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Thermoresponsive starch-based particle-stabilized Pickering high internal phase emulsions as nutraceutical containers for controlled release
International Journal of Biological Macromolecules 146 (2020) 171–178
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Thermoresponsive starch-based particle-stabilized Pickering high internal phase emulsions as nutraceutical containers for controlled release Chao Wang a,b, Xiaopeng Pei a,b, Junling Tan c, Tongwu Zhang c, Kankan Zhai a,b, Fan Zhang a,b, Yungang Bai a, Yukun Deng a, Baichao Zhang a, Yinchuan Wang a, Ying Tan a,⁎, Kun Xu a,⁎, Pixin Wang a a b c
Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China University of Science and Technology of China, Hefei 230026, PR China Oil &Gas Technology Research Institute of Changqing Oilfield Company, Yanan 716000, PR China
a r t i c l e
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Article history: Received 16 November 2019 Received in revised form 24 December 2019 Accepted 31 December 2019 Available online 03 January 2020 Keywords: Starch-based particle High internal phase emulsions Thermoresponsive Controlled release Pickering emulsions
a b s t r a c t Pickering high internal phase emulsions (HIPEs) stabilized solely by bioderived starch-based particles hold potential for application in the food and pharmaceutical fields. This paper reports the use of a thermoresponsive 2-hydroxy-3-butoxypropyl starch (HBPS) particle as a representative natural biocompatible material for use as an effective stabilizer for HIPE formation. HBPS is synthesized by using butyl glycidyl ether as a hydrophobic reagent to change the hydrophobic–hydrophilic balance of starch, and then starch-based particles are fabricated by a simple nanoprecipitation procedure. The size of particles increased with an increase in temperature, and the particles are essentially monodisperse with a PDI of about 0.1 when the temperature was above 15 °C. These HBPS particles were subsequently used as an effective stabilizer to fabricate stable oil-in-water (o/w) Pickering HIPEs with an internal phase volume of 80% at different stabilizer concentrations. The results demonstrated that increasing the particle concentration is conducive to the formation of stable Pickering HIPEs with greater stiffnesses. In addition, the nutraceutical material (β-carotene) was encapsulated into HIPEs and in vitro release experiments revealed that the release in this system can be controlled by adjusting the temperature. © 2020 Elsevier B.V. All rights reserved.
1. Introduction High internal phase emulsions (HIPEs) are often defined as very concentrated emulsions in which the volume fraction of the internal phase is larger than 0.74 [1]. Similar to ordinary dilute emulsions, HIPEs can be classified as either the normal oil-in-water (o/w) or the reverse waterin-oil (w/o) form. In the past few years, HIPEs have shown excellent suitability in a great number of applications including as porous materials, [2–4] templates for gels, [5] scaffolds [6] foams, [7] nutraceutical containers [8,9] and in capillary electrochromatography [10]. Generally, HIPEs can be stabilized by a large number of molecular surfactants (5–50 vol%), such as Hypermer 2296, [11] sorbitan monooleate (Span 80), [12] poly(vinyl alcohol), [13] and trimethylammonium bromide (CTAB) [14], to prevent droplet coalescence and phase inversion. In addition to surfactants, solid particles have also been used to stabilize HIPEs, which are specifically termed Pickering HIPEs [15–17]. The concept of using particles to stabilize emulsions provides a number of benefits compared to the use of ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Tan), [email protected] (K. Xu).
https://doi.org/10.1016/j.ijbiomac.2019.12.269 0141-8130/© 2020 Elsevier B.V. All rights reserved.
conventional molecular surfactants. First, the particles irreversibly adsorb on or self-assemble at the oil–water interface, thus forming rigid layers which hinder droplet coalescence, Ostwald ripening, and creaming [18,19]. Pickering emulsions are extremely stable with shelf life stabilities of months to years [20–22]. Also, colloidal particles are far less toxic to humans and more environmentally friendly than surfactants [23,24]. Furthermore, compared to particles, the large amount of surfactant required to stabilize HIPEs is not cost effective and limits the properties of the resulting emulsions [2,11]. Recently, a wide variety of Pickering HIPE systems have been produced using inorganic and organic particles as stabilizers. The inorganic particles, such as silica, [25] metal oxide, [26] clay, [27] and carbon [28], often need chemical modification or to be mixed with molecular surfactants to achieve the desired emulsifying capacity [25]. On the other hand, polymeric particles, such as poly (divinylbenzene-methacrylic acid) (P(DVB-MAA)), [29] poly{(styrene-alt-maleic acid)-co-[styrene(N-3,4-dihydroxyphenylethylmaleamic acid)]} (P(SMA-dopa)), [30] and polystyrene (PS) particles, [31] have excellent emulsifying properties for the efficient stabilization of emulsions, though their poor biocompatibility has greatly limited their applicability in the cosmetic, pharmaceutical, and food industries. Another interesting approach to
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the design of Pickering emulsifiers is the utilization of natural biopolymers which circumvent the aforementioned issues and shortcomings. Monodispersed gliadin/chitosan hybrid particles were successfully used as particulate emulsifiers for Pickering HIPEs [32]. Gelatin particles synthesized through a two-step desolvation method have also been used to stabilize Pickering HIPEs·[33]. In another work, the synthesis of “green” polyHIPEs was made possible through the use of w/o Pickering HIPEs stabilized with modified bacterial cellulose whiskers [34]. Starch, one of the most abundant biopolymers in nature, is an excellent substrate candidate to meet the food and biomedical requirements due to its biodegradability, derivability, high availability, and low cost. While our group has previously demonstrated the feasibility of preparing starch-based particles for stabilizing Pickering emulsions [35,36] to the best of our knowledge, there is almost no paper reporting the use of environmentally friendly biocompatible starch-based particles as Pickering HIPE stabilizers. In this study, thermoresponsive starchbased nanoparticles derived from 2-hydroxy-3-butoxypropyl starches (HBPS) were used as stabilizers for Pickering HIPEs. The effects of particle concentration on the properties of the HIPE were investigated in detail. Furthermore, the starch nanoparticle-stabilized HIPEs were applied as nutraceutical containers to encapsulate β-carotene. 2. Material and methods 2.1. Material Acidified wax cornstarch with a macromolecular weight of 200,000 was obtained according to a previously reported method [37]. Butyl glycidyl ether (BGE) was provided by Tokyo Chemical Industries (Tokyo, Japan). Natural β-carotene was purchased from Aladdin Chemistry Co., Ltd. Fluorescein isothiocyanate (FITC) and dibutyltin-dilaurate (DBTDL) were supplied by Sigma-Aldrich Co., Ltd. All the other chemicals and soybean oil (Arawana) were used as received. Deionized water was used in all processes. 2.2. Synthesis of 2-hydroxy-3-butoxypropyl starches (HBPS) and fluorescein labelled HBPS Acidified wax cornstarch (8.10 g) was dispersed in distilled water (60 mL) in a 250 mL three-neck flask. NaOH (1.50 g) was added and the mixture was stirred at 75 °C for 1 h in an oil bath. Then, butyl glycidyl ether (6.50 g) was added to the flask and the mixture was allowed to react at 75 °C for 5 h. The resulting suspension was cooled in ice water and the pH was adjusted to 7.0 with hydrochloric acid. Next, the solution was added to acetone in a dropwise fashion to facilitation precipitation. This mixture was then dispersed in a moderate amount of distilled water. This operation was repeated three times. Finally, the products were purified by dialysis in deionized water for three days and dried by lyophilizer. Fluorescein labelled HBPS was synthesized according to a previous method [36,38]. 1.0 g of HBPS was dissolved in 10 mL of DMSO and reacted with 25 mg of FITC in the presence of 0.1 mL of DBTDL for 5 h at 100 °C. Then, the FITC labelled HBPS was precipitated with water and purified by dialysis in deionized water until the filtrate was not fluorescent. 2.3. Preparation of HBPS particles The preparation of starch-based particles was based on a nanoprecipitation method. Typically, HBPS (1 g) was dissolved in 20 mL of N,N-Dimethylformamide (DMF). Then, the polymer solution was added dropwise to 20 mL of distilled water. The resultant particle suspensions were dialyzed to remove all DMF. The suspension was adjusted to the desired concentration by adding or removing deionized water. The fluorescent starch-based particles were also prepared by the same process.
2.4. Preparation of Pickering high internal phase emulsions The Pickering HIPEs were produced by mixing a certain proportion of an aqueous dispersion of HBPS particles with hexane or soybean oil by mechanical shearing with an IKA Ultra Turrax T18 homogenizer operating at 4000 rpm for 120 s. Immediately after homogenization, the emulsion type was determined by conductivity measurement and a drop test. 2.5. In vitro release The in vitro release capabilities of the HBPS particle-stabilized HIPEs were studied on the basis of a membrane-free model [39] to determine their potential as nutraceutical containers, and at the same time, to obtain a thermosensitive controlled release system. First, β-carotene (10 mg) was dissolved in 10 mL of acetone and then mixed with 12 mL of soybean oil. The resulting solution was stirred at room temperature until the acetone was completely vaporized. The HIPEs were prepared by homogenizing 2 mL of 1 wt% HBPS particle suspension with 8 mL of the β-carotene infused soybean oil at 4000 rpm for 120 s. Subsequently, 2.0 g of HIPEs were placed in a conical flask, followed by the careful addition of 8 mL of an aqueous solution of Tween 80 (3 wt %) and 20 mL of hexane. The flasks were kept at certain temperature in a shaking chamber at 70 rpm, and 200 μL of the supernatant was periodically removed for measurement of the absorbance at 450 nm with a UV–vis spectrophotometer (HITACHI, Japan) to determine the amount of β-carotene that was released. 2.6. Characterization The 1H NMR spectra were measured on a Bruker AV400 spectrometer (400 MHz, Ettlingen, Germany). The spectra of the HBPS samples were recorded in d6-DMSO at 25 °C. In addition, 1H NMR was used to calculate the degree of substitution (DS) of HBPS. Since the substituent group contains an –OH, the number of protons (OH-X and H-1) is constant depending on the DS of the modified starch. Thus, we determined the DS by the following equation (Eq. (1)): B B DS ¼ .3 ¼ 1:33 A A
ð1Þ
4
where A is the integral of the peaks representing the hydroxyl and H-1 protons at 4.5–6.6 ppm and B is the integral of the area of methyl protons represented at 0.8 ppm. The size and polydispersity of HBPS particles present in aqueous dispersions at different temperatures (from 10 °C to 35 °C) were measured by dynamic light scattering (DLS) using a 90 Plus particle size analyzer (PSA; Brookhaven) with dilute suspensions (concentration approximately 0.01% in distilled water). The samples were dropped on a silica surface and dried at room temperature to obtain the microscopic appearance of the HBPS nanoparticles by scanning electron microscopy (SEM) using a Merlin FE-SEM (ZEISS). The lower critical solution temperature (LCST) was measured with a UV–vis spectrophotometer (PerkinElmer Lambda 35, America). The transmittance of HBPS particles in aqueous suspensions (5%, w/w) was measured at 500 nm under a heating rate of 2 °C/min. The fluorescein labelled particles were observed at the emulsion droplet interface using confocal laser scanning microscopy (CLSM) with a Leica TCS SP2 CLSM, and the samples were excited by using a 488 nm He/Ne laser. The morphology of the emulsion droplets was observed with digital optical microscopy (MOTIC, China), and the image was processed by microscopic analysis software to obtain the size of the emulsion droplets. Rheological analysis was performed for HBPSstabilized Pickering emulsions formulated using four different nanoparticle concentrations (0.5, 1, 2 and 4 wt%). The rheological analysis of the
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3.2. Characterization of HBPS particles
Fig. 1. 1H NMR spectra of HBPS, where R = H or substituent (depending on the DS).
resulting emulsions was performed at room temperature with a rheometer (AR 2000ex, TA) by a plate-plate mode to measure the storage modulus (G′) and the loss modulus (G″) at varied frequencies (0.1 to 10 Hz). The strain-temperature profile of the 1 wt% HBPS particlestabilized Pickering HIPE was measured at a constant frequency of 1 Hz.
3. Results and discussion 3.1. Synthesis of 2-hydroxy-3-butoxypropyl starches Starch is strongly hydrophilic and thus is difficult to present at the interface of two phases without hydrophobic modification. In this work, butyl glycidyl ether (BGE) was used as hydrophobic modifier to tailor the hydrophilic/hydrophobic nature of starch [40]. The structural features of HBPS were characterized by 1H NMR spectroscopy as illustrated in Fig. 1. Based on previous reports of peak assignment for proton species in starch [41], we assigned the peaks at 4.5–5.6 ppm to OH-2, 3, 6, and H-1 of the anhydroglucose units (AGUs) and OH-8 of the substituent. The peaks at 3.0–4.0 ppm were assigned to the six remaining protons of the AGUs (H-2, 3, 4, 5 and 6), the seven protons of the BGE substituent (–O–CH2–CHOH–CH2–O–CH2–), and H2O (at 3.3 ppm). It is reasonable to assign the peaks at 1.3 ppm and 1.45 ppm to the remaining methylene groups of the butyl portion of the substituent (H11, H-12). Obviously, the peaks at 2.5 and 0.8 ppm correspond to DMSO and the methyl group (H-13), respectively. The degree of substitution (DS) of HBPS can be controlled by the feed amount of BGE. It was noted that HBPS with a low DS was not capable of stabilizing emulsions, while those exhibiting a high DS were unable to disperse very well in aqueous solutions. Therefore, HBPS with a suitable DS of 0.54 was selected for further experimentation.
HBPS particles were prepared through a simple nanoprecipitation procedure in which the polymer solution was added in a dropwise fashion to a nonsolvent, water. This method is essentially based on the interfacial deposition of polymers following the displacement of a semi-polar water-miscible solvent from a lipophilic solution. After the complete removal of DMF, starch nanoparticles with size of ~120 nm were obtained according to DLS characterization. As shown in Fig. 2, SEM images indicated that the size of the HBPS particles was congruent with those measured by DLS. It was also noted that the obtained starch nanoparticles present irregular microscopic morphologies. The introduction of the BGE substituent endowed the starches with certain degree of temperature responsiveness, thus the size and polydispersity of particles in aqueous dispersions at different temperatures (0 °C to 40 °C) were characterized by DLS. As shown in Fig. 3(a), the diameter of the HBPS particles increased from 40 nm to 134 nm with the temperature rises from 0 °C to 40 °C. In particular, the rate of increase between 10 and 30 °C was significantly fast, with rate increases of about 4 nm/°C. In addition, we could get the volume phase transition temperature (VPTT) of particles is about 18 °C. At the same time, the PDI decreased with an increase in temperature, and the particle size distribution was very narrow (PDI ~ 0.1) at temperatures above 15 °C, indicating that the particles were essentially monodispersed. The transmittance changes seen for the 0.5% (w/v) aqueous suspensions of HBPS particles at 500 nm with a heating process suggest that the LCST of the HBPS particles is about 20 °C (Fig. 3(b)), which was almost consistent with the DLS measurements. Fig. 3(c) shows the changes of interfacial tension at the n-hexane– water interface with HBPS particles at different temperature. As we can see, the interfacial tension increased with the increase of temperature, which was opposite to the general situation. It was worth noting that the interfacial tension curve with temperature is consistent with the temperature phase transition curve of the HBPS particles (Fig. 3 (b)). This suggested that temperature affects the hydrophilicity of particles and thus changes the interface properties. As the temperature increased, the HBPS particles became more hydrophobic, resulting in a big interfacial tension. Moreover, it has been reported in relevant literatures that the interfacial tension increases with the increase of particle size, so the increasing particle size with the temperature (Fig. 3(a)) also plays a crucial role in this system [42]. The hydrophobic BGE substituent played an important role in the thermal response of the particles. When the temperature was lower than the LCST, the hydrophilic groups of the macromolecules interacted with surrounding water molecules. Due to the effects of hydrogen bonds and van der Waals forces, the water molecules formed a solvated shell around the macromolecules, connected by hydrogen bonds, so that the hydrophilic chains of macromolecules were fully solvated while the hydrophobic groups condensed to form the nucleus in water. Therefore, the particles were smaller and the transmittance was higher at lower temperatures. With an increase in temperature, the hydrogen bonds formed between the hydrophilic groups and the
Fig. 2. SEM image of HBPS particles with varied magnification: (a) 15 K ×. (b) 50 K ×.
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Fig. 3. (a) The size and polydispersity of HBPS particles in aqueous solution at different temperatures (0 °C to 40 °C) as measured by DLS. (b) Transmittance changes for 0.5% (w/v) aqueous solutions of HBPS particles as obtained by UV–vis spectrophotometry at 500 nm with a heating rate of 1.0 °C/min. (c) Interfacial tension for 0.5% (w/v) aqueous dispersion of HBPS particles in hexane at different temperature.
water molecules were gradually weakened, and the solvation shell was destroyed. In a poor solvent, a polymer optimizes its free energy via intermolecular association among polymer chains, which results in a decrease of the chain translational entropy. Thus, the particle size increased, narrow size distribution and the transmittance decreased at higher temperatures [43]. 3.3. Pickering HIPEs stabilized by HBPS particles Images of the Pickering HIPEs with different oil volume fractions stabilized by 1 wt% HBPS particles (based on external phase) are shown in Fig. 4(a). In this study, the volume fraction of the continuous phase of the emulsions ranges from 10 to 90%. It is obvious to note that the samples with the continuous phase volume fraction of 10% quickly separated into two layers, indicating that the amount of HBPS particles
was insufficient to fully stabilize the emulsion. As the continuous phase volume fraction was increased from 30 to 90%, the cream layer (top) decreased and the aqueous phase (bottom) increased. It was worth noting that all cream layers were o/w type emulsions as determined by observing the outcome of adding a drop of each emulsion to either pure oil or pure water, indicating that emulsion type did not change with the changing oil phase volume fraction. Furthermore, the clear and transparent aqueous phase indicated to some extent that most of particles in the aqueous phase have been sequestered to stabilize the emulsion. Interestingly, stable gel-like Pickering emulsions were obtained when the disperse phase volume fraction was up to 80% (Fig. 4(b)). The results of conductivity measurements (σ = 1.70 S/m) and drop test experiments both suggested that the emulsions were o/w type. In order to investigate the distribution of the starch-based particles in
Fig. 4. Photograph of Pickering HIPEs stabilized by HBPS particles. a. Different continuous phase volume fractions in emulsions, ranging from 10 to 90% (from left to right) stabilized by 1 wt % (based on external phase) HBPS particles. b. Emulsions with varied particle concentration (0.5 to 4 wt% with respect to external phase) and emulsions after one-month storage.
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Fig. 5. CLSM image of prepared emulsion droplets using 2 wt% (based on external phase) fluorescent starch-based particles as an emulsifier for hexane-in-water emulsions with an internal phase fraction of 80%.
these emulsions, fluorescent groups were introduced into the particles by covalent modification of starch with FITC [36]. As shown in Fig. 5, there was a green halo and dense film seen around the emulsion droplets when the emulsion was prepared using a fluorescently labelled emulsifier. This provided direct evidence for the adsorption of particles at the interface between the oil and water phases. Particle concentration is an important parameter governing the stability of particle-stabilized emulsions against coalescence. Therefore, the effects of HBPS particle concentration on the properties of the emulsions were investigated. As shown in Fig. 4(b), o/w HIPEs were obtained as the nanoparticle concentration increased from 0.5 to 4.0 wt% at a fixed oil:water volume ratio of 8:2. Moreover, all emulsions not only displayed prominent stability but also remained stable after one
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month, which further indicated that HBPS particles are suitable for stabilizing Pickering HIPEs. Furthermore, at HBPS concentrations between 1 and 4 wt%, the emulsions exhibited illiquidity and inhibition of creaming. The novel interfacial structure and compressed deformed droplets present in the HIPEs formed a percolating network framework, which ensured that the emulsions were stable against creaming and coalescence [44]. In addition, the droplet size decreased as the concentration of particles increased. Fig. 6 shows the microstructure and droplet size of the HIPEs. The droplet size for 0.5 to 4.0 wt% HBPS particles was 11.14, 8.22, 6.68, and 5.89 μm, respectively. When the internal volume fraction was held constant, a decrease in droplet size means an increase in interfacial area. This is one possible reason why increasing the particle concentration was conducive to the formation of stable Pickering HIPEs with greater stiffnesses. Analogously, this phenomenon has also been reported for gelatin-stabilized HIPEs [45]. The rheological characterization of HIPEs stabilized by different HBPS particle concentrations is shown in Fig. 7. On one hand, the storage modulus of all the measured Pickering HIPEs was significantly higher than the corresponding loss modulus regardless of the particle concentration in the test frequency range (0.1–100 rad/s), indicating that all of the prepared emulsions exhibit elastic or solid-like behavior. However, it was observed that the modulus of these Pickering HIPEs generally increased with an increase in particle concentration from 0.5 to 4.0 wt% at a specific test frequency; which confirmed the enhancement of the gel network. The primary determinant of the rheological behavior of these materials was the extent of particle loading, wherein the gel-like nature became stronger with an increase in concentration of particles available to interact at the interface. 3.4. Release of nutraceutical materials from Pickering HIPEs It has been previously shown that large interstices between particles at the interface give encapsulated compounds the opportunity to diffuse out of the internal phase of emulsions [46]. Pickering HIPEs are advantageous in that they are permeable and can retain lipophilic compounds for long time periods. Furthermore, the o/w emulsifier system is
Fig. 6. Optical microscope images and droplet size distributions of the HIPEs with an internal phase volume of 80% stabilized by different concentration HBPS particles. (a) 0.5 wt% (b) 1 wt% (c) 2 wt% (d) 4 wt%.
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almost no release at 35 and 37.5 °C, the release percentages of βcarotene at 32.5, 30, 27.5, and 25 °C were 32.2, 52.4, 62.1, and 69.0%, respectively. Mathematical modelling was applied to evaluate the kinetics of the β-carotene release. Data were fitted by Eq. (2) using linear regression analysis. Peppas equation : F ¼ kt
Fig. 7. Representative frequency dependence profiles of storage moduli (filled markers) or loss moduli (open markers) for the corresponding fresh Pickering HIPEs stabilized by HBPS particles.
considered to be an effective way to improve the dispersion, stability, and bioavailability of nutrients [47]. In this work, β-carotene, which is of interest as a valuable nutrient, serves as a controlled-release model to gain insight into the release process of an encapsulated bioactive lipid from Pickering HIPEs. Fig. 8(a) shows the β-carotene release curves of Pickering HIPEs stabilized by HBPS particles at different temperatures. It was observed that the rate and amount of β-carotene release were controlled by temperature. That is, both the rate and amount of β-carotene diffusing out of the emulsion were inhibited as the temperature increased. Compared to
n
ð2Þ
where F refers to the fractional drug release, k refers to the release rate constant, t refers to release time, and n refers to the diffusional exponent. The results of fitting the experimental data to the mathematical model were displayed in Fig. 8(b). As we can see, all of the experimental data curves fit the Peppas equation well. The release mechanism and matrix geometry can be judged by the value of n obtained with the Peppas model [48]. All values for n in this system were lower than 0.43, indicating that the β-carotene release mechanism in this system was spherical matrix Fickian diffusion. Moreover, the values of the release rate constant (k) were decreased with an increase in temperature, which confirmed the potential of HBPS particle-stabilized HIPEs as a controlled release platform. The release of encapsulated compounds depends on factors related to the polymer network (composition and network structure), the guest molecule (chemical structure and molecular weight), and the release conditions (temperature and pH) [49]. In this system, temperature played an important role in controlled release. However, in contrast to the general release system where increasing temperature had a positive effect, the rate and amount of release decreased with an increase in temperature. The following explanation has been put forth to explain the relationship between the temperature sensitivity of HBPS particles and the release mechanism. As the temperature increases, the extended threadlike hydrophilic chains coated on the hydrophobic core began to contract, thereby increasing the size of the particles at the phase
Fig. 8. (a) Release curves for β-carotene diffusing from HIPEs stabilized by 1 wt% HBPS particles at different temperatures. (b) The results of fitting experimental data to mathematical models. (c) Rheological strain-temperature profiles of 1 wt% HBPS particle-stabilized Pickering HIPEs measured at a constant frequency of 1 Hz. (d) Release percentages of β-carotene at different temperatures at 100 min.
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Fig. 9. Schematic for temperature-controlled release of β-carotene.
interface such that the pores of the hydrophilic shell become narrower. This further inhibits diffusion of β-carotene out of the inner phase (Fig. 9). To further verify this mechanism, rheology was used to measure the strain-temperature profiles of 1 wt% HBPS particle-stabilized Pickering HIPEs at a constant frequency of 1 Hz (Fig. 8(c)). The important observation that storage modulus of prepared HIPEs gradually increased with temperature can readily be made, reflecting that the gel-like network of emulsions was enhanced by the contraction and aggregation of particles on the interface. In addition, when compared with Fig. 3 and the curve for β-carotene release percentage at different temperatures over the same time period (Fig. 8(d)), it can be found that the release percentage of β-carotene changed rapidly when the particle suspension transmittance and emulsion storage modulus changed greatly (from 25 to 35 °C), while the release percentage of β-carotene changed slightly when the particle suspension transmittance and emulsion storage modulus changed slightly. This interesting phenomenon indicated that the change in interface particles has a significant influence on the release system and supported the above explanation. The internal phase volume of HBPS-particle-stabilized HIPEs is higher (80%) than other starch-based nanoparticles stabilized emulsions with loading drug or nutrient [50,51], therefore they have higher carrying capacity under the same volume of emulsion. Furthermore, compared to other food-grade particle-stabilized emulsion release system, this release behavior shows a faster release rate [9,33]. Specifically, the release percentage can reach 60% in about 80 min at 27.5 °C, which is close to the emulsion stabilized by inorganic solid particles [45]. More importantly, the release behavior of this system is thermoresponsive and can be controlled by adjusting the temperature. In conclusion, the food-grade and biocompatible HBPS-stabilized HIPEs demonstrated excellent performance in release. 4. Conclusions Thermally responsive HBPS particles were prepared by nanoprecipitation and used as emulsifiers to prepare o/w Pickering HIPEs with a stable internal phase volume of 80%. The size of the particles increased with an increase in temperature, and the particles were essentially monodisperse with a PDI of less than 0.1 at temperatures above 20 °C. The experimental results showed that the particle concentration has a great influence on the droplet size distribution and the formation of emulsions. The droplet size decreased from 11.14 to 5.89 μm when HIPEs were formed at particle concentrations ranging from 0.5 to
4 wt%. Increasing the particle concentration was found to be conducive to the formation of stable Pickering HIPEs with greater stiffnesses. Nutraceutical material (β-carotene) was successfully encapsulated by HIPEs and the release could be controlled by adjusting the temperature of the system. The release profiles indicated that a high extent of release of β-carotene occurred at low temperatures (below 30 °C) while almost no release occurred at 37.5 °C. The Peppas model fit well with this release system, suggesting that the β-carotene release mechanism of this system is spherical matrix Fickian diffusion. Moreover, unlike many other synthetic materials, HIPEs stabilized by HBPS particles are based on completely universal food-grade starches and biocompatible natural renewable resources, which provides a new opportunity for the application of starch-stabilized Pickering HIPEs in functional foods and pharmaceuticals. Declaration of competing interest No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed. Acknowledgements Financial support from the National Natural Science Foundation of China (grant numbers 51673191, 21774124, U1762106), special funds of the Science and Technology Cooperation of the Chinese Academy of Sciences (grant number 2018SYHZ0008), and the Changchun Science and Technology Bureau Project (grant no. 18YJ023) is gratefully acknowledged. References [1] B.P. Binks, Chapter 1. Emulsions — recent advances in understanding, Modern Aspects of Emulsion Science 2007, pp. 1–55. [2] N.R. Cameron, High internal phase emulsion templating as a route to well-defined porous polymers, Polymer 46 (5) (2005) 1439–1449. [3] N.C. Grant, A.I. Cooper, H. Zhang, Uploading and temperature-controlled release of polymeric colloids via hydrophilic emulsion-templated porous polymers, ACS Appl. Mater. Interfaces 2 (5) (2010) 1400–1406. [4] Z. Zheng, X. Zheng, H. Wang, Q. Du, Macroporous graphene oxide-polymer composite prepared through Pickering high internal phase emulsions, ACS Appl. Mater. Interfaces 5 (16) (2013) 7974–7982.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Thermoresponsive starch-based particle-stabilized Pickering high internal phase emulsions as nutraceutical containers for controlled release Chao Wang a,b, Xiaopeng Pei a,b, Junling Tan c, Tongwu Zhang c, Kankan Zhai a,b, Fan Zhang a,b, Yungang Bai a, Yukun Deng a, Baichao Zhang a, Yinchuan Wang a, Ying Tan a,⁎, Kun Xu a,⁎, Pixin Wang a a b c
Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China University of Science and Technology of China, Hefei 230026, PR China Oil &Gas Technology Research Institute of Changqing Oilfield Company, Yanan 716000, PR China
a r t i c l e
i n f o
Article history: Received 16 November 2019 Received in revised form 24 December 2019 Accepted 31 December 2019 Available online 03 January 2020 Keywords: Starch-based particle High internal phase emulsions Thermoresponsive Controlled release Pickering emulsions
a b s t r a c t Pickering high internal phase emulsions (HIPEs) stabilized solely by bioderived starch-based particles hold potential for application in the food and pharmaceutical fields. This paper reports the use of a thermoresponsive 2-hydroxy-3-butoxypropyl starch (HBPS) particle as a representative natural biocompatible material for use as an effective stabilizer for HIPE formation. HBPS is synthesized by using butyl glycidyl ether as a hydrophobic reagent to change the hydrophobic–hydrophilic balance of starch, and then starch-based particles are fabricated by a simple nanoprecipitation procedure. The size of particles increased with an increase in temperature, and the particles are essentially monodisperse with a PDI of about 0.1 when the temperature was above 15 °C. These HBPS particles were subsequently used as an effective stabilizer to fabricate stable oil-in-water (o/w) Pickering HIPEs with an internal phase volume of 80% at different stabilizer concentrations. The results demonstrated that increasing the particle concentration is conducive to the formation of stable Pickering HIPEs with greater stiffnesses. In addition, the nutraceutical material (β-carotene) was encapsulated into HIPEs and in vitro release experiments revealed that the release in this system can be controlled by adjusting the temperature. © 2020 Elsevier B.V. All rights reserved.
1. Introduction High internal phase emulsions (HIPEs) are often defined as very concentrated emulsions in which the volume fraction of the internal phase is larger than 0.74 [1]. Similar to ordinary dilute emulsions, HIPEs can be classified as either the normal oil-in-water (o/w) or the reverse waterin-oil (w/o) form. In the past few years, HIPEs have shown excellent suitability in a great number of applications including as porous materials, [2–4] templates for gels, [5] scaffolds [6] foams, [7] nutraceutical containers [8,9] and in capillary electrochromatography [10]. Generally, HIPEs can be stabilized by a large number of molecular surfactants (5–50 vol%), such as Hypermer 2296, [11] sorbitan monooleate (Span 80), [12] poly(vinyl alcohol), [13] and trimethylammonium bromide (CTAB) [14], to prevent droplet coalescence and phase inversion. In addition to surfactants, solid particles have also been used to stabilize HIPEs, which are specifically termed Pickering HIPEs [15–17]. The concept of using particles to stabilize emulsions provides a number of benefits compared to the use of ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Tan), [email protected] (K. Xu).
https://doi.org/10.1016/j.ijbiomac.2019.12.269 0141-8130/© 2020 Elsevier B.V. All rights reserved.
conventional molecular surfactants. First, the particles irreversibly adsorb on or self-assemble at the oil–water interface, thus forming rigid layers which hinder droplet coalescence, Ostwald ripening, and creaming [18,19]. Pickering emulsions are extremely stable with shelf life stabilities of months to years [20–22]. Also, colloidal particles are far less toxic to humans and more environmentally friendly than surfactants [23,24]. Furthermore, compared to particles, the large amount of surfactant required to stabilize HIPEs is not cost effective and limits the properties of the resulting emulsions [2,11]. Recently, a wide variety of Pickering HIPE systems have been produced using inorganic and organic particles as stabilizers. The inorganic particles, such as silica, [25] metal oxide, [26] clay, [27] and carbon [28], often need chemical modification or to be mixed with molecular surfactants to achieve the desired emulsifying capacity [25]. On the other hand, polymeric particles, such as poly (divinylbenzene-methacrylic acid) (P(DVB-MAA)), [29] poly{(styrene-alt-maleic acid)-co-[styrene(N-3,4-dihydroxyphenylethylmaleamic acid)]} (P(SMA-dopa)), [30] and polystyrene (PS) particles, [31] have excellent emulsifying properties for the efficient stabilization of emulsions, though their poor biocompatibility has greatly limited their applicability in the cosmetic, pharmaceutical, and food industries. Another interesting approach to
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the design of Pickering emulsifiers is the utilization of natural biopolymers which circumvent the aforementioned issues and shortcomings. Monodispersed gliadin/chitosan hybrid particles were successfully used as particulate emulsifiers for Pickering HIPEs [32]. Gelatin particles synthesized through a two-step desolvation method have also been used to stabilize Pickering HIPEs·[33]. In another work, the synthesis of “green” polyHIPEs was made possible through the use of w/o Pickering HIPEs stabilized with modified bacterial cellulose whiskers [34]. Starch, one of the most abundant biopolymers in nature, is an excellent substrate candidate to meet the food and biomedical requirements due to its biodegradability, derivability, high availability, and low cost. While our group has previously demonstrated the feasibility of preparing starch-based particles for stabilizing Pickering emulsions [35,36] to the best of our knowledge, there is almost no paper reporting the use of environmentally friendly biocompatible starch-based particles as Pickering HIPE stabilizers. In this study, thermoresponsive starchbased nanoparticles derived from 2-hydroxy-3-butoxypropyl starches (HBPS) were used as stabilizers for Pickering HIPEs. The effects of particle concentration on the properties of the HIPE were investigated in detail. Furthermore, the starch nanoparticle-stabilized HIPEs were applied as nutraceutical containers to encapsulate β-carotene. 2. Material and methods 2.1. Material Acidified wax cornstarch with a macromolecular weight of 200,000 was obtained according to a previously reported method [37]. Butyl glycidyl ether (BGE) was provided by Tokyo Chemical Industries (Tokyo, Japan). Natural β-carotene was purchased from Aladdin Chemistry Co., Ltd. Fluorescein isothiocyanate (FITC) and dibutyltin-dilaurate (DBTDL) were supplied by Sigma-Aldrich Co., Ltd. All the other chemicals and soybean oil (Arawana) were used as received. Deionized water was used in all processes. 2.2. Synthesis of 2-hydroxy-3-butoxypropyl starches (HBPS) and fluorescein labelled HBPS Acidified wax cornstarch (8.10 g) was dispersed in distilled water (60 mL) in a 250 mL three-neck flask. NaOH (1.50 g) was added and the mixture was stirred at 75 °C for 1 h in an oil bath. Then, butyl glycidyl ether (6.50 g) was added to the flask and the mixture was allowed to react at 75 °C for 5 h. The resulting suspension was cooled in ice water and the pH was adjusted to 7.0 with hydrochloric acid. Next, the solution was added to acetone in a dropwise fashion to facilitation precipitation. This mixture was then dispersed in a moderate amount of distilled water. This operation was repeated three times. Finally, the products were purified by dialysis in deionized water for three days and dried by lyophilizer. Fluorescein labelled HBPS was synthesized according to a previous method [36,38]. 1.0 g of HBPS was dissolved in 10 mL of DMSO and reacted with 25 mg of FITC in the presence of 0.1 mL of DBTDL for 5 h at 100 °C. Then, the FITC labelled HBPS was precipitated with water and purified by dialysis in deionized water until the filtrate was not fluorescent. 2.3. Preparation of HBPS particles The preparation of starch-based particles was based on a nanoprecipitation method. Typically, HBPS (1 g) was dissolved in 20 mL of N,N-Dimethylformamide (DMF). Then, the polymer solution was added dropwise to 20 mL of distilled water. The resultant particle suspensions were dialyzed to remove all DMF. The suspension was adjusted to the desired concentration by adding or removing deionized water. The fluorescent starch-based particles were also prepared by the same process.
2.4. Preparation of Pickering high internal phase emulsions The Pickering HIPEs were produced by mixing a certain proportion of an aqueous dispersion of HBPS particles with hexane or soybean oil by mechanical shearing with an IKA Ultra Turrax T18 homogenizer operating at 4000 rpm for 120 s. Immediately after homogenization, the emulsion type was determined by conductivity measurement and a drop test. 2.5. In vitro release The in vitro release capabilities of the HBPS particle-stabilized HIPEs were studied on the basis of a membrane-free model [39] to determine their potential as nutraceutical containers, and at the same time, to obtain a thermosensitive controlled release system. First, β-carotene (10 mg) was dissolved in 10 mL of acetone and then mixed with 12 mL of soybean oil. The resulting solution was stirred at room temperature until the acetone was completely vaporized. The HIPEs were prepared by homogenizing 2 mL of 1 wt% HBPS particle suspension with 8 mL of the β-carotene infused soybean oil at 4000 rpm for 120 s. Subsequently, 2.0 g of HIPEs were placed in a conical flask, followed by the careful addition of 8 mL of an aqueous solution of Tween 80 (3 wt %) and 20 mL of hexane. The flasks were kept at certain temperature in a shaking chamber at 70 rpm, and 200 μL of the supernatant was periodically removed for measurement of the absorbance at 450 nm with a UV–vis spectrophotometer (HITACHI, Japan) to determine the amount of β-carotene that was released. 2.6. Characterization The 1H NMR spectra were measured on a Bruker AV400 spectrometer (400 MHz, Ettlingen, Germany). The spectra of the HBPS samples were recorded in d6-DMSO at 25 °C. In addition, 1H NMR was used to calculate the degree of substitution (DS) of HBPS. Since the substituent group contains an –OH, the number of protons (OH-X and H-1) is constant depending on the DS of the modified starch. Thus, we determined the DS by the following equation (Eq. (1)): B B DS ¼ .3 ¼ 1:33 A A
ð1Þ
4
where A is the integral of the peaks representing the hydroxyl and H-1 protons at 4.5–6.6 ppm and B is the integral of the area of methyl protons represented at 0.8 ppm. The size and polydispersity of HBPS particles present in aqueous dispersions at different temperatures (from 10 °C to 35 °C) were measured by dynamic light scattering (DLS) using a 90 Plus particle size analyzer (PSA; Brookhaven) with dilute suspensions (concentration approximately 0.01% in distilled water). The samples were dropped on a silica surface and dried at room temperature to obtain the microscopic appearance of the HBPS nanoparticles by scanning electron microscopy (SEM) using a Merlin FE-SEM (ZEISS). The lower critical solution temperature (LCST) was measured with a UV–vis spectrophotometer (PerkinElmer Lambda 35, America). The transmittance of HBPS particles in aqueous suspensions (5%, w/w) was measured at 500 nm under a heating rate of 2 °C/min. The fluorescein labelled particles were observed at the emulsion droplet interface using confocal laser scanning microscopy (CLSM) with a Leica TCS SP2 CLSM, and the samples were excited by using a 488 nm He/Ne laser. The morphology of the emulsion droplets was observed with digital optical microscopy (MOTIC, China), and the image was processed by microscopic analysis software to obtain the size of the emulsion droplets. Rheological analysis was performed for HBPSstabilized Pickering emulsions formulated using four different nanoparticle concentrations (0.5, 1, 2 and 4 wt%). The rheological analysis of the
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3.2. Characterization of HBPS particles
Fig. 1. 1H NMR spectra of HBPS, where R = H or substituent (depending on the DS).
resulting emulsions was performed at room temperature with a rheometer (AR 2000ex, TA) by a plate-plate mode to measure the storage modulus (G′) and the loss modulus (G″) at varied frequencies (0.1 to 10 Hz). The strain-temperature profile of the 1 wt% HBPS particlestabilized Pickering HIPE was measured at a constant frequency of 1 Hz.
3. Results and discussion 3.1. Synthesis of 2-hydroxy-3-butoxypropyl starches Starch is strongly hydrophilic and thus is difficult to present at the interface of two phases without hydrophobic modification. In this work, butyl glycidyl ether (BGE) was used as hydrophobic modifier to tailor the hydrophilic/hydrophobic nature of starch [40]. The structural features of HBPS were characterized by 1H NMR spectroscopy as illustrated in Fig. 1. Based on previous reports of peak assignment for proton species in starch [41], we assigned the peaks at 4.5–5.6 ppm to OH-2, 3, 6, and H-1 of the anhydroglucose units (AGUs) and OH-8 of the substituent. The peaks at 3.0–4.0 ppm were assigned to the six remaining protons of the AGUs (H-2, 3, 4, 5 and 6), the seven protons of the BGE substituent (–O–CH2–CHOH–CH2–O–CH2–), and H2O (at 3.3 ppm). It is reasonable to assign the peaks at 1.3 ppm and 1.45 ppm to the remaining methylene groups of the butyl portion of the substituent (H11, H-12). Obviously, the peaks at 2.5 and 0.8 ppm correspond to DMSO and the methyl group (H-13), respectively. The degree of substitution (DS) of HBPS can be controlled by the feed amount of BGE. It was noted that HBPS with a low DS was not capable of stabilizing emulsions, while those exhibiting a high DS were unable to disperse very well in aqueous solutions. Therefore, HBPS with a suitable DS of 0.54 was selected for further experimentation.
HBPS particles were prepared through a simple nanoprecipitation procedure in which the polymer solution was added in a dropwise fashion to a nonsolvent, water. This method is essentially based on the interfacial deposition of polymers following the displacement of a semi-polar water-miscible solvent from a lipophilic solution. After the complete removal of DMF, starch nanoparticles with size of ~120 nm were obtained according to DLS characterization. As shown in Fig. 2, SEM images indicated that the size of the HBPS particles was congruent with those measured by DLS. It was also noted that the obtained starch nanoparticles present irregular microscopic morphologies. The introduction of the BGE substituent endowed the starches with certain degree of temperature responsiveness, thus the size and polydispersity of particles in aqueous dispersions at different temperatures (0 °C to 40 °C) were characterized by DLS. As shown in Fig. 3(a), the diameter of the HBPS particles increased from 40 nm to 134 nm with the temperature rises from 0 °C to 40 °C. In particular, the rate of increase between 10 and 30 °C was significantly fast, with rate increases of about 4 nm/°C. In addition, we could get the volume phase transition temperature (VPTT) of particles is about 18 °C. At the same time, the PDI decreased with an increase in temperature, and the particle size distribution was very narrow (PDI ~ 0.1) at temperatures above 15 °C, indicating that the particles were essentially monodispersed. The transmittance changes seen for the 0.5% (w/v) aqueous suspensions of HBPS particles at 500 nm with a heating process suggest that the LCST of the HBPS particles is about 20 °C (Fig. 3(b)), which was almost consistent with the DLS measurements. Fig. 3(c) shows the changes of interfacial tension at the n-hexane– water interface with HBPS particles at different temperature. As we can see, the interfacial tension increased with the increase of temperature, which was opposite to the general situation. It was worth noting that the interfacial tension curve with temperature is consistent with the temperature phase transition curve of the HBPS particles (Fig. 3 (b)). This suggested that temperature affects the hydrophilicity of particles and thus changes the interface properties. As the temperature increased, the HBPS particles became more hydrophobic, resulting in a big interfacial tension. Moreover, it has been reported in relevant literatures that the interfacial tension increases with the increase of particle size, so the increasing particle size with the temperature (Fig. 3(a)) also plays a crucial role in this system [42]. The hydrophobic BGE substituent played an important role in the thermal response of the particles. When the temperature was lower than the LCST, the hydrophilic groups of the macromolecules interacted with surrounding water molecules. Due to the effects of hydrogen bonds and van der Waals forces, the water molecules formed a solvated shell around the macromolecules, connected by hydrogen bonds, so that the hydrophilic chains of macromolecules were fully solvated while the hydrophobic groups condensed to form the nucleus in water. Therefore, the particles were smaller and the transmittance was higher at lower temperatures. With an increase in temperature, the hydrogen bonds formed between the hydrophilic groups and the
Fig. 2. SEM image of HBPS particles with varied magnification: (a) 15 K ×. (b) 50 K ×.
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Fig. 3. (a) The size and polydispersity of HBPS particles in aqueous solution at different temperatures (0 °C to 40 °C) as measured by DLS. (b) Transmittance changes for 0.5% (w/v) aqueous solutions of HBPS particles as obtained by UV–vis spectrophotometry at 500 nm with a heating rate of 1.0 °C/min. (c) Interfacial tension for 0.5% (w/v) aqueous dispersion of HBPS particles in hexane at different temperature.
water molecules were gradually weakened, and the solvation shell was destroyed. In a poor solvent, a polymer optimizes its free energy via intermolecular association among polymer chains, which results in a decrease of the chain translational entropy. Thus, the particle size increased, narrow size distribution and the transmittance decreased at higher temperatures [43]. 3.3. Pickering HIPEs stabilized by HBPS particles Images of the Pickering HIPEs with different oil volume fractions stabilized by 1 wt% HBPS particles (based on external phase) are shown in Fig. 4(a). In this study, the volume fraction of the continuous phase of the emulsions ranges from 10 to 90%. It is obvious to note that the samples with the continuous phase volume fraction of 10% quickly separated into two layers, indicating that the amount of HBPS particles
was insufficient to fully stabilize the emulsion. As the continuous phase volume fraction was increased from 30 to 90%, the cream layer (top) decreased and the aqueous phase (bottom) increased. It was worth noting that all cream layers were o/w type emulsions as determined by observing the outcome of adding a drop of each emulsion to either pure oil or pure water, indicating that emulsion type did not change with the changing oil phase volume fraction. Furthermore, the clear and transparent aqueous phase indicated to some extent that most of particles in the aqueous phase have been sequestered to stabilize the emulsion. Interestingly, stable gel-like Pickering emulsions were obtained when the disperse phase volume fraction was up to 80% (Fig. 4(b)). The results of conductivity measurements (σ = 1.70 S/m) and drop test experiments both suggested that the emulsions were o/w type. In order to investigate the distribution of the starch-based particles in
Fig. 4. Photograph of Pickering HIPEs stabilized by HBPS particles. a. Different continuous phase volume fractions in emulsions, ranging from 10 to 90% (from left to right) stabilized by 1 wt % (based on external phase) HBPS particles. b. Emulsions with varied particle concentration (0.5 to 4 wt% with respect to external phase) and emulsions after one-month storage.
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Fig. 5. CLSM image of prepared emulsion droplets using 2 wt% (based on external phase) fluorescent starch-based particles as an emulsifier for hexane-in-water emulsions with an internal phase fraction of 80%.
these emulsions, fluorescent groups were introduced into the particles by covalent modification of starch with FITC [36]. As shown in Fig. 5, there was a green halo and dense film seen around the emulsion droplets when the emulsion was prepared using a fluorescently labelled emulsifier. This provided direct evidence for the adsorption of particles at the interface between the oil and water phases. Particle concentration is an important parameter governing the stability of particle-stabilized emulsions against coalescence. Therefore, the effects of HBPS particle concentration on the properties of the emulsions were investigated. As shown in Fig. 4(b), o/w HIPEs were obtained as the nanoparticle concentration increased from 0.5 to 4.0 wt% at a fixed oil:water volume ratio of 8:2. Moreover, all emulsions not only displayed prominent stability but also remained stable after one
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month, which further indicated that HBPS particles are suitable for stabilizing Pickering HIPEs. Furthermore, at HBPS concentrations between 1 and 4 wt%, the emulsions exhibited illiquidity and inhibition of creaming. The novel interfacial structure and compressed deformed droplets present in the HIPEs formed a percolating network framework, which ensured that the emulsions were stable against creaming and coalescence [44]. In addition, the droplet size decreased as the concentration of particles increased. Fig. 6 shows the microstructure and droplet size of the HIPEs. The droplet size for 0.5 to 4.0 wt% HBPS particles was 11.14, 8.22, 6.68, and 5.89 μm, respectively. When the internal volume fraction was held constant, a decrease in droplet size means an increase in interfacial area. This is one possible reason why increasing the particle concentration was conducive to the formation of stable Pickering HIPEs with greater stiffnesses. Analogously, this phenomenon has also been reported for gelatin-stabilized HIPEs [45]. The rheological characterization of HIPEs stabilized by different HBPS particle concentrations is shown in Fig. 7. On one hand, the storage modulus of all the measured Pickering HIPEs was significantly higher than the corresponding loss modulus regardless of the particle concentration in the test frequency range (0.1–100 rad/s), indicating that all of the prepared emulsions exhibit elastic or solid-like behavior. However, it was observed that the modulus of these Pickering HIPEs generally increased with an increase in particle concentration from 0.5 to 4.0 wt% at a specific test frequency; which confirmed the enhancement of the gel network. The primary determinant of the rheological behavior of these materials was the extent of particle loading, wherein the gel-like nature became stronger with an increase in concentration of particles available to interact at the interface. 3.4. Release of nutraceutical materials from Pickering HIPEs It has been previously shown that large interstices between particles at the interface give encapsulated compounds the opportunity to diffuse out of the internal phase of emulsions [46]. Pickering HIPEs are advantageous in that they are permeable and can retain lipophilic compounds for long time periods. Furthermore, the o/w emulsifier system is
Fig. 6. Optical microscope images and droplet size distributions of the HIPEs with an internal phase volume of 80% stabilized by different concentration HBPS particles. (a) 0.5 wt% (b) 1 wt% (c) 2 wt% (d) 4 wt%.
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almost no release at 35 and 37.5 °C, the release percentages of βcarotene at 32.5, 30, 27.5, and 25 °C were 32.2, 52.4, 62.1, and 69.0%, respectively. Mathematical modelling was applied to evaluate the kinetics of the β-carotene release. Data were fitted by Eq. (2) using linear regression analysis. Peppas equation : F ¼ kt
Fig. 7. Representative frequency dependence profiles of storage moduli (filled markers) or loss moduli (open markers) for the corresponding fresh Pickering HIPEs stabilized by HBPS particles.
considered to be an effective way to improve the dispersion, stability, and bioavailability of nutrients [47]. In this work, β-carotene, which is of interest as a valuable nutrient, serves as a controlled-release model to gain insight into the release process of an encapsulated bioactive lipid from Pickering HIPEs. Fig. 8(a) shows the β-carotene release curves of Pickering HIPEs stabilized by HBPS particles at different temperatures. It was observed that the rate and amount of β-carotene release were controlled by temperature. That is, both the rate and amount of β-carotene diffusing out of the emulsion were inhibited as the temperature increased. Compared to
n
ð2Þ
where F refers to the fractional drug release, k refers to the release rate constant, t refers to release time, and n refers to the diffusional exponent. The results of fitting the experimental data to the mathematical model were displayed in Fig. 8(b). As we can see, all of the experimental data curves fit the Peppas equation well. The release mechanism and matrix geometry can be judged by the value of n obtained with the Peppas model [48]. All values for n in this system were lower than 0.43, indicating that the β-carotene release mechanism in this system was spherical matrix Fickian diffusion. Moreover, the values of the release rate constant (k) were decreased with an increase in temperature, which confirmed the potential of HBPS particle-stabilized HIPEs as a controlled release platform. The release of encapsulated compounds depends on factors related to the polymer network (composition and network structure), the guest molecule (chemical structure and molecular weight), and the release conditions (temperature and pH) [49]. In this system, temperature played an important role in controlled release. However, in contrast to the general release system where increasing temperature had a positive effect, the rate and amount of release decreased with an increase in temperature. The following explanation has been put forth to explain the relationship between the temperature sensitivity of HBPS particles and the release mechanism. As the temperature increases, the extended threadlike hydrophilic chains coated on the hydrophobic core began to contract, thereby increasing the size of the particles at the phase
Fig. 8. (a) Release curves for β-carotene diffusing from HIPEs stabilized by 1 wt% HBPS particles at different temperatures. (b) The results of fitting experimental data to mathematical models. (c) Rheological strain-temperature profiles of 1 wt% HBPS particle-stabilized Pickering HIPEs measured at a constant frequency of 1 Hz. (d) Release percentages of β-carotene at different temperatures at 100 min.
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Fig. 9. Schematic for temperature-controlled release of β-carotene.
interface such that the pores of the hydrophilic shell become narrower. This further inhibits diffusion of β-carotene out of the inner phase (Fig. 9). To further verify this mechanism, rheology was used to measure the strain-temperature profiles of 1 wt% HBPS particle-stabilized Pickering HIPEs at a constant frequency of 1 Hz (Fig. 8(c)). The important observation that storage modulus of prepared HIPEs gradually increased with temperature can readily be made, reflecting that the gel-like network of emulsions was enhanced by the contraction and aggregation of particles on the interface. In addition, when compared with Fig. 3 and the curve for β-carotene release percentage at different temperatures over the same time period (Fig. 8(d)), it can be found that the release percentage of β-carotene changed rapidly when the particle suspension transmittance and emulsion storage modulus changed greatly (from 25 to 35 °C), while the release percentage of β-carotene changed slightly when the particle suspension transmittance and emulsion storage modulus changed slightly. This interesting phenomenon indicated that the change in interface particles has a significant influence on the release system and supported the above explanation. The internal phase volume of HBPS-particle-stabilized HIPEs is higher (80%) than other starch-based nanoparticles stabilized emulsions with loading drug or nutrient [50,51], therefore they have higher carrying capacity under the same volume of emulsion. Furthermore, compared to other food-grade particle-stabilized emulsion release system, this release behavior shows a faster release rate [9,33]. Specifically, the release percentage can reach 60% in about 80 min at 27.5 °C, which is close to the emulsion stabilized by inorganic solid particles [45]. More importantly, the release behavior of this system is thermoresponsive and can be controlled by adjusting the temperature. In conclusion, the food-grade and biocompatible HBPS-stabilized HIPEs demonstrated excellent performance in release. 4. Conclusions Thermally responsive HBPS particles were prepared by nanoprecipitation and used as emulsifiers to prepare o/w Pickering HIPEs with a stable internal phase volume of 80%. The size of the particles increased with an increase in temperature, and the particles were essentially monodisperse with a PDI of less than 0.1 at temperatures above 20 °C. The experimental results showed that the particle concentration has a great influence on the droplet size distribution and the formation of emulsions. The droplet size decreased from 11.14 to 5.89 μm when HIPEs were formed at particle concentrations ranging from 0.5 to
4 wt%. Increasing the particle concentration was found to be conducive to the formation of stable Pickering HIPEs with greater stiffnesses. Nutraceutical material (β-carotene) was successfully encapsulated by HIPEs and the release could be controlled by adjusting the temperature of the system. The release profiles indicated that a high extent of release of β-carotene occurred at low temperatures (below 30 °C) while almost no release occurred at 37.5 °C. The Peppas model fit well with this release system, suggesting that the β-carotene release mechanism of this system is spherical matrix Fickian diffusion. Moreover, unlike many other synthetic materials, HIPEs stabilized by HBPS particles are based on completely universal food-grade starches and biocompatible natural renewable resources, which provides a new opportunity for the application of starch-stabilized Pickering HIPEs in functional foods and pharmaceuticals. Declaration of competing interest No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed. Acknowledgements Financial support from the National Natural Science Foundation of China (grant numbers 51673191, 21774124, U1762106), special funds of the Science and Technology Cooperation of the Chinese Academy of Sciences (grant number 2018SYHZ0008), and the Changchun Science and Technology Bureau Project (grant no. 18YJ023) is gratefully acknowledged. References [1] B.P. Binks, Chapter 1. Emulsions — recent advances in understanding, Modern Aspects of Emulsion Science 2007, pp. 1–55. [2] N.R. Cameron, High internal phase emulsion templating as a route to well-defined porous polymers, Polymer 46 (5) (2005) 1439–1449. [3] N.C. Grant, A.I. Cooper, H. Zhang, Uploading and temperature-controlled release of polymeric colloids via hydrophilic emulsion-templated porous polymers, ACS Appl. Mater. Interfaces 2 (5) (2010) 1400–1406. [4] Z. Zheng, X. Zheng, H. Wang, Q. Du, Macroporous graphene oxide-polymer composite prepared through Pickering high internal phase emulsions, ACS Appl. Mater. Interfaces 5 (16) (2013) 7974–7982.
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