All-Natural Food-Grade HydrophilicâHydrophobic CoreâShell

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Applications of Polymer, Composite, and Coating Materials

All-Natural Food-Grade Hydrophilic-Hydrophobic Core-Shell Microparticles: Facile Fabrication Based on Gel-Network-Restricted Antisolvent Method Bing Hu, Lingyu Han, Ruixiang Ma, Glyn O. Phillips, Katsuyoshi Nishinari, and Yapeng Fang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b00980 • Publication Date (Web): 07 Mar 2019 Downloaded from http://pubs.acs.org on March 11, 2019

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ACS Applied Materials & Interfaces

All-Natural Food-Grade Hydrophilic-Hydrophobic Core-Shell Microparticles: Facile Fabrication Based on Gel-Network-Restricted Antisolvent Method Bing Hu‡1, Lingyu Han‡1, Ruixiang Ma1, Glyn O. Phillips1, Katsuyoshi Nishinari1, and Yapeng Fang2* 1. Phillips Hydrocolloid Research Centre, School of Food and Biological Engineering, Hubei University of Technology, Wuhan 430068, China 2. Department of Food Science and Engineering, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China KEYWORDS: core-shell microparticle, hydrophilic-hydrophobic structure, restricted antisolvent precipitation, moisture resistance, controlled release

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ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

Hydrophilic-hydrophobic core-shell microparticles are highly appealing for a variety of industrial applications (foods, pharmaceutics, cosmetics, biomedicines, etc.) owing to their unique properties of moisture resistance and controlled release. However, the fabrication of such structured microparticles proves to be nontrivial due to the difficulty in assembling two materials of distinctly different hydrophilicity and hydrophobicity. This paper reports a facile method to fabricate hydrophilic-hydrophobic core-shell microparticles using all-natural food-grade polysaccharide and protein, based on a novel principle of gel-network-restricted antisolvent precipitation. Immersion of microgel beads prepared from hydrophilic polysaccharide (i.e., alginate, -carrageenan, agarose) into a hydrophobic protein solution (i.e., zein in 70% aqueous ethanol), enables slow and controllable antisolvent precipitation of a protein layer around the microbead surface, leading to the formation of hydrophilic-hydrophobic core-shell structure. The method applies to various gelling systems and can easily tailor the particle size and shell thickness. The resulting freeze-dried microparticles demonstrate restricted swelling in water, improved moisture resistance and sustained release of encapsulants, with great potential in applications such as protection of unstable and/or hygroscopic compounds, delivery and controlled release of drugs, bioactives and flavors, etc. The method is rather universal, and can be extended to prepare more versatile core-shell structures using a large variety of hydrophilic and hydrophobic materials.


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INTRODUCTION Core-shell microparticles are structured composite particles consisting of at least two different components, one in principle forming the core and the other forming the shell of the particles.1 Due to their unique structural properties, they are highly appealing for a variety of applications such as in foods,2, 3 pharmaceutics,4, 5 cosmetics6 and biomedicines.7, 8 They are used to enhance the physical and chemical stabilities of encapsulated compounds and to control their release kinetics.9-11 Core-shell microparticles exhibit superior properties through integration of the characteristics of both the core and shell materials, thus bringing novel and synergistic functionalities.8, 12 Materials of different hydrophilicity and hydrophobicity are often combined to formulate core-shell microparticles of different types, such as hydrophobic-hydrophobic, hydrophobic-hydrophilic, hydrophilic-hydrophilic, hydrophilic-hydrophobic structures. This allows the encapsulation and protection of functional compounds of different natures, and tailoring their delivery and releasing properties. Various methods have been proposed to fabricate microparticles with core-shell structures. Chemical methods, e.g., emulsion or dispersion polymerizations, usually involves multiple steps where seed particles are first synthesized or introduced as the core and subsequently a second monomer is polymerized or grafted on to the seed particles to form the shell. Interfacial polymerization of active surfactants that stabilize emulsions also results in the formation of coreshell microparticles. Physical methods normally involve the deposition/solidification of wall materials on to the surface of emulsion droplets or particles by antisolvent precipitation, electrostatic layer-by-layer adsorption or coaxial electrospray,13 etc. For instance, Filippidi et al. prepared oil-filled core-shell microcapsules by antisolvent precipitation of zein protein from the continuous phase of an oil-in-(water/ethanol) emulsion onto the oil droplets.14 Patel et al.


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reported the synthesis of cellulose ether-polyphenol core-shell beads by utilizing the strong interactions

between

epigallocatechin

gallate

and

methyl

cellulose/hydroxylpropyl

methylcellulose.15 Qin et al. fabricated chitosan-alginate core-shell gel beads by one-step dripping of Ca2+ containing chitosan solution into pH 10.0 alginate solution.16 The chitosan core and alginate shell were solidified simultaneously by pH-responsive and ionotropic gelations, respectively. Methods based on microfluidic technique have also been used to prepare core-shell microparticles, which are characterized by their monodispersity.17, 18 However, many of the approaches mentioned above suffer from low biocompatibility or low efficiency of production. The application of chemical approaches or synthetic materials seriously limits their use in food, drug and personal care products. In particular, the fabrication of biocompatible hydrophilic-hydrophobic core-shell microparticles proves to be a challenge, owing to the difficulty in creation and stabilization of a hydrophobic outer shell in aqueous environments that are typically required for biocompatibility and edibility. In addition, some approaches like microfluidic fabrication are difficult to be scaled up and lacks of production efficiency. In view of these, development of novel methods for facile and scalable fabrication of biocompatible and/or edible hydrophilic-hydrophobic core-shell microparticles, is highly demanded. Herein, we report the synthesis of biocompatible core-shell microparticles based on a novel principle of gel-network-restricted antisolvent precipitation. It is realized simply by immersing pre-formed hydrogel beads into a solution containing the water-insoluble hydrophobic shell material. Slow solvent exchange taking place at the surface of hydrogel beads, restricted and controlled by gel network, leads to a gradual antisolvent precipitation of the shell material around the gel beads, generating structurally well-defined core-shell microparticles. We further


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show that the resulting microparticles have unique properties of moisture resistance and releasing functional encapsulants. A method to prepare pectin-zein complex microspheres has been attempted to load drugs, by dropwise addition of pectin aqueous solution into zein-ethanol solution (75%) containing drugs and solidifying agents such as CaCl2 and ZnSO4.19 However, due to uncontrolled antisolvent process, the method was incapable of generating core-shell morphology. The method is generally applicable to a large variety of hydrophilic and hydrophobic materials. To showcase, we here produce biocompatible and edible core-shell microparticles using allnature food-grade materials, i.e., gelling polysaccharides including alginate,20 -carrageenan21 and agarose22 as the hydrophilic core, and a prolamin zein23 as the hydrophobic shell. These polysaccharides have been long used in the food industry for stabilizing, thickening and gelling applications. They undergo sol-gel transitions upon addition of ions such as Ca2+ , K+ ions or by cooling.24-26 Due to excellent biocompatibility and biodegradability, they have been extensively used for pharmaceutical and biomedical applications such as tissue engineering.27, 28 Zein is an FDA-approved GRAS material derived from corn.29 It is composed of a high proportion (>50%) of hydrophobic amino acids (proline, alanine, and leucine).30 Zein is water insoluble, but can be solubilized in binary mixture of water and ethanol if the ethanol content is in the range of 60%95%.31 This unique feature makes zein a good candidate for design of colloidal delivery systems for encapsulation of drugs,32 nutraceuticals,33 pigments34, 35 and flavors,36 and for modifying food optical properties.37 Additionally, owing to its film-forming and hydrophobic properties, zein is widely used as a packaging material to provide moisture impervious barrier.29


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RESULTS AND DISCUSSION Fabrication of Core-Shell Microparticles The process to fabricate core-shell microparticles is schematically illustrated in Figure 1. It involves the antisolvent precipitation of zein from water-ethanol solution (70% v/v ethanol) upon addition of pre-formed hydrogel microparticles. Solvent exchange takes place at the surface of hydrogel microparticles, where water diffuses out of the gel microparticles and in opposite direction ethanol diffuses into the microparticles. The outflowed water, as an antisolvent of zein, decreases the ethanol content in close vicinity of hydrogel surface, and reduces the solubility of zein and triggers its precipitation around the surface of gel microparticles. This results in the formation of a hydrophobic zein layer coated around the hydrophilic gel microparticles (Figure 1). It is crucial that the amount of hydrogel microparticles added, thus the water contained therein, should be controlled so that the overall content of ethanol after mixing is below ca. 45 v/v %. Insufficient amount of hydrogel microparticles cannot initiate zein precipitation, or the formed zein shell is again eroded away. (a)

H2O

Pre-formed hydrogel microbeads

Zein in 70% EtOH

Shell formation

EtOH Restricted antisolvent precipitation

Zein

(b)

(c)

200mm


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Figure 1. Schematic illustration of the fabrication of hydrophilic-hydrophobic core-shell microparticles using gel-network-restricted antisolvent precipitation: mixing pre-formed hydrogel microbeads with a zein solution (1-3% w/v) in 70% v/v aqueous ethanol leads to a slow solvent exchange of ethanol/water in and out of the gel beads. The zein gradually precipitates around the surface of hydrogel microbead, forming a hydrophobic shell around the hydrophilic core. The transparent hydrogel beads (a) turn completely opaque (b) upon shell formation (c), as shown by light microscopy and confocal scanning laser microscopy. The kinetics of this process is controlled by the gel network of the hydrogel microbeads. The seeding gel microparticles can be produced beforehand using various methods such as electrospray/dripping into crosslinking solution,16, 38 and water-in-oil emulsification followed by solidification of the internal dispersed phase.39 In the present work, templated gelation using water-in-oil emulsion was adopted. Various natural polysaccharides including alginate, carrageenan and agarose were selected to verify the universality of the method. Alginate has a calcium-induced sol-gel transition since it can bind with Ca2+ to form egg-box junction zones. κcarrageenan and agarose form thermo-reversible gels upon cooling, as a result of coil-to-helix transition and the ensuing helix aggregation. K+ ions specifically promote the gelation of κcarrageenan. These polysaccharide solutions were firstly emulsified with 80% v/v middle chain triglyceride (MCT) containing 2% w/v L-- phosphatidylcholine, to form water-in-oil emulsions. The internal droplets of the emulsions were then gelled by internal or external introduction of Ca2+ and K+ ions or by cold-setting, and harvested by phase inversion or filtration. The preparation of hydrogel microparticles and the subsequent antisolvent precipitation, therefore, involve purely biocompatible and food-grade materials and agents.


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Figure 2 shows the optical images of the core-shell microparticles prepared using the different polysaccharides as hydrophilic core. The polysaccharide gel microparticles are initially transparent under light microscopy (column I), and become completely opaque after mixing with zein solution in 70% v/v aqueous ethanol (column II). This is due to the precipitation and formation of a solid and compact zein shell at the surface of the gel microparticles. The shells look homogenous and well defined under fluorescence and confocal microscopies (column III and IV), and are several tens of micrometers in thickness. The shells are formed in all the polysaccharide gel systems, regardless of their gelation mechanisms. The method, therefore, should apply principally to any material capable of forming a hydrogel. (I)

(IV)

(III)

(II)

Alginate

-carrageenan

Agarose

200mm

200mm

200mm

200mm

Figure 2. Hydrophilic-hydrophobic core-shell microparticles prepared using gel-networkrestricted antisolvent precipitation of 2% w/v zein in 70% v/v aqueous ethanol against hydrogel microparticles of 3% w/v alginate (upper panel), 1.5% w/v-carrageenan (middle panel) and 0.5%


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w/v agarose (bottom panel): before antisolvent precipitation (column I) and after antisolvent precipitation (column II, III and IV). Light microscopic images (column I and II); Fluorescence microscopic images (column III, Rhodamine B-stained); Confocal scanning laser microscopic images (column IV, Nile Blue-stained). The overall ethanol content was kept at 45% v/v by taking account of the water contained in hydrogel microparticles. A stirring speed of 500, 300 and 200 rpm was used to prepare the seeding alginate,-carrageenan and agarose gel microparticles, respectively. Note that the images in different columns were not necessarily taken on the same microparticles. Underlying Mechanism To unveil the formation mechanism of the core-shell microparticles, we monitor in situ the structural evolution of the zein shell upon antisolvent precipitation using confocal scanning laser microcopy (CSLM). Figure 3a displays the CSLM images of an individual κ-carrageenan (1.5% w/v) gel microparticle, taken at different time points upon mixing with 2% w/v zein in 70% v/v aqueous ethanol. The images reveal clearly a gradual process of shell formation around the gel microparticle. Within the first 2 mins, no visible shell structure is formed. A blurred shell profile starts to appear at t = 2.5 min. This indicates that the precipitation of zein at the gel microparticle surface has created a shell thickness enough to be resolved by the microscope. With prolonged incubation time, the shell become increasingly distinguishable and grows in thickness. A complete zein shell encasing the gel microparticle is achieved approximately at 10 min. Afterwards, little change is observed with the structure of the shell. The shell formation kinetics was quantified by measuring the average shell thickness as a function of time. The average shell thickness was obtained by CLSM image analysis using Image J software at different locations of the shell profile (>8 for a single microparticle) and over


9


a sufficient number of microparticles (>100). Figure 3b compares the kinetics of shell formation around an individual hydrogel microparticle prepared with different

κ-carrageenan

concentrations. The onset of shell formation is postponed from 1.0 min, to 2.5 mins and further to 3.5 mins, when κ-carrageenan concentration is increased from 1.0%, 1.5% to 2.5% w/v. The growth of shell thickness fits reasonably well to the logistic function:

𝐿=

𝐿𝑒 −𝑘(𝑡−𝑡 0.5 ) 1+𝑒

(eq. 1)

Where Le is the equilibrium shell thickness, k is the shell growth rate, and t0.5 is the time required to reach the half of the equilibrium shell thickness. Le only slightly decreases from 24.7 to 22.8 mm with increasing κ-carrageenan concentration from 1.0% to 2.5% w/v. The kinetic parameters k and t0.5, however, are greatly affected by increasing κ-carrageenan concentration, with t0.5 being more than doubled from 3.1 to 7.4 min. This indicates that the kinetics of the precipitation of zein shell is controlled by hydrogel network concentration. 0 min

(a)

3.5 min

7.5 min

11.0 min

0.5 min

4.0 min

8.5 min

12.0 min

1.5 min

5.5 min

9.0 min

13.0 min

2.0 mins

30

2.5 min

6.0 min

6.5 min

10.0 min

10.5 min

13.5 min

14.5 min

200mm

(b) Average shell thickness (mm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.0% carrageenan

25

1.5% carrageenan

20 2.5% carrageenan

15

10

5

0 0

5

10 Time (min)

15

20

Figure 3. (a) In-situ CSLM observation of the zein shell formation around an individual carrageenan (1.5% w/v) hydrogel microparticle upon mixing with 2% w/v zein in 70% v/v aqueous ethanol. The zein shell is stained with Nile Blue. (b) Kinetics of the zein shell formation


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as affected by -carrageenan concentration of the hydrogel microparticle: (□) 1.0% w/v; (△) 1.5% w/v; (○) 2.5% w/v. The solid lines represent the curve fittings to Eq. 1: Le=24.7+0.4 mm, k=0.88+0.06 min-1, t0.5=3.1+0.1 min for 1.0% w/v -carrageenan; Le=24.3+0.4 mm, k=0.67+0.05 min-1, t0.5=4.9+0.1 min for 1.5% w/v -carrageenan; Le=22.8+0.2 mm, k=0.54+0.02 min-1, t0.5=7.4+0.1 min for 2.5% w/v -carrageenan. Hydrogel microparticle is a three-dimensional network swollen by the solvent water. When it comes in contact with zein solution in water/ethanol, osmotic pressure would drive the outflow of water molecules. This on one hand induces the antisolvent precipitation of zein in the close vicinity of microparticle surface, forming a shell, and on the other hand causes the shrinking of the gel network. The latter would counterbalance the former due to the presence of bulk modulus exerted by the gel network.40 Therefore, the kinetics of antisolvent precipitation and shell formation is indirectly controlled and restricted by the gel network. The higher the gel bulk modulus, the slower the shell formation. Indeed, κ-carrageenan gels with concentrations of 1.0%, 1.5% and 2.5% w/v exhibited significantly different storage moduli of about 5000, 20000, and 120000 Pa (Figure S1), respectively, which can account for the great difference in shell formation kinetics observed in Figure 3. Moreover, ethanol is expected to flow in an opposite direction. The diffusion of ethanol into the gel microparticles could decrease the solubility of the gel network and cause its collapse. This also leads to a restricted-antisolvent precipitation of zein. It should be noted that not only the kinetics of antisolvent precipitation but also its equilibrium extent is limited by the gel network. The limited extent of antisolvent precipitation of zein, hence the reduction in Le, is due to a less amount of water that diffuses out of the hydrogel microparticle with a higher gel bulk modulus. Therefore, gel-network-restricted antisolvent precipitation of zein is the principal mechanism that governs the formation of


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hydrophilic-hydrophobic core-shell structure. Considering one extremity, i.e., in the absence of gel network, the mixing of polysaccharide solution with zein aqueous ethanol elicits an instantaneous antisolvent process that kinetically is too fast to be controlled. It leads to irregular precipitates of zein rather than a structurally well-defined shell (Figure S2). It should be pointed out that judging visually from Figure 3a, no significant shrinking of the inner gel microparticle occurs during antisolvent process. It means that only a very small amount of water from the inner gel phase is sufficient for inducing the precipitation and formation of a zein shell locally at the gel microparticle surface. Microparticle Morphology The morphology of the hydrophilic-hydrophobic core-shell microparticles prepared using gelnetwork-restricted antisolvent precipitation was characterized by field-emission scanning electron microscopy (SEM). Alginate-zein core-shell microparticles, formed by mixing 2% w/v zein in 70% v/v aqueous ethanol with hydrogel microparticles of 3% w/v alginate, are taken as an example (Figure 4). Microparticles with and without shell formation are both spherical. Compared with the seeding alginate microparticle (Figure 4a), the zein-coated alginate microparticle exhibits a much rougher surface (Figure 4b and d). Under careful examination, the surface has a granular texture and seems to be made of heterogenous aggregates of numerous small zein particles. Similar structures have been observed in core-shell microcapsules prepared by antisolvent precipitation of zein onto the oil droplets of an oil-in-water emulsion,14 and in carbon nanotubes/protein core-shell hybrids prepared by heating-induced aggregation and deposition of -lactoglobulin.41 It implies that the shell formation may proceed via heterogenous growing mechanism where separated zein protein clusters nucleate and expand at the surface of the seeding hydrogel microparticles, and ultimately coalesce into a continuous shell with


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structural integrity. Meanwhile, compression test shows that the presence of a zein shell adds to the mechanical strength of alginate microparticles (Figure S3). (a)

(b)

200mm

200mm

(c)

(d) (d)

200mm

(e)

40mm

40mm

Figure 4. (a) Representative SEM images of a freeze-dried alginate hydrogel microparticle without (a) and with (b) zein shell. Cross-sectional SEM image of a core-shell microparticle is shown in (c). The indicated area in (b) and (c) are expanded and shown in (d) and (e), respectively. The core-shell microparticles are fabricated using gel-network-restricted antisolvent precipitation of 2% w/v zein in 70% v/v aqueous ethanol mixed with hydrogel microparticles of 3% w/v alginate. Cross-sectional image of the microparticles confirms the core-shell structure (Figure 4c and e). The core is sponge-like, while the shell is much more solid and compact. The sponge-like structure results from the hydrogel network of the core after water is removed by freeze drying. The compact shell structure differs from porous shells formed by more conventional antisolvent precipitation. This might indicate an interesting feature of gel-network-restricted antisolvent precipitation where slow kinetics allows gradual precipitation and more intimate deposition of


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zein. The shell has a thickness of ~ 20 mm (Figure 4e), which is comparable to that determined by CSLM (Figure 2). It should be noted that although the fabrication of core-shell structure is demonstrated for spherical particles, the proposed method of gel-network-restricted antisolvent precipitation should apply to a range of morphologies, such as prolates, fibers, and films, which are mainly determined by the shapes of seeding hydrogel particles. Furthermore, the inner hydrogel phase can be melted out by heating or using other physical/chemical methods to prepare hollow microparticles.42 Regulation of Size and Shell Thickness The size of the hydrophilic-hydrophobic core-shell microparticles is dictated by seeding hydrogel microparticles, and can be tuned by adjusting experimental conditions. In this work, the stirring/homogenization speed during emulsification step was varied to control the size of seeding hydrogel microparticles, and thus that of final hydrophilic-hydrophobic core-shell microparticles. The particle size was analyzed by using Nano Measurer (version, 1.2) software over a sufficient number of microparticles (>100). Figure 5 demonstrates that the particle size is easily tailored within the range of several hundreds to several tens of microns, simply by changing stirring/homogenization speed. The average particle size of alginate-zein core-shell microparticles is reduced from 666 to 98 mm when stirring/homogenization speed is increased from 500 to 10000 rpm. Irrespective of the change in microparticle size, well-defined zein shells are created in all the cases. Table 1 summarizes the effect of stirring/homogenization speed on the average size of different polysaccharide-zein core-shell microparticles. Due to the limited ability of reducing particle size by emulsification, only micron-sized core-shell particles are produced in the work. However, the method using gel-network-restricted antisolvent


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precipitation should work similarly for fabrication of nano-sized core-shell particles, as long as further smaller seeding hydrogel microparticles are employed. Moreover, the fabrication of monodisperse core-shell microparticles could also be possible if hydrogel microparticles with low polydispersity index, e.g., those produced by microfluidic technique43 or microchannel emulsification44, are used as seeding particles. (a)

(II)

(I)

200mm

200mm

(III)

(IV)

25

500 rpm

200mm

Frequency

20

200mm

15 10 5 0 0

300

600

900

1200

Particle diamter (mm) 25

Frequency

20

5000 rpm

15 10 5 0 0

300

600

900

1200

Particle diamter (mm) 25 20

Frequency

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10000 rpm

15 10 5 0

200mm

(b)

I.0% Zein

200mm

0

200mm

200mm

2.0% Zein

200mm

300

600

900

1200

Particle diamter (mm)

3.0% Zein

200mm

Figure 5. (a) Regulation of the size of core-shell microparticles by stirring/homogenization speeds at 500, 1000, and 10000 rpm: light microscopic images (column I); fluorescence microscopic images (column II, Rhodamine B-stained); confocal scanning laser microscopic


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images (column II, Nile Blue-stained); particle size distribution (column IV). The core-shell microparticles were prepared by antisolvent precipitation of 2.0% w/v zein in 70% aqueous ethanol against hydrogel microparticles of 3.0% w/v alginate. (b) Regulation of the shell thickness of core-shell microparticles by zein concentration, as shown by confocal scanning laser microscopy. The core-shell microparticles were prepared by antisolvent precipitation of 1.0-3.0% w/v zein in 70% aqueous ethanol against hydrogel microparticles of 1.5% w/v -carrageenan. The thickness of the shell of the hydrophilic-hydrophobic core-shell microparticles is controllable either by the hydrogel network concentration as already shown in Figure 3b, or by the zein concentration. Here, we demonstrate the effect of zein concentration on the resulting shell thickness (Figure 5). For the same seeding hydrogel microparticles of 1.5% w/v κcarrageenan, increasing zein concentration leads to a roughly proportional increase in shell thickness. The zein shell thickness is 8, 18, and 22 mm, respectively, for a zein concentration of 1%, 2% and 3% w/v. Similar trends are observed for other polysaccharide-zein core-shell microparticles (Table 1). It should be pointed out that at the same zein concentration, increasing stirring/homogenization speed during the emulsification step leads to a decrease in shell thickness (Table 1). This should be explained by an increase in the specific surface area of finer microparticles at higher homogenization speed. Therefore, core-shell microparticles with different shell thickness can be easily realized by antisolvent precipitation against zein solutions of different concentrations. The control of shell thickness by zein concentration seems to be much more effective than by hydrogel network concentration, i.e., the concentration of polysaccharides.


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Table 1. Control of the size and shell thickness of hydrophilic-hydrophobic core-shell microparticles by stirring/homogenization speed during emulsification and zein concentration. Core materials

3% w/v alginate

1.5% w/v -carrageenan

0.5% w/v agarose

a)

Zein

Stirring speed

[% w/v]

[rpm]

Average particle Average size thickness [mm]

[mm]

1.0

500a)

653±139

11±0.8

2.0

500a)

666±147

23±1.0

3.0

500a)

671±149

27±1.2

2.0

5000b)

160±42

15±0.9

2.0

10000b)

98±28

10±0.6

1.0

300a)

368±85

8±0.6

2.0

300a)

378±94

18±0.8

3.0

300a)

384±99

22±0.6

2.0

400a)

293±76

16±0.9

2.0

800a)

121±32

12±0.5

1.0

200a)

408±93

9±0.7

2.0

200a)

418±95

16±0.7

3.0

200a)

423±95

21±1.0

2.0

500a)

164±38

15±0.5

2.0

1000a)

99±30

10±0.6

shell

(achieved by a low-speed mechanical blender); b) (achieved by a high-speed homogenizer)

Swelling and Moisture Resistance Due to its hydrophobic nature, zein is a well-known moisture barrier. The zein-coated hydrophilic-hydrophobic core-shell microparticles are expected to possesses distinctly different swelling and moisture resistance properties from the bare hydrophilic core. As shown in Figure


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6a, the freeze-dried alginate hydrogel microparticle, without the zein shell, swells gradually from the edge to the core when exposed to water, and turns transparent at 24 h. In contrast, the freezedried alginate-zein core-shell microparticle shows little sign of swelling even at 24 h. The swelling of inner alginate network is completely inhibited by the presence of an outer zein shell. This is attributed to the compact structure of zein shell that blocks the diffusion of water molecules. We also compare the moisture sorption behaviors of the microparticles by conducting dynamic vapor sorption (DVA) measurements. The microparticles were allowed to equilibrate at different relative humidity (RH), and the change in equilibrium mass was recorded as a function of RH during sorption-desorption circles. For freeze-dried alginate hydrogel microparticles without zein shell, the mass increase reaches about 20% when RH is elevated from 0 to 90%. Significant amount of moisture is adsorbed by the microparticles. Moreover, the desorption curve exhibits a pronounced hysteresis when RH is decreased from 90% to 0%. This indicates a retention of the moisture by the alginate hydrogel network. In comparison, the freeze-dried alginate-zein coreshell microparticles only have a < 3% mass increase when RH increases from 0 to 90%, and almost no hysteresis occurs during the sorption-desorption circle. The negligible amount of moisture uptake can be attributed to the capillary effect arising from the rough surface of the core-shell microparticles as revealed in Figure 4b. The results manifest an excellent moisture resistance property of the core-shell microparticles, conferred by the hydrophobic zein shell. The present method using gel-network-restricted antisolvent precipitation, therefore, provides a new route to creating hydrophobic surface layers or shells on top of a gel network, for enhanced moisture resistance and effective protection of hydroscopic compounds encapsulated therein.


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(a) Without shell 1h

8h

1h

8h

16 h

24 h

With shell 16 h

24 h

200mm

(b) 20

Change In Mass (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15 Without shell

10

5 With shell

0 0

20

40 60 Relative Humidity (%)

80

100

Figure 6. Swelling in water (a) and moisture sorption-desorption (b) of freeze-dried alginate hydrogel microparticles without and with zein shell. The core-shell microparticles were prepared by antisolvent precipitation of 2.0% w/v zein in 70% aqueous ethanol against hydrogel microparticles of 3% w/v alginate. In Vitro Release of Encapsulants We further investigate the potential applications of the hydrophilic-hydrophobic core-shell microparticles for encapsulation and controlled release of food functional factors and flavors. Pyridoxine and ethyl acetate were chosen as representative encapsulants. The former is known as vitamin B6, and has been often incorporated in food products as dietary supplement for preventative treatment of pyridoxine deficiency, sideroblastic anaemia, epilepsy, and certain


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metabolic disorders, etc. The latter is widely used as a model volatile compound for flavor release. Figure 7a compares the release in water of 0.2% w/v pyridoxine from bare alginate hydrogel microparticles and alginate-zein core-shell microparticles. The release is well approximated by the equation of first-order kinetics45:

𝑷𝒕 = 𝑷𝒊𝒏𝒇 (𝟏 − 𝒆−𝒌𝒕 )

(eq. 2)

where Pt and Pinf are the release percentage at time t and infinite time, respectively, and k is the rate constant. For bare alginate hydrogel microparticles, pyridoxine is nearly fully released at prolonged time (Pinf = 98.9%). In comparison, a much lower percentage of pyridoxine is released from alginate-zein core-shell microparticles (Pinf = 29.9%). The rate constant k is also reduced from 0.0754 to 0.062 min-1when the zein shell is present. The considerable difference in Pinf can be attributed to the rather solid and compact structure of the zein shell (Figure 4e), which greatly restricts the penetration of pyridoxine and water as observed in Figure 6a. Figure 7b displays the release profiles of 1.0% w/v ethyl acetate from alginate-zein core-shell microparticles of different shell thickness. The release percentage is normalized by the equilibrium headspace concentration of 1.0% w/v ethyl acetate aqueous solution. The core-shell microparticles prepared with 1.0% w/v and 2.0% w/v zein solutions have an average shell thickness of 11 and 23 mm (Table 1), respectively. Ethyl acetate is found to be released in a shell-thickness-dependent manner. The process is relatively faster at the initial stage and then reaches pseudo-equilibrium at about 10 min. The initial release rate (IRP),46 obtained from the slope of the release curve in the first 5 mins, decreases from 0.16 to 0.11 and then to 0.06 min-1 when shell thickness is increased from 0 to 11 and then to 23 mm. Correspondingly, the percentage of release at equilibrium also decreases markedly from about 80% to 50% and further down to 40% with increasing shell thickness. It should be pointed out that the slight drop in


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release percentage at later stage of the release could be caused by constant GC samplings of the headspace, which decreases the equilibrium flavor concentration. Similar phenomenon has also been observed by Chen et al.47 (a) 100% Pinf = 98.9%±1.2% k = 0.0754±0.0036

Release %

80%

No zein

60%

Pinf = 29.9%±0.7% k = 0.0682±0.0061

40% 20%

2.0% zein

0% 0

20

40

60

80

100

Time (min)

(b) 100%

No zein

IRR =0.16

80% Release %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.0% zein

IRR =0.11

60%

40%

2.0% zein IRR =0.06

20% 0% 0

5

10

15

20

25

30

Time (min)

Figure 7. Release profiles of pyridoxine (a) and ethyl acetate (b) from alginate hydrogel microparticles with and without zein shell: without zein shell (●); shell formed with 1.0% w/v zein (■); shell formed with 2.0% w/v zein (▲). The core-shell microparticles were prepared by antisolvent precipitation of zein solutions in 70% aqueous ethanol against hydrogel


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microparticles of 3% w/v alginate. The solid lines in (a) represent curve fittings to the first-order kinetics, and those in (b) guides the initial slopes of the release curves during the first 5 mins. The above results clearly indicate a retention and sustained release of pyridoxine and ethyl acetate. This is apparently due to the presence of the outer zein shell around alginate hydrogel microparticles. It is also demonstrated that the core-shell structure can effectively protect sensitive compounds such as vitamin C from oxidation (Figure S4). Therefore, the hydrophilichydrophobic core-shell microparticles, prepared using gel-network-restricted antisolvent precipitation, can be utilized to encapsulate functional and active components for stabilization and controlled release. Furthermore, it has been reported that zein exhibits slow digestibility by proteases such as trypsin and pepsin.14 In combination with this property, the core-shell microparticles as proposed in this work might be potential systems for controlled digestion of lipids and starches, and for oral delivery and sustained release of nutraceuticals/pharmaceuticals that are vulnerable in gastrointestinal conditions.48 CONCLUSION The work demonstrates a facile method to fabricate hydrophilic-hydrophobic core-shell microparticles using all-natural food-grade materials, based on a novel principle of gel-networkrestricted antisolvent precipitation. The method involves no use of toxic chemical agents and/or synthetic substances, and thus is biocompatible and edible. The method is highly extensible, and in principal applies to any core material that is capable of forming a hydrogel and any shell material that is insoluble and precipitates in water. The morphology, size and shell thickness of the core-shell microparticles can be easily controlled by the geometry of seeding hydrogel microparticles and the conditions of antisolvent precipitation, e.g., the concentration of shell material. Due to a gradual and restricted dynamics of antisolvent precipitation, the resulting shell


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has a rather compact structure, which greatly impacts on the swelling, moisture resistance and releasing properties of the core-shell microparticles. The method therefore can be used to create hydrophilic-hydrophobic core-shell structure for protection of unstable and hydroscopic compounds, enhancement of moisture resistance, and encapsulation and controlled release of drugs, bioactives, and flavors etc. In view of its biocompatibility and edibility, the method is particularly useful in formulating delivery systems for applications in food, cosmetics, pharmaceutics and biomedicines. EXPERIMENTAL SECTION Chemical and Materials. Zein from corn (Z3625) and L-- phosphatidylcholine from soybean (P5638) were purchased from Sigma Aldrich. Sodium type alginate and -carrageenan were kindly provided by FMC BioPolymer (Norway) and used without further purification. Agarose was purchased from BioFroxx (Germany). Medium Chain Triglyceride (MCT) was purchased from KLK Oleo, Ltd. (Malaysia). Ethanol, glacial acetic acid, calcium carbonate, and potassium chloride were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ethyl acetate, pyridoxine and ascorbic acid were purchased from Macklin Reagent Co., Ltd. (Shanghai, China). Milli-Q water was used in all experiments. Preparation of Seeding Hydrogel Microparticles. The seeding hydrogel microparticles were produced by using water-in-oil emulsion as a template followed by solidification of the internal dispersed phase. Stock solution of 3% w/w sodium alginate containing 50 mM CaCO3, 0.5% w/w agarose or 1.5% w/w -carrageenan in water was prepared. 1 part in weight of each stock solution was mixed with 4 parts of MCT containing 2% w/w L-- phosphatidylcholine. Depending on the targeted particle size, the mixtures were emulsified with a low-speed mechanical blender (Eurostar 20 Digital, IKA, German) at 200-1000 rpm for 15 min or a high-


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speed homogenizer (T25 digital ULTRA TURRAX®, IKA, German) at 5000-10000 rpm for 3 min, to produce W/O emulsions. Different methods were adopted to solidify the internal dispersed phases. Solidification of alginate seeding gel microparticles was achieved by Ca2+induced gelation. One part in weight of MCT containing 2.7% w/w glacial acetic acid was slowly added into alginate-in-MCT emulsion. The release of calcium ions from CaCO3 induced by acidification triggers the gelation of alginate. Solidification of agarose seeding gel microparticles was done by cold-setting. The agarose-in-MCT emulsion prepared at 80oC was quenched in an ice bath at 4 oC to induce the gelation of agarose. Solidification of -carrageenan seeding gel microparticles was carried out by K+-induced gelation. One part in weight of MCT mixing with 2g KCl solution (1 M) was slowly added into -carrageenan-in-MCT emulsion to induce the gelation of -carrageenan internal phase. Constant stirring around 50 rpm was necessary for all solidifications in order to prevent gel microparticles from coalescing. Fabrication of Hydrophilic-Hydrophobic Core-Shell Microparticles. Depending on the targeted shell thickness, 1% w/v, 2% w/v and 3% w/v zein in 70% v/v aqueous ethanol stock solutions were prepared. The pre-formed seeding gel microparticles were added into the zein aqueous ethanol to induce the antisolvent precipitation of zein. The water contained the seeding gel particles were considered so that the total ethanol content was adjusted to 45% v/v. The antisolvent precipitation process usually took tens of minutes under constant stirring at 50 rpm. Optical Microscopy. The optical microscope images of microparticles were taken using an Olympus optical microscope (BX50, Olympus, and Tokyo, Japan) with 4 × objective. Size distribution of the microparticles was measured by statistical analysis of optical microscopic images using Nano Measurer (version, 1.2) software. At least 500 randomly selected microparticles were used for each statistical analysis.


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Fluorescence Microscope. Fluorescent images were acquired on an IX73 inverted microscope (Olympus, Tokyo, Japan) equipped with a 4 × objectives with excitation at 530-550 nm. The zein protein was stained with rhodamine B (10−3 parts of 1% fluorophore in water solution). Confocal Laser Scanning Microscopy. Nile Blue (10−3 parts of 1% fluorophore in water solution) was used to stain the zein protein. CLSM images of the microparticles were obtained on an inverted laser-scanning confocal microscope (Leica TCS-SP8) with 10 × objective and excitation at 633 nm probing the Nile Blue local environment of the protein. Shell thickness of the microparticles was measured by analysis of CLSM images using Image J software. The shell thickness was determined at different locations of the shell profile (>8 for a single microparticle) and over a sufficient number of microparticles (>100). Average values with standard deviation were reported. Scanning Electron Microscopy (SEM). Microparticle morphology was observed by fieldemission SEM (Helios NanoLab G3, FEI, United States). Microparticles with and without shell were frozen by liquid nitrogen and then lyophilized. For cross-sectional imaging, the lyophilized samples were again treated with liquid nitrogen, and microtomed immediately. The samples then were mounted on a metal stub with the aid of double-sided tape and sputter coated with gold before being photographed with a scanning electron microscope at 15 kV. Dynamic Vapor Sorption. The moisture sorption behavior of the microparticle samples were measured using a dynamic vapor sorption system (DVS-1, Surface Measurement Systems Ltd., London, UK). 5–10 mg of powder (lyophilized microparticles with and without shell) was placed in the measurement chamber under continuous N2 gas flow and at 25 oC. The RH inside the chamber were step-changed from 0 to 90%, with 10% increments or decrements for sorption and


25


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desorption cycles, respectively. Equilibrated mass was recorded when the value of dm/dt was less than 0.002% per minute. Swelling experiment. Lyophilized pure sodium alginate and core-shell gel microparticles were submerged in Milli-Q water. In situ swelling of the microparticles was observed by an optical microscope (BX50, Olympus, Tokyo, Japan) with 4 × objective at different time intervals (1 h, 8 h, 16 h, 24 h). Encapsulation and Release of Pyridoxine. Pyridoxine-loaded microparticles were prepared using 3% w/w sodium alginate containing 50 mM CaCO3 and 0.2% w/v pyridoxine, according to the method afore-mentioned using a stirring speed of 500 rpm. The microparticles with and without shell containing pyridoxine were harvested and frozen by liquid nitrogen and then lyophilized. The release of pyridoxine in water was determined by ultraviolet spectroscopy based on the absorbance at 290 nm. 0.03 g microparticles with and without zein shell containing 0.2% w/v pyridoxine were mixed with 20 g water, and absorbance at 290 nm was measured on a UVVis spectrophotometer (TU-1900, Beijing, China) at different time intervals. The release percentage was calculated by normalization using the theoretical amount of pyridoxine contained in the microparticles. Encapsulation and Release of Ethyl Acetate. Ethyl acetate-loaded microparticles with and with zein shell were fabricated similarly using 3% w/w sodium alginate containing 50 mM CaCO3 and 1% w/v ethyl acetate. Zein solutions were also incorporated with 1% w/v ethyl acetate. The as-prepared microparticles were harvested and then immersed in 1% w/v ethyl acetate solution at 4oC to prevent the loss of the encapsulated before release measurements. The release kinetics of ethyl acetate was measured using dynamic headspace analysis. It was carried out by adding 1 g of the wet microparticles into a closed vessel of 100 mL at 40 oC. 1.0 mL of


26

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the headspace gas was collected by Combi-PAL autosampler (CTC Analytics AG, Zwingen, Switzerland) using a 2.5 mL thermostatic gastight syringe (Hamilton, Bonaduz, Switzerland) at prescribed time intervals (1.5 min) and then injected into Agilent 7890B gas chromatograph (GC) system for detection. The release percentage was calculated by normalization against the equilibrium headspace concentration of 1 g of 1.0% w/v ethyl acetate aqueous solution. The initial release rate (IRP) was obtained from the slope of the release curve within the first 5 mins by linear regression. ASSOCIATED CONTENT Supporting Information. Rheological characterization of hydrogel modulus, CLSM image of unrestricted antisolvent precipitation of zein, compression profiles of core-shell microparticles, encapsulation and protection of ascorbic acid. AUTHOR INFORMATION Corresponding Author *

Email: [email protected]

Author Contributions ‡These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The work is funded by the grants from the National Natural Science Foundation of China (No. 31671811 and No. 31701555), the State Key Research and Development Plan “Modern food


27


processing

and

food

storage

and

transportation

technology and

Page 28 of 35

equipment”

(No.

2017YFD0400200), and the Science and Technology Commission of Shanghai Municipality (No. 18JC1410801) ABBREVIATIONS DVS, dynamic vapor sorption system; RH, relative humidity; SEM, scanning electron microscopy; CLSM, confocal laser scanning microscopy; IRP, initial release rate; GC, gas chromatograph; MCT, medium chain triglyceride. REFERENCES 1.

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29. Elisangela, C.; Curti, P. S.; Meniqueti, A. B.; Martins, A. F.; Rubira, A. F.; Edvani Curti, M., Recent Advances in Food-Packing, Pharmaceutical and Biomedical Applications of Zein and Zein-Based Materials. Int. J. Mol. Sci. 2014, 15, 22438-22470. 30. Patel, A. R.; Velikov, K. P., Zein as a Source of Functional Colloidal Nano- and Microstructures. Curr. Opin. Colloid Interf. Sci. 2014, 19, 450-458. 31. Chen, G. Y.; Ali, F.; Dong, S.; Yin, Z. L.; Li, S. H.; Chen, Y., Preparation, Characterization and Functional Evaluation of Chitosan-Based Films with Zein Coatings Produced by Cold Plasma. Carbohydr. Polym. 2018, 202, 39-46. 32. Chen, S.; Han, Y. H.; Sun, C. X.; Dai, L.; Yang, S. F.; Wei, Y.; Mao, L. K.; Yuan, F.; Gao, Y. X., Effect of Molecular Weight of Hyaluronan on Zein-Based Nanoparticles: Fabrication, Structural Characterization and Delivery of Curcumin. Carbohydr. Polym. 2018, 201, 599-607. 33. Hu, S. Q.; Wang, T. R.; Fernandez, M. L.; Luo, Y. C., Development of Tannic Acid Cross-Linked Hollow Zein Nanoparticles as Potential Oral Delivery Vehicles for Curcumin. Food Hydrocoll. 2016, 61, 821-831. 34. Kasaai, M. R., Zein and Zein-Based Nano-Materials for Food and Nutrition Applications: A Review. Trends In Food Sci. Tech. 2018, 79, 184-197. 35. Patel, A. R.; Heussen, P. C. M.; Dorst, E.; Hazekamp, J.; Velikov, K. P., Colloidal Approach to Prepare Colour Blends from Colourants with Different Solubility Profiles. Food Chem. 2013, 141, 1466-1471.


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43. Li, W.; Zhang, L. Y.; Ge, X. H.; Xu, B. Y.; Zhang, W. X.; Qu, L. L.; Choi, C. H.; Xu, J. H.; Zhang, A.; Lee, H. M.; Weitz, D. A., Microfluidic Fabrication of Microparticles for Biomedical Applications. Chem. Soc. Rev. 2018, 47, 5646-5683. 44. Bhatti, H. S.; Khalid, N.; Uemura, K.; Nakajima, M.; Kobayashi, I., Formulation and Characterization of Food Grade Water-in-Oil Emulsions Encapsulating Mixture of Essential Amino Acids. Eur. J. Lipid Sci. Technol. 2017, 119, 1600202. 45. Peleg, M.; Normand, M. D.; Corradini, M. G., A New Look at Kinetics in Relation to Food Storage. Annual Rev. Food Sci. Technol. 2017, 8, 135-153. 46. Mao, L. K.; Roos, Y. H.; Miao, S., Study on the Rheological Properties and Volatile Release of Cold-Set Emulsion-Filled Protein Gels. J. Agric. Food Chem. 2014, 62, 11420-11428. 47. Chen, X. W.; Guo, J.; Wang, J. M.; Yin, S. W.; Yang, X. Q., Controlled Volatile Release of Structured Emulsions Based on Phytosterols Crystallization. Food Hydrocoll. 2016, 56, 170179. 48. Patel, A. R.; Remijn, C.; Cabero, A. M.; Heussen, P. C. M.; Seijen ten Hoorn, J. W. M.; Velikov, K. P., Novel All-Natural Microcapsules from Gelatin and Shellac for Biorelated Applications. Adv. Funct. Mater. 2013, 23, 4710-4718.


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Table of Contents Graphic For Table of Contents Only

(a)

H2O

Pre-formed hydrogel microbeads

Zein in 70% EtOH

Shell formation

EtOH Restricted antisolvent precipitation

Zein

(b)

(c)

200mm


35

All-Natural Food-Grade HydrophilicâHydrophobic CoreâShell

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