). In both cases, the number of homogenization cycles exhibited a stronger effect than pres-

Figure 3. Response surface plots (a, b) and Pareto charts (c, d) describing the effect of pressure (X1) and homogenization cycles (X2) on particle size and PDI of NLCTPGS.

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4 Conclusion [3] Chemistry of Vegetable Oils: Valorization of the Amazon Bio- diversity, 1st ed. (Eds: L. R. B. M. Morais, E. Gutjahr), Author Ucuu`ba fat presents an excellent resistance to oxidative stress Press, Bele´m 2012. which, combined with optimal physicochemical properties, [4] R. M. Cordeiro, A. P. Ana, R. H. H. Pinto, W. A. da Costa, make it a good candidate for the production of NLCs. The pro- S. H. M. da Silva, W. B. de Souza Pinheiro, M. S. P. Arruda, duction of NLCs by HPH, using Ucuu`ba fat in a mixture with R. N. Carvalho Junior, Chem. Eng. Commun. 2019, 206 (1), CapryolTM90, demonstrated that the number of cycles and the 86–97. DOI: https://doi.org/10.1080/00986445.2018.1474741 [5] R. R. Pereira, M. Testi, F. Rossi, J. O. C. Silva Junior, homogenization pressure can be factors that directly affect the R. M. Ribeiro-Costa, R. Bettini, P. Santi, C. Padula, F. Son- nanomaterial particle size distribution, depending also on the vico, Pharmaceutics 2019, 11 (6), 284. DOI: https://doi.org/ stabilizing surfactant used. In summary, the proposed Ucuu`ba 10.3390/pharmaceutics11060284 fat nanoformulations appear to be highly promising for the [6] Lipid Nanoparticle: Production, Characterization and Stabil- production of cosmetic or pharmaceutical products for topical ity, 1st ed. (Eds: R. Shah, D. Eldridge, E. Palombo, I. Har- use. ding), Springer, Geneva 2015. [7] A. P. Nikalje, Med. Chem. 2015, 5 (2), 81–89. DOI: https:// Acknowledgment doi.org/10.4172/2161-0444.1000247 [8] E. Di Cola, E. Cantu, P. Brocca, V. Rondelli, G. C. Fadda, E. Canelli, P. Martelli, A. Clementino, F. Sonvico, R. Bettini, The authors are grateful to the Brazilian Federal Agency for E. Del Favero, Colloids Surf., B 2019, 183, 110439. DOI: Support and Evaluation of Graduate Studies (CAPES) and https://doi.org/10.1016/j.colsurfb.2019.110439 Dean of Graduate Studies and Research (PROPESP/UFPA). [9] G. G. Pereira, T. Rawling, M. Pozzoli, C. Pazderka, Y. Chen, C. R. Dunstan, M. Murray, F. Sonvico, Nanomaterials 2018, The authors have declared no conflict of interest. 8 (10), 825. DOI: https://doi.org/10.3390/nano8100825 [10] I. Telo`, E. Favero, L. Cantu`, N. Frattini, S. Pescina, C. Padula, P. Santi, F. Sonvico, S. Nicoli, Mol. Pharm. 2019, 14 (10), Supporting Information 3281–3289. DOI: https://doi.org/10.1021/acs.molpharma- ceut.7b00348 Supporting Information for this article can be found under [11] J. Pardeike, A. Hommoss, R. H. Mu¨ller, Int. J. Pharm. 2009, DOI: https://doi.org/10.1002/ceat.202000404. 366 (1–2), 170–184. DOI: https://doi.org/10.1016/ j.ijpharm.2008.10.003 [12] G. R. Vaz, A. Clementino, J. Bidone, M. A. Villetti, M. Fal- Abbreviations kembach, M. Batista, P. Barros, F. Sonvico, C. Dora, Nano- materials 2020, 10 (9), 1650. DOI: https://doi.org/10.3390/ ANOVA analysis of variance nano10091650 AOCS American Oil Chemistry Society [13] P. Severino, M. H. Santana, E. B. Souto, Mater. Sci. Eng., C DoE design of experiments 2012, 32 (6), 1375–1379. DOI: https://doi.org/10.1016/ DSC differential scanning calorimetry j.msec.2012.04.017 FAME fatty acid methyl ester [14] Farmacopeia Brasileira, 6th ed., Ageˆncia Nacional de Vigi- HPH high-pressure homogenization laˆncia Sanita´ria, Ministe´rio da Sau´de, Brasilia 2019. IP induction period [15] Official Methods and Recommended Practices of the AOCS, MUFA monounsaturated fatty acid 6th ed. (Ed: D. Firestone), American Oil Chemists’ Society, NLC nanostructured lipid carrier Urbana 2009. OSI oxidative stability index [16] A. M. C. Rodrigues, S. Darnet, L. M. Da Silva, J. Braz. Chem. PDI polydispersity index Soc. 2010, 21 (10), 2000–2004. DOI: https://doi.org/10.1590/ PUFA polyunsaturated fatty acid S0103-50532010001000028 SFA saturated fatty acid [17] E. Pereira, M. C. Ferreira, K. A. Sampaio, R. Grimaldi, A. J. SLN solid lipid nanoparticle A. Meirelles, G. J. Maximo, Food Chem. 2019, 278, 208–215. DOI: https://doi.org/10.1016/j.foodchem.2018.11.016 [18] T. T. A. Gomes, R. R. Pereira, A. P. Duarte Junior, A. M. C. References Rodrigues, C. M. R. Reme´dios, D. S. B. Brasil, L. R. B. Mor- ais, J. O. C. Silva Ju´nior, R. M. R. Costa, J. Therm. Anal. [1] D. M. L. Contente, R. R. Pereira, A. M. C. Rodrigues, E. S. Calorim., in press. DOI: https://doi.org/10.1007/s10973-020- Oliveira, R. M. R. Costa, J. O. C. Silva Costa, Chem. Eng. 10352-3 Technol. 2020, 43 (7), 1424–1432. DOI: https://doi.org/ [19] Standard for Named Vegetabel Oils, Codex Alimentarius, 10.1002/ceat.201900627 FAO, Rome 1999. [2] J. J. R. Pardauil, F. A. de Molfetta, M. Braga, L. K. C. de Sou- [20] J. L. Serra, L. H. M. Silva, A. J. A. Meirelles, S. H. Darnet, za, G. N. R. Filho, J. R. Zamian, C. E. F. da Costa, J. Therm. R. A. de Freitas, A. M. C. Rodrigues, Int. Food Res. J. 2018, Anal. Calorim. 2016, 115 (3), 1–9. DOI: https://doi.org/ 116, 12–19. DOI: https://doi.org/10.1016/ 10.1007/s10973-016-5605-5 j.foodres.2018.12.028

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Research Article 9

Research Article: Ucuu`ba fat presents Ucuu` ba Fat Characterization and Use an excellent resistance to oxidative to Obtain Lipid Nanoparticles by High- stress which combined with optimal Pressure Homogenization by Full physicochemical properties make it a Factorial Design good candidate for the production of nanostructured lipid carriers. These R. R. Pereira, A. T. A. Gomes, M. Testi, were successfully generated using two A. Bianchera, R. M. R. Costa, C. Padula, different surfactants. The proposed J. O. C. Silva Ju´nior, F. Sonvico* Ucuu`ba fat nanoformulations are highly promising for the production of Chem. Eng. Technol. 2021, 44 (6), cosmetic or pharmaceutical products XXX K XXX for topical use. DOI: 10.1002/ceat.202000404

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