Lignin–Quercetin Hybrid Colloidal Particles as Sustainable Pickering Emulsifiers: A Bio-Based and Functional Approach

Abstract

Lignin, the second-most-abundant polymer on Earth, has attracted attention for its value-added applications. Colloidal lignin particles can overcome handling and compatibility issues, offer antioxidant, antimicrobial, and UV-protective properties, and serve as Pickering stabilizers. Plant extracts rich in bioactive compounds, such as polyphenols and flavonoids (e.g., quercetin), can further enhance lignin-based formulations. In this context, colloidal lignin–quercetin particles (CLQPs) were produced for the first time via antisolvent precipitation and used as Pickering emulsion stabilizers. CLQP dispersions (30 g/L) were prepared by solubilizing lignin and quercetin in 80% (v/v) aqueous acetone solution, followed by precipitation with a pH 8 buffer. A quercetin content of 50% (w/w) (CLQP-50) resulted in predominantly round-shaped lignin–quercetin particles (<1 µm) with a small fraction of quercetin crystals. Both structures contributed to emulsion stabilization, as evidenced by confocal microscopy, a three-phase contact angle of 91.6 ± 0.1°, and a zeta potential of −52.8 ± 2.7 mV. CLQP-50 successfully stabilized Pickering emulsions at a 60/40 oil/water ratio, showing high physical stability (stability index 0.01) and shear-thinning behavior with gel-like consistency. These findings demonstrate the pioneering development of lignin–quercetin hybrid colloidal particles as sustainable and functional Pickering stabilizers, opening new opportunities for advanced cosmetic and pharmaceutical formulations.

1. Introduction

Pickering emulsions address the use of solid particles to improve the stability between the two phases of an emulsion [1,2]. This type of system proved to be a more sustainable and safer alternative to conventional emulsions, as it is free from synthetic molecular surfactants associated with environmental impacts and health issues, such as poor biodegradability and irritations or allergic responses [3,4,5]. One of the most recent pathways explored involves using particles of natural origin (e.g., lignin, cellulose, and chitosan) as alternatives to synthetic surfactants for emulsion stabilization [1,2,6].

Lignin, one of the most abundant aromatic biopolymers on Earth, is present in the tissues of all vascular plants and forms lignocellulosic biomass [7]. Isolated lignins are primarily obtained as byproducts of the pulp and paper industries, where approximately 95% of the black liquor is used as an energy source [7,8]. However, lignin’s remarkable properties, such as antioxidant, UV protection, and antimicrobial activity, can be valorized into high-value-added applications [7]. The direct application of lignin is limited by its heterogeneous chemical structure and low compatibility with aqueous systems. Therefore, colloidal lignin particles (CLPs), due to the reorganization of lignin’s functional groups, offer a straightforward strategy to overcome these limitations, as they form consistent supramolecular structures exhibiting higher dispersibility in water, regardless of the used raw material. Additionally, CLPs have been shown to enhance lignin’s intrinsic properties, such as antioxidant power, which was attributed to their high surface-area-to-volume compared to the lignin in its pristine form [7,8].

Quercetin is widely recognized for its potent antioxidant activity, primarily due to the multiple hydroxyl groups in its structure, which can donate hydrogen atoms to neutralize free radicals. It is a plant-derived flavonoid found in fruits, vegetables, herbs, and oils, e.g., apples, onions, and grapes [9,10,11]. Its poor solubility in aqueous solutions, limited stability, and low bioavailability have led to the development of various strategies to enable its use in commercial products, such as nutraceuticals and cosmetics [10,12]. In this context, the production of quercetin particles has been shown to improve both its solubility and bioavailability [9,10,13]. These include applications targeting therapeutic purposes in Alzheimer’s disease [13] and enhancing the dissolution of quercetin-based drugs [14]. In addition, Liu et al. [15] investigated the synergistic enhancement of antioxidant properties in lignin–quercetin mixtures, targeting applications in the food, cosmetics, and pharmaceutical industries.

Quercetin particles were also studied in the context of Pickering emulsion stabilization. Li et al. [16] used quercetin–starch complexes to improve both the emulsifying capacity and oxidative resistance of food-grade Pickering emulsions. Yin et al. [17] conducted experiments with composite particles composed of zein, quercetin, and quaternary ammonium chitosan, which served as promising Pickering stabilizers and exhibited potent antibacterial activity. Li et al. [18] synthesized a carboxymethyl curdlan–quercetin conjugate to stabilize Pickering emulsions loaded with curcumin. They obtained a multifunctional emulsion (antioxidant, pH-responsive, and prebiotic) showing capacity for colon-targeted delivery of functional ingredients. In this context, the production of colloidal lignin–quercetin particles (CLQPs) can add value through their synergistic antioxidant and emulsifying effects, thereby enhancing the value of the resulting emulsions and offering evident benefits for innovative and advanced formulations in cosmetic and healthcare applications. To the best of our knowledge, the use of lignin–quercetin particles has never been explored as a Pickering stabilizer.

In this work, CLQPs were produced by antisolvent precipitation, in which lignin and quercetin were dissolved in an acetone–water solution and precipitated with an alkaline buffer at pH 8, as previously described [19]. The objective was to combine lignin and quercetin to enhance antioxidant and emulsifying properties. Various lignin-to-quercetin ratios were studied, including particles composed exclusively of lignin (CLP) and quercetin (CQP). The resulting particles were systematically evaluated in terms of size, zeta potential, morphology, and interfacial properties. Pickering emulsions were produced from the most promising CLPQs and compared for droplet size, stability, morphology, and color. Confocal laser scanning microscopy (CLSM) was used to gain insights into the mechanisms of CLQPs’ stabilization. Additionally, the rheological behavior and antioxidant activity of the emulsions were assessed for potential cosmetic applications.

2. Results and Discussion

2.1. Effect of the Quercetin Content on Lignin–Quercetin Particle Properties

The impact of quercetin content on colloidal particles’ properties was evaluated in terms of visual aspect, morphology, size, and particle charge, which is also an indicator of dispersion stability. Digital images (Figure 1) show homogeneous particle dispersion, with the color becoming more yellowish and less brownish as the quercetin concentration increased. Except for CQP, which showed large longitudinal structures, optical microscopy (OM) images (Figure 1) revealed the prevalence of spherical structures across all samples. For CLP and CLQP-25 samples, smaller sizes and less agglomeration were observed. For the CLQP-50 and CLPQ-75, larger and more agglomerated structures were observed.

Figure 1. Optical microscopy of the colloidal particles with different quercetin contents: (a) CLP (0%), (b) CLP-25 (25%), (c) CLP-50 (50%), (d) CLP-75 (75%), and (e) CQP (100%). Each micrograph is accompanied by the photographic image of the respective particle dispersion.

In terms of particle size (Table 1), a gradual decrease in the number-averaged size was observed as the quercetin content increased, i.e., from CLP (0% quercetin) to CLQP-75 (75% quercetin). The same trend is observed for the volume-averaged size, with relatively high values attributed to agglomeration, as shown in the analysis of Figure 1 images. These observations were further supported by statistical analysis, which demonstrated that the progressive increase in quercetin content resulted in significant differences among the samples. Previous studies have shown that a decrease in the initial lignin concentration results in smaller colloidal particles [20,21], an effect attributed to the increased quercetin content in these formulations.

Table 1. Median number-averaged and volume-averaged sizes, and zeta potential.

	Number-Averaged Size (nm)	Volume-Averaged Size (µm)	Zeta Potential (mV)
CLP	502 a ± 1	8.4 a ± 0.1	−49.2 a ± 2.4
CLQP-25	492 ab ± 1	7.0 ab ± 0.1	−49.2 a ± 1.5
CLQP-50	463 bc ± 1	6.9 b ± 0.1	−52.8 b ± 2.7
CLQP-75	444 bc ± 1	5.2 c ± 0.1	−56.0 c ± 1.5
CQP	543 c ± 1	3.7 d ± 0.1	−57.1 d ± 3.4

Values are presented as mean ± standard deviation. Particle sizes were obtained from n = 5 measurements, and zeta potential from n = 3. Different letters indicate statistically significant differences (p < 0.05, one-way ANOVA followed by Tukey’s post hoc test).

For the particles formed with only quercetin (CQP), the largest particle size was obtained. Note that these particles have a rod-like structure, indicating lignin’s influence on the round shape. Moreover, care must be taken when comparing the sizes of rod-like structures measured by laser diffraction with those of spherical ones, especially in suspension, which can lead to an averaged or misleading size for the former. Given these constraints, the particles have been characterized by transmission electron microscopy (TEM).

TEM analysis (Figure 2) confirmed the structural type observed by OM, revealing that CLPs are spherical particles with heterogeneous sizes. Indeed, lignin particles are usually reported to have a spherical shape, a behavior attributed mainly to their structural reorganization upon contact with an aqueous medium [20]. In contrast, CQPs are rod-like structures, reflecting their crystallinity, as shown in the X-ray diffraction (XRD) results (Figure S1), with quercetin characteristic peaks at 2θ = 10.44°, 12.38°, 26.46°, and 27.20°, indicating a highly crystalline structure [22]. The morphological analysis revealed a clear evolution in particle shape with increasing quercetin content. As shown in Figure 2, a coexistence of the two structures was observed in the hybrid CLQP samples: lignin-rich spherical particles and quercetin-derived rod-like crystals. The proportion of these rod-like structures increases progressively with the quercetin content, being clearly visible in CLQP-50 and dominant in CQP (Figure 2c–e), which validates the influence of quercetin’s morphology on the final particle shape. Quercetin forms complexes with other chemical species due to its polyhydroxylated chemical structure, which justifies its incorporation into lignin structures through physical entrapment and non-covalent interactions (hydrogen bonds, hydrophobic interactions, and π-π stacking). Quercetin is classified as a polyphenolic crystal, with particles exhibiting a shape resembling that obtained upon crystallization in contact with acetone, as shown in OM in the Supplementary Materials (Figure S2) [23]. Interestingly, the spherical lignin-rich particles were observed to adhere to the quercetin crystals, indicating a potential affinity between their surface functional groups. TEM analysis also revealed that the round structures, which were prevalent in the CLQP-25 and CLQP-50, are roughly smaller than 1 μm.

Figure 2. TEM images of the colloidal particles with different quercetin content: (a) CLP (0%), (b) CLP-25 (25%), (c) CLP-50 (50%), (d) CLP-75 (75%), and (e) CQP (100%).

The zeta potential was measured as an indicator of particle dispersion stability, as values above +30 mV or below −30 mV indicate that particles have sufficient electrostatic repulsion to prevent aggregate formation [24]. Lignin and quercetin have negatively charged surfaces due to the presence of carboxylic and hydroxyl groups [23,25]. Table 1 presents the zeta potential of the particles, which ranged from −49.2 to −57.1 mV, with values showing statistically significant differences as quercetin content increased. The increase in zeta potential values with quercetin content increase may be correlated to the number, position, and arrangement of the carboxylic and hydroxyl groups of the components [11,26].

2.2. Effect of the Quercetin Content on Lignin–Quercetin Particle Interfacial Properties

Particle wettability indicates the type of Pickering emulsion they can form, as it reflects the degree of affinity between the particles and the continuous and dispersed phases [24]. Three-phase contact angle (CA) values lower than 90° indicate more hydrophilic particles and tend to form oil-in-water (O/W) emulsions, whereas particles with CA values higher than 90° indicate a more hydrophobic character and tend to stabilize water-in-oil (W/O) emulsions. Particles with CA values close to 90° exhibit the highest adsorption energy at the oil–water interface, sterically preventing droplet coalescence and providing the ideal contact angle for stabilizing Pickering emulsions [24].

Figure 3 shows that the lignin–quercetin particles (CLQP-25, CLQP-50, and CLQP-75) presented similar CA values between 91.6 and 93.1°. The proximity of the obtained CA values to 90°, indicative of an equally amphiphilic state, indicates that the particles are prone to stabilize O/W interfaces. The CLP sample showed a slightly lower value, but still close to 90. The CQP sample showed a value of 75.0°, i.e., a more hydrophilic character and a greater affinity to stabilize O/W emulsions [20,24].

Figure 3. Three-phase contact angle of the colloidal particles with different quercetin content: (a) CLP (0%), (b) CLP-25 (25%), (c) CLP-50 (50%), (d) CLP-75 (75%), and (e) CQP (100%).

CA analysis also revealed that the transformation of pristine lignin and quercetin, with CAs of 109.9° and 107.2°, respectively (Figure S3, Supplementary Materials), into a particulate form imparted a more hydrophilic character, associated with the reorganization of their functional groups during particle formation. This change led to an amphiphilic behavior, thus improving their Pickering stabilizing capacity [24].

The interfacial tension (IT) reflects the efficiency of particle adsorption at the O/W interface, where lower IT values indicate stronger interfacial coverage and more effective particle migration, which are generally associated with improved emulsion stabilization [27]. Figure 4 shows that the lignin–quercetin particles exhibit a reduction in IT as the quercetin content increases up to CLQP-50, the sample with the lowest IT value. In this sample, quercetin and lignin are present in equal amounts, which may have optimized their cooperative behavior and promoted a synergistic effect. Lignin contributes to colloidal stability by forming a robust particulate framework, while quercetin enhances interfacial activity through its amphiphilic structure, thereby promoting adsorption at the oil–water interface [28,29]. The CQP sample containing only quercetin particles had the highest IT, a behavior attributed to quercetin’s poor aqueous solubility and limited colloidal stability. Excessive quercetin content in CLQP-75 likely contributed to reduced interfacial efficiency.

Figure 4. Interfacial tension between CLQPs with different lignin–quercetin ratios and Miglyol 812 versus time.

2.3. Pickering Emulsions Development

To first inspect the ability of quercetin–lignin particles to stabilize Pickering emulsions, different O/W ratios (50/50, 60/40, and 70/30, v/v) were tested using CLQP-50 particles. These particles consistently exhibited near-optimal performance across all characterizations and were therefore selected as the most representative ones. The CLQP-50 particles formed emulsions across the tested O/W ratios, demonstrating their effectiveness as a Pickering stabilizer. All the emulsions were confirmed to be O/W type, as shown in the drop test (Figure S4), in which the droplets remained intact in the oil and promptly dispersed in water.

Figure 5 presents digital photographs and microscopy images of the emulsions, highlighting the formation of spherical droplets with well-defined shapes. Emulsions with O/W ratios of 50/50 and 60/40 exhibited similar droplet sizes, whereas larger droplets were observed for the 70/30 ratio. These findings were further confirmed by laser diffraction analysis, yielding values of 31 ± 3 µm, 26 ± 1 µm, and 46 ± 2 µm for E50_50, E60_40, and E70_30, respectively.

Figure 5. Optical microscopy of the Pickering emulsions stabilized with CLQP-50 using different O/W ratios (×400 magnification): (a) 50/50; (b) 60/40; (c) 70/30. Each micrograph is accompanied by an image of the corresponding emulsion.

In general, emulsions with smaller droplet sizes tend to exhibit improved stability due to higher interfacial coverage efficiency [20,30]. Among the tested formulations, the emulsions with an O/W ratio of 60/40 resulted in the smallest average droplet size and superior visual stability. Although creaming formation occurred, there was no apparent separation of oil. Therefore, this O/W ratio was chosen for subsequent studies. For comparison purposes, the CLPs (100% lignin) and the CQPs (100% quercetin) were also used under the same emulsion preparation conditions.

Figure 6 shows visual and microscopy images of the emulsions prepared with CLQP-50, CLP, and CQP particles after 1 and 30 days of storage. In agreement with the microscopy images, the emulsions stabilized with CLP exhibited the smallest average droplet size (18 ± 2 µm), while those stabilized with CQP showed the largest (94 ± 3 µm). The particles combining lignin and quercetin (CLQP-50) resulted in an intermediate droplet size (27 ± 1 µm) close to the CLP-based emulsion. Well-defined spherical droplets were observed in emulsions prepared with lignin-containing particles (CLP and CLQP-50), resulting in only a slight increase in droplet size after 30 days of storage (18 to 24 µm and 27 to 33 µm for CLP and CLQP, respectively). For the emulsion stabilized with CQP, larger, touching but non-merging droplets were formed, indicating minimal coalescence. In fact, after 30 days, only a subtle increase in droplet size was observed (from 94 to 96 µm). These results highlight the primary role of lignin in stabilizing Pickering emulsions with lignin and lignin–quercetin particles (CLPs and CLQPs), giving the best performance. Nevertheless, quercetin itself (CQPs) was able to prevent coalescence, which can be attributed to its surface properties, such as charge and hydrophobicity, highlighting its relevant role in emulsion stabilization.

Figure 6. Optical microscopy of Pickering emulsions stabilized with (a,d): CLP, (b,e): CLQP-50, and (c,f): CQP, after 1 and 30 days of storage. Each micrograph is accompanied by a photograph of the respective emulsion.

The high stability of the emulsions was also evidenced by the emulsion instability index (EII) values (0.01 for all emulsions), which fall within the range 0–0.05, associated with good stability [31].

Colorimetric changes can be used to corroborate the establishment of molecular interactions and the structural organization of emulsified systems, as exemplified in the work of Wackerbarth et al. [32], where a color change in a cream was associated with the formation of carotenoid-protein complexes. According to the data shown in Table 2, CLP-stabilized emulsions exhibited lower L* values (brownish) and CQP-stabilized emulsions higher b* values, consistent with the intrinsic properties of lignin and quercetin, respectively (Figure S5). The mixed system showed intermediate color values between pure lignin and quercetin, with lignin more strongly affecting the a* coordinate. This deviation from simple color mixing suggests molecular interactions between the components.

Table 2. Color parameters of the Pickering emulsions stabilized with different particle systems.

Particle System	L*	a*	b*	RGB Color
CLP	48.12 ± 0.11	8.88 ± 0.05	18.47 ± 0.09
CLQP-50	53.23 ± 0.02	9.47 ± 0.02	27.10 ± 0.06
CQP	58.13 ± 0.31	13.38 ± 0.49	36.66 ± 0.19

2.3.1. Stabilization Mechanism

The stabilization mechanism of the Pickering emulsions was analyzed using CLSM, which enables direct visualization of the particles within the emulsion system, in this case, by exploring the intrinsic autofluorescence of lignin and quercetin [33]. This approach allows not only to identify particle location but also to infer their contribution to interfacial versus bulk stabilization phenomena [34]. Figure 7 shows the CLSM images of the emulsions stabilized with CLP, CLQP-50, and CQP, highlighting the autofluorescence of lignin and quercetin (red) and the hydrophobic oil stained with Nile red (green). The images showed the stained green oil droplets, confirming their O/W type, as observed by the drop test.

Figure 7. Confocal microscopy images of the superimposition of oil phase (dyed with Nile red) with different solid particles (without dye) of Pickering emulsions at 40×/1.30 oil: (a) CLP; (b) CLQP-50; (c) CQP.

Figure 7a shows that for the CLP-stabilized emulsions, the particles are predominantly distributed within the continuous aqueous phase. The formation of a particle network in the continuous phase can be advantageous for reducing droplet motion, thereby increasing viscosity and stabilizing emulsions, while minimizing aggregation and coalescence [35]. This three-dimensional structuring of particles in the bulk phase is known to restrict droplet collision frequency and mechanically hinder coalescence events, contributing to long-term physical stability [34]. For the CQP-stabilized emulsions (Figure 7c), the frequency of the particles at the droplets’ interface increased, even though some of them remained dispersed in the continuous aqueous phase. The quercetin particles are reported to be primarily driven by capillary pressure, with a large surface area and non-spherical structure, which enhances their adsorption energy at the oil–water interface [35]. Such morphology-dependent interfacial anchoring favors the formation of a dense protective layer surrounding the droplets [34,36]. The stabilization mechanism of CLQP-50 emulsions, as visualized by CLSM in Figure 7b, points out a synergistic effect. The key evidence presented in the images shows particles both surrounding the oil droplets (interfacial anchoring) and forming a network in the continuous aqueous phase (bulk stabilization). This dual behavior, in which quercetin favors interfacial adsorption while lignin promotes the formation of a protective particulate network, is explicitly illustrated by the distribution of the red autofluorescence from the hybrid particles and the green oil droplets in Figure 7b. This result highlights the combined stabilization mechanism imparted by lignin and quercetin in the particles and indicates a synergistic mechanism in which interfacial adsorption and continuous-phase network formation occur concurrently.

CLSM analysis further revealed a uniform particle distribution across the emulsified system. This homogeneous distribution, both at the interface and in the continuous phase, indicates the formation of a structured particulate network, thereby enhancing emulsion stability. Although CLSM is inherently qualitative, it enables direct visualization of particle localization and interfacial structuring, providing mechanistic insights into Pickering stabilization. Similar qualitative approaches have been extensively employed in the literature to describe adsorption behavior, particle networking, and bridging phenomena, including studies by D. McClements [37] and other established authors in the field [3,38].

2.3.2. Rheological Behavior

The rheology of the emulsions was analyzed to determine how the final product will behave during transportation, packaging, and application to the skin. The rheological behavior of the emulsions was analyzed using viscosity and frequency sweeps, measuring the energy storage due to deformation with a rheometer. When emulsions are compressed, the droplets deform, leading to an increase in interfacial and elastic energy stored within the system due to the resistance of the interface and geometric constraints at high packing fractions [39]. This deformation-dependent energy storage can be associated with droplet–droplet interactions and the structuring imposed by stabilizing particles within the emulsion matrix [40]. Figure 8 shows the decrease in viscosity with increasing shear rate, characteristic of shear-thinning non-Newtonian fluid behavior, typically observed in cosmetic emulsions that deform when applied to the skin [39,41]. This flow profile reflects the progressive disruption and alignment of droplet aggregates and particle networks under shear, thereby facilitating flow at higher deformation rates [40].

Figure 8. Apparent viscosity of the Pickering emulsions stabilized with different lignin–quercetin particle systems.

The most significant difference between the samples was observed at low shear rates (0.1–1.0 s⁻¹). In this range, viscosity decreased for the CLQP-50-based samples compared to those prepared with lignin (CLP) and quercetin (CQP). This trend may be tentatively associated with an effect analogous to the Farris effect, originally described for polydisperse suspensions of rigid particles, in which a broader particle size distribution can lead to lower apparent viscosity by enabling more efficient packing and reduced hydrodynamic interactions [42]. In the present case of emulsions, the reduction in viscosity can be attributed to a broader droplet size distribution, which enables more efficient packing and diminished hydrodynamic interactions. Additionally, improved droplet accommodation may reduce frictional resistance during flow, contributing to the observed lower apparent viscosity [40]. This mechanism cannot be directly visualized but can only be inferred from the rheological response. In the mixed system, the coexistence of lignin and quercetin particles may have affected droplet organization, potentially facilitating their accommodation and improving flow at low shear. Such structural rearrangements under weak deformation are typical of a heterogeneous Pickering system (containing particles with different interfacial properties) [40]. Similar trends have been reported in colloidal dispersions and emulsions, where heterogeneity in particle size leads to lower apparent viscosity than in systems containing monodisperse components [43].

In the frequency sweep measurements of Figure 9, a gel-like behavior was observed for all the samples since the storage modulus (G′) remained higher than the loss modulus (G″) over the evaluated frequency range [39]. This dominance of the elastic modulus indicates that weak solid-like structures govern the mechanical response of the emulsions within the linear viscoelastic region (LVE). This viscoelastic profile reflects the formation of weak elastic networks that contribute to emulsion stability but do not prevent flow under gravity or macroscopic deformation, as may occur during container inversion. In the present case, all formulations exhibited similar viscoelastic trends, indicating that the type of stabilizing particle did not significantly alter the balance between elastic and viscous contributions within the explored frequency window [41]. Furthermore, the higher absolute moduli of G′ and G″ indicate that the emulsion containing lignin is more rigid and resistant to deformation than the others. This increased rigidity can be associated with the greater propensity of lignin particles to form a continuous-phase network, reinforcing the emulsion microstructure [40]. These findings are consistent with similar Pickering emulsion systems reported in the literature, where the type of stabilizing particle modulates the relative balance between elastic and viscous contributions across the frequency spectrum [3,44].

Figure 9. Frequency sweep measurements for the Pickering emulsions stabilized with different lignin–quercetin particle systems.

2.3.3. Antioxidant Activity

The antioxidant activity of the emulsions was analyzed using the IC50, with lower values indicating greater antioxidant activity [35]. Lignin and quercetin are compounds with phenolic hydroxyl groups [7,10], and their amounts are directly linked to their antioxidant capacity, as reported by Dizhbite et al. [45]. The emulsion stabilized with CQPs exhibited considerably higher antioxidant activity, with an IC50 value of 0.13 mg/mL, compared to both lignin-based particles (0.60 mg/mL for CLP and 0.22 mg/mL for CLQP-50), even though they showed a strong antioxidant capacity. In the literature, nanoemulsions prepared with quercetin have been reported to exhibit antioxidant activity in the range of 0.056–0.106 mg/mL [46,47]. In the samples prepared in this work, the strong antioxidant power of quercetin prevailed over that of lignin, resulting in a low IC50 value (0.22 mg/mL).

3. Materials and Methods

LIGNOVA™ Pure, a high-purity hydrolysis lignin from Betula pendula (Mw = 10,000 Da), was kindly provided by Fibenol (Tallinn, Estonia). Quercetin anhydrous was purchased from Apollo Scientific (Manchester, UK). Acetone (purity ≥ 99.8%) was purchased from Carlo Erba Reagents (Val-de-Reuil, France). A citrate–phosphate buffer (pH 8) at 0.1 mol/L was prepared using disodium hydrogen phosphate anhydrous (Panreac, Barcelona, Spain) and citric acid (Merck, Darmstadt, Germany). Miglyol 812 was acquired from Acofarma (Barcelona, Spain), and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and dimethyl sulfoxide (DMSO) from Thermo Fisher Scientific (Waltham, MA, USA). Distilled water was used for all procedures.

3.1. Particle Production

CLQPs were prepared by antisolvent precipitation, as described in a previously published group procedure with minor modifications [19]. Briefly, dried lignin and quercetin were solubilized in an 80% (v/v) acetone aqueous solution at a final total concentration of 30 g/L. The tested quercetin contents were 25%, 50%, and 75% (w/w), corresponding to the CLQP-25, CLQP-50, and CLQP-75 formulations, respectively. Additionally, 100% lignin formulations, designed by CLP, and 100% quercetin formulations, designed by CQP, were produced for comparison purposes. The antisolvent, a citrate phosphate buffer at pH 8, was dripped at a flow rate of 14 mL/min using a syringe pump (KDS 200, KD Scientific, Holliston, MA, USA) into the lignin–quercetin solution, which was continuously stirred at 500 rpm. Finally, the acetone was removed through rotary evaporation (R-114, Waterbath B-480, and Vacuum Controller B-721, Büchi, Flawil, Switzerland) at 60 °C, followed by particle dispersion concentration (water evaporation) to achieve the initial lignin–quercetin concentration (30 g/L).

3.2. Particle Characterization

3.2.1. Particle Morphology and Crystallographic Analysis

The morphology of the particles was analyzed using OM, TEM, and their crystalline structure was characterized by XRD. OM was performed using a Nikon Eclipse 50i microscope (Nikon Corporation, Tokyo, Japan), equipped with a Nikon Digital camera. For that, the particle dispersions were placed on a microscope slide and covered with a coverslip. Image acquisition (400× magnification) and processing were performed using NIS-Elements Br software (version 5.01, Nikon Corporation, Tokyo, Japan). For the TEM analysis, the particles were diluted at a 1:100 (v/v) ratio with distilled water, and 10 µL were dropped onto Formvar/carbon film-coated nickel grids (Electron Microscopy Sciences, Hatfield, PA, USA), which were then left standing for 2 min. The excess liquid was removed using filter paper. Visualization was conducted by a JEM-1400 Flash Electron Microscope (JEOL Ltd., Tokyo, Japan) at 120 kV, and the images were acquired using a CCD digital camera Orious 1100 W (Tokyo, Japan). XRD analyses were performed to confirm the crystalline behavior of the quercetin particles using an X-ray diffractometer (D2 Phaser, Bruker, Karlsruhe, Germany) with 1.54 Å/8.047 keV (Cu-Kα1) radiation. Measurements were performed at 30 kV and 10 mA. The selected angle was 2° ≤ 2θ ≥ 70°, and the scanning rate was 0.02°/s, as per the equipment restrictions [22].

3.2.2. Particle Size

The particle size was measured using a Mastersizer 3000 equipped with a Hydro MV dispersion unit (Malvern Instruments Ltd., Worcestershire, UK). Five measurements were taken after adding the sample to the dispersing medium (distilled water), until an obscuration of approximately 3.5% was achieved. The used apparatus is installed in a climate-controlled room (22 °C). The particle size was characterized by the D50, which represents the median diameter, i.e., the diameter at which 50% of the particles are smaller, based on number-based distributions [48]. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test, with significance set at p < 0.05 (STATISTICA 7).

3.2.3. Particle Surface Charge

The surface charge of the particles was determined through zeta potential using a Zetasizer Ultra instrument (Malvern Panalytical Ltd., Worcestershire, UK). For the assays, samples were diluted 1:100 (v/v) in distilled water, resulting in an approximate ionic strength of 1 mM+. Zeta potentials were automatically calculated by Zetasizer software, ZS XPLORER (version 3.3.1.5, Malvern Panalytical, Worcestershire, UK) from electrophoretic mobility data using Smoluchowski approximation, according to the instrument’s theoretical model for aqueous dispersions. All measurements were performed at 25 °C and represent the mean of three consecutive measurements (n = 3). Due to the polydisperse nature of the particles, these values are descriptive averages and do not capture the full distribution of electrophoretic mobilities. One-way ANOVA followed by Tukey’s post hoc test was used for statistical analysis, with p < 0.05 considered significant (STATISTICA, version 7, StatSoft Inc., Tulsa, OK, USA).

3.2.4. Particle Interfacial Properties

The interfacial properties, including the CA (angle formed between the particle surface, the oil, and the water phase) and the IT, were measured using a Goniometer (model 210, Ramé-Hart Instrument Co., Succasunna, NJ, USA). For the CA, particles were dried at 50 °C for 72 h to produce pellets with a diameter of 13 mm and a thickness of 2 mm using a hydraulic press (Specac Ltd., Orpington, UK) at 9 tons for 1 min. The pellets’ surface was previously examined by OM (Nikon Eclipse 50i, Nikon Corporation, Tokyo, Japan) to discard those with visible surface cracks [21]. For CA measurement, particle pellets (in triplicate) were placed in a quartz cuvette containing Miglyol 812 (30 mL), followed by the deposition of a water drop (5 μL) on the surface. The CA was recorded by a camera coupled to the goniometer after 10 s of the drop deposition. The dynamic IT between the particle dispersion and the oil was analyzed using the curvature of the pendant droplet with the Young-Laplace equation. For this, the particle dispersions were suspended as pendant drops (volume: 20 µL) in a cuvette filled with oil (Miglyol 812), and the droplet’s shape was monitored using the goniometer. The IT was calculated over 1800 s using the equipment software. Experiments were conducted at room temperature.

3.3. Pickering Emulsion Production

Pickering emulsions were prepared according to a described group procedure [24], with minor modifications. Briefly, Miglyol 812 was added dropwise to the particle dispersion for approximately 2 min and kept under stirring (13,500 rpm) with a rotor-stator homogenizer (CAT, Unidrive X 1000, Ballrechten-Dottingen, Germany). The oil-to-water ratios were calculated to yield 20 mL of emulsion. After adding the oil, the emulsion was further homogenized for 5 min. To select the best formulation, different O/W ratios (50/50, 60/40, and 70/30, v/v) were tested using the most promising particles at a concentration of 30 g/L.

3.4. Pickering Emulsion Characterization

3.4.1. Emulsion Type

The emulsion type was verified by a drop test. Briefly, three drops of emulsion were dropped into a beaker filled with distilled water, and another three into a beaker filled with oil. If the drops disperse quickly in water, they are classified as (O/W) type, whereas if they disperse quickly in the oil, they are classified as W/O type [49].

3.4.2. Emulsion Morphology

Emulsion morphology was examined using the microscope described for the particles, following a procedure similar to that described in Section 2.3.1. The emulsified phase of each system was analyzed at a 400× magnification.

3.4.3. Emulsion Droplet Size

The emulsion droplet size was measured using the equipment described for particle size analysis, following a similar procedure (Section 2.3.2), with an obscuration of 7%. The droplet size was represented by the D4,3 volume-weighted mean diameter, also known as the De Brouckere mean diameter, a commonly used parameter for estimating the average droplet diameter in emulsions from laser diffraction measurements [48].

3.4.4. Emulsion Instability Index

The microscopic stability of emulsions was measured by calculating the EII. This parameter enables analyzing the change in the droplet size over time (Equation (1)) [31,49], where d(0) refers to the initial droplet diameter and d(t) indicates the droplet diameter at time t, expressed in terms of D4,3:

$$\mathrm{E}\mathrm{I}\mathrm{I}={\displaystyle \frac{\mathrm{d}\left(\mathrm{t}\right)-\mathrm{d}\left(0\right)}{\mathrm{d}\left(0\right)\times \mathrm{t}}}$$

(1)

The assay time was 30 days. An increase in EII indicated greater emulsion instability, as evidenced by increased droplet aggregation. According to the literature, emulsions can be classified as highly stable (0 < EII < 0.05), moderately stable (0.05 < EII < 0.5), moderately unstable (0.5 < EII < 5.0), and highly unstable (EII > 5.0) [31].

3.4.5. Emulsion Color

The color of the emulsions was measured using a CR-400 colorimeter (Konica Minolta, Tokyo, Japan). The L*, a*, and b* parameters were determined, where L* indicated the luminosity ranging from dark (0) to light (100), a* refers to the green-red component, and b* the blue-yellow component [48].

3.4.6. Emulsion Microstructure

The interfacial microstructure of the formed droplets was investigated using CLSM with Leica TCS-SP5 AOBS (Leica Microsystems Inc., Heidelberg, Germany). The emulsions were stained with a Nile Red fluorescent dye solution prepared in isopropyl alcohol at 0.1% (w/v). The stained emulsions were placed on concave slides and analyzed using an argon laser (excitation 488 nm, emission 649–757 nm). Images were acquired with a 40×/1.30 oil immersion objective and digitally recorded and processed with LAS X software (version 1.4.7, Leica Application Suite X, Leica Microsystems Inc., Heidelberg, Germany) [24].

3.4.7. Emulsion Rheology

The produced emulsions were analyzed for their viscous and viscoelastic behavior. The tests were performed using a Kinexus Prime lab+ (NETZSCH Group, Selb, Germany) in a parallel-plate configuration with a 1 mm gap at 25 °C. The dynamic viscosities were determined as a function of the shear rate in the 0.1 to 1000 1/s range. The frequency sweep ranged from 0.1 to 10 Hz, and all measurements were performed within the LVE determined by amplitude-sweep tests (0.05% strain). Data were reported as storage modulus (G′) and loss modulus (G″) as a function of frequency.

3.4.8. Emulsion Antioxidant Activity

The antioxidant activity of the emulsions was evaluated using the DPPH radical scavenging assay according to an established procedure [48,50]. Briefly, the emulsions were diluted at different concentrations (0.005 to 2000 µg/mL) using DMSO. Then, 30 µL of the diluted samples were transferred to a 96-well microplate and mixed with 270 µL of DPPH methanol solution (6 × 10⁻⁵ mol/L). The mixture was incubated for 30 min in the dark at room temperature. The reduction in the DPPH radical was determined by measuring the absorption at 517 nm using a microplate reader (Epoch, Agilent, Santa Clara, CA, USA). The antioxidant activity was calculated by the percentage of DPPH scavenging using Equation (2), where Ac is the absorbance of DPPH without the emulsion sample and As is the absorbance of DPPH with the sample [48].

$$\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H} \mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\mathrm{\%}\right)={\displaystyle \frac{(\mathrm{A}\mathrm{c}-\mathrm{A}\mathrm{s})}{\mathrm{A}\mathrm{c}}}\times 100$$

(2)

The antioxidant activity results were expressed as the half-maximal inhibitory concentration (IC50), defined as the sample concentration required to achieve a 50% reduction in the initial DPPH concentration.

4. Conclusions

In this work, CLQPs were developed for the first time and explored in Pickering emulsion stabilization. The more effective lignin–quercetin ratio was 50:50 (CLQP-50), where spherical and crystal-like morphologies were observed. These particles exhibited the highest interfacial activity, as measured by contact angle measurements and interfacial tension, suggesting the most promising Pickering stabilizing capacity. For Pickering emulsion stabilization, the optimal oil/water ratio was 60/40, which yielded stable emulsions with intermediate droplet characteristics between those prepared with lignin or quercetin particles alone. Confocal laser scanning microscopy revealed a combined stabilization mechanism, with lignin-rich structures contributing to the formation of a particle network within the continuous phase, and quercetin-enriched domains favoring interfacial adsorption at the droplet interface. The hybrid particles, therefore, provided simultaneous steric barrier effects and restriction of droplet mobility, resulting in enhanced resistance to coalescence and long-term physical stability (over 30 days). This synergistic behavior highlights the functional complementarity between lignin and quercetin, demonstrating how bio-derived polyphenolic systems can be engineered to tailor interfacial performance. Overall, this study demonstrates that combining lignin and quercetin produces bio-based colloidal particles capable of stabilizing Pickering emulsions, offering promising strategies for innovative delivery systems in cosmetic, healthcare, and pharmaceutical applications.