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Protein Cages as Containers for Gold Nanoparticles Aijie Liu, Martijn Verwegen, Mark Vincent de Ruiter, Stan J. Maassen, Christoph H.-H. Traulsen, and Jeroen J.L.M. Cornelissen J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b03066 • Publication Date (Web): 02 May 2016 Downloaded from http://pubs.acs.org on May 3, 2016

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The Journal of Physical Chemistry

Protein Cages as Containers for Gold Nanoparticles Aijie Liu, Martijn Verwegen, Mark V. de Ruiter, Stan J. Maassen, Christoph H.-H. Traulsen, and Jeroen J.L.M. Cornelissen* Department of Biomolecular Nanotechnology, MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands.

ABSTRACT Abundant and highly diverse, viruses offer new scaffolds in nanotechnology for the encapsulation, organization, or even synthesis of novel materials. In this work the coat protein of the cowpea chlorotic mottle virus (CCMV) is used to encapsulate gold nanoparticles with different sizes and stabilizing ligands yielding stable particles in buffered solutions at neutral pH. The size of the virus-like particles correspond to T=1, 2 and 3 Caspar-Klug icosahedral triangulation

numbers.

We developed a simple one step process enabling the encapsulation of commercially available gold nanoparticles without prior modification with up to 97% efficiency. The encapsulation efficiency, is further increased using bis-p-(sufonatophenyl) phenyl phosphine surfactants up to 99%. Our work provides a simplified procedure for the preparation of metallic particles stabilized in CCMV protein cages. The presented results are expected to enable the preparation of a variety of similar virus-based colloids for current focus areas.

1 ACS Paragon Plus Environment


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Introduction Metallic colloids such as gold nanoparticles (AuNPs) have been studied intensively due to their potential applications in optics, electronics, catalysis, and biomedicine.1-4 Especially for biomedical applications1-2,

5

their stability is crucial since particle aggregation significantly increases toxicity.

Furthermore, particles lose their unique properties upon coagulation.6 Nanoparticle stabilization can be obtained either sterically by attaching organothiols and dendrimers or electrostatically by physisorption of highly polar or charged compounds such as acids or tertiary amines.7-8 Core-shell system, which use polymer or silica as stabilizer, have also been studied.9-12 Besides synthetic nanocarrier, virus protein cages hybrid bioassemblies have recently gained attention because of their straightforward preparation and biocompatibility.5, 13-18 Viruses exhibit a symmetrical architecture with a high degree of monodispersity. They occur in a variety of different shapes and sizes and their coat proteins can be modified with biologically active ligands to use them for drug delivery rendering them ideal building blocks for nanotechnology .17 The self-assembly of virus-like particles (VLPs) around artificial colloids is driven by the maximization of protein-protein interactions as well as protein-cargo interactions. Thus, encapsulation of AuNPs into VLPs is facilitated by modifying their surface, e.g. by coupling RNA, DNA or thiol-based ligands to the nanoparticles. Biohybrids can be created with precise control over structure, properties and functionality at the nanometer scale.19 The encapsulation efficiency

strongly dependents on electrostatic interactions and the relative concentrations of coat protein and cargo. However, the diameter of the particles which can be encapsulated in a specific VLPs is limited by the curvature of the coat protein and polymorphism is observed by the encapsulation of larger particles.14, 20 Alternatively, nanoparticles can be grown inside empty virus capsids. The size and shape of the resulting particle is templated by the virus.21-22


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Dragnea and coworkers recently developed a straightforward strategy for the encapsulation of metallic particles in BMV.23 Anionic gold nanoparticles are incubated with coat proteins at neutral pH forming electrostatically bound protocapsids. After dialysis to a lower pH value AuNP-VLPs have been obtained.23 In further studies the encapsulation efficiencies was increased to 95%.21 Besides BMV, cowpea chlorotic mottle virus (CCMV) can be used as protein cage for the encapsulation of AuNPs.13 CCMV is a well-studied bromovirus with a native icosahedral conformation and a T=3 Caspar-Klug symmetry. Cargos can be encapsulated in CCMV including polyanions, proteins, enzymes and micelles.24-27 Due to a flexible N-terminus CCMV can be assembled at will into a wide variety of geometries such as tubes, multi-layered structures or dumbbells by controlling the pH and ionic strength of the assembly buffer.28-31 In this work we investigate the encapsulation of gold nanoparticles of different sizes and surfactants in cowpea chlorotic mottle virus cages to establish conditions which are applicable for different cargos. The encapsulation is performed through a straightforward one-step process and is applicable for similar virus-like particles under various conditions.

Experimental Methods Materials. All chemicals were purchased from Sigma Aldrich and used without further purification. Gold NPs were obtained from nanoComposix. Milli-Q water used for preparation as well

as

analysis

has

been

obtained

by

ultrafiltration

(Millipore

Adv.

A10).

Isolation of the CCMV coat protein dimers. CCMV coat protein dimers (CP) were isolated as described by Verduin and Comellas Aragones et al.25, 32 CCMV capsids (8 mg/mL) are dissolved in 500 µL aqueous virus buffer (pH 5.0; 100 mM sodium acetate, 1 mM EDTA, 1 mM NaN3;) and dialyzed 2 times 3 hours against 500 mL RNA buffer (pH 7.5; 50 mM Tris, 500 mM CaCl2). Precipitated RNA is removed by ultracentrifugation at 40000 rpm and the supernatant is dialyzed



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2 times 3 hours against 500 mL cleaning buffer (pH 7.5; 50 mM Tris, 500 mM NaCl). For storage, the CP solution can be dialyzed for 2 times 3 hours to capsid storage buffer (pH 5.0; 100 mM NaAc, 500 mM NaCl, 1 mM NaN3). Bis-p-(sufonatophenyl) phenyl phosphine (BSPP) ligand exchange. BSPP capped nanoparticles are prepared according to literature procedures.18 Briefly, BSPP (4 mg, 7.5 µmol) is dissolved in Milli-Q (1 mL) added to an aqueous solution of citrate stabilized gold nanoparticles (10 mL) and stirred overnight at r. t. Citric acid is removed using a 30 kD MWCO centrifuge filter and washing three times with aqueous BSPP (1 mM). The particles were concentrated (1.5 mL, 0.33 mg/mL). Hybrid particle formation. In a typical experiment AuNP solution (400 µL, 0.5 mg/ml; H2O) is added to a solution of CCMV coat protein (100 µL, 15 mg/mL; pH 7.5; 50 mM Tris, 500 mM NaCl) and allowed to incubate for 5 min at r.t. The reaction mixture is subsequently dialyzed overnight to the coat protein buffer (pH 7.5; 50 mM Tris, 500 mM NaCl). The resulting VLPs are purified using preparative FPLC. FPLC. FPLC size exclusion chromatography samples, ranging from 100 µL up to 500 µL were measured on an Aktapurifier (Box-900) equipped with a 24 mL Superose 6 10/100 GL column (GE Healthcare) at 0.5 mL/min flow and collected by fractionation (Frac-950). UV-Vis. Samples are prepared from 500 µL fresh sample solutions. They were measured in a 1 cm quartz cuvette in a PerkinElmer Lambda 850 UV/VIS Spectrometer. (S)TEM. The TEM samples are prepared by drop casting 5 µL of a freshly made sample solution onto a formvar carbon coated copper grid. After 5 min incubation the remaining liquid is removed by tipping the grid onto low lint paper (Kimtech science precision wipes). The samples are stained using 5 µL of a 1% uranyl acetate solution which is removed after 30 s to provide


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optimal contrast. Samples are imaged using a Philips CM300ST-FEG TEM or a Zeiss Merlin (S)TEM. The resulting images are analyzed using ImageJ software to determine the sizes of the total particles and the gold cores.33 DLS. Each sample is measured five times for 120 seconds using an Anaspec nanotrack wave dynamic light scattering instrument, using a refractive index of 1.54 for the hybrid particles and the viscosity of water. The average of 5 measurements is used for further analysis. Zeta-potential. The ζ-potential of the AuNPs with different ligands at pH 7.5 was characterized by a Zetasizer Nano ZS ZEN3600 instrument (Malvern Instruments) at 25 ̊C with 633 nm laser. Results and Discussion. The assembly of CCMV coat proteins into monodisperse capsids is systematically investigated (Figure 1). We determined three parameters to significantly influence this process: 1. The molar ratio of coat protein and cargo, 2. the size of the metallic nanoparticle and 3. the electrostatic properties of the organic stabilizer at the gold core. By variation of those parameters we will demonstrate the capabilities as well as the limitations of CCMV as nanocontainer. Due to the large amount of templating particles and resulting VLPs we used a systematic nomenclature: cowpea chlorotic mottle virus (CCMV) loaded with gold nanoparticles (Au) of 12 nm core diameter (12) and citrate surfactant (c) are abbreviated with CCMVAu12c. Data obtained using TEM on the smallest VLPs have been confirmed by DLS measurements (ESI). These data are not used for estimation of the VLP dimension because of solvent shells which are not included in Casper-Klug icosahedral triangulation numbers.



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Figure 1. The assembly procedure applied for the preparation of hybrid gold nanoparticle cowpea chlorotic mottle virus like particles. Potassium 4,4'-(phenylphosphanediyl)dibenzenesulfonate is abbreviated with BSPP. Determination of the CP/AuNP ratio. The influence of the molar ration between coat protein and gold nanoparticle is investigated. Gold nanoparticles with a core diameter of 12 nm stabilized by citrate ligands are chosen as a model template. Under neutral pH the carboxylic acids of citric acid are deprotonated and negatively charged (pKa: 3.13-6.434) enabling interaction with the coat proteins (CP). We disassembled wild-type CCMV and isolated the coat proteins and subsequently reassembled the capsid at r.t. by combining CPs and AuNPs in aqueous solutions. To ensure at least T=3 capsid formation can take place we chose molar CPAuNP ratios of 180:1, 360:2, and 720:1. The resulting CCMVAu12c hybrid virus like particles are analyzed using FPLC, TEM and DLS (Figure 2 and ESI).


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Figure 2. Analytical data of the assembly of CCMVAu12c. A) FPLC chromatogram of reaction solutions containing the coat protein and gold nanoparticles in ratios of 180:1, 360:1 and 720:1 (CP to AuNP, molar). Only the plasmon absorption band at λ=520 nm is depicted enabling the detection of gold nanoparticles in the eluted fraction. The data are normalization (11.2 mL). B) TEM image of the FPLC fraction eluted at 8.0 mL revealing encapsulated aggregated AuNPs. C) TEM image of the FPLC fraction eluted at 11.2 ml showing encapsulated isolated AuNPs. The FPLC used for particle purification and analysis is equipped with a UV-Vis detector. Fractions are detected at elution volumes of 8.0 and 11.2 mL. The fraction eluting at 18 mL absorbs only at 260 and 280 nm. Earlier studies revealed this compound to be the non-covalent assembly of capsid protein dimers.35 No absorption band at 520 nm is detected for this fraction. In order to investigate particles containing gold nanoparticles, we focused on fractions containing species with absorption bands at 520 nm corresponding to the plasmon resonance of



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the nanoparticle cargo (Figure 2A). All investigated product solutions exhibit two fractions with elution volumes of V=8.0 mL and 11 mL colored purple and red respectively. We investigated those fractions using uranyl acetate stained TEM images and revealing the V=8 mL fraction peak to be composed of large hybrid assemblies of coagulated AuNPs surrounded by a protein shell (Figure 2B). TEM analysis of the second fraction eluted at V=11 mL shows isolated gold particles encapsulated in an organic shell (Figure 2C). This result enables us to evaluate the success of an encapsulation because the 11 mL fraction can clearly be assigned to the desired particles. We now estimate the success of AuNP encapsulation of different CP-AuNP ratios by comparing the relative heights of the plasmon absorption bands of the fractions detected at 8 mL and 11 mL. The values obtained exclude the protein material and do not take empty VLP formation into account. As a result, 33% of the gold nanoparticles are encapsulated and isolated at a ratio CP-AuNP of 180:1. A much larger value is obtained at 720:1, 70% of the gold particles are encapsulated in single spheres without multiple particle aggregates. Two processes compete during the encapsulation. 1. AuNPs aggregate under the applied condition due to reduced electrostatic repulsion at high salt concentrations and 2. the desired assembly of the AuNPs and coat proteins which prevents aggregation. Consequently, a higher amount of coat protein in the reaction solution results is reduced aggregation. We obtained more isolated, monodisperse CCMVAu assemblies by using excess of coat protein. We varied ratio of coat protein to gold nanoparticle to increase the efficiency of encapsulation. Larger amounts of coat protein yield more isolated particles with defined size and shape. Consequently, all further assemblies presented are carried out using a large access of coat proteins. 8 ACS Paragon Plus Environment

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Core size and ligand variation. In order to determine the size effect of AuNPs upon encapsulation at neutral pH we used commercial citrate stabilized gold nanoparticles with core diameters of 7, 12 and 17 nm and investigated the formation of hybrid capsids using FPLC and STEM (Figure 3A and B). In line with the preliminary investigations, the FPLC runs of each crude solution elutes fractions at 8, 11 and 18 mL corresponding to aggregated particles and larger assemblies, intact and isolated hybrid particles as well as pristine capsomers, respectively (Figure 3A). The highest encapsulation efficiency is obtained using Au7c particles. The corresponding TEM as well as STEM image reveals monodisperse particles to have formed (Figure 3B). The gold encapsulation efficiency is 90, 53 and 75% for 7, 12 and 17 nm core sized particles, respectively. We also conducted the same experiment using particles which are stabilized with tannic acid instead of citric acid. Tannic acid is polyphenol incorporating 25 aromatic hydroxyls. Their acidity is significantly lower than citric acid and therefore, the main interaction between the coat protein and the surfactant is related to electrostatics rather that ionic interactions. Because electrostatics are less influenced by variation of the pH we expect different encapsulation behavior with this ligand. The preparation procedure using tannic acid stabilized gold nanoparticles remains unchanged.



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Figure 3. Encapsulation of citrate (A and B) or tannic acid (C and D) stabilized gold nanoparticles with different core diameters. The size of the gold core as received is 7, 12 and 17 nm. A) FPLC chromatograms obtained after injecting assembly solutions templated by citrate stabilized AuNPs. B) STEM image of the hybrid particles obtained using 12 nm citrate stabilized AuNPs. C) FPLC chromatograms obtained after injecting assembly solutions templated by tannic acid stabilized AuNPs. D) negatively stained TEM image of the hybrid particles obtained using 7 nm tannic acid stabilized AuNPs. The black particles correspond to AuNPs and the white/grey areas to the capsid. Tannic acid stabilized particles yield isolated hybrid nanoparticles at a particle diameter of 7 nm, 12 and 17 nm. Larger core-particles facilitate the encapsulation of multiple particles as well


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as particle coagulation as it can be observed by the fractions detected at 8 mL (Figure 3C). Clearly spherical hybrid particles are detected by TEM analysis of the VLPs obtained from differently sized AuNPs. The average diameter of encapsulated 7 nm, 12 nm, and 17 nm cored AuNPs in the protein shell is 18 nm, 23 nm and 28 nm respectively. Caspar-Klug Icosahedral virus structure triangulation numbers (T) explain these differences. Wild-type CCMV (T=3) capsids are 28 nm in diameter and have an 18 nm cavity. T=2 CCMV capsid have a diameter close to 23 nm with a cavity of around 13 nm, but according to Caspar-Klug symmetry this is not as stable as the T=1 or T=3 capsids. T=1 confirmations are approximately 18 nm in diameter and have an 8 nm cavity.24 The gold encapsulation efficiency is 97, 54 and 82% for 7, 12 and 17 nm core sized particles, respectively. The preferred Caspar-Klug symmetry of WT CCMV is T=3 but those nanoparticles are less stable in solutions of a high pH because the protein-protein interaction is smaller. Thus the influence of the protein-cargo interactions becomes dominant and determines the hybrid VLP size. The higher stability T=1 VLPs compared to T=3 is in accordance with previous observations.36-37 CCMV hybrid particles can be obtained by using differently stabilized gold nanoparticles with a broad range of core diameters. The coat proteins assemble into capsids which differ in triangulation. The T=1 and T=3 hybrid VLPs are stable and do not degenerate or coagulate during storage. In contrast, the yield of T=2 particles is significantly lower. This observation is in line with expectation derived from literature.38 Gold nanoparticles with a core diameter of 7 nm stabilized with tannic acid yield highly monodisperse particles with superior encapsulation efficiency (Figure 3C and D). The assembly of hybrid VLPs using tannic acid stabilized AuNPs yielded a significant amount of capsids without AuNPs cargo (Figure 3D). The formation of these particles is likely templated by tannic



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acid molecules which are no longer bound by the nanoparticle core but free in solution. Due to the large amount of hydroxyl groups strong non-covalent interactions such as hydrogen bonds with the CCMV coat protein are expected.39 Thus we speculate, that a protein-tannic acid seeds forms which acts as a nucleating center for further self-assembly. We investigated this issue in more detail by using only tannic acid during the capsid formation. According to DLS, FPLC and TEM (ESI) tannic acid filled capsids can be formed. Although this remarkable property of tannic acid has not been observed before, it hampers the efficient formation of gold-protein hybrid VLPs. Ligand exchange. Aiming at an improved encapsulation, we now introduce bis-p-(sulfonatophenyl) phelylphosphine (BSPP) as stabilizing ligand for gold nanoparticles (Au7b).40 The binding of BSPP to the gold NPs is stronger compared to tannic acid and citric acid. In contrast to tannic acid, a single negatively charged BSPP molecule is too small to be a nucleating center for coat protein self-assembly. Ligand exchange of commercially available citric acid stabilized gold nanoparticles is carried out by spin filtration with excess of BSPP. Excess BSPP is removed by dialysis and the resulting particles are used for the formation of CCMVAu7b as stated above. Analysis is carried out using FPLC and TEM (Figure 4).


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Figure 4. Encapsulation of bis-p-(sulfonatophenyl) phelylphosphine (BSPP) stabilized gold nanoparticles with a core diameters of 7 nm. A) FPLC chromatogram of CCMVAu7b. B) TEM image of CCMVAu7b. CCMVAu7b are formed without pronounced formation of nanoparticle aggregates because no fraction could be collected with an elution volume of 8 mL using FPLC (Figure 4A). Nicely uniform particles of 17 nm diameter are assembled as determined by TEM (Figure 4B). In contrast to tannic acid stabilized templates, BSPP as surfactant exclude the formation capsids without AuNPs. In this context, the issue of charge matching between capsids and cargo of e.g. bromovirus has been addressed by the group of Dragnea and others in great detail.41-43 To assess the charge match between the particles and the capsid we estimated the surface area of gold nanoparticle with a diameter of 7 nm (A=154 nm2) that is covered by BSPP (A=0.5 nm2) by MM@ force field calculations yielding 306 BSPP molecules per nanoparticle (ESI). Due to the doubly charged BSPP about 612 negative charges are present at the AuNP interface. This number nicely matches the number of positively charged n-termini inside a T=1 particle (600).



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Gold nanoparticles with a diameter of 7 nm and stabilized by BSPP very effectively template the formation of hybrid virus like particles. This final experiment concludes the series of encapsulation experiments. Our results are summarized in Table 1. Table 1. Experimental data obtained for the formation of hybrid virus-like particles. AuNP

Hybrid Virus-like Particle

ligand

diameter [nm]

zeta potential [mV]

efficiency [%]

CAu7t

tannic acid

7.0

-25.0

97

12.3

17.8

1

CAu7c

citric acid

7.0

-6.0

(90)¥

11.9

15.2

1

CAu7b

BSPP

7.3

-25.0

99

12.1

17.4

1

CAu12t

tannic acid

12.

-42.7

54

10.6

23.5

2

CAu12c

citric acid

12.3

-17.6

53

11.2

22.0

2

CAu17t

tannic acid

16.1

-32.7

82

10.3

28.5

3

CAu17c

citric acid

15.5

-18.4

75

10.3

25.1

3

FPLC Diameter* Triangulation$ [mL] [nm] number

* Diameter based on EM data; $suspected triangulation number based on the diameter; ¥exact determination was hampered by sample coagulation. Conclusion. Gold nanoparticles with a diameter of 7 nm are encapsulated significantly more efficient than larger particles due to an optimal size match for the formation of stable T=1 CCMV capsids. The formation of T=2 particles is less favorable, resulting in a low efficiency of the encapsulation of 12 nm particles. Further increasing the particles size to 17 nm leads to the formation of T=3 particles and an improved encapsulation efficiency. Additional to particle size, the electrostatic potential of the surfactants strongly influences the particle formation. Within a specific particle size range, larger zeta-potential values increase successful particle formation. The efficient encapsulation of Au7t and Au7b suggests that a zeta14 ACS Paragon Plus Environment

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potential of a -25 mV enables a complete particle encapsulation. Furthermore, the BSPP stabilized gold nanoparticles display hardly any protein cages without a gold core, in that way proving to be a more efficient template. By tracing down specific values of core diameters for the formation of stable particles as well as zeta-potential values we provide the tools for straightforward preparation of biohybrid assemblies using CCMV capsids as nanocontainers with inorganic cargos at neutral pH.

Supporting Information. Materials, Methods, Instrumentation and Data processing as well as additional analytical data can be found in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author * [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Acknowledgements This research forms part of the research program of the Dutch Polymer Institute (DPI), Project #777t (DPI, P.O. Box 902, 5600 AX Eindhoven, the Netherlands) and financial support from the ERC (Consolidator Grant ProtCage #616907) is gratefully acknowledged. Abbreviations



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FPLC, fast protein liquid chromatography; DLS, dynamic light scattering; TEM, transmission electron microscopy.

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16. Loo, L.; Guenther, R. H.; Lommel, S. A.; Franzen, S., Encapsidation of Nanoparticles by Red Clover Necrotic Mosaic Virus, J. Am. Chem. Soc., 2007, 129, 11111-11117. 17. Ma, Y.; Nolte, R. J. M.; Cornelissen, J. J. L. M., Virus-Based Nanocarriers for Drug Delivery, Adv. Drug Delivery Rev., 2012, 64, 811-825. 18. Pulsipher, K. W.; Dmochowski, I. J., Ferritin Encapsulation and Templated Synthesis of Inorganic Nanoparticles. In Methods in Molecular Biology, 2015; Vol. 1252, pp 27-37. 19. Soto, C. M.; Ratna, B. R., Virus Hybrids as Nanomaterials for Biotechnology, Current Opinion in Biotechnology, 2010, 21, 426-438. 20. Sun, J., et al., Core-Controlled Polymorphism in Virus-Like Particles, Proc. Natl. Acad. Sci., 2007, 104, 1354-1359. 21. Chen, C.; Daniel, M. C.; Quinkert, Z. T.; De, M.; Stein, B.; Bowman, V. D.; Chipman, P. R.; Rotello, V. M.; Kao, C. C.; Dragnea, B., Nanoparticle-Templated Assembly of Viral Protein Cages, Nano Lett., 2006, 6, 611-615. 22. Li, F.; Wang, Q., Fabrication of Nanoarchitectures Templated by Virus-Based Nanoparticles: Strategies and Applications, Small, 2014, 10, 230-245. 23. Tsvetkova, I.; Chen, C.; Rana, S.; Kao, C. C.; Rotello, V. M.; Dragnea, B., Pathway Switching in Templated Virus-Like Particle Assembly, Soft Matter, 2012, 8, 4571-4577. 24. Sikkema, F. D.; Comellas-Aragonès, M.; Fokkink, R. G.; Verduin, B. J. M.; Cornelissen, J. J. L. M.; Nolte, R. J. M., Monodisperse Polymer-Virus Hybrid Nanoparticles, Organic and Biomolecular Chemistry, 2007, 5, 54-57. 25. Aragonès, M. C. The Cowpea Chlorotic Mottle Virus as a Building Block in Nanotechnology. Radboud Universiteit Nijmegen, 2010. 26. Minten, I. J.; Claessen, V. I.; Blank, K.; Rowan, A. E.; Nolte, R. J. M.; Cornelissen, J. J. L. M., Catalytic Capsids: The Art of Confinement, Chem. Sci., 2011, 2, 358-362. 27. Hu, Y.; Zandi, R.; Anavitarte, A.; Knobler, C. M.; Gelbart, W. M., Packaging of a Polymer by a Viral Capsid: The Interplay between Polymer Length and Capsid Size, Biophys. J., 2008, 94, 1428-1436. 28. Bancroft, J. B.; Hiebert, E.; Bracker, C. E., The Effects of Various Polyanions on Shell Formation of Some Spherical Viruses, Virology, 1969, 39, 924-930. 29. Lavelle, L.; Gingery, M.; Phillips, M.; Gelbart, W. M.; Knobler, C. M.; Cadena-Nava, R. D.; Vega-Acosta, J. R.; Pinedo-Torres, L. A.; Ruiz-Garcia, J., Phase Diagram of Self-Assembled Viral Capsid Protein Polymorphs, J. Phys. Chem. B, 2009, 113, 3813-3819. 30. Speir, J. A.; Munshi, S.; Wang, G.; Baker, T. S.; Johnson, J. E., Structures of the Native and Swollen Forms of Cowpea Chlorotic Mottle Virus Determined by X-Ray Crystallography and Cryo-Electron Microscopy, Structure, 1995, 3, 63-78. 31. Douglas, T.; Strable, E.; Willits, D.; Aitouchen, A.; Libera, M.; Young, M., Protein Engineering of a Viral Cage for Constrained Nanomaterials Synthesis, Adv. Mater., 2002, 14, 415-418. 32. Verduin, B. J. M., The Preparation of CCMV-Protein in Connection with Its Association into a Spherical Particle, FEBS Lett., 1974, 45, 50-54. 33. Schneider, C. A.; Rasband, W. S.; Eliceiri, K. W., Nih Image to Imagej: 25 Years of Image Analysis, Nat Meth, 2012, 9, 671-675. 34. Lide, D. R., Crc Handbook of Chemistry and Physics, 84 ed.; CRC Press, 2003. 35. Verwegen, M.; Cornelissen, J. J. L. M., Clustered Nanocarriers: The Effect of Size on the Clustering of CCMV Virus-Like Particles with Soft Macromolecules, Macromol. Biosci., 2015, 15, 98-110. 17 ACS Paragon Plus Environment


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Table of Contents Graphic.



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The assembly procedure applied for the preparation of hybrid gold nanoparticle cowpea chlorotic mottle virus like particles. Potassium 4,4'-(phenylphosphanediyl)dibenzenesulfonate is abbreviated with BSPP. 98x56mm (300 x 300 DPI)

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Analytical data of the assembly of CCMVAu12c. A) FPLC chromatogram of reaction solutions containing the coat protein and gold nanoparticles in ratios of 180:1, 360:1 and 720:1 (CP to AuNP, molar). Only the plasmon absorption band at λ=520 nm is depicted enabling the detection of gold nanoparticles in the eluted fraction. The data are normalization (11.2 mL). B) TEM image of the FPLC fraction eluted at 8.0 mL revealing encapsulated aggregated AuNPs. C) TEM image of the FPLC fraction eluted at 11.2 ml showing encapsulated isolated AuNPs. 96x109mm (300 x 300 DPI)



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Encapsulation of citrate (A and B) or tannic acid (C and D) stabilized gold nanoparticles with different core diameters. The size of the gold core as received is 7, 12 and 17 nm. A) FPLC chromatograms obtained after injecting assembly solutions templated by citrate stabilized AuNPs. B) STEM image of the hybrid particles obtained using 12 nm citrate stabilized AuNPs. C) FPLC chromatograms obtained after injecting assembly solutions templated by tannic acid stabilized AuNPs. D) negatively stained TEM image of the hybrid particles obtained using 7 nm tannic acid stabilized AuNPs. The black particles correspond to AuNPs and the white/grey areas to the capsid. 172x117mm (300 x 300 DPI)


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Encapsulation of bis-p-(sulfonatophenyl) phelylphosphine (BSPP) stabilized gold nanoparticles with a core diameters of 7 nm. A) FPLC chromatogram of CCMVAu7b. B) TEM image of CCMVAu7b. 64x27mm (300 x 300 DPI)



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Table of Contents Graphic 47x27mm (300 x 300 DPI)


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Protein Cages as Containers for Gold Nanoparticles - The Journal of

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