Silk Nanofibers as Robust and Versatile Emulsifiers - ACS Publications

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Silk Nanofibers as Robust and Versatile Emulsifiers Qingqing Cheng, Bingbo Zhang, Yao He, Qiang Lu, and David L Kaplan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13460 • Publication Date (Web): 29 Sep 2017 Downloaded from http://pubs.acs.org on October 3, 2017

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ACS Applied Materials & Interfaces

Silk Nanofibers as Robust and Versatile Emulsifiers Qingqing Chenga,b, Bingbo Zhangc, Yao Hed, Qiang Lua,b,e,*, David L. Kaplanf

a

College of Chemistry, Chemical Engineering and Materials Science & Collaborative Innovation

Center of Suzhou Nano Science and Technology, Soochow University, Suzhou 215123, People’s Republic of China b

Key Laboratory of Stem Cells and Biomedical Materials of Jiangsu Province and Chinese

Ministry of Science and Technology, Soochow University, Suzhou 215123, People’s Republic of China c

The Institute for Advanced Materials & Nano Biomedicine, Tongji University, Shanghai

200092, People’s Republic of China d

Institute of Functional Nano & Soft Materials (FUNSOM) and Jiangsu Key Laboratory for

Carbon-Based Functional Materials & Devices, Soochow University, Suzhou 215123, People’s Republic of China e

National Engineering Laboratory for Modern Silk, Soochow University, Suzhou 215123,

People’s Republic of China f

Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155,

United States

KEYWORDS: silk, nanofibers, emulsifiers, stability, biocompatibility

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ABSTRACT: Peptides have been extensively studied as emulsifiers due to their sequence and size control, biocompatibility, versatility and stabilizing capacity. However, cost and mass production remain challenges for broader utility for these emulsifiers. Here we demonstrate the utility of silk fibroin nanofibers as emulsifiers, with superior functions to the more traditional peptide emulsifiers. This silk nanofiber system is universal for different oil phases with various polarities, and demonstrates control of microcapsule size through tuning the ratio of silk fibroin nanofiber solutions to oils. Besides the improved stabilizing capacity to peptides, these silk fibroin nanofibers endow additional stability to the emulsions formed under high salt concentration and low pH. Highly efficient encapsulation of biomarkers through interfacial networks suggests potential applications in therapeutics, food and cosmetics. Compared to peptide emulsifiers, these silk fibroin nanofibers offer advantages in terms of cost, purification and production scale, without compromising biocompatibility, stabilizing capacity and versatility.


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1. INTRODUCTION Emulsifiers play a central role in forming emulsions that are widely used in food, cosmetic, catalysis, encapsulation, drug delivery, materials and biomedical fields.1-9 Traditional amphiphilic surfactants are most often used to stabilize emulsions and are well understood, but they can also exhibit some disadvantages, including potential toxicity, inflammation and inferior stability toward pH, salts and temperature.10-12 The development of new emulsifiers with well-defined stability, biocompatibility, and biodegradability is therefore desirable for the food, drug and biomedical industries. Copolymers,13-15 lipids,16-18 proteins,19, 20 polymersomes,21, 22 and solid particles23-26 have been developed to complement traditional surfactants, with proteins desirable due to their biocompatibility, biodegradability, and intrinsic amphiphilic properties. Extensive studies have focused on exploring various water-soluble27 and water-insoluble protein28-31 emulsifiers and clarifying the influence of their physical-chemical properties on emulsion formation and stability. However, physical or chemical cross-linking, or coupling with surfactants and synthetic polymers, is required for soluble proteins to provide stable emulsions, while additional solvents are a prerequisite to increase the solubility of water-insoluble proteins in emulsion forming systems.32-35 All of these procedures result in intricate processes and can decrease biocompatibility and degradability. The challenge remains for protein emulsifiers to actively tailor weak intermolecular interactions (salt bridges, hydrophobic interactions, hydrogen bands) involved in emulsion formation, which can impact the application-specific performance of the emulsions. Peptides are emerging as promising biocompatible emulsifiers since they possess the advantages of protein-based emulsifiers, while also demonstrating superior versatility through



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the active design, size and sequence control of peptide building blocks with diverse chemical functionalities.36,

37

Theoretically, the formation and stability of emulsions can be controlled

through tuning peptide sequences and amino acid chemistries. Enhanced stability of emulsions has also been achieved by the formation of nanofiber peptide networks at interfacial layers, as a different strategy for stable encapsulation, with potential applications in therapeutics and tissue engineering.38-40 However, compared to traditional surfactants, the scalability and costs are key challenges for peptide emulsifiers, restricting their potential applications. Silk fibroin (SF), a natural protein derived from Bombyx mori silkworm, shows encouraging potential in a broad range of applications from biomaterials, tissue engineering and drug release, to microdevices, due to its biocompatibility, mechanical properties, tunable biodegradability and minimal inflammatory reactions, as well as its mass production availability and simple purification processes.41-44 The coexistence of alternating hydrophobic and hydrophilic blocks endows SF amphiphilic capacity, suggesting the possibility as a protein emulsifier. Several attempts have been reported to prepare emulsions stabilized by SF and assess stability and modulation under different conditions.45-50 These emulsions did not show enhanced stability or tunability compared to the stabilization afforded by proteins. Therefore, SF has not been fully considered as a promising emulsifier to date. Recently, we demonstrated that SF formed stable nanofibers (SNF) in aqueous solution through a controllable self-assembly process.51 Unlike traditional SF in aqueous solution, the SNF had high beta-sheet content and charge density, hydrophobic properties and water dispersibility.52,

53

Inspired largely by this confluence of

unusual properties (water-dispersibility, hydrophobicity, stable nanofiber structures), we hypothesized that the SNF would offer an opportunity to prepare versatile, stable and controlled oil-in-water emulsions.


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Herein, we demonstrate how assembled SNF can be utilized as emulsifiers to generate stabilized emulsions with versatile properties. The approach achieves tunable properties by adjusting SNF concentration and ratios of water to oil. The control of SNF conformation provides additional control of the process. The results suggest that SNF are versatile emulsifiers with long-term stability at high temperature, near isoelectric points and with high salt concentrations. 2. MATERIALS AND METHODS 2.1. Preparation of Silk Nanofibers Preparation of silk fibroin solution followed previous procedures.51 Briefly, Bombyx mori cocoons were boiled 30 min in a sodium carbonate solution (0.02 M), and rinsed thoroughly with distilled water three times to remove the sericin proteins. The extracted silk was loosened and dried at 60oC overnight. The dried fibers were dissolved in lithium bromide solution (9.3 M) at 60°C for 4 hours, yielding a 20% (w/v) solution. This solution was cooled to 25oC and dialyzed against distilled water for 72 h to remove the salt. The solution was optically clear after dialysis and was centrifuged at 9,000 rpm 3 times at 4°C to remove silk insoluble fibrous debris formed during the process. The final concentration of aqueous silk solution was about 6 wt%, determined by weighing the remaining solid after drying. To prepare SNF, fresh silk fibroin solution was treated by a concentration-dilution process to induce silk assembly in aqueous solution.53 The solution (6 wt%) was concentrated to about 12 wt% at 60oC within 24 hours, slowly further concentrated to about 20 wt% to obtain metastable nanoparticles with size of about 100 nm,53 and then diluted to 2 wt% with ultrapure water. The diluted silk solution was incubated for about 24 h at 60oC to induce the nanofiber formation. 2.2. Preparation of Emulsions The emulsion droplets were prepared by emulsifying SNF solutions with oils. Typically, SNF (1ml, 2 wt%) was stirred by a magnetic stirrer and diluted



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with water to increase its fluidity, and then added to the dodecane solution (5ml). The ratio of aqueous phase to dodecane (with Oil Red O) was 5:5, and the final concentration of the SNF was adjusted to 0.2 wt%. Homogenization under 10,000 rpm for 10 min was carried out by a Highspeed dispersion machine (XHF-DY, Scientz Biotechnology Co., Ningbo, China). The formed emulsions were stored for 12 h to ensure stability. To reveal the effect of various conditions, silk fibroin with different conformations (high beta-sheet and amorphous), various SNF final concentrations (0.2, 0.5, 1 wt%), different mixing speeds (3,000, 6,000, 10,000 rpm) and time (1, 3, 10 min) were chosen to form emulsions. Different ratios of aqueous phase to oil phase (1:9, 3:7, 5:5, 7:3, 9:1) and various oil phases (hexane, hexanol, dodecane, butyl butyrate) were selected to assess the universality of the system. 2.3. Fluorescence Microscope An inverted fluorescence microscope equipped with a CCD video camera (AxioVert A1, Carl Zeiss, Germany) was used to measure the size of the emulsions. Each sample was gently diluted with distilled water. A drop of diluted sample was transferred onto the microscope slide and covered with a cover slip prior to measure. At least 15 pictures were measured for each sample. 2.4. Analysis of Morphology The interfacial structures of samples were observed using a scanning electron microscopy (SEM, S-4800, Hitachi, Tokyo, Japan) at 3 kV. Before SEM examination, the samples were freeze-dried and coated with platinum. 2.5. Fourier Transform Infrared Spectroscopy The secondary structures of the freeze-dried samples were analyzed with Fourier Transform Infrared Spectroscopy (FTIR 5700, Thermo Scientific, FL, United states). For each measurement, 64 scans were recoded with a resolution of 4 cm−1, with the wavenumber ranging from 1200 to 1800 cm−1.


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2.6. Zeta Potential Measurements The surface charges of the droplets were determined using zeta potential measurements at a refractive index (RI) of 1.450. The laser obscuration was about 10%. Before measurements, droplets and SNF were diluted with ultrapure water. One milliliter of the solution was loaded to a Zetasizers (Nano ZS, Malvern, Worcesteshire, UK) at 25oC. The surface charges of each sample were measured at least five times. 2.7. Mechanical Properties of Microcapsules The mechanical properties of the microcapsules were measured with Atomic Force Microscopy (AFM, Dimension Icon, Bruker, Germany). For AFM measurements, SNF-stabilized emulsions were diluted to below 0.1 wt% and 2 µL of the diluted emulsions were dropped onto silicon surfaces. The nominal force constant is 0.4 N m-1 and the actual spring constant of the probe was calibrated before each test. 2.8. Determination of Emulsion Stability To analyze thermal stability, each sample was incubated in a water bath at room temperature and 60oC. The samples were also incubated at different concentration (0M, 0.01M, 0.1M) of PBS and pHs (4.5, 7.4, 11.0) at 37oC to determine salt and pH stability. 2.9. Fluorescence Stability The human breast cancer cell lines, MCF-7 cells, are usually used to assess fluorescence stability of the quantum dots (QDs) inside cells,54 were also used in our study. The cells were obtained from the cell bank of Chinese Academy of Science (Shanghai, China). MCF-7 cells were seeded on coverslips in 24-well plates (3×104cells/well) supplemented DMEM with fetal bovine serum (10% (V/V)), penicillin (100 U/mL), streptomycin (100 µg/mL), and cultured at 37oC using a humidified 5% CO2 incubator, and allowed to recover overnight. Fluorescent stability was determined by adding free water-soluble CdSSe/ZnS (Free-QDs, gift of Bingbo Zhang, Tongji University) and silk encapsuled oil-soluble CdSSe/ZnS (SNF-QDs) into the cell-seeded plates, with a QDs concentration of 0.03mg.mL-1, and then the cells accompanied



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with the QDs were incubated for 3 days. At different time points, the cells were washed three times with PBS, and imaged by laser scanning confocal microscopy (LSCM, Olympus Fluoview FV1000, Japan) with excitation/emission at 460/610nm.

3. RESULTS AND DISCUSSION The study of SNF as emulsifiers started from the discovery that these nanofibers had hydrophobic properties, but form solutions and gels in aqueous solvents at different concentrations. Recent studies on aromatic peptide emulsifiers39, 40 suggested the possibility that SNF networks might be able to stabilize emulsions at the oil/water interface. Figure 1 shows a schematic of the process for the formation of oil-in-water emulsions using these SF nanofibers. A traditional emulsifying process was used to form the emulsions, except that SF nanofibers replaced traditional surfactants. Oil Red O was used to label the oil phase. By adding various volumes of dodecane to SF nanofiber water solutions at room temperature (the volume ratio of SF nanofiber solution to dodecane (W/O) was altered from 1:9, 3:7, 5:5, 7:3, to 9:1), after emulsification (agitation) for 10 min, emulsions formed (Figure 2A). At a low volume of SF nanofiber solution (W/O, 1:9), the pink emulsions appeared in the aqueous phases and the dodecane phases remained transparent, suggesting the formation of oil-in-water emulsions. The dodecane phases were occupied by milky emulsions following an increased W/O ratio, due to the lower density of emulsions dominated by the oil core and their smaller size. Interestingly, the aqueous layers below the emulsion layers were light pink rather than transparent, indicating the existence of smaller dodecane-filled microcapsules.


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Figure 1. Schematic of the process for the formation of oil-in-water emulsions. A traditional emulsifying process was used to form the emulsions. Oil Red O was used to label the oil phase.

Microscopy images confirmed the formation of oil-filled microcapsules inside the milky emulsion layers and below the water phases. At W/O ratios from 1:9 to 9:1, the average size of the microcapsules inside the milky emulsion phase increased at first and then decreased, where the largest microcapsules sized between 20~30 µm appeared at the W/O ratio of 3:7. The smallest microcapsules with a size range of 5~10 µm were achieved at the ratio of 9:1 (Figure 2B, C). Similar to peptide-stabilized emulsions previously reported, a fraction of smaller capsules appeared in all cases.39, 40 Significantly smaller microcapsules sized between 200-800 nm were distributed below the water phases (Figure 2D), presumably due to the higher density of these smaller droplets when compared with the larger sizes in the emulsion phase. Maintaining a fixed W/O ratio of 5:5, we next investigated the morphological changes of the microcapsules under various emulsifying conditions. Higher SF nanofiber concentrations, higher shear speed and longer shear time resulted in the formation of smaller microcapsules, suggesting control of size 9 ACS Paragon Plus Environment


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with SF nanofibers as emulsifiers (Figure S1). The organic phase and SF nanofibers were labeled with Oil Red O and Rhodamine 6G, respectively, to visualize the interfacial assemblies of the capsules (Figure 2E). Fluorescent microscopy images showed that the SF nanofibers localized to the interface of the capsules and coated the oils.

Figure 2. The morphology and structure of the emulsions formed at various volume ratios (W/O) of SNF solution to dodecane. (A) Optical photographs,(B) Size distribution, (C) Fluorescence micrographs of the emulsions at various W/O ratios, (D) Fluorescence micrographs of emulsions


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formed in the water phase at W/O ratio of 9:1, (E) Fluorescence micrographs of emulsions labeled with Oil Red O and Rhodamine 6G.

Scanning electron microscopy (SEM) was used to visualize the assembled network of SF nanofibers at the interface (Figure 3). All the samples were freeze-dried for SEM imaging to minimize changes in the microcapsule structure, also suggesting stability of these networks (Figure 3A). Higher magnification of the interface showed fibrous networks with pore sizes of 38 nm(Figure 3B). This result confirmed that the SF nanofibers formed interconnected networks to stabilize the capsules, similar with that previously observed for various peptide emulsifiers.27, 39

A fractured freeze-dried microcapsule showed a hollow internal structure (Figure 3C). Infrared

spectroscopy was used to assess the conformational changes of SF nanofibers at the interface (Figure 3D). The SF nanofibers showed typical beta-sheet structures with an amide I peak at 1623 cm-1 without conformational change after microcapsule formation. This result suggests that the microcapsules were likely stabilized by physically entangled nanofiber networks rather than a conformational transition. Zeta potential (Table 1) changes were also investigated to confirm the stability of SF nanofibers in the emulsion formation process, without significant changes after microcapsule formation. Uncontrollable conformational transitions are a significant drawback with previously SF-stabilized emulsions.49 Here, the hydrophobic SF nanofibers with stability provided a controllable and reliable method to prepare SNF-stabilized emulsions.



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Figure 3. Representative microstructures and conformations of the emulsions after freeze-drying: (A) SEM images, (B) High magnification of the interface of the emulsions, (C) SEM micrograph of a fractured SNF microcapsule, and (D) FTIR spectra of the SNF emulsions before and after emulsification.

A useful feature of peptides as emulsifiers is the enhanced stability of the emulsions when compared to traditional surfactants and peptides. Considering the similar nanofiber network structures to those formed by peptides, the stability of SNF-stabilized emulsions was assessed and comparisons were made to emulsions stabilized by KYF (tripeptide that forms nanofibers) as a control (Figure 4).39 All the emulsions were prepared under same conditions with SNF and KYF as emulsifiers. The peptide-stabilized emulsions lost emulsification and phase-separated after 24 hours at room temperature, while the emulsions stabilized by SNF were still stable after 12 ACS Paragon Plus Environment

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6 months at room temperature. Compared to previous studies,39 the KYF-stabilized emulsions in our study showed inferior stability, partly due to the storage conditions. However, the results suggested that SNF-stabilized emulsions achieved significantly better stability. The SNFstabilized emulsions also showed tolerance for heat. There was no visible phase separation for SNF emulsions after exposure to 60oC for 6 months (Figure 4A). Fluorescent microscopy images of the emulsions confirmed that all of the microcapsules in the emulsions were stable after 6 months at room temperature and 60oC (Figure S2).

Table 1. Zeta potential of SNF and SNF-stabilized emulsions at various W/O ratios. ratio of water to oil

zeta potential ± SD(mV)

SNF

-33.7 ± 0.2

1:9

-36.4 ± 0.9

3:7

-35.4 ± 2.4

5:5

-37.3 ± 1.3

7:3

-35.4 ± 1.8

9:1

-35.9 ± 1.9

When 2 ml emulsions were added into 8 ml aqueous solutions with different salt concentrations and pH values, the SF nanofiber stabilized emulsions exhibited different stabilities. The SF nanofiber-stabilized emulsions remained more stable than that stabilized by peptide as control, and maintain emulsification after 3 months at the higher pH value and lower salt concentrations (Figure 4B, C). Interestingly, in contrast to emulsions stabilized by surfactants, peptides and traditional SF that usually showed inferior stability at high salt concentration and lower pH value,11,

40, 46

the SNF-stabilized emulsions achieved higher stability at similar conditions,



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without loss of emulsification after 3 months. This phenomenon was similar to that in chargedistribution regulated SF assembling processes.52 Due to the force balance between the charge repulsion inside and outside the microcapsules, and the physical entanglements, the microcapsules showed superior stability without loss of emulsification at room temperature and 60oC. The addition of emulsions into neutral aqueous solutions without salts decreased the charge repulsion outside the microcapsules, which weakened the interaction balance and resulted in a gradual loss of emulsification of the SF nanofiber stabilized emulsions. Since the added salts or acids could shield the charge repulsion inside and outside the microcapsules, the physical entanglements and interactions became the main factor involved in stabilizing the emulsions, resulting in the recovery of stability (Figure 5). The high modulus of the SF nanofiber microcapsules (27.4 MPa ±1.7) measured by AFM under compression of extension model further confirmed the strong physical interactions of the nanofibers (Figure S3). SEM images of the SF nanofiber microcapsules also revealed the influence of the charge repulsion on stability. Compared to that in neutral solutions, the pore sizes on the interface decreased at high salt concentration and lower pH values, confirming the reduction of charge repulsion among the SF nanofibers (Figure 4D). Maintaining stability of the emulsions in harsh conditions remains a challenge for current emulsion systems. The additional stability of the SF nanofiber stabilized emulsions in high salt concentrations and low pH values suggests utility in various applications.


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Figure 4. The stability of emulsions prepared with 0.2 wt% SNF solution and 20 mmol.L-1 KYF solution when incubated for a few days at (A) room temperature and 60oC, (B) different concentration of salts (0M, 0.01M, 0.1M) and pHs (4.5, 7.4, 11.0), (C) Fluorescence micrographs of the different layers at various conditions after 3 months in (B), and (D) SEM images of the interface of the SNF microcapsules after 3 months. Freeze-drying was used to prepare the samples. 15 ACS Paragon Plus Environment


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Figure 5.The stability mechanism for the SNF emulsions in different aqueous solutions: (i) aqueous solution with higher salt concentration and lower pH value; (ii) Neutral aqueous solution without salts.

Next dodecane was replaced with hexanol, butyl butyrate and hexane to assess the generality of the strategy with SF nanofibers as emulsifiers (Figure S4). The emulsification process was performed under similar conditions as used with dodecane. Stable emulsions quickly formed with these organic phases having significantly different polarities, supporting the universality of this strategy and the robustness of the SF nanofiber system. The microcapsules showed similar variations in capsule sizes at the various W/O ratios but had different capsule sizes at the same W/O ratios. This finding further implies the versatility of the SF nanofiber system. Further modification of SF nanofibers including its conformations55 could also have significant influence


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on the microcapsule size of emulsions (Figure S5). The universality and versatility improve the applicability of SNF-stabilized emulsions in various fields.

Figure 6. The impact of Free-QDs and SNF-QDs on the fluorescent stability: (A) Schematic of oil-soluble quantum dots coated inside SNF microcapsules and adhered to the surface of the MCF-7 cells; and (B) Confocal fluorescence images of MCF-7 cells incubated with Free QDs and SNF-QDs (QDs concentration of 0.03mg.mL-1) for 3days.

Another advantage of SF nanofibers to stabilize emulsions is the inherent biocompatibility of SF materials. As an example of a biomedical application, oil soluble quantum dots were coated 17 ACS Paragon Plus Environment


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inside SF nanofiber microcapsules (SNF-QDs) efficiently (82%) and facilitated fluorescent stability than water soluble quantum dots (Free-QDs), which resulted in optimized cell imaging when compared to a soluble control (Figure 6).

4. CONCLUSIONS SF nanofibers were useful emulsifiers to stabilize emulsions. Unlike peptide emulsifiers, stable nanofibers were used directly to stabilize emulsions rather than self-assembled in the emulsifying process, providing better control in emulsion systems. The systems were universal for diverse oil phases with different polarity and versatile where tunable microcapsules were prepared through tuning oil phases, W/O ratio, and the concentration, conformation and size of SNF. The emulsions exhibited stability superior to the performance of peptides. Compared with a traditional surfactant model, the SNF formed emulsions with additional stability at high salt concentrations and low pH values, enhancing potential utility. Due to the biocompatibility, relatively easy purification process, and the mass production already available for silk, the SF nanofibers have a promising future in cosmetics, food, drug delivery and biomedicine applications.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Size distribution and Fluorescence micrographs of the emulsions formed at various SNF concentrations, shearing speeds and times; Fluorescent microscopy images of the SNF-stabilized emulsions at room temperature and 60oC; AFM images of the microcapsules; Optical photographs and Fluorescence micrographs of Emulsions formed with different organic phases; Fluorescence micrographs of emulsions formed with amorphous silk nanofibers (PDF)

AUTHOR INFORMATION Corresponding Author *Tel: (+86)-512-67061649; E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors thank the National Key Research and Development Program of China, Strategic International Innovation Key Grants (2016YFE0204400), NSFC (81272106), the NIH (R01NS094218, R01AR070975) and the AFOSR. We also thank the Natural Science Foundation of Jiangsu Province (Grants No BK20140397, BK20140401) and the second affiliated hospital of Soochow university preponderant clinic discipline group project funding (NO.XKQ2015010) for support of this work.



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Silk Nanofibers as Robust and Versatile Emulsifiers - ACS Publications

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