Programming Self-Assembly of Tobacco Mosaic Virus Coat Proteins at

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Programming Self-Assembly of Tobacco Mosaic Virus Coat Proteins at Pickering Emulsion Interfaces for Nanorod-Constructed Capsules Zhaocheng Wang,†,‡ Sijia Gao,†,‡ Xiangxiang Liu,†,‡ Ye Tian,*,† Man Wu,† and Zhongwei Niu*,†,§ †

Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, 29 Zhongguancun East Road, Beijing 100190, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China § School of Future Technology, University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: Pickering emulsion constructions on nanorods with high aspect ratio are a great challenge because of the geometry restrictions. On the basis of the theory that the stability of Pickering emulsion is highly dependent on the size and amphiphilicity of the nanoparticle at fluid interfaces, we report a novel strategy to fabricate long-time stable Pickering emulsion consisting of tobacco mosaic virus (TMV)-like nanorods through the programming self-assembly of TMV coat protein (TMVCP). The first step is the self-assembly of amphiphilic TMVCP at Pickering emulsion interfaces, and the second step is the in situ interfacial self-assembly of TMVCP into nanorods with increased particle size. The robust capsules can be further fabricated through cross-linking of the proteins. By taking advantage of both the amphiphilicity of TMVCP and the subsequent size growth induced by TMVCP self-assembly, this work provides a powerful strategy for constructing robust capsules consisting of nanorods with high aspect ratio, which may show potential applications for drug delivery and virus recognition. KEYWORDS: self-assembly, Pickering emulsion, virus-like nanorods, robust capsule, virus coat protein

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bionanoparticle-hydrophobic polymer conjugates, were prepared for fabricating Pickering emulsions with improved stability.13,14 Tobacco mosaic virus (TMV), measuring 300 × 18 nm, is a classic rod-like plant virus with high aspect ratio. It is composed of 2130 identical coat proteins (TMVCP) self-assembling around single-stranded RNA. In a mono TMVCP, there are both hydrophilic parts and hydrophobic parts, whereas during the self-assembly process of TMVCP, only relatively hydrophilic parts are exposed on external surfaces, making wild-type TMV a highly hydrophilic nanoparticle with homogeneous wettability. Thus, although wild-type TMV has been studied in detail of its assembly behaviors at interfaces,15−17 researchers still failed to form long-time stable Pickering emulsions based on wild-type TMV because of its low amphiphilicity. The TMVCP shows amphiphilic property and surface activity to form Pickering emulsion, yet the small size makes it quite easy to be detached from the interfaces. An efficient way to increase the particle size of TMVCP is through self-assembly. As previously reported, in the absence of RNA, TMVCP can self-assemble into a variety of well-defined structures.18,19 At high pH value and low ionic strength, TMVCP will form a mixture of monomer and small

ynthetic capsules consisting of nanoparticles with anisotropic shape have received great attention because of the specific optical, electronic, and mechanical properties.1−3 Pickering emulsion provides an efficient approach for capsule production.4 With the reduction of the interfacial energy between two fluids, nanoparticles spontaneously assembled at liquid−liquid interfaces,5−7 and these interfaces of emulsion droplets provide templets to form capsules with designed structures and functionalities.8,9 However, for nanofibers or nanorods with high aspect ratio, because of the geometry restrictions, the fabrication of stable Pickering emulsions remains a great challenge.10 The stability of Pickering emulsions is highly dependent on the particle size (R). T. P. Russell et al. have proved that the energy change induced by the placement of one nanoparticle at interface depends on R2, thus at a fluid interface, the smaller nanoparticles show less stability than larger ones and can even be preferentially replaced by larger nanoparticles.11 By taking use of this sizedependence theory, Ulyana Shimanovich and Thomas C.T. Michaels group fabricated stretchable and semipermeable capsules with cross-linked networks of protein nanofibrils.10 Another important factor influencing the stability of Pickering emulsion is the particle amphiphilicity. Through theoretical calculation, the desorption energy of a nanoparticle with uniform wettability may be increased 3-fold by maxing its amphiphilicity.12 Thus, various amphiphilic nanoparticles, like “Janus” particles with two parts exhibiting different wettability, or © XXXX American Chemical Society

Received: June 8, 2017 Accepted: August 7, 2017 Published: August 7, 2017 A

DOI: 10.1021/acsami.7b08186 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Letter

ACS Applied Materials & Interfaces Scheme 1. Schematic representation of the programming self-assembly of TMVCP at oil−water interfaces for nanorodconstructed capsules

the perfluorodecalin/water interfacial tension (only from 47.5 to 43.3 mN/m). Whereas TMVCP behaved a better effect in reducing interfacial tensions comparing to wild-type TMV (from 47.0 to 30.0 mN/m at pH 8.0). At different pH values, TMVCP showed different capacity in reducing interfacial tension and the capacity was strongest at pH 8.0. This phemomenon was probably associate with the different amphiphilicity of the TMVCP assemblies at different pH value. Through reducing the pH value from 8.0 to 5.5, TMVCP can self-assemble from monomers to disks, stacked disks and rods. TMVCP monomer was a kind of amphiphilic protein. During the self-assembly process, TMVCP assembled helically with only its hydrophilic parts exposed on external surfaces, leading to a decreased amphiphilicity. Thus, at pH 8.0, when it behaved as amphiphilic monomer, TMVCP showed highest capacity in reducing interfacial tension. TMVCP concentration also has influences on the emulsion properties. As previously reported,21 an increase in particle concentration will lead to a decrease in droplet size, an improvement in surface coverage and further a promotion in emulsion stability. As Figures S3 and S4 showed, when TMVCP concentration increased, TMVCP coverage at the o/w interfaces augmented, making the emulsion droplets hard to coalescence with each other. This can lead to larger emulsion volume (Figure S3) and smaller droplet size (Figure S4) at higher TMVCP concentration. We measured at least 300 different emulsion droplets stabilized by 0.1, 0.3, and 0.5 mg/mL TMVCP aqueous solution under different pH values for a droplet diameter distribution statistics. From Figure 1D−F, it was shown that at TMVCP concentration of 0.1, 0.3, or 0.5 mg/mL, the emulsion droplets were more uniform under pH 8.0 which may result from its higher capacity to reduce the interfacial energy. The stability of formed emulsions were evaluated against time. For 0.1 mg/mL TMVCP, the emulsion prepared at pH 5.5 showed obvious droplets coalescence within 2 weeks whereas the corresponding emulsion stabilized at pH 7.0 and 8.0 remained stable for at least 2 months (Figure S5). The results were also in accordance with the interfacial tension reduction experiments. The influence of ionic strength and temperature on the emulsion formation was also tested. Because high ionic strength will help TMVCP to form disks or stacks of disks,19 which have low amphiphilicity, increased ionic strength greatly hindered TMVCP’s interfacial assembly and emulsion formation (see Figure S6). Because of the nontemperature sensitivity of TMVCP, temperature showed little effect on the emulsion formation (see Figure S7). TEM images showed the assembly pattern of TMVCP at emulsion droplet interfaces. Under pH 8.0 and pH 7.0, no rodshaped assembly structures were observed, yet at pH 5.5 closely packed rod-shaped TMVCP assemblies were observed (Figure

aggregates known as A-protein. With reduced pH value, TMVCP will assemble helically first into a two-layer disk structure then into helix rod. The rod-shaped assembly at pH 5−6 has similar or increased size as wild-type TMV, may perform enhanced stability at fluid interfaces than TMVCP. Herein, inspired by the amphiphilicity and self-assembly behavior of TMVCP, we report a simple yet novel strategy to prepare robust capsules consisted of TMV-like nanorods. As shown in Scheme 1, the amphiphilic TMVCP were first assembled at oil−water interfaces, forming Pickering emulsions with high coverage. Then through a pH-induced self-assembly process, the interfacial TMVCP assemble into nanorods in situ, which performed larger particle size and higher interfacial stability. Through the subsequent cross-linking, robust capsules consisted of TMV-like nanorods could be prepared. This work will combine the benefits of amphiphilicity of TMVCP and the subsequent size growth induced by TMVCP self-assembly, and will provide a powerful strategy for constructing robust capsules consisting of virus-like nanoparticles with high aspect ratio, which have great potential for drug delivery and virus recognition. Emulsions were produced with perfluorodecalin as oil phase as previously reported.17,20At room temperature, TMVCP aqueous solution (TMVCP concentration 0.1, 0.3, and 0.5 mg/mL) and perfluorodecalin were mixed with a volume ratio of 9:1, then the mixture was shaken by hand vigorously for 2 min. Then Pickering emulsion was formed and emulsion droplets could be observed at the bottom layer by eye. To track TMVCP in formed emulsions, Rhodamine B isothiocyanate (RBITC) was conjugated to the amine groups of TMVCP. The chemical conjugation of RBITC onto TMVCP was confirmed by SDS-PAGE analysis and UV− vis spectra (Figure S1). The formed TMVCP-RB was mixed with perfluorodecalin following the same procedure and calcein was added as aqueous phase dye. Derived emulsions were visualized directly by confocal laser scanning microscopy (CLSM). In CLSM images it was clearly revealed that TMVCP-RB mostly located at o/w interfaces and an oil-in−water emulsion was formed (Figure 1A−C). Results showed that because of its amphiphilicity, TMVCP could self-assemble at o/w interfaces to stabilize the droplets from coalescence, which was in accordance with the Pickering emulsion theory mentioned above.5 During the formation of Pickering emulsion, the reduction of the interfacial energy between two fluids plays a key role to the uniformity and stabilization of the emulsions. To evaluate TMVCP’s ability to stabilize o/w interfaces under different conditions, a dynamic interfacial tension measurement was introduced and wild-type TMV’s interfacial tension was also measured for comparison (see Figure S2). The measurements showed that during the 2500 s, wild-type TMV can rarely reduce B


Letter

ACS Applied Materials & Interfaces

Figure 1. (A−C) TMVCP-RB stabilized perfluorodecalin-in-water emulsion imaged by CLSM, with (A) 570−610 nm channel (RBITC), (B) 510−550 nm channel (calcein) and (C) overall channel. Scale bars depict 100 μm. (D−F) Normal distribution of emulsion droplet diameter stabilized by (D) 0.1 mg/mL, (E) 0.3 mg/mL, and (F) 0.5 mg/mL TMVCP under different pH values. (G) Bar graph of emulsion droplet diameter stabilized by TMVCP at different pH values.

phobic directional assembly at o/w interfaces that prevented the

S8). The nanorods were rather short (about 100 nm in length), assembling densely at the droplet interfaces which was different from its assembling pattern in solution. This assembly pattern at o/w interfaces was probably caused by the geometry restriction of curved interfaces and the preferential hydrophilic−hydro-

TMVCP from assembling into longer rods as in solution. Taking simple thermodynamic considerations into account of the surface energies in the various phases, the energy change caused by placing one nanoparticle at an interface is given by11 C


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Figure 2. (A) Optical micrographs of emulsions prepared by 0.1, 0.3, and 0.5 mg/mL TMVCP with pH value subsequently decreasing from 8.0 to 5.5. Scale bars: 10 μm. (B−D) Normal distribution of emulsion droplet diameter stabilized by (B) 0.1 mg/mL, (C) 0.3 mg/mL, and (D) 0.5 mg/mL TMVCP with pH value subsequently decreasing from 8.0 to 5.5 (E) Bar graph of emulsion droplet diameter stabilized by TMVCP with pH value subsequently decreasing.

ΔE = −

π R2 [γ − (γP/W − γP/O)]2 γO/W O/W

where γO/W, γP/W, and γP/O are the interfacial tensions of the oil− water interface, particle−water interface, and particle−oil interface, respectively; R is the effective nanoparticle radius. The assembly of nonspherical particles can also be explained by a

(1)

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Figure 3. TEM images of the perfluorodecalin-in-water emulsions stabilized by 0.1 mg/mL TMVCP, pH was subsequently decreased from (A) 8.0 to (B) 7.0, (C) 6.0, and (D) 5.5. Scale bars depict 200 nm for original images (the left lane) and 100 nm for magnified images (the right lane).

interfaces under pH 8.0, to form a uniform Pickering emulsion with high interfacial coverage, and we subsequently decrease the system pH value to 7.0, 6.0, and 5.5 by dropwise adding diluted hydrochloric acid, to lead TMVCP assembling into nanorods at o/w interfaces (as Scheme 1 shows). We assume that through the in situ interfacial assembling of TMVCP into nanorods, we could combine the amphiphilicity and high interfacial coverage of TMVCP with the higher interfacial energy change and stability of

similar mechanism with corresponding modification, where the energy changes when the nanorods are parallel or perpendicular to the interface have been separately calculated by He et. al.22 We can learn from the eq 1 that the energy change depends on R2 and larger particles tend to behave higher energy change, resulting in more stable localization at the interfaces. Yet larger rod-like particles often come with geometrical restrictions.23 Inspired by the polymorphic assembly pattern of TMVCP under different pH values, we first assemble the amphiphilic TMVCP at o/w E


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Figure 4. SEM images of protein capsules formed by the perfluorodecalin-in-water emulsions stabilized by 0.1 mg/mL TMVCP after pH was changed from 8.0 to (A) 7.0, (B) 6.0, and (C) 5.5 then cross-linked by glutaraldehyde. Scale bars depict 5 μm.

In conclusion, based on the theory that the stability of Pickering emulsion can be improved by increasing the amphiphilicity and the size of the nanoparticle at fluid interfaces, we fabricated long-time stable Pickering emulsions constructed by nanorods through the programming self-assembly of TMVCP. The amphiphilic TMVCP were first assembled at oil−water interfaces, forming Pickering emulsions with high coverage. Then through a pH-induced self-assembly process, the interfacial TMVCP can self-assemble into nanorods, which perform larger particle size and higher interfacial stability. The robust protein capsules were produced by subsequent crosslinking. This work provides a simple yet novel strategy combining the benefits of amphiphilicity of TMVCP and the subsequent size growth induced by TMVCP self-assembly, for fabricating long-time stable Pickering emulsion consisting of virus-like nanoparticles with high aspect ratio that have great potential for drug delivery and virus recognition.

TMV-like nanorod, forming long-time stable Pickering emulsions stabilized by TMV-like nanorods. During the pH value decreasing process, optical microscopy images were taken at each pH value condition. More than 300 different droplets were measured at each condition for droplet diameter distribution statistics (Figure 2). Results showed that the droplets in general could keep their sizes during the pH decreasing process, with only a little increase in the droplet size. By measuring TMVCP concentration remained in aqueous phase through UV−vis spectrum, we can calculate the amount of TMVCP attached to emulsion droplet surface. Results showed that during the pH decreasing process, the amount of TMVCP attached to emulsion droplet surface increased (Table S1). This phenomenon may result from the self-assembly of TMVCP into nanorods, during which the nanoparticle number at interfaces decreased, leading to more TMVCP adsorbing onto the interfaces. TEM was performed to characterize the interfacial morphology and the assembling patterns of TMVCP during the process. In TEM images (Figure 3 and Figure S9), it was clearly shown that during the pH decreasing process, TMVCP gradually assembled into short nanorod structures at the emulsion droplet interfaces. At the meantime, with high interfacial coverage induced by TMVCP nanorods assembly at emulsion droplet interfaces, the thin and soft emulsion interfaces gradually became thick and robust. From emulsion stability evaluation (see Figure S5), we can see that at 0.1 mg/mL TMVCP concentration, Pickering emulsions produced by the pH decreasing method kept stable during the first 2 weeks and showed droplets coalescence at 2 months, whereas the emulsions produced at pH 5.5 showed seriously droplets coalescence just at 2 weeks. This indicates that the Pickering emulsions produced by the pH decreasing method had better stability. To achieve more robust capsules consisting of TMV-like nanorods, TMVCP were cross-linked at o/w interfaces with glutaraldehyde (2.5 wt %) at 4 °C for 3 h. SEM images showed that cross-linking after the pH-induced nanorod growth could produce robust protein capsules composed of TMV-like nanorods (Figure 4 and Figure S10). Although the capsules collapsed during the drying process and vacuum SEM observation, we can still observe a rather intact and robust structure. TEM was also used to observe the protein capsule structure. We could clearly see that the protein capsules were composed of TMV-like nanorods (Figure S11). Emulsions stabilized by TMVCP under pH 5.5 were directly cross-linked with glutaraldehyde for comparison. As shown in Figure S12, formed protein capsules were not robust enough. They completely collapsed during the drying process, which could be due to the low coverage ratio at o/w interfaces.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b08186. Digital photos of SDS-PAGE analysis, time-dependent interfacial tension curves, emulsions prepared using TMVCP, TEM images of perfluorodecalin-in-water emulsions, SEM images of protein capsules formed by perfluorodecalin-in-water emulsion (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Z.N.). *E-mail: [email protected] (Y.T.). ORCID

Ye Tian: 0000-0002-4607-2785 Zhongwei Niu: 0000-0002-6119-3227 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21474123, 51303191, 21304103 and 51173198), the Ministry of Science and Technology of China (2013CB933800), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2017039) and the Presidential Foundation of Technical Institute of Physics and Chemistry. F


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(23) Balakrishnan, G.; Nicolai, T.; Benyahia, L.; Durand, D. Particles Trapped at the Droplet Interface in Water-in-water Emulsions. Langmuir 2012, 28 (14), 5921−5926.

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Programming Self-Assembly of Tobacco Mosaic Virus Coat Proteins at

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