Bioorganometallic ferrocene-tripeptide nanoemulsions

Xuejiao Yang a, Yuefei Wang ab, Wei Qi *abc, Rongxin Su abc and Zhimin He a
aState Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China. E-mail: qiwei@tju.edu.cn
bTianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin 300072, P. R. China
cCollaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, P. R. China

Received 2nd June 2017 , Accepted 16th July 2017

First published on 20th July 2017


We demonstrate an effective strategy to prepare highly stable nanoemulsions using ferrocene-modified tripeptides. Compared with traditional nanoemulsions, bioorganometallic peptide nanoemulsions are appealing for a number of reasons, including long-term and outstanding thermal stability, redox activity and biocompatibility. The formed nanoemulsions could remain stable for more than four months at room temperature, which is the highest stability reported so far for peptide and protein emulsifiers. The phase behaviour and size distribution of the emulsions could be precisely tailored by altering the temperature, solvent ratio and redox state of the ferrocene moiety. In this process, we observed a unique enthalpy-driven phase transition from nanoemulsions to hydrogels, which could be attributed to the competition between the interfacial free energy and the association energy among the self-assembling peptides. Moreover, we could impart catalytic activity to the nanoemulsions through rationally altering the sequence of the tripeptides. The structurally tunable, functional bioorganometallic nanoemulsions offer new opportunities in many areas including drug delivery, and the food and cosmetic industries.


Introduction

Nanoemulsions have been intensively studied in the food,1 pharmaceutical,2 cosmetic,3 and materials industries to encapsulate,4 protect,5 and deliver bioactive components.6 Compared with microemulsions, the smaller size of nanoemulsion droplets has a lot of advantages, including long-term stability, high optical clarity, and better bioavailability,7 which are beneficial for the delivery system and other certain applications. At present, the majority of the commercial nanoemulsions is commonly developed based on lipophilic components, copolymers,8 polypeptides,9 and some solid particles.10 However, exorbitant price and inferior biocompatibility make the wider applications of nanoemulsions difficult; therefore, it is necessary to develop a new strategy to prepare nanoemulsions.

Self-assembling peptides are a kind of versatile building block to create nanoscale materials. Since diphenylalanine (FF) as a self-assembling motif has been established by Reches and Gazit,11 FF based nanostructures have been widely studied over a decade, and it is demonstrated to be an excellent building block to obtain versatile materials.12–14 Moreover, by modification at the amino terminus of FF with functional groups such as fluorenylmethoxycarbonyl (Fmoc), butoxycarbonyl (Boc), porphyrin, and naphthalene (Nap),15–18 new types of self-assembling molecules are obtained, leading to the formation of materials with additional functionalities such as stimuli responsiveness, chiral amplification and photocatalysis. Recently, Ulijn et al. reported the use of dipeptide derivatives and tripeptides as surfactants to form microemulsions.19,20 However, due to the high interfacial energy cost, the preparation of functional nanoemulsions with precisely controlled phase behaviour using short peptides as emulsifiers has rarely been reported.

A ferrocene (Fc) modified peptide is a kind of attractive functional molecule for the fabrication of highly ordered materials, due to the unique molecular configuration, ultra-strong hydrophobicity and redox activity of the Fc moiety.21–25 In this work, we reported a considerable strategy to prepare highly functional nanoemulsions using simple ferrocene-tripeptide amphiphiles. The formed nanoemulsions could remain stable for more than four months at room temperature, and showed high thermal stability even at 70 °C. The formation of stable nanoemulsions at the aqueous/organic interface could be attributed to a delicate balance between the interfacial free energy and the association energy among the self-assembling peptides, which was an enthalpy-driven process. Through altering the temperature, solvent ratio and redox state of the Fc moiety, we can precisely control the phase behaviour and size distribution of the emulsions. Moreover, we can fabricate nanoemulsions with catalytic activity through rationally altering the sequence of the tripeptides.

Results and discussion

General strategy for the design of peptide emulsifiers

We designed four ferrocene modified tripeptides, which were defined by three structural regions: the ferrocenyl functionality (Fc) affording the steric hindrance and strong hydrophobicity, the structural peptide sequence of the diphenylalanine (FF), which provided necessary hydrogen-bonding interactions and exhibited high propensity for β-sheet formation, and the C-terminal amino acid with a negatively charged carboxylate group (histidine, aspartic acid, phenylalanine, and serine) serving as the hydrophilic tail (Scheme 1a). At the aqueous/organic interface, the tripeptide amphiphiles arranged in the specific configuration, self-assembling into thin shells to stabilize the nanoemulsion droplets (Scheme 1b, I). The formed nanoemulsions could remain stable at room temperature (T = 25 °C) for more than four months. However, when using Fc-FFH as emulsifiers, an enthalpy-driven phase transition from nanoemulsions to hydrogels occurred by the decrease in temperature (Scheme 1b, II), while the other three ferrocene-tripeptide nanoemulsions were still stable (Scheme 1b, III).
image file: c7nr03932h-s1.tif
Scheme 1 (a) Chemical structure of the ferrocene-tripeptides: ferrocene-phenylalanine–phenylalanine-histidine (Fc-FFH), ferrocene-phenylalanine–phenylalanine-aspartic acid (Fc-FFD), ferrocene-phenylalanine–phenylalanine–phenylalanine (Fc-FFF), and ferrocene-phenylalanine–phenylalanine-serine (Fc-FFS). The four molecules consisted of three regions, the steric region was the ferrocene (Fc) motif (green part), the structural peptide region was diphenylalanine (FF) (blue part), and the tail was a C-terminal amino acid with a negatively charged carboxylate group (yellow part). (b) Schematic illustration of the nanoemulsion formation and phase transition based on the self-assembly of ferrocene-tripeptides. I. At the aqueous/organic interface, the monomeric tripeptide amphiphiles arranged in the specific configuration, self-assembling into thin shells to stabilize the nanoemulsions. The formed nanoemulsions could remain stable for more than four months at room temperature. II. At T < 25 °C, the enthalpy-driven phase transition from nanoemulsions to hydrogels occurred in Fc-FFH nanoemulsions. III. At T < 25 °C, the Fc-FFD, Fc-FFF, and Fc-FFS nanoemulsions remained stable still.

Formation of bioorganometallic peptide nanoemulsions

To prepare the nanoemulsions, 1 mg Fc-FFH was dissolved in 50 mM, pH 7.20 phosphate buffer at 60 °C, which was then mixed with ethyl acetate at different volume ratios at room temperature. The mixtures were homogenized for 30 s, leading to the formation of stable nanoemulsions (Fig. 1a). The milky layers below were nanoemulsions, and the transparent layers above were the ethyl acetate phase. The static water contact angle of such Fc-FFH nanoemulsions on a glass coverslip was 63.1° (Fig. S1a), indicating the hydrophilicity of the nanoemulsions, and the emulsions were oil-in-water. We investigated the microstructure within the Fc-FFH nanoemulsions prepared at a volume ratio of 7[thin space (1/6-em)]:[thin space (1/6-em)]3 (phosphate buffer/ethyl acetate) using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM). CLSM (Fig. S2a), high-magnification SEM (Fig. 1b) and AFM images (Fig. 1e) showed that the nanoemulsions consisted of nanovesicles with diameters of ∼150 nm. Transmission electron microscopy (TEM) (Fig. 1c and d) confirmed that the nanoemulsions contained large amounts of hollow nanovesicles. Dynamic light scattering (DLS) revealed that the size distribution of Fc-FFH nanoemulsions ranged from 120 to 180 nm (Fig. 1f), which was consistent with the results of SEM analysis.
image file: c7nr03932h-f1.tif
Fig. 1 (a) Photograph showing the oil-in-water nanoemulsions (yellow milky layer) prepared by homogenizing ethyl acetate with 1 mg mL−1 Fc-FFH phosphate buffer solution (50 mM, pH 7.20). From left to right, the volume ratio of phosphate buffer to ethyl acetate was altered from 1[thin space (1/6-em)]:[thin space (1/6-em)]9, 3[thin space (1/6-em)]:[thin space (1/6-em)]7, 5[thin space (1/6-em)]:[thin space (1/6-em)]5, 7[thin space (1/6-em)]:[thin space (1/6-em)]3 to 9[thin space (1/6-em)]:[thin space (1/6-em)]1, and the total volume was 1 mL. High-magnification SEM (b), TEM (c, d), and AFM (e) images of Fc-FFH nanoemulsions prepared at a 7[thin space (1/6-em)]:[thin space (1/6-em)]3 aqueous/organic volume ratio. (f) Size distribution of the Fc-FFH nanoemulsions prepared in different aqueous/organic volume ratios derived from the DLS analysis.

It has been reported that the tripeptides and 9-fluorenylmethoxycarbonyl (Fmoc) modified dipeptides could form microemulsions at aqueous/organic interfaces.19,20 However, our results showed that the ferrocene modified tripeptides could stabilize nanoemulsions. We proposed that the ferrocene (Fc) motif played a leading role in this formation process. Its irregular shape afforded large steric hindrance and strong hydrophobicity, making the ferrocene modified tripeptides much more like a surfactant, and thus self-assembled at the aqueous/organic interface. To demonstrate this speculation, we tested the ability of the phenylalanine–phenylalanine–histidine (FFH) tripeptide to prepare nanoemulsions at the volume ratio of 7[thin space (1/6-em)]:[thin space (1/6-em)]3 (phosphate buffer/ethyl acetate) following the same procedure as Fc-FFH. However, no stable emulsions were formed as indicated by the CLSM and SEM analysis (Fig. S3). The results proved the necessity of Fc in the preparation of nanoemulsions. On the basis of this strategy, we successfully prepared another three kinds of ferrocene-tripeptide nanoemulsions using Fc-FFD, Fc-FFF, and Fc-FFS as emulsifiers at the 7[thin space (1/6-em)]:[thin space (1/6-em)]3 aqueous/organic volume ratio, respectively (Fig. S4). The static water contact angle of such Fc-FFD, Fc-FFF, and Fc-FFS nanoemulsions on a glass coverslip was 53.3°, 44.5°, and 54.4°, respectively (Fig. S1b–d), indicating that the three kinds of emulsions were oil-in-water. CLSM (Fig. S2b–d), high-magnification SEM (Fig. S5) and TEM (Fig. S6) images showed that the three kinds of tripeptide nanoemulsions consisted of nanovesicles. Furthermore, DLS analysis indicated that the average diameters of Fc-FFD, Fc-FFF, and Fc-FFS nanoemulsions were 159 nm, 181 nm, and 217 nm (Fig. S7), respectively. As expected, these results proved the efficiency of ferrocene-tripeptides as emulsifiers to stabilize nanoemulsions.

Precisely controlling the size distribution of nanoemulsions

The volume ratio of phosphate buffer to ethyl acetate had a great influence on the phase behaviour of the ferrocene-tripeptide nanoemulsions. As shown in Fig. 1a, the volume of nanoemulsion layers increased with the increase of aqueous/organic volume ratio. Moreover, the nanoemulsion layers changed from milky to transparent, indicating the decrease of the emulsion droplets’ size. DLS analysis proved that with the increase of volume ratio (phosphate buffer/ethyl acetate) from 1[thin space (1/6-em)]:[thin space (1/6-em)]9 to 9[thin space (1/6-em)]:[thin space (1/6-em)]1, the average diameter distribution of the nanoemulsions decreased from 5585 nm, 836 nm, 255 nm, 136 nm to 108 nm (Fig. 1f and Fig. S8a), indicating that the emulsions evolved from the microscale to the nanoscale. Then we investigated the average diameter of nanoemulsions in the TEM image (Fig. 1c). The average diameter derived from the TEM image was ∼145 nm, which agreed strongly with the data derived from DLS (Fig. S8b).

Furthermore, the diameter of Fc-FFD, Fc-FFF, and Fc-FFS nanoemulsions could also be tailored using a similar strategy. When we altered the aqueous/organic volume ratio from 1[thin space (1/6-em)]:[thin space (1/6-em)]9 to 9[thin space (1/6-em)]:[thin space (1/6-em)]1, as with the Fc-FFH nanoemulsions, the emulsion layers became bigger, and the average diameter increased (Fig. S9). The results demonstrated that we could precisely regulate the size distribution of ferrocene-tripeptide nanoemulsions by simply adjusting the volume ratio of the aqueous/organic phase.

High stability of bioorganometallic peptide nanoemulsions

The ability to control the separation of an emulsion with external stimuli is of great importance for the practical applications in various fields.26–29 Based on this point, we investigated the stability of the ferrocene-tripeptide nanoemulsions as a function of time and temperature. The tripeptide nanoemulsions were incubated at room temperature for 4 months. Surprisingly, compared with freshly prepared nanoemulsions, demulsion and phase separation did not occur. The emulsion layer and organic layer still remained separate, indicating the high long-term stability of the ferrocene-tripeptide nanoemulsions (Fig. S4). We used TEM and DLS to characterize the nanoemulsions incubated for 4 months. As shown in Fig. S10, nanoemulsions also remained hollow nanovesicles, but the shells of nanovesicles became thicker. We supposed that at a specific temperature, with the increase of incubation time, the ferrocene-tripeptide monomers dissolved in the aqueous phase would continuously self-assemble on the external surface of the nanoemulsion droplets, thereby leading to the formation of thick shells. DLS analysis revealed that the average size of Fc-FFH, Fc-FFD, Fc-FFF, and Fc-FFS nanoemulsions was 556 nm, 480 nm, 632 nm, and 498 nm (Fig. S11), respectively, in accordance with the results of TEM images. In view of the excellent long-term stability, the ferrocene-tripeptide nanoemulsions may have a grander application prospect in many fields.

In order to investigate the thermal stability of the ferrocene-tripeptide nanoemulsions, the samples prepared in a 7[thin space (1/6-em)]:[thin space (1/6-em)]3 aqueous/organic volume ratio were incubated in water baths at different temperatures for 3 hours. The appearance of the nanoemulsions was monitored at 10 °C intervals (Fig. 2). Across the temperature range tested, it was clear that the Fc-FFH nanoemulsions remained in the emulsified state above 25 °C, according to the optical photographs (Fig. 2a), SEM (Fig. S12b and c) and TEM (Fig. 2c–e) images. This indicated that the Fc-FFH nanoemulsions had a strong tolerance to high temperature. What's more, we discovered that with the increase of temperature, the average diameter of nanoemulsions increased, and the shells of nanoemulsions grew thicker (Fig. 2c–e). We used DLS analysis to investigate the change in the average diameter of Fc-FFH nanoemulsions. It could be observed that the average size distribution of Fc-FFH nanoemulsions increased from 184 nm (30 °C), 233 nm (40 °C), 310 nm (50 °C), 392 nm (60 °C) to 457 nm (70 °C) (Fig. 2f). We supposed that with the increase of temperature, Fc-FFH monomers had a higher solubility in the water phase, and then more monomers self-assembled at the aqueous/organic interface, leading to the expansion of droplets’ interfaces, as well as the increase of the average diameter and shell thickness of the nanoemulsion droplets.


image file: c7nr03932h-f2.tif
Fig. 2 (a) Optical photographs of Fc-FFH nanoemulsions prepared in a 7[thin space (1/6-em)]:[thin space (1/6-em)]3 aqueous/organic volume ratio at different temperatures. From left to right, the temperature was altered from 10 °C, 30 °C, 40 °C, 50 °C, 60 °C to 70 °C. TEM images of Fc-FFH assemblages prepared in a 7[thin space (1/6-em)]:[thin space (1/6-em)]3 aqueous/organic volume ratio incubated at (b) 10 °C, (c) 30 °C, (d) 50 °C, and (e) 70 °C for 3 hours. The inset of (b) is the histogram of the diameter distribution of the Fc-FFH fibers, derived from the SEM image (Fig. S12a). (f) Phase diagram for the Fc-FFH self-assemblies on the basis of change in temperature. The data of average size are derived from DLS analysis. The red stars correspond to the emulsion state, tested at 30 °C, 40 °C, 50 °C, 60 °C, and 70 °C; and the blue rings correspond to the hydrogels state, tested at 5 °C, 10 °C, 15 °C and 20 °C.

In addition, we investigated another three kinds of tripeptide nanoemulsions. As expected, Fc-FFD, Fc-FFF, and Fc-FFS nanoemulsions also had a good stability across the temperature range tested from 20 °C to 70 °C (Fig. S13). TEM images showed that even at 70 °C, the three kinds of ferrocene-tripeptide nanoemulsions still maintained a hollow nanovesicle structure (Fig. S14). Moreover, as with the Fc-FFH nanoemulsions, the average diameter of the Fc-FFD, Fc-FFF, and Fc-FFS nanoemulsions increased along with the increase of temperature (Fig. S15).

Enthalpy-driven phase transition from nanoemulsions into hydrogels

During the thermal stability experiment, we found that the Fc-FFH nanoemulsions prepared in a 7[thin space (1/6-em)]:[thin space (1/6-em)]3 aqueous/organic volume ratio could transform into hydrogels below 25 °C (Fig. 2a and f). SEM (Fig. S12a) and TEM (Fig. 2b) images showed that the hydrogels consisted of three-dimensional staggered nanofibers with an average diameter of 34.4 nm (Fig. 2b and inset). To probe the molecular packing within the mixture solutions before and after the phase transition, in situ synchrotron wide angle X-ray diffraction (WAXD) was performed. The nanoemulsions showed no obvious reflections in the 2D WAXD pattern (Fig. S16) and the 1D profile (Fig. 3b, red curve), indicating the disordered arrangement of the peptides at the aqueous/organic interfaces. However, after the transformation of nanoemulsions into hydrogels, we observed reflections corresponding to the d spacing of 4.60 and 4.41 Å, respectively (Fig. 3a and b). These reflections could be attributed to the periodic intermolecular hydrogen bonding between adjacent peptide segments within the β-sheet secondary structure.30,31 We also observed a reflection in the high q range corresponding to the d spacing of 3.75 Å, which could be assigned to the π–π stacking distance.
image file: c7nr03932h-f3.tif
Fig. 3 (a) 2-D X-ray diffraction pattern of Fc-FFH hydrogels assembled at 10 °C. (b) Wide angle X-ray diffraction (WAXD) curves of Fc-FFH hydrogels and nanoemulsions. (c) FTIR spectra in the amide I region corresponding to the carbonyl stretching mode of amino acid residues. (d) FTIR spectra in the range of 3200–3500 cm−1, corresponding to the N–H stretching bands.

Fourier transform infrared spectroscopy (FTIR) was used to probe the secondary structure of aggregates within the nanoemulsions and hydrogels. As shown in Fig. 3c, the Fc-FFH nanoemulsions showed a dominant peak located at 1654 cm−1 in the amide I region, which might be attributed to the alpha-helix conformation. However, after the transformation of nanoemulsions into hydrogels, the FTIR spectrum showed an absorption peak at 1629 cm−1, characteristic of the β-sheet arrangement. In the N–H stretching band, the nanoemulsions showed a broad band at 3258 cm−1, while the hydrogels showed two sharp peaks at 3357 cm−1 and 3282 cm−1 (Fig. 3d). This indicated that the intermolecular hydrogen bonding between the peptides became stronger during the phase transition from nanoemulsions to hydrogels. Combining the results of synchrotron WAXD and FTIR, we demonstrated that the peptides probably underwent a molecular rearrangement at the aqueous/organic interfaces with secondary structural transitions from a relatively disordered configuration to much more ordered β-sheets.

To have an in-depth understanding about this process, we determined the concentration of peptide monomers within the mixture solutions during the phase transition at 10 °C using nuclear magnetic resonance (NMR) spectroscopy (Fig. S17, Table S1). The concentration of Fc-FFH monomers in nanoemulsions and hydrogels was calculated as 0.71 mM and 0.33 mM, respectively (Table S1). This indicated that more monomeric peptide molecules within solution self-assembled with each other during the phase transition process. We supposed that in the Fc-FFH nanoemulsion system, two kinds of energy existed. One was the interfacial free energy between Fc-FFH monomer and solvents, which tended to achieve stable nanoemulsion droplets. The other was the association energy amongst monomeric Fc-FFH themselves, which was likely to promote the self-assembly of monomers into much more ordered structures. We speculated that the formation of stable nanoemulsions could be attributed to a delicate balance between the interfacial free energy and the association energy among the self-assembling peptides. When the temperature decreased, the association energy increased, and the Fc-FFH monomers rearranged or disassociated on the aqueous/organic interface and reassembled with each other into well-defined nanofibers (Scheme 2). Moreover, due to the small size of the nanoemulsions (∼145 nm) and the high geometric constraints, it is unlikely for the fibers (diameters of ∼34.4 nm) to stagger on the surface of the droplets. This led to the phase transition of nanoemulsions into self-supporting hydrogels.


image file: c7nr03932h-s2.tif
Scheme 2 Schematic illustration of the temperature-triggered, phase transition from nanoemulsions to hydrogels via a two-step process: (1) peptide disassembly at the aqueous/organic interface; (2) monomers reassemble into the β-sheet secondary structure. Due to the steric hindrance of the ferrocene moiety, the sheet probably has a twisted architecture.22 The β-sheets further assemble into the staggered nanofibers.

From the thermodynamic standpoint, the hydrogels were more stable than the nanoemulsions, and the phase transition was governed by Ostwald's rule of stages.32 This rule has been used in many polymer systems in both experimental and computational studies, and a few examples of this phenomenon have been reported for supramolecular peptide polymers.32–35 Ostwald's rule of stages elucidates that a system goes through several phases, and each successive phase possesses the smaller Gibbs free energy relative to its predecessor phase, before finally reaching the most thermodynamically stable form. According to the NMR experiments, the critical concentration of both the nanoemulsions and hydrogels was calculated as 0.71 mM and 0.33 mM, respectively, and they could further be converted to the Gibbs free energy through the thermodynamic relationship: C = exp(−βΔG).32 As calculated, ΔG of the phase transition from nanoemulsions to hydrogels at 10 °C was −1.78 kJ mol−1, indicating that the Gibbs free energy of the system decreased during the phase transition, and a more thermodynamically stable structure was achieved. Furthermore, we investigated the thermodynamic characteristics during the phase transition from nanoemulsions to hydrogels by using differential scanning calorimetry (DSC) in constant temperature mode. The data showed that the phase transition from nanoemulsions to hydrogels was an exothermic process across the temperature range investigated (5–15 °C), with the corresponding enthalpy change being −26.51 J mol−1 (5 °C), −24.68 J mol−1 (10 °C), and −20.84 J mol−1 (15 °C), respectively (Fig. 4a). Combining with ΔG obtained at 10 °C, the entropy change was calculated as 6.20 J mol−1 K−1 (Fig. 4b). It could be found in this phase transition that the enthalpy change at 5 °C was bigger than that at 10 °C and 15 °C. What's more, the starting time of the phase transition happened at 5 °C was earlier than that at 10 °C and 15 °C. We supposed that this phenomenon was caused by the change of monomeric Fc-FFH concentration. When the phase transition occurred at lower temperature, the solubility of monomers would decrease, leading to more Fc-FFH monomers incorporated in the reassembly process, and the increase of enthalpy change. The thermodynamic analysis suggested that the phase transition of the nanoemulsions into hydrogels was an enthalpy-driven process. Moreover, it supplied us a unique way to control the separation of the tripeptide nanoemulsions by simply changing the temperature on demand.


image file: c7nr03932h-f4.tif
Fig. 4 (a) Differential scanning calorimetry (DSC) measurement of the Fc-FFH phase transition from nanoemulsions to hydrogels at three constant temperatures. Inset: Magnified image of the dotted box region. (b) Schematic free energy landscape during the Fc-FFH phase transition from nanoemulsions to hydrogels at 10 °C.

Other properties with potential application

Redox activity. The ability to control the characteristics of nanoemulsions under external stimuli is of practical value for controlled drug delivery.36 Here we demonstrated that the microstructure and surface charge of the nanoemulsions could be further tailored by altering the redox state of the ferrocene moiety. The Fc-FFH molecule could be oxidized from the state having a net charge of 0 (reduced) to a net charge of +1 (oxidized) by using the electrochemical oxidation of the ferrocene unit (Fig. 5a). SEM and TEM revealed that the oxidation of Fc-FFH molecules within the nanoemulsions led to the transformation of hollow vesicles into smaller micellar aggregates roughly 60 nm in diameter (Fig. 5b and Fig. S18). DLS revealed that the size distribution of the oxidized nanoemulsions was measured to be 54.78 nm (Fig. 5b, inset), which was in accordance with the data derived from SEM and TEM images. Furthermore, we investigated the ζ-potential of Fc-FFH nanoemulsions before and after electrochemical oxidation, and the results revealed that the ζ-potential typically changed from negative (−16.58 mV) to positive values (8.36 mV) (Fig. 5c). As expected, reduced Fc-FFH nanoemulsions could be transformed into the oxidized state using the electrochemical method, with the morphological transition from nanovesicles to smaller micellar aggregates. In theory, the oxidized aggregates could be reduced to the nanoemulsions; however, as the formation of nanoemulsions needed energy, and the self-assembly process must start from the monomer state, the aggregates could not transform to nanoemulsions by the electrochemical method. The tunable structure and surface charge of the nanoemulsions may offer an opportunity as carriers to convey some targeted medicine to treat disease efficiently by controlled fusing with cell membranes in the negative state.
image file: c7nr03932h-f5.tif
Fig. 5 (a) Molecular structures of reduced and oxidized Fc-FFH. In theory, the charge density of Fc-FFH can be oxidized from a 0 state (reduced) to a +1 state (oxidized) by the application of the electrochemical method. (b) High-magnification TEM image of oxidized Fc-FFH nanoemulsions. Inset: Size distribution of the oxidized Fc-FFH nanoemulsions derived from the DLS analysis. (c) ζ-Potential values of Fc-FFH nanoemulsions before and after electrochemical oxidation.
Catalytic activity. It has been shown that the peptides containing histidine residues could serve as a platform for the construction of artificial hydrolase.37–40 Based on this point, the specific catalytic activity of the obtained Fc-FFH nanoemulsions was studied with the hydrolysis of oil-soluble p-nitrophenyl butyrate (PNPB) (Fig. 6a). The Fc-FFH nanoemulsions were prepared at a 7[thin space (1/6-em)]:[thin space (1/6-em)]3 aqueous/organic volume ratio. When the nanoemulsions were mixed as a catalyst with PNPB, the reaction happened immediately, and the colour of the reaction system became luminous yellow, indicating the catalytic activity of nanoemulsions toward the hydrolysis of PNPB, as well as the generation of the p-nitrophenolate anion (PNP). Moreover, according to the standard curve of PNP (Fig. S19), UV-visible spectroscopy was utilized to monitor the formation of PNP (Fig. 6b), it could be calculated that within 27 minutes, 10 mM PNPB totally conversed to PNP, and the specific activity of nanoemulsions was calculated as 48.54 U mL−1, which proved that we have produced artificial hydrolase nanoemulsions. Furthermore, because of the long-term stability, the Fc-FFH nanoemulsions had a very good recyclability with 82.58% of conversion efficiency maintained even after 15 cycles (Fig. 6c, grey column). In addition, the specific activity of nanoemulsions remained 39.31 U mL−1 after 15 cycles (Fig. 6c, orange column). So we were very delighted to find that the nanoemulsions self-assembled by Fc-FFH as the artificial enzymes possessed excellent storage stability and hydrolysis activity.
image file: c7nr03932h-f6.tif
Fig. 6 (a) Nanoemulsions catalyzed PNPB to convert into PNP and butyric acid. (b) UV-visible monitoring the formation of PNP; the hydrolysis reaction catalyzed by Fc-FFH nanoemulsions was finished within 27 minutes. (c) The conversion and specific activity of Fc-FFH nanoemulsions in 15 cycles. The grey region corresponds to the conversion, and the orange region corresponds to the specific activity.

Experimental

Materials

Ferrocene-L-Phe-L-Phe-L-His (Fc-FFH, 98% in purity), ferrocene-L-Phe-L-Phe-L-Asp (Fc-FFD, 98% in purity), ferrocene-L-Phe-L-Phe-L-Phe (Fc-FFF, 98% in purity), and ferrocene-L-Phe-L-Phe-L-Ser (Fc-FFS, 98% in purity) were synthesized by GL Biochem Ltd (China). All other chemicals were of analytical grade, which were obtained from commercial sources. Phosphate buffer (50 mM, pH 7.20) was prepared by mixing a certain volume of 50 mM NaH2PO4 solution (prepared by dissolving 600 mg NaH2PO4 in 100 mL water) and 50 mM Na2HPO4 solution (prepared by dissolving 1.79 g Na2HPO4·12 H2O in 100 mL water).

Methods

Preparation of nanoemulsions. In a typical experiment, 1 mg Fc-FFH (Fc-FFD, Fc-FFF, and Fc-FFS) was first dissolved in 50 mM pH 7.20 phosphate buffer at 60 °C, then different volumes of ethyl acetate were added into Fc-FFH (Fc-FFD, Fc-FFF, and Fc-FFS) buffer solution (the volume ratio of phosphate buffer to ethyl acetate was altered from 1[thin space (1/6-em)]:[thin space (1/6-em)]9, 3[thin space (1/6-em)]:[thin space (1/6-em)]7, 5[thin space (1/6-em)]:[thin space (1/6-em)]5, 7[thin space (1/6-em)]:[thin space (1/6-em)]3, to 9[thin space (1/6-em)]:[thin space (1/6-em)]1, and the total volume was always 1 mL) at room temperature. The mixtures were homogenized for 30 s, leading to the formation of stable nanoemulsions at different aqueous/organic volume ratios.
Preparation of hydrogels. Fc-FFH nanoemulsions were firstly prepared as described above. The nanoemulsions were incubated at temperatures below 25 °C without any disturbance, leading to the formation of self-supporting hydrogels.
Determination of Fc-FFH monomers’ concentration during the phase transition. In the experiment, we used D2O to prepare pH 7.20 phosphate buffer. Then the Fc-FFH nanoemulsions and hydrogels were prepared as described above. The two kinds of samples were tested by nuclear magnetic resonance (NMR) analysis. The characteristic peaks of D2O and Fc could be integrated, and the ratio corresponded to the monomers’ concentration in two phases.
Electrochemistry oxidation experiment. Electrochemical oxidation of Fc-FFH nanoemulsions prepared in 7[thin space (1/6-em)]:[thin space (1/6-em)]3 aqueous/organic volume ratio was conducted at room temperature using an LK2005A electrochemical workstation (LANLIKE, China). The amperometric it curve was measured under a nitrogen atmosphere, and a three-electrode cell was used to maintain a constant potential of 500 mV between the working electrode and reference electrode. A platinum plate electrode, a platinum wire electrode, and an Hg/Hg2Cl2 electrode were used respectively as the working, counter and reference electrodes, and 50 mM pH 7.20 phosphate buffer was used as the supporting electrolyte. The progress of oxidation was followed by monitoring the current.
Catalytic experiment. Fc-FFH nanoemulsions as catalysts were prepared at a 7[thin space (1/6-em)]:[thin space (1/6-em)]3 aqueous/organic volume ratio as described above. 10 mM p-NPB was mixed with 850 μL nanoemulsions, 122.5 μL 50 mM phosphate buffer, and 17.5 μL acetonitrile. The mixture solution was homogenized for 30 s and then added into a quartz cuvette. The production of p-NP was determined based on the absorbance at 400 nm. In addition, at the end of each catalytic reaction, the nanoemulsions were recycled simply by injecting another equivalent of PNPB to the next batch.

Characterization

Contact angle analysis. A 100 μL aliquot of nanoemulsions was deposited neatly on a microscope glass coverslip. The static contact angle of nanoemulsions was measured with a contact angle measuring device OCA15EC (Data Physical Instruments, Germany) equipped with SCA 202 software.
Laser scanning confocal microscope (LSCM). 100 μL of nanoemulsions was deposited neatly on a microscope glass coverslip, and the image was obtained using a laser scanning confocal microscope (LSCM, FV-1000, Olympus, Japan), with an excitation wavelength of 488 nm.
Scanning electron microscopy. 10 μL of prepared nanoemulsions and a slice of hydrogel were deposited neatly on a microscope glass coverslip and dried in air. All samples were sputter-coated with platinum using an E1045 Pt-coater (Hitachi High-technologies CO., Japan), and then imaged with an S-4800 field emission scanning electron microscope (SEM, Hitachi High-technologies CO., Japan) at an acceleration voltage of 3 keV.
Transmission electron microscopy. The morphology of the tripeptide assemblies was further assessed using a JEOL 100CX-II transmission electron microscope (TEM, JEOL Ltd, Japan) operated at 80 keV. To prepare samples, a 10 μL aliquot of nanoemulsions was placed onto a 200 mesh carbon-coated copper grid, and air dried. And for hydrogels, the sample was dispersed in ddH2O, and 10 μL aliquot of aqueous dispersion was placed onto a 200 mesh carbon-coated copper grid, and air dried.
Dynamic light scattering. The nanoemulsions prepared as described above were diluted in ddH2O and left to stand without any disturbance at 20 °C, 25 °C, 30 °C, 40 °C, 50 °C, 60 °C, and 70 °C. The size distribution of the samples was measured using a Zetasizer Nano AS (Malvern Instruments, British) at the given temperature.
X-ray scattering measurements. In situ X-ray scattering measurements were carried out at beamline 1W2A of the Beijing Synchrotron Radiation Facility (Beijing, China). The wavelength of the radiation source was λ = 0.154 nm. Mar165-CCD was set at 146 mm sample–detector distance in the direction of the beam for WAXS data collections, respectively.
Fourier transform infrared spectroscopy. The as-prepared nanoemulsions and hydrogels were frozen at −45 °C and subsequently dried under vacuum. The spectra of samples were recorded on a Nicolet-560 FTIR spectrometer (Nicolet Co., USA) with a KBr pellet method across the range of 400–4000 cm−1. A total of 16 scans were accumulated with a resolution of 4 cm−1 for each spectrum.
Differential scanning calorimetry. Thermal analysis of Fc-FFH nanoemulsions prepared in the 7[thin space (1/6-em)]:[thin space (1/6-em)]3 aqueous/organic volume ratio was performed by differential scanning calorimetry (DSC 1/500, Mettler Toledo, Switzerland), under the protection of a nitrogen atmosphere at constant temperatures of 5, 10 and 15 °C. The sample cell was filled with 20 μL Fc-FFH nanoemulsions. The sample should be prepared immediately prior to its introduction into the instrument. The area of the exothermic peak was integrated to yield the enthalpy of the phase transition from nanoemulsions to hydrogels.
Ultraviolet–visible spectroscopy. Optical absorption measurements were performed on a TU-1810 UV/vis spectrophotometer (Shanghai Metash Instruments, China) using a 1 mm path length quartz cuvette.
ζ-Potential measurement. ζ-Potential measurement was performed in a Malvern disposable folded capillary cell with the additional specification of the samples at 25 °C, using a Zetasizer Nano AS (Malvern Instruments, British).

Conclusions

In summary, we have demonstrated that the bioorganometallic ferrocene-tripeptide could self-assemble into thin shells at the aqueous/organic interface to stabilize the oil-in-water nanoemulsions. The formed nanoemulsions could remain stable for more than 4 months at room temperature, and showed a strong tolerance to heat even at 70 °C. Furthermore, through subtle modulations in the aqueous/organic volume ratio, temperature, and sequence of ferrocene-tripeptide, we are able to precisely control the size distribution, phase behaviour (emulsion–hydrogel transition) and functionality (e.g., specific catalytic activity) of the self-assembled nanoemulsions on demand. In addition, the ferrocene-tripeptide nanoemulsions could be oxidized to smaller micellar aggregates by using the electrochemical method, which offers an opportunity to be used as the carrier in the drug delivery field. To the best of our knowledge, the ferrocene-tripeptides are the most effective emulsifiers to prepare peptide nanoemulsions with excellent stability, tunable structure, and multifunctionality, which might find more applications in drug delivery, and the food and cosmetic industries.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This work was supported by the Natural Science Foundation of China (no. 21476165, 21606166, 21621004), the 863 Program of China (no. 2013AA102204), the Ministry of Science and Technology of China (no. 2012YQ090194), the Beiyang Young Scholar of Tianjin University (2012) and the Program of Introducing Talents of Discipline to Universities of China (no. B06006). The authors thank Prof. Zhonghua Wu and Dr Guang Mo of BSRF for assistance with the X-ray diffraction measurement.

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Footnotes

Electronic supplementary information (ESI) available: Additional morphology characterization (optical photographs, confocal laser scanning microscopy, SEM and TEM images) of the peptide self-assemblages, DLS and 2-D WAXS analysis of nanoemulsions in various environments, and physical calculation of monomer concentrations within different self-assembling systems. See DOI: 10.1039/c7nr03932h
These authors contributed equally to this work.

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