The Effect of Alginate and κ-Carrageenan on the Stability of Pickering Emulsions Stabilized by Shellac-Based Nanoparticles

Abstract

We developed highly stable shellac-based emulsions that incorporated alginate (Al) and κ-carrageenan (Kcar), two anionic polysaccharides capable of undergoing in situ crosslinking for various applications. The stability, droplet size distribution, and microstructure of these emulsions were assessed. Fluorescence microscopy confirmed nanoparticle accumulation at the oil–water interface, which enhanced stability. By leveraging the crosslinking potential of the polysaccharides, we created Pickering emulsion hydrogels (PEH) loaded with curcumin, a model food supplement with poor water solubility, and evaluated their release profiles in an in vitro gastrointestinal model. The results demonstrated two distinct release behaviors: full release in the small intestine and targeted release in the large intestine. Further study revealed fundamental differences in how Al and Kcar influence creaming, which led to a deeper investigation into the mechanisms behind these differences. Rheology measurements showed that a more complex mechanism governs the system’s viscosity. Small angle X-ray scattering (SAXS), Fourier transform infrared spectroscopy (FTIR), and further viscosity measurements revealed that hydrogen bonding in the Kcar emulsions formed unique structures, which provided superior resistance to creaming. This study highlights the potential of tailoring emulsion hydrogels for specific applications in food and drug delivery systems and offers new insights into the structural dynamics of biopolymer-stabilized emulsions.

1. Introduction

Polysaccharides are a widely studied class of biopolymers that play a pivotal role in numerous systems due to their non-toxic, biocompatible, and biodegradable properties [1]. These abundant carbohydrate macromolecules, which are composed of repeating monosaccharide units, are used extensively in biomedical, pharmaceutical, and food applications [1,2]. Recently, the incorporation of polysaccharides into complex systems, like emulsions, has garnered significant attention. Polymers, such as chitin nanocrystals [3], chitosan [4], starch [5], cellulose [6], and microcrystalline cellulose [7], have shown potential as stabilizers in Pickering emulsions, which rely on solid particles rather than surfactants to stabilize oil–water interfaces. This particle-based stabilization offers exceptional emulsion stability and safety and substantial loading capacity [8]. Unlike traditional surfactant-based emulsions, Pickering emulsions form a robust barrier at the oil–water interface through solid particle adsorption and thus prevent droplet coalescence. These properties, coupled with their “surfactant-free” nature, have made them attractive for diverse applications, from traditional food technology to innovative fields like 3D food printing, where emulsion stability, flow properties, and solidification are critical [9,10,11]. For example, Pickering emulsions stabilized by food-grade particles have been suggested for the encapsulation of sensitive bioactive ingredients such as curcumin, vitamins and probiotic molecules [12].

Shellac, a natural resin secreted by the lac insect, is widely utilized in the food, pharmaceutical, and cosmetic industries due to its excellent film-forming properties, biocompatibility, and low toxicity [13]. It is used, for example, to glaze sweets and pharmaceutical tablets, to coat apples and as an enteric coating. Shellac is generally recognized as safe (GRAS status) by the Food and Drug Administration [14]. In the context of Pickering emulsions, shellac-based nanoparticles (Sh-NPs) have shown promise as effective stabilizers. Our previous work demonstrated the efficacy of NPs formed from shellac grafted with Jeffamine^® to create Pickering emulsions with high oil concentrations of up to 65%, which maintained high stability for up to six months [15]. These emulsions were stable against coalescence, namely the merging of oil drops, leading to an increase in their size with time and eventually phase separation. However, they exhibited creaming, which could be suboptimal for certain applications. This creaming occurs due to the upward movement of oil droplets that are driven by density differences between the phases [16].

A well-established strategy to inhibit creaming and enhance emulsion stability is the addition of water-soluble polysaccharides to Pickering emulsions. These polymers increase the viscosity of the continuous phase and may interact with stabilizing particles, thereby reducing droplet mobility and preventing their aggregation [12,17]. Several studies have shown that polysaccharides, such as xanthan gum [18,19], chitosan [20], alginate (Al) [21], curdlan [22], carrageenan [23], and pectin [24], are effective in stabilizing Pickering emulsions through such mechanisms. While enhanced viscosity is inherently associated with polysaccharide addition, the establishment of biopolymer-particle interactions depends on the chemical nature of the components. For example, elevated viscosity due to alginate addition was reported as the sole stabilization mechanism in a Pickering emulsion based on starch nanocrystals [21]. On the contrary, xanthan gum was shown to interact with silica [18] and zein [19] nanoparticles, hence providing a second stabilization mechanism.

Certain polysaccharides solidify in the presence of ions due to physical gelation induced by polymer–ion interactions. We thus sought to utilize physical gelation induced by calcium and potassium ions to create Pickering emulsions suitable for applications that require curing, such as 3D bioprinting for food applications and tissue engineering [25,26] and advanced drug delivery systems [27,28]. Our hypothesis was that introducing a gelling polysaccharide post-emulsification into the continuous phase of a Pickering emulsion has the potential to simultaneously enhance resistance to creaming while providing a means to create emulsion hydrogels composed of stable oil droplets embedded in a crosslinked network made from the hydrophilic polymer.

Accordingly, we selected two anionic polysaccharides capable of undergoing in situ gelation: Al and κ-carrageenan (Kcar). The ability of these polysaccharides to limit creaming in shellac-based Pickering emulsions and provide crosslinked Pickering emulsion hydrogels (PEHs) was investigated. Al is a naturally occurring polysaccharide extracted from brown seaweed and is widely used for its ability to form hydrogels in the presence of divalent cations, such as calcium [29]. In addition, the biocompatibility and non-toxicity of Al make it an attractive option for food and pharmaceutical applications, where a controlled release ability and stability are essential. Al has been investigated extensively as a costabilizer in Pickering emulsions [21,29,30] and as a gelling agent crosslinked into hydrogel-containing emulsions [26,29] and bead-containing emulsions [31].

Kcar, which is derived from red seaweed, is known for its gelling, thickening, and stabilizing properties [32]. It forms gels in the presence of potassium ions and has been studied for its role in enhancing emulsion stability, including in Pickering emulsions. Kcar’s ability to interact with proteins and other polysaccharides makes it a versatile ingredient, as it can provide texture and stability to a wide range of food products [33,34]. Kcar was previously investigated as a costabilizer in Pickering emulsions [23,34] and as a gelling agent in emulsion hydrogels [12,35].

While these natural polymers are well known for enhancing emulsion stability, a direct comparison of their effects in combination with Sh-NPs has yet to be explored. During our study, we discovered a surprising phenomenon: a significant difference in stability against creaming between the two polysaccharides. Notably, Kcar provided better protection against creaming in Sh-NP-stabilized oil in water (O/W) Pickering emulsions compared to Al via a combination of increased viscosity and interaction with Sh NPs, despite Al’s higher viscosity. This unexpected finding highlighted the fact that viscosity cannot be used as a single predictor for creaming prevention and led to a systematic investigation into the underlying mechanisms behind creaming prevention, aiming to identify the importance of enhancing viscosity and Kcar–Sh interactions. To achieve this, we employed a range of experimental techniques to ensure the reliability of the results, including small angle X-ray scattering (SAXS), rheological measurements, and Fourier transform infrared spectroscopy (FTIR) analysis, to characterize the interactions and structural variations of the polysaccharides in the presence of Sh-NPs. Furthermore, we examined the stability and microstructure of the Pickering emulsions with added polysaccharides and explored the formation of stable PEHs by incorporating curcumin, a model food supplement with poor water solubility [36]. We investigated its release in an in vitro gastrointestinal model and leveraged the gelling properties of Kcar and Al for potential application in food and drug delivery systems. Overall, this work not only introduces a novel approach to constructing stable Sh-NP emulsions with Al and Kcar but also highlights the potential of these PEHs in drug delivery and 3D printing applications, particularly where region-specific release and in situ crosslinking are advantageous. These findings advance the understanding of Sh-Kcar interactions and their structural implications and lay a foundation for future innovations in Kcar and Sh-based systems.

2. Materials and Methods

2.1. Materials

Wax-free shellac (55 Astra FL, Batch # 268990) was donated by SSB Stroever GmbH & Co. KG (Bremen, Germany). Jeffamine^® ED-2003 (Batch# BCCB1323), medium molecular weight Al (Mw 250–350 KDa, Batch# SLCM9527), fluorescein (Batch# BCBV9534), and Nile red (NR) (Batch# SLBP9326V) were purchased from Sigma (Rehovot, Israel). Kcar (Mw 200–400 KDa, Batch#AGN21-524452-1) was purchased from Angene International Limited (Nanjing, China). Medium chain triglyceride (MCT) was procured from Omega 3 Galil (Misgav, Israel). Absolute ethanol (CAS# 64-17-5) was obtained from Gadot Group Ltd. (Haifa, Israel) and curcumin (Batch# FZ180817, ≥95%) from FZBIOTECH, Xi’an Fengzu Biological Technology Co., Ltd. (Xianyang City, China). Aqueous solutions were prepared using double-deionized water (DDW). Taurocholic acid sodium salt (Lot# SLBL1190V, ≥95%) and glycodeoxycholate acid sodium salt (CAS# 16409-34-0, ≥97%) were purchased from Merck (Rehovot, Israel) and Holland Moran (Yehud, Israel), respectively. All the materials were used as received without further purification.

2.2. Nanoparticle Preparation

The Sh-NPs were prepared as previously described [15]. Briefly, modified shellac was prepared through a condensation reaction of wax-free shellac dissolved in absolute ethanol (200 mg/mL) with 20% Jeffamine^® ED-2003 added. After mixing at room temperature for 15 min, the solutions were cast in silicon molds and then dried at 60 °C for 2 h, followed by 100 °C for 4 h to enhance the condensation reaction. The modified shellac was then dissolved in absolute ethanol (100 mg/mL) and added dropwise to a DDW vial at a constant rate of 20 µL per 10 s under constant magnetic stirring at 1300 rpm until a concentration of 31.2 mg/mL (w/v) was reached. The particle dispersions were centrifuged at 3300× g for 10 min to separate any large aggregates and precipitates that may have formed. The dispersions were kept at 4 °C until further use. For fluorescent imaging, the labeled NPs were prepared similarly, with 0.15 mg/mL fluorescein added to the shellac solution in ethanol.

2.3. Nanoparticle Characterization

Dynamic light scattering measurements were performed to determine the intensity average particle size, particle size distribution, and zeta potential of the Sh-NPs using a Zetasizer Nano Series (Malvern Instruments Ltd., Worcestershire, UK) equipped with an He–Ne laser (633 nm) at 25 °C and an angle of 173°. The size and zeta potential were analyzed using the Zetasizer software version 7.03 provided with the instrument. The particle dispersions were diluted by 100 before the experiment to ensure proper measurement. All the measurements were carried out in triplicate. These measurements revealed that Sh NPs have a diameter of 200 ± 3 nm and zeta potential of 6.7 ± 0.7 mV. In addition, the NP’s spherical shape and size were verified by cryo-TEM, as described in our previous publication [22], and found to be consistent with the DLS findings.

2.4. Emulsion Preparation

O/W emulsions were prepared as previously described by Delmar and Bianco-Peled [15]. Briefly, MCT oil was added to the Sh-NP dispersions to achieve a final concentration of 1% (w/v) NP and an oil percentage of 40% (v/v). The mixtures were premixed with Vortex-Genie 2 (Scientific Industries Inc., Bohemia, NY, USA) for a few seconds and immediately homogenized using a Bio-Gen PRO200 homogenizer (PRO Scientific, Inc., Oxford, CT, USA) at a constant speed (~20,000 rpm) for 1 min. This emulsion was labeled a stock emulsion. A series of polymer stock solutions were prepared separately by dissolving Al or Kcar in DDW to achieve concentrations in the range 0.5–2% (w/v). Kcar solutions at concentrations of 1.5% and 2% (w/v) were mildly heated to 40 °C to allow proper sampling and mixing, as at these concentrations, Kcar creates hydrogels at room temperature [37].

The studied emulsions were then prepared by mixing equal volumes of the stock emulsion and Al or Kcar solution to achieve concentrations of 0.25–1% (w/v) polymer, 20% (v/v) MCT, and 0.5% (w/v) Sh-NPs in the final emulsion. The control emulsions were prepared by mixing equal volumes of the stock emulsion and DDW. For fluorescent imaging, the emulsions were prepared similarly with 0.0027% (w/v) NR in MCT, and the fluorescein NPs were prepared as described in Section 2.3. Finally, 0.1% (w/v) curcumin in MCT was used as the oil phase to prepare PEH loaded with curcumin.

2.5. Emulsion Characterization

2.5.1. Emulsion Stability

Long-Term Emulsion Appearance

The appearance of the emulsions was assessed visually and recorded with a digital camera at room temperature for 28 days. The long-term emulsion stability was quantified using the creaming index (CI) calculated from Equation (1) [16]:

$$CI\left(\%\right)=100{\displaystyle \frac{H_S}{H_E}}$$

(1)

where H_E is the total height of the emulsion and H_S is the height of the serum layer (the water-rich layer). All the measurements were carried out in triplicate.

Accelerated Stability Testing

The physical stability of the emulsion was tested under accelerated conditions using the centrifuge method [38]. The emulsions stabilized by Sh-NPs were centrifuged using an Eppendorf centrifuge (5418R, Eppendorf, Enfield, CT, USA) at 10,000× g at 4 °C for 45 min. The appearance of emulsions before and after centrifugation was recorded using an iPhone 13 Pro (Apple Inc., Cupertino, CA, USA) built-in camera.

2.5.2. Emulsion Microstructure

Emulsion Droplet Size Analysis

The oil droplet size was measured using a Mastersizer 2000 laser diffraction particle size analyzer (Malvern Instruments Ltd., Worcestershire, UK). An appropriate amount of emulsion was added to the flow system containing DDW and pumped through the optical chamber until the desired obscuration was achieved. The reported diameter is the volume-weighted mean diameter:

$$d_{\mathrm{4,3}}=\sum _i{n}_i{d}_i^4/\sum _i{n}_i{d}_i^3$$

where n_i is the number of droplets with diameter d_i. All the measurements were carried out in triplicate.

Optical Microscopy

The emulsions were visualized by light microscopy using an Olympus BX53M microscope equipped with a complementary metal oxide semiconductor (CMOS) color camera (Sony, Tokyo, Japan). A drop of the emulsion was transferred to a slide, covered with a coverslip, and the oil droplets were visualized using the CMOS camera.

Fluorescent Microscopy

Fluorescent microscopy was employed to examine the spatial arrangement of the emulsions and the location of the Sh-NPs within them. An LSM 880 laser scanning confocal microscope (Zeiss, Oberkochen, Germany) equipped with a plan-apochromat 63× oil objective (Numerical Aperture (NA) = 1.4) was used to image a control emulsion, which contained 20% (v/v) MCT. The imaging was performed with a 5 mW 543 nm NeHe laser at 1% power and a 30 mW 488 nm multiline argon laser at 1.5% power to visualize the structural arrangement of the oil droplets (stained with NR) and the distribution of the Sh-NPs (stained with fluorescein) within the emulsion, respectively.

2.6. Pickering Emulsion Hydrogel

2.6.1. Preparation of the Pickering Emulsion Hydrogel

PEHs based on Al or Kcar were fabricated using the ionotropic gelation method [26]. A volume of 600 µL emulsion containing 20% (v/v) MCT emulsion and 0.75% (w/v) Al or Kcar was loaded into a Teflon mold (diameter 13 mm, thickness 3 mm). Gelation was promoted by casting an appropriate ion solution (0.1 M CaCl₂ solution for Al containing emulsions or 0.1 M KCl solution for Kcar containing emulsions) on the molds. The molds were left at room temperature for 30 min. The gelation process was then stopped by removing the ion solution.

The diameters of the PEHs with either Al or Kcar before and after in vitro digestion were measured with a digital caliper, and the averages of at least five different hydrogels were recorded.

2.6.2. Digestion Experiments

One use for the emulsions could be oral applications that undergo digestion. Hence, the emulsion gels were studied under in vitro gastrointestinal conditions of a healthy adult, which were recreated using an auto titration system (Titrando 902, Metrohm, Herisau, Switzerland). This semidynamic system is controlled by software (TIAMO 2.05) to emulate postprandial gastric pH gradients according to physiological data relevant to food research and as previously described [39,40]. Ion solutions mimicking the physiological composition and concentration and bile salts (5 mM each of sodium glycodeoxycholate and taurocholic acid) were applied, all according to the INFOGEST protocol for in vitro digestion [39]. Dynamic gastric pH gradients shifted from a pH of 4.5 to 1.5, followed by a static intestinal phase at pH = 6.25, stirring rate of 250 rpm, and the temperature was set to 37 °C using an external circulation bath, as also applied previously [41,42]. Briefly, 10 PEHs were placed in a 50 mL tube and mixed with simulated saliva fluid to an effective final volume of 50 mL. The tube was then shaken for 2 min to simulate mastication, followed by rapid mixing with 50 mL of simulated gastric fluid. The formed bolus pH was rapidly adjusted to 4.5 (with 1 M NaOH) as needed, and a gastric pH gradient was initialized via computer control by auto-titration of 0.25 M HCl and followed for 2 h. Subsequently, an intestinal phase was initiated via rapid pH elevation to 6.25 by the addition (1:1 v/v) of simulated intestinal fluid (SIF) and bile salts (5 mM sodium glycodeoxycholate and 5 mM taurocholic acid sodium salt), which was kept at a constant pH for 2 h using 0.25 M NaOH. Aliquots of 2 mL were aspirated during the digestion experiments as follows: from the gastric phase at 10, 60, and 120 min (abbreviated as G10, G60, and G120) and from the intestinal phase at 10, 20, 60, and 120 min (abbreviated as I15, I30, I60, and I120). All the digestive aspirates were kept on ice until further analysis was performed at the end of each digestion experiment.

Curcumin Release Measurements

The curcumin concentration in the release media and the Pickering emulsion was determined similarly to Han et al. with minor changes [43]. The curcumin was recovered from the release media and the emulsion by dissolving the substance in ethanol. A 100 µL sample was added to 900 µL absolute ethanol, and the mixture was then centrifuged at 10,000× g at 4 °C for 10 min. The supernatant was collected, and the concentration was measured using an Infinite 200 PRO M Nano UV–Vis spectrophotometer (Tecan, Männedorf, Switzerland) to measure the absorbance at 426 nm based on a calibration curve. The measurements were carried out in triplicate.

2.7. Polymer–Nanoparticle Interactions

2.7.1. Rheology

The apparent shear viscosity of the Al and Kcar solutions (0.25–1% (w/v)), 0.5% (w/v) NP solutions, and the mixtures of 0.5% and 0.75% (w/v) Al or Kcar were measured using an MCR 302 rheometer (Anton Paar GmbH, Graz, Austria) with a 50 mm parallel plate geometry (sanded surface) and a gap of 0.5 mm. The mixtures were free-flowing solutions. All measurements were performed at a constant temperature of 25 °C. The apparent shear viscosity was measured by decreasing the shear rate from 100 to 1 s⁻¹. All the measurements were carried out in triplicate.

2.7.2. Fourier Transform Infrared Spectroscopy Measurements

FTIR analysis was used to characterize the interactions between the Sh-NPs and Al or Kcar. The samples were lyophilized using an Alpha 1-2 LD plus lyophilizing machine) Martin Christ, Osterode, Germany) for 48 h at −22 °C and a pressure of 0.85 mbar.

The FTIR measurements were performed using a Bruker Tensor 27 spectrometer (Bruker, Billerica, MA, USA) equipped with a TGS detector and a Bruker Platinum ATR, diamond, single reflection, accessory. The spectra were collected in ATR mode over the wavenumber range of 4000–600 cm⁻¹ at a resolution of 4 cm⁻¹. Microsoft^® Excel was used to perform the analysis.

2.7.3. Small Angle X-Ray Scattering

The structures of Al (0.75% w/v), Kcar (0.75% w/v), Sh-NP (0.5% w/v), and the mixtures of Al and Sh-NP and Kcar and Sh-NP (with the same final concentrations as the individual solutions) in DDW were characterized by SAXS. The sample was poured into a thin-walled glass capillary with a 0.01 mm wall thickness and 2 mm diameter. The SAXS measurements were performed using a Xeuss 3.0 (Xenocs, Grenoble, France) equipped with a GeniX^3D beam delivery system (Cu Kα high-intensity X-ray beam, wavelength λ = 1.5419 Å⁻¹, 50 kV, 0.6 mA) and EIGER2 R 1M DECTRIS detector (75 µm pixel size). The scattered intensity I(q) was recorded in the scattering vector q range 0.007 < q < 0.16 Å⁻¹, where q is defined as q = (4π/λ) sin(θ), 2θ is the scattering angle, and λ is the radiation wavelength (1.542 Å).

I(q) was normalized to time, solid angle, primary beam intensity, sample dimensions, transmission, and the Thompson factor. Scattering from the solvent, electronic noise, and empty capillary was subtracted from the sample scattering. Fitting to different models was performed using software previously developed by our group [44].

For the NP dispersion, the best fit of the measured intensity I_NP(q) was obtained using a power law, Equation (2):

$$I_{NP}\left(q\right)=k_1{q}^n$$

(2)

where $k_1$ is a prefactor that depends on the concentration of the scattering objects and their contrast, and n is an exponent. For n = −4, Equation (2) takes the form of Porod’s law [45].

For the Kcar and Al solutions, the scattering from the polymer solution I_pol(q) was fitted to Equations (3)–(6) [46]:

$$I_{pol}\left(q\right)=k_{2}P\left(q\right)S\left(q\right)$$

(3)

where k₂ is a prefactor, P(q) is the form factor related to the size and shape of the individual scattering objects, and S(q) is the structure factor arising from the interactions between the scattering objects [45]. The best fits were obtained using a model of semiflexible chains with excluded volume effects for the form factor, which was originally developed by Pedersen and Schurtenberger [47] and modified by Chen et al. [46]:

$$P\left(q,b,L\right)=P_{exv}\left(q,b,L\right)+C\left({\displaystyle \frac{L}{b}}\right){\displaystyle \frac{b}{15L}}\left[4+{\displaystyle \frac{7}{u}}-\left(11+{\displaystyle \frac{7}{u}}\right)e^{-u}\right]$$

(4)

where P_exv is given by

$$P_{exv}\left(q,b,L\right)=\left[1-w\left(q{R}_g\right)\right]{·P}_{debye}\left(q,b,L\right)+w\left(q{R}_g\right)\left[C_1{\left(q{R}_g\right)}^{-1/\nu }{C}_2{\left(q{R}_g\right)}^{-2/\nu }{C}_3{\left(q{R}_g\right)}^{-3/\nu }\right]$$

(5)

where L is the contour length, b is the Kuhn’s length, and R_g is the radius of gyrations. Further details and definitions of the other functions in Equations (3)–(5) can be found elsewhere [44,46]. For the structure factor, the best fits were obtained using a function that describes a Gaussian-type repulsion potential between charges of objects with a correlation length ξ_e [44,48] as follows:

$$S\left(q\right)={\displaystyle \frac{1}{C_e·exp\left(-{\xi }_e^2{q}^2\right)}}$$

(6)

where C_e depends on the concentration, the second viral coefficient, and the molecular weight.

The scattering patterns from the solutions of polysaccharide containing NPs reflect the contributions of both components: the chains and the NPs. For the Al mixed with NPs dispersion, a good fit was obtained with Equation (7):

$$I\left(q\right)=I_{NP}\left(q\right)+I_{pol}\left(q\right)$$

(7)

However, for the Kcar mixed with NPs dispersion, Equation (7) could not provide an adequate fit. Therefore, a third contribution, which describes the scattering from a rod-like (cylinder) particle approximated using Guinier’s law, was added to the model [49]:

$$I\left(q\right)=I_{NP}\left(q\right)+I_{pol}\left(q\right)+I_{Cylinder}\left(q\right)$$

(8)

where I_Cylinder(q) is given by

$$I_{Cylinder}\left(q\right)=k_3{q}^{-1}exp\left(-{\displaystyle \frac{R_{g,C}^2{q}^2}{2}}\right)$$

(9)

where k₃ is a prefactor, and R_g,c is the cross-sectional radius of gyration.

2.8. Statistical Analysis

The statistical analysis was performed using GraphPad Prism version 10.2.3. The data from the independent experiments were quantified and analyzed for each variable. Two group comparisons were performed using Student’s t-test, while comparisons between multiple treatments were analyzed via one-way analysis of variance with Tukey’s test as a post hoc analysis. A p value < 0.05 was considered statistically significant. The standard errors of the mean were calculated and are presented for each treatment group. All the experiments were performed in triplicate.

3. Results and Discussion

In this study, we investigated the impact of incorporating Al and Kcar into stable O/W shellac-based Pickering emulsions to explore their influence on emulsion stability and structural properties and their potential for forming crosslinked hydrogels.

3.1. Stability of Shellac-Based Pickering Emulsion Following Polysaccharide Addition

To investigate the effects of adding two types of polysaccharides, Al and Kcar, to Pickering emulsions stabilized by Sh-NPs, we first screened a range of polymer concentrations. The polysaccharides were introduced into the continuous phase post-emulsification by mixing an emulsion with a polymer solution to achieve final concentrations of 0.25–1.0% (w/v) polymer, 20% (v/v) MCT, and 0.5% (w/v) Sh-NPs. Our objective was to evaluate how the incorporation of varying concentrations of these polysaccharides influenced the physical characteristics, stability, and resistance to creaming of the emulsions over time compared to a control sample. The appearance and stability of the emulsions were systematically monitored and analyzed over a period of 28 days (Figure 1).

Figure 1A shows that the control emulsion exhibited creaming immediately upon preparation, with the cream layer remaining stable throughout the monitoring period. This behavior was consistent with previous observations of Pickering emulsions that contained isopropyl myristate stabilized by Sh-NPs, which also exhibited creaming [15]. Creaming was also observed in all the emulsions with added Al. Qualitatively, the height of the serum layer increased with the Al concentration on the day of preparation, but differences between the samples seemed to diminish over time, with the exception of the highest Al concentration, for which the cream layer was somewhat thicker. Thus, Al delayed creaming for a limited time but could not effectively prevent it. In contrast, when examining the emulsions with added Kcar, creaming was only evident at the lowest concentration of 0.25% (w/v) Kcar, while the emulsions that contained higher concentrations did not exhibit any creaming throughout the observation period. Hydrophilic polymers, such as Al and Kcar, inhibit creaming mainly due to the restricted movement of oil droplets under gravity, which is caused by the higher viscosity of the continuous phase [12]. Since the dissimilar effects of Al and Kcar could have arisen from variations in viscosity, we conducted further investigations to assess whether this was the mechanism, as described in Section 3.3.

It should be noted that creaming does not necessarily lead to phase separation (oiling out), and an emulsion is considered stable as long as coalescence and phase separation do not occur [50]. All the studied emulsions, both with and without the added polymers, maintained stability over the 28-day period without any oiling-out phenomenon.

To further challenge the high stability exhibited by the emulsions at ambient conditions, we examined their stability under accelerated conditions (Figure 1B,C). We chose to test the emulsions with 0.5% (w/v) and 0.75% (w/v) added Kcar, as creaming was observed at concentrations below 0.5% (w/v), while an emulsion with 0.75% (w/v) added Kcar remained stable without any creaming during the observation period. For comparison, we selected emulsions with the same concentrations of added Al that had exhibited creaming. As centrifugation is known to expedite the creaming process, we subjected the emulsions to centrifugation at 10,000× g for 45 min, which is considered to be an extreme condition [38]. As shown in Figure 1C, none of the emulsions experienced oiling out, thus indicating the superior stability of these Pickering emulsions both with and without added polysaccharides. While no creaming was observed in the emulsions with added Kcar under ambient conditions (Figure 1A), under centrifugation, forced creaming eventually occurred, which underlines the kinetic nature of the stability of these emulsions.

To further assess the influence of the added polymers on creaming and stability, the CI was calculated for the aforementioned emulsions, and its value was followed over time. The CI (Figure 2A) increased with time for all the Al-containing emulsions and reached a maximum value of 60–67% depending on the composition of the emulsion. The control emulsion and the emulsion with 0.25% (w/v) added Al reached the equilibrium CI on the day of preparation without any significant changes over a longer period. The emulsions with 0.5% and 0.75% (w/v) added Al reached the equilibrium CI after a week, with no significant changes over time after that point. The single emulsion containing Kcar (0.25% (w/v)) that exhibited creaming reached equilibrium shortly after preparation (1 day).

To quantify the different extents of the CI, the values were compared after 7 days. No significant changes were observed for any of the studied emulsions thereafter (Figure 2B). The CI of the control was the highest (66.5% ± 5.6%), which hints at a lesser resistance against creaming. When 1% (w/v) Al was added, the creaming decreased significantly to 53.1% ± 11%. For all the other Al concentrations in the range of 0.25–0.75% (w/v), the CI did not change significantly compared to the control, which means that the polymer presence did not hinder the oil droplets’ upward movement. When examining the emulsions with added Kcar, only the lowest concentration of 0.25% (w/v) manifested a creaming phenomenon (CI = 59.4% ± 0.5%, which was not significantly different from the CI observed for the control emulsion). None of the other emulsions containing Kcar, which had concentrations ranging from 0.5–1% (w/v), exhibited any creaming phenomenon.

The quantitative analysis strengthened the qualitative observation described above: Kcar significantly enhanced the emulsion’s resistance to creaming, while Al proved to be considerably less effective. The addition of Al (0.25% and above) has been shown to prevent creaming when used as a costabilizer in Pickering emulsions stabilized by salmon byproduct protein [29] and montmorillonite [51]. Conversely, the addition of Kcar to myosin-based Pickering emulsions reduced but did not entirely prevent creaming [23]. However, when Kcar was incorporated as a gelling agent in zein/carboxymethyl dextrin [52] and xanthan gum/lysozyme [53] based Pickering emulsions, no creaming occurred. It is worth noting that relatively few studies have included Kcar in Pickering emulsions.

To further investigate the influence of the polymers added to the emulsions on the creaming and stability of the latter, the same screening process was implemented for emulsions containing paraffin oil (PO). These emulsions were monitored (Supplementary Figure S1), and their CI% was measured over time, as summarized in Figure 2C,D. These emulsions exhibited the same trends as the MCT emulsions (Figure 1A). When comparing the CI of the emulsions consisting of the same polymer at the same concentration with that of the PO and MCT, no significant differences in the creaming extent were detected for any of the examined emulsions. This observation led to the assumption that the creaming phenomenon is governed by the polymer addition, type, and concentration and not the oil type. The creaming process results from the upward movement of oil droplets because of density differences between the dispersed and the continuous phase [16]. Since the incorporation of different oil types with different densities (0.839 g/cm³ for PO and 0.94 g/cm³ for MCT at 25 °C) and chemical structures resulted in a consistent CI, we hypothesized that either the viscosity of the continuous phase or a more complex mechanism related to intermolecular interactions between the Sh-NPs and the added polymer govern the creaming mechanism. This hypothesis was further evaluated, as described in Section 3.3.

Changes in oil droplet size are considered a sensitive measure of emulsion stability [54]. We therefore performed size analysis to investigate whether the lack of oiling out during storage and centrifugation was indicative of stability on a microscopic scale. We chose to conduct this analysis for emulsions with 0.5% (w/v) and 0.75% (w/v) added polysaccharide, which according to our initial screening were representative of other compositions, namely, added Al did not prevent creaming while added Kcar did. To assess the impact of polymer addition on the emulsion characteristics, we first measured the oil droplet size of the stock emulsion before and after the addition of the polymer solutions (or DDW as a control). As shown in Figure 3G, the average droplet size of the Pickering emulsions, determined by d(4,3) analysis, did not significantly change with the addition of the polymer, both for the added Al and Kcar.

To further explore the emulsion characteristics, we utilized light microscopy with the aim of providing additional insights into the microstructures of the emulsions. The images are summarized in Figure 3A–F alongside the measured size distribution of each emulsion (Figure 3G). All the emulsions exhibited similar monomodal distributions regardless of the added polysaccharide. When examining the microstructures of the emulsions, the control emulsion (without the polymer) and those with added Al or Kcar exhibited comparable microstructures, which supports the d(4,3) measurements, indicating that the polymer addition did not significantly alter the droplet size or distribution. When observing the stock emulsion (containing 40% MCT before dilution), the micrograph revealed densely packed oil droplets. Upon dilution, the droplet density decreased slightly, although the droplet size remained unchanged.

To further investigate the microstructures of the emulsions, we imaged a control emulsion using fluorescent microscopy. The MCT was dyed with NR and appeared red in the image (Figure 3H), while the Sh-NPs were dyed with fluorescein and appeared green in the image (Figure 3I). As shown in Figure 3H,I, the NPs accumulated at the oil–water interface (white arrows). The accumulation of particles at the interface is a key feature of Pickering emulsions, where the adsorbed solid particles act as a physical barrier, thereby preventing the coalescence of the droplets and stabilizing the system against phase separation. The superior stability is attributed to the high detachment energy from the interface, which leads to the irreversible adsorption of the NPs [8]. The accumulation of Sh-NPs in the oil–water interface highlights their role in the emulsion’s superior stability and is in line with our previous study, which focused on emulsions prepared with 40% isopropyl myristate [15]. Even upon dilution of the emulsion, the NPs remained attached to the interface, thereby providing superior stability.

The observation that accelerated conditions did not induce oiling out and that dilution had no significant impact on droplet size or stability indicates that the structural integrity of the Pickering emulsions was well preserved even after dilution and highlights the robustness and resilience of this emulsion system.

3.2. Pickering Emulsion Hydrogels for Curcumin Release

In situ crosslinking of a polymer added to the continuous phase of an emulsion can potentially enhance its functional versatility and thereby make it suitable for applications such as ink formulations for bioprinting and controlled release systems. Pickering emulsions serve as effective encapsulation systems as they enhance the storage stability of active compounds and increase their bioavailability [55]. For example, Wang et al. demonstrated that crosslinking protein–Al based Pickering emulsions significantly improved the viability of encapsulated probiotics compared to uncrosslinked emulsions [29]. Inspired by that study, we investigated the potential of crosslinking the polysaccharides added to the emulsions to create PEH and to further utilize them as controlled release systems for a model bioactive compound, curcumin, in simulated gastric conditions.

The gelation of Kcar starts with a transition from a random coil configuration to an ordered helical structure, which can be promoted by lowering the temperature. Monovalent cations, such as K⁺ and Rb⁺, facilitate the clustering of Kcar double helices, which lead to the formation of aggregated domains [37]. Al gelation occurs primarily through interactions with divalent cations, mainly Ca²⁺ ions, leading to the formation of distinctive “egg-box” structures, which stabilize the gel network [56].

As shown in Figure 4A and the Supplementary Videos, emulsions with added polysaccharide crosslink instantly when injected into a bath containing a solution of an appropriate salt and form stable PEHs when cast and soaked in a crosslinker solution for a set period.

PEH based on 0.75% (w/v) Kcar or Al were obtained by crosslinking cast Pickering emulsions (Figure 4B). The release of curcumin from the aforementioned PEHs in an in vitro gastrointestinal model was investigated and is summarized in Figure 4D. We selected the standardized INFOGEST protocol, which incorporates a pH gradient to more accurately simulate the gastric conditions typical of male digestion [39,40]. Given the critical role of the initial interactions with saliva—which facilitates the ion exchange that influences the behavior of the Al hydrogel during the subsequent digestive phases—we included this salivary step in our experimental design [57].

As summarized in Figure 4D, the Al-based PEH showed no curcumin release in the gastric phase and only released up to 29.8% ± 4.9% curcumin by the end of the small intestinal phase. The absence of curcumin release in the gastric phase was likely due to the additional crosslinking of Al in the acidic conditions through hydrogen bonding [58,59], which may have restricted curcumin diffusion into the surrounding medium. This was further supported by the reduction in the hydrogel diameter from 12.6 ± 0.6 mm to 9.8 ± 0.6 mm after exposure to the gastric conditions, thereby indicating shrinkage due to the increased crosslinking density. In contrast, within the small intestine phase, the hydrogels swelled and lost their structural integrity (see Supplementary Figure S2), likely due to the chelation of Ca²⁺ with ions from the SIF, which lead to the crosslinking density decrease [56] and subsequent partial curcumin release. This behavior is consistent with the well-known characteristics of Al, which typically shrinks in gastric environments and swells in the intestine [60].

The Kcar-based PEH exhibited a significantly larger diameter (13.4 ± 0.1 mm) compared to those with added Al (12.6 ± 0.6 mm) despite being cast in the same mold (Figure 4C). This size difference could be attributed to the syneresis effect during the crosslinking of Al, which resulted in smaller gel sizes [61]. The curcumin release profile from the Kcar PEH differed from that of the Al-based PEH. After 2 h in the gastric phase, only 4.1% ± 1.9% of curcumin had been released, whereas in the small intestine phase, the majority of curcumin (82.3% ± 10.3%) was released within the first 20 min, with the hydrogels disintegrating by the end of this phase (Supplementary Figure S3). The dissolution of Kcar in the SIF is linked to the increased ionization of sulfate groups, thereby causing electrostatic repulsion, which expands the polymer network and allows greater water penetration until complete dissolution occurs [62]. Furthermore, the destabilization of the physically crosslinked network occurred due to the substitution of the monovalent ions with the divalent ions or proteins present in the SIF [63], along with the rise in temperature, which disrupted the hydrogel’s physical junctions [64].

Based on the resulting release profile, we suggest that both types of PEH can serve as enteric vesicle systems and prevent release in the stomach. The Al-based PEHs enabled a small portion of the bioactive ingredient to be released in the small intestine while delivering most of the curcumin to the large intestine. This targeted release may support gut health, as curcumin has been shown to modulate gut microbiota in irritable bowel syndrome models by reducing the levels of harmful bacteria, such as Bacteroidetes [65], and to restore microbial balance in colorectal cancer models [66], which indicates its potential for managing intestinal dysbiosis and related conditions. On the other hand, the Kcar-based PEHs released most of their cargo primarily in the small intestine, making them particularly suitable for pH-sensitive drugs, such as aspirin and mycophenolic acid. This targeted delivery approach enhances drug bioavailability by protecting sensitive drugs from gastric degradation and ensures their release at the optimal absorption site [67,68].

3.3. The Effect of Added Polysaccharide on Shellac-Based Pickering Emulsions

Our objective was to develop stable Pickering emulsions that could be crosslinked and serve as a platform for various applications. We demonstrated that PEHs with added Al can function as stable enteric release systems, while those with added Kcar offer dual-phase release, first in gastric conditions and then in the small intestine. These shellac-based Pickering emulsions exhibited high resistance to oiling out under both ambient and accelerated conditions, although they differed significantly in terms of stability against creaming. This distinction has practical implications, as creaming affects the homogeneity of emulsions, which could impact their ability to be printed or cast without requiring premixing. To better understand this creaming behavior, we performed a series of experiments aimed at revealing the importance of the different mechanisms that may be involved in creaming prevention.

Increasing the viscosity of the continuous phase can prevent creaming, as it hinders the upward movement of oil droplets [69]. To investigate the role of this mechanism, we analyzed the viscosity of the polymer solutions used in the Pickering emulsions, as detailed in Section 3.1. Since all the emulsions contained the same oil (20% (v/v)) and NP (0.5% (w/v)) content, we focused on the viscosity of the polymer solutions alone (Figure 5A,B). The Kcar solutions (Figure 5A) exhibited Newtonian behavior at concentrations up to 0.5% (w/v), which is similar to the findings of previous reports [70], while at higher concentrations, the samples showed shear-thinning behavior. This type of non-Newtonian behavior is consistent with the results of previous studies and is attributed to the rearrangement of entangled Kcar chains [71]. In contrast, all the Al solutions displayed Newtonian behavior, as observed in Figure 5B. Due to the non-Newtonian nature of part of the polymer solutions, further comparisons were made at the same shear rate of 1 s⁻¹.

When examining the results, it became clear that effective creaming prevention cannot be correlated with viscosity alone. For example, as demonstrated in Figure 5C, the viscosities of the 0.5% (w/v) Kcar and Al solutions were fairly close and insignificantly different—53 ± 2 mPas and 40 ± 4 mPas, respectively, at a shear rate of 1 s⁻¹. However, adding 0.5% (w/v) Kcar to the Pickering emulsion effectively prevented creaming, whereas emulsions with 0.5% (w/v) Al added exhibited creaming within a day. As another example, the viscosity of the 1% (w/v) Al solution (177 ± 26 mPas) was about three times higher than that of the 0.5% (w/v) Kcar solution at the same shear rate. Despite the higher viscosity, the addition of 1% (w/v) Al to the emulsions did not prevent creaming, while a cream layer was not observed in the emulsions with 0.5% (w/v) Kcar added. These observations suggest that the polymer solution’s viscosity alone cannot fully account for the differences in creaming behavior. We note that viscosity is highly temperature-dependent, and therefore the results described above may not be valid at other temperatures.

Based on these findings, we further hypothesized that interactions between the Kcar polymeric chains and the Sh-NPs polymer in the continuous phase and surrounding the oil droplets govern the creaming mechanism. As observed in our earlier study on a similar Pickering emulsions system [15], Sh-NPs are present in both the continuous phase and at the interface. Interactions between the NPs in the continuous phase could hinder oil droplet movement and contribute to creaming inhibition. To test this hypothesis, we examined the viscosities of polymer solutions combined with a 0.5% (w/v) NP dispersion. The viscosity of the NP dispersion alone was similar to that of water and exhibited Newtonian behavior (Figure 5D–I). When Al was mixed with the NP dispersion, the viscosity for all examined shear rates increased and matched that of the Al solution without NPs, as shown in Figure 5D,E. The viscosity of the Al–NP mixture at a shear rate of 1 s⁻¹ was not significantly different from that of the Al solution alone (Figure 5F). Due to the low concentration and compact structure of the NPs, the viscosity of their dispersion was almost two orders of magnitude lower than that of the Al solution. Therefore, the contribution of the NPs to the viscosity of the mixture was negligible compared to that of Al.

In contrast, the behavior of the Kcar solutions varied considerably when mixed with NPs, as detailed in Figure 5G–I. The addition of NPs transformed the behavior of the 0.5% (w/v) Kcar solution from Newtonian to non-Newtonian, while the 0.75% (w/v) Kcar solution remained non-Newtonian following the NP addition. For both Kcar concentrations, the viscosity of the mixtures across all shear rates was significantly higher than that of the Kcar solutions alone. As depicted in Figure 5I, the viscosities at a shear rate of 1 s⁻¹ of the Kcar–NP mixtures containing 0.5% and 0.75% (w/v) Kcar increased by 1500% and 660%, respectively. This substantial increase suggests that the NPs had a strong influence on the Kcar viscosity, which could have arisen from the interactions between the Kcar polymer chains and Sh-NPs. Previous studies have shown that interactions of Kcar with casein micelles [72], cyclodextrins [73], iron oxyhydroxide NPs [74], and protein–Kcar complexation [75] induce helix formation and gelation of Kcar chains. Furthermore, synergistic hydrogen bond interactions between Kcar NPs and konjac glucomannan chains have been shown to enhance viscosity in such mixtures compared to the individual components [76]. Nevertheless, the interactions of polymers with NPs can potentially enhance viscosity even when the chain conformation is not affected. Yang et al. reported a similar increase in the viscosity of hydrolyzed polyacrylamide in the presence of silica NPs and attributed it to hydrogen bond interactions between the NPs and polymer chains [77]. Similarly, Buitrago-Rincon et al. reported a 21% increase in the viscosity of xanthan gum solutions after the addition of silica NPs, which was attributed to synergistic interactions, likely involving hydrogen bonding between the components [78].

Thus, the significant viscosity increase in the Kcar–NP mixture in our study was due to specific interactions between the Sh-NPs and Kcar, which were likely driven by both electrostatic and hydrogen bonds and led to a more structured and viscous system. To evaluate this suggestion, FTIR and SAXS measurements were performed, as described below.

To evaluate our assumption that intermolecular interactions occurred between the Sh-NPs and Kcar, an FTIR analysis was performed. Figure 6A shows the FTIR spectrum of the Sh-NPs as well as the spectra of Al and Kcar alongside their corresponding mixtures with Sh–NPs. All the studied materials exhibited a broad band of between 3100–3550 cm⁻¹, which was attributed to –OH stretching, with distinct maxima for each material. The Sh-NPs showed characteristic peaks at 2928 cm⁻¹ and 2858 cm⁻¹, which corresponded to the C–H stretching vibrations of the –CH₃ and –CH₂ groups, respectively [79]. A strong peak at 1710 cm⁻¹ was assigned to the carbonyl (C=O) groups present in the carboxyl groups on the shellac backbone [80,81]. Additionally, a peak at 1556 cm⁻¹ indicated the amide bond (N–H bending [82]) between the shellac backbone and the Jeffamine^® amine groups [15].

The Kcar spectrum exhibited distinctive bands at 1226 cm⁻¹, which could be attributed to the sulfate ester (S–O) bond, and at 925 cm⁻¹ and 842 cm⁻¹, which corresponded to the 3,6-anhydro-D-galactose (C–O) and D-galactose-4-sulfate (C–O–SO₃) bonds, respectively [76]. The Al spectrum exhibited peaks at 1597 and 1406 cm⁻¹, which corresponded to the asymmetric and symmetric modes of the stretching vibration of the carboxyl groups, respectively [83,84].

In the spectrum of the NP mixture with Kcar, no new peaks appeared, but there was a slight red shift in the O–H stretching band from 3396 cm⁻¹ to 3389 cm⁻¹ and broadening of the peak, which was indicative of hydrogen bond formation [84,85].

Conversely, the spectra of NPs and Al showed a blue shift, which indicated fewer hydrogen bonds between the NPs and Al. This difference in hydrogen bonding may explain the observed variations in the creaming extent of the Al-containing emulsions compared to those with Kcar. It was previously shown that Kcar can react with casein proteins via hydrogen bonds through their sulfate and hydroxyl groups [23,86]. Here, the interactions between the carboxyl groups on the Sh-NPs and the hydroxyl and sulfate groups on the Kcar backbone were likely involved.

To further understand whether the hydrogen bonding revealed by the FTIR analysis was related to the viscosity enhancement and creaming prevention, we introduced urea (10% (w/v)) into the emulsion with 0.75% (w/v) Kcar, as urea is known to disrupt hydrogen bonds [87,88]. Prior to the addition of urea, this emulsion displayed high viscosity and gel-like properties, which were evidenced by its resistance to flow when the vial was flipped (Figure 6B). Upon the addition of urea, this gel-like behavior transitioned to a less viscous state. Thus, gel formation was inhibited due to the hindering of hydrogen bond formation. Furthermore, we monitored the emulsion with added Kcar over time—with and without added urea—compared to a control emulsion (Figure 6C). After 10 days, the Pickering emulsion with 0.75% (w/v) added Kcar with urea showed creaming similar to that of the control, whereas the identical emulsion without urea remained stable. This experiment highlights the crucial role of the hydrogen bonds between the Kcar and NPs, which led to viscosity enhancement and creaming prevention.

Once we established that the viscosities of the Kcar and Sh-NP solutions had increased due to the interactions between them, we aimed to decipher the underlying mechanism controlling this phenomenon. Two explanations are possible. First, it is known that Kcar chains have a self-gelation ability thanks to the creation of helixes via chain–chain hydrogen bonds. Here, perhaps the presence of the NPs facilitated these structures via interactions with the NPs, which led to an increase in the viscosities of the solutions. A second possible mechanism is polymer chains bridging between the NPs accompanied by chain jamming [89]. This mechanism is not specific to Kcar and has been described for other systems of polymers and interacting NPs, where the mixed solutions displayed very high viscosities and even formed hydrogels [90,91,92].

To distinguish between the mechanisms, we conducted structural analysis using SAXS, as summarized in Figure 7. The experimental SAXS pattern of the NP suspension (Figure 7A) displayed linearity on a log–log scale. Accordingly, the data were fitted to a power law (Equation (2)), yielding a value of n = −4.08. This aligned closely with the predicted n = −4 by Porod’s law, which describes the scattering from objects with sharp interfaces at high q values [45]. The Porod approximation was applicable since the NP diameter (200 ± 3 nm) was large, thus satisfying the condition qR >> 1 across the entire q-range.

The scattering from both the Al (Figure 7A) and Kcar (Figure 7B) solutions was best modeled as semiflexible chains with excluded volume effects along with an electrostatic structure factor (Equations (3)–(6)). This model has previously been used to fit scattering from Al solutions [44] and is consistent with the semiflexible conformation of Kcar [93]. The best-fit parameters, summarized in

Table 1. Best-fit parameters for Al, Kcar, and the Kcar–NP mixture.

	Al	Kcar	Kcar and NP
L (Å)	1638	20073	20073
b (Å)	42.4	48.2	50.3
R (Å)	4.1	8.6	11.2
Ce	15.0	6.4	5.1
ξe (Å)	38.7	42.4	56.4
R2	0.908	0.994	0.997

, demonstrate that both polysaccharides exhibited similar conformations and dimensions. However, the impact of adding NPs to the polysaccharide solutions differed significantly between Al and Kcar.

For Al in the presence of NPs (Figure 7A), the scattering appeared as a simple superposition of the scattering from Al and NPs, with a negligible cross-term (Equation (7)). In contrast, the scattering from the NP–Kcar mixture was substantially higher than a mere superposition of the components’ scattering (Figure 7B). After testing various models, we concluded that the excess scattering was best explained by the presence of long cylindrical objects with a cross-sectional radius of gyration 70 Å, which contributed to the scattering from the chains and were absent without NPs. Fitting the experimental data to Equation (8), which accounted for both the cylindrical objects and the semiflexible chain contribution, revealed notable changes in the chain conformation. Specifically, the chain rigidity increased slightly, as evident by an increase in the Kuhn’s length from 48.2 to 50.3 Å upon the addition of the NPs to the Kcar solution (Table 1). The chain radius also increased from 8.6 Å in the Kcar alone to 11 Å in the NP–Kcar mixture, while the correlation length increased from 42.4 to 56.4 Å with the addition of NPs.

It is widely accepted that the gelation of Kcar involves a coil-to-helix transition, followed by the aggregation of these helices into a 3D network [93]. Previous SAXS studies have described the crosslinking domains formed by aggregated helices as long cylindrical objects [94]. Further, additional studies have shown that interactions between Kcar and nanometric entities [72,73,74,75] induce the helix formation and gelation of Kcar chains. Therefore, the structural changes observed in the scattering patterns upon the addition of NP to the Kcar in this study indicated helix formation, with the helix aggregation manifested through the formation of cylindrical objects.

4. Conclusions

This study marks the first exploration of highly stable Sh-based Pickering emulsions through the addition of Al and Kcar polysaccharides. Both polymers imparted remarkable stability to the emulsions under accelerated conditions, although their impact on creaming stability differed significantly. Kcar at concentrations of 0.5–1% (w/v) effectively prevented creaming, whereas Al did not exhibit the same efficacy at equivalent concentrations. The observed stability differences were attributed to specific interactions between the Kcar chains and Sh-NPs. FTIR and urea addition investigations confirmed the presence of hydrogen bonds, while SAXS measurements indicated structural changes and helical formation, presumably due to the intermolecular interactions. These changes collectively increased viscosity and mitigated creaming. In contrast, the addition of Al did not induce similar structural or stability enhancements.

The physical crosslinking of both polymer types resulted in PEHs with distinct release profiles for encapsulated curcumin in an in vitro model. The Al-based PEHs demonstrated a targeted release primarily in the large intestine, which is potentially beneficial for microbiome modulation and gut health. Conversely, the Kcar-based PEHs disintegrated in the small intestine, which aligns with the targeted delivery needs for pH-sensitive drugs absorbed in this region.