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Cite This: Biomacromolecules XXXX, XXX, XXX−XXX

Polyelectrolyte Complex Inclusive Biohybrid Microgels for Tailoring Delivery of Copigmented Anthocyanins Chen Tan,† Giovana B. Celli,† Michelle Lee,† Jonathan Licker,‡ and Alireza Abbaspourrad*,† †

Department of Food Science, Cornell University, Stocking Hall, Ithaca New York 14853, United States PepsiCo Research and Development, Plano, Texas 75024, United States

‡

S Supporting Information *

ABSTRACT: This study fabricated a novel biohybrid microgel containing polysaccharide-based polyelectrolyte complexes (PECs) for anthocyanins. Herein, anthocyanins were encapsulated into PECs composed of chondroitin sulfate and chitosan, followed by incorporation into alginate microgels using emulsification/internal gelation method. We demonstrated that PECs incorporation strongly affected the properties of microgels, dependent on the polysaccharide concentration and pH in which they were fabricated. The dense internal network surrounded by an alginate shell was clearly visualized in crosssectioned PECs-microgels. Stability studies carried out under varying ionic strength and pH conditions demonstrated the stimuli-responsiveness of the PECs-microgels. Additionally, the presence of PECs conferred microgels with high rigidity during freeze-drying and excellent reconstitution capacity upon rehydration. These observations were attributed to the modulation of electrostatic and hydrogen-bonding cross-linking between PECs and the alginate gel matrix and suggest the PECs inclusive microgels hold promise as delivery vehicles for the controlled release of hydrophilic bioactive compounds.

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INTRODUCTION Increased consumer demand and regulatory pressure has driven interest in the food industry for substitution of synthetic colorants with natural pigments. In addition to color, natural pigments are often associated with health-promoting properties, such as the high antioxidant potential noted of anthocyanins.1,2 However, a common limitation to the use of natural colorants is their susceptibility to degradation by various processing and storage conditions, such as heat, changes in pH, and exposure to light and oxygen, 3 which ultimately impairs their functionality, color, and overall product’s sensory attributes. Anthocyanins are examples of such compounds; their stability is improved in acidic environments, where they are mostly found in the red flavylium cation form. Color variations toward purple/blue hues are observed at pH values close to neutral as a result of molecular changes that also increase the pigment susceptibility to degradation by nucleophilic attack and hydrolysis reactions.4 In this context, micro/nanoencapsulation is often referred to as an alternative to improve the stability of natural ingredients.5 Encapsulation can extend anthocyanin stability by providing a physicochemical barrier against harsh environmental conditions. For instance, Khazaei et al.6 showed that the entrapment of anthocyanins from saffron petals in a freeze-dried maltodextrin or gum arabic matrix resulted in higher retention of these compounds during storage in comparison to an unencapsulated control. However, a limitation often observed with these techniques (e.g., spray drying, freeze-drying, and © XXXX American Chemical Society

ionotropic gelation) is the low encapsulation efficiency of hydrophilic compounds that tend to leach from the particles during processing.7 Another alternative that can improve the stability of anthocyanins is by copigmentation, a phenomenon in which a copigment (e.g., phenolic acids, flavonoids, metal ions) stabilizes and protects the flavylium cation from nucleophilic attack of water.8 Copigmentation also promotes color variations in the form of hyperchromic and bathochromic shifts. Depending on the compounds involved, a different mechanism can explain the interaction between anthocyanins and copigments, such as self-association, intra- or intermolecular copigmentation, and metal complexation.9 However, these complexes are mostly formed by noncovalent bonds and depend on certain environmental conditions that will favor their formation.10 For instance, variations in pH can affect the complexation of anthocyanins with polysaccharides, such as noted by Fernandes et al.11 The copigmented complex might also dissociate when expose to high temperatures or a different solvent system.12 Overall, these alternatives still face challenges with respect to anthocyanin encapsulation efficiency and preservation, making multistrategy approaches highly attractive for many applications. In this study, we explored a unique biohybrid structure as delivery system for anthocyanin, as a model of hydrophilic colorant. Our multistrategy was to combine copigmentation Received: February 28, 2018 Published: March 27, 2018 A

DOI: 10.1021/acs.biomac.8b00352 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 1. Schematic representation of the synthesis of anthocyanin-loaded CS-CHS PECs through sequential copigmentation and encapsulation (A) and PECs-incorporated microgels via emulsification/internal gelation technique (B). cP) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Chondroitin sulfate (CHS) type A from bovine trachea cartilage (Mw = 5−10 kDa) was purchased from Bulk Supplements (Henderson, NV, U.S.A.). Sodium alginate (Manugel GHB, M/G ratio of 0.59) was kindly donated by FMC BioPolymer (Philadelphia, PA, U.S.A.). The anthocyanin source used in all experiments herein was an blueberry extract obtained from Bulk Supplements; anthocyanin content in this extract was measured to be approximately 25% (w/w) according to the protocol outlined using the pH differential method.13 All other chemicals used were analytical grade. Preparation of Polyelectrolyte Complexes (PECs). The anthocyanin-loaded PECs were prepared following the procedure illustrated in Figure 1A. Briefly, the anthocyanin-rich extract was dissolved in deionized water and adjusted pH to 4.2 and 5.5 with acetic acid. The solution was then filtered using a 0.22 μm syringe filter to remove insoluble particles. CHS and CS were dissolved separately in acidified deionized water (1% acetic acid, v/v), and the pH was adjusted to 4.2 or 5.5. The concentrations of polysaccharide ranged from 0.5 to 1.5 mg/mL. The CHS/anthocyanin complex was first prepared by adding anthocyanin to CHS solution at an equal volume. After incubation for 30 min, the copigmented complexes were added dropwise into CS solution of different concentrations (0.5, 1.0, and 1.5 mg/mL). For all formulations, the final concentration of anthocyanin was fixed at 1.67 mg/mL. Anthocyanin-loaded CS-CHS PECs were stored at 4 °C in the dark until further analysis. Preparation of PECs-Inclusive Hybrid Microgels. Microgels were synthesized by emulsification/internal gelation technique according to the procedure by Ching et al.,14 with some modifications. A schematic of the procedure is illustrated in Figure 1B. The aqueous phase consisted of PEC suspension and 10 mL of 1% (w/v) alginate solution at pH 4.2 or 5.5. A total of 40 mL of corn oil with 0.8 mL of Span 80 was used as the oil phase. After adding the aqueous solution into the oil phase, the mixture was homogenized at 6000 rpm for 2 min using an Ultra-Turrax T-25 homogenizer (IKA T25-Digital Ultra-

and encapsulation by fabricating a composite system consisting of polysaccharide-based polyelectrolyte complex-containing microgel, as illustrated in Figure 1. The polyelectrolyte complexes (PECs) of chondroitin sulfate−chitosan, where anthocyanin was preloaded, were prepared through sequential copigmentation and encapsulation methods. These complexes were then incorporated into alginate microgel particles by emulsification/internal gelation technique. The large internal volume and cross-linking of microgels were the key issues for increasing the encapsulation efficiency and preservation of anthocyanins. Another interesting feature is that the incorporation of polyelectrolyte complexes can tune the interactive driving forces between polysaccharides and, therefore, interconnecting networks, which could confer the system with different responsiveness against environmental conditions, such as pH and ionic strength. Herein, the PECs and microgels were investigated with respect to the chitosan concentration and pH in which the systems were formulated. The physical properties were systematically characterized including morphology, size, zeta potential, and anthocyanin encapsulation efficiency. As a potential delivery system in food and pharmaceutical fields, the color change, degradation, and release of anthocyanin under different pH and ionic strength conditions were carried out. Additionally, the excellent reconstitution capacity of microgels after freeze-drying without cryoprotectant was demonstrated.

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EXPERIMENTAL SECTION

Materials. Chitosan (CS) of medium molecular weight (Mw = 190−310 kDa, 75−85% degree of deacetylation, viscosity 200−800 B


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Figure 2. Characterization of copigmented complexes and CS-CHS PECs. UV/vis spectra of CHS/anthocyanin and PECs formed at pH 4.2 (A) and 5.5 (B). FTIR spectra (C). SEM images of copigmented complex formed at pH 4.2 (D) and 5.5 (E), and PECs formed at pH 4.2 (F) and 5.5 (G). Turrax, Staufen, Germany). This was followed by the dropwise addition of 0.5 mL of calcium chloride solution (20 mg/mL) at the same homogenization rate for an additional minute. The dispersion was then transferred into a centrifuge tube and was left to stand for 1 h to allow further reaction of calcium ions with the dispersed PECs/ alginate. The oil phase was removed by centrifugation (6000 × g, 10 min). Microgels were washed by redispersion in water (pH 4.2 or 5.5), followed by centrifugation at 4000 × g for 2 min. Blank alginate microgels were prepared in the same way without addition of PECs. Physicochemical Characterization. The formation of copigmented complexes and PECs were monitored by visible spectra scanned in the range of 460−620 nm using a UV-2600 spectrophotometer (Shimadzu, Japan). Average particle size and zeta potential were carried out using a commercial zeta-sizer (Nano-ZS90, Malvern Instruments Ltd., U.K.) with a He/Ne laser (λ = 633 nm) and scattering angle of 90°. Zeta potential of single polyelectrolyte solution was also measured to determine the effect of pH on net charge. Before measurements, aliquots of samples were diluted with the same buffer solution to avoid multiple scattering phenomena due to particle interaction.15 The measurements were repeated in triplicate and reported as averages. Scanning electron microscopy (SEM) was used to characterize the morphology of copigmented complexes, PECs, freeze-dried microgels, and redispersed samples, which were dropped onto a stamp, air-dried, and sputtered with gold. Observations were performed on a LEO 1550 SEM equipped with a GEMINI field emission column (LEO Electron Microscopy, Thornwood, NY). The morphology of fresh microgels was characterized by cryo-focused ion beam (cryo-FIB). In this technique, the sample was snap-frozen by immediately plunging it into slush nitrogen and transferred under vacuum into an FEI Strata 400 FIB fitted with a Quorum PP3010T

Cryo-FIB/SEM Preparation System (Quorum Technologies, Newhaven, U.K.). The sample was then maintained at −165 °C for the duration of the experiment and coated with gold−palladium. To obtain a cross section of interface, a focused gallium ion beam was used to mill through the microgel particle. FTIR spectra were recorded on an IRAffinity-1S spectrometer equipped with a single-reflection attenuated total reflectance (ATR) accessory (Shimadzu Corp., Kyoto, Japan). The FTIR spectra were scanned between 500 and 4000 cm−1, with a resolution of 4 cm−1. Determination of Local Alginate Amount inside Microgels. The amount of alginate in the microgels was determined based on the phenol-sulfuric acid method as described previously.16 This test is an indirect method to determine the amount of alginate by measuring the concentration of polysaccharide in the supernatant after centrifugation. CS and CHS cannot react with phenol and sulfuric acid to yield furfural, and therefore they do not interfere with the alginate determination. The microgel samples were centrifuged at 17000 × g for 20 min. A 30 μL aliquot of the supernatant of unknown alginate concentration was taken and placed in a tube. Then, 0.16 mL of freshly prepared 5% (v/v) phenol solution and 0.8 mL of concentrated sulfuric acid were added. After incubation for 20 min at room temperature, the absorbance was recorded at 490 nm with a UV-2600 spectrophotometer (Shimadzu, Japan). Distilled water was used as blank. Determination of Anthocyanin Encapsulation Efficiency. To determine anthocyanin encapsulation efficiency in PECs and microgels, the samples were centrifuged at 17000 × g for 20 min, and the supernatant containing free anthocyanins was collected. The amount of anthocyanin in the supernatant was quantified spectrophotometrically at 519 nm13 against a standard curve using a UV-2600 C


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Biomacromolecules spectrophotometer (Shimadzu, Japan). Each experiment was carried out in triplicate. The anthocyanin encapsulation efficiency (EE, %) was then calculated using the following equation:

EE(%) =

and hyperchromic shifts are generally associated with copigmentation,20 owing to the formation of ionic complexes between the sulfate groups of CHS and the cationic moieties of anthocyanins. The copigmentation of CHS was more pronounced at pH 5.5, since the maximum absorbance was 1.5-fold greater than that at pH 4.2. Afterward, these copigmented complexes were dropwise transferred to chitosan solutions with different concentrations. The maximum absorbance wavelength further shifted to 522 nm, whereas the absorbance decreased considerably. Previous studies suggest that encapsulation in counterionic capsules could impact anthocyanin color21 or result in the disappearance of quinoidal species.22 Differences in FTIR spectra were used to assess the formation of CHS/anthocyanin copigmented complexes and CS-CHS PECs (Figure 2C). The spectra of pure CS and CHS were also provided for comparison (Figure S1). After copigmentation with anthocyanin, the characteristic peaks of CHS at 1253 cm−1 related to SO stretching became weak and shifted to 1249 cm−1 at both pH 4.2 and 5.5, suggesting the interaction between the sulfate groups of CHS and anthocyanins.17 Because of the polyelectrolytes interaction, the FTIR spectra of CS-CHS changed greatly in the carbonylamide region. For PECs formed at pH 4.2, the 1656 cm−1 peak of the amine I band in CS spectrum shifted to 1647 cm−1, and the 1573 cm−1 peak of N−H deformation shifted to 1558 cm−1. The characteristic peak of CHS at 1249 cm−1 (SO stretching) is not visible in these spectra. These observations indicated the electrostatic interaction between sulfate group of CHS and amino group of CS. It was noted that at pH 5.5 the stretch vibrations of C−O at 1041 cm−1 in CHS spectrum became weaker and shifted to 1045 cm−1, whereas the intensity of amide I band of CS at 1647 cm−1 reduced substantially, demonstrating the reaction of the carboxyl group of CHS with CS.23 The band intensity of −OH and −NH stretching at 3359 cm−1 considerably decreased at higher pH, which suggested the low degree of hydration in PECs formed at pH 5.5.24 In relation to the morphology, SEM images presented in Figure 2D,E show the diamond-like appearance of CHS/ anthocyanin copigmented complexes at both pH 4.2 and 5.5. This morphology was also observed in an earlier study17 and could be suggestive of tight intermolecular stacking of anthocyanins induced by CHS and formation of an anthocyanin core inside the polyelectrolyte shell. After encapsulation by CS, the particles became larger (Figure 2F,G). The particle size observed from the SEM images was consistent with the data using DLS technique (Table S2). The physical properties of anthocyanin-loaded CS-CHS PECs are summarized in Table S2. For all the formulations, PECs had positive zeta potential values, suggesting an excess of positively charged chitosan on the surface of complexes. The uncomplexed sites (−NH3+) of chitosan was expected to interact ionically with alginate matrix. PECs prepared at pH 4.2 had higher positive charges than those at pH 5.5 due to the relatively high amount of protonated amine groups (−NH3+) on the surface of CS. It was also noted that higher pH and lower concentrations of CS led to smaller PECs. The increase of pH decreased the density of positive charges and the repulsive force among the free −NH2 groups, contributing to the formation of smaller particles. Similarly, at low concentration of CS there were less free NH2 groups, resulting in a more compact CS-CHS network. However, PECs with various physical properties could display different encapsulation

total amt of anthocyanin − free anthocyanin × 100 total amt of anthocyanin

Stability Assays. For the ionic strength stability, 0.1 mL of NaCl solution of different concentrations was added into 1.5 mL microgel samples; the final salt concentrations were 0.05, 0.1, 0.2, and 0.3 M. After incubation for 24 h at 4 °C in the absence of light, an aliquot of the salt-treated samples was removed to measure the particle size and alginate amount in the microgels. The anthocyanin retention rate was determined by calculating the amount of leaked anthocyanin after centrifugation in the same way as described previously. The retention rate was calculated as the amount of anthocyanin encapsulated in microgels after the salt treatment divided by the initial encapsulated amount of anthocyanin in the microgels. For pH stability, phosphate buffer was prepared at pH 1, 3, 5, and 7. Then, a 0.5 mL sample was mixed with 0.5 mL of buffer with different pH values in a 24-well plate. After incubation for 5 h, samples were taken for determining alginate amount in microgels, and anthocyanin degradation and retention rate. The degradation of anthocyanin was measured based on an earlier study.17 The samples were freeze-dried, redissolved in ethanol, and filtered to obtain pure anthocyanin in solution. The remaining anthocyanin amount was determined using a UV/vis spectrophotometer at 519 nm. Freeze-Drying and Reconstitution. Freshly prepared microgels were poured into 50 mL centrifuge tubes without addition of cryoprotectants. The tubes were frozen at −80 °C for 5 h and then transferred to a Labconco FreeZone 2.5 L Bench-top Freeze-Dry System (Kansas City, MO, U.S.A.) at 0.09 mbar for 24 h, with the condenser temperature kept at −60 ± 5 °C. After freeze-drying, the lyophilizate was rehydrated by slowly adding distilled water at a corresponding pH (4.2 or 5.5) and stirred for approximately 10 min. The encapsulation efficiency of anthocyanin in the reconstituted microgels was determined in the same way as described previously. To evaluate the amount of anthocyanin lost during freeze dying, the lyophilizate was dissolved in ethanol and the remaining anthocyanin was determined using a UV/vis spectrophotometer at 519 nm. The long-term stability of freeze-dried samples was investigated during storage at room temperature in the dark. Samples were rehydrated after 30 days of storage for measurement of anthocyanin loss and encapsulation efficiency. Statistical Analysis. Results were reported as mean ± standard deviation. The experiments were done in triplicate. Data were analyzed by one-way analysis of variance (ANOVA) test. Duncan’s test and independent samples t-test at P = 0.05 were used to assess significant differences between treatment means using SPSS 19.0 (IBM, Chicago, IL, U.S.A.).

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RESULTS AND DISCUSSION Formation of Polyelectrolyte Complexes (PECs). Polyelectrolyte complexation is mainly triggered by electrostatic interaction. In this study, pH 4.2 and 5.5 were selected to prepare PECs based on the pKa values of CS (≈6.2)18 and CHS (≈2.6 and 4.57).19 Electrokinetic measurement showed that CHS had a stronger negative charge at pH 5.5, while CS had a stronger positive charge at pH 4.2 (Table S1). This is because an increase of pH induces the deprotonation of ionizable groups, which converts the −NH3+ groups of CS into −NH2 and the −COOH groups of CHS into −COO−. The zeta potential of chitosan solution varied slightly at different concentrations, regardless of pH. UV absorbance spectra show that after mixing anthocyanin and CHS solutions, there was a bathochromic shift in maximum absorbance wavelength from 515 to 518 nm (Figure 2A,B). An increased intensity of maximum absorbance was also observed. Such bathochromic D


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Table 1. Particles Size (μm), Zeta Potential (mV), Encapsulation Efficiency (EE), and Incorporated Alginate Percentage of PECs-Microgels as a Function of Chitosan Concentrationa pH

CS concentration (mg/mL)

4.2 4.2 4.2 4.2 5.5 5.5 5.5 5.5

0 0.5 1.0 1.5 0 0.5 1.0 1.5

particle size (μm) 1.9 2.4 3.3 3.7 1.5 1.8 2.0 2.1

± ± ± ± ± ± ± ±

zeta potential (mV) −36.4 −33.6 −31.8 −29.5 −42.2 −39.7 −38.4 −38.1

0.2abc 0.3c 0.3d 0.5d 0.1a 0.1ab 0.1abc 0.2bc

± ± ± ± ± ± ± ±

1.2af 2.5ab 1.4bc 0.4c 1.9d 1.8de 2.1ef 1.3ef

EE (%) 61.0 66.5 73.1 70.8 70.3 74.5 81.5 82.2

± ± ± ± ± ± ± ±

local alginate (%)

1.2a 2.1b 1.6cd 2.5c 1.7c 1.8d 1.5e 1.6e

65.9 69.0 73.9 70.9 74.0 78.3 82.3 82.6

± ± ± ± ± ± ± ±

1.3a 1.4b 1.4c 1.9b 1.1c 1.2d 1.7e 1.4e

a Results are expressed as mean value ± standard deviation (n = 3). Different letters in the same column indicate a significant difference (P < 0.05, Duncan’s test).

Figure 3. Microstructure of microgels. Cryo-SEM images of microgels (A, C) and PECs-microgels (B, D) formed at pH 4.2 (A, B) and 5.5 (C, D). Cross-section of microgels (E−H) produced by cryo-FIB milling, E−H samples corresponding to those in A−D. The arrows in panel F indicate porous structure and the inset in panel H shows high magnification of core. FTIR spectra (I). Schematic (J) illustrates the potential cross-linking in microgels and PECs-incorporated microgels formed at pH 4.2 and 5.5.

observed at pH 5.5. The influence of pH on EE can be explained by the fact that at pH 4.2, the residual NH3+ with the increase of chitosan concentration would cause electrostatic repulsion resulting in loosened polymer structure of PECs,

capacities. The anthocyanin EE values for PECs prepared at pH 5.5 was approximately 10% higher than those at pH 4.2. Interestingly, increasing CS concentration caused a significant decrease in EE at pH 4.2, whereas an opposite trend was E


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Figure 4. pH-responsive stability of PECs-microgels formed at pH 4.2 and 5.5 evaluated by incorporated alginate (%), remaining anthocyanin (%), and anthocyanin retention rate (%). The images on the left show the corresponding color change as a function of pH. The labels a−e refer to the pure anthocyanins (a), microgels formed at pH 4.2 (b), and PECs-microgels prepared at pH 4.2 with chitosan at 0.5 mg/mL (c), 1.0 mg/mL (d), and 1.5 mg/mL (e). Labels f−i refer to the microgel form at pH 5.5 (f), and PECs-microgels prepared at pH 5.5 with chitosan of 0.5 mg/mL (g), 1.0 mg/mL (h), and 1.5 mg/mL (i). Error bars represent the standard deviations from triplicate analyses.

into the microgel (P < 0.05), depending on the chitosan concentration. When chitosan concentration was increased to 1.5 mg/mL, EE value was almost steady at pH 5.5 but significantly decreased at pH 4.2 (P < 0.05). This observation suggested that excess of chitosan in the PECs did not favor anthocyanin entrapment in the microgels. To explain such variation trend of EE, we determined the local amount of alginate in the microgels, and found that incorporation of PECs and high pH significantly improved the participation of alginate in the microgel (P < 0.05). Moreover, the amounts of alginate in the microgels was the highest for the formulations with moderate concentration of chitosan (1.0 mg/mL). Figure 3A−D shows the cryo-SEM images of microgels with and without incorporation of PECs. The microgels had a homogeneous spherical or disk-shaped morphology with no obvious aggregation or fusion. The microgel size observed from the microscopic images was consistent with that determined using DLS technique. The cross-section of cryo-SEM (Figure 3E−H) revealed that the microgels exhibited a core−shell-like structure, which could indicate the gelation of alginate on the interface as calcium ions diffused into the droplets. Note that the cross-linking inside microgels in the absence of PECs was not apparent, while a dense network was clearly visualized when PECs were incorporated. Interestingly, PECs-microgels at pH 4.2 showed a thick shell with porous structure, while those at pH 5.5 had a dense network inside the core (insert in panel H). The highly cross-linked microgels at pH 5.5 were more compact, leading to smaller particle size.25 Further insights into the microstructure of microgels were obtained by FTIR spectra (Figure 3I and Figure S1). The characteristic peaks at 1743, 1608, and 1411 cm−1 present in

whereas at pH 5.5 more charged sulfate group in CHS can interact with chitosan. Altogether, we fabricated the CS-CHS complexes as carriers for anthocyanin by a combination of copigmentation and encapsulation techniques. It was noticed that pH can affect the formation of PECs by controlling the ionization degree of functional groups such as −NH2, −COOH−, and −SO3. Consequently, PECs displayed different physical properties, especially anthocyanin EE. These findings were expected to help us better understand the behavior of PECs incorporated into microgels, which is discussed in the following section. PECs Incorporation into Microgels. To synthesize PECsincorporated alginate microgels, the emulsification/internal gelation method was used. Before emulsification, alginate was cross-linked with PECs through electrostatic interaction between positively charged CS and negatively charged alginate. Herein, we used serial concentrations of CS for PECs (0.5−1.5 mg/mL) to tune the cross-linking density of the alginate gel. The addition of Ca2+ solution further enhanced the internal cross-linking of sodium alginate, resulting in homogeneous microgel particles. We found that independent of pH in which the microgels were prepared, the incorporation of PECs led to an increase in microgel size (Table 1). More extended structure was observed for PECs-microgel at pH 4.2 with larger size compared to those at pH 5.5. The surface charge of the microgel, on the other hand, decreased with incorporation of PECs. Zeta potential values of PECs-microgels formed at pH 4.2 ranged from −36 to −29 mV and were lower than those at pH 5.5 (−43 to −38 mV). Note that the microgels had higher anthocyanin EE when formulated at pH 5.5. Incorporation of PECs significantly enhanced the entrapment of anthocyanin F


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As the pH decreased, more alginate was present in the microgels, which could indicate more cross-linking of polyelectrolytes by increased cation ions at low pH. The system exhibited hydrogel-like structure at pH 1 and 3. In contrast, the incorporated alginate in microgel considerably decreased at pH 7 due to the dissociation of alginate-Ca2+ network. Note that the local concentration of alginate decreased to a lower extent in the presence of PECs. It was hypothesized that the additional complexation between PECs and alginate inhibited the dissociation of the microgel structure. We also found that microgels effectively protected anthocyanins from pH-related degradation. At pH 5 and 7, approximately 21% and 40% of pure anthocyanins degraded after a 5 h incubation period, respectively, while these values were around 18% and 30% in alginate microgels. The protective effect was more pronounced for PECs-microgels, especially those formulated pH 5.5. Microgels with chitosan concentration of 1.0 mg/mL maintained more than 94% and 85% anthocyanins at pH 5 and 7, respectively, after a 5 h period. Analysis on the retention rate showed that the pH-responsive release of anthocyanin greatly depended on the formulated pH. Compared to the microgels prepared at pH 5.5, those prepared at pH 4.2 had lower release at pH 1 and 3, despite high release at pH 5 and 7. Incorporation of increased chitosan reduced the release of anthocyanins at high pH, and is likely due to a stronger interaction during complexation formation between the cationic chitosan and the alginate uronic acids which would be more fully deprotonated at pH 5.5 over pH 4.2. The tunable pH responsiveness demonstrated the potential of these microgels as controlled delivery system for anthocyanins. Figure 5 shows the salt-responsive stability of microgels. It is well-known that high ionic strength can effectively screen out the surface charges on particles and increase osmotic pressure of the counterions.28,29 In this study, the size of microgels and PECs-microgels formed with 0.5 mg/mL of CS at pH 4.2 progressively increased with increasing ionic strength, possibly due to the aggregation of microgel particles resulting from screened repulsive electrostatic force. However, decreased particle sizes were observed for those with higher concentration of CS (1.0−1.5 mg/mL). When prepared at pH 5.5, all microgels underwent a slight size increase and then leveled off when the salt concentration was >1.0 mg/mL. With addition of salt, the zeta potential of microgels exhibited an appreciable decrease from a net positive charge to approximately neutral, demonstrating the existence of electrostatic screening effect. It was also observed that the local concentration of alginate was independent of ionic strength, indicating that the microgel structure was relatively stable against dissociation at the tested salt concentration. Despite that, there was a considerable release of anthocyanin from microgels: 53.4% of anthocyanins were released from microgels formed at pH 4.2 in the presence of 0.3 M NaCl. It was believed that the decreased degree of cross-linking between polysaccharides by ionic strength reduced the energy barrier for anthocyanin to diffuse through the interior of microgels.30 Such salt-responsive release of anthocyanin was effectively inhibited when PECs were incorporated in the microgels. The PECs-microgels displayed the lowest anthocyanin release at 1.0 mg/mL CS, with retention rate higher than 78% and 86% at pH 4.2 and 5.5, respectively. Note that PECs-microgels formed at pH 5.5 possessed higher stability owing to the hydrogen bonding between polysaccharides. Considering these observations, it was demonstrated that a simple modification of pH and

the IR spectrum of sodium alginate were assigned to carboxylic acids (−COOH), carboxylate ions (COO−) asymmetric and symmetric stretching peaks, respectively. In the PECs-microgels prepared at pH 4.2, a shift was observed from 1608 to 1604 cm−1, indicating the interaction between amino groups of CS and carboxyl groups of alginate. The relative intensity of peak at 1743 cm−1 was higher in PECs-microgels, suggesting a lower degree of ionization of −COOH groups. The probable reason was that the incorporation of positively charged PECs inhibited the ionization of −COOH groups on alginate. This observation also indicated the formation of hydrogel-bond between −COOH and −OH groups.26 At pH 5.5, a new peak was observed at 1573 cm−1, which was explained in terms of the unreacted -NH2 groups of CS.27 In addition, the intensity of peak at 1743 cm−1 was higher than that at pH 4.2. The larger amounts of −COOH and −NH2 groups suggested stronger hydrogen bonding in microgels formed at pH 5.5. Based on these findings, we proposed a schematic representation of the formation of microgels in the absence and presence of PECs (Figure 3J). For the microgels with PECs, the excessive CS in the PECs led to additional polyelectrolyte network with alginate matrix through electrostatic interactions between −NH3+ and −COO−, which improved the entrapment of anthocyanins. At pH 4.2, which is lower than the pKa of CS (≈ 6.5), −NH2 groups of chitosan were highly protonated, while only the sulfate groups on CHS were partially ionized considering the pKa values of 2.6 (sulfate group) and 4.5 (carboxylic group). The excessive −NH3+ groups caused electrostatic repulsion between CS chains and, therefore, an increase in the diameter of PECs and microgels (Table S1−2). Although more charged sites (−NH3+) in protonated CS were available for interaction with alginate at pH 4.2, the amount of ionized alginate in microgels was less than that at pH 5.5 (Table 1), possibly due to the low degree of deprotonation. At pH 5.5, both the sulfonic and carboxylic groups of CHS were deprotonated, suggesting a stronger interaction with the partially ionized −NH3+ groups from CS. The complexes would become more compacted when considering strong hydrogen bonding. Although the degree of protonation of chitosan decreased at pH 5.5, the incorporation of PECs significantly increased the amount of alginate in the microgels. On the other hand, a high concentration of CS (1.5 mg/mL) might disturb the structural organization of microgels probably by expanding the polymer network (especially at pH 4.2), and therefore, the ability to encapsulate anthocyanins decreased. Stability Studies. The stability of PECs-microgels was investigated in response to the changes in pH and ionic strength. Anthocyanins are unique compounds that can rearrange and form resonant structures with changes on the pH, which affects their color and stability.4 Figure 4 shows the color variation of microgels formulated at pH 4.2 and 5.5 and subjected to varying pHs. In the case of control samples (pure anthocyanins), an increase in pH induces a change in color toward purple hues and a reduction of color intensity, most considerably after incubation for 5 h at room temperature. Encapsulated anthocyanins, on the other hand, retained their color even after incubation. It was interesting to observed that PECs-microgels produced at pH 5.5 and maintained at pH 3 presented redder hues after incubation, when compared to the same initial samples. Moreover, color fading was not obvious at pH 7 for microgels formed at pH 5.5. G


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integrity and reconstitution capacity. Freeze-drying is a valuable method used to improve the long-term stability of colloidal particles, in contrast to their poor stability in aqueous medium during storage. In this study, we compared the performances of different formulations during freeze-drying without addition of any cryoprotectant. At pH 4.2, a polymeric matrix was observed in the freeze-dried samples, although no particles were clearly distinguished (Figure 6). This could be explained by various stresses generated during freezing and drying steps that could destabilize colloidal suspensions.31 Compared to pure microgels, those containing PECs exhibited smooth and intact plates with a porous structure, especially when the concentration of chitosan was 1.0 mg/mL. Such morphology could retain encapsulated compounds more efficiently than those with loose structure and rough surface.24,31 A marked difference in structure was observed in freeze-dried microgels formed at pH 5.5. All the formulations displayed a continuous matrix without pores. Interestingly, in formulations with 0.5 and 1.0 mg/mL chitosan it was possible to visualize some wellpreserved spherical particles even after freeze-drying. This observation suggested that incorporation of PECs conferred high rigidity to microgels against freeze-drying. It was also noted that with a concentration of 1.5 mg/mL of chitosan, the structure became loosen at pH 4.2, whereas at pH 5.5, the matrix surface was rough without the occurrence of particles. The destabilization of particles inevitably occurs by the freezing and drying stresses; thus, the reconstitution ability after rehydration must be evaluated. The SEM micrographs of the formulations after resuspension are shown in Figure 7. For pure microgels, there were more reconstituted particles observed at pH 5.5 than at pH 4.2. However, it is possible to see the amorphous matrix into which the microgels were embedded, suggesting that the polyelectrolytes did not fully reconstitute after rehydration. In the case of freeze-dried PEC-containing microgels, the particles were well reconstituted with a clean background (Figure 7C,D). Although they were in a collapsed state possibly due to the drying step before SEM imaging, the spherical shape and size of microgels and PECs were maintained after reconstitution. Surprisingly, a large proportion of PEC particles were reloaded into the microgels after rehydration (Figure 7E), demonstrating their excellent

Figure 5. Salt-responsive stability of PECs-microgels formed at pH 4.2 (left) and 5.5 (right) evaluated by hydrodynamic size (nm), zeta potential (mV), incorporated alginate (%), and anthocyanin retention rate (%). Error bars represent standard deviations from triplicate analyses.

polysaccharide concentration during preparation would allow us to tune the pH- and salt-responsive release of anthocyanins from PECs-microgels. Freeze-Drying and Reconstitution. Samples were also subjected to freeze-drying process to evaluate their structural

Figure 6. SEM micrographs of freeze-dried samples formed at pH 4.2 (A−D) and 5.5 (E−H): freeze-dried microgels (A, E), freeze-dried PECsmicrogels at the chitosan concentration of 0.5 mg/mL (B, F), 1.0 mg/mL (C, G), and 1.5 mg/mL (D, H). H


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Biomacromolecules

Figure 7. SEM micrographs of resuspension of freeze-dried samples: microgels at pH 4.2 (A) and 5.5 (B), PECs-microgels at pH 4.2 (C) and 5.5 (D), a single microgel showing PECs aggregation inside (E), partially collapsed PECs-microgels (F).

reconstitution capacity. Similar phenomenon was also observed in an incomplete collapsed microgel (Figure 7F). The freeze-drying process can also result in the degradation of encapsulated compounds or their leakage from the particles (Figure 8). More than 82% of anthocyanins were maintained in all formulations after freeze-drying. The presence of PECs further inhibited anthocyanin loss by freeze-drying pressure (Figure 8A,B). However, the storage stability of freeze-dried samples showed pH- and chitosan concentration-dependency. The remaining anthocyanins at pH 4.2 decreased significantly (P < 0.05) during storage (except at 1.5 mg/mL), but the remaining anthocyanin still exceeded 74% after 30 days. By contrast, the loss of anthocyanin was not significant (P > 0.05) in the formulation containing PECs at pH 5.5, where more than 82% of anthocyanin was retained. This observation can be explained by the intact structure of freeze-dried samples containing PECs (Figure 6F−H). The reconstitution of the microgel during hydration was accompanied by the reloading of anthocyanins. The anthocyanin EE values significantly decreased after freeze-drying (Figure 8C,D), possibly because of the reconstitution of microgels and

losses during freeze-drying itself. However, the degree of reduction of EE was relatively low in the presence of PECs. For example, the EE decreased by 21% and 28% for pure microgel at pH 4.2 and pH 5.5, respectively, but only by 12% and 10% at the corresponding formulation with 1.0 mg/mL chitosan. Higher encapsulation efficiency was probably attributed to the fact that PECs were incorporate more effectively into microgels after reconstitution, as evidenced in SEM images (Figure 8). Leakage of anthocyanins was also effectively inhibited during the storage period by the incorporation of PECs, especially at 1.0 mg/mL chitosan. Besides, less anthocyanins leaked at pH 5.5.

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CONCLUSION This study demonstrates a unique composite polysaccharide system consisting of PECs incorporated into microgels for the delivery of anthocyanins. The approach includes sequential copigmentation, encapsulation, and emulsification with internal gelation techniques. The observations above show the physical properties of microgels can be controlled by incorporation of PECs and the pH in which the system was formulated. As an I


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Biomacromolecules

Figure 8. Anthocyanin remaining (A, B) and encapsulation efficiency (C, D) in reconstituted microgels at pH 4.2 and 5.5 after rehydration of freezedried microgels. The freeze-dried microgels were resuspended immediately after freeze-drying or after 30 days of storage at room temperature in dark, respectively. For each column group, different letters represent a significant difference (P < 0.05, Duncan’s test). In exhibit B and D, when marked with *, results are significantly (P < 0.05, independent samples t test) different from the corresponding formulations at pH 4.2.

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ACKNOWLEDGMENTS Authors are grateful for the financial support from PepsiCo, PepsiCo Research and Development, Plano, TX 75024, U.S.A. We thank the Cornell Center for Materials Research (CCMR) for use of their facilities. CCMR facilities are supported by the National Science Foundation under Award Number DMR1719875. Any opinions or scientific interpretations expressed in this manuscript are those of the authors and do not necessarily reflect the position or policy of PepsiCo, Inc.

example, incorporation of PECs with moderate CS content improved anthocyanin EE in the microgels and also tuned the ionic strength and pH responsiveness of the anthocyanin carriers. Similar effects were also observed for microgels prepared at different pHs. Furthermore, incorporation of PECs effectively protected the microgel against various freeze-drying stresses without the addition of cryoprotectant and contributed to excellent reconstitution capacity after rehydration. These findings should be of interest to industries working toward more robust systems for the controlled delivery of anthocyanins and other water-soluble bioactive compounds.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.8b00352. Zeta potential of anthocyanin and single polyelectrolyte solution at pH 4.2 and pH 5.5; particle size, zeta potential, and encapsulation efficiency of polyelectrolyte complex as a function of chitosan concentration; and FTIR spectra of pure chitosan, chondroitin sulfate, and alginate (PDF).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Chen Tan: 0000-0002-2428-9449 Alireza Abbaspourrad: 0000-0001-5617-9220 Notes

The authors declare no competing financial interest. J


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