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PLGA nanoparticles optimized by Box-Behnken for efficient encapsulation of therapeutic Cymbopogon citratus essential oil
Colloids and Surfaces B: Biointerfaces 181 (2019) 935–942
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PLGA nanoparticles optimized by Box-Behnken for efficient encapsulation of therapeutic Cymbopogon citratus essential oil
T
Kessiane B. Almeidaa,b, Aline S. Ramosc, Júlia B.B. Nunesa, Bianca O. Silvad, Elisa R.A. Ferraza, Andreia S. Fernandese, Israel Felzenszwalbe, Ana Claudia F. Amaralc, V. Gaëlle Roullinf, ⁎ Deborah Q. Falcãoa,b,f, a
Faculdade de Farmácia, Universidade Federal Fluminense, Brazil Pós-graduação em Ciências Aplicadas a Produtos para Saúde, Universidade Federal Fluminense, Brazil c Laboratório de Plantas Medicinais e Derivados, FarManguinhos, Fiocruz, Brazil d Instituto de Biofísica, Universidade Federal do Rio de Janeiro, Brazil e Departmento de Biofísica e Biometria, Universidade do Estado do Rio de Janeiro, Brazil f Laboratoire de Nanotechnologie Pharmaceutique, Faculté de Pharmacie, Université de Montréal, Canada b
ARTICLE INFO
ABSTRACT
Keywords: Nanotechnology Polymeric nanoparticles Essential oil Cymbopogon citratus Box-Behnken design Cytotoxicity
This study aimed to optimize Cymbopogon citratus essential oil loaded into PLGA-nanoparticles by investigating the effect of processing variables (sonication time, ultrasound power, and essential oil/polymer ratio) on encapsulation efficiency and particle mean hydrodynamic diameter using Box-Behnken design. Nanoparticles were prepared by an emulsification/solvent diffusion method and physicochemically characterized by FTIR, DSC and TGA/DTA. Cytotoxicity was evaluated in human HaCat keratinocytes by WST-1 and LDH assays. The optimized formulation had a hydrodynamic mean diameter of 277 nm, a polydispersity index of 0.18, a Zeta potential of −16 mV and an encapsulation efficiency of 73%. Nanoparticle characterization showed that only citral was incorporated in nanocarriers, with some amount adsorbed on their surface, and highlighted the potential in increasing the oil thermal stability. The drug release profile demonstrated a biphasic pattern with a substantial sustained release depending on diffusion from the polymeric matrix. Toxicity effects on cell viability of pure essential oil at low concentrations were significantly eliminated when encapsulated. Results revealed the ability of PLGA-nanoparticles to improve essential oil physicochemical characteristics, by controlling release and reducing toxicity, suggesting their potential use in pharmaceutical preparations.
1. Introduction Essential oils (EOs) are complex mixtures of volatile secondary metabolites, extracted from different parts of aromatic plants. They mainly comprise mono- and sesquiterpenoids, phenylpropanoids and short-chain aliphatic hydrocarbon derivatives [1,2]. Due to their unique physicochemical characteristics, EOs have been successfully used in various industries, including the medical field [3] due to a broad spectrum of biological activities to treat many pathological conditions [4,5]. Cymbopogon citratus (DC) Stapf (Poaceae), more commonly known as lemongrass, is a perennial tropical and subtropical herb [6]. The major compound of C. citratus essential oil (CcEO) is citral, a mixture of two stereoisomers: neral (cis-citral, 21.4–39.7%) and geranial (transcitral, 29.4–60.3%) [7]. CcEO is known to display many
⁎
pharmacological effects, such as anti-inflammatory, antifungal, sedative, antibacterial, antiviral and anticarcinogenic properties [8]. However, the use of CcEO in pharmaceutical preparations is limited due its chemical characteristics. In fact, EOs are easily degraded by oxidation, volatilization, heating and light, if they are not protected from environmental factors [5,9]. Furthermore, their low aqueous solubility prevents oral administration. Such characteristics are barriers to scale up EOs products at the industrial level. Different strategies have been designed to overcome these issues, such as emulsification, aerosolization and encapsulation in nanocarriers [10]. EOs encapsulation in polymeric nanoparticles has enhanced their physical stability, reduced their volatility and improved their aqueous solubility, while providing a sustained release of active compounds [10,11]. Moreover, due to their nanometric size, these colloidal systems may increase cellular absorption and potentiate bioefficiency [1].
Corresponding author at: Faculdade de Farmácia, Universidade Federal Fluminense, Brazil. E-mail addresses: [email protected], [email protected] (D.Q. Falcão).
https://doi.org/10.1016/j.colsurfb.2019.06.010 Received 14 March 2019; Received in revised form 3 June 2019; Accepted 5 June 2019 Available online 05 June 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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Appropriate selection of a polymeric matrix is essential in order to develop a successful nanoparticulate delivery system. Poly(D,L-lacticco-glycolic) acid (PLGA), one of the most extensively used biodegradable polymers, is widely endorsed to encapsulate natural products due its excellent biocompatibility and biodegradation [12,13], well-known safety, its ability to control drug release kinetics and its possible functionalization [14,15]. The emulsification/solvent diffusion (ESD) technique is a suitable method to encapsulate lipophilic substances [16,17], resulting in high encapsulation efficiency and narrow size distribution [18]. However, several process variables can affect the physicochemical properties of the formed nanoparticles [19]. Design of experiment (DoE) is a useful tool to investigate the influence of different factors on the selected responses, by running a small number of experiments, thus saving time and reducing costs [20]. Box–Behnken design (BBD) is a fractional factorial design of second-order based on three levels, widely applied for optimization of nanoparticle synthesis. This technique is suitable to explore quadratic response surfaces and constructs according to a mathematical model, assisting to determine a system optimal solution [21–24]. This study aimed to optimize the ESD synthesis and encapsulation efficiency (EE) of previously developed CcEO-loaded nanoparticles intended for anti-herpetic topical treatment [22]. The effects of some parameters, such as sonication time, ultrasound power and essential oil/polymer ratio were analyzed using BBD and response surfaces. Optimized experimental conditions were used to obtain nanoparticles with reduced mean hydrodynamic size (Z-ave) and high EE. The optimized nanoparticles were physicochemically characterized and their cytotoxicity evaluated in HaCat cells by WST-1 and LDH assays.
Table 1 Variables employed in BBD. Independent variable/factor
X1: Sonication time (min) X2: Ultrasound power (W) X3: Essential oil/polymer ratio (w/w) Dependent variable/response Y1: Encapsulation efficiency (%) Y2: Mean hydrodynamic size (d.nm)
Levels −1
0
1
5 50 0.50 Constraints
10 100 1.25
15 150 2.00
Maximize Minimize
medium and high levels, respectively (Table 1). The medium level was chosen as follows: sonication time (10 min), ultrasound power (100 W) and essential oil/polymer ratio (1.66:1.33), where corresponding amounts of oil and PLGA were 194 mg and 156 mg, respectively. EE (Y1) and Z-ave (Y2) were used as dependent variables (responses). The interaction of independent variables and responses were evaluated using the following quadratic mathematical model (Eq. 1): Yi = b0 + b1X1 + b2X2 + b3X3 + b1,2X1X2 + b1,3X1X3 + b2,3X2X3 + b1,1X12 + b2,2X22 + b3,3X32
(1)
where Yi is the dependent variable, b0 is the arithmetic mean response of 17 runs, bi is the estimated regression coefficient for each factor, and Xi represents the coded independent variables. Statistical analysis from formulation design was performed using Statistica software 7.0 (Stafsoft, Inc., USA). The experimental design matrix generated by software and corresponding observations for the dependent variables are shown in Table S1.
2. Materials and methods 2.1. Materials
2.4. Statistical and checkpoint analyses
Cymbopogon citratus essential oil (L192 lot, Laszlo Aromaterapia, Brazil). Poly (vinyl alcohol) (PVA; Mw 85,000–124,000 Da), poly (D,Llactide-co-glycolide) (PLGA; Mw 50,000–75,000 Da, 85:15), dichloromethane (DCM), trifluoroacetic acid (TFA) and citral standard reference (Sigma-Aldrich, USA). Carbopol® Ultrez 10 NF (Fagron, Brazil). Triethanolamine (Vetec, Brazil). Dimethyl sulfoxide (DMSO), nhexane and acetonitrile HPLC grade (Tedia, Brazil), Dulbecco’s Modified Eagle’s Medium (DMEM) (Life technologies, EUA), WST-1 reagent (Roche Co., USA). Phosphate-buffered saline (PBS) pH 6.8 [23] was prepared using sodium phosphate dibasic anhydrous, sodium phosphate monobasic anhydrous and sodium dodecyl sulfate (SDS) (Vetec, Brazil).
Analysis of variance (ANOVA) and lack of fit were performed to ensure the model fitting. The variables which significantly affected Y1 and Y2 were identified and shown through Pareto charts using Student’s t-test. Results were considered statistically significant at p-value < 0.05. Response surfaces were plotted and critically exploited to determine the impact of factors (X1, X2 and X3) on nanoparticles preparation. CcNP theoretical optimal experimental conditions were obtained by response surface analysis and mathematical model aiming to formulate nanoparticles with maximum EE and smallest Z-ave. The resulting statistical models were checked in triplicate and experimental responses (Yi) were compared with those predicted.
2.2. Preparation of CcEO-loaded nanoparticles (CcNP)
2.5. Characterization of nanoparticles
CcNP were synthesized by the ESD method with slight modifications [22]. An appropriate amount of PLGA was dissolved in DCM saturated with distilled water and CcEO was added. In parallel, PVA (1.0%, w/v) was solubilized in distilled water saturated with DCM. The organic phase was added dropwise to the aqueous phase, maintained in an ice bath, under continuous ultrasonic homogenization (Sonic Ruptor 250, Omni International, USA). Subsequently, the oil-in-water emulsion was diluted in distilled water under magnetic stirring to allow solvent diffusion. The nanoparticle suspension was lyophilized (Terroni Enterprise II, LT 1000, Brazil) to obtain CcNP. Blank nanoparticles (NP) were prepared without addition of CcEO.
Synthesized nanoparticles were characterized by Z-ave using dynamic light scattering (DLS) and Zeta potential (ZP) by electrophoretic mobility, using a Zetasizer Nano ZS90 (Malvern Instruments, UK). Lyophilized samples were suspended in ultra-purified water (1:25) and analyzed at a scattering angle of 173°, 25 °C, using water as the reference medium. Measurements were performed in triplicate and results expressed as mean ± standard deviation [22]. Nanoparticles shape and surface morphology were examined by transmission electronic microscopy (TEM) (Vega 3LMU Tescan, Czech Republic) (acceleration voltage 80 kV). A drop of nanoparticles, suspended in distilled water, was spread on a 200-mesh copper grid covered with formvar film, dried at room temperature and followed by double staining with uranyl acetate and lead citrate solution. The amount of CcEO incorporated into CcNP was recovered by solvent extraction method [25] for EE evaluation. CcNP were dissolved in DMSO, vortexed and centrifuged (2000 x g) for 1 min. Supernatants were diluted with DMSO and analyzed by UV–vis spectrophotometry (Shimadzu® UV-2600, Japan) at 260 nm. Blank NP were used as a
2.3. Experimental design A 3-factor, 3-level BBD was used to optimize CcNP previously developed [22]. The investigated independent variables (factors) were: sonication time (X1), ultrasound power (X2), and CcEO/polymer massic ratio (X3), which were represented by −1, 0 and +1, analogous to low, 936
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reference. Total CcEO content was quantified from an average of three standard curves in the range of 0.002–0.04 mg/mL (r2 = 0.9999, y = 22.537x - 0.0176). The extraction procedure and analysis were carried out in triplicate and EE (%) was calculated by Eq. 2:
keratinocytes HaCat by water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) assays and compared with CcEO [29]. 2.9. HaCaT cells and culture conditions
EE = 100 × (measured CcEO content/theoretical total CcEO content) (2)
HaCaT cells were acquired from the Cell Bank of Rio de Janeiro (Rio de Janeiro, Brazil). DMEM completed with 10% of fetal bovine serum (FBS), sodium bicarbonate 3.7 g/L and 1.0% of antibiotic solution (100 IU/mL penicillin and 100 μg/mL streptomycin, Life, EUA) was used to grow the cells at 37 °C and 5% CO2 and 95% relative humidity. The cells were subcultured, under the same conditions, every 2–3 days until reaching a density of ˜1 × 106cells/mL.
CcNP were characterized by Fourier transformed infrared spectroscopy (FTIR) over the spectral region from 400 to 4000 cm−1 using a Shimadzu IR Tracer-100 FTIR spectrometer with Attenuated Total Reflectance (ATR) acessory (MIRacle, 10) and compared with NP, PLGA and CcEO. Thermal characterizations were also performed by differential scanning calorimetry (Shimadzu Co., Japan) using a DSC-60 system and by Thermogravimetric (TGA) and Differential Thermal analyses (DTA) using a TGA Q500 V20 Thermogravimetric Analyzer (TA Instruments, USA). Samples (3.5 mg) were placed in aluminium pans and heated from 25 to 200 °C at a scanning rate of 10 °C/min under constant nitrogen purge of 80 mL/min for DSC analyses. TGA and DTA were carried out at 10 °C/min from 25 to 600 °C at a flow rate of 60 mL/min and compared with NP, PLGA and PVA.
2.10. WST-1 assay About 2 × 104 HaCaT cells were seeded into each well of sterile flatbottomed 96-well plates, incubated with either CcEO (1.9–250.0 μg/ mL) or CcNP (21.28–2800.0 μg/mL, corresponding to 1.9–250.0 μg/mL of CcEO) for 24, 48 and 72 h. To allow CCEO detection, samples were suspended in DMEM containing DMSO (1.0%, v/v). This medium was used as a negative control whereas Triton X-100 (10.0%, v/v) was used as a positive control. At the end of the treatment period, the culture media in each well were replaced by 100 μL of WST-1 reagent (10.0%, v/v, in DMEM) and incubated at 37 °C and 5% CO2 for 3 h. The assay measures cell viability by assessing the activity of mitochondrial dehydrogenases. The plate absorbance was measured at 450 nm using a Polaris Microplate Reader (Celer, Brazil) according to kit protocol and literature [29]. The cell viability in each well was calculated as a ratio to the one of negative control cells. The concentration that caused 50% growth inhibition (IC50) was determined by plotting the log10 of the percentage of viability versus CcEO concentrations after 72 h of treatment.
2.6. CcEO chemical composition Qualitative analyses, before and after encapsulation, were performed by a GCMS-QP5000 (Shimadzu) gas chromatograph equipped with a mass spectrometer using electron ionization. Gas chromatographic (GC) conditions were as follows: injector temperature, 250 °C; carrier gas, helium; flow rate, 1 mL/min and split injection with split ratio 1:40. The oven temperature was initially 80 °C and raised to 290 °C at a rate of 12 °C/min. One microliter of each sample, dissolved in hexane (1:100 mg/μl), was injected onto a DB-5 column (i.d. = 0.25 mm, length 30 m, film thickness = 0.25 μm). The mass spectrometer was monitored to scan m/z 35–650 with an ionizing voltage at 70 eV and scan rate 1 scan/s [26]. Retention indices (RI) were determined in relation to a homologous series of n-alkanes (C10C40) analyzed under the same operating conditions. Identification of the constituents was performed by comparison of their RI and mass spectra from literature [27] and checked against NIST mass spectra libraries. Component relative percentages were calculated based on GC peak areas without using correction factors.
2.11. LDH assay LDH assay, which measures cytotoxicity by assessing the integrity of the plasma membrane, was evaluated using a commercial kit (Roche, Germany) according to the manufacturer’s protocol. After treatment, 100 μL supernatant and 100 μL reaction mixture (freshly prepared) were transferred from each well of a 96-well flat-bottomed plate. The plates were incubated for 30 min at 20 °C in the dark and absorbance was measured using a Polaris Microplate Reader at 492 nm. The cytotoxicity percent was calculated according to the kit protocol [29].
2.7. In vitro release studies CcEO in vitro release from nanoparticles was evaluated by the dialysis membrane diffusion technique [25]. In brief, 35 mg of lyophilized nanoparticles (117 mg CcEO/g CcNP) were added in the donor chamber and 12.5 mL of PBS (pH 6.8) with SDS (1.0%, w/v) in the acceptor chamber. CcEO solubility in the dissolution medium was previously determined to be 0.33 mg/mL. The system was placed in a shaking bath (37 °C ± 0.5) under constant agitation (100 rpm). At several points (0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 3.0, 5.0 and 24.0 h, and then once a day for 8 days), an aliquot of 2.5 mL was withdrawn and replaced with an equal volume of fresh dissolution medium to ensure sink conditions. Samples were analyzed by UV–vis spectrophotometry at 240 nm using blank NP as references. The CcEO content was calculated from an average of three standard curves prepared with the medium, ranging from 0.0025-0.04 mg/mL (r2 = 0.9996, y = 71.862x + 0.0047). The release study was carried out in triplicate. The cumulative drug release percent was plotted against time. Results were fitted to various kinetic equations to evaluate the mechanism of release from CcNP: Zero Order, First Order, Higuchi, Hixson-Crowell and Korsmeyer-Peppas [28].
2.12. Statistical analysis Statistical evaluation of data was performed using one-way analysis of variance (ANOVA) and Student s̓ t-test, employing GraphPad Prism software 5.0 (GraphPad Software Inc., USA). In the cytotoxicity assays, the statistical analysis was calculated using one-way ANOVA and Dunnett’s post hoc test. Data were expressed as the mean value ± standard deviation (mean ± S.D.) (n = 3). The differences were considered statistically significant when p-value < 0.05. 3. Results and discussion In drug delivery systems development, it is crucial to consider EE and Z-ave, in order to ensure that enough drug is delivered to target tissue to produce a therapeutic effect. These properties can be controlled during the early stages of development by analysis of certain formulation and process variables [20]. In the present study, BBD was applied to optimize previously developed CcNP using the ESD technique [22]. A total of 17 runs with 5 centre points was carried out to evaluate the effect of 3 independent variables on 2 dependent variables.
2.8. CcNP in vitro cytotoxicity CcNP in vitro cytotoxicity was evaluated in human skin 937
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Fig. 1. Response surface plots showing (A) effect of X2 and X3 on encapsulation efficiency, (B) effect of X1 and X2 on average particle size with X3 set at 0.5 and, (C) effect of X1 and X3 on average particle size with X2 set at 150 W. *X1: sonication time; X2: power of ultrasound; X3: essential oil/polymer ratio.
3.1. Effect of independent variables on EE (Y1)
polymer ratio) exhibited a negative linear effect on EE. An increase in PLGA concentration enhanced CcEO content into polymeric nanoparticles (Fig. 1A) since it increased the organic phase viscosity, resulting in higher resistance to CcEO diffusion into the aqueous phase [16,30]. Furthermore, increasing PLGA concentration favored the precipitation process, reducing the timespan available for CcEO diffusion from the nanoparticle matrix, resulting in higher encapsulation rates [31]. According to Eq. 3, the ultrasound power had a positive effect in EE. Enhancing ultrasound power leads to smaller droplets during the emulsification step, thus globally increasing nanoparticle surface area [20], and ultimately leading to high encapsulation content. Furthermore, increasing ultrasound power provided a turbulent and unidirectional flow, avoiding loss of CcEO from the organic phase [31].
All encapsulation efficiencies ranged between 17.33% (F-11) and 86.97% (F-6) (Table S1). A Pareto chart was constructed to evaluate which factors influenced the Y1 response (Figure S1A). Only X2 and X3 variables had significant effects on EE at a 95% confidence level. There was no significant interaction between the studied factors. The model proposed for predicting the effect of factors on the Y1 response is presented in Eq. 3: Y1 = 56.6 + 15.0 X2 – 17.1 X3
(3)
The predicted (0.662) and adjusted (0.614) R-square values obtained from the ANOVA test suggested that some other important variables for the EE parameter were not considered in this study. However, residues showed a normal distribution according to Kolmogorov-Smirnov (p > 0.20) and Shapiro-Wilk tests (p = 0.51766) and the lack of fit term was not significant. Thus, this model could still be used to navigate the design space. A positive or negative value is related to a positive or negative effect on the studied response, respectively. According to Eq. 3, factor X3 (EO/
3.2. Effect of independent variables on Z-ave (Y2) Particle size plays a crucial role in transport and absorption of bioactive molecules into the body as well as the drug release rate [18]. CcNP average hydrodynamic diameters were found in the range of 938
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167 nm (F-20) to 318 nm (F-12) (Table S-1), in agreement with other studies reporting the use of the ESD method to synthetize nanoparticles [17,19]. Analysis of data by ANOVA and Pareto chart (Figure S-1B) showed the significant effect of all factors and their interactions on Y2 (p < 0.05), except for X2, with a quadratic effect and a linear interaction between X2 and X3. The effect on Z-ave can be explained by the polynomial Eq. 4:
order to obtain nanoscale particles with high content of CcEO: a sonication time of 8 min, an ultrasound power of 150 W and an essential oil/polymer ratio of 0.5. For model validation, nanoparticles were prepared in triplicate following these conditions and results were compared with the values predicted by model, with an acceptable error of less than 5%. Applying these conditions, EE and Z-ave predicted by the calculated model were 88.74% and 173.56 nm, respectively. The experimental results are presented in Table 2. The experimental value for response Y1 (73.29%) of optimized formulation is consistent with the predicted confidence interval (71.51–105.98%). This result confirmed its adequate precision for predicting optimum conditions in the chosen domain of levels for the independent variables. Therefore, by applying BBD it was possible to incorporate an amount 2.6 times greater of CcEO then previously described (EE 28.48%) [22]. Regarding the mean hydrodynamic diameter, the average replicates synthesized were 277 nm (Table 2), a result which was not predicted by the mathematical model (174 nm), suggesting that other important factors controlling the nanocarrier size were not considered in this study. However, it is known that the tensions generated during lyophilization can destabilize systems, leading to aggregation or even particle coalescence [33]. The concentration of the stabilizing agent may also influence particle size [19], since it tends to position itself on droplet surface, reducing free energy at the interface between organic and aqueous phases, stabilizing the droplets [31]. Thus, it can be suggested that the lack of standardization in the lyophilization step and/or an insufficient amount of PVA may have contributed to not having achieved the predicted particle size. The size distribution profiles (Fig. 2) suggested that the CcEO presence favored size distribution homogeneity, showing unimodal profiles different than that of unloaded NP which showed a bimodal profile. These patterns were corroborated by polydispersity index (PDI) results which were statistically different for both samples (Table 2, Student s̓ ttest, p < 0.05). Moreover, optimized CcNP showed more homogeneous size distribution than those previously reported for unoptimized nanoparticles [22], highlighting the success of BBD. Nanoparticle morphological evaluation (Fig. 2B and D) showed spherical shapes and smooth surfaces with size distribution consistent with results obtained by DLS. EO incorporation did not alter the particle shape. However, a strong aggregation tendency was observed in both samples, probably related to insufficient steric stabilization by the PVA [34] or lyophilization step. This aggregation was expected for both samples due their low Zeta potential which suggests that the attractive force surpasses the repulsive force between particles. The negative charge is related to free ionized PLGA terminal carboxyl groups on the surface [19,20,30]. The addition of CcEO enhanced the Zeta potential values (Student's t test, p < 0.05), which seemed to minimize the aggregation observed for NP, according to its unimodal particle size distribution and morphological evaluation. The more negative charge of CcNP seemed to be related to the presence of ionized carbonyl groups from citral adsorbed on the nanocarrier surface, as showed by FTIR characterization (Figure S-2). Absorption bands of carbonyl groups from α, β-unsaturated aldehyde (C]CeCOH, 1672 cm−1) and CeH axial deformation of aldehydes (2968–2856 cm−1 and 1377 cm−1), characteristics of CcEO [23,35], can be observed in both CcNP and CcEO FTIR spectra, with different intensities. It indicated that only a
Y1 = 183.8 +15.9 X1 – 9.5 X12 + 13.2 X2 – 38.9 X3 – 11.6 X32 + 13.9 X1X2 + 15.1 X1X22 – 11.6X12X2 – 28.6 X1X3 + 18.9X12 X3 (4) The values for predicted (0.994) and adjusted (0.984) R-square showed the adequacy of the model to predict the response in the optimization process. Moreover, residues showed a normal distribution according to Kolmogorov-Smirnov (p > 0.20) and Shapiro-Wilk tests (p = 0.61824) and the lack of fit term was not significant. Thus, the statistical analysis demonstrated model suitability to describe the behavior of the independent variables and their interactions on Y2 in the experimental domain. Eq. 4 shows that X3 presented negative, linear and quadratic effects on Y2, as well as interaction effects with the different factors evaluated in the study. Therefore, increasing PLGA concentration, i.e. enhancing the organic phase viscosity, reducing the stirring shear efficiency required for droplet breakdown, led to larger diameter droplets during the emulsification process. A more viscous organic phase could also decrease the diffusion rate from the organic to the aqueous phase, promoting the Ostwald ripening phenomenon, causing an increase in particle size [19,30]. Such increase could be also explained by polymerpolymer interactions in a more concentrated phase, which could lead to particle aggregation [16,20]. As described before, the variable X1 showed no effect on EE. In order to evaluate the X1 value, the X2 factor was set at its maximum level (+1) and X3 at its minimum (-1), as previously determined optimal conditions to obtain particles with maximum EE (Y1). The respective 3D response surfaces were plotted (Fig. 1B and C) and the X1 value was graphically determined as 8 min, to be used in the optimization process. The influence of the X1 factor on the Y2 response showed a positive linear effect, negative quadratic effect and interaction effects with the other factors (Equ. 4). Fig. 1B and 1C highlights the relation between the sonication time and the particle size. Increasing sonication time from 0 to 10 min decreased particle size. However, the reverse effect was verified when increasing the sonication time to 15 min. This may be due to aggregation of small particles resulting from prolonged sonication time [16]. Various studies have reported that increasing ultrasound power led to the formation of smaller particles, since more released energy increased shear stress and, consequently, decreased the viscosity of the organic phase during the emulsification step [32]. Eq. 4 shows that X2 had a positive linear effect on Y2 and interaction effects on the studied factors. However, by setting X3 factor to 0.5, small particles were obtained throughout the experimental domain, employing minimal or maximal levels (50–150 W). 3.3. Optimized polymeric nanoparticles The application of statistical tools and critical analysis of response surfaces allowed to determine the optimal experimental conditions in
Table 2 Results of mean hydrodynamic size (Z-ave), polydispersity index (PDI), Zeta potential (ZP) and encapsulation efficiency (EE) of optimized nanoparticles. Formulation
Z-ave (nm)
PDI
ZP (mV)
EE (%)
NP CcNP
280.8 ± 19.3* 277.0 ± 5.5*
0.27 ± 0.02** 0.18 ± 0.04**
−13.3 ± 1.7** −16.1 ± 1.8**
– 73.29 ± 8.96
Mean value ± S.D. (n = 9); *(p > 0.05), **(p < 0.05) by Student’s t-test. NP: blank nanoparticles; CcNP: CcEO load nanoparticles. 939
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Fig. 2. Typical size distribution (A) and TEM image of NP (B); size distribution (C) and TEM image of CcNP (D). *TEM image x180,000 magnification. Scale bar: 250 nm.
small CcEO amount was adsorbed on the CcNP surface. Small changes were observed in bands of −OH (3317 cm−1), –C]O (1755cm−1) and C–O stretching of ester groups (1086 cm−1) when compared to NP and CcEO spectra, suggesting interaction between the citral and the nanocarrier and its successful encapsulation. The spectra obtained for PLGA agreed with literature data [30,36]. The presence of the CcEO component adsorbed in CcNP surface and its encapsulation were confirmed by thermal analysis. DSC thermograms (Figure S-3) of CcNP and NP exhibited two endothermic peaks. The peak temperature observed in CcNP first event (133 °C) was higher than observed for NP and CcEO, demonstrating the nanocarrier’s ability in increasing EO thermal stability. CcEO showed two endothermic peaks at 102.4 and 141.7 °C, relative to volatilization and/or thermooxidative decomposition. It is well-known that EO thermal profiles are influenced by their chemical composition, since each substance exhibits different characteristics, such as molecular weight and boiling/volatilization temperature [37]. DSC thermal profiles of PVA and PLGA 85:15 were consistent with previously reported data [38,39]. CcNP thermogravimetric results (TGA and DTA) (Figure S-4 and Table S-3) showed four thermal events. The first event, observed in range 30 to 60 °C (Δm = 2.69%), should be linked to the volatilization of citral molecules adsorbed on CcNP surface, since Tdeg max (39.4 °C) was superior to that obtained for the NP, corroborating the results previously discussed. The second thermal event (60–130 °C: Δm = 10.26%) referred to thermo-oxidative volatilization and/or decomposition of encapsulated CcEO. The higher Tdeg max (104 °C) compared to that obtained for CcEO highlighed its encapsulation and protection by nanoencapsulation. Subsequent thermal events related to nanostructure degradation showed maximum mass loss at temperatures of 298 and 452.4 °C as observed for the NP, in agreement with literature [40]. CcEO thermogram showed a fast mass loss ranging from 30 to 94 °C, with a maximum rate observed at 72 °C, attributed to the volatilization and/or thermo-oxidative decomposition. Mass loss of 5.78% was still verified up to 260 °C related to oil volatilization.
compounds represented 99.45% of the total oil (Table S-2). Citral components, geranial (42.67%) and neral (33.03%), were the major constituents. Citral total content (75.70%) agreed with literature data [6]. Other substances with appreciable relative abundance were myrcene (10.68%) and geraniol (4.99%), also previously reported in CcEO [41]. CcEO chemical profile after encapsulation showed only citral (neral - 42.14% and geranial - 57.86%) incorporated into the nanocarrier matrix. Turbulent flow and heating generated by the ultrasound probe during the emulsification process may contribute to loss of minor compounds by volatilization. Similar results were previously described in the development of CcEO-loaded PCL nanoparticles by the ESD method [25], highlighting the presence of citral in CcNP and its interaction with the matrix material. 3.5. In vitro CcEO release studies The release behavior of CcEO from optimized nanoparticles (Fig. 3) showed a biphasic pattern, with an initial burst (24.8 ± 1.7% in 3 h), followed by a sustained release for 8 days, reaching a maximum oil release of 83.9 ± 3.5%. The initial burst might be attributed to the immediate desorption of citral molecules adsorbed onto the particle surface, as previously discussed. The slower release phase corresponded to diffusion of citral molecules through interconnected pores and channels in the polymeric matrix, from the particle core to the dissolution medium [42]. PLGA with a high lactic acid content and molecular weight, such as the one used in this study, is more hydrophobic and, consequently, exhibits slower water uptake, decreasing polymer degradation rate [34]. Furthermore, the increment in lactic acid content increases polymer hydrophobicity, favoring the partition of citral molecules in the polymer matrix compared to the aqueous external medium [43]. The results were analysed by different mathematical models in order to determine CcEO release mechanisms from CcNP (Table 3). Analysis of correlation coefficients revealed that the Korsmeyer-Peppas model as the best fit (R2 = 0.9445). However, the behavior proposed by Higuchi (R2 = 0.9155) must be considered. It suggested citral release was mainly based on diffusion and agreed with the diffusional exponent
3.4. Chemical composition of CcEO GC-MS analysis of CcEO before encapsulation showed that 14 940
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Fig. 3. In vitro CcEO release profile in saline-phosphate buffer with sodium lauryl sulfate (1.0%, w/v), pH 6.8, from optimized nanoparticles (n = 9).
value (n = 0.3933). Citral release was governed by Fickian transport mechanism (n = 0.3933), only dependent on citral diffusion through the polymeric matrix. Both models have been efficiently applied to describe the release behavior of hydrophobic drugs from PLGA nanoparticles [42,30]. The release profile reflected that CcNP allowed CcEO sustained release for several days. Hence, the therapeutic actions of CcEO could definitively be enhanced by reducing the number of administrations and the patient’s compliance and comfort to treatment.
viability at concentrations greater than 3.9 μg/mL (Figure S-5A). Cell viability was increased by CcEO encapsulation, showing cytotoxic effects in concentrations higher than 31.25 μg/mL (Figure S-5C). The average IC50 of CcEO and CcNP were 39.40 and 66.28 μg/mL, respectively. CcEO altered integrity of HaCaT cell membranes in a time- and dose-dependent manner from 125 μg/mL after 24 h of exposure (Figure S-5C). On the other hand, CcNP induced apparent LDH leakage from cells at 31.25 μg/mL after 48 h of incubation (Figure S-5D). However, there was no significant difference between IC50 values for CcEO (137.9 μg/mL) and CcNP (130.3 μg/mL). CcEO and CcNP induced cell cytotoxicity by affecting mitochondrial dehydrogenase activity, as detected by the WST-1 assay and by destroying cell membrane integrity, as showed by the LDH assay. CcEO toxic effects for HaCaT cells were attributed to its citral content [44]. However, the encapsulation in CcNP greatly decreased citral toxic effect on cell mitochondrial dehydrogenase activity compared to free CcEO
3.6. In vitro cytotoxicity Toxicity is an important issue to consider in nanocarrier development, since their reduced dimensions favor cell penetration. The effect of CcEO and CcNP on cell viability (% of control) was evaluated by the WST-1 assay [29]. CcEO showed time- and dose-dependent reduction in HaCaT cell Table 3 Kinetic behavior of in vitro CcEO release from CcNP. Mathematical model Zero Order k0 0.0066
First Order 2
R 0.7726
k1 0.0002
Higuchi 2
R 0.5599
kH 0.7547
Hixson-Crowell 2
R 0.9155
R2: correlation coefficient; k: release rate constant; n: diffusional release exponent. 941
kHC −0.0002
Korsmeyer-Peppas 2
R 0.8639
kK 2.6133
R2 0.9445
n 0.3933
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Funding This work was supported by Fundação de Amparo à Pequisa do Estado do Rio de Janeiro (FAPERJ) - Brazil [grant numbers JCNE 2016, E-26/202,759/2017 and E26/202,256/2018]; (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CAPES) - Brazil [grant number 001] and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) - Brazil [grant number 302345/2017-5]. Acknowledgment The authors thank the staff members of the Multiuser Laboratory of Material Characterization (www.uff.br/lamate) for training and assistance pertaining to the Zetasizer Nano ZS90 results and Rodolpho Albino University Laboratory (www.lura.uff.br) for the use of the freeze-dryer. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.06.010. References [1] [2] [3] [4] [5] [6] [7] [8] [9]
Fig. 4. Effect on mitochondrial dehydrogenase activity (A) and cell membrane integrity (B) of CcEO and CcNP in HaCaT cell. (mean ± S.D., n = 3).
(Fig. 4). Several in vitro studies have emphasized the ability of polymeric nanoparticles to increase cell viability in the presence of natural molecules. Chen et al. [45] demonstrated the reduction of cytotoxicity of carvacrol and eugenol in mouse 3T3 fibroblasts, when grafted on chitosan nanoparticles. The neutral red dye-uptake method, evaluated by Almeida et al. [22] on Vero cells viability, showed decreased EOassociated cytotoxicity after encapsulation in PLGA nanoparticles, with a maximum non-toxic concentration 5-fold lower than that of free CcEO.
[10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]
4. Conclusion
[24] [25] [26] [27]
CcEO-loaded PLGA nanoparticles were prepared by the ESD technique and physicochemically characterized. The 3-factor, 3-level BBD used provided fitting polynomial equations for the evaluated responses (EE and Z-ave) and was subsequently employed with success to optimize the nanoparticle formulation. In comparison to previous report, CcNP developed in this study provided higher EE and lower PDI. Optimized CcNP physical characterization evidenced the citral interaction with the polymeric matrix, highlighting the nanocarrier potential in increasing its thermal stability. CG-MS showed that only citral was incorporated into the nanostructure. Citral was released from CcNP according to a biphasic pattern, with an initial burst due to the fraction adsorbed onto the particle surface, followed by a sustained release dependent on its diffusion from the polymeric matrix. Moreover, nanoencapsulation attenuated CcEO toxic effects on the mitochondrial dehydrogenase activity of human keratinocytes. These results revealed the ability of PLGA-nanoparticles to improve the physicochemical characteristics of CcEO, to control its release and reduce its toxicity, suggesting a promising potential for pharmaceutical uses.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
PLGA nanoparticles optimized by Box-Behnken for efficient encapsulation of therapeutic Cymbopogon citratus essential oil
T
Kessiane B. Almeidaa,b, Aline S. Ramosc, Júlia B.B. Nunesa, Bianca O. Silvad, Elisa R.A. Ferraza, Andreia S. Fernandese, Israel Felzenszwalbe, Ana Claudia F. Amaralc, V. Gaëlle Roullinf, ⁎ Deborah Q. Falcãoa,b,f, a
Faculdade de Farmácia, Universidade Federal Fluminense, Brazil Pós-graduação em Ciências Aplicadas a Produtos para Saúde, Universidade Federal Fluminense, Brazil c Laboratório de Plantas Medicinais e Derivados, FarManguinhos, Fiocruz, Brazil d Instituto de Biofísica, Universidade Federal do Rio de Janeiro, Brazil e Departmento de Biofísica e Biometria, Universidade do Estado do Rio de Janeiro, Brazil f Laboratoire de Nanotechnologie Pharmaceutique, Faculté de Pharmacie, Université de Montréal, Canada b
ARTICLE INFO
ABSTRACT
Keywords: Nanotechnology Polymeric nanoparticles Essential oil Cymbopogon citratus Box-Behnken design Cytotoxicity
This study aimed to optimize Cymbopogon citratus essential oil loaded into PLGA-nanoparticles by investigating the effect of processing variables (sonication time, ultrasound power, and essential oil/polymer ratio) on encapsulation efficiency and particle mean hydrodynamic diameter using Box-Behnken design. Nanoparticles were prepared by an emulsification/solvent diffusion method and physicochemically characterized by FTIR, DSC and TGA/DTA. Cytotoxicity was evaluated in human HaCat keratinocytes by WST-1 and LDH assays. The optimized formulation had a hydrodynamic mean diameter of 277 nm, a polydispersity index of 0.18, a Zeta potential of −16 mV and an encapsulation efficiency of 73%. Nanoparticle characterization showed that only citral was incorporated in nanocarriers, with some amount adsorbed on their surface, and highlighted the potential in increasing the oil thermal stability. The drug release profile demonstrated a biphasic pattern with a substantial sustained release depending on diffusion from the polymeric matrix. Toxicity effects on cell viability of pure essential oil at low concentrations were significantly eliminated when encapsulated. Results revealed the ability of PLGA-nanoparticles to improve essential oil physicochemical characteristics, by controlling release and reducing toxicity, suggesting their potential use in pharmaceutical preparations.
1. Introduction Essential oils (EOs) are complex mixtures of volatile secondary metabolites, extracted from different parts of aromatic plants. They mainly comprise mono- and sesquiterpenoids, phenylpropanoids and short-chain aliphatic hydrocarbon derivatives [1,2]. Due to their unique physicochemical characteristics, EOs have been successfully used in various industries, including the medical field [3] due to a broad spectrum of biological activities to treat many pathological conditions [4,5]. Cymbopogon citratus (DC) Stapf (Poaceae), more commonly known as lemongrass, is a perennial tropical and subtropical herb [6]. The major compound of C. citratus essential oil (CcEO) is citral, a mixture of two stereoisomers: neral (cis-citral, 21.4–39.7%) and geranial (transcitral, 29.4–60.3%) [7]. CcEO is known to display many
⁎
pharmacological effects, such as anti-inflammatory, antifungal, sedative, antibacterial, antiviral and anticarcinogenic properties [8]. However, the use of CcEO in pharmaceutical preparations is limited due its chemical characteristics. In fact, EOs are easily degraded by oxidation, volatilization, heating and light, if they are not protected from environmental factors [5,9]. Furthermore, their low aqueous solubility prevents oral administration. Such characteristics are barriers to scale up EOs products at the industrial level. Different strategies have been designed to overcome these issues, such as emulsification, aerosolization and encapsulation in nanocarriers [10]. EOs encapsulation in polymeric nanoparticles has enhanced their physical stability, reduced their volatility and improved their aqueous solubility, while providing a sustained release of active compounds [10,11]. Moreover, due to their nanometric size, these colloidal systems may increase cellular absorption and potentiate bioefficiency [1].
Corresponding author at: Faculdade de Farmácia, Universidade Federal Fluminense, Brazil. E-mail addresses: [email protected], [email protected] (D.Q. Falcão).
https://doi.org/10.1016/j.colsurfb.2019.06.010 Received 14 March 2019; Received in revised form 3 June 2019; Accepted 5 June 2019 Available online 05 June 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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Appropriate selection of a polymeric matrix is essential in order to develop a successful nanoparticulate delivery system. Poly(D,L-lacticco-glycolic) acid (PLGA), one of the most extensively used biodegradable polymers, is widely endorsed to encapsulate natural products due its excellent biocompatibility and biodegradation [12,13], well-known safety, its ability to control drug release kinetics and its possible functionalization [14,15]. The emulsification/solvent diffusion (ESD) technique is a suitable method to encapsulate lipophilic substances [16,17], resulting in high encapsulation efficiency and narrow size distribution [18]. However, several process variables can affect the physicochemical properties of the formed nanoparticles [19]. Design of experiment (DoE) is a useful tool to investigate the influence of different factors on the selected responses, by running a small number of experiments, thus saving time and reducing costs [20]. Box–Behnken design (BBD) is a fractional factorial design of second-order based on three levels, widely applied for optimization of nanoparticle synthesis. This technique is suitable to explore quadratic response surfaces and constructs according to a mathematical model, assisting to determine a system optimal solution [21–24]. This study aimed to optimize the ESD synthesis and encapsulation efficiency (EE) of previously developed CcEO-loaded nanoparticles intended for anti-herpetic topical treatment [22]. The effects of some parameters, such as sonication time, ultrasound power and essential oil/polymer ratio were analyzed using BBD and response surfaces. Optimized experimental conditions were used to obtain nanoparticles with reduced mean hydrodynamic size (Z-ave) and high EE. The optimized nanoparticles were physicochemically characterized and their cytotoxicity evaluated in HaCat cells by WST-1 and LDH assays.
Table 1 Variables employed in BBD. Independent variable/factor
X1: Sonication time (min) X2: Ultrasound power (W) X3: Essential oil/polymer ratio (w/w) Dependent variable/response Y1: Encapsulation efficiency (%) Y2: Mean hydrodynamic size (d.nm)
Levels −1
0
1
5 50 0.50 Constraints
10 100 1.25
15 150 2.00
Maximize Minimize
medium and high levels, respectively (Table 1). The medium level was chosen as follows: sonication time (10 min), ultrasound power (100 W) and essential oil/polymer ratio (1.66:1.33), where corresponding amounts of oil and PLGA were 194 mg and 156 mg, respectively. EE (Y1) and Z-ave (Y2) were used as dependent variables (responses). The interaction of independent variables and responses were evaluated using the following quadratic mathematical model (Eq. 1): Yi = b0 + b1X1 + b2X2 + b3X3 + b1,2X1X2 + b1,3X1X3 + b2,3X2X3 + b1,1X12 + b2,2X22 + b3,3X32
(1)
where Yi is the dependent variable, b0 is the arithmetic mean response of 17 runs, bi is the estimated regression coefficient for each factor, and Xi represents the coded independent variables. Statistical analysis from formulation design was performed using Statistica software 7.0 (Stafsoft, Inc., USA). The experimental design matrix generated by software and corresponding observations for the dependent variables are shown in Table S1.
2. Materials and methods 2.1. Materials
2.4. Statistical and checkpoint analyses
Cymbopogon citratus essential oil (L192 lot, Laszlo Aromaterapia, Brazil). Poly (vinyl alcohol) (PVA; Mw 85,000–124,000 Da), poly (D,Llactide-co-glycolide) (PLGA; Mw 50,000–75,000 Da, 85:15), dichloromethane (DCM), trifluoroacetic acid (TFA) and citral standard reference (Sigma-Aldrich, USA). Carbopol® Ultrez 10 NF (Fagron, Brazil). Triethanolamine (Vetec, Brazil). Dimethyl sulfoxide (DMSO), nhexane and acetonitrile HPLC grade (Tedia, Brazil), Dulbecco’s Modified Eagle’s Medium (DMEM) (Life technologies, EUA), WST-1 reagent (Roche Co., USA). Phosphate-buffered saline (PBS) pH 6.8 [23] was prepared using sodium phosphate dibasic anhydrous, sodium phosphate monobasic anhydrous and sodium dodecyl sulfate (SDS) (Vetec, Brazil).
Analysis of variance (ANOVA) and lack of fit were performed to ensure the model fitting. The variables which significantly affected Y1 and Y2 were identified and shown through Pareto charts using Student’s t-test. Results were considered statistically significant at p-value < 0.05. Response surfaces were plotted and critically exploited to determine the impact of factors (X1, X2 and X3) on nanoparticles preparation. CcNP theoretical optimal experimental conditions were obtained by response surface analysis and mathematical model aiming to formulate nanoparticles with maximum EE and smallest Z-ave. The resulting statistical models were checked in triplicate and experimental responses (Yi) were compared with those predicted.
2.2. Preparation of CcEO-loaded nanoparticles (CcNP)
2.5. Characterization of nanoparticles
CcNP were synthesized by the ESD method with slight modifications [22]. An appropriate amount of PLGA was dissolved in DCM saturated with distilled water and CcEO was added. In parallel, PVA (1.0%, w/v) was solubilized in distilled water saturated with DCM. The organic phase was added dropwise to the aqueous phase, maintained in an ice bath, under continuous ultrasonic homogenization (Sonic Ruptor 250, Omni International, USA). Subsequently, the oil-in-water emulsion was diluted in distilled water under magnetic stirring to allow solvent diffusion. The nanoparticle suspension was lyophilized (Terroni Enterprise II, LT 1000, Brazil) to obtain CcNP. Blank nanoparticles (NP) were prepared without addition of CcEO.
Synthesized nanoparticles were characterized by Z-ave using dynamic light scattering (DLS) and Zeta potential (ZP) by electrophoretic mobility, using a Zetasizer Nano ZS90 (Malvern Instruments, UK). Lyophilized samples were suspended in ultra-purified water (1:25) and analyzed at a scattering angle of 173°, 25 °C, using water as the reference medium. Measurements were performed in triplicate and results expressed as mean ± standard deviation [22]. Nanoparticles shape and surface morphology were examined by transmission electronic microscopy (TEM) (Vega 3LMU Tescan, Czech Republic) (acceleration voltage 80 kV). A drop of nanoparticles, suspended in distilled water, was spread on a 200-mesh copper grid covered with formvar film, dried at room temperature and followed by double staining with uranyl acetate and lead citrate solution. The amount of CcEO incorporated into CcNP was recovered by solvent extraction method [25] for EE evaluation. CcNP were dissolved in DMSO, vortexed and centrifuged (2000 x g) for 1 min. Supernatants were diluted with DMSO and analyzed by UV–vis spectrophotometry (Shimadzu® UV-2600, Japan) at 260 nm. Blank NP were used as a
2.3. Experimental design A 3-factor, 3-level BBD was used to optimize CcNP previously developed [22]. The investigated independent variables (factors) were: sonication time (X1), ultrasound power (X2), and CcEO/polymer massic ratio (X3), which were represented by −1, 0 and +1, analogous to low, 936
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reference. Total CcEO content was quantified from an average of three standard curves in the range of 0.002–0.04 mg/mL (r2 = 0.9999, y = 22.537x - 0.0176). The extraction procedure and analysis were carried out in triplicate and EE (%) was calculated by Eq. 2:
keratinocytes HaCat by water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) assays and compared with CcEO [29]. 2.9. HaCaT cells and culture conditions
EE = 100 × (measured CcEO content/theoretical total CcEO content) (2)
HaCaT cells were acquired from the Cell Bank of Rio de Janeiro (Rio de Janeiro, Brazil). DMEM completed with 10% of fetal bovine serum (FBS), sodium bicarbonate 3.7 g/L and 1.0% of antibiotic solution (100 IU/mL penicillin and 100 μg/mL streptomycin, Life, EUA) was used to grow the cells at 37 °C and 5% CO2 and 95% relative humidity. The cells were subcultured, under the same conditions, every 2–3 days until reaching a density of ˜1 × 106cells/mL.
CcNP were characterized by Fourier transformed infrared spectroscopy (FTIR) over the spectral region from 400 to 4000 cm−1 using a Shimadzu IR Tracer-100 FTIR spectrometer with Attenuated Total Reflectance (ATR) acessory (MIRacle, 10) and compared with NP, PLGA and CcEO. Thermal characterizations were also performed by differential scanning calorimetry (Shimadzu Co., Japan) using a DSC-60 system and by Thermogravimetric (TGA) and Differential Thermal analyses (DTA) using a TGA Q500 V20 Thermogravimetric Analyzer (TA Instruments, USA). Samples (3.5 mg) were placed in aluminium pans and heated from 25 to 200 °C at a scanning rate of 10 °C/min under constant nitrogen purge of 80 mL/min for DSC analyses. TGA and DTA were carried out at 10 °C/min from 25 to 600 °C at a flow rate of 60 mL/min and compared with NP, PLGA and PVA.
2.10. WST-1 assay About 2 × 104 HaCaT cells were seeded into each well of sterile flatbottomed 96-well plates, incubated with either CcEO (1.9–250.0 μg/ mL) or CcNP (21.28–2800.0 μg/mL, corresponding to 1.9–250.0 μg/mL of CcEO) for 24, 48 and 72 h. To allow CCEO detection, samples were suspended in DMEM containing DMSO (1.0%, v/v). This medium was used as a negative control whereas Triton X-100 (10.0%, v/v) was used as a positive control. At the end of the treatment period, the culture media in each well were replaced by 100 μL of WST-1 reagent (10.0%, v/v, in DMEM) and incubated at 37 °C and 5% CO2 for 3 h. The assay measures cell viability by assessing the activity of mitochondrial dehydrogenases. The plate absorbance was measured at 450 nm using a Polaris Microplate Reader (Celer, Brazil) according to kit protocol and literature [29]. The cell viability in each well was calculated as a ratio to the one of negative control cells. The concentration that caused 50% growth inhibition (IC50) was determined by plotting the log10 of the percentage of viability versus CcEO concentrations after 72 h of treatment.
2.6. CcEO chemical composition Qualitative analyses, before and after encapsulation, were performed by a GCMS-QP5000 (Shimadzu) gas chromatograph equipped with a mass spectrometer using electron ionization. Gas chromatographic (GC) conditions were as follows: injector temperature, 250 °C; carrier gas, helium; flow rate, 1 mL/min and split injection with split ratio 1:40. The oven temperature was initially 80 °C and raised to 290 °C at a rate of 12 °C/min. One microliter of each sample, dissolved in hexane (1:100 mg/μl), was injected onto a DB-5 column (i.d. = 0.25 mm, length 30 m, film thickness = 0.25 μm). The mass spectrometer was monitored to scan m/z 35–650 with an ionizing voltage at 70 eV and scan rate 1 scan/s [26]. Retention indices (RI) were determined in relation to a homologous series of n-alkanes (C10C40) analyzed under the same operating conditions. Identification of the constituents was performed by comparison of their RI and mass spectra from literature [27] and checked against NIST mass spectra libraries. Component relative percentages were calculated based on GC peak areas without using correction factors.
2.11. LDH assay LDH assay, which measures cytotoxicity by assessing the integrity of the plasma membrane, was evaluated using a commercial kit (Roche, Germany) according to the manufacturer’s protocol. After treatment, 100 μL supernatant and 100 μL reaction mixture (freshly prepared) were transferred from each well of a 96-well flat-bottomed plate. The plates were incubated for 30 min at 20 °C in the dark and absorbance was measured using a Polaris Microplate Reader at 492 nm. The cytotoxicity percent was calculated according to the kit protocol [29].
2.7. In vitro release studies CcEO in vitro release from nanoparticles was evaluated by the dialysis membrane diffusion technique [25]. In brief, 35 mg of lyophilized nanoparticles (117 mg CcEO/g CcNP) were added in the donor chamber and 12.5 mL of PBS (pH 6.8) with SDS (1.0%, w/v) in the acceptor chamber. CcEO solubility in the dissolution medium was previously determined to be 0.33 mg/mL. The system was placed in a shaking bath (37 °C ± 0.5) under constant agitation (100 rpm). At several points (0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 3.0, 5.0 and 24.0 h, and then once a day for 8 days), an aliquot of 2.5 mL was withdrawn and replaced with an equal volume of fresh dissolution medium to ensure sink conditions. Samples were analyzed by UV–vis spectrophotometry at 240 nm using blank NP as references. The CcEO content was calculated from an average of three standard curves prepared with the medium, ranging from 0.0025-0.04 mg/mL (r2 = 0.9996, y = 71.862x + 0.0047). The release study was carried out in triplicate. The cumulative drug release percent was plotted against time. Results were fitted to various kinetic equations to evaluate the mechanism of release from CcNP: Zero Order, First Order, Higuchi, Hixson-Crowell and Korsmeyer-Peppas [28].
2.12. Statistical analysis Statistical evaluation of data was performed using one-way analysis of variance (ANOVA) and Student s̓ t-test, employing GraphPad Prism software 5.0 (GraphPad Software Inc., USA). In the cytotoxicity assays, the statistical analysis was calculated using one-way ANOVA and Dunnett’s post hoc test. Data were expressed as the mean value ± standard deviation (mean ± S.D.) (n = 3). The differences were considered statistically significant when p-value < 0.05. 3. Results and discussion In drug delivery systems development, it is crucial to consider EE and Z-ave, in order to ensure that enough drug is delivered to target tissue to produce a therapeutic effect. These properties can be controlled during the early stages of development by analysis of certain formulation and process variables [20]. In the present study, BBD was applied to optimize previously developed CcNP using the ESD technique [22]. A total of 17 runs with 5 centre points was carried out to evaluate the effect of 3 independent variables on 2 dependent variables.
2.8. CcNP in vitro cytotoxicity CcNP in vitro cytotoxicity was evaluated in human skin 937
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Fig. 1. Response surface plots showing (A) effect of X2 and X3 on encapsulation efficiency, (B) effect of X1 and X2 on average particle size with X3 set at 0.5 and, (C) effect of X1 and X3 on average particle size with X2 set at 150 W. *X1: sonication time; X2: power of ultrasound; X3: essential oil/polymer ratio.
3.1. Effect of independent variables on EE (Y1)
polymer ratio) exhibited a negative linear effect on EE. An increase in PLGA concentration enhanced CcEO content into polymeric nanoparticles (Fig. 1A) since it increased the organic phase viscosity, resulting in higher resistance to CcEO diffusion into the aqueous phase [16,30]. Furthermore, increasing PLGA concentration favored the precipitation process, reducing the timespan available for CcEO diffusion from the nanoparticle matrix, resulting in higher encapsulation rates [31]. According to Eq. 3, the ultrasound power had a positive effect in EE. Enhancing ultrasound power leads to smaller droplets during the emulsification step, thus globally increasing nanoparticle surface area [20], and ultimately leading to high encapsulation content. Furthermore, increasing ultrasound power provided a turbulent and unidirectional flow, avoiding loss of CcEO from the organic phase [31].
All encapsulation efficiencies ranged between 17.33% (F-11) and 86.97% (F-6) (Table S1). A Pareto chart was constructed to evaluate which factors influenced the Y1 response (Figure S1A). Only X2 and X3 variables had significant effects on EE at a 95% confidence level. There was no significant interaction between the studied factors. The model proposed for predicting the effect of factors on the Y1 response is presented in Eq. 3: Y1 = 56.6 + 15.0 X2 – 17.1 X3
(3)
The predicted (0.662) and adjusted (0.614) R-square values obtained from the ANOVA test suggested that some other important variables for the EE parameter were not considered in this study. However, residues showed a normal distribution according to Kolmogorov-Smirnov (p > 0.20) and Shapiro-Wilk tests (p = 0.51766) and the lack of fit term was not significant. Thus, this model could still be used to navigate the design space. A positive or negative value is related to a positive or negative effect on the studied response, respectively. According to Eq. 3, factor X3 (EO/
3.2. Effect of independent variables on Z-ave (Y2) Particle size plays a crucial role in transport and absorption of bioactive molecules into the body as well as the drug release rate [18]. CcNP average hydrodynamic diameters were found in the range of 938
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167 nm (F-20) to 318 nm (F-12) (Table S-1), in agreement with other studies reporting the use of the ESD method to synthetize nanoparticles [17,19]. Analysis of data by ANOVA and Pareto chart (Figure S-1B) showed the significant effect of all factors and their interactions on Y2 (p < 0.05), except for X2, with a quadratic effect and a linear interaction between X2 and X3. The effect on Z-ave can be explained by the polynomial Eq. 4:
order to obtain nanoscale particles with high content of CcEO: a sonication time of 8 min, an ultrasound power of 150 W and an essential oil/polymer ratio of 0.5. For model validation, nanoparticles were prepared in triplicate following these conditions and results were compared with the values predicted by model, with an acceptable error of less than 5%. Applying these conditions, EE and Z-ave predicted by the calculated model were 88.74% and 173.56 nm, respectively. The experimental results are presented in Table 2. The experimental value for response Y1 (73.29%) of optimized formulation is consistent with the predicted confidence interval (71.51–105.98%). This result confirmed its adequate precision for predicting optimum conditions in the chosen domain of levels for the independent variables. Therefore, by applying BBD it was possible to incorporate an amount 2.6 times greater of CcEO then previously described (EE 28.48%) [22]. Regarding the mean hydrodynamic diameter, the average replicates synthesized were 277 nm (Table 2), a result which was not predicted by the mathematical model (174 nm), suggesting that other important factors controlling the nanocarrier size were not considered in this study. However, it is known that the tensions generated during lyophilization can destabilize systems, leading to aggregation or even particle coalescence [33]. The concentration of the stabilizing agent may also influence particle size [19], since it tends to position itself on droplet surface, reducing free energy at the interface between organic and aqueous phases, stabilizing the droplets [31]. Thus, it can be suggested that the lack of standardization in the lyophilization step and/or an insufficient amount of PVA may have contributed to not having achieved the predicted particle size. The size distribution profiles (Fig. 2) suggested that the CcEO presence favored size distribution homogeneity, showing unimodal profiles different than that of unloaded NP which showed a bimodal profile. These patterns were corroborated by polydispersity index (PDI) results which were statistically different for both samples (Table 2, Student s̓ ttest, p < 0.05). Moreover, optimized CcNP showed more homogeneous size distribution than those previously reported for unoptimized nanoparticles [22], highlighting the success of BBD. Nanoparticle morphological evaluation (Fig. 2B and D) showed spherical shapes and smooth surfaces with size distribution consistent with results obtained by DLS. EO incorporation did not alter the particle shape. However, a strong aggregation tendency was observed in both samples, probably related to insufficient steric stabilization by the PVA [34] or lyophilization step. This aggregation was expected for both samples due their low Zeta potential which suggests that the attractive force surpasses the repulsive force between particles. The negative charge is related to free ionized PLGA terminal carboxyl groups on the surface [19,20,30]. The addition of CcEO enhanced the Zeta potential values (Student's t test, p < 0.05), which seemed to minimize the aggregation observed for NP, according to its unimodal particle size distribution and morphological evaluation. The more negative charge of CcNP seemed to be related to the presence of ionized carbonyl groups from citral adsorbed on the nanocarrier surface, as showed by FTIR characterization (Figure S-2). Absorption bands of carbonyl groups from α, β-unsaturated aldehyde (C]CeCOH, 1672 cm−1) and CeH axial deformation of aldehydes (2968–2856 cm−1 and 1377 cm−1), characteristics of CcEO [23,35], can be observed in both CcNP and CcEO FTIR spectra, with different intensities. It indicated that only a
Y1 = 183.8 +15.9 X1 – 9.5 X12 + 13.2 X2 – 38.9 X3 – 11.6 X32 + 13.9 X1X2 + 15.1 X1X22 – 11.6X12X2 – 28.6 X1X3 + 18.9X12 X3 (4) The values for predicted (0.994) and adjusted (0.984) R-square showed the adequacy of the model to predict the response in the optimization process. Moreover, residues showed a normal distribution according to Kolmogorov-Smirnov (p > 0.20) and Shapiro-Wilk tests (p = 0.61824) and the lack of fit term was not significant. Thus, the statistical analysis demonstrated model suitability to describe the behavior of the independent variables and their interactions on Y2 in the experimental domain. Eq. 4 shows that X3 presented negative, linear and quadratic effects on Y2, as well as interaction effects with the different factors evaluated in the study. Therefore, increasing PLGA concentration, i.e. enhancing the organic phase viscosity, reducing the stirring shear efficiency required for droplet breakdown, led to larger diameter droplets during the emulsification process. A more viscous organic phase could also decrease the diffusion rate from the organic to the aqueous phase, promoting the Ostwald ripening phenomenon, causing an increase in particle size [19,30]. Such increase could be also explained by polymerpolymer interactions in a more concentrated phase, which could lead to particle aggregation [16,20]. As described before, the variable X1 showed no effect on EE. In order to evaluate the X1 value, the X2 factor was set at its maximum level (+1) and X3 at its minimum (-1), as previously determined optimal conditions to obtain particles with maximum EE (Y1). The respective 3D response surfaces were plotted (Fig. 1B and C) and the X1 value was graphically determined as 8 min, to be used in the optimization process. The influence of the X1 factor on the Y2 response showed a positive linear effect, negative quadratic effect and interaction effects with the other factors (Equ. 4). Fig. 1B and 1C highlights the relation between the sonication time and the particle size. Increasing sonication time from 0 to 10 min decreased particle size. However, the reverse effect was verified when increasing the sonication time to 15 min. This may be due to aggregation of small particles resulting from prolonged sonication time [16]. Various studies have reported that increasing ultrasound power led to the formation of smaller particles, since more released energy increased shear stress and, consequently, decreased the viscosity of the organic phase during the emulsification step [32]. Eq. 4 shows that X2 had a positive linear effect on Y2 and interaction effects on the studied factors. However, by setting X3 factor to 0.5, small particles were obtained throughout the experimental domain, employing minimal or maximal levels (50–150 W). 3.3. Optimized polymeric nanoparticles The application of statistical tools and critical analysis of response surfaces allowed to determine the optimal experimental conditions in
Table 2 Results of mean hydrodynamic size (Z-ave), polydispersity index (PDI), Zeta potential (ZP) and encapsulation efficiency (EE) of optimized nanoparticles. Formulation
Z-ave (nm)
PDI
ZP (mV)
EE (%)
NP CcNP
280.8 ± 19.3* 277.0 ± 5.5*
0.27 ± 0.02** 0.18 ± 0.04**
−13.3 ± 1.7** −16.1 ± 1.8**
– 73.29 ± 8.96
Mean value ± S.D. (n = 9); *(p > 0.05), **(p < 0.05) by Student’s t-test. NP: blank nanoparticles; CcNP: CcEO load nanoparticles. 939
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Fig. 2. Typical size distribution (A) and TEM image of NP (B); size distribution (C) and TEM image of CcNP (D). *TEM image x180,000 magnification. Scale bar: 250 nm.
small CcEO amount was adsorbed on the CcNP surface. Small changes were observed in bands of −OH (3317 cm−1), –C]O (1755cm−1) and C–O stretching of ester groups (1086 cm−1) when compared to NP and CcEO spectra, suggesting interaction between the citral and the nanocarrier and its successful encapsulation. The spectra obtained for PLGA agreed with literature data [30,36]. The presence of the CcEO component adsorbed in CcNP surface and its encapsulation were confirmed by thermal analysis. DSC thermograms (Figure S-3) of CcNP and NP exhibited two endothermic peaks. The peak temperature observed in CcNP first event (133 °C) was higher than observed for NP and CcEO, demonstrating the nanocarrier’s ability in increasing EO thermal stability. CcEO showed two endothermic peaks at 102.4 and 141.7 °C, relative to volatilization and/or thermooxidative decomposition. It is well-known that EO thermal profiles are influenced by their chemical composition, since each substance exhibits different characteristics, such as molecular weight and boiling/volatilization temperature [37]. DSC thermal profiles of PVA and PLGA 85:15 were consistent with previously reported data [38,39]. CcNP thermogravimetric results (TGA and DTA) (Figure S-4 and Table S-3) showed four thermal events. The first event, observed in range 30 to 60 °C (Δm = 2.69%), should be linked to the volatilization of citral molecules adsorbed on CcNP surface, since Tdeg max (39.4 °C) was superior to that obtained for the NP, corroborating the results previously discussed. The second thermal event (60–130 °C: Δm = 10.26%) referred to thermo-oxidative volatilization and/or decomposition of encapsulated CcEO. The higher Tdeg max (104 °C) compared to that obtained for CcEO highlighed its encapsulation and protection by nanoencapsulation. Subsequent thermal events related to nanostructure degradation showed maximum mass loss at temperatures of 298 and 452.4 °C as observed for the NP, in agreement with literature [40]. CcEO thermogram showed a fast mass loss ranging from 30 to 94 °C, with a maximum rate observed at 72 °C, attributed to the volatilization and/or thermo-oxidative decomposition. Mass loss of 5.78% was still verified up to 260 °C related to oil volatilization.
compounds represented 99.45% of the total oil (Table S-2). Citral components, geranial (42.67%) and neral (33.03%), were the major constituents. Citral total content (75.70%) agreed with literature data [6]. Other substances with appreciable relative abundance were myrcene (10.68%) and geraniol (4.99%), also previously reported in CcEO [41]. CcEO chemical profile after encapsulation showed only citral (neral - 42.14% and geranial - 57.86%) incorporated into the nanocarrier matrix. Turbulent flow and heating generated by the ultrasound probe during the emulsification process may contribute to loss of minor compounds by volatilization. Similar results were previously described in the development of CcEO-loaded PCL nanoparticles by the ESD method [25], highlighting the presence of citral in CcNP and its interaction with the matrix material. 3.5. In vitro CcEO release studies The release behavior of CcEO from optimized nanoparticles (Fig. 3) showed a biphasic pattern, with an initial burst (24.8 ± 1.7% in 3 h), followed by a sustained release for 8 days, reaching a maximum oil release of 83.9 ± 3.5%. The initial burst might be attributed to the immediate desorption of citral molecules adsorbed onto the particle surface, as previously discussed. The slower release phase corresponded to diffusion of citral molecules through interconnected pores and channels in the polymeric matrix, from the particle core to the dissolution medium [42]. PLGA with a high lactic acid content and molecular weight, such as the one used in this study, is more hydrophobic and, consequently, exhibits slower water uptake, decreasing polymer degradation rate [34]. Furthermore, the increment in lactic acid content increases polymer hydrophobicity, favoring the partition of citral molecules in the polymer matrix compared to the aqueous external medium [43]. The results were analysed by different mathematical models in order to determine CcEO release mechanisms from CcNP (Table 3). Analysis of correlation coefficients revealed that the Korsmeyer-Peppas model as the best fit (R2 = 0.9445). However, the behavior proposed by Higuchi (R2 = 0.9155) must be considered. It suggested citral release was mainly based on diffusion and agreed with the diffusional exponent
3.4. Chemical composition of CcEO GC-MS analysis of CcEO before encapsulation showed that 14 940
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Fig. 3. In vitro CcEO release profile in saline-phosphate buffer with sodium lauryl sulfate (1.0%, w/v), pH 6.8, from optimized nanoparticles (n = 9).
value (n = 0.3933). Citral release was governed by Fickian transport mechanism (n = 0.3933), only dependent on citral diffusion through the polymeric matrix. Both models have been efficiently applied to describe the release behavior of hydrophobic drugs from PLGA nanoparticles [42,30]. The release profile reflected that CcNP allowed CcEO sustained release for several days. Hence, the therapeutic actions of CcEO could definitively be enhanced by reducing the number of administrations and the patient’s compliance and comfort to treatment.
viability at concentrations greater than 3.9 μg/mL (Figure S-5A). Cell viability was increased by CcEO encapsulation, showing cytotoxic effects in concentrations higher than 31.25 μg/mL (Figure S-5C). The average IC50 of CcEO and CcNP were 39.40 and 66.28 μg/mL, respectively. CcEO altered integrity of HaCaT cell membranes in a time- and dose-dependent manner from 125 μg/mL after 24 h of exposure (Figure S-5C). On the other hand, CcNP induced apparent LDH leakage from cells at 31.25 μg/mL after 48 h of incubation (Figure S-5D). However, there was no significant difference between IC50 values for CcEO (137.9 μg/mL) and CcNP (130.3 μg/mL). CcEO and CcNP induced cell cytotoxicity by affecting mitochondrial dehydrogenase activity, as detected by the WST-1 assay and by destroying cell membrane integrity, as showed by the LDH assay. CcEO toxic effects for HaCaT cells were attributed to its citral content [44]. However, the encapsulation in CcNP greatly decreased citral toxic effect on cell mitochondrial dehydrogenase activity compared to free CcEO
3.6. In vitro cytotoxicity Toxicity is an important issue to consider in nanocarrier development, since their reduced dimensions favor cell penetration. The effect of CcEO and CcNP on cell viability (% of control) was evaluated by the WST-1 assay [29]. CcEO showed time- and dose-dependent reduction in HaCaT cell Table 3 Kinetic behavior of in vitro CcEO release from CcNP. Mathematical model Zero Order k0 0.0066
First Order 2
R 0.7726
k1 0.0002
Higuchi 2
R 0.5599
kH 0.7547
Hixson-Crowell 2
R 0.9155
R2: correlation coefficient; k: release rate constant; n: diffusional release exponent. 941
kHC −0.0002
Korsmeyer-Peppas 2
R 0.8639
kK 2.6133
R2 0.9445
n 0.3933
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Funding This work was supported by Fundação de Amparo à Pequisa do Estado do Rio de Janeiro (FAPERJ) - Brazil [grant numbers JCNE 2016, E-26/202,759/2017 and E26/202,256/2018]; (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CAPES) - Brazil [grant number 001] and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) - Brazil [grant number 302345/2017-5]. Acknowledgment The authors thank the staff members of the Multiuser Laboratory of Material Characterization (www.uff.br/lamate) for training and assistance pertaining to the Zetasizer Nano ZS90 results and Rodolpho Albino University Laboratory (www.lura.uff.br) for the use of the freeze-dryer. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.06.010. References [1] [2] [3] [4] [5] [6] [7] [8] [9]
Fig. 4. Effect on mitochondrial dehydrogenase activity (A) and cell membrane integrity (B) of CcEO and CcNP in HaCaT cell. (mean ± S.D., n = 3).
(Fig. 4). Several in vitro studies have emphasized the ability of polymeric nanoparticles to increase cell viability in the presence of natural molecules. Chen et al. [45] demonstrated the reduction of cytotoxicity of carvacrol and eugenol in mouse 3T3 fibroblasts, when grafted on chitosan nanoparticles. The neutral red dye-uptake method, evaluated by Almeida et al. [22] on Vero cells viability, showed decreased EOassociated cytotoxicity after encapsulation in PLGA nanoparticles, with a maximum non-toxic concentration 5-fold lower than that of free CcEO.
[10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]
4. Conclusion
[24] [25] [26] [27]
CcEO-loaded PLGA nanoparticles were prepared by the ESD technique and physicochemically characterized. The 3-factor, 3-level BBD used provided fitting polynomial equations for the evaluated responses (EE and Z-ave) and was subsequently employed with success to optimize the nanoparticle formulation. In comparison to previous report, CcNP developed in this study provided higher EE and lower PDI. Optimized CcNP physical characterization evidenced the citral interaction with the polymeric matrix, highlighting the nanocarrier potential in increasing its thermal stability. CG-MS showed that only citral was incorporated into the nanostructure. Citral was released from CcNP according to a biphasic pattern, with an initial burst due to the fraction adsorbed onto the particle surface, followed by a sustained release dependent on its diffusion from the polymeric matrix. Moreover, nanoencapsulation attenuated CcEO toxic effects on the mitochondrial dehydrogenase activity of human keratinocytes. These results revealed the ability of PLGA-nanoparticles to improve the physicochemical characteristics of CcEO, to control its release and reduce its toxicity, suggesting a promising potential for pharmaceutical uses.
[28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45]
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