Advanced emulsions via noncovalent interaction

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Feb 27, 2018 - increasing importance toward advanced emulsions.16–18 ..... emulsions, breaking occurred when the temperature increased to 60 1C (Fig.


Cite this: Chem. Commun., 2018, 54, 3174 Received 2nd January 2018, Accepted 27th February 2018

Advanced emulsions via noncovalent interaction-mediated interfacial self-assembly† Songling Han,ab Huijie An,ac Hui Tao,a Lanlan Li,a Yuantong Qi,a Yongchang Ma,a Xiaohui Li,b Ruibing Wang *c and Jianxiang Zhang *a

DOI: 10.1039/c8cc00016f

We demonstrate that the traditional emulsification theory can be enriched by a self-assembly approach, in which hydrophilic copolymers with one block exhibiting noncovalent forces with the oil phase self-assemble at the oil–water interface, thereby reducing interfacial tension and forming emulsions. This approach was established using affinity diblock copolymers that can interact with oil molecules through electrostatic interactions or hydrogen-bonding. Nanoemulsions with excellent stability were successfully obtained simply via vortexing. The self-assembled emulsions showed unexpected catastrophic phase inversion, further extending the phase structures to bicontinuous and reverse emulsions. Complex emulsions could also be fabricated by this strategy. In addition, the thus prepared nanoemulsions can be used to engineer different nanomaterials.

Emulsions have been widely utilized in the food,1 pharmaceutical,2 and cosmetic industries,3 as well as in biomedical engineering4–6 and materials science.7–9 Emulsification generally relies on high-energy approaches, such as high-pressure homogenization and ultrasonication.10 Also, phase inversion emulsification and microfluidics have been developed as low energy methods.10–13 In most cases, however, high concentrations of surfactants are used to obtain stable nanoemulsions.2,10,13–15 Moreover, complicated emulsification processes are frequently adopted to realize complex emulsions. Consequently, the development of conceptually creative emulsification approaches is of increasing importance toward advanced emulsions.16–18 Although emulsions can be stabilized with solid nanoparticles,19–22 molecular surfactants are most frequently used a

Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, Chongqing 400038, China. E-mail: [email protected] b Institute of Materia Medica, College of Pharmacy, Third Military Medical University, Chongqing 400038, China c State Key Laboratory of Quality Research in Chinese Medicine, and Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau, China. E-mail: [email protected] † Electronic supplementary information (ESI) available: Details of materials and methods as well as additional data. See DOI: 10.1039/c8cc00016f

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to achieve effective emulsification.1–5,8,9,23 According to the traditional theory of emulsification,24,25 emulsifiers with hydrophilichydrophobic balance (HLB) values of 8–18 or 3.5–6 are required to form oil-in-water (o/w) or water-in-oil (w/o) emulsions, respectively. Herein we report the formation of diverse emulsions using highly hydrophilic copolymers with HLB values far beyond the proposed limiting range, by interfacial self-assembly-mediated emulsification (Fig. S1, ESI†). As a proof of concept, we first examined electrostatic interaction-mediated emulsification (Fig. S2, ESI†). A series of hydrophilic cationic copolymers bearing one polyethylene glycol (PEG) block and another segment of polyaspartamide, including PEG-PEDA, PEG-PDETA, and PEG-PTETA were synthesized by aminolysis of PEG-block-poly(b-benzyl L-aspartate) (PEG with Mw of 5 or 2 kDa) with an excess amount of ethylenediamine (EDA), diethylenetriamine (DETA), and triethylenetetramine (TETA), respectively (Fig. S2A and S3, ESI†).26 1H NMR spectra revealed the comparable polyaspartamide length for the three cationic copolymers (Table S1, ESI†), while measurement by MALDI-TOF mass spectrometry indicated the narrow Mw distribution profiles for the synthesized copolymers (Fig. S4, ESI†). All these copolymers can be easily dissolved in water. Typical liquid and hydrophobic aliphatic acids of n-valeric acid (VAL), n-hexanoic acid (HEX), and n-heptanoic acid (HEP) served as oil phase molecules. After aqueous solutions with different concentrations of PEGPEDA and various volumes of HEX were mixed and vortexed for 30 s, measurement by dynamic light scattering revealed notable changes in the particle diameters (Fig. 1A), while the polydispersity index (PDI) was comparable (Fig. 1B). Following an initial increase in the mean diameter, it decreased dramatically. Nevertheless, a significant phase separation was observed at 2.5 mg ml 1 PEG-PEDA, when the relative HEX volume was above 0.05. At 10 mg ml 1 PEG-PEDA, almost similar changing profiles were observed for VAL, HEX, and HEP (Fig. 1C and D), although two separated phases were also observed in the case of HEP when its relative volume was higher than 0.1. To a certain degree, these size changing curves are consistent with those of

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Fig. 2 Characterization of assembled HEX/PEG-PEDA emulsions. (A and B) Size distribution profiles (A) and mean diameter (B) of emulsions formed by HEX and various concentrations of PEG-PEDA at an oil–water volume ratio of 0.04 : 1. (C and D) Digital photos (C) and changes in the mean size and PDI (D) of HEX/PEG-PEDA nanoemulsions at 0.04 : 1. Data in (D) are mean  s.d. (n = 3).

Fig. 1 Self-assembly of aliphatic acids in aqueous solutions of PEG-PEDA. (A and B) The average diameter (A) and PDI values (B) of emulsions formed by various concentrations of PEG-PEDA with increased HEX. (C–E) The mean size (C), PDI values (D), and scattering intensities (E) of 10 mg ml 1 PEG-PEDA with increased aliphatic acids. In these cases, aliphatic acids were added to 1 ml of an aqueous solution containing PEG-PEDA. (F) Size distribution profiles of emulsions assembled by 10 mg ml 1 PEG-PEDA and various aliphatic acids at an oil–water volume ratio of 0.04 : 1. Data in (A–D) are mean  s.d. (n = 3).

light scattering intensities (Fig. 1E). At a specific volume ratio of oil–water phases, the assembled nanoemulsions showed relatively narrow size distributions, regardless of the different aliphatic acids (Fig. 1F). The similar changes were found for cationic copolymers with different polyaspartamide blocks and PEG of either 5 or 2 kDa, upon addition of various volumes of HEX (Fig. S5A and B, ESI†). According to these results, we proposed a self-assembly process for the aqueous solution of cationic block copolymers and liquid aliphatic acids (Fig. S6A, ESI†). At low contents of aliphatic acid, its binding with the cationic segment gives rise to supramolecular amphiphiles that may assemble into micellelike aggregates.27 Further increasing the content of aliphatic acid leads to a morphological transition into vesicles.28,29 With additionally enhanced aliphatic acid, nanoemulsions are formed, which are stabilized by an assembled layer of supramolecular amphiphilic molecules at the oil–water interface. Of note, the magnitude of the critical transition volume (CTV) is closely related to the chemical structures of the aliphatic acids and the copolymers. Observation by super-resolution fluorescence microscopy (SRFM) supported this aliphatic acid content-dependent transition for HEX/PEG-PEDA (Fig. S6B, ESI†). Small nanoparticles were found at an oil–water volume ratio of 0.005 : 1, while we observed vesicles at 0.01 : 1. By contrast, relatively large nanoparticles appeared at 0.04 : 1. This was further affirmed by transmission electron microscopy (TEM), after PEG-PEDA was cross-linked via glutaraldehyde (Fig. S6C, ESI†). For the vesicular structure, statistical analysis showed a high yield of B49% (Fig. S7, ESI†). Vesicles were also observed at relatively high contents of HEX, mainly due to its evaporation under vacuum. Accordingly, nanoscale emulsions can be successfully assembled by mixing aliphatic acids (the o phase) and aqueous solutions of cationic block copolymers (the w phase), when the oil phase volume is above the CTV. The thus formed o/w nanoemulsions

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displayed relatively narrow size distribution profiles for different aliphatic acid/copolymer pairs (Fig. 1F and Fig. S5C and D, ESI†), when the oil volume was below a certain value that is determined by their chemical structures. Notably, the nanoemulsions based on HEX and different copolymers exhibited a significantly higher viscosity than deionized water (Fig. S5E, ESI†). For the HEX/PEG-PEDA-based o/w emulsions, their size notably increased with a decrease in copolymer concentration (Fig. 2A and B), and we observed microscale emulsions below 5 mg ml 1 PEG-PEDA. Of note, the stability of the assembled nanoemulsions was strongly associated with the aliphatic acids. For the o/w nanoemulsion assembled by HEX/PEG-PEDA at an oil–water volume ratio of 0.04 : 1, its mean size and PDI changed mildly after 10 days of incubation at room temperature (Fig. 2C and D). Only a slight phase separation occurred after 33 days of storage at room temperature, and nanoemulsions were attained again after vortexing. Comparatively, we observed a remarkable size increase with subsequent phase separation for the VAL/PEG-PEDA emulsions with the same oil content (Fig. S5F and G, ESI†). Also, we found that interfacial self-assembly-mediated nanoemulsification can be realized for long-chain aliphatic acids. Nanoemulsions were successfully formed by oleic acid and PEG-PEDA when the volume ratio of the oil phase to the water phase was 0.04 : 1 (Fig. S5H–J, ESI†). These findings demonstrated that o/w nanoemulsions can be assembled by liquid hydrophobic aliphatic acids and aqueous solutions of cationic copolymers at defined oil phase contents, which may be easily achieved by vortexing. For the oil phase of VAL, HEX, and HEP, the theoretically required HLB values were estimated to be 11.0, 11.5, and 12.0, respectively.30 The calculated HLB values were 209, 168, and 133 for PEG-PEDA, PEG-PDETA, and PEG-PTETA, respectively (Table S1, ESI†).24 Despite the significant mismatch considering the well-established effective HLB range,24,25 our findings substantiated that these hydrophilic copolymers may serve as emulsifiers to afford o/w nanoemulsions. Importantly, this strategy only involves mild emulsification using relatively low contents of polymers. Nanoemulsions based on traditional techniques, however, are generally attained by either high-energy approaches or low-energy methods combined with the use of extremely high contents of surfactants and co-surfactants.10,14,15 We subsequently investigated interactions between cationic copolymers and aliphatic acids. Measurement by isothermal titration calorimetry (ITC) revealed thermodynamically favorable binding between PEG-PEDA and VAL, HEX, or HEP (Fig. S8, ESI†),

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with the binding constants exceeding 1.2  105 M 1 for the three examined aliphatic acids, which is the same order of magnitude as previously reported for proteins and peptides.31 In the presence of HEX, proton signals due to the EDA units of PEG-PEDA exhibited a high-field chemical shift (Fig. S9A, ESI†). Likewise, shifts toward high magnetic field were observed for the aliphatic protons of HEX in the presence of PEG-PEDA (Fig. S9B, ESI†). 1 H–1H COSY NMR spectroscopy indicated correlation signals between HEX and the EDA unit of PEG-PEDA (Fig. S9C, ESI†). FT-IR spectrometry also showed significant interactions between PEG-PEDA and HEX (Fig. S9D, ESI†). Additional fluorescence spectroscopy using pyrene suggested that hydrophobization occurred in the aqueous solution of PEG-PEDA with increased HEX (Fig. S9E and F, ESI†). These results demonstrated that there are strong interactions between PEG-PEDA and aliphatic acids. For the primary amine of EDA, its pKa is about 10.7, while the used deionized water had a pH value of 6.9 (Fig. S10, ESI†). At 10 mg ml 1, the aqueous solutions of different copolymers displayed slightly alkaline pH (Fig. S10, ESI†). As for HEX, its pKa is B4.9, and the saturated aqueous solution of HEX showed a pH value of 3.4. Therefore PEG-PEDA is positively charged, and HEX is negatively charged in deionized water. Electrostatic interactions are mainly responsible for the formation of affinitymediated nanoemulsions, through the effective binding of PEDA chains with HEX molecules (Fig. S2B, ESI†). By driving the cationic copolymer assembly at the oil–water interface, the interfacial tension between HEX and the aqueous solution of PEG-PEDA was dramatically and rapidly reduced (Fig. S11A and B, ESI†). Consistent with this result, demulsification occurred upon the addition of water soluble propionic acid or free EDA (Fig. S11C–F, ESI†), both of which can impair electrostatic forces between HEX and PEG-PEDA. Also, HEX could not be emulsified into an aqueous solution of PEG-PEDA containing 0.9% NaCl (Fig. S11G, ESI†), while the addition of NaCl immediately demulsified the HEX/PEG-PEDA nanoemulsions (Fig. S11H, ESI†). This should be due to electrostatic screening by electrolytes, resulting in dramatically decreased electrostatic forces between HEX and PEG-PEDA. These results also suggested that the assembled emulsions are switchable, which is beneficial for specific applications such as nanoparticle synthesis and emulsion polymerization.32 It should be noted that interfacial assembly of these cationic copolymers is different from a recent finding on polyelectrolyte assembly at the oil–water interface,33,34 where the charged moieties cannot directly interact with the oil-phase molecules. Our assembled emulsions are also different from another system, in which the component of a hydrophilic graft copolymer does not penetrate into the oil phase, despite its pH-regulatable HLB.35 Additionally, in the latter case the emulsion is stabilized by preventing coalescence rather than by reducing interfacial tension. Interestingly, we observed phase inversion of the assembled emulsions from o/w to the bicontinuous phase and finally to w/o upon increasing the oil content (Fig. 3A). To give a clear illustration, Nile red-doped HEX and an aqueous solution of PEG-PEDA were mixed via gentle shaking. At 10 mg ml 1 PEG-PEDA, o/w emulsions, a bicontinuous phase, and w/o emulsions were

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Fig. 3 Diverse phase structures by the self-assembly of HEX and an aqueous solution of PEG-PEDA. (A and B) Sketch (A) and representative fluorescence images (B) showing catastrophic phase inversion upon increasing HEX fractions at 10 mg ml 1 PEG-PEDA. (C) The binary phase diagram of the HEX/PEG-PEDA system. (D) Engineering of complex emulsions. Scale bars, 10 mm.

separately formed when the oil volume fraction (f) was enhanced from 9.1%, to 60%, to 90.9% (Fig. 3B). A full spectrum of the phase transition can be observed with increasing f (Fig. S12A, ESI†). Also, catastrophic phase inversion was found at other concentrations of PEG-PEDA (Fig. S12B and C, ESI†). The binary phase diagram revealed that the bicontinuous phase exists in a relatively broad range of oil fractions at low concentrations of PEG-PEDA (Fig. 3C). Whereas this phenomenon has been reported for emulsions stabilized by colloidal particles,19,21,36,37 herein our finding for the first time demonstrated that the emulsion transition and bicontinuous emulsions can be achieved using cationic block copolymers. Furthermore, complex emulsions, including waterin-oil-in-water (w/o/w) and oil-in-water-in-oil (o/w/o) emulsions could be realized (Fig. 3D). Furthermore, we explored whether other non-covalent interactions can be utilized to form emulsions by interfacial assembly. Hydrogen-bonding (H-bonding) was examined using a hydrophilic copolymer PEG-block-poly(N-isopropylacrylamide) (PEGPNIPAm) (Table S2 and Fig. S13A, ESI†). ITC measurement suggested the favourable affinity of PEG-PNIPAm with VAL, HEX, or HEP (Fig. S13B and C, ESI†). Our previous studies demonstrated the presence of both H-bonding and hydrophobic interactions between amide-containing polymers and carboxyl-bearing compounds.38,39 Independent of aliphatic acids, o/w nanoemulsions formed when they were mixed with an aqueous solution of PEG-PNIPAm by vortexing at oil–water volume ratios of 0.01 : 1–0.04 : 1 (Fig. S13D–F, ESI†). Nevertheless, phase separation occurred when the oil–water ratio was above 0.04 : 1. These results revealed that o/w nanoemulsions can be obtained at defined oil fractions, using a hydrophilic copolymer that has synergistic H-bonding and hydrophobic forces with the oil-phase molecules. For the HEX/PEG-PNIPAm based nanoemulsions, breaking occurred when the temperature increased to 60 1C (Fig. S14, ESI†). This is in accordance with the temperature-induced disruption of H-bonding and thermosensitivity of PNIPAm.38,40 These assembled emulsions are anticipated to have broad applications. We found that the HEX/PEG-PEDA-based o/w nanoemulsions could be applied to solubilize either Au or Ag nanocrystals, giving rise to aqueous colloidal Au or Ag nanoparticles

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(Fig. S15 and S16A and B, ESI†). Also, nanoemulsions assembled via HEX/PEG-PEDA may be used to prepare nanoparticles of biodegradable polyesters such as poly(lactide-co-glycolide) (PLGA5050, Fig. S16C, ESI†). The conventional knowledge suggests that emulsions should be stabilized by surfactants including amphiphilic molecules or solid particles.25,41 In addition, theoretical studies have revealed that multiblock copolymers are more efficient surfactants than diblock copolymers, since the latter may extend more chains into the bulk phase.42 Herein we demonstrate that these limitations can be circumvented via non-covalent force-mediated selfassembly of hydrophilic copolymers at the oil–water interface. In this new emulsification strategy, hydrophilic copolymers with HLB values far beyond the range proposed by traditional theory can be used as effective surfactants as long as they have strong noncovalent forces with the oil phase molecules. Despite the limitation of relatively low oil contents for emulsions by selfassembly, this conceptually new emulsification approach provides a facile, mild, and low-energy strategy toward diverse emulsions with wide applications in the engineering of functional materials as well as for the development of cosmetic and biomedical products. This study was financially supported by the National Natural Science Foundation of China (No. 81471774 & 81520108029) and the Graduate Student Research Innovation Project of Chongqing to LLL.

Conflicts of interest There are no conflicts to declare.

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