Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Solid lipid nanoparticles optimized by 22 factorial design for skin administration: Cytotoxicity in NIH3T3 fibroblasts Roberta Balansin Rigona,1, Maíra Lima Gonçaleza,1, Patrícia Severinob, Danilo Antonini Alvesc, Maria H.A. Santanad, Eliana B. Soutoe,f,⁎, Marlus Chorillia,⁎⁎ a Faculty of Pharmaceutical Sciences, UNESP – São Paulo State University, Campus Araraquara, Departamento de Fármacos e Medicamentos, Araraquara, SP 14800-850, Brazil b Centre of Biological Sciences and Health, Tiradentes University, Aracaju, Sergipe 49010-390, Brazil c Laboratory of Biotechnology, Institute of Biology, Campinas University (UNICAMP), Campinas, São Paulo 13083-862, Brazil d Faculty of Chemical Engineering, Campinas University (UNICAMP), Campinas, São Paulo 13083-970, Brazil e Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), Polo das Ciências da Saúde Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal f REQUIMTE/LAQV, Group of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Portugal A R T I C L E I N F O Keywords: Solid lipid nanoparticles Factorial design Fibroblasts MTT assay Skin administration A B S T R A C T The present study focuses on the characterization of the cytotoxic profile on NIH3T3 mouse embryonic fi- broblasts of solid lipid nanoparticles (SLN) optimized by a 22 full factorial design for skin administration. To build up the surface response charts, a design of experiments (DoE) based on 2 independent variables was used to obtain an optimized formulation. The effect of the composition of lipid and water phases on the mean particle size (z-AVE), polydispersity index (PdI) and zeta potential (ZP) was studied. The developed formulations were composed of 5.0% of lipid phase (stearic acid (SA), behenic alcohol (BA) or a blend of SA:BA (1:1)) and 4.7% of surfactants (soybean phosphatidylcholine and poloxamer 407). In vitro cytotoxicity using NIH3T3 fibroblasts was performed by MTT reduction assay. This factorial design study has proven to be a useful tool in optimizing SLN (z-AVE ∼ 200 nm), which were shown to be non-cytotoxic. The present results highlight the benefit of applying statistical designs in the preparation and optimization of SLN formulations. 1. Introduction Solid lipid nanoparticles (SLN) have been widely used for skin de- livery of active pharmaceutical ingredients (APIs) due to their safe in- teraction with stratum corneum and other skin layers, and improved skin permeation [1,2]. Additional attributes include the possibility of controlled release of the loaded APIs, drug protection, biodegradability and safety profile, and low cost of the production process [3–5]. For this purpose, a large number of lipid ingredients may be used, including glycerol behenate, glycerol palmitostearate, cetyl palmitate, glycerol trilaurate, and stearic acid as solid lipids, which are known to have approved safety profile for a set of clinical applications [3,4]. For the physicochemical stability of SLN, several surfactants may be employed e.g. sodium chlorate, polysorbates, phospholipids and poloxamers, polyvinyl alcohols, which should also be of generally recognized as safe status. The development of a new formulation encounters numerous vari- ables which have to be taken into account when loading a new API. Factorial design experiments offer a simple, efficient and statistically valid approach which can simultaneously analyze the influence of dif- ferent variables on the properties of drug delivery systems [18]. In this way, optimization with respect to the selection of ingredients and methodology parameters to prepare the formulation can be achieved by factorial design, with the ultimate aim of reducing the time for the development of a new formulation, as well as the production costs [19]. The aim of this study has been the optimization by factorial design of an innovative SLN formulation composed of stearic acid (SA) and/or behenic alcohol (BA) as solid lipids and stabilized by a combination of https://doi.org/10.1016/j.colsurfb.2018.07.065 Received 3 July 2017; Received in revised form 27 July 2018; Accepted 28 July 2018 ⁎ Corresponding author at: Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal. ⁎⁎ Corresponding author at: Faculty of Pharmaceutical Sciences, UNESP – São Paulo State University, Campus Araraquara, Departamento de Fármacos e Medicamentos, Araraquara, SP 14800-850, Brazil. 1 These authors contributed equally to this work. E-mail addresses: souto.eliana@gmail.com, ebsouto@ff.uc.pt (E.B. Souto), chorilli@fcfar.unesp.br (M. Chorilli). Colloids and Surfaces B: Biointerfaces 171 (2018) 501–505 Available online 30 July 2018 0927-7765/ © 2018 Elsevier B.V. All rights reserved. T http://www.sciencedirect.com/science/journal/09277765 https://www.elsevier.com/locate/colsurfb https://doi.org/10.1016/j.colsurfb.2018.07.065 https://doi.org/10.1016/j.colsurfb.2018.07.065 mailto:souto.eliana@gmail.com mailto:ebsouto@ff.uc.pt mailto:chorilli@fcfar.unesp.br https://doi.org/10.1016/j.colsurfb.2018.07.065 http://crossmark.crossref.org/dialog/?doi=10.1016/j.colsurfb.2018.07.065&domain=pdf poloxamer 407 (P407) and soy phosphatidylcholine (SP), for skin ap- plication. The dependent variables were set as the mean hydrodynamic diameter, polydispersity index (PdI) and zeta potential (ZP), while the ratio of lipids and surfactants were defined as the independent vari- ables. 2. Materials and methods 2.1. Materials Stearic acid (SA) was obtained from Via Farma (São Paulo, Brazil), behenic alcohol (BA) and poloxamer 407 (Pluronic® F127) were pur- chased from Sigma-Aldrich (St. Louis, USA), and soybean phosphati- dylcholine (Lipoid® S100) from Lipoid GmbH (Ludwigshafen, Germany). NIH 373 (ATCC® CRL-1658™) were obtained from LGC Standards S.L.U. (Barcelona, Spain). RPMI 1640 was purchased from Lonza (Verviers, Belgium), and Fetal Bovine Serum from Biowest (Nuaillé, France). Double distilled water was used after filtration in a Millipore system (home supplied). 2.2. Methods 2.2.1. Preparation of solid lipid nanoparticles SLN composed of 5.0% of lipid phase (SA or BA, or a blend of SA and BA) and 4.7% of surfactants (SP and P407) were produced by high shear homogenization. Briefly, the lipid phase (solid lipid+ SP) was heated up to approximately 5–10 °C above its melting point, before being added to the aqueous surfactant solution of poloxamer P407 of the same temperature. The formulations were stirred for 1min using a magnetic stirrer. Then, the SLN mixture was sonicated using an ultra- sonic processor (Q700 Sonicator, QSonica Sonicators, USA) for 20min (amplitude 6 μm, 14–19W power, 20 kHz frequency, ½" probe). During sonication, samples were maintained in a cooling bath. Since the so- nication process encounters the risk of titanium contamination, samples were centrifuged at 5000 rpm for 10min to remove any metal traces [6]. As SLN are covered with surfactant, they re- mained in suspension with the pre-set centrifugation conditions. Factorial design was used to optimize the production yield, re- quiring a minimum of experiments. The influence of the lipid nano- particles’ composition on their mean hydrodynamic diameter (z-AVE), polydispersity index (PdI) and zeta potential (ZP) was evaluated using a 22 factorial design, i.e. 2 variables which were set at 2-levels each, with central point for estimating the experimental error requiring a total of 5 experiments. The lower and higher values of the lower and upper levels are represented by (− 1) and (+ 1) and the central point by (0), as summarized in Table 1. 2.2.2. Particle size parameters and zeta potential The mean hydrodynamic diameter (z-AVE), polydispersity index (PdI) and zeta potential (ZP) of SLN were determined by photon correlation spectroscopy i.e. Dynamic Light Scattering (DLS, Zetasizer Nano NS, Malvern Instruments, Malvern, UK). The samples were di- luted in ultra-purified water (10 μL/mL) to attenuate their opalescence placed in scintillation vials to maintain cleanliness. All the analyses were done in triplicate (n=3) and data are given as the average values and standard deviations. 2.2.3. In vitro cytotoxicity assay In vitro cytotoxicity assays of SLN composed of 5.0% of SA and 3.1:1.4 P407:SP was performed using NIH 373 mouse fibroblasts as cell model. Cells were seeded in 96-well plates at a density of 10×103 cells/well with different concentrations of SLN (7.0–125.0 μg/mL) at 37 °C and 5.0% of CO2 for 24 h, or medium without SLN as vehicle control or medium without nutrient as negative control. The cells were washed with phosphate-buffered saline (PBS) and cell viability was assessed by MTT reduction assay [7,8]. After removal of the treatment media and cells washed with PBS, the MTT solution (0.5 mg/mL of culture medium) was added to each well and cells were seeded at 37 °C for 3 h. MTT solution was then removed and formazan crystals solubi- lized in 100 μL of ethanol. The plates were shaken for 10min on a plate shaker and the absorbance was measured at 570 nm in a microplate reader (Spectrophotometer Infinite® 200 PRO series). 3. Results and discussion The effect of the lipid composition (i.e. SA, BA or a blend of SA:BA at ratio 1:1) and surfactant ratio on the mean hydrodynamic dia- meter of SLN is shown in Fig. 1, with recorded z-AVE values between 180 and 300 nm. At a first glance, the lowest mean particle size was recorded for the SLN produced with a combination of SA:BA at ratio 1:1, stabilized by P407:SP (3.7:1.0). Table 2 shows the influence of the lipid (SA:BA) and surfactant (P407:SP) ratio on the polydispersity index (PdI) and zeta potential (ZP) of SLN. To determine the mean hydrodynamic diameter responses, the fol- lowing equation has been used: Hydrodynamic diameter (nm)= 250.92+58.15*[lipid]a + 5.7[surfactants]bwhere a stands for the ratio between the lipid phases (SA:BA) and b for ratio between the surfactants (P407:SP) [9]. Indeed, a frequent problem observed in pre-formulation studies is optimizing the excipients concentration in order to obtain a final formulation with the required attributes [10]. From the equation, the mean particle size was 58.15-fold more influenced by the ratio between the two selected lipids (SA:BA), whereas the ratio between both surfactants influenced only 5.7 times. These results are corroborated by those shown in Fig. 2a, which demonstrate that the ratio between the percentage of surfac- tants (P407:SP) exhibited low influence on the mean hydrodynamic diameter of the obtained SLN, but nevertheless also statistically sig- nificant (Fig. 3a). With respect to the ratio between both lipids (SA:BA), the increase on the mean hydrodynamic diameter of nanoparticles was shown to be directly proportional to BA concentration, i.e. a higher mean hydrodynamic diameter was obtained when SLN were composed Table 1 The 22 factorial design used for the development of an optimized SLN for- mulation, providing the lower (− 1), upper (+ 1) and (0) central point level. Variable Level −1 0 +1 SA:BAa 1:0 1:1 0:1 P407:SPb 3.7:1.0 3.5:1.2 3.3:1.4 a 5.0% of solid lipid, which a mixture of stearic acid: behenic alcohol. b 4.7% of surfactant in aqueous solution, which a mixture of poloxamer 407: soybean phosphatidylcholine. Fig. 1. Effect of the lipid composition (i.e. SA or BA or a blend of SA:BA at ratio 1:1) and surfactant ratio on mean hydrodynamic diameter of SLN. Error bars stand for standard deviation (SD, n= 3). R.B. Rigon et al. Colloids and Surfaces B: Biointerfaces 171 (2018) 501–505 502 only of BA as solid lipid (Equation 1). These results were attributed to the different viscosities of lipids used, i.e. SA (C18H36O2) has 7.79mPa/s and BA (C22H46O) has 40.4mPa/s. BA can increase the formulation viscosity and when it is combined with polymers the viscosity is even higher [11]. Viscous formulations usually show higher polydispersity and larger mean particle size, attributed to the lower capacity for energy distribution during the homogenization process [12,13]. On the other hand, it has also been reported that the increased viscosity in nano-formulations might not have a direct influence on the increase of the particle size, but rather the enhanced risk of formation of agglomerates and/or aggregates [14]. With respect to the production of SLN, the particle size distribution is highly dependent on the lipid and surfactant composition, viscosity of the aqueous phase and pro- duction parameters [15]. Jenning et al. conducted a study that aimed to verify the influence of the viscosity of lipid on the particle size [16]. Leaving all other parameters constant, and varying only the composi- tion of the lipid matrix, the results demonstrate that lipid phase of low viscosity improved the homogenization process. In addition, the pro- cessing of lipids of low viscosity at room temperature could ameliorate the particle size distribution when using an incompletely thermos- regulated homogenizer with colder spots, an effect that has not been observed when homogenizing the lipid phase at 85 °C. Hu et al. de- monstrated that the addition of a liquid lipid (e.g. oleic acid) to stearic acid SLN could decreased the viscosity of the lipid phase thereby re- ducing the PdI in a proportional fashion with respect to the oleic acid content [17]. None of our tested variables had a significant effect on the PdI of the SLN (Figs. 2b and 3b). For all SLN matrices, the range of PdI was be- tween 0.190 and 0.217, which can be classified as a monomodal dis- tribution (Figs. 2b and 3b). Indeed, a nanoparticle formulation can display a monomodal (only one population) versus plurimodal (mul- tiple populations), and monodisperse (narrow distribution) versus polydisperse (wide distribution). As PdI is used to describe particle size distribution, its values vary between 0 and 1. Thus, PdI lower than 0.1 is associated to a higher uniformity in particle size distribu- tion, whereas PdI higher than 0.5 suggests a wide particle size dis- tribution or the presence of multiple populations [15,18]. Decreasing the P407:SP ratio (i.e. decrease of %P407 and increase of %SP) increased the absolute values of the ZP for formulation com- posed only of SA (Fig. 2c), but without a significant effect on the mean particle size (Fig. 3c). On the other hand, when increasing the %BA (and decreasing %SA) a decrease of the ZP was observed. Particles with zeta potential higher than+30mV or lower than −30mV are con- sidered physically stable [19]. Nevertheless, good electrostatic stabili- zation could also be achieved with absolute values of zeta potential between 8 and 9mV [20]. While all formulations presented a minimum value for electrostatic stabilization, that composed only of SA and 3.3:1.4 P407:SP ratio demonstrated better results with respect to the zeta potential values (close to ± 30mV). Cytotoxicity of the selected formulation (5.0% of SA and 4.7% of surfactants P407:SP at a ratio 3.1:1.4) was determined by calculating total number of viable cells in SLN-treated wells compared to the total number of viable cells in untreated control wells (positive control). High percentage of cellular viability indicates low toxicity (10%, high Table 2 Influence of the lipid (SA:BA) and surfactant (P407:SP) ratio on the poly- dispersity index (PdI) and zeta potential (ZP) of SLN. SA:BA P407:SP PdI SD ZP (mV) SD −1 −1 0.173 0.010 −22.2 0.289 +1 −1 0.187 0.022 −19.2 0.115 0 0 0.276 0.018 −33.1 0.252 −1 +1 0.194 0.021 −23.5 0.200 +1 +1 0.174 0.018 −13.6 0.173 SD: standard deviation. Fig. 2. Surface response charts of experimental design: (a) mean hydrodynamic diameter (nm); (b) polydispersity index; (c) zeta potential as a function of SA:BA (stearic acid: behenic alcohol) and P407:SP (poloxamer 407:soybean phosphatidylcholine) ratio. R.B. Rigon et al. Colloids and Surfaces B: Biointerfaces 171 (2018) 501–505 503 toxicity; 11%–40%, moderate toxicity; 40%–70%, low toxicity; ≥70% without toxicity) [21]. NIH3T3 mouse embryonic fibroblast cells served as standard cell line for evaluating in vitro cytotoxicity of skin delivery formulations [22]. Results are shown as the percentage of vi- able cells (Fig. 4). Cytotoxicity of the tested SLN was shown to follow a concentration- dependent profile, i.e. SLN composed of 5.0% SA and 3.3:1.4 P407:SP was not cytotoxic up to 31.25 μg/mL with 76.55%±0.009 of cellular viability. Duplicating the concentration (62.50 μg/mL), cell viability was reduced down to ca. 47%, while with the highest tested con- centration (125 μg/mL) the percentage of viable cells was as low as 34.95%±0.0171 (moderate toxicity). The observed reduction of viable cells was attributed to cells hypoxia, as the high concentration of lipid promotes insufficient oxygen supply. However, any concentration de- monstrated high toxicity, only moderate toxicity was observed (34.95%±0.0171 of cellular viability). 4. Conclusions We have demonstrated that a simple factorial design approach, based on two dependent variables, was useful for the optimization of solid lipid nanoparticles produced by sonication. Nanoparticles with higher percentage of stearic acid and lower P407:SP ratio (3.3:1.4) presented the smallest hydrodynamic diameter, with 0.200 of PdI and ZP values of |26 mV|. When treating NIH3T3 fibroblast cells, SLN did not demonstrate relevant cytotoxic events at concentrations up to 39 μg/mL, translating the suitability of the developed formulation for skin administration. Acknowledgments This work was supported by grant#2011/16888-5, grant#2012/ 19568-4, grant#2013/21319-5 and grant#2013/21500-1 Fundação de Amparo À Pesquisa do Estado de São Paulo (FAPESP), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and PADC-FCF-UNESP (Programa de Apoio ao Desenvolvimento Científico da Faculdade de Ciências Farmacêuticas). 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