Solid Lipid Nanoparticles from Platonia insignis Seeds, a Brazilian Amazon Fruit: Characterization, In Vitro and In Vivo Toxicological and Antioxidant Activities

Abstract

Solid Lipid Nanoparticles (SLNs) are drug delivery systems with important advantages over conventional nanosystems. Considering previously reported pharmacological and physicochemical properties of Platonia insignis seed butter (BBI), this work aimed at developing, characterizing and performing toxicological and antioxidant studies of SLNs produced from BBI. The GC-MS analysis identified palmitic and oleic acids as the major compounds. Three SLN prototypes were developed through high-shear homogenization followed by ultrasonication. During a 180-day stability evaluation, the formulation SLN/TW-1.5 presented greater stability since pH was around 6.0, as well as a lesser variation of the PdI (Polydispersity Index), particle size and Zeta Potential (ZP), confirmed with Raman Spectroscopy and Atomic Force Microscopy (AFM). The CC₅₀ in macrophages was around 249.4 µg∙mL⁻¹ for BBI, whereas the CC₅₀ value for SLN/TW-1.5 was 45.2 µg∙mL⁻¹. Electron Paramagnetic Resonance (EPR) showed a marked in vitro antioxidant activity for BBI and SLN/TW-1.5. After in vivo SLN/TW-1.5 administration in Zophobas morio larvae, assessment of reduced glutathione (GSH), nitrite (NO₂⁻) and myeloperoxidase (MPO) demonstrated antioxidant activity. Thus, the intrinsic physicochemical properties of BBI allowed the development of an optimized nanoformulation with high stability indexes, besides the great potential for antioxidant applications.

1. Introduction

Currently, the application of nanotechnologies for pharmaceuticals and nutraceuticals is a great challenge [1,2,3,4]. The interest in nanostructures such as Solid Lipid Nanoparticles (SLNs) has gained prominence in recent years, given the high potential for the development of new drug delivery systems. SLNs have advantages over conventional pharmaceutical forms, such as their unique size (ranging from 40 to 1000 nm) and large contact surface, as well as the possibility of controlling drug release and vectorization, leading to greater therapeutic efficacy and fewer side effects [5].

The use of complex Solid Lipid Matrices (SLM) for the development of nanosystems allows the formation of amorphous crystals with solid lipid cores at room temperature capable of incorporating drugs. In this sense, plant butters are a suitable matrix for the development of nanosystems due to their diverse lipid composition [6,7]. Among the potential matrices, the bacuri (Platonia insignis Mart.) seed butter can be cited [8,9] due to its important physicochemical and pharmacological characteristics that favor the development of SLN and even the best employment of its intrinsic biological potential [10,11,12].

The genus Platonia is rich in natural substances, such as xanthones, fatty acids and triglycerides. The phytochemical study of the lipid fractions of bacuri seeds and the presence of fatty acids such as palmitic, oleic and linoleic acids were detected, as well as dipertenes and polyprenylated benzophenones, such as garcinielliptone FC (GFC) [9,13,14]. Among the previously reported properties, the antioxidant [15,16], leishmanicidal, immunomodulatory and anti-inflammatory activities [17,18,19] stand out, also presenting vasorelaxant activity induced by GFC, the major constituent of the dried seeds [20]. It is worth mentioning the current work of Lima Nascimento et al. [21] on the production and characterization of a β-cyclodextrin inclusion complex with Platonia insignis seed extract as a proposal for a gastroprotective system.

In addition to the use of SLNs for the development of nanostructures, the strategic selection of emulsifiers is a critical step, given the biphasic characteristic of the system. Depending on the lipid concentration, an average of 0.5 to 5% of surfactants is used, which are responsible for decreasing the interfacial tension, increasing the surface area and originating smaller particles. The presence of surfactants in the formulation facilitates emulsification between the phases by forming a mechanical or electrical barrier that prevents coalescence over time [22]. Moreover, energy supply through physical and chemical processes is required for SLN formulation. Among the techniques used on a laboratory scale are solvent replacement, phase inversion, membrane filtration, ultrasound, high-shear or through the chemical energy of the system components [23]. The most commonly used devices are high-shear agitators, high-pressure homogenizers and ultrasonic generators. In this context, the smaller the desired particle size is, the greater must be the required energy force or amount of surfactant used [6,7].

The characterization of the matrix used, as well as the physicochemical and toxicological studies, especially in the early stages of the development of new drug delivery systems, stands out as an important issue. Another key issue is the toxicity evaluation of nanostructures, related to the safety of the new pharmaceutical formulation [24], estimating the range of concentrations that will be used in the preclinical stages of development, as well as the maximum dose at which the product can be used without causing toxicity [25,26]. The search for less toxic therapeutic alternatives that reduce oxidative stress and are safer is of great value.

Considering the above-mentioned and the potential presented by the P. insignis seed butter, a plant species native to the Brazilian Amazon with high added therapeutic potential, one can clearly understand the utmost importance and the potential economic and social impacts of pharmaceutical studies involving the species, aiming at the complete understanding and utilization of its pharmacological activities [27]. Also, it is possible to make the most of the plant matter, considering the possibility of using bacuri shells and seeds, which are considered agroindustrial residues that account for more than 58% of the total weight of the fruit [28]. Thus, this biomass rich in biological properties could be used to improve the local bioeconomy and develop new bioproducts [29].

Therefore, this study aimed to develop, characterize and investigate the stability of Solid Lipid Nanoparticles produced from bacuri (Platonia insignis Mart.) seed butter, as well as to evaluate the antioxidant potential and perform toxicological studies.

2. Materials and Methods

2.1. Plant Material

The bacuri seed butter or BBI (Industrialized Bacuri Butter) used to produce the Solid Lipid Nanoparticles was obtained from Amazon Oil Industry (Ananindeua, PA, Brazil), a producer of the butter in Brazil, by the cold-pressing method. The plant material was stored at room temperature of approximately 25 °C.

2.2. Precipitation of Lipids from Bacuri Seed Butter (BBI)

For the analysis of the lipid composition of BBI, the butter components were precipitated for subsequent derivatization and analysis in gas chromatography coupled with mass spectrometer (GC-MS). In the precipitation step, 60 mL of acetone was added to 8.0 g of butter. The mixture was heated to 50 °C and then kept under refrigeration (2 to 8 °C) for 24 hours and filtered in a separating funnel to remove the supernatant. After evaporation of the acetone, 60 mL of methanol was added to the supernatant residue, with subsequent heating, followed by cooling in a refrigerator for 24 h. From this second process, the precipitate was obtained and prepared for the analysis.

2.3. Obtaining Methyl Derivatives of the Triacylglycerols from the Bacuri Seed Butter (BBI) Precipitate

In order to identify the fatty acids present in the BBI precipitate, derivatization was performed according to the methodology described by Hartman and Lago [30], based on a saponification reaction of fatty acids followed by esterification (methylation). The esterifying reagent was prepared by dissolving 2 g of ammonium chloride (NH₄Cl) in 60 mL of methanol, with the subsequent addition of 3 mL of concentrated sulfuric acid (H₂SO₄). After that, this mixture was subjected to the reflux system for 15 min.

Next, 50 mg of the BBI precipitate was saponified with 5 mL of a 0.5 mol·L⁻¹ NaOH methanolic solution−1 under heating for 5 min. Then, 10 mL of the esterifying reagent was added to the still-hot saponified mixture, and it was subjected to a reflux system for 5 min. After the reaction, the mixture was taken to a separation funnel, where 30 mL of water was added, and then the mixture was extracted thoroughly with hexane. The organic phase was concentrated in a rotary evaporator and stored in a desiccator.

2.4. Analysis of the Methyl Derivatives by Gas Chromatography Coupled with Mass Spectrometer (GC-MS)

The methyl derivatives were analyzed on a Shimadzu GC-17A/MS-QP505A gas chromatograph equipped with a mass spectrometer. The chromatographic separation was performed using an Rxi-5HT 30 m × 0.25 mm capillary column. The heating schedule of the oven had an initial temperature of 70 °C (maintained for 2 min), followed by a heating ramp of 6 °C·min⁻¹ to a final temperature of 310 °C (maintained for 10 min). The quadrupole mass spectrometer was operated in scan mode in the mass range 57–650 Da. The ion source was set for operation in electron ionization (EI) mode at 70 eV. The total scan time for the chromatographic run was 52 min, including 5 min of solvent delay. The identification was made by comparison with the fragmentation pattern available in the WILEY library^®.

2.5. The Production and Characterization of Solid Lipid Nanoparticles (SLNs)

SLNs were produced by the high-shear homogenization method described by Neves et al. [31] and Soldati (2015) [32], with adaptations followed by ultrasonication. Two phases were prepared: the organic phase, composed of BBI, and the aqueous phase, consisting of water and surfactant. Three prototypes of SLN were made with 1.0% (w/w) bacuri butter, differing only in the type and concentration of the surfactant (

Table 1. Composition of solid lipid nanoparticle samples produced from bacuri seed butter.

Abbreviation	Surfactant Type	Surfactant Concentration
SLN/TW-1.5	Tween 80	1.5
SLN/PL-1.0	Pluronic 127	1.0
SLN/PL-1.5	Pluronic 127	1.5

Note: SLN/TW-1.5: Solid Lipid Nanoparticles with Tween 80 at 1.5%; SLN/PL-1: Solid Lipid Nanoparticles with Pluronic 127 at 1%; SLN/PL-1.5: Solid Lipid Nanoparticles with Pluronic 127 at 1.5%.

).

Both phases were heated (50 °C), and the aqueous phase was poured into the oily phase. The system was subjected to high-speed stirring (15,000 rpm) in an Ultra-Turrax (IKA® T18 basic) for 2 min, followed by sonication in a Qsonica ultrasonicator model Q500, with amplitude of 40%, On-cycle of 10 s, and Off-cycle of 5 s for a time of 5 min.

The SLNs were analyzed for particle size (PS), Polydispersity Index (PdI) and Zeta Potential (ZP) at 25 °C using the equipment Nanopartica SZ-100 (Horiba Scientific, Sao Paulo, Brazil). The PS and PdI parameters were measured by dynamic light scattering (DLS), while the ZP was determined through the evaluation of electrophoretic mobility.

2.6. Storage Stability Study of SLNs

For the stability study, the formulation SLN/TW-1.5 (Solid Lipid Nanoparticles with Tween 80 at 1.5%) was selected, with the production of new batches of SLNs under optimized conditions (5 or 10 min ultrasonication). The dispersions were subjected to high agitation (15,000 rpm) in an IKA ULTRA-TURRAX T18 device for 2 min, followed by ultrasonication for 5 or 10 min, which characterizes the T5 and T10 formulations, respectively, with an intensity of 40%, aided by an ultrasound tip (6 mm diameter), in a QSONICA Sonicator/Ultrasonic processor–Q500 equipment.

The stability study was conducted based on the maintenance of the formulations in Falcon tubes at 25 °C. Stability indicators were evaluated at times 0 (day of production), 7, 15, 30, 60, 90, 120 and 180 days. The parameters investigated were the organoleptic characteristics, pH, average particle size (PS), Polydispersity Index (PdI) and Zeta Potential (ZP).

Throughout storage, the SLN/TW-1.5 T5 and T10 formulations were analyzed for homogeneity (phase separation, color, gloss, turbidity, presence of foreign body or any other changes). At each analysis time, photographic records of the formulations were obtained. The pH measurements were performed in INSTRUTHERM (Brazil) pH meter equipment.

The SLN/TW-1.5 T5 and T10 were analyzed for particle size (PS), Polydispersity Index (PdI) and Zeta Potential (ZP) at 25 °C using the Nanopartica SZ-100 equipment (Horiba Scientific). The PS and PdI parameters were measured by Dynamic Light Scattering (DLS), while the ZP was determined by electrophoretic mobility evaluation. Before the readings, the formulations were homogenized and diluted in the proportion of 1 mL to 3 mL of deionized water and, with a 5 mL syringe, transferred to the reading cell of the equipment (until it was filled), without forming bubbles, to perform the DLS readings.

2.7. Raman Spectroscopy

The analysis of Raman microspectroscopy was performed with a resolution of 0.7 cm⁻¹ in the range from 500 to 3100 cm⁻¹ by the LabRam HR Evolution, Horiba, equipped with a 1800 lines/mm grid and CCD detector. Measurements were performed with a He-Ne laser source tuned to the 633 nm line (17 mW) focused on the samples by an OLYMPUS microscope with a 50× objective and N.A. (0.55). The isolated components of the solution of SLN/TW-1.5, bacuri seed butter seed and Tween 80 were evaluated.

2.8. In vitro Antioxidant Evaluation by Electron Paramagnetic Resonance (EPR) Spectroscopy

The antioxidant proprieties represent the evaluation of the combined action of the bioactive component of the food matrix and can be viewed as an indicator of potentially beneficial properties of a food matrix [33,34]. Evaluation of antioxidant activity for this study followed the parameters of time, reading and concentration using the protocol of Ferreira-Nunes et al. [35]. The purpose of this technique, electronic paramagnetic resonance spectroscopy (EPR), is to determine the antioxidant activity of molecules because the EPR has a higher sensitivity than existing methods, enabling a better assessment of antioxidant activity even at concentrations considered low by other methodologies. For the evaluation of a free radical standard, the DPPH (1,1-diphenyl-2-picrylhydrazyl) was used in this assay, which was prepared in a solution with 5 mL of ethanol (PA) at a concentration of 500 µmol·L⁻¹. The NPB and MFB samples were used in the EPR and prepared as aliquots with the addition of bacuri butter to the original DPPH solution until concentrations of 50, 25 and 12.5 µg·mL⁻¹ of each solution were reached. The samples were added only with ethanol. After the addition of bacuri butter, the samples were kept at room temperature for 60 min in the dark, and then 50 µL of each sample was placed in microcapillaries and frozen at liquid nitrogen temperature. They were then thawed at the time of being placed in the EPR device. The spectra were collected in a Bruker Bio Spin spectrometer, EMX Plus, in X-band (9.35 GHz), with 10 G and 100 kHz modulation field, 20 mW microwave power, with 30 dB receiver gain, 10 s scan time, 100 G scan width and 6 scans. With the application of 10 as modulation field, overmodulation of the spectrum occurred, implying its deformation of the spectrum, but without any change in the value of the double integral, whose value depends on the number of paramagnetic centers present in the samples or otherwise, on the number of free DPPH molecules present in the samples. The Amplitude x Concentration plot presents the percentage result of the ratio of the amplitude of the double integral obtained from the spectra of the samples with different concentrations of bacuri butter by the double integral of the DPPH sample with ethanol. The decrease in the values presented in the graph shows the decrease in the number of free DPPH molecules as a function of bacuri butter concentration. For the statistical evaluation of this assay, which was performed in triplicate reading of each sample for all concentrations under analysis, the data were stored in spreadsheets for later statistical analysis.

2.9. Morphological Analysis of the SLN by Atomic Force Microscopy

The morphology of the nanoparticles was examined using AFM (atomic force microscopy). The analysis was performed on the samples in vibrating (beating) mode. The images were taken with a TT-AFM instrument (AFM Workshop, South Carolina, USA) with a Tap 300-G cantilever (Ted Pella, California, USA) at a resonance frequency of about 3248 kHz. Images were collected at a resolution of 512 pixels and 10 × 10 µm. Briefly, 20 µL of the nanoparticles (diluted 1:100 in ultrapure water) were deposited onto the cleaned mica, and after 30 min, the samples were gently washed with deionized water and dried again for further analysis under the TT-AFM microscope. Images were processed, analyzed and displayed using Gwyddion 2.58 software.

2.10. The Cellular Viability of Macrophages by the MTT Test

The evaluation of cytotoxicity of SLN on murine macrophages by MTT ((3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)) test was performed as previously described [36]. In a 96-well plate, 100 µL of RPMI 1640 medium (Sigma, St. Louis, MO, USA) and about 2 × 10⁵ macrophages per well were added. The plate was incubated at 37 °C in 5% CO₂ for cell adhesion. Then, concentrations of the samples (400, 200, 100, 50, 25, 12.5, 6.25 and 3.125 µg∙mL⁻¹) of BBI 1%, Pluronic 1.5%, Tween 1.5%, SLN/PL-1.5 and SLN/TW-1.5 were added. Subsequently, 10 μL of MTT (3-[4,5-dimethylthiazzo-2-yl]-2,5-diphenyltetrazolium bromide) diluted in PBS was added to a final concentration of 5 mg∙mL⁻¹ and incubated for the period of 4 h at 37 °C and 5% CO₂. After that, the supernatant was discarded, and 100 μL of DMSO (dimethyl sulfoxide) was added to each well. The plate was kept on stirring for 30 min for complete dissolution of formazan crystals. Spectrophotometric reading was carried out at the wavelength of 550 nm in an ELISA plate reader (ELx800™, BioTek® Instruments, CA, USA). The results were expressed as the mean inhibitory concentration for 50% of cells (IC₅₀).

2.11. The Evaluation of the In Vivo Toxicity and Antioxidant Potential of SLN/TW-1.5 Formulations on Zophobas Morio Larvae

For toxicity evaluation, different groups of Z. morio larvae were prepared, in duplicate, containing 10 Zophobas morio larvae. One group received 1.5% Tween80 (10 mg/g), named TWEEN 80; three groups received the physical mixture (MF) of the fusion between BBI (1%) and Tween 80 (1.5%) at doses of 16.5, 8.25 and 4.125 mg/g, named MF, MF1:2 and MF1:4; and three other groups received SLN/TW-1.5 T10 at doses of 16.5, 8.25 and 4.125 mg/g, named, respectively, SLN, SLN1:2 and SLN1:4.

The administration was performed with a 0.3-mL syringe, and the substances were inoculated into the third false leg of the larvae of the respective groups. Five days after the administration, photographic records were obtained for visual evaluation of the larvae, in addition to the evaluation of the survival rate of the groups and the degree of melanization they presented [37]. The melanization ratio was observed by skin pigmentation and applied at the ratio of

$$melanizated ratio=\frac{number of melanizated larvae}{total number of larvae per group}\times 100$$

For antioxidant evaluation, the larvae were separated in duplicate, weighed, stored at −40 °C and then homogenized with a tissue homogenizer in a sufficient volume of buffer. The buffer used was specific for the parameter to be analyzed. Then, homogenates were aliquoted into eppendorfs and centrifuged into a microcentrifuge (1460 rpm for 30 min, 4 °C). Then, the supernatant was separated and centrifuged again at 1370 rpm for 90 min (4 °C) [37]. This second supernatant, diluted in the respective buffer specific for each analysis, was used to follow up on the methodologies.

2.11.1. Reduced Non-Protein Sulfhydryl Groups—Reduced Glutathione (GSH)

Larval homogenate was produced using 0.02 M ethylenediaminetetraacetic acid (EDTA) buffer solution (ratio of 100 mg:1 mL). Then, the supernatant of the second centrifugation was diluted with an equal volume of 10% trichloroacetic acid (TCA); the mixture was stirred for 30 s and centrifuged (3000 rpm for 15 min). Finally, it was aliquoted for the addition of Tris buffer (composed of 0.4 M tris(hydroxymethyl)aminomethane (Tris) and 0.2 M EDTA buffered at pH 8.9).

The reading was performed in an SP-220 biospectro spectrophotometer at 412 nm. To each 3 mL of solution to be analyzed, 50 μL of 0.01 M 5,5-dithio-bis-(2-nitobenzoic) acid (DNTB) was added. Also, 2 mL of distilled water, 4 mL of Tris buffer and 100 μL of 0.01 M DNTB as blank solution were used. The concentrations in μM of reduced sulfhydryl groups are given by multiplying the absorbance obtained in the test by the factor 317.8 [38].

2.11.2. Nitrite by Griess Method

Nitrite concentration was determined with Griess method as described by Green et al. [39]. Previously, the tissue samples were deproteinized following the method of Moshage et al. [40] and Romitelli et al. [41]. The homogenate of the larvae was produced using phosphate buffer at pH 7.4 (ratio of 50 mg:1 mL). Then, to deproteinize the sample, 300 μL of the second supernatant, 180 μL of Zinc Sulfate (ZnSO₄) solution (0.15 M) and 3 μL of Sodium Hydroxide (NaOH) solution (10 M) were added in an eppendorf. The mixture was incubated on ice for 15 min and again centrifuged (14,600 rpm for 5 min). In a microplate, 100 mL was added per well of the deproteinized supernatant, along with 100 μL of Griess reagent, with a 10 min interval for reading in a GloMax spectrophotometer at 560 nm.

The results were obtained by comparing a calibration curve and the absorbance values obtained for a given sample. The calibration curve was obtained by spectrophotometric reading (560 nm) of serial dilutions (100, 50, 25, 12.5, 6.25, 3.125 and 1.56 μM) of a 10 mM sodium nitrite (NaNO₂) stock solution that reacted with Griess reagent under the same conditions as the samples.

2.11.3. Superoxide Dismutase (SOD) Enzyme Activity

The measurement of SOD activity was based on the ability to consume the O₂⁻ radical, decreasing the autoxidation ratio of pyrogallol (1,2,3-trihydroxybenzene). Homogenate of the larvae was produced using sodium phosphate buffer pH 7.5 (ratio of 50 mg:1 mL). Then, 30 μL of the supernatant from the second centrifugation of the homogenate was added to an eppendorf, along with 99 μL of the phosphate buffer, 15 μL of pyrogallol solution (0.2 mM) and 6 μL of dimethylthiazol-diphenyltetrazolium bromide (MTT) (5 mg/mL). Then, the mixture was incubated for 5 min at 37 °C. After this period, the reaction was stopped by the addition of 150 μL of DMSO for subsequent reading of absorbance in the GloMax spectrophotometer at 560 nm. The result was expressed in units of SOD activity per mg of tissue [42].

2.11.4. Myeloperoxidase (MPO) Enzyme Activity

The measurement of MPO activity was performed by the method of oxidation speed of the substrate o-dianisidine in the presence of H₂O₂. The buffer solution used to produce the homogenate of the larvae was 0.5% potassium phosphate hexadecyltrimethylammonium buffer (HTAB) pH 6.0 (ratio of 50 mg:1 mL). For the reading, 10μL of the second supernatant was added to a microplate well, and 200μL of the reading solution (3 mL pH 6 phosphate buffer, 15 mL hydrogen peroxide (1%) and 5 mg o-dianisidine in distilled water up to 50 mL of solution). Two spectrophotometric readings were performed, observing the difference between the absorbance at 450 nm. The first was performed one minute after the addition of the reading solution and the second after two minutes. The results are expressed in units of MPO per mg of tissue [43].

2.12. Statistical Analysis

The stability values of the PS, PdI and ZP formulations and the antioxidant activity parameters such as nitrite, GSH, SOD and MPO were expressed as means ± standard deviation (SD). In the stability and cytotoxicity study, the significances of compared values were analyzed with Student’s t-test or analysis of variance (ANOVA). For the cytotoxicity test, in the analysis of variance (ANOVA), significance was considered when p < 0.05 regarding the control group with 100% viable cells. The significances for compared values in the antioxidant activity parameters were analyzed by the one-way ANOVA method of variance, and differences with p < 0.05 were considered significant.

All procedures were performed using the Graph Pad Prism 8.0.1™ software (Graph Pad Software, Inc., San Diego, CA, USA).

3. Results and Discussion

3.1. The Chemical Composition of the Fatty Acids in Bacuri Seed Butter (BBI)

Qualitative determination of the fatty acids present in BBI was carried out by preparing methyl esters by saponification followed by esterification and fractionation of the esters—the most rapid and common way. Rapid saponification with methanolic alkali hydroxide avoids the inadvertent isomerization of polyunsaturated fatty acids, which can interfere with the process [30].

Thus, in the analysis of the methyl derivatives of fatty acids in GC-MS, six signals were identified in the chromatogram of the precipitate (Figure 1), equivalent to six methyl derivatives. Considering the principles of absorption spectroscopy, the greater the number of molecules capable of absorbing light of a certain wavelength, the greater the extent of this absorption; therefore, the intensity of the peak is related to the number of molecules [44].

Table 2. Methyl derivatives of the fatty acids present in the bacuri seed butter obtained by GC-MS.

Peak	Chemical Constituents	Molecular Formula	[M+•]	Retention Time (min)	Relative Intensity (%)
1	Methyl (Z)-hexadec-9-enoate	C17H32O2	268	27.245	6.29
2	Methyl hexadecanoate (palmitic acid)	C16H32O2	270	27.635	62.08
3	(9Z,12Z)-Octadec-9,12-methyl dienoate	C19H34O2	294	30.470	0.86
4	Methyl (Z)-octadec-9-enoate (oleic acid)	C18H34O2	296	30.580	28.33
5	(E)-octadec-9-methyl acetate	C19H36O2	296	30.660	0.89
6	Methyl octadecenoate (stearic acid)	C19H38O2	298	30.970	1.55

reports the methyl compounds inherent in the peaks shown in the chromatogram of Figure 1. As shown in Figure 1, the esters representing palmitic (methyl hexadeconate) and oleic (methyl Z-octadec-9-enoate) acids are the most abundant fatty constituents in the BBI precipitate (62.08% for palmitic acid and 28.33% for oleic acid).

The identification of the fatty acid composition of BBI is important for the experimental design of the production of SLN, considering how the physicochemical characteristics of the lipids in butter influence the preparation and properties of nanostructures [45]. Methyl hexadecanoate (Peak 2), a methyl derived from palmitic acid (a component of tripalmitin), is the most prevalent compound. Tripalmitin is a triacylglycerol in abundance in bacuri seed butter [46]. The (Z)-octadec-9-methylenoate (Peak 4) corresponds to the oleic acid, which is related to the reduction of risks to human health, such as the decrease in the loss of cognitive rate and reduction of effects from the ingestion of diets rich in saturated fatty acids that are a great risk to the cardiovascular system [42,47]. Saturated fatty acids promote the emergence of chronic diseases such as dyslipidemia and atheroma formation, in addition to the elevation of the cholesterol rate [48]. In a general evaluation, the saturated compounds (structures relative to peaks 2 and 6) account for 63.63% of the total fatty acids, and the unsaturated compounds (peaks 1, 3, 4 and 5) comprise 36.37% of the total lipids present in the precipitate (Table 2). In this context, the fatty acid profile in BBI enables the production of SLN and drug loading.

During the process of solidification and crystallization of SLNs, triglycerides formed by a single type of fatty acid tend to crystallize in a highly organized manner (lowest energy system), giving rise to a structure that is difficult to carry actives. The high organization produces a system with few sites to accommodate the drug, which is, therefore, prone to be expelled from the solid lipid matrix [45,49]. On the other hand, the presence of different fatty acids in the BBI allows the lipid matrix to crystallize more imperfectly, leading to the accommodation of bioactive compounds in the spaces between the lipid chains and in the crystal imperfections [45,50]. The formation of this system is due to the difference in intermolecular interactions between fatty acids of different carbon chain sizes, as well as the presence of cis-isomeric carbon-carbon double bonds that force a curvature in the lipid chain. Thus, spaces appear between the chains of fatty compounds by reducing the intermolecular interaction, which is advantageous because it facilitates the accommodation of the possible active principle to be conveyed [45,51,52].

Therefore, the production of SLN from a complex matrix maximizes the efficiency of encapsulation of assets [53]. Kim, Na and Choi [54] obtained an incorporation efficiency of verapamil higher than 75% in SLN produced from cocoa butter, which also has a complex lipid matrix consisting mainly of palmitic (C16) (25.1–27.9%), stearic (C18:0) (33.3–33.8%) and oleic (C18:1) (34.5–36.5%) acids and, in smaller amounts, linoleic (C18:2), lauric (C12) and myristic (C14) acids [55].

3.2. The Production and Physicochemical Characterization of Solid Lipid Nanoparticles (SLNs)

Two steps followed: high-shear homogenization and ultrasonication. The first process promotes emulsification by high-shear of the mixture of aqueous and oily phases to produce particles in the micrometer range; the second, through ultrasonic waves, originates cavitation of the mixture [31,56]. This results from alternating high- and low-pressure sound waves produced by ultrasound, which can enable the reduction of micrometric particles to the nanometer order [31]. These steps allowed the production of the BBI SLN.

The surfactants Tween 80 and Pluronic F127 were chosen in different concentrations for the production of SLN derived from BBI. Such choices were guided by the review work by Coelho et al. [23], where both were identified as surfactants with good performance in producing SLN-containing vegetable butters. Furthermore, Pluronic F127 belongs to a group of non-ionic surfactants, presenting high biocompatibility and solubility, as well as stability and low toxicity in formulations in aqueous vehicles. In parallel, Tween 80 is characterized as a non-ionic hydrophilic surfactant widely used in the preparation of pharmaceutical, cosmetics and food formulations. Among its characteristics, the solubilizing, emulsifying and stabilizing functions stand out, in addition to being effective in controlling fat agglomeration, which contributes to the stabilization of the formulation [23,32].

3.2.1. Organoleptic Characteristics

The visual aspect is important in order to verify eventual phase separation, predictive of instability, possibly associated with insufficient surfactant concentration. In this context, the samples of SLN/PL-1.0, SLN/PL-1.5 and SLN/TW-1.5 were visually homogeneous, with a translucent aspect and yellowish color (Figure 2).

3.2.2. Particle Size, Polydispersity Index and Zeta Potential of SLN

According to Legrand and collaborators [57], the particle size (PS) of SLN is influenced by a multitude of factors, i.e., the chemical composition and concentration of the organic phase, nature and concentration of the active and surfactant, preparation method and stirring speed. As shown in

Table 3. Size distribution of Solid Lipid Nanoparticles produced from bacuri seed butter (BBI).

Sample	PSD 1 (nm)	Intensity (%)	PSD 2 (nm)	Intensity (%)
SLN/PL-1.0	75.0 ± 37.2	33.0 ± 4.6	563.2 ± 292.1	67.0 ± 12.8
SLN/PL-1.5	44.9 ± 6.7	33.7 ± 11.5	456.8 ± 69.8	66.3 ± 10.3
SLN/TW-1.5	20.7 ± 3.5	44.0 ± 8.7	134.4 ± 32.5	56.0 ± 8.7

Note: PSD 1: Particle Size Distribution 1; PSD 2: Particle Size Distribution 2; SLN/TW-1.5: Solid Lipid Nanoparticles with 1.5% Tween 80; SLN/PL-1: Solid Lipid Nanoparticles with 1% Pluronic 127; SLN/PL-1.5: Solid Lipid Nanoparticles with 1.5% Pluronic 127.

, the DLS analysis of the SLN produced from BBI showed a bimodal character, indicating the presence of two populations of nanostructures with distinct particle size distributions (PSD). Moreover, the samples with larger particle sizes (PSD 2) showed higher intensity compared to the population of smaller nanostructures (PSD 1).

Moreover, Gaussian curves of density probability by size distribution were obtained for SLN produced from BBI (Figure 3). SLN/TW-1.5, prepared with Tween 80, showed a markedly higher probability density in the function of size distribution when compared with SLN prepared with the surfactant Pluronic 127. These findings support that smaller SLN from BBI are able to be prepared with Tween80.

Comparing the PS of the SLN produced with Pluronic, the sample SLN/PL-1.0 had a larger particle size compared to SLN/PL-1.5, which contained a larger amount of the surfactant Pluronic. This corroborates Lippacher, Muller and Mader [58], in which the surfactant/lipid ratio in dispersions is inversely proportional to the particle size of the system. Thus, a higher surfactant concentration gives rise to a lower PS.

Due to the presence of a polydisperse system (populations of nanoparticles with two different sizes), the PdI values obtained for all surfactant samples were considered high (greater than 0.40), indicating heterogeneity amongst SLN sizes (

Table 4. Zeta Potential (ZP) and Polydispersity Index (PdI) values of Solid Lipid Nanoparticles produced from bacuri seed butter.

Scheme	PdI	ZP (mV)
SLN/PL-1.0	0.536 ± 0.017	−38.37 ± 1.45
SLN/PL-1.5	0.5955 ± 0.047	−24.89 ± 0.70
SLN/TW-1.5	0.532 ± 0.036	−30.25 ± 0.70

Note: SLN/TW-1.5: Solid Lipid Nanoparticles with Tween 80 at 1.5%; SLN/PL-1: Solid Lipid Nanoparticles with Pluronic 127 at 1%; SLN/PL-1.5: Solid Lipid Nanoparticles with Pluronic 127 at 1.5%.

). This parameter may range from 0 to 1 and express how homogeneous the nanosystem is; the closer it is to 1, the more uniform the particle size [59]. The system in which the lipid nanoparticles have PdI less than or equal to 0.30 is considered homogeneous. However, values up to 0.40 are accepted for SLN [60].

In a study by Soddu and colleagues [61], producing SLN with different types of surfactants from cocoa butter as an organic phase and high-shear double homogenization method, a better surfactant activity of Tween 80 compared to other surfactants (polyethylene glycol 400 and polyglyceryl-6-diesterate) was revealed, obtaining nanocomposites with particle size around 300 nm. The result by Soddu and colleagues [61] was in line with the results herein presented, where the surfactant Tween 80 was proven to be more effective for the preparation of BBI nanosystems.

Another key parameter in the characterization of nanosystems is the Zeta Potential (ZP). The ZP is attributed to the surface charge of particles, responsible for giving rise to electrostatic forces of repulsion and favoring the increased stability of the dispersive system [62]. As shown in Table 4, the ZP of the produced SLN had a negative surface charge, varying in modulus from 24.89 mV to 38.37 mV. Moreover, regarding Zeta Potential, the dispersions can be classified as highly unstable (±0–10 mV), relatively stable (±10–20 mV), moderately stable (±20–30 mV) and highly stable (greater than ±30 mV). Thus, samples SLN/PL-1 and SLN/TW-1.5 had a suitable stability since SLN/PL-1.5 and SLN/TW-1.5 are classified as moderately and highly stable, respectively [63].

3.3. Stability Study of SLNs

Considering its lower size and PdI values related to better stability, the formulation SLN/TW-1.5 (Table 1) was selected for the stability study, producing new batches of Solid Lipidic Nanoparticles of bacuri seed butter (SLN/TW-1.5). However, in order to optimize the production process regarding the processing time by ultrasonication, the new batches were developed in two different conditions, being submitted to 5 or 10 min of ultrasonication. These formulations will, therefore, be referred to as T5 or T10.

3.3.1. Organoleptic Characteristics

The results described below point to the stability analysis (up to 180 days of storage). It is worth considering that the evaluation of organoleptic characteristics is subjective, and there may be variations according to the evaluators [64]. In order to minimize this bias, the evaluations were performed under standardized conditions of place, time and researcher.

It is possible to observe, through photographic records (Figure 2), that there were no visible changes in the appearance of the formulations over time when kept at room temperature (25 °C) and without agitation. It is also noted that the T10 formulation presented the best visual stability in the evaluated period, considering that the presence of sediments or precipitates was not observed, and it remained without turbidity, being characterized by a clear, homogeneous formulation and without the presence of particulate material visible to the naked eye, while the T5 formulation was more turbid and with subtle formation of sediment.

The organoleptic characteristics are indispensable because they provide parameters for the acceptance of the product by the consumer [65]. Therefore, obtaining a product with pleasant and consistent characteristics is proven to be very important, besides the functional evaluation that can be made about this product.

A number of reasons, such as oxidation, can cause instability and alter macroscopic characteristics. Lipids can undergo degradation reactions, such as acid hydrolysis and oxidative rancidity [64]. Thus, it can be inferred that intense organoleptic changes in formulations are mediated by oxidative reactions, and the source of these reactions can be the degradation of the compounds against oxygen and/or light or by microorganisms that use lipids as substrate.

It is known that the increase in ultrasonication time reduces the average diameter and the PdI of nanoparticles and that there is a direct association between the turbidity of the formulation and the increase in the average size [66]. This could explain the formation of aggregates and higher qualitative turbidity index in samples with shorter ultrasonication time, T5.

3.3.2. pH Evaluation

The two main factors causing the destabilization of aqueous nanodispersions are ionic strength and/or pH reduction [67]. As for the formulations produced, the gradual decrease in pH values with an increasing number of days of storage was observed; moreover, the pH decrease ratio of sample T5 was higher than the decrease ratio for sample T10 (Figure 4A). This may demonstrate a greater stability of sample T10 compared to T5 in relation to the pH parameter.

The pH data of the formulations evaluated here are not in line with the results by Weilin et al. [68], who reported no significant variation for their control lipid nanoparticles during 90 days of storage (at 4 °C). This pattern was only observed for formulation T10 on days 7, 15, 30, 60, 90 and 180 storage analyses. Even with the variations observed, formulation T10 showed pH values that remained close to 6 in six of eight evaluations, which indicates maintenance at pH and then a suitable stability compatible with biological systems [69].

From the pH values, it is possible to identify alterations that are not always perceived visually, such as oxidation, chemical hydrolysis of functional groups present in the components of the formulations, integrity of the emulsion phases, or microbiological contamination. It is known that the stability of the pharmaceutical product is compromised with high microbial loads, considering that the active ingredient can be degraded, the pH of the formulation changed, and the appearance harmed [70]. One of the main sources of pH change in pharmaceutical forms is microbiological contamination. Although a little variation was observed in the organoleptic characteristics of the SLN/TW-1.5 overall after 180 days, a possible microbiological origin for this observation is less likely yet worthy of further investigation.

3.3.3. Particle Size

Particle size is one of the main parameters for evaluating product quality, and this can vary depending on the formulation composition, the methods and the conditions used for manipulation, such as time, temperature and pressure [22,64]. For T5 and T10 formulations, the ultrasonication time clearly changed the particle TM of each formulation and its pattern of variation with time when the two batches were compared (Figure 4B).

As reported in Table 3, the particle size of T10 is smaller compared to T5 since it presents a size smaller than 200 nm in the first four times of analysis (0, 30, 60 and 90 days of storage) (Figure 4B) and is constant over time, since the statistical evaluation did not determine any significant difference in this period.

The evaluation of the T5 formulation (Figure 4B) showed values ranging from 400 nm to 2000 nm, then returned to values close to the initial ones, observing absolute variation in nanoparticle size values. However, no statistically significant difference was found over time. It is emphasized that the lifetime of SLN depends on the initial particle size and the PdI since smaller sizes are correlated with a reduction in particle aggregation [71].

The statistical analysis confirms that comparing the size between T5 and T10 formulations in the same period, there is an inversely proportional relationship (p < 0.05) between the ultrasonication time and the size of the nanoparticles formed. Therefore, the increase in ultrasonication time was able to induce a reduction in particle size, which is a key factor for the stability of the formulations.

The same behavior was reported by Weilin et al. [68], where no significant variations in TM were observed after 7, 14, 30 and 90 days of their lipid nanoformulation. Moreover, the average particle size of T10 was close to the control particles reported by the same study [68] in the same evaluation period. The increase in the particle size of T10 after 180 days of storage was not considered significant since the standard deviation was very high.

3.3.4. Polydispersion Index (PdI)

The PdI allows the analysis of the level of homogeneity of the particle size distribution [49]. The lower the PdI value, the more homogeneous the particle population. PdI with values greater than 0.4 indicates a polydisperse distribution [72]. As reported in Figure 4C, samples T5 and T10 obtained values greater than 0.4 for PdI throughout all evaluation days, except for formulation T10 on days 0 and 180. Values above 0.5 are obtained in case of wide distribution [22,64], and this high was not identified on days 0 and 180 for formulation T10. In contrast, a strong increase in the PdI of sample T5 was observed on day 180.

The tendency to aggregation and sedimentation, as a function of time, can be monitored by determining changes in particle size distribution [73]. Thus, high values for PdI (above 0.4) indicate greater particle size distribution and the presence of larger particles that would destabilize the formulation. Therefore, the formulation T10 presents a more ideal pattern for the values of PdI, indicating greater stability because it does not present an increasing ratio in the values (Figure 4C). A difference between the pattern of PdI of the formulations can also be observed, considering that on day 180, a strong increase in the PdI of the T5 formulation was observed, determining a lower stability of this formulation compared to T10. On the other hand, there is conflicting information since Sousa [74] and Meyagusku [75] observed a gradual increase in PdI over time under similar conditions, an increase that was not observed for formulation T10.

3.3.5. Zeta Potential (ZP)

The Zeta Potential reflects the surface potential of the particles and is influenced by changes at the interface with the dispersant medium due to the dissociation of functional groups on the particle surface or adsorption of ionic species present in the aqueous dispersion medium [73]. The ZP is an important parameter in assessing stability in the storage of nanoparticulate systems. ZP values that are greater than 50 mV in modulus are good indicators of stability [71]. For both formulations, T5 and T10 values greater than 50 mV were observed during all days of evaluation (Figure 4D).

Considering the results, the production method and the ultrasonication time used in the production of both formulations allow for a storage time with good stability assured in relation to ZP. As shown in Figure 4D, a difference between T5 and T10 is not determined, with higher and relatively constant values observed for both formulations, according to Weilin et al. [68] regarding ZP variation over time, in addition to Meyagusku [75], who observed no increase in the ZP values over time.

Among the factors influencing ZP are, for example, the composition of the particles, the dispersant medium, the pH and the ionic strength of the medium. Also, the influence of changes at the interface with the dispersant medium due to the dissociation of functional groups or the adsorption of ionic species present in the aqueous dispersion medium should be taken into account [64,76].

Furthermore, no association between the pH of the formulations and ZP was observed. Possibly, the larger particle size of the T5 formulation and/or its higher PDI may have correlated with the increase in ZP. Overall, relatively stable and considerably high values for T5 and T10 formulations were obtained during the 180 days, which is a good indicator of stability.

3.4. Raman Spectroscopy

Figure 5 shows the Raman spectra of the bacuri seed butter (i), Tween 80 (iii) and SLN/TW-1.5 (ii) samples. The Raman spectrum of the SLN/TW-1.5 sample shows the typical feature of lipid, more specifically, bacuri seed butter. Evidence of the presence of the Tween 80 is observed around 1250 and 1650 cm-1 due to the cis-unsaturated bonds present mainly in the surfactant. Vibrations associated with the methyl group, present in all samples, are found around 890 cm⁻¹ (𝜌(𝐶𝐻₂)), 1300 cm⁻¹ (𝑟(𝐶𝐻₂)), 1450 cm⁻¹ (𝛿(𝐶𝐻₂)) and in the region of 2800–3000 cm⁻¹ (𝜈(𝐶𝐻₂)) [77]. The 1000–1200 cm⁻¹ Raman region is associated with 𝐶−𝐶 skeletal stretching [78]. Finally, the stretching modes of the 𝐶 = 𝐶 and 𝐶=𝑂 bonds (carbonyl ester group) are found around 1650 and 1740 cm⁻¹, respectively [79].

It is known that Raman spectroscopy is highly sensitive to conformational changes in the hydrocarbon chains of lipids [78]. Conformational changes in the lateral packing of cis-unsaturated fatty acid chains lead to significant changes in vibrational energies and the shape of the Raman lines. For example, the 1000–1200 cm⁻¹ region provides information on 𝐶−𝐶 skeletal stretches, with sharp Raman peaks for the solid-state phase and broad bands for the liquid-state phase [78]. This occurs due to the increased number of gauche defects present in the liquid phase. Additionally, the conformational arrangement of lipids is also reflected in the profile of the vibrational band associated with carbonyl stretch modes ((𝐶=𝑂)). Finally, it is well documented that the intensity of the vibrational bands found in the region of 2800–3100 cm⁻¹ are sensitive to the interchain packing of lipids in general.

Note from Figure 5 that, although they have the same feature, Raman spectra of the bacuri seed butter and SLN/TW-1.5 samples are much better resolved than the spectrum of the Tween 80 sample. This statement is most evident for the stretching modes of the −𝐶 skeletal, carbonyl ester (𝐶=𝑂) and 𝐶−𝐻 bonds. In lipids in the solid phase, stretching modes of carbon-carbon bonds in the gauche conformation (𝐶−𝐶)_𝑔 are found around 1100 cm⁻¹, while the symmetrical and antisymmetric (𝐶−𝐶)_𝑡 stretching modes in the all-trans conformation are found around 1130 and 1060 cm⁻¹, respectively. On the other hand, in the liquid phase, these bands cannot be resolved anymore, and a broad band associated with the (𝐶−𝐶) gauche stretching vibrations dominates the spectrum in this region. Likewise, in lipids in the ordered phase, the 𝜈(𝐶=𝑂) modes split into two bands at ~1730 and ~1740 cm⁻¹, while a single broad band is observed around 1735 cm⁻¹ when in the liquid phase [78]. Additionally, it is known that the ratio between the intensities of the (𝐶𝐻2) and 𝜈(𝐶𝐻2) bands rise with the increase in conformational order of the hydrocarbon chains [80].

Thus, SLN/TW-1.5 formulation in the presence of bacuri seed butter, where initially the hydrocarbon chains are highly organized, induces an improvement in the lateral packing of the hydrocarbon chains of the Tween 80 molecules, which explains the high stability of this formulation.

3.5. In Vitro Antioxidant Evaluation by Electron Paramagnetic Resonance Spectroscopy (EPR)

The EPR technique has been widely used for assessing antioxidant properties in oils. Figure 6 shows the antioxidant potential of the physical mixture (BPM: bacuri physical mixture) and the solid lipid nanoparticle (SLN/TW-1.5) from bacuri seed butter. The bacuri butter presents a significant antioxidant potential in either of the two formulations tested: a physical mixture and a nanolipid solution. However, there is no significant difference in the antioxidant potential between the two formulations since the two curves have very close decay rates.

Antioxidant compounds of natural origin have been studied in recent years as a worldwide trend and have high potential for applications in industry, being its main source of obtaining co-products from fruits [81]. The bacuri seed butter has benzophenones, polyprenylated and xanthones in its composition [29,82]. These plant metabolites [27,83] have documented antioxidant activity and potential beneficial properties [84,85,86,87].

In order to present an antioxidant level considered satisfactory, as shown by the curves in the EPR technique, the presence of signal decay is suggestive of a kinetics of the disappearance of the free radicals present with the elapsed time. This finding suggests that the formulation may be evaluated in screening methods for the best way to consume the antioxidant activity present. According to Bidzenska et al. [88], when using the EPR technique to prove radicals that have a short duration, they can be used for purposes as probes for starch-type foods.

3.6. Morphological Analysis of the SLN/TW-1.5 by Atomic Force Microscopy

The atomic force microscopy (AFM) technique is widely used to provide high-resolution and accurate information about the size, size distribution, morphology and surface topography of lipid nanoparticles. The force acts between a surface and a probing tip, resulting in a special resolution of up to 0.01 nm for the images [89,90]. For this technique, the influence of sample preparation should be taken into account; the sample preparation (such as fixing and drying) can cause artifacts that make it difficult to obtain information from the analysis of the images. Also, the use of high-energy electron beams or vacuum conditions can cause changes in the structure of the particles, impairing the analysis of the results [89,90]. In this study, morphological analysis of the SLN/TW-1.5 (Figure 7) showed the spheroidal shape of the nanoparticles produced. Usually, the shapes of the SLNs are directly influenced by the lipid composition of the matrix and can be spheroidal, anisometric or flattened. The existence of a specific shape is determined by the particle size. Smaller particles tend to form more compact crystalline arrangements, and larger ones exhibit polymorphic shapes [91].

The AFM analysis also indicates that T5 presents an average diameter of 46 nm and T10 of 36 nm (Figure 7). This result corroborates the observations of average size by DLS (Figure 4), where T5 presented a size mostly larger than T10 in the 180 days of storage. Despite the gradual increase, already expected, in particle size of T10, the diameter remained smaller than T5 in the analysis period. Eaton et al. [92], when comparing different techniques to characterize nanoparticles in terms of size, explain the result found here, in which the size through AFM is smaller when compared to the DLS technique since the DLS measures the hydrodynamic radius of the particle in solution, involving the ions and solvent molecules close to nanoparticle at the time of measurement.

3.7. Cytotoxicity Evaluation of Solid Lipid Nanoparticles

The evaluation of cytotoxicity in murine peritoneal macrophages by the MTT assay is widely used in toxicity studies. It is based on a colorimetric test and allows the assessment of mitochondrial function through the enzymatic reduction of the tetrazolium salt by dehydrogenases present in the mitochondria of viable cells, giving rise to an insoluble, purple-colored salt, formazan [93]. The cytotoxic effect of bacuri butter, SLN and surfactants used in its production is shown in Figure 8.

BBI caused a significant decrease in cell viability from the concentration of 200 µg∙mL⁻¹ (Figure 8a), with a mean inhibitory concentration (IC50) of 249.4 µg∙mL⁻¹. Meanwhile, the surfactant Tween80 used in the preparation of SLN caused a significant decrease in macrophage cell viability starting at 12.5 µg∙mL⁻¹, having IC50 of 273.1 µg∙mL⁻¹ (Figure 8b). SLN/TW-1.5 also caused a significant minimization of macrophage cell viability starting at the concentration of 12.5 µg∙mL⁻¹ (p < 0.05), as seen in Figure 8c.

The cytotoxic effect of the nanosystems was higher than that of the surfactants alone and BBI, with IC50 of 45.2 µg∙mL⁻¹ for the SLN/TW-1.5 and 54.9 µg∙mL⁻¹ for the SLN/PL-1.5. The higher cytotoxic activity of the nanoparticles could possibly be related to byproducts derived from the degradation of the lipid matrix [26,94]. A study by Scholer et al. [94] states that the lipid matrix influences the toxic activity of macrophages, which they attribute to stearic acid (octadecanoic acid), released after enzymatic degradation—the cytotoxic effect. It is important to note that stearic acid was also found as a fatty constituent of bacuri seed butter, as observed in Figure 1 (signal no. 6), but with a low percentage of 1.55% in the butter precipitate.

Moreover, the size of the SLN probably may have influenced the cytotoxic activity since macrophages, as cells of the Mononuclear Phagocytic System (MPS), are equipped with a receptor system that allows them to detect particles unknown to the organism and thus promote phagocytosis and consequent processing [95,96]. Thus, nanoparticles with a diameter greater than 200 nm are easily recognized and phagocytosed; this process of nanoparticle internalization and intracellular degradation can trigger toxic effects on such a cell type [26,97]. On the other hand, Pluronic block copolymers contain hydrophilic polyethylene oxide (PEO) (hydrophilic) and hydrophobic polypropylene oxide (PPO) (hydrophobic) fractions, which act to reduce in vitro and in vivo uptake by macrophages [98]. This may explain the greater cytotoxicity caused by SLN/TW-1.5 when compared to SLN/PL-1.5.

Another possible hypothesis for this response could be related to the potent antioxidant action of BBI [12,16,21], which would interpose the reduction of MTT to formazan, not necessarily indicating cell inviability. It is also known that there may be a decrease in formazan production, even in viable cells, after modification of the culture medium composition, such as by decreasing the concentration of D-glucose, NADH or NADPH [99]. Furthermore, Lustosa et al. [17] reported that BBI presents low cytotoxicity, given the occurrence of less than 10% hemolysis at the highest concentration tested (800 µg/ml). Similarly, when investigating the hemolytic activity of the bacuri seed hexanic extract, the induced cytotoxic effect promoted less than 3% hemolysis at the highest concentration tested (400 µg/ml), also being considered a low cytotoxicity extract [100].

Thus, the application of the MTT assay to natural products to detect their influence on cell viability requires care, considering that the complex reaction medium involved can influence the oxireduction reactions inherent to the assay [101]. Therefore, further studies are needed to elucidate this activity induced by the SLN produced from bacuri seed butter.

Furthermore, the putative influence of PBS on stability and cytotoxic activity is discussed. Henderson et al. [102] have reported the negative effects of PBS on the crystallization of buffering solutions upon freeze–thaw cycle, which can severely affect the properties of biologics and can induce SLNs rupture and aggregation, as captured in recent reports. On the other hand, the in vitro cell viability of HeLa and HEK cells was not affected when SLNs were diluted in PBS. Interestingly, Midekessa et al. [103] measured ZP and showed a significant shift toward less negative values only in the presence of higher concentrations of PBS, which was not observed for differences in SLN concentrations. In the present work, PBS was used exclusively to dissolve MTT salt for the cytotoxic test. From this stock solution, only 10 microliters was added to each microplate well with the previous volume of 200 microliters of cell culture medium (RPMI). Then, a dilution of 1:20 (v/v) of PBS was only incubated with our solid lipid nanoparticles (SLNs) at concentrations ranging from 400 to 3125 micrograms per mililiter. Therefore, we do not assume that a possibility of disruption of the stability of SLNs might occur in very low concentrations. Furthermore, the putative influence of PBS on stability and cytotoxic activity is discussed.

3.8. The Evaluation of SLN/TW-1.5 Toxicity in Zophobas Morio Larvae

The photographic records of larvae obtained are shown in Figure 9 and demonstrate the state of melanization related to an increased response to a xenobiotic agent and then a degree of toxicity as well as a possible activation of the innate immune response [104].

Table 5. Rate of melanization of Zophobas morio larvae in the groups after administration of the formulations.

Group	Melanized Larval Rate (%)
TWEEN 80	5
MF	15
MF 1:2	15
MF 1:4	5
SLN	25
SLN 1:2	25
SLN 1:4	15

TWEEN 80: group received administration of 1.5% Tween 80; MF, MF1:2, MF1:4: groups that received administration of physical mixture through between bacuri seed butter (1%) and Tween 80 (1.5%) at doses of 16.5, 8.25 and 4.125 mg/g, respectively; SLN, SLN 1:2, SLN 1:4: groups that received SLN/TW-1.5 at doses of 16.5, 8.25 and 4.125 mg/g.

shows the percentage of larvae in each group that were considered melanized after 5 days of substance administration. No larvae died during the toxicity evaluation, and this is a good indication of the reduced toxicity presented by the tested formulations.

From the data in Table 5, the groups that received the SLN/TW-1.5 showed a higher rate of melanization and, therefore, are the groups that had the highest elevation in immune response, and this is associated with greater tissue response to these formulations. This greater tissue response is understandable as being an indicator of greater biological activity, mediated either by greater pharmacological or toxicological activity or by better kinetic action of the particles of the formulation in question [37].

3.9. The Evaluation of the Antioxidant Activity in Zophobas Morio Larvae

Vertebrates and invertebrates possess innate immunity that is limited by the number of effective pathways available, and detoxifying enzymes act in an essential manner in the survival of insects exposed to endogenous or exogenous xenobiotics [105,106]. Major components of the insect antioxidant defense system, such as superoxide dismutase (SOD), peroxidases and catalases, assist in converting radicals into less reactive and, therefore, fewer damaging molecules. Thus, an imbalance between oxidative stress and the antioxidant response can lead to disease and death in these insects [107]. Hence, it is understood that the exacerbation of these pathways after substance administration is related to the emergence of a toxic response, and the correlation to be found against control with the vehicle can provide important parameters regarding both toxicokinetics and toxicodynamics.

3.9.1. Reduced Non-Protein Sulfhydryl Groups—Reduced Glutathione (GSH)

Pizzorno [108] reports that intracellular levels of glutathione are as high as glucose levels. Therefore, although the method used for its quantification is not very specific for this molecule, we can assume that being it at such high levels, the interferences would be minimized. Moreover, the increase in the ratio between the concentration of reduced glutathione (GSH) and oxidized glutathione (GSSG) is a great indicator that the body is under oxidative stress [108]. Thus, the association between higher levels of GSH is understood as valid since this would increase the ratio between the concentrations of the reduced and oxidized form and greater oxidative stress in the body.

Results reported in Figure 10A indicate that larvae receiving lower concentrations of the active ingredient in the formulation by physical mixture were in a situation of oxidative stress gradually higher when evaluating the GSH content; no relationship is observed in the larvae that received the nanoparticle formulation, what is observed in these is actually a significant average reduction in GSH levels compared to the control. One explanation for such a result may lie in the better kinetics that nanoparticulate formulations exhibit [109,110], as these would be able to better deliver the antioxidant-active ingredients [111] present in bacuri seed butter, which in turn would meet the need for exacerbation of the larval antioxidant activity and contribute to less oxidative stress overall.

Therefore, the data obtained regarding the evaluation of GSH levels show that the SLN formulations behaved as less taxative in the animal study, most likely for their better kinetic characteristics. Comparatively, Nascimento et al. [16,21] show that the hexanic extract of bacuri seed complexed with cyclodextrin seems to have a better antioxidant effect than the non-complexed extract, with the non-complexed extract being comparable to the physical mixture under study, which could indicate that these complexations and nanostructuring processes can improve the delivery of actives to the sites of action and thus improve the activity.

3.9.2. Nitrite Evaluation by Griess Method

According to Csonka et al. [112], nitrite determination is commonly used to assess total nitric oxide (NO) production since, due to its short half-life time, its direct measurement is challenging. In addition, Bhatia et al. [113] determined a significant increase in nitrite under pathological conditions of intense oxidative stress. We observed, as shown in Figure 10B, a significant difference between the groups and the TWEEN-80 control group, in a pattern relatively similar to that observed for GSH; an increased nitrite concentration is observed in groups that received lower doses of MF, whereas a decreased mean concentration was observed in groups that received SLN when compared to the control, except for the SLN1:2 group.

3.9.3. Superoxide Dismutase (SOD) Enzyme Activity

Li et al. [114] found that when faced with more intense infections in Zophobas morio larvae, SOD activity showed a tendency to increase. From our results, the administration of the formulations did not stimulate this response. As shown in Figure 10C, since no increase in SOD activity was observed, indeed, none of the formulations showed a significantly worse response when compared with the control (TWEEN 80).

Younus [115] described how the action of SOD in the removal of superoxide (O₂⁻) and peroxynitrite (ONOO⁻), a precursor of nitrite, can help in the prevention of cellular energy failure and tissue damage. Thus, as the levels of SOD activity did not show a significant difference and a reduction in nitrite concentrations was observed, it may mean that the mechanism through which the bacuri butter formulations act in reducing nitrite concentrations involves another substrate than SOD, and the medium by which this change is induced is not altered by changes in the kinetics of the formulations.

3.9.4. Evaluation of Myeloperoxidase (MPO) Enzyme Activity

Li et al. [114] found that when facing more intense infections in Zophobas morio larvae, the activity of peroxidase enzymes also showed a tendency to increase. Significant increases, compared to the control, in MPO activity were only observed in the MF and MF1:4 groups, as exposed in Figure 10D, which could indicate the induction of greater oxidative stress for formulations produced from the physical mixture. In contrast, no significant difference was observed between the control and SLN formulations.

4. Conclusions

The fat composition of P. insignis Mart. seed butter enabled the development of SLN, reaffirming the potential of the matrix used for the development of promising biosystems, allowing the maximum use of the richness of properties presented by the fruit of P. insignis Mart.

In the production process, the formulation with Tween 80 at 1.5% was proven to be more stable and promising. Moreover, the characterization of the formulations allowed us to conclude that the increase in the ultrasonication time contributes positively to a greater stability, considering that the T10 formulation obtained greater organoleptic stability, pH, particle size and PdI. On the contrary, the T5 formulation showed greater instability aspects, as in the atomic force microscopy, in which the presence of particle coalescence was observed. Regarding the ZP of both formulations, high and constant values are observed, with these being good indicators. The data presented in the EPR test demonstrate that the molecule under study from the bacuri stone butter has antioxidant activity and, as the technique used to determine this activity has different sensitivity compared to others, there is a significant difference in the nanostructured molecule compared to its physical mixture.

The cytotoxicity study revealed a higher inhibitory activity of the SLN, requiring further studies to elucidate the possible cause. As for the toxicity of the formulations in T. molitor larvae, the formulations composed of SLN were found to be greater inducers of the larval immune system, and no death was observed after the induction period. Evaluations of GSH, nitrite, SOD and MPO confirm the antioxidant activity of these formulations. Thus, future microbiological assays for stability evaluation, where pH source varies, need to be further investigated, and comparative cell viability assays between the physical mixture and the nanoparticles will be of great interest for further elucidation about the possible induction of oxidative stress mediated by the formulations and their toxic characteristics and stability.