Letter pubs.acs.org/macroletters
Dried and Redispersible Cellulose Nanocrystal Pickering Emulsions Zhen Hu, Heera S. Marway, Hesham Kasem, Robert Pelton, and Emily D. Cranston* Department of Chemical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4L7, Canada S Supporting Information *
ABSTRACT: The effect of tannic acid (TA) and water-soluble cellulose derivatives on the properties of Pickering emulsions stabilized by cellulose nanocrystals (CNCs) was investigated. The potential to both fully dry CNC-stabilized emulsions and to redisperse the dried emulsions in water is demonstrated. When CNCs are mixed with excess adsorbing polymer, either methyl cellulose or hydroxyethyl cellulose, followed by emulsification with corn oil, oil-in-water emulsions can be transformed without oil leakage into solid dry emulsions via freeze-drying. However, these dry emulsions exhibit droplet coalescence within the solid matrix and thus cannot be redispersed. Addition of TA (after emulsification) imparts dispersibility to the dried emulsions due to complexation between the cellulose derivatives and TA which condenses the “shell” around the oil droplets. When dried emulsions with TA are placed in water, the emulsion droplets redisperse readily without the need for high energy mixing, and minimal change in emulsion droplet size is observed by Mastersizer and confocal microscopy. Therefore, the simple addition of two sustainable components to CNC Pickering emulsions (i.e., TA and methyl cellulose or hydroxyethyl cellulose) has led to the first dried and redispersible CNC-based emulsions with oil content as high as 94 wt %. These processing abilities will likely extend the use of these surfactant-free, “green”, and potentially edible emulsions to new food, cosmetic, and pharmaceutical applications.
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indicating that these systems are not redispersible, as shown in this work.6−8 Compared to surfactant-stabilized emulsions, particle-stabilized emulsions (also called Pickering emulsions) should be more attractive to produce dry emulsions as a result of their enhanced stability.21 Surprisingly, only silica nanoparticles and starch granules have been reported as Pickering stabilizers in dry emulsions to date.1,3,4 Furthermore, hydrophobic modification and thermal treatment of these particles were necessary to allow for successful production of dry emulsions. By contrast, herein we demonstrate that stabilizing o/w emulsions with cellulose nanocrystals (CNCs) preadsorbed with uncharged water-soluble cellulose derivatives with only 2 wt % of tannic acid (TA) enables both freeze-drying of the emulsions to produce an oil powder and easy redispersion of the emulsion oil droplets in water. CNCs have been used to stabilize emulsions.22−27 The novel aspect of the current work is the inclusion of TA with the water-soluble cellulosic derivatives and CNCs. TA is known to form complexes with a wide range of macromolecules including carbohydrates, proteins, enzymes, and other synthetic polymers due to hydrogen bonding, hydrophobic, and cation−π interactions.28,29 For example, the complexation of methylcellulose (MC) with tea catechins (similar in structure to TA) to form a coacervate via hydrophobic interactions and hydrogen bonding was shown to convert the normally water-
il-in-water (o/w) emulsions are often dried in the food, cosmetic, and pharmaceutical industries to increase the shelf life of oil (and encapsulated components) against degradation and oxidation, limit microbial growth, or facilitate transportation.1−4 The dried products are solid emulsions (also called dry emulsions or oil powders), and they are usually made by spray-drying or freeze-drying.1,3,5 Some of the potential applications of these soft solids with high liquid oil content include oil structuring in foods, encapsulation in drug delivery, stability improvement in cosmetics, and lubrication in mechanical devices.6−8 Unfortunately dehydration of emulsions often leads to disruption of emulsion droplets and oil leakage.9,10 Upon freezing the emulsions, the water and oil phases crystallize, which can lead to destabilization following a number of different mechanisms.2,3,11−16 In order to prevent coalescence of the emulsion droplets and oil leakage, hydrophilic carrier compounds, such as lactose, glucose, maltodextrin, and cellulose, can be added to the formulations after emulsification. The minimum amount of carrier required typically ranges between 30 and 80 wt % of the final dry emulsion mass,1,4,17 and only a few literature examples have demonstrated the redispersion of dried emulsions back into water.3,6−8,18−20 Currently for food applications, one of the most promising approaches to make structured oil solids is to use o/w emulsions with polysaccharide carriers, dry the emulsions, and then shear them to form oil gels; while this avoids the use of solid fats and oil-soluble gelation agents, they use high energy re-emulsification to reform emulsions from the oil solids, © XXXX American Chemical Society
Received: December 15, 2015 Accepted: January 11, 2016
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DOI: 10.1021/acsmacrolett.5b00919 ACS Macro Lett. 2016, 5, 185−189
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ACS Macro Letters soluble MC film into an insoluble film.30 In our work, TA was used to strengthen the CNC-polymer shell around the oil droplets which enhances stability during freeze-drying, prevents coalescence after drying, and allows for redispersion. On the basis of our previous work, we hypothesized that o/w emulsions stabilized by CNCs and MC (or hydroxyethyl cellulose, HEC) would be amenable to drying due to their longterm colloidal stability during thermal cycling.24 We recognized that CNCs could be rendered more surface active and better emulsifiers through simple in situ adsorption23,24 and that the oil droplets are small and require notably less emulsifier than for other nanoparticle-stabilized systems.1,31,32 However, our CNC−MC-stabilized emulsions could not be dried and redispersed, thus we included TA as an additional stabilizing agent. Figure 1a shows photographs of a corn oil-in-water emulsion (1:4 oil to water with 20 mM HEPES buffer) stabilized by 0.25
Table 1. Formulations Demonstrating That All Three Stabilizing Components (CNCs, MC, and TA) Are Required to Achieve Redispersible Dry Emulsionsa stable emulsion upon CNCs (wt %)
MC (wt %)
TA (wt %)
freeze-drying?
redispersion?
0.25 0.25 0.25 0.25 0.25 0 0 0
0.25 0 0 0.25 0.75 0.25 0.5 0.25
0.5 0.5 0.75 0 0 0.5 0.5 0.75
yes no no no yes no no no
yes no no no no no no no
a
Emulsions are 20 vol % oil, 80 vol % water, with 20 mM HEPES buffer, and the indicated wt % of added components refers to the concentrations in the water phase.
(Supporting Information Figure S1). With polymer but without TA, successful freeze-dried emulsions were achieved if the MC concentration was increased to 0.75 wt % (Supporting Information Figure S1). However, oil leakage was observed when water was added to the dry CNC−MC emulsions (Supporting Information Figure S2). High-magnification CLSM images of freeze-dried CNC−MC emulsions imply that ice crystals may force the aggregation of emulsion droplets and induce the formation of macroporous structures inside the dry emulsions during freezing (Supporting Information Figure S3), while some emulsion droplets were intact after freezedrying and most droplets broke, leaving oil dispersed within the polymer/TA carrier. Therefore, redispersion of the dry emulsions resulted in extensive oil leakage/phase separation and could not form stable emulsions unless significant sonication energy was used to reform the emulsion droplets. TA is known to form dispersed or precipitated hydrogel complexes with MC.33,34 TA also adsorbs onto cellulose.35 Therefore, emulsion quality was likely to depend upon the order of addition. CNC−MC−TA emulsions were prepared by two different routes: in route 1, emulsions stabilized by CNCs and MC were prepared, and TA was added afterward (and mixed for 6 h before analysis was carried out). In route 2, TA was premixed with either all or 0.23 wt % of the MC and then added to the CNC-only or CNC−0.02 wt % MC stabilized emulsions, respectively. On the basis of our previous work, 0.02 wt % MC should be sufficient to fully cover the surface of the CNCs at 0.25 wt %, making them surface active and good emulsion stabilizers.24 Any excess MC partitions at the oil− water interface to further enhance emulsion stability. As shown in Table 2, only the sample in which all of the MC was mixed with CNCs before emulsification and TA was added later to the CNC−MC emulsion (route 1) could be successfully freezedried and redispersed. By route 2, TA was consumed by forming colloidal complexes with the MC in the aqueous phase before having a chance to migrate to oil-water interface or cellulose surfaces. In addition, we used a quartz crystal microbalance with dissipation monitoring (QCM-D) to confirm complexation between MC and TA (Figure 2). A QCM-D sensor was spincoated with CNCs, and then MC was adsorbed in situ followed by addition of TA. There is a large frequency decrease upon injection of TA which is due to the binding/adsorption of TA on MC-coated CNCs. Approximately 1.5 times more TA adsorbed than MC onto the sensor. The strong adsorption of
Figure 1. (a) Photographs showing the effect of freeze-drying and redispersion on the appearance of emulsions (20 vol % corn oil stained with Nile red) stabilized by CNCs (0.25 wt %), MC (0.25 wt %), and TA (0.5 wt %). (b) CLSM image of the initial emulsion. (c) SEM image of the same emulsion after freeze-drying. (d) CLSM image of the same emulsion after freeze-drying and redispersion in water. Mean droplet diameter is indicated below the corresponding CLSM image.
wt % CNCs, 0.25 wt % MC, and 0.5 wt % tannic acid (wt % in the water phase), before and after freeze-drying, and finally, as a redispersed emulsion. The freeze-dried emulsion has an oil content of 94 wt %. These dried emulsions can be readily redispersed back into water with only gentle shaking by hand and are stable to coalescence over months. The comparison of the confocal laser scanning microscopy (CLSM) images of CNC−MC−TA emulsions before freeze-drying (Figure 1b) and after redispersion (Figure 1d) shows that the drying and rehydrating processes do not significantly affect the emulsion droplet diameter, which was also confirmed by Malvern Mastersizer (mean droplet diameter and span shown in Figure 1). Similarly, spherical-shaped dry emulsions with diameters smaller than 10 μm can be viewed under scanning electron microscopy (SEM) (Figure 1c). Therefore, we believe that CNC−MC−TA emulsions are stable to both the freeze-drying and redispersion processes. To determine which components are necessary to formulate stable dry emulsions that can be redispersed, different amounts of CNC, MC, and TA were added to prepare the emulsions. As shown in Table 1, all components are required. Without the polymer and TA, emulsions stabilized by CNCs alone displayed extensive emulsion breaking and oil leakage after freeze-drying 186
DOI: 10.1021/acsmacrolett.5b00919 ACS Macro Lett. 2016, 5, 185−189
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ACS Macro Letters Table 2. Influence of Stabilizer Mixing Order on Dry Emulsion Properties stable emulsion upon
route 1 route 2 route 2
CNCs (g) 0.02 0.02 0.02
MCa (g)
TA (g)
freezedrying?
redispersion?
0.02 + 0.00 0.00 + 0.02 0.0016 + 0.0184
0.04 0.04 0.04
yes no no
yes no no
a
MC was added in two portions, in the amounts indicated on either side of the + symbol. The first portion was added to the CNC suspension before emulsification, and the second portion was added to the TA solution and then mixed with the CNC-only or CNC−MC emulsion. Emulsions are 10 mL: 2 mL of oil and 8 mL of water with total concentrations of 0.25 wt % CNCs, 0.25 wt % MC, and 0.5 wt % TA in the water phase.
Figure 3. AFM height images of the air-dried surfaces after QCM-D experiments: (a) CNC-coated substrate before MC injection, (b) after injection of MC and rinsing, (c) after injection of TA and rinsing (green arrows are highlighting some of the MC−TA complexes), and (d) CNC-coated substrate after TA injection and rinsing (with no MC).
surface active and may contribute to further emulsion stabilization.34 The binding between MC, TA, and CNCs at the oil−water interface was also visualized through AFM images of dried dodecane-in-water emulsions on silicon wafers (Supporting Information Figures S4 and S5). To ensure that the nanoparticles in Figure 3c are complexed MC and TA (and not TA alone), a control experiment whereby TA solution was passed over a CNC sensor (without MC) was carried out, and no nanoparticles were observed (Figure 3d). Transmission electron microscopy (TEM) of MC−TA colloidal complexes formed after mixing MC and TA solutions together also supports the belief that nanoparticles in Figure 3c are MC−TA complexes (Supporting Information Figure S6). In addition to MC, HEC also showed colloidal complex formation upon mixing with TA (Supporting Information Figure S6). Interestingly, CNC−HEC−TA dry emulsions could also be redispersed in water (data not shown), implying that the complex formation between water-soluble cellulose derivatives and TA benefits the production of redispersible dry emulsions with various chemistries possible. In conclusion, we have shown that adding TA to emulsions stabilized by CNCs and MC allows for the preparation of stable dry emulsions with high oil content (94 wt %) that can be readily redispersed in water to again form stable o/w emulsions. This is a new encapsulation method for oil and oil-soluble compounds whereby only 6 wt % of the dry emulsion mass is due to CNCs, MC, and TA (all biobased macromolecules) indicating very low carrier concentrations compared to previous reports. Without addition of MC, emulsions stabilized by CNCs alone are broken upon freeze-drying. Without addition of TA, the CNC−MC emulsions are not able to withstand the redispersion process, displaying extensive oil leakage after adding water to the dried emulsions. In our previous work, we showed that the physical interactions between adsorbing polymers and CNCs are important in making emulsions and emulsion gels which are stable to coalescence. In this study, we envision that the combination of CNCs, MC, and TA provides more control over emulsion properties and applications due to
Figure 2. Δf 3/3 (black) and ΔD (gray) versus time for QCM-D study of a CNC-coated silica sensor exposed to MC (1 g/L in 20 mM HEPES buffer) and TA (2 g/L in 20 mM HEPES buffer), with buffer rinsing steps in between. MC adsorbs on the CNC-coated sensor, and TA subsequently binds to the MC-coated CNC surface. MC binding is associated with an increase in ΔD, whereas TA binding shows a decrease in ΔD, implying dense MC/TA complexes are formed.
TA is accompanied by a steep decrease in dissipation, which suggests a stiffening and condensing of the MC layer on CNCs. We believe the decrease in dissipation is related to the complexation of the MC layer and the weak deposition of colloidal MC−TA complexes formed in the aqueous phase.33,34,36 Further rinsing with buffer induces an increase in dissipation which is associated with some mass loss, likely the weakly bound MC−TA complexes (but significant amounts of MC and TA still remain on the substrate). When the complexes are rinsed away, the CNC−MC−TA layer appears to expand and soften. The formation of colloidal complexes between MC adsorbed on CNC-coated QCM-D sensors and TA was confirmed by AFM imaging. No change was observed for the CNC substrate before and after MC adsorption (Figure 3a and 3b, respectively). However, adsorption of TA onto MC-covered CNC substrates resulted in the appearance of sub-100 nm nanoparticles (Figure 3c) which agrees with the particle size analysis included in the Supporting Information. Although both QCM-D measurements and AFM only detect the changes of 2D films in the presence of MC and TA, we believe that this is representative of what occurs in 3D at the oil−water interface. TA complexes with both the MC bound to the CNCs and the free MC at the oil−water interface, creating a dense, insoluble shell and stabilizing the emulsions during freeze-drying and redispersion processes. Any free MC−TA nanoparticles are also 187
DOI: 10.1021/acsmacrolett.5b00919 ACS Macro Lett. 2016, 5, 185−189
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ACS Macro Letters
Marway thank NSERC for funding this work through the CREATE Biointerfaces Training Program Grant. R. Pelton holds the Canada Research Chair in Interfacial Technologies.
the complexation between MC and TA leading to an insoluble emulsion shell. For food formulation specifically, the potential to structure liquid edible oils to produce soft solids without the use of significant amounts of solid fats is a clear nutritional benefit. We believe our findings offer an exciting opportunity to extend CNC-stabilized emulsions to food, cosmetic, pharmaceutical, and agricultural applications where solid dry emulsions are desirable either to be used dry or to facilitate shipping and redispersion.
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Methyl cellulose (MC, 40 kDa, degree of substitution of 1.6−1.9), 2hydroxyethyl cellulose (HEC, 90 kDa, molar substitution of 2.5), tannic acid (TA), corn oil, Nile red, and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES buffer) were all purchased from Sigma-Aldrich. All water used was deionized and further purified with a Barnstead Nanopure Diamond system (Thermo Scientific). CNCs were prepared by sulfuric acid hydrolysis of cotton filter aid (Whatman ashless filter aid, GE Healthcare Canada), as described previously.37 Generally, CNCs from cotton and wood are highly crystalline rigid rodlike particles that are 5−10 nm in cross section and 100−200 nm long, and the average dimensions of the CNCs used here were 128 × 7 nm according to TEM with a surface charge density of 0.33 ± 0.02 e/nm2. Emulsions were prepared using a probe sonicator (Sonifier 450, Branson Ultrasonics), as described in our previous work.23,24 In emulsions with TA, TA was added to the emulsions and mixed for 6 h. Emulsions were imaged by CLSM, and Nile red was added to color the oil phase. The dried oil powders were visualized by SEM (JEOL 7000F SEM, JEOL Ltd., Japan). Adsorption measurements were performed with an E4 QCM-D instrument from Q-Sense AB (Sweden) by procedures previously reported.37 QCM-D surfaces before and after adsorption experiments were imaged using a MFP-3D AFM (Asylum Research an Oxford Instrument Company, Santa Barbara, CA) using FMR cantilevers (NanoWorld Technologies, Neuchatel, Switzerland) with a nominal spring constant of 2.8 N/m and resonance frequencies of 75 kHz. The AFM data were collected and processed using the Asylum research AFM software built on Igor Pro (Wavemetrics, Portland, OR). S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.5b00919. Experimental section detailing the preparation of CNCs, AFM imaging, and QCM-D analysis, photographs and CLSM images of emulsions stabilized without TA after freeze-drying and rehydration, 3D AFM height images of CNC−MC and CNC−MC−TA emulsions, and TEM images of MC-TA and HEC-TA colloidal complexes (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank X. Dong for technical help with SEM imaging. Useful discussions with Dr. A. Marangoni are gratefully acknowledged. This work was carried out using instruments in McMaster’s Biointerfaces Institute and the Canadian Centre for Electron Microscopy. Z. Hu and H. S. 188
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