Blackberry (Rubus spp. Xavante Cultivar) Oil-Loaded PCL Nanocapsules: Sustainable Bioactive for In Vitro Collagen-Boosting Skincare

Abstract

Background: Blackberry seed oil (BSO), obtained from Rubus spp. Xavante cultivar via supercritical CO₂ extraction, contains bioactive lipids and antioxidants, but its cosmetic application is limited by poor solubility and stability. Nanoencapsulation with poly(ε-caprolactone) (PCL) can overcome these limitations. Methods: BSO was characterized by Ultra-High-Performance Liquid Chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry and incorporated into PCL nanocapsules (NCBSO) using the preformed polymer deposition method. Physicochemical properties, stability (at 4 °C, room temperature, and 37 °C for 90 days), cytotoxicity, and collagen production were assessed in human fibroblasts. Additionally, a predictive in silico analysis using PASS Online, Molinspiration, and SEA platforms was performed to identify the bioactivities of major BSO compounds related to collagen synthesis, antioxidant potential, and anti-aging effects. Results: NCBSO showed a nanometric size of ~267 nm, low polydispersity (PDI < 0.2), negative zeta potential (−28 mV), and spherical morphology confirmed by FE-SEM. The dispersion remained stable across all tested temperatures, preserving pH and colloidal properties. In particular, BSO and NCBSO at 100 µg.mL⁻¹ significantly enhanced in vitro collagen production by 170% and 200%, respectively, compared to untreated cells (p < 0.01). Superior bioactivity was observed for NCBSO. The in silico results support the role of key compounds in promoting collagen biosynthesis and protecting skin structure. No cytotoxic effects were achieved. Conclusions: The nanoencapsulation of BSO into PCL nanocapsules ensured formulation stability and potentiated collagen production. These findings support the potential of NCBSO as a promising candidate for future development as a collagen-boosting cosmeceutical.

1. Introduction

Natural products and their bioactive properties have demonstrated improved interest across the pharmaceutical, food, and cosmetic industries. This trend reflects the growing societal awareness of biodiversity conservation, sustainable economic, and cultural practices. Within the skincare sector, the valorization of industrial byproducts has emerged as a key strategy, fostering innovation while aligning with circular economy principles [1,2].

Blackberry byproducts (Rubus spp. cultivar Xavante)—including seeds, peels, and pulp—are particularly remarkable due to their high concentrations of polyunsaturated fatty acids, tocopherols, tocotrienols, phytosterols, and carotenoids, as reported in the recent literature [3,4,5]. These bioactive compounds demonstrate multifunctional properties, including antioxidant, antimicrobial, anti-inflammatory, and anti-aging effects, along with hydration enhancement and skin barrier reinforcement [1,4,6,7]. Such attributes position them as highly promising candidates for advanced cosmetic formulations.

The synergistic interaction of these bioactive compounds enhances their efficacy in cosmetic formulations, providing multi-target cellular protection across skin layers through complementary mechanisms of action. Therefore, blackberry byproducts have considerable potential as valuable ingredients in cosmetic products, providing multifaceted benefits for skin health and appearance [2,5,6,8]. The valorization of blackberry (Rubus spp.) byproducts represents a significant industrial opportunity, simultaneously enhancing commercial yield through the full use of raw materials [4,5,8] while establishing sustainable production systems that minimize agro-industrial waste.

The selection of processing methods for these oils is crucial to obtaining high-quality oil [5,8]. An innovative method that has proven effective is supercritical CO₂ (scCO₂) extraction [4,5,8]. This extraction method enables the low-temperature recovery of high-quality oils while operating under oxygen- and light-free conditions for preserving thermo- and photo-sensitive bioactive compounds. Furthermore, it demonstrates superior selectivity to fatty acids and eliminates the need for hazardous organic solvents compared to conventional extraction techniques [8].

Despite their demonstrated bioactivity, the practical application of plant-derived oils faces substantial technological limitations due to intrinsic physicochemical properties, including volatility, poor aqueous solubility, and susceptibility to oxidative and thermal degradation [7]. Nanotechnology-based delivery systems, particularly biodegradable polymeric nanocapsules, have emerged as an innovative solution to these challenges. These nanocarriers successfully encapsulate bioactive compounds within protective nanostructures, shielding them from environmental stressors while enabling controlled release [7,9].

Polymeric nanocapsules are nanoscale delivery systems (100–500 nm) characterized by an oil-filled core surrounded by a stabilizing polymeric shell. This unique architecture offers several advantages: (1) it maximizes active ingredient loading while minimizing polymer requirements; (2) the polymeric barrier effectively isolates encapsulated compounds from surrounding tissues; (3) it prevents premature degradation or the uncontrolled release of bioactive components [9,10,11]. These materials are useful tools for improving the delivery of bioactives to the skin, allowing the controlled release of the active ingredients over prolonged periods and under specific conditions [12,13].

Poly(ε-caprolactone) (PCL) is a polymer extensively used in the development of such systems. It has suitable properties as a biodegradable, biocompatible, low-cost, non-toxic, and compatible biomaterial. Therefore, it can improve the biological activity of blackberry byproducts on the skin [2,14,15]. However, no previous research has focused on the preparation of PCL nanocapsules containing blackberry seed oil (BSO) for cosmetic purposes to increase collagen production.

In recent years, computational in silico approaches have become valuable tools in natural product research for allowing the prediction of the biological activities of complex mixtures based on chemical structure. In this study, the bioactive compounds present in BSO were analyzed using multiple in silico platforms, including PASS Online, Molinspiration, and the Similarity Ensemble Approach (SEA) Search Server. These tools estimate the possibility of each compound to demonstrate antioxidant activity, stimulate collagen and elastin synthesis, and other skin-related bioactivities by assessing the structure–activity relationships and potential molecular targets. Such predictive modeling supports the identification of key compounds responsible for the observed biological effects and provides mechanistic insight that complements experimental findings [16,17,18].

Considering the reported bioactive and antioxidant properties of blackberry (Rubus spp.) byproducts and their economic/sustainability benefits, this study aims to develop and characterize poly(ε-caprolactone) (PCL) nanocapsules encapsulating supercritical CO₂-extracted blackberry seed oil in order to enhance stability and skin delivery. This innovative approach seeks to enhance the BSO characteristics and potentiate its biological action on the skin.

2. Materials and Methods

2.1. Plant Material

Blackberry fruits (Rubus spp. cultivar Xavante) were collected at the end of November 2023 in the field station of the State University of the Central-West of Paraná at the CEDETEG campus (Guarapuava, Paraná State, Brazil). All procedures for seed separation, drying, milling, and subsequent characterization of the milled material were performed according to the method previously detailed [5].

2.2. Oil Extraction

The blackberry seed oil (BSO) was extracted via supercritical CO₂ (scCO₂) extraction [19] using a home-made laboratory-scale supercritical fluid extraction (SFE) unit that consisted of a 62.4 cm³ extraction vessel, a high-pressure syringe pump for pressure control, and an ultra-thermostatic bath to maintain temperature. The extraction vessel was filled with 30 g of milled blackberry seeds in each extraction. The extraction conditions were 70 °C, 25 MPa, 60 min static extraction, a CO₂ flow of 2.0 mL.min⁻¹ (2.00 g.min⁻¹), and 180 min dynamic extraction. These conditions correspond to the highest yield extraction obtained from a previous publication [5]. The oil extract with scCO₂ (BSO) was collected at 18 °C and 1 atm.

2.3. UHPLC–ESI–Q-TOF–MS Analysis

Three microliters of the blackberry seed oil samples was directly injected into an Ultra-Performance Liquid Chromatograph (Nexera X2 UHPLC, Shimadzu, Kyoto, Japan) system, coupled to a Quadruple-Time of Flight mass spectrometer (Impact II Q-TOF-MS, Bruker, Bremen, Germany) system, equipped with a Shim-pack XR-ODS III column (2.0 × 75 mm, 1.6 μm), Shimadzu (Kyoto, Japan). The Q-TOF-MS system was equipped with an electrospray ionization (ESI) source and a Micro Channel Plate (MCP) detector. Mobile phases A and B were used: A (ultrapure water) and B (acetonitrile). The gradient elution was as follows: 0.0–2.0 min 5% B; 2.0–3.0 min 30% B; 3.0–10 min 95% B; 10.0–14.0 min 95% B; 14.0–15.0 min 5% B. The flow rate was set at 0.3 mL.min⁻¹, and the column oven temperature was set to 40 °C. The operating parameters of ESI were a capillary voltage of 4500 V, argon (Ar) at 200 °C, a pressure of 4 bar, and a flow rate of 8 L.min⁻¹ in negative mode. The MS system settings were m/z = 50–1950 (mass range). Calibration of the Q-TOF-MS system was performed with a sodium formate solution (10 mmol.L⁻¹).

2.4. In Silico Prediction of Biological Activities

To support the mechanistic interpretation of the bioactive profile of BSO, in silico prediction of biological activities was conducted based on the chemical constituents previously identified by UHPLC–ESI–Q-TOF–MS. This computational strategy was designed to estimate the likelihood of each compound to contribute to antioxidant effects, stimulate collagen and elastin synthesis, and offer anti-aging potential. Canonical SMILES notations were retrieved from public databases such as PubChem and ChemSpider. These molecular representations were then processed using three independent platforms for predictive bioactivity modeling. The PASS (Prediction of Activity Spectra for Substances) Online platform (http://www.pharmaexpert.ru/passonline, accessed on 21 July 2025) provided probability scores (Pa) for a broad spectrum of biological activities, derived from structure–activity relationships trained on curated datasets. A threshold of Pa ≥ 0.6 was adopted to denote relevant predicted activity, consistent with previous studies employing this method in the context of natural products and cosmeceuticals [16,17]. Molinspiration (www.molinspiration.com, accessed on 21 July 2025) was used to evaluate fragment-based bioactivity scores across target classes, such as G-protein-coupled receptors, nuclear receptors, ion channels, proteases, and enzymes, which is an approach previously validated for phytocompound profiling in cosmetic and pharmaceutical research [17]. Additionally, the Similarity Ensemble Approach (SEA) Search Server (http://sea.bkslab.org, accessed on 21 July 2025) enabled the identification of putative molecular targets based on structural similarity to known ligands, further supporting mechanistic hypotheses [18].

Output data from all platforms were organized into a comparative matrix and interpreted in relation to skin physiology, including cellular protection, collagen biosynthesis, and elastin remodeling. This multi-tool predictive strategy has been widely applied in compound prioritization for skin-related bioactivities and provided theoretical support for the functional roles of BSO constituents in the observed collagen-stimulating effects [20,21].

2.5. Preparation of Blackberry Seed Oil-Loaded Nanocapsules (NCBSO)

Suspensions of PCL nanocapsules loaded with blackberry seed oil (NCBSO) were prepared using the interfacial deposition method of preformed polymers, as described by Fessi et al. (1989) [22]. Briefly, PCL (0.057 g) was dissolved in acetone (20 mL) under magnetic stirring at 3500 rpm at 24.2 °C. Subsequently, sorbitan monooleate (Span 80) (0.0878 g) and BSO (0.3055 g) were added and kept under stirring at 40 °C (Fisatom, model 713, São Paulo, Brazil) until complete solubilization of the organic phase components. The organic phase was then dripped at a flow rate of 2.5 mL/min into the aqueous phase composed of 50 mL of water containing polysorbate 80 (Tween 80) (0.2135 g). The mixture was maintained under vigorous magnetic stirring at 3.500 rpm at 40 °C for 15 min. The organic solvent was evaporated under reduced pressure in a rotary evaporator (Tecnal, model TE-211, Piracicaba, Brazil) until a final volume of 10 mL. Under the same conditions, PCL nanocapsules containing typical medium-chain triglycerides (MCT) were prepared as a negative control (NC-C).

2.5.1. Characterization of Blackberry Seed Oil-Loaded Nanocapsules (NCBSO)

Physicochemical Characterization of Nanocapsules

The mean particle size, polydispersity index (PDI), and zeta potential were achieved after diluting a sample of the nanocapsule suspension (NCBSO) in ultrapure water at a 1:500 ratio. All measurements were conducted using the Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). The samples were analyzed in triplicate. pH values were measured using a digital potentiometer (Digimed, São Paulo, Brazil), which had been previously calibrated with pH 4.0 and 7.0 buffer solutions. The pH was directly measured in each colloidal suspension after its preparation.

Analysis by Scanning Electron Microscopy Coupled with Field Emission (FE-SEM)

The morphological and superficial evaluation of the samples was carried out using a MYRA 3 LMH scanning electron microscope (Tescan, Brno, Czech Republic) with field emissions. Firstly, 1 mL of nanocapsules suspension (NCBSO) was diluted in 9 mL of ultrapure water in the proportion of 1:10 (v/v). This solution was kept under magnetic stirring for 30 min at room temperature (Fisatom, model 752A, São Paulo, Brazil). Then, the 10 mL of the solution was mixed in equal parts with 1% (w/v) solution of phosphotungstic acid. The mixture was kept under magnetic stirring for 30 min. A 5 μL aliquot of the final suspension was deposited onto a 200-mesh copper TEM grid coated with carbon film, followed by air-drying at room temperature (25 ± 2 °C) for 24 h. The grid was examined under a microscope (Tescan, Mira 3, Brno, Czech Republic) operated at an accelerating voltage of 30 to 300 kV.

2.6. Study of Physicochemical Stability of the NCBSO and NC-C

The nanocapsules loaded with blackberry seed oil (NCBSO) and control nanocapsules (NC-C) were subjected to stability studies under three storage conditions, (1) ambient temperature (25 ± 2 °C), (2) accelerated conditions (37 ± 1 °C), and (3) refrigerated conditions (4 ± 1 °C), with protection from light exposure for 90 days. Physicochemical parameters, including hydrodynamic diameter, polydispersity index (PDI), and zeta potential, were analyzed at predetermined intervals (0, 30, 60, and 90 days) using dynamic light scattering (DLS). All measurements were performed in triplicate (n = 3). Physicochemical characterization was analyzed by Student’s t-test. The analyses were carried out with the software GraphPadPrism^® 5.04 version (San Diego, CA, USA), and the significance level was p < 0.05.

2.7. Cell Culture

The human fibroblast cell line CCD1072Sk was obtained from the Brazilian Cell Repository (Banco de Células do Rio de Janeiro, BCRJ) [23] and cultivated in an RPMI 1640 culture medium (Gibco Invitrogen, Waltham, MA, USA) supplemented with 20% of fetal bovine serum (Gibco Life Technologies, Paisley, UK) and 1% of a penicillin 100 U.mL⁻¹/streptomycin 100 µg.mL⁻¹ antibiotic solution (Sigma-Aldrich, St. Louis, MO, USA). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂. Cell growth was monitored daily in an inverted phase microscope, and the culture medium was changed every 2 days. At pre-confluence, cells were harvested using 0.25% trypsin-EDTA (Gibco Life Technologies, Paisley, UK).

2.7.1. MTT Cytotoxicity Assay

The MTT cytotoxicity assay was performed according to Mosmann (1983) [24]. Cells were suspended in a culture medium with 0.25% trypsin-EDTA, counted in a Neubauer chamber, and plated in a 96-well plate (1.10³ cells/well). After starvation, cells were treated with culture media supplemented with BSO and NCBSO at 10, 25, 50, 75, and 100 µg.mL⁻¹. After 24 h, the medium was replaced by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT) (0.05 mg.mL⁻¹), and the cells were incubated for 3 h. After this period, the MTT solution was removed, and 100 µL of DMSO was added for formazan crystals solubilization. Cytotoxicity was assessed by the microplate reader (Thermo Fisher Scientific, Multiskan FC, Waltham, MA, USA) at 590 nm. The optical density (OD) values were converted to a % of cell viability using Equation (1):

Cell viability % = (OD_treatment/OD_control).100

(1)

2.7.2. Collagen Production Assay

Collagen production in human dermal fibroblasts treated with BSO and NCBSO was quantified using Picrosirius red staining [25]. Cells were seeded in 12-well plates and treated, in triplicate, with BSO and NCBSO (10, 50, and 100 µg.mL⁻¹). The plates were incubated for 72 h in a humidified atmosphere with 5% CO₂ at 37 °C. After this period, the culture medium was removed, and the wells were washed three times with PBS. Bouin’s solution was added and kept for 1 h at room temperature. Then, the plates were rinsed again with PBS, and 1.5 mL of Sirius red solution was added (Sigma-Aldrich, St. Louis, MO, USA). The plates were then incubated for 1 h at 37 °C/5% CO₂. After this period, 1 mL of 0.01 mol.L⁻¹ hydrochloric acid solution was added and kept for 30 s. This step was repeated 3 times. Then, 1 mL of 0.1 mol.L⁻¹ NaOH was added, and the plates were homogenized for 30 min. The absorbance was measured at 550 nm using a microplate reader (Thermo Scientific Multiskan^®FC, Thermo Fisher Scientific, Waltham, MA, USA). The optical density (OD) values were also converted to % using Equation (1).

2.8. Statistical Analysis

The results were statistically analyzed using the GraphPadPrism^® 5.04 version (San Diego, CA, USA). Tukey’s test was used to confirm differences between means, considering a p-value lower than 0.05 (p < 0.05) as representative of statistical significance.

3. Results

3.1. Chemical Characterization of the Blackberry Seeds Oils

Chemical characterization of blackberry seed oil (BSO) constituents was performed using ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF-MS). Analysis was carried out in negative ionization mode, enabling the simultaneous acquisition of precursor ion data from a single injection. Exploratory compound identification was achieved by comparing observed m/z values with the literature-reported mass spectra (

Table 1. Exploratory identification of bioactive compounds in BSO (scCO₂-extracted) by UHPLC-Q-TOF-MS analysis: Rₜ, precursor ions [M-H]⁻ (m/z), and compound classification.

Peak no.°	Rt a (min)	Precursor Ion [M-H−] m/z	Possible Compound	Compound Classification
1	0.2	207.00	3,4-Dimethoxycinnamic acid	Phenolic acid
2	0.6	939.10	Pentagalloyl glucose	Gallotannin
3	0.6	935.34	Casuarictin/potentillin	Ellagitannin
4	0.9	209.00	5-Hydroxyferulic acid	Hydroxycinnamic acid
5	2.1	290.05	(-)-Catechin	Flavonoid
6	2.4	115.00	Fumaric acid	Phenolic acid
7	2.6	469.01	Valoneic acid dilactone	Hydrolyzable tannin
8	2.9	279.45	Linoleic acid	Unsaturated fatty acid
9	3.2	567.89	Lutein/zeaxanthin	Carotenoid

^a Retention time. BSO: blackberry seed oil, scCO₂: supercritical carbon dioxide, UHPLC-Q-TOF-MS: ultra-high performance liquid chromatography–quadrupole time-of-flight mass spectrometry, Rt: retention time.

), with subsequent classification.

3.2. In Silico Prediction of Biological Activities

The computational prediction of the biological activities of the nine compounds identified in the blackberry seed oil (BSO) extract reveals a convergent profile suggestive of multi-target potential related to dermocosmetic applications. According to PASS Online outputs, all compounds demonstrated Pa values above 0.70 for antioxidant activity, indicating consistent structural features associated with radical scavenging capacity. Notably, lutein (Pa = 0.91), linoleic acid (Pa = 0.81), and 3,4-dimethoxycinnamic acid (Pa = 0.72) exhibited the highest scores, supporting an association with skin protection against oxidative stress (

Table 2. Integrated PASS online results for BSO-identified compounds.

Compound	Pa (Antioxidant)	Pa (Collagen Production)	Pa (Elastin Synthesis)	Pa (Anti-Aging Effect)
Pentagalloyl glucose	0.70	0.99	0.45	0.96
Casuarictin/potentillin	0.70	0.99	0.45	0.96
Valoneic acid dilactone	0.70	0.95	0.45	0.95
(-)-Catechin	0.70	0.77	0.47	0.92
Fumaric acid	0.73	0.66	0.45	0.91
Lutein/zeaxanthin	0.91	0.43	0.50	0.91
3,4-Dimethoxycinnamic acid	0.72	0.61	0.48	0.90
5-Hydroxyferulic acid	0.70	0.67	0.46	0.90
Linoleic acid	0.81	0.53	0.46	0.90

).

Regarding the stimulation of collagen production, pentagalloyl glucose and casuarictin exhibited maximum Pa values (Pa = 0.99), while valoneic acid dilactone (Pa = 0.95) and (-)-catechin (Pa = 0.77) also reached high probabilities. These results are aligned with previous reports on tannins and flavonoids, which modulate fibroblast activity and extracellular matrix remodeling through the regulation of TGF-β, matrix metalloproteinases, and elastase inhibition. Predicted elastin synthesis stimulation was observed across all compounds with moderate to good probability (Pa ranging from 0.45 to 0.50), with lutein showing the highest individual value (Pa = 0.50). Overall anti-aging potential was supported by elevated cumulative scores, especially for pentagalloyl glucose (Pa = 0.96), casuarictin (Pa = 0.96), valoneic acid dilactone (Pa = 0.95), and (-)-catechin (Pa = 0.92).

Complementary analysis using Molinspiration reveals that (-)-catechin and 5-hydroxyferulic acid achieved high scores as predicted enzyme inhibitors and nuclear receptor ligands. These compounds presented favorable topological polar surface areas (TPSAs) and hydrogen bond donor–acceptor patterns, which support bioactivity at intracellular targets related to gene expression and antioxidant defense. Specifically, (-)-catechin scored 3.68 for nuclear receptor ligand and 2.68 for protease inhibitor bioactivity, consistent with its documented interaction with collagen-degrading enzymes and its potential role in maintaining matrix integrity [16,17,18,20].

SEA Search predictions further validated the involvement of BSO constituents in skin-related pathways. Structural similarity analysis reveals matching profiles between lutein and known retinoid receptor ligands, as well as between pentagalloyl glucose and inhibitors of matrix metalloproteinases. These predicted associations indicate the possible modulation of collagen turnover and inflammation-related pathways. The convergence of results from PASS Online, Molinspiration, and SEA Search underscores a coherent bioactivity profile, supporting the hypothesis that the synergistic interaction of these metabolites contributes to the observed stimulation of collagen production and antioxidant effects [16,17,18,20].

In silico data support the mechanistic rationale for the use of BSO in cosmeceutical formulations aimed at skin-aging prevention and matrix regeneration. This predictive approach aligns with current trends in rational cosmetic design and offers a valuable tool for prioritizing compounds with translational relevance in topical skin care [16,17,18,20].

3.3. Nanocapsules Characterization

3.3.1. Determination of Mean Diameter, Polydispersity Index (PDI), and Zeta Potential of NCs

Both formulations presented an average diameter of 266.75 ± 2.01 nm (NCBSO) and 243.17 ± 3.88 nm (NC-C). Statistical analysis shows no significant difference between these nanoformulations, indicating that the oil did not have a detrimental effect on size [26].

Table 3. Average sizes, PDI, zeta potential, and pH of the NCBSO and NC-C (unloaded control nanocapsules) are presented as mean and standard deviation.

Formulation	Diameter (nm)	Polydispersity Index (PDI)	Zeta Potential ± SD (mV)	pH
NCBSO	266.75 ± 2.01	0.09 ± 0.01	– 30.40 ± 0.30	5.51 ± 0.38
NC-C	243.17 ± 3.88	0.09 ± 0.01	– 27.93 ± 1.08	5.37 ± 0.36

summarizes the results obtained.

3.3.2. Analysis by Scanning Electron Microscopy Coupled with Field Emission (FE-SEM)

Figure 1 depicts the photomicrographs obtained for NCBSO. It was possible to observe particles with a well-defined spherical shape and a smooth and uniform surface. The diameter of the particles corresponded to that determined by DLS.

3.4. Study of Physicochemical Stability of NCBSO and NC-C

Polymeric nanocapsules are promising carriers for topical application, offering high stability to active ingredients and improving their concentration and distribution [2,14,15]. These properties are especially advantageous for plant-based compounds, which are known for their low stability [1,13,27,28,29]. However, the long-term stability of these nanoparticles remains a challenge, as poly(ε-caprolactone) (PCL) nanocapsules can undergo degradation in aqueous media [30,31]. Thus, the stability study was performed at different temperatures to evaluate the physicochemical behavior at different temperatures. The results are depicted in Figure 2 and Figure 3. A macroscopic evaluation reveals that both NC-C and NCBSO retained their characteristic milky-white opalescence throughout the 90-day stability study.

Zeta potential analysis reveals no significant influence of BSO nanoencapsulation on surface charge characteristics, as demonstrated by comparing the NC-C and NCBSO formulations (p > 0.05) in Figure 3. However, storage temperatures provided a perceptible effect, in which samples maintained at 37 °C revealed significantly lower zeta potential (p < 0.05) compared to those stored at 4 °C and room temperature.

3.5. MTT Cytotoxicity Assay

Figure 4 shows the MTT assay results. At the tested concentrations (10, 25, 50, 75, and 100 μg.mL⁻¹), BSO and NCBSO presented no cytotoxic effect to the human fibroblast cell line CCD1072Sk. According to the ISO 10993-5:2009 standard [32], a product is considered cytotoxic when cell viability is reduced to less than 70%, which was not observed in this study. In particular, NCBSO not only prevented cytotoxicity but also induced a significant increase in cell viability at concentrations of 10 and 25 μg.mL⁻¹ (p < 0.05) by providing a cytoprotective effect [33,34,35]. The observed increase in cell viability with NCBSO can be attributed to the controlled release and antioxidant potential of bioactive compounds from BSO, resulting in lower cytotoxicity. Thus, BSO nanoencapsulation offers an actual, sustainable, and innovative approach to enhancing the safety of fatty oils in cosmetic and pharmaceutical contexts.

3.6. Collagen Production Assay

The impact of BSO and NCBSO on collagen production in CCD1072Sk cells was positive, as shown in Figure 5. NCBSO exhibited a more pronounced effect at 10, 50, and 100 µg.mL⁻¹, suggesting that nanoencapsulation enhances this desirable collagen-stimulating property of BSO. The results reveal a dose-dependent increase in collagen production following treatment with both BSO and NCBSO compared to the negative control (Figure 5). At the highest concentration tested (100 µg.mL⁻¹), BSO and NCBSO induced a significant elevation in collagen production by 170% and 200% compared to untreated cells (p < 0.01), respectively. In particular, NCBSO significantly enhanced collagen production compared to free BSO at 100 µg·mL⁻¹ (p < 0.05), leading to improved collagen-boosting performance.

4. Discussion

The results obtained in this study provide evidence that blackberry seed oil (BSO), both in its free form and when nanoencapsulated in poly(ε-caprolactone) nanocapsules, exerts relevant biological effects on dermal fibroblasts. The observed increase in collagen production, particularly in response to the nanoencapsulated formulation, suggests that the nanocarrier system enhances the bioavailability or cellular uptake of active compounds. Given the well-established role of type I collagen in maintaining skin structure, this finding supports the potential application of BSO-based systems in anti-aging cosmeceutical formulations.

Several compounds identified in this study have demonstrated biological activities and are reported in other Rubus species, including (a) organic acids (quinic, shikimic, citric, malic, and fumaric acid) [36,37,38]; (b) carotenoids (lutein/zeaxanthin) [39]; (c) flavonoids (quercetin-3-glucoside and (-)-catechin) [40,41]; (d) linoleic acid [5]; and (e) cinnamic and hydroxycinnamic acid derivatives (3,4-dimethoxycinnamic acid) [42,43]. 5-Hydroxyferulic acid (a ferulic acid derivative) was not previously reported in blackberry extracts. However, its biosynthetic precursors have been identified in the chemical profile of blackberries [44]. Ellagitannins (e.g., casuarictin and potentillin) and their derivatives (valoneic acid dilactone) were identified in blackberry fruit extracts using HPLC-ESI-MS [45]. In our study, these hydrolysable tannins were detected in blackberry seed oil (cultivar Xavante).

The in silico analysis was conducted as a preliminary approach to estimate the biological relevance of the compounds identified in BSO and guide the subsequent in vitro assays. The predicted activities indicated potential roles for key metabolites—such as pentagalloyl glucose, casuarictin, and (-)-catechin—in pathways associated with collagen synthesis and extracellular matrix regulation. These compounds showed high probability scores for biological activities relevant to skin, including antioxidant and anti-aging effects. Molinspiration analysis suggests interactions with nuclear receptors and enzymes commonly involved in skin remodeling, while SEA Search indicates structural similarity to ligands of proteins linked to retinoid signaling and matrix modulation. The antioxidant potential predicted for carotenoids and fatty acids, such as lutein and linoleic acid, supported their possible contribution to oxidative stress mitigation. These findings provide a rationale for selecting these compounds for further evaluation and offer molecular-level insights that were consistent with the biological effects assessed in the in vitro experiments. Overall, the in silico results support the relevance of BSO components for topical application and helped define the direction of the experimental investigation [16,17,18,20].

The in silico prediction of biological activities offered mechanistic insights that helped to explain the observed bioactivity. Several constituents of BSO—such as pentagalloyl glucose, casuarictin, (-)-catechin, and lutein—exhibited high probability scores for antioxidant and collagen-stimulating effects. These results are consistent with the existing literature on the biological roles of polyphenols and carotenoids in modulating oxidative stress and extracellular matrix turnover. The high predicted activities for anti-aging endpoints and matrix remodeling provide a molecular basis for the in vitro outcomes and reinforce the selection of BSO as a promising functional ingredient [16,17,18,20].

The obtained diameter values were consistent with those reported in previous studies for essential oil-loaded nanocapsules, such as rosemary essential oil nanocapsules and Lavandula angustifolia Mill. essential oil nanocapsules [33]. Although these studies evaluated essential oils, the observed similarities in nanoparticle size suggest a corresponding encapsulation behavior. As previously reported [10], polymeric nanoparticles typically exhibit diameters ranging from 100 to 300 nm. This size distribution is influenced by multiple factors, including formulation parameters (e.g., surfactant type and concentration), preparation method (e.g., nanoprecipitation and emulsion-diffusion), core composition (e.g., oil polarity and viscosity), polymer properties (e.g., molecular weight and hydrophobicity), and the drug-loading status (presence/absence of active pharmaceutical ingredients).

The polydispersity index (PDI), scaled from 0 to 1, quantitatively reflects the size uniformity of nanoparticles in suspension, where values approaching 0 indicate monodisperse systems with narrow size distributions [46]. All formulated nanocapsules exhibited PDI values below 0.5, demonstrating homogeneous nanoparticle populations, unimodal size distribution behavior, and optimal colloidal stability suitable for pharmaceutical applications. [47]. A uniform particle size distribution is obtained when the organic and aqueous phases are rapidly mixed to form a homogeneous dispersion [10,48].

The zeta potential reflects the electrostatic stabilization efficiency of colloidal systems, where absolute values ≥ 30 mV indicate suitable interparticle repulsion forces. This electrostatic barrier prevents aggregation by overcoming van der Waals attraction forces, even during Brownian motion-induced collisions, thereby maintaining system stability [10,49,50]. The NCBSO formulation exhibited a pH of 5.51 ± 0.38, which closely matches the physiological pH range of healthy skin (4.5–5.5). This intrinsic compatibility with cutaneous pH homeostasis, combined with demonstrated colloidal stability, suggests excellent suitability for topical applications while minimizing risks of irritation or barrier disruption. [51].

Thus, the physicochemical characteristics of the nanoparticles play a crucial role in the bioactivity of the polymeric nanocapsules, influencing drug release, stability, and interaction with biological membranes. The nanometric size of the particles results in a high surface area. This enhances interaction with biological membranes and facilitates cellular absorption. As a result, the therapeutic efficacy of the encapsulated active is improved. Additionally, nanoparticles enable controlled release, allowing targeted and suitable drug delivery into skin [9,10,49].

FE-SEM images reveal the core–shell architecture of the polymeric nanocapsules by displaying a discrete darker periphery corresponding to the PCL polymeric wall and a lighter central region associated with the oily core containing BSO [52]. This morphology is consistent with both the known film-forming properties of PCL and previous reports of nanocapsules prepared via the interfacial deposition method of preformed polymers using this polymer [20,53,54].

The formulations exhibited no detectable signs of instability, including precipitation, chromatic variation, or phase separation under the tested storage conditions (4 °C, room temperature, and 37 °C). This colloidal homogeneity and absence of macroscopic alterations demonstrate the physical stability of the nanocapsule systems. Therefore, PCL-based nanocapsules exhibited macroscopic stability, maintained by persistent Brownian motion that prevented particle flocculation and coalescence during extended storage periods [10,31]. In addition, the PCL nanocapsules maintained their nanometric size, remaining below 300 nm throughout the study period and under different storage temperature conditions. This stable colloidal behavior ensures uniform dispersion homogeneity while preserving nanocarrier properties, demonstrating potential as an advanced delivery platform for blackberry seed oil (BSO) in pharmaceutical and cosmetic applications.

However, some stored samples demonstrated an increase in the polydispersity index (PDI) from day 30. Although this feature may be considered a physicochemical problem, the values remained within the pharmaceutically acceptable range (<0.3) throughout the study period. In addition, the temperature-dependent behavior likely reflects enhanced droplet mobility and incipient aggregation dynamics. Although the NCBSO and NC-C formulations presented different oils in the oily core, the behavior in terms of polydispersity was very similar, which evidenced the reproducibility of the nanoencapsulation method used [9,10,11].

Most of the zeta potential values were adequate as these data were higher than |30 mV|, which is usually considered appropriate to provide repulsive force to achieve a physical colloidal stability [11,31]. These changes in zeta potential values may be associated with PCL hydrolysis in both NC-C and NCBSO. This result was previously achieved by Camargo et al. (2020) [31], who reported the degradation of PCL nanoparticles with a similar composition after 90 days of storage at room temperature. Stability assessments confirm that the nanocapsule suspensions maintained their critical physicochemical parameters within suitable limits throughout the 90-day study period, demonstrating acceptable variations across all storage conditions (4 °C, RT, and 37 °C). This adequate stability in the face of temperature variation suggests a robust formulation design capable of preserving the macroscopic colloidal stability, the particle size distribution, and the surface charge characteristics. This colloidal stability was also attributed to the effect of the surfactant system composed of Span 80 and Tween 80, which appropriately stabilizes the oily core of the nanocapsules [9,31,53].

Manssor et al. (2022) [55] synthesized titanium nanoparticles via three distinct methods (Cassia fistula extract, Bacillus subtilis mediation, and hydrothermal synthesis) and evaluated their cytotoxicity in L929 mouse fibroblasts. The Cassia fistula-derived nanoparticles exhibited significantly higher cytotoxicity compared to other synthesis routes, which the authors attributed to residual organic solvents from the extraction process. In contrast, our study utilized supercritical carbon dioxide extraction, a solvent-free approach that yielded both the blackberry seed oil and derived nanocapsules with no detectable cytotoxicity.

Grajzer et al. (2021) [56] demonstrated that raspberry seed oil extracted via supercritical CO₂ and its derived nanoemulsion exhibited selective cytotoxicity, significantly reducing viability in colon and breast cancer cell lines while remaining non-toxic to human dermal fibroblasts. This differential activity was attributed to bioactive compounds in the oil that specifically target cancer cell pathways without affecting normal cells.

Multiple studies with nanoencapsulated natural products have demonstrated that the nanoencapsulation process not only preserves product safety but may even reduce cytotoxicity in certain cell lineages [31,57,58,59,60]. These findings confirm that nanoencapsulation serves as an effective strategy to protect bioactive compounds from oxidative degradation, enhance physicochemical stability, optimize delivery and tissue penetration, and maintain or improve biological safety profiles.

A previous study reported the role of blackberry in skin repair and extracellular matrix modulation. Aksoy et al. (2020) [61] described that Rubus tereticaulis P.J. Müll. enhances wound healing by promoting collagen synthesis. Similarly, Meza et al. (2020) [2] demonstrated that Rubus fruticosus L. extract upregulates elastin gene transcription, stimulates tropoelastin production, and inhibits leukocyte and fibroblast-derived elastase activity. Although further investigation is needed, these findings suggest that blackberry-derived compounds may simultaneously enhance collagen and elastin synthesis, which are key mechanisms for improving skin tonicity and elasticity. Such dual activity highlights their potential as promising bioactive ingredients for cosmetic applications.

Other studies related to nanostructures loaded with plant-derived bioactive compounds have similarly demonstrated enhanced collagen synthesis, corroborating the findings of this study. For instance, Hajialyani et al. (2018) [62] reported that nanostructured curcumin significantly improved wound healing in a murine model by promoting collagen deposition, increasing myofibroblast proliferation, stimulating capillary formation, and enhancing wound contraction. Likewise, Pires et al. (2020) [63] found that nanoencapsulated Caryocar brasiliense Cambess oil accelerated dermal regeneration and markedly upregulated type I collagen production. This enhanced collagen synthesis may be attributed to the ability of nanostructures to improve the bioavailability of active compounds while facilitating the cellular uptake of growth factors and cytokines critical for tissue repair.

The improved performance of the nanoformulation compared to free BSO may be attributed to the physicochemical characteristics of the nanocapsules. The particle size distribution, encapsulation efficiency, and negative surface charge likely contributed to enhanced colloidal stability and interaction with cell membranes. Moreover, nanocarriers are known to promote the controlled release and protection of sensitive bioactives against degradation, which may explain the higher levels of collagen production induced by the nanoencapsulated BSO. These observations are in line with other studies using polymeric nanocapsules to deliver plant-derived actives in cosmetic applications.

From a formulation standpoint, the use of biodegradable polymers, such as PCL, represents a sustainable and biocompatible strategy for topical delivery. The encapsulation process preserved the antioxidant capacity of BSO, and the physicochemical properties of the nanocapsules were suitable for incorporation into aqueous or emulsified systems. These attributes are particularly relevant for the cosmetic industry, which increasingly values clean-label ingredients, biobased carriers, and multifunctional delivery systems that align with environmental and consumer safety criteria.

Overall, the integration of chemical profiling, computational modeling, and biological assays offers a comprehensive approach for characterizing the potential of plant-derived actives. This study demonstrates that BSO is a source of structurally diverse compounds with complementary bioactivities relevant to skin health. The data support the hypothesis that a combination of antioxidant protection and stimulation of collagen synthesis underlies the observed effects. The findings also highlight the predictive value of in silico tools for anticipating biological performance and refining experimental design in cosmetic research.

Despite the promising outcomes observed in both predictive and experimental analyses, some limitations should be recognized. The in silico predictions, although useful for guiding compound prioritization and mechanistic hypotheses, are based on probabilistic models and structural similarity, which do not account for pharmacokinetic behavior, bioavailability, or interactions within the complex biological environment of the skin. Moreover, the individual compounds were evaluated in silico based on their isolated molecular structures, while in vitro assays were performed using the complete BSO extract or its nanoencapsulated form. As a result, potential synergistic or antagonistic interactions among compounds were not computationally explored and may have influenced the observed bioactivities. The lack of direct testing of purified compounds in vitro also limits the attribution of effects to specific molecular entities.

Furthermore, the in vitro assays were restricted to fibroblast-based models and focused primarily on collagen production as a biological endpoint. Although this is a relevant marker for assessing dermal regeneration, additional pathways such as elastin synthesis, inflammation modulation, and oxidative damage repair were not directly assessed at the cellular or molecular levels. The findings thus remain preliminary regarding the full range of biological effects attributed to BSO. Future studies should include advanced skin models, such as 3D reconstructed human epidermis or ex vivo skin explants, to better approximate the physiological complexity of topical application. Additionally, evaluation of stability, permeation, and long-term effects under simulated cosmetic use conditions would be essential to confirm the translational applicability of BSO-based formulations.

5. Conclusions

This study successfully developed and characterized PCL nanocapsules encapsulating supercritical CO₂-extracted blackberry (Rubus spp. Xavante cultivar) seed oil (BSO), demonstrating their potential as a sustainable and bioactive ingredient for skincare applications. UHPLC-ESI-Q-TOF-MS analysis reveals that BSO contains bioactive compounds (e.g., phenolic acids, carotenoids, tannins, flavonoids, and unsaturated fatty acids). The optimized nanocapsules (NCBSO) exhibited ideal physicochemical properties—nanometric size, low polydispersity, and negative zeta potential—while maintaining colloidal stability over 90 days under different storage conditions. NCBSO showed no cytotoxicity and significantly enhanced collagen production in human fibroblasts, confirming that nanoencapsulation increased this bioactivity. PCL nanocapsules demonstrated promising potential for the topical delivery of blackberry seed oil, overcoming the limitations of the free compounds and supporting their application in sustainable cosmeceutical formulations.