Nanoencapsulated buriti oil: toxicological assessment in Caenorhabditis elegans , simulated digestion and enhanced biological potential

Abstract

BACKGROUND

Buriti oil (Mauritia flexuosa) (BO) possesses high nutritional and functional value due to its carotenoids, phenolics, and unsaturated fatty acids, which exhibit antioxidant and antimicrobial properties. However, its lipophilic nature and susceptibility to oxidation limit its industrial application. Nanoencapsulation overcomes these obstacles by increasing oxidative stability and water dispersibility and by enhancing bioactivity. This study investigated the effects of BO nanoencapsulation in porcine gelatin (OPG) on toxicity (cell cultures and Caenorhabditis elegans), simulated digestion, as well as redox‐modulating, antioxidant, and antifungal properties.

RESULTS

The OPG nanoformulation had a diameter of 69.72 nm, a high encapsulation efficiency (95.79%), and high water dispersibility (79.91%). No activity was observed against human cervical cancer cells (HeLa) or Chinese hamster ovary cells (CHO‐K1) for either OPG or BO, but both exhibited activity against 3T3 cells (P < 0.05) at 500 μg mL⁻¹. In C. elegans assays, no toxicity on egg hatching or body size was observed at the evaluated concentrations (0.1, 0.2, and 1.0 mg mL⁻¹). Linoleic acid was preferentially released in the intestinal phase (56.12%) during simulated digestion. Compared with BO (2.24 ± 0.003 mg ascorbic acid equivalent [AAE] g⁻¹), OPG exhibited a significantly higher total antioxidant capacity (23.40 ± 0.004 mg AAE g⁻¹), greater antifungal activity against Candida spp. (minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) = 5 mg mL⁻¹), and an enhanced redox‐modulating dose–response in C. elegans.

CONCLUSION

Buriti oil nanoparticles in porcine gelatin were safe in cellular and animal models in a dose‐dependent manner while demonstrating antifungal efficacy against Candida spp. The nanoencapsulation technology protects the oil from gastric digestion, ensuring intestinal release and enhancing antioxidant capacity. These results demonstrate that nanoencapsulation enhanced the bioactive properties of BO. © 2026 Society of Chemical Industry.

INTRODUCTION

Buriti oil (BO) originates from the buriti palm (Mauritia flexuosa), an important Brazilian resource found primarily in the Amazon and Cerrado regions, as well as other areas of South America. ¹ This oil possesses valuable nutritional and functional characteristics, being rich in bioactive compounds, especially oleic acid (72.7%), which classifies it as a monounsaturated oil with recognized antioxidant potential.^{2, 3, 4, 5, 6} Despite this potential, the bioactive constituents of BO are highly susceptible to degradation during processing and storage due to exposure to light, heat, oxygen, and pH variations.^{7, 8, 9}

In this context, nanoencapsulation has been widely explored as a technological strategy to protect vegetable oils against lipid oxidation and promote the controlled release of bioactive content, such as carotenoids and phenolic compounds.^{10, 11} The reduction in particle size through nanoencapsulation increases the solubility, bioaccessibility, bioavailability, and stability of the active ingredients while prolonging the product's shelf life. ¹²

Several studies have demonstrated that nanoencapsulated BO exhibits superior biological activity compared with the non‐encapsulated oil, with particular emphasis on its antioxidant and antibacterial effects.^{7, 13, 14, 15, 16} Thus, in vitro studies, based on standardized protocols such as the method proposed by Brodkorb et al., ¹⁷ are fundamental for evaluating the release and stability of the encapsulated compounds in the gastrointestinal environment.

However, the specific characteristics of nanoparticles, such as high surface reactivity, a tendency to agglomerate, and small size, may represent toxicological risks that are still poorly understood. ¹⁸ The scarcity of studies on the physiological effects of nanoparticles highlights the need for detailed toxicological evaluations using suitable biological models. In vitro assays using cell cultures have proven effective for identifying potential adverse effects and clarifying the interactions of nanoparticles with biological systems.^{19, 20}

Caenorhabditis elegans has been used as a complementary tool for studies on the toxicity, bioavailability, and antioxidant effects of bioactive compounds.^{21, 22}

To evaluate the bioactive properties of nanoencapsulated oil systems, it is essential to assess the release profile of fatty acids under simulated in vitro digestion conditions – a fundamental test for mimicking release in the gastrointestinal tract. Assessing the toxicity (in vitro and/or in vivo) of the nanoparticles of interest is also crucial for determining the safety of these systems and ensuring compliance for future applications across various industries, including the food, pharmaceutical, and cosmetic industries. In‐depth knowledge of specific biological effects, such as antioxidant and antifungal activity, highlights the technological potential of these lipid nanoparticles for the development of new value‐added products. The authors’ research group has previously studied BO and nanoencapsulated BO in a porcine gelatin matrix.^{7, 13} Those studies included detailed characterization of these nanoparticles, along with evaluation of their biological properties, to guide future applications.

The present study aimed to evaluate toxicity using different cell cultures and the C. elegans model, and to investigate simulated digestion and bioactive properties, including antioxidant and antifungal activity.

MATERIALS AND METHODS

Materials

Buriti oil was supplied by Plantus S.A. (Nísia Floresta, RN, Brazil). The use of BO in this work was registered with the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under registration number A679EA3. Porcine gelatin (Sigma‐Aldrisch, Cajamar, BR), Tween 20 (Sigma‐Aldrisch, Cajamar, BR), α‐amylase (Sigma‐Aldrisch, Cajamar, BR), pepsin (Sigma‐Aldrich, Cajamar, BR), pancreatin (Sigma‐Aldrich, Cajamar, BR), bile salts (Sigma‐Aldrich, Cajamar, BR), dimethyl sulfoxide (DMSO) ‐ (Sigma‐Aldrich, Cajamar, BR), nystatin (Sigma‐Aldrich, Cajamar, BR), Roswell Park Memorial Institute 1640 medium (RPMI medium) (Sigma‐Aldrich, Cajamar, BR), and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT)_(Sigma‐Aldrich, Missouri, USA).

Preparation of the nanoformulation containing BO

The nanoformulation based on porcine gelatin containing BO (OPG) was obtained through the oil‐in‐water (O/W) emulsification technique, followed by dispersion of a solution containing the encapsulating agent in the resulting emulsion, as described by Medeiros et al., ²³ with modifications. ¹³

Buriti oil (10 mL) was used for the oil phase. The two aqueous phases were formulated and prepared as follows:

• Aqueous phase 1 (AP1) (90 mL) – consisted of 1.5% Tween 20 solubilized in distilled water under magnetic stirring at room temperature for 30 min.

• Aqueous phase 2 (AP2) (100 mL) – consisted of 4% (w/v) of the encapsulating agent and 1.5% Tween 20 solubilized in distilled water. Material solubilization was performed under magnetic stirring for 1 h at 40 °C.

The emulsion was obtained by homogenizing AP1 with the oil phase at 17 000 rpm for 10 min using an ultra‐disperser (Ultra‐Turrax, IKA T18 basic, Satufen im Breisgau, DE). Subsequently, AP2 was homogenized with the resulting emulsion under the same conditions. After homogenization, the final emulsion was freeze‐dried (LioTop L101, São Carlos, São Paulo, Brazil) at −57 °C and 43 μHg. The encapsulation process was performed in triplicate.

Characterization of nanoparticles

The new batches of formulations used in this study were characterized using scanning electron microscopy (SEM), dynamic light scattering (DLS), Fourier‐transform infrared (FTIR) spectroscopy, encapsulation efficiency, and the dispersibility test.

Scanning electron microscopy

The OPG nanoformulation was suspended in acetone, and the dispersion was drop‐cast onto silicon wafers attached to stubs using carbon tape. The samples were subsequently analyzed at different magnifications under high vacuum at 2–3 kV and without metallization using a Zeiss field emission (FE)‐SEM microscope (Auriga) (Oberkochen, Baden‐Württemberg, Germany).

Dynamic light scattering

Particle diameters were obtained by DLS using a ZetaSizer Nano ZS (Malvern, ZEN3601, Egham, Surrey, United Kingdom). Samples were diluted 100‐fold in distilled water (pH 7.0) using an ultrasonic bath. Measurements were performed in triplicate (60 s each) using 2 mL of suspension.

Fourier‐transform infrared spectroscopy

Buriti oil, porcine gelatin, Tween 20, and OPG were homogenized separately with potassium bromide (KBr), macerated, and pressed to obtain pellets. Spectra were recorded in transmittance mode in the mid‐infrared region (400 to 4000 cm⁻¹) using a Shimadzu spectrometer (FTIR‐8400S, IRaffinity‐1 series, IR solution software version 1.60, Ettlingen, DE) with 32 scans and a resolution of 4 cm⁻¹.

Encapsulation efficiency (%)

The encapsulation efficiency was determined as described by El‐Messery et al. ²⁴ with modifications. Hexane (15 mL) was added to 1.5 g of the nanoformulation under agitation in a TE‐421 rotary incubator (Tecnal, São Roque, SP, Brazil) for 2 min, and the mixture was then filtered through Whatman No. 1 filter paper. Subsequently, the content retained on the filter paper was washed twice with 20 mL of hexane. The liquid phase was collected, and the hexane was removed in an incubator at 60 °C for 48 h. The remaining extract corresponded to the non‐encapsulated oil on the particles' surfaces.

The amount of nanoencapsulated oil was determined by the difference between the initial amount of oil used to promote the nanoencapsulation process and the free oil present on the surface of the nanoparticles after hexane evaporation, according to Eqn (1). The average mass of OPG obtained from the triplicate formulations and measured with an analytical balance (Edutec, EEQ9003F‐B, São Bernanrdo do Campo, SP, Brazil) was used for calculations.

$$\mathit{EO}\left(g\right)=\mathit{OUE}-\mathit{RO}$$ (1)

where EO is the quantity of encapsulated oil, OUE is the weight of oil used in nanoencapsulation, and RO is the weight of the remaining oil.

The encapsulation incorporation was calculated using Eqn (2), as described by El‐Messery et al., ²⁴ based on the average OPG quantity obtained from triplicate samples and measured with the analytical balance.

$$\mathit{EI}\left(\%\right)=\left[\frac{\mathit{EO}}{\mathit{IO}}\right]x100$$ (2)

where EI is encapsulation incorporation, EO is the amount of encapsulated oil, and IO is the initial oil quantity.

Water dispersibility

The water dispersibility of the gelatin‐based particles was determined according to the methodology described by Silva et al. ²⁵ with modifications. Encapsulated BO (1.0 g) was added at room temperature to a centrifuge tube containing 10 mL of distilled water. The mixture was agitated at 100 rpm for 30 min, followed by centrifugation at 1362 g for 5 min at the same temperature. Subsequently, a 5 mL aliquot of the supernatant was removed and dried in an oven at 105 °C until a constant weight was achieved. Dispersibility was expressed as a percentage, as shown in Eqn (3):

$$\text{dispersibility}\left(\%\right)=\frac{\mathrm{m}1\mathrm{x}2}{\mathrm{m}0}\times 100$$ (3)

where m₁ is the mass of the sample in the 5 mL aliquot after oven drying (g), m₀ is the initial mass of the sample incorporated into 10 mL of water (g), and 2 represents the correction factor as the aliquot volume was half of the initial volume.

Toxicity assays

Cytotoxicity

The cytotoxicity of the samples was evaluated using the MTT reduction assay, according to the protocol of Chave Filho, ²⁶ with modifications, to assess potential cytotoxic effects on Chinese hamster ovary cells (CHO‐K1), human cervical cancer cells (HeLa), and 3T3 cells. The cells were previously cultured in a basal culture medium consisting of Dulbecco's Modified Eagle's Medium (DMEM) enriched with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic in a humidified incubator at 37 °C and 5% CO₂.

First, the cells were seeded in 96‐well plates (2.5 × 10³ cells per well) for 24 h. The samples (50, 100, 500 and 1000 μg mL⁻¹) were applied to the cells for 24 h and 72 h. After the 24 h and 72 h exposure periods, the samples were removed, 100 μL of 1 mg mL⁻¹ MTT (Sigma‐Aldrich, Missouri, USA) was applied to the wells, and the plates were incubated for 4 h. The resulting formazan crystals were then dissolved with 100 μL of DMSO, and the absorbance was measured at 570 nm. Cells maintained in basal culture medium were used as a negative control and cell viability was calculated according to Eqn (4):

$$\mathit{MTT}\mathit{\text{reduction}}\left(\%\right)=\frac{\mathit{\text{treatment absorbance}}}{\mathit{\text{control absorbance}}}\times 100$$ (4)

In vivo toxicity using the C. elegans model

Maintenance of the C. elegans strain

The wild‐type strain (N2) used for all experiments was maintained at 20 °C on nematode growth medium (NGM) (NaCl 3.0 g L⁻¹, peptone 2.5 g L⁻¹, bacteriological agar 17.0 g L⁻¹), supplemented with cholesterol (1.0 mg), CaCl₂ (1 mol.L⁻¹), MgSO₄ (1 mol.L⁻¹), and KPO₄ buffer (25 mol.L⁻¹), seeded with Escherichia coli (OP50) as a food source.

Chronological synchronization of animals

Gravid adult hermaphrodites were subjected to an alkaline lysis solution consisting of 1.0 mL of 10.0 mol.L⁻¹ NaOH solution, 4.0 mL of 2% sodium hypochlorite, and 5.0 mL of deionized water. Embryos resistant to the lysis treatment were collected and placed in liquid M9 solution (KH₂PO₄, Na₂HPO₄, NaCl, MgSO₄, and water) for a period of 12 h, in the absence of food (E. coli OP50), to allow hatching and arrest at the dormant L1 stage. For subsequent experiments, the animals were transferred onto NGM medium containing E. coli OP50 and incubated for 48 h at 20 °C to allow development to the L4 larval stage.

Egg‐hatching test

Synchronized embryos were incubated in M9 medium supplemented with liquid‐cultured E. coli as a food source. Approximately 30 embryos were incubated under the following treatment conditions: each sample (OPG and BO) at concentrations of 0.1, 0.2, and 1.0 mg mL⁻¹; 10% DMSO as the vehicle control for BO dissolution; and an untreated group that served as the negative control (CTN). The concentrations selected for this study were established based on previously described protocols for nanoparticles, ranging from 0.05 to 2.5 mg mL⁻¹.^{27, 28}

The eggs were incubated for 24 h, and the effect on hatching was measured as the percentage of hatched eggs.

Assessment of body size

Body size assessment was performed according to the method described by Boasquívis et al. ²⁹ with modifications. Following chronological synchronization, approximately 300 wild‐type nematodes (N2) at the L1 larval stage were treated with concentrations of 0.1, 0.2, and 1.0 mg mL⁻¹ of each sample (OPG and BO), alongside the vehicle control and untreated control groups, and incubated for 48 h at 20 °C to allow development to the L4 stage. Thirty animals from each group were randomly selected, and body size was individually assessed using images captured under a stereomicroscope (Zeiss, Axio Imager Z2, NewYork, USA) and analyzed using NIH ImageJ software (version 1.53t, Bethesda, MD, USA).

Quantification of reactive oxygen species

To quantify reactive oxygen species (ROS), a synchronized population of wild‐type N2 animals obtained from alkaline lysis was used. Larvae at the L1 stage were transferred onto treatment and control plates and incubated at 20 °C for approximately 48 h until they reached the L4 stage. Subsequently, the animals were washed with M9 solution and centrifuged. The resulting pellet was transferred to new microtubes containing M9 solution and a 20% Tween solution, followed by the addition of the fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate (H₂DCF‐DA) to a final concentration of 20 μmol L⁻¹. The animals were incubated in an orbital shaker at 37 °C for 30 to 40 min. Agarose slides were prepared for observation under an Olympus BX51 fluorescence microscope (Tokyo, Japan), and fluorescence intensity was quantified using the ImageJ software.

Release of nanoencapsulated oil during simulated in vitro digestion

Simulated digestive fluids

Simulated digestive fluids representing the three stages of the digestive process (oral, gastric, and intestinal) were prepared for the in vitro digestion experiment. Simulated salivary fluid (SSF) was prepared as described by Wang et al., ³⁰ with modifications. NaHCO₃ (521 mg), K₂HPO₄·3H₂O (137 mg), NaCl (88 mg), KCl (48 mg), and CaCl₂ (41 mg) were dissolved in 90 mL of deionized water. Subsequently, 20 000 units of α‐amylase was added, the pH was adjusted to 7.0 using 1.0 mol L⁻¹ HCl or 1.0 mol L⁻¹ NaOH, and the final volume was adjusted to 100 mL.

Simulated gastric fluid (SGF) was prepared according to the Pharmacopoeia (2000), ³¹ by dissolving 200 mg of NaCl and 0.7 mL of concentrated HCl (36%) in 90 mL of deionized water. Subsequently, 320 mg of pepsin was added, and the mixture was stirred until completely dissolved. The pH was adjusted to 1.6 using 1.0 mol L⁻¹ HCl, and the final volume was adjusted to 100 mL. Gastric lipase was omitted because it does not exhibit significant activity at acidic pH. ³²

Simulated intestinal fluid (SIF) was prepared according to the protocol described by Leong et al. ³³ K₂HPO₄ (680 mg), NaCl (876 mg), bile salts (500 mg), and pancreatin (640 mg) were added to 90 mL of Milli‐Q water. The pH was adjusted to 7.0 using 1.0 mol L⁻¹ HCl or 1.0 mol L⁻¹ NaOH, followed by stirring for 1 h at room temperature. The solution was then stored overnight at 4 °C to complete pancreatin solubilization, and the final volume was adjusted to 100 mL.

All simulated digestive fluids were used within 1 h after preparation or stored at −20 °C until use.

Determination of encapsulated oil release and digestion

To evaluate the oil release profile from the nanoformulations, 10 mL of pre‐equilibrated SSF was mixed with an amount of nanoparticles equivalent to 1 g of oil in a Schott flask. The pH of the mixture was adjusted to 7.0 using 1.0 mol L⁻¹ HCl or 1.0 mol L⁻¹ NaOH, and the mixture was agitated at 95 rpm for 2 min at 37 °C. After 2 min of exposure to SSF, 10 mL of pre‐equilibrated SGF was added to the mixture, and agitation was maintained at 95 rpm for an additional 2 h. Following the completion of the gastric phase, 10 mL of SIF was added to the mixture, which was agitated at 95 rpm for another 2 h at 37 °C.

After each phase, 2 mL of the sample was withdrawn for analysis. The withdrawn samples were filtered through Whatman No. 1 filter paper and washed twice with 10 mL of hexane. The hexane layer containing the released oil was separated, and the released oil was esterified following the protocol described by Hartman and Lago. ³⁴ A 50 mg sample was added to a capped test tube along with 2.5 mL of 0.5 mol L⁻¹ NaOH in methanol, and the mixture was incubated in a water bath (Quimis, Q334M‐28, Diadema, São Paulo, Brazil) at 70 °C for 15 min. The mixture was then cooled to room temperature, and 7.5 mL of the esterification reagent (96 parts methanol to 4 parts HCl) was added.

The tubes were incubated again in a water bath at 70 °C for 10 min and cooled to room temperature. To extract the fatty acid methyl esters from the sample, 2 mL of HPLC‐grade hexane and 5 mL of a saturated NaCl solution (20%) were added. The mixture was vortexed for 1 min, and 1 mL of the upper hexane phase was transferred to a previously identified tube. The extraction was then repeated by adding an additional 1 mL of hexane and vortexing again to collect another 1 mL of the hexane phase, which was transferred to the same Eppendorf tube. Following hexane evaporation in a fume hood, the Eppendorf tube was weighed, the final mass was recorded, and the sample was stored frozen at −20 °C for gas chromatography analysis.

The fatty acid profile of the released oil was analyzed using an Agilent 8860 gas chromatograph with an Agilent 5977 mass selective detector (MSD) mass spectrometer and an Agilent G4513A autosampler. The chromatographic column employed was the Agilent HP‐5MS model (30 mm × 0.25 mm × 0.25 μm). The injection volume was 1 μL with a split ratio of 1:2, and electron ionization was performed at 70 eV. Data acquisition began approximately 3 min after injection. The temperature gradient started at 50 °C and was raised at a rate of 3 °C min⁻¹ to 240 °C. ³⁵ The injector temperature was set to 250 °C, the carrier gas (helium) flow was approximately 1 mL min⁻¹, and the linear velocity was 36.4 cm s⁻¹. Data were evaluated using total ion count (TIC) to obtain retention times, peak areas, and substance identities. Analysis was performed in scan mode for ions with m/z values from 40 to 550. Constituents were identified based on the spectral similarity index, and fragmentation profiles were compared with the National Institute of Standards and Technology (NIST) mass spectra database library. ³⁶

The percentage of fatty acids released was calculated by subtracting the percentage released in each phase from the total percentage of fatty acids in the system, then dividing the result by the total percentage of fatty acids in the system and multiplying by 100.

Bioactive potential investigation

Total antioxidant capacity

The total antioxidant capacity (TAC) of the free and nanoencapsulated oils in this study was evaluated using the method described by Prieto et al., ³⁷ with modifications. Each test tube received 100 μL of 40 mmol L⁻¹ sulfuric acid–ammonium molybdate, 100 μL of 280 mmol L⁻¹ sodium phosphate, and 100 μL of the sample solution.

Distilled water was added, and the tubes were incubated in a water bath at 90 °C for 90 min. The absorbance of the solutions was measured at 695 nm using a spectrophotometer (Bioespectro, Mod. SP‐220, Curitiba, SC, Brazil). The experiment was performed in triplicate, with water as the negative control. Antioxidant activity was expressed as mg ascorbic acid g⁻¹ (mg AA g⁻¹) of sample using a standard ascorbic acid curve with concentrations of 25–250 mg mL⁻¹.

Antifungal activity

The antifungal activity of the free and nanoencapsulated oils was evaluated according to the Clinical and Laboratory Standards Institute (CLSI, 2017) ³⁸ guidelines with adaptations, using the microdilution technique in 96‐well U‐bottom microtiter plates with RPMI medium. The strains used were Candida albicans ATCC 90028, C. tropicalis ATCC 750, both obtained from the American Type Culture Collection (ATCC), and C. krusei CBS 573, obtained from the Central Bureau voor Schimmelcultures (CBS).

The strains were inoculated in RPMI medium. The fungal suspension was adjusted to obtain a fungal inoculum equivalent to 2.5 × 10³ CFU mL⁻¹, with absorbance ranging from 0.08 to 0.1 at 530 nm. Fungal inoculum (100 μL) was added to the microplates, followed by 100 μL of BO and OPG at serial concentrations from 0.078 to 10 mg mL⁻¹. For the positive control, nystatin (Sigma‐Aldrich) was used at concentrations from 48 μg mL⁻¹ to 0.75 μg mL⁻¹. Negative and sterility controls were performed simultaneously with the assay. The encapsulating materials, porcine gelatin and Tween 20, were also tested.

The microdilution assay was performed in triplicate across three independent experiments, and the data were analyzed with results expressed as the mode of the minimum inhibitory concentration (MIC) values. The microplate was incubated for 24 h at 35 °C and results were assessed by observing cell aggregates at the bottom of the wells. The MIC was the lowest concentration capable of visually inhibiting fungal growth, compared with the growth control. In parallel, to confirm the cell viability, 50 μL of 1% 2,3,5‐triphenyl tetrazolium chloride (a colorimetric oxide‐reduction indicator) was added and the plates were incubated at 35 °C for 24 h again.

To define the minimum fungicidal concentration (MFC), 20 μL aliquots corresponding to the MIC and the two concentrations above (MIC × 2 and MIC × 4) were subcultured on Sabouraud dextrose agar (KASVI, Curitiba, Brazil). The plates were incubated for 24 h at 35 °C, and the results were determined by observing fungal growth on the culture medium.

Statistical analysis

Data were analyzed using GraphPad Prism software (version 8.0.2). For egg‐hatching and body size assays, where the primary objective was to compare treatment groups against an untreated control, a one‐way analysis of variance (ANOVA) followed by Dunnett's post hoc test was applied. For ROS and cell viability assays, a one‐way ANOVA followed by Tukey's post hoc test was used to allow for multiple comparisons between all experimental groups and concentrations. Results are expressed as means ± standard deviations (SDs). A P‐value < 0.05 was considered statistically significant for all analyses.

RESULTS AND DISCUSSION

Nanoparticle characterization

These analyses were conducted to confirm that the new nanoparticle batches (OPG) matched the physicochemical characteristics reported by Morais et al., ⁷ thereby ensuring material standardization and functionality (Fig. 1).

Figure 1.

Characterization of buriti oil (BO) nanoencapsulated in porcine gelatin (OPG). (A) Scanning electron microscopy (SEM) micrograph. (B) Dynamic light scattering (DLS). (C) Fourier transform infrared (FTIR) spectroscopy of (a) nanoencapsulated BO, (b) BO, (c) Tween 20, and (d) porcine gelatin.

Scanning electron microscopy images confirmed that the particles were spherical, had a smooth, intact surface, and were nanometric in size (<100 nm) (Fig. 1(A)). These characteristics demonstrate effective encapsulation of BO and the method's reproducibility. Dynamic light scattering data indicated an average OPG diameter of 69.72 ± 15.20 nm. The OPG nanoparticles also exhibited a polydispersity index (PDI) of 0.48 ± 0.012, indicating a nanometric size and unimodal distribution (Fig. 1(B)).

Observing the OPG spectrum (Fig. 1(C) – (a)), an interaction was noted between the BO (Fig. 1(C) – (b)), Tween 20 surfactant (Fig. 1(C) – (c)), and the porcine gelatin (Fig. 1(C) – (d)), evidenced by the attenuation and/or shifting of the vibrational bands that characterize the presence of BO (3007, 2923, 2856, and 1746 cm⁻¹), indicating the protection of the oil within the particles. The formation of new bands (1552, 1343, and 1097 cm⁻¹), absent in the raw material spectra, may indicate hydrophobic interactions between BO and the nonpolar amino acids of the porcine gelatin. In summary, the characterization confirmed that the OPG nanoparticles produced in this study exhibited the same physicochemical characteristics as previous formulations, validating the method's standardization and reproducibility.

The encapsulation efficiency was 95.79 ± 4.35%, which is considered high (i.e., ≥ 80%).^{39, 40} The nanoparticles also exhibited a water dispersibility of 79.91 (±9.01)%. Nanoencapsulation, especially at the nanoscale (<100 nm), increases the solubility of lipophilic substances such as BO, in aqueous matrices. This reduction in particle size increases the surface area for interactions with water, which contributes directly to the oil's dispersion and, consequently, to faster, more efficient absorption in the gastrointestinal tract, potentially resulting in greater bioaccessibility and bioavailability of the encapsulated bioactive compounds.^{38, 41, 42}

Cytotoxicity

The cytotoxicity assay was performed using CHO‐K1 cells (Fig. 2) with BO and nanoencapsulated BO (OPG) at varying concentrations. Buriti oil and the encapsulated oil did not show cytotoxicity in CHO‐K1 cells, with cell viability above 70%. ⁴³

Figure 2.

Cytotoxicity evaluation of buriti oil (BO) (Fig. 2(a), (b)) and BO nanoencapsulated in porcine gelatin (OPG) (Fig. 2(c), (d)) samples in Chinese hamster ovary (CHO‐K1) cells at 24 h and 72 h. Data were evaluated using analysis of variance (ANOVA). Cells were treated with samples at concentrations of 50, 100, 500, and 1000 μg mL⁻¹. The negative control is represented as a 100% reduction in 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT). Results with a statistically significant difference compared to the negative control are indicated with * (P < 0.05).

Consistent with the present study, Marques et al. ⁴⁴ evaluated free and nanoencapsulated quinoa oil in porcine gelatin at concentrations of 100, 500, and 1000 μg mL⁻¹. No cytotoxicity was observed in the cells tested.

HeLa cells are human cervical carcinoma cells that have been widely used for in vitro cytotoxicity assays of various agents. Buriti oil and OPG also showed no reduction in cell viability (Fig. 3). No studies in the literature have used HeLa cells and BO, making this the first report of its kind.

Figure 3.

Cytotoxicity evaluation of buriti oil (BO) (Fig. 3(a), (b)) and BO nanoencapsulated in porcine gelatin (OPG) (Fig. 3(c), (d)) samples in human cervical cancer (HeLa) cells at 24 h and 72 h. Data were evaluated using analysis of variance (ANOVA). Cells were treated with samples at concentrations of 50, 100, 500, and 1000 μg mL⁻¹. The negative control is represented as a 100% reduction in 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT). Results with a statistically significant difference compared with the negative control are indicated with * (P < 0.05).

In 3T3 fibroblast cells (Fig. 4), a different behavior was observed in comparison with the other cells in the study. Buriti oil did not show a decrease in cell viability after 24 h of exposure. However, after 72 h, cell viability decreased to 56% at the highest concentration (1000 μg mL⁻¹). However, OPG maintained cell viability for 24 h; at 72 h cell viability dropped to 63% at 500 μg.mL^‐1 and further decreased to 32% at the highest concentration of 1000 μg.mL^‐1.

Figure 4.

Cytotoxicity evaluation of buriti oil (BO) (Fig. 4(a), (b)) and BO nanoencapsulated in porcine gelatin (OPG) (Fig. 4(c), (d)) samples in 3T3 fibroblast cells at 24 and 72 h. Data were evaluated using analysis of variance (ANOVA). Cells were treated with samples at concentrations of 50, 100, 500, and 1000 μg mL⁻¹. The negative control is represented as a 100% reduction in 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT). Results with a statistically significant difference compared with the negative control are indicated with * (P < 0.05).

Zanatta et al. ⁴⁵ tested nanoemulsions containing BO at concentrations of 5% to 10% in 3T3 cells and observed no cytotoxicity using the neutral red assay. According to the literature, ⁴⁶ 3T3 cells are considered more sensitive to irritants than other cell types. The concentration of nanoparticles and the exposure time also play a decisive role in the toxicity observed. The internalization of nanoparticles via endocytosis at high concentrations can result in damage to plasma membranes, changes in mitochondrial potential, and the release of intracellular calcium, affecting essential metabolic pathways, ⁴⁷ which may explain the cytotoxic effect observed at higher concentrations of the OPG nanoparticles.

High concentrations or prolonged exposure can cause toxic effects, mainly by saturating the cells’ antioxidant defense mechanisms and leading to cell damage.^{48, 49} Smaller nanoparticles tend to be more cytotoxic due to their high surface‐to‐volume ratio, which increases their reactivity and ability to interact directly with cell membranes and intracellular organelles.

Evaluation of in vivo toxicity using the C. elegans model

The nematode C. elegans has established itself as an alternative in vivo model, widely used to evaluate the toxicological effects of nanoparticles. It is especially relevant in nanotoxicology due to its simplicity, low cost, and high genetic homology with humans. ²⁷ No reports on the toxicity of BO, free or nanoencapsulated, using this in vivo model have been found in the literature. In this context, the results of this study provide novel and essential data for understanding the possible effects of these formulations in multicellular organisms and for extrapolating to more complex animal models.

Previous studies with C. elegans treated with vegetable oils or their isolated constituents have demonstrated outstanding potential for toxicological applications, reinforcing its utility as a predictive tool in evaluating bioactive compounds. ⁵⁰

In the present work, no significant changes were observed in the egg‐hatching rate of C. elegans exposed to BO or the nanoencapsulated formulation (OPG) (Fig. 5) at any of the tested concentrations.

Figure 5.

Caenorhabditis elegans egg‐hatting test. Results are expressed as mean ± standard deviation (SD). Data were analyzed using one‐way analysis of variance (ANOVA) followed by Dunnett's post hoc test for comparisons against the negative control (animals on plates without treatment). No statistically significant differences were observed between the treated groups and the control (P > 0.05).

Pascoli et al. ²⁸ evaluated zein nanoparticles containing neem oil and observed no impact on egg hatching at concentrations from 0.05 to 0.75 mg mL⁻¹. The absence of effects on hatching suggests that neither BO nor OPG interferes with the initial mechanisms of nematode embryonic development. These data are particularly relevant, as more than 70% of human lipid metabolism genes are conserved in C. elegans, making the model highly predictive for safety studies of lipophilic compounds. ⁵¹

Analysis of nematodes’ body size (Fig. 6) revealed that only the highest concentration of the OPG nanoparticle tested (1 mg mL⁻¹) promoted a statistically significant reduction (8.04%) in nematode length.

Figure 6.

Caenorhabditis elegans body size test. The results represent the mean ± standard deviation (bars in columns). Data were evaluated using Dunnet's post hoc test. Control: negative control corresponding to untreated animals. P < 0.05 indicates statistical significance relative to the control.

This finding agrees with Charão et al., ²⁷ who observed a similar effect with lipid nanocapsules containing melatonin, in which only the highest concentration (2.5 mg mL⁻¹) induced a significant reduction in body size. Pascoli et al. ²⁸ did not identify alterations in the growth of C. elegans exposed to neem oil and the corresponding nanoparticles (0.05–0.75 mg mL⁻¹), suggesting that changes in this parameter depend on the formulation's composition and concentration. The reduction in body size observed at high concentrations may indicate interference in conserved genetic pathways that regulate the organism's growth and development, possibly related to lipid homeostasis and metabolic signaling. ⁵² Measuring body length is a sensitive and widely used parameter for screening potential toxic effects in C. elegans.

The results suggest that BO, in both free and nanoencapsulated forms, exhibited low general toxicity in the C. elegans model, with only slight alterations observed at higher concentrations. These findings reinforce the safety of using these formulations in biological systems and support their potential for future nutraceutical or pharmacological formulation applications.

In the ROS quantification analysis (Fig. 7), the concentrations of BO and OPG used did not differ significantly from the control (P > 0.05), indicating that neither increased ROS production in the animals.

Figure 7.

Quantification of reactive oxygen species in the Caenorhabditis elegans test. Results represent the mean ± standard deviation (bars in columns). Data were evaluated using Tukey's test. Control: Negative control corresponding to animals placed in a plate without treatment, P < 0.05 (statistical significance relative to the control).

The OPG showed a reduction in ROS with increasing concentration, demonstrating a dose–response relationship relative to BO when evaluated. However, a concentration of 1 mg mL⁻¹ of nanoparticles is equivalent to 0.475 mg mL⁻¹ of oil within the nanoparticles, indicating a higher antioxidant potential than oil alone. This result indicates that nanoencapsulation enhanced the redox‐modulating potential found in BO.

Moniz et al. ⁵⁰ found that various concentrations of Lippia origanoides oil showed a favorable dose–response for ROS reduction in C. elegans. This finding reinforces the finding that OPG has a redox‐modulating potential, especially at the highest concentrations.

The current study investigated ROS production in the in vivo model of C. elegans but did not investigate oxidative stress markers, limiting insight into the actions of the bioactive compounds present in BO. For a comprehensive investigation of oxidative stress, it would be important to determine the oxidation markers of biomolecules, as well as the state of antioxidant enzymes. ⁵³

In this context, these investigations represent future directions for subsequent studies involving free and nanoencapsulated BO. It is important to reinforce the pioneering role of the present study in investigations related to toxicity in an in vivo model using C. elegans, controlled release, and the antifungal potential of nanoencapsulated BO. These findings will contribute to the scientific community regarding the development of promising ingredients for the food and pharmaceutical industries.

Release assay

The release profile of an active compound from nanoencapsulated systems is fundamental for understanding its behavior in biological environments and the mechanisms that regulate its release. Assays conducted under simulated conditions that mimic the human gastrointestinal environment allow researchers to evaluate formulation stability and provide insights for the development of products with targeted applications.

In this study, OPG was subjected to in vitro simulations of the oral, gastric, and intestinal phases of human digestion, with the aim of investigating the oil release pattern from the encapsulating matrix (Fig. 8).

Figure 8.

Free fatty acid (FFA) release profiles during in vitro digestion in the gastric (green) and intestinal (blue) phases.

During the salivary phase, no significant release of the oil encapsulated with porcine gelatin was observed. This can be attributed to reduced exposure time and the limited capacity of the SSF to disintegrate the encapsulating matrix. The main enzyme in SSF, α‐amylase, primarily hydrolyzes polysaccharides but is ineffective in degrading proteins, such as the gelatin used in encapsulation. ⁵⁴

Previous studies have corroborated this finding. Gholivand et al., ⁵⁴ when investigating the release of encapsulated hemp oil, also observed insignificant release in the oral phase, with small quantities attributed to surface oil that was not completely encapsulated. A significant release was observed in the gastric phase, as evidenced in this study. This release can be explained by protein degradation induced by the acidic environment of gastric juice and by enzymes such as pepsin, which act on the protein matrix, facilitating the rupture of the nanoparticles and the diffusion of the oil. ⁵⁵

During the gastric phase, palmitic acid (22.35%), oleic acid (2.84%), and stearic acid (38.08%) – all fatty acids – were detected, suggesting that the degradation of the encapsulating matrix allowed for the release of lipid components from the core. However, unsaturated fatty acids, such as oleic and linoleic acid, are susceptible to oxidation under pro‐oxidant conditions, characteristic of the gastric environment (acidic pH, presence of oxygen and metal ions).^{56, 57} In this study, linoleic acid was not released in the gastric phase, indicating good protection for the system.

During the simulated intestinal phase, palmitic acid (35.15%) was continuously present. Being saturated, palmitic acid shows greater oxidation resistance, maintaining its integrity throughout the digestive process. ⁵⁸ The detection of linoleic acid (56.12%) in this phase suggests that encapsulation effectively protected it from degradation during the gastric step, keeping it available for intestinal absorption. This indicates that the nanoencapsulation conferred efficient protection for this polyunsaturated fatty acid, as reported by Sehl et al. ⁵⁹

Oleic acid (2.84%) and stearic acid (38.08%) were identified in the gastric and intestinal phases, respectively, with different liberation percentages. The oleic acid (31.53%) indicates that its release occurred predominantly at this stage. Lauric acid was observed only during the intestinal phase, possibly released by the progressive degradation of the encapsulating matrix under alkaline conditions and by pancreatic enzymes in intestinal fluid.

These results confirm that nanoencapsulation promotes targeted, phase‐dependent release of the fatty acids in BO. It protects more sensitive compounds in the gastric phase and favors their release in the intestinal phase, where absorption is more effective. This behavior reinforces the potential of nanoencapsulation as an effective strategy for the transport and controlled release of lipophilic compounds in complex biological systems.

Total antioxidant capacity

The TAC values for BO and OPG were 2.24 ± 0.00 and 23.40 ± 0.00 mg AA g⁻¹, respectively. Based on these values, the OPG nanoparticles demonstrated a significantly higher total antioxidant capacity (P < 0.05). Some studies indicate that the nanoencapsulation of vegetable oils can potentiate or maintain their antioxidant activity,^{15, 44, 60, 61} as seen in a study evaluating soybean oil pigmented with astaxanthin extracted from shrimp, in which the nanoencapsulation with porcine gelatin potentiated the total antioxidant capacity of the pigmented oil ⁶⁰ .

Marques et al. ⁴⁴ analyzed the total antioxidant activity of quinoa oil encapsulated with porcine gelatin compared to crude quinoa oil. Consistent with this study, the nanoparticles showed a higher value (38.61 ± 2.00 mg AA g⁻¹) than the crude quinoa oil (25.64 ± 2.56 mg AA g⁻¹). The oil concentration in the nanoparticle is 4.70 mg, indicating that nanoencapsulation potentiates the antioxidant effect of BO. The results indicate that the encapsulation process increased the antioxidant capacity of the nanoparticle.

Evaluation of antifungal activity in Candida strains

Table 1 indicates that neither the free BO nor the wall material used for nanoencapsulation showed antifungal activity. No reports in the literature exist on the antifungal action of BO, free or nanoencapsulated. A study using olive oil showed a 17% inhibition of C. albicans growth. ⁶²

Table 1.

Antifungal activity of buriti oil and nanoparticle

Sample	Strains	MIC (mg mL−1)	MFC (mg mL−1)
BO	Candida albicans	nd	nd
	Candida tropicalis	nd	nd
	Candida krusei	nd	nd
OPG	Candida albicans	5	5
	Candida tropicalis	5	5
	Candida krusei	5	5

Note: BO, buriti oil; OPG, buriti oil encapsulated with porcine gelatin; nd, not detected.

On the other hand, the nanoparticles containing BO exhibited effective antifungal activity against all tested strains, with MIC and MFC showing similar values of 5 mg mL⁻¹, indicating strong fungicidal power. Often, when MIC and MFC values overlap, it suggests that the mechanism of action disrupts critical, irreparable processes, such as cell wall integrity or membrane function. ⁶³

Encapsulation improves the stability and efficacy of active compounds, enabling sustained release and facilitating their interaction with fungal pathogens. Studies have demonstrated that these nanoparticles can compromise the integrity of the fungal cell membrane, leading to the leakage of intracellular content and cell death. ⁶⁴ This effect is associated with the induction of ROS and the release of potassium ions, factors that compromise cellular homeostasis.^{64, 65, 66} The nanoencapsulation of BO also favors the solubilization of hydrophobic compounds in an aqueous medium, facilitating their penetration into the fungal cells and resulting in the loss of cell membrane integrity. ⁶⁷

CONCLUSION

The nanoparticles were found to be safe in CHO‐K1 and HeLa cells and at low concentrations in 3T3 cells. Similarly, no toxicity was observed in C. elegans in the hatching or ROS tests, demonstrating consistency between these assays. However, for body size, toxicity was observed only at the highest concentration tested, indicating that this effect is distinct from those measured in hatching and ROS assays. This study was pioneering in investigating the release and antifungal potential of BO encapsulated in OPG. The results demonstrated that nanoencapsulation protects the oil in the gastric environment, ensuring its effective release in the intestinal phase during simulated gastrointestinal digestion. Nanoencapsulation also increased the oil's antioxidant potential and conferred inhibitory activity against Candida sp. These findings highlight the biotechnological potential of BO nanoparticles as a promising ingredient for the food and pharmaceutical industries.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.