research.utwente.nl · i Table of Contents Chapter 1 ................................................................................................................... 1 General

CONTROLLING THE ASSEMBLY OF

PROTEIN CAGES TOWARDS FUNCTIONAL

SUPRAMOLECULAR MATERIALS

Shuqin Cao

Graduation Committee:

The research described in this thesis was performed within the laboratories of the

Biomolecular Nanotechnology (BNT) group, the MESA+ Institute for Nanotechnology,

and the Department of Science and Technology (TNW) of the University of Twente

(UT). This research was supported by the European Research Council (ERC) via the

consolidator grant ‘Portcage’ (616907).

Controlling the Assembly of Protein Cages towards Functional Supramolecular

Materials

Copyright ©2020 Shuqin Cao, Enschede, The Netherlands. No parts of this thesis

may be reproduced, stored in a retrieval system or transmitted in any form or by any

means without permission of the author.

Cover design: Luca Ricciardi, Shuqin Cao Printed by: Ipskamp Printing- The Netherlands ISBN: 978-90-365-4954-7

DOI: 10.3990/1.9789036549547

Chairman: Prof. dr. J. L. Herek University of Twente

Promoter: Prof. dr. J. J. L. M. Cornelissen University of Twente

Members: Prof. dr. T. Weil Max Planck Institute

for Polymer Research

Dr. R.J. de Vries Wageningen University

Prof. dr. ir. J. Huskens University of Twente

Prof. dr. ing. N.H. Katsonis University of Twente

Dr.ir. S. Lindhoud University of Twente

CONTROLLING THE ASSEMBLY OF

PROTEIN CAGES TOWARDS FUNCTIONAL

SUPRAMOLECULAR MATERIALS

DISSERTATION

to obtain

the degree of doctor at the Universiteit Twente,

on the authority of the rector magnificus,

Prof.dr. T.T.M. Palstra,

on account of the decision of the graduation committee

to be publicly defended

on Friday the 28th of February 2020 at 14.45 hours

by

Shuqin Cao

born on the 30th of August 1989

in Chongqing, China

This dissertation has been approved by:

supervisor

Prof. dr. J.J.L.M. Cornelissen

i

Table of Contents

Chapter 1 ................................................................................................................... 1

General Introduction .............................................................................................. 1

Reference ............................................................................................................... 2

Chapter 2 ................................................................................................................... 5

2.1 Introduction ............................................................................................... 6

2.2 Virus-like-particles and their applications ................................................. 7

2.3 Induce assembly of capsid proteins into virus-like particles by

supramolecular interactions ........................................................................................ 9

2.3.1 Electrostatic interaction induced assembly ......................................... 10

2.3.2 Metal-coordinated assembly of virus-like-particles …………………12

2.3.3 Other supramolecular interactions induced assembly of virus-like

particles ……………………………………………………….…………………15

2.4 Self-assembly of protein cages into 3D structures via supramolecular

interactions ................................................................................................................ 16

2.4.1 Hierarchical lattices of protein cages via electrostatic interactions……17

2.4.2 Assembly of virus-like-particles into superlattices through other

supramolecular interactions .................................................................................. 19

2.5 Summary and future perspectives ...................................................... 21

2.6 References ............................................................................................... 21

Chapter 3 ................................................................................................................. 29

3.1 Introduction ............................................................................................. 30

3.2 Results and discussion ............................................................................. 32

3.3 Conclusions ............................................................................................. 41

3.4 Acknowledgments ................................................................................... 42

3.5 Materials and methods ............................................................................ 42

3.6 References ............................................................................................... 49

Chapter 4 ................................................................................................................. 53

4.1 Introduction ............................................................................................. 54


4.3 Conclusions………………………………………………………………61

ii

4.4 Acknowledgments ………………………………………………………61

4.5 Materials and methods …………………………………………………..62

4.6 References .............................................................................................. 64

Chapter 5 ................................................................................................................. 67

5.1 Introduction ............................................................................................. 68


5.3 Conclusions ............................................................................................. 82

5.4 Acknowledgments ................................................................................... 82

5.5 Materials and methods ............................................................................ 83

5.6 References ............................................................................................... 88

Chapter 6 ................................................................................................................. 91

6.1 Introduction ............................................................................................. 92


6.3 Conclusions ........................................................................................... 104

6.4 Acknowledgements ............................................................................... 105

6.5 Materials and methods .......................................................................... 105

6.6 References ............................................................................................. 111

6.7 Appendix ............................................................................................... 113

Chapter 7 ............................................................................................................... 117

7.1 Introduction ........................................................................................... 118

7.2 Achievement and challenges ................................................................. 118

7.3 Outlook .................................................................................................. 120

7.4 References ............................................................................................. 121

Summary ............................................................................................................... 123

Samenvatting ......................................................................................................... 125

Acknowledgments ................................................................................................. 127

About the author .................................................................................................... 132

Publications ........................................................................................................... 133

1

Chapter 1

General introduction

In natural as well as synthetic systems, supramolecular interactions plays a vital role

in essential life processes, such as catalysis,1 protein synthesis,2 cell replication and

differentiation,3 as well as molecule transport and delivery.4 These supramolecular

interactions are reversible, stimuli responsive and dynamic. Understanding and

modulating these interactions would thus offer an effective manner to control the

organization of a range of complexes which would benefit their potential properties and

applications. Protein cages such as viruses and bacterial nano-compartment are perfect

examples of natural assemblies formed by such supramolecular interactions.5-8

Recent years have witnessed an increasing attempt to understand the assembly

process of virus-like particles, in that way using these capsid proteins to construct

functional materials for various applications.9, 10 More specifically, plant viruses, as an

example, consist of capsid proteins which envelop their genome inside highly symmetric

cages via both electrostatic interactions and hydrophobic interaction. Furthermore, these

viruses are uniform in size and hollow, which allows the encapsulation of functional

cargos. These hollow nanometer-sized protein structures provide a template for the study

of host-guest interactions in confined space, which in theory should be different from

bulk solution, in addition, the specific interactions parameters could also provide vital

information of the physical microenvironment inside protein cages. The work described

in this thesis combined supramolecular chemistry and physical virology, by studying

host-guest interactions inside the cowpea chlorotic mottle virus (CCMV) capsid, and

ultimately utilize these interactions to induce the self-assembly of virus-based protein

into structures with control in multi dimension.

Chapter 2 provides an overview of recently reported assembly of virus-like particles

induced by supramolecular interactions, that have been used as nanocontainers,

nanoreactors and nano-templates for inorganic or organic material synthesis.

Furthermore, the assembly of protein cages via supramolecular interactions into highly

ordered 2D and 3D super-structures is described.

Introduction

2

1

Chapter 3 introduces a generic bio-inspired template to facilitate encapsulation of

non-spherical inorganic materials into CCMV-based protein cages. Both gold nanorods

and single-walled carbon nanotubes were successfully coated with capsid protein.

In Chapter 4 the coating of, so-called, nano-diamonds with capsid proteins through

electrostatic interactions is discussed, in that way stabilizing these nano-diamonds while

keeping their intrinsic physical properties for biological application.

As for the natural structure of viruses, the interior of CCMV or its capsids can be used

as a model to mimic the nano-compartments. To better understand the host-guest

interactions in these confinement space, in Chapter 5 cyclodextrin modified polymer

was encapsulated inside CCMV. We studied the host-guest interactions via 19F-NMR

and fluorescence spectrometry, which showed the complexity of CCMV system.

Nevertheless, after chemically modifying the N-terminus of the capsid protein with

azobenzene moiety via Sortase A enzymes, Azo-CP was able to self-assemble into virus

like particles (VLPs) through hydrophobic interactions at neutral pH. The assembled

VLPs are reversible by adding different chemical reagents.

Chapter 6 describes the use of CCMV and TM encapsulin nanoparticles as building

blocks for the construction of 2-D or 3-D structures. These highly ordered assemblies

can be modulated via electrostatic interactions or metal coordination.

Chapter 7 presents a brief discussion and outlook based on the data in this thesis.

Reference

1. W. R. Cannon, S. F. Singleton and S. J. Benkovic, Nature Structural Biology,

1996, 3, 821-833.

2. A. Mackiewicz, T. Speroff, M. K. Ganapathi and I. Kushner, The Journal of

Immunology, 1991, 146, 3032-3037.

3. P. Anversa, A. Leri, M. Rota, T. Hosoda, C. Bearzi, K. Urbanek, J. Kajstura and

R. Bolli, STEM CELLS, 2007, 25, 589-601.

4. E. Miyako, K. Kono, E. Yuba, C. Hosokawa, H. Nagai and Y. Hagihara, Nature

Communications, 2012, 3, 1226.

5. A. Liu, M. V. de Ruiter, W. Zhu, S. J. Maassen, L. Yang and J. J. L. M.

Cornelissen, Adv. Funct. Mater., 2018, 28, 1801574.

6. T. Douglas and M. Young, Nature, 1998, 393, 152-155.

Chapter 1

3

1

7. R. M. Putri, J. J. L. M. Cornelissen and M. S. T. Koay, ChemPhysChem, 2015,

16, 911-918.

8. W. M. Aumiller, M. Uchida and T. Douglas, Chemical Society Reviews, 2018,

47, 3433-3469.

9. I. Barwal, R. Kumar, S. Kateriya, A. K. Dinda and S. C. Yadav, Sci. Rep., 2016,

6, 37096.

10. J. G. Millan, M. Brasch, E. Anaya-Plaza, A. de la Escosura, A. H. Velders, D.

N. Reinhoudt, T. Torres, M. S. T. Koay and J. J. L. M. Cornelissen, J. Inorg.

Biochem., 2014, 136, 140-146.

4

Chapter 2

5

2

Chapter 2

Self-assembly of Virus-like Particles Induced

by Supramolecular Interactions

Natural protein assemblies provide unique characteristics compared to

traditional synthetic materials. Moreover, with the development of

supramolecular chemistry, a better understanding of thermodynamics, the

behavior of multivalent systems has been made. Thus allowing an improved

understanding of the assembly pathway and mechanisms of virus -like

particles. Using such mechanisms to control and design supramolecular

assemblies would open up the possibility to construct protein -cage based

functional materials . Here, we provide a concise review of recent advances

on the strategies developed and used to fabricate vi rus-like particles and

protein cage super lattices by supramolecular interactions.

Part of this chapter will be submitted

Self-assembly of Virus-like Particles Induced by Supramolecular Interactions

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2.1 Introduction

Supramolecular chemistry mainly focusses on non-covalent interactions,1, 2 such as

hydrogen-bonding interaction,3 π-π stacking interaction,4 electrostatic interaction,5 van

der Waals forces,6 and hydrophobic/hydrophilic attraction. This field in chemistry has

now matured and has attracted a lot of attention since its discovery back in the 1960s.

The development of methodology for the creation of new molecules based on covalent

bonds, has made it possible to synthesize a vast array of compounds. As a result, there

are infinite possibilities for the self-assembly of these compounds with ineffable

combinations into larger structures, particles and entities, based on non-covalent

interactions. Chemists, biologists and material scientists are brought together towards

multidisciplinary subjects achieving bioinspired self-assembled advanced functional

materials.7 All these considerate efforts have been made to investigate their applications

in catalysis, functional materials, electronic devices, sensors, nanomedicine, and so on.

In biology, self-assembly has been considered for decades as one of the major structuring

and compartmentalization force in nature. Proteins are the most widely used building

blocks,8 which are also responsible for the structural and functional complexity of life.

Natural protein assemblies are at the basis of numerous biological machines, such as

ATPase,9 the cytoskeleton,10 microtubules,11 intracellular microcompartments,12

bacterial S-layer lattices,13 and capsids of viruses.14 All these biological machines are

highly organized assemblies of their building blocks. Inspired by nature, scientists have

developed in recent years self-assembled virus-like-particles (VLPs) and highly

organized larger assemblies based on these VLPs, yielding improved understanding of

the designing discipline for VLPs and their applications as advanced functional

materials.

This chapter reviews the formation and assembly of VLPs by different

supramolecular interactions. The co-assembly of the capsid proteins from native viruses

and different functional cargos into VLPs, might find application as biocatalyst,

biosensors, cancer therapeutics, bioimaging agents, etc. Furthermore, larger-sized,

higher-ordered structures by using VLPs or their subunits as building blocks were

employed towards artificial biological machines.

Chapter 2

7

2

2.2 Virus-like-particles and their applications

Protein-based viruses are nanosized entities composed of coat proteins and genome,

which infect plants, bacteria or animal cells in order to replicate and form new virus

particles. These are well-defined structures that occur in different shapes and sizes

depending on the virus species and are often highly symmetrical and monodisperse

(Figure 2.1). Some examples of icosahedral particles ranging from 18-500 nm are:

cowpea chlorotic mottle virus (CCMV), 15 cowpea mosaic virus (CPMV),16,17 red clover

necrotic mosaic virus (RCNMV),18 human hepatitis B virus (HBV),19 simian vacuolating

virus 40 (SV40),20 MS2,21 E. coli bacteriophage Qβ22 and salmonella typhimurium

bacteriophage P22.23 Some examples of rod-shaped particles are bacteriophage M13,24

tobacco mosaic virus (TMV)25 and potato virus X (PVX)26 with lengths of >2µm.

In material science, these building blocks of viruses are prodigious components for

the construction of virus like particles (VLPs). VLPs are formed by empty shells and

cargos without viral genomes. While the inherently polished properties remain, such as

their perfectly defined structure, stability, biocompatibility, homogeneity, self-assembly,

and low toxicity for application purposes27 (Figure 2.2). To realize the function of VLPs,

it is important to keep the native conformation that is morphologically identical to the

infectious virion. Development of commercial VLPs production has been realized with

bacteria, yeast, insect, mammalian and avian, and plant expression systems. However,

synthesizing these VLPs in the intended downstream applications with controlled quality

and high yield at low cost is still a major challenge.

In a different approach, noncovalent interactions can be employed by modification

of specific residues at the protein surface or N-terminals to enable π – π stacking, metal

ion coordination or electrostatic interactions. The VLPs with specific symmetries and

superstructures can be obtained by controlling the position and orientation of the

modification site. To this end, we will discuss the state-of-the-art in the field of

controlling the assembly of capsid proteins into VLPs by noncovalent interactions, and

the precise control of nanoscale ordered assemblies of VLPs via supramolecular

interactions by using advanced design and synthesis methods.8, 28-31


8

2

Figure 2.1. An overview of the physical structures of various viruses. Structures of the icosahedral viruses

are reproduced from the protein database (https://www.wwpdb.org/), PDB ID: CCMV (1CWP), CPMV

(1NY7), RCNMV (6MRM), HBV (3J2V), SV40 (1SVA), MS2 (2B2G), BMV (3J7L), and P22 (5UU5). M13

is adapted from Yoo et al.,24 copyright © 2014 Yoo et al. (open access). TMV is adapted with permission from

Schlick et al.,25 copyright © 2005 American Chemical Society.

Chapter 2

9

2

Figure 2.2. diverse applications of VLPs within nanotechnology.

2.3 Induce assembly of capsid proteins into virus-like

particles by supramolecular interactions

One of the main features of VLPs is the highly uniform and regular shape from

nanoparticle to nanoparticle. However, the design and production of these VLPs still

remain challenging due to low efficiency of self-assembly, especially for complex VLPs.

The process of VLPs assembly in vitro are one of the best examples of supramolecular

interaction induced assembly. To induce the encapsulation of functional materials to the

interior of VLPs, there are mainly two approaches, one is to genetically fuse the

functional proteins or peptides to the (N-)terminus of the capsid protein, which restrains

the application of other synthesized organic or inorganic materials. Another approach is


10

2

to induce the assembly of VLPs together with downstream cargo in vitro either via

covalent modification or noncovalent interactions.

VLPs assembly is a complex process which involves protein-protein interactions and

protein-cargo interactions. This part will mainly focus on the assembly of VLPs by

noncovalent protein-cargo interactions which are designed to be reproducible and

programmable.

2.3.1 Electrostatic interaction induced assembly

As mentioned before, a natural virus is composed of capsid protein and its genome

which often also serves as a stabilizer through electrostatic interactions between capsid

proteins and nucleic acids. To utilize this natural property of capsids, a vast amount of

researches applied electrostatic interactions to introduce negatively charged cargo such

as non-viral nucleic acids,32, 33 polyacids,34-37 gold nanoparticles,38-42 organic

complexes,43, 44 luminescence molecules45, 46 and enzymes.33, 47-49

To realize the application of the interior cavity of VLPs, insight of the assembly

mechanism of capsid protein and genome is required. The first question that needs to be

answered is whether the sequence specificity of genome contributes to its three-

dimensional arrangement. Karyn and colleagues did pioneering work in this regard50

where they showed similar lengths of nonviral RNA was able to construct similar

morphology and arrangement of native Pariacoto virus (PaV). Synthetic polyacids such

as poly(styrene sulfonic acid) (PSS), polyferrocenylsilane (PFS) and poly(acrylic acid)

(PAA) were successfully used as multivalent guests to induce reassembly of VLPs from

Hibiscus chlorotic ringspot virus (HCRSV)35 and Cowpea chlorotic mottle virus

(CCMV) .36 Because of the feasibility, more and more systems were built up to construct

VLPs through electrostatic interactions between capsid protein and negatively charged

cargos. However, the question remains: How long does the negatively charged template

need to be, what is the limit of the length of polyacids or nucleic acid? Can we control

the assembly of VLPs in shape and size by using different cargo? Can the assembly of

VLPs through electrostatic interaction be controlled?

With these questions in mind, Hu et al.37 investigated the interplay between polymer

length and capsid size of CCMV. They managed to use different lengths of PSS to induce

assembly of CCMV VLPs, nevertheless, only two icosahedral-symmetry structures

(T=2 and T=3) were found when various lengths and charged PSS were applied. This

Chapter 2

11

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paper revealed the first attempt of understanding the criteria needed for VLPs assembly.

Further research is required as it has been demonstrated that the charge ratio is not the

only factor. Maassen et al.51 attempted to study the assembly of CCMV VLPs with

different lengths of short single stranded DNA (ssDNA) at neutral pH to gain insight into

the assembly pathway, as multivalent electrostatic interactions are involved in VLPs

assembly. The assembly process occurred when the length of ssDNA was longer than 14

nucleotides, which indicates that the charge number on individual PSS need to reach a

minimal length. With a better understanding of the electrostatic interaction induced

assembly of VLPs, attempts at constructing various shaped VLPs and encapsulating

functional cargos were carried out.40, 43, 46, 52, 53 Sinn et al.46 managed to utilize assembly

of water-soluble Pt(Ⅱ) amphiphiles to template CCMV VLPs assembly. This formation

of VLPs can be tailored by modulating the supramolecular interactions between

negatively charged template and capsid proteins (Figure 2.3).

Compared to other supramolecular systems, the assembly of VLPs are rather

complicated which makes it challenging to study the assembly mechanism. Electrostatic

interactions between positively charged capsid proteins and negatively charged

polyelectrolytes provide an important thermodynamic driving force for the assembly

process54-56. Theoretical models have been built to gain more insight into the assembly

mechanism and the optimal length required for encapsulation of linear polyelectrolyte.

However, most intermediates on assembly pathways remain challenging to characterize.

It is becoming increasingly more urgent to obtain a thorough understanding of the

multivalent interactions between oppositely charged components based on experimental

facts and theoretical investigation.


12

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Figure 2.3. Formation of differently shaped virus-like particles depending on the chosen Pt(II)

monomer. The proposed mechanism includes a preceding self-assembly of the Pt(II) amphiphiles featuring a

subsequent self-assembly process with the CCMV coat proteins to spherical (icosahedral) or tubular

luminescent virus-like particles. Inset: Schematic formation of tubular structures composed of subunits of

“uncapped” icosahedrons. Copyright© 2018 Sinn et al..46 with permission from American Chemical Society

(https://pubs.acs.org/doi/10.1021/jacs.7b12447, further permissions related to the material excerpted should

be directed to the ACS).

2.3.2 Metal-coordinated assembly of virus-like-particles

Supramolecular coordination chemistry has developed at a tremendous rate through

the last few decades. This allowed chemists to synthesize various supramolecular

complexes with well-defined nanoscale structures57-59 such as enzymes, chemical

sensors, gas adsorption etc. Metal-directed protein assembly has been used to form

designed nanostructures for biomedical applications. Ferritin,60 for example, is a widely

used protein cage for the construction of hybrid materials due to its ability to store ion.

The cage is composed of 24 subunits with a diameter of ~12 nm. To encapsulate

molecular cargo in vitro, multi-subunit protein cages are disassembled at extremely

acidic pH and results in low yield.61 Sana et al.62 managed to induce assembly of

https://pubs.acs.org/doi/10.1021/jacs.7b12447

Chapter 2

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2

Archaeoglobus fulgidus open-pore ferritin (AfFtn) and its closed-pore mutant (AfFtn-

AA) through the process of metal ion mineralization via metal-coordinated interaction.

The disassembly of AfFtn and AfFtn-AA cages could be realized by dissolving the ion

core. Despite its potential application for metal ion storage and release, the diameter of

the inner cavity of ferritin is only 8 nm, which is challenging to encapsulate larger cargos.

Another widely studied metal-ion-binding peptide to construct protein assembly are

Histidine-tagged peptide or proteins which is conventionally applied for protein

purification63 Inspired by this, Minten et al.64 genetically modified the N-terminus of

CCMV CP with a histidine tag. Upon the addition of metal ions to the His-tag-modified

capsid proteins (His-CPs), VLPs were formed and stabilized at neutral pH. Based on this

research, Van Eldijk et al.65 synthesized multi-responsive protein-based block

copolymers by extending the ELP-CP with His-tag. This H6-ELP-CP can be employed

to induce the assembly and encapsulation of functional cargos by adding divalent metal

ions (Figure 2.4).

Figure 2.4. A: Overview of metal ion-induced self-assembly of H6 -ELP-CP into 60-mer protein cages

with T = 1 icosahedral symmetry. B: encapsulation of hexa-histidine-tagged proteins into these nano-capsules.

Copyright© 2016 Eldijk et al.65 with permission from Wiley.

Next to inducing assembly of VLPs at the inner phase of capsid proteins, directional

metal coordination between protein surfaces is an alternative to protein-protein

interactions.66 The first attempt to induce VLPs assemblies in this manner is a

genetically engineered ferritin that can assemble into protein-cage like structures upon

CuⅡ binding at the interface of ferritins.67 Later on, Malay et al.68 designed an ultra-

stable gold-coordinated protein cage with reversible assembly properties. They

genetically engineered a double-mutant trp RNA-binding attenuation protein (TRAP)

which is prone to form an 11-mer ring with 11 equally spaced thiol groups residing

along the outer rim of it. The addition of gold (Ⅰ)-triphenylphosphine generates


14

2

monodispersed protein cages around 22 nm in diameter (Figure 2.5). This work

demonstrates a novel approach for designing protein components assembled into VLPs

with unexplored geometries.

Figure 2.5. Formation of TRAP-cage. A: Structure of the double mutated TRAP(K35C/R64S) (based

on PDB ID: 4V4F). Substituted residues Cys35 (red) and Ser64 (grey) are modelled as spheres. B: Chemical

structure of Au-TPPMS. C: size-exclusion chromatography of assembled TRAP(K35C/R64S) cage. D:

Transmission electron microscopy (TEM) images of unreacted TRAP(K35C/R64S) (left) and cages after

mixing TRAP(K35C/R64S) and Au-TPPMS (right). Scale bars, 100 nm. E: Top, single-molecule mass

photometry. Insets: single-particle images of partially (left) and fully assembled (right) cages. Scale bars, 1

μm. Bottom, extracted assembly kinetics. F: Cryo-electron microscopy density maps of the left-handed and

right-handed forms of TRAP-cage, refined to 3.7 Å resolution. G: Cutaway view of the left-handed map,

showing a hollow interior. H: Snub cube (left-handed and right-handed forms) consisting of 32 regular

triangles and 6 square faces. The four-, three- and two-fold rotational axes are represented in blue, yellow and

red, respectively. I: Magnified view of the left-handed map showing 11-fold rotational symmetry of ring

elements and prominent density bridges connecting adjacent rings. J: Refined left-handed cage model,

consisting of 24 TRAP(K35C/R64S) rings. Three views are indicated, centered on the (from left to right)

two-, three- and four-fold symmetry axes. Image taken from Malay et al..68 Copyright© 2019 with

permission from Springer Nature.

Chapter 2

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2.3.3 Other supramolecular interactions induced assembly of virus-

like particles

Apart from the electrostatic interaction and metal coordination mentioned in the

previous two sections, other interactions such as hydrophobic interaction,49, 69-71 coil-

coil interaction72-76 and specific icosahedral symmetry protein-protein interactions77, 78

can also be used to induce assembly of VLPs at neutral pH.

Conjugation of function fragments to capsid proteins enables the incorporation of

hydrophobic entities for the construction of VLPs via hydrophobic interactions. Taking

CCMV as an example, capsid protein equipped with elastin-like polypeptide (ELP) at

its N-terminal (ELP-CCMV) was constructed. This block copolypeptide is able to

assemble into VLPs at pH 7.5 with high salt concentration.49, 70, 71 This method

facilitates the attachment of functional moieties, such as enzymes, to the interior of the

VLPs (Figure 2.6 A). Another approach to induce assembly of higher ordered

nanoscale cages is to incorporate de novo-designed coiled-coil domain into the subunit

of protein cages (Figure 2.6 B) which results in symmetrical icosahedral VLPs.72 With

the development of the understanding of assembly pathway and mechanism,

computational design of proteins based on symmetry principles, have been widely used

in the last decades to create ordered assembly of icosahedral protein cages 79-81 (Figure

2.6 C).

Diverse strategies and interactions to obtain highly ordered symmetry VLPs are

available and are still developing, but solid mechanisms and designing principles to

meet specific requirements for application purposes is essential to promote progress in

the field that deals with protein assemblies.


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Figure 2.6. schematic representation of various supramolecular interaction induced assembly of VLPs

mentioned in the chapter. A: Sortase A mediated N-teminal functionalization of ELP-CCMV with enzyme

and the formation of VLPs.49 Copyright© 2016 with permission from Nanoscale. B: Ico 8 fusion protein

trimers genetically fusioned with pentameric coiled-coil assemble into an icosahedral cage.72 Copyright ©

2019, with permission from American Chemical Society. C: atomic structure ilustration of a designed protein

cage.80 Permission was provided by The American Association for the Advancement of Science.

2.4 Self-assembly of protein cages into 3D structures via

supramolecular interactions

In general, there are two methods to construct nanoscale structures, top-down82 and

bottom-up.83 Fabrication of nanostructures with the top-down method is undoubtedly

efficient but reached its limits in further reducing size. Bottom-up methods provide the

versatility to incorporate different types of small particles into large assembly systems.

Protein cages are one of the most versatile building blocks for self-assembling systems,

due to their uniform size and feasibility of controlled functionalization. Supramolecular

multivalent interactions are the most commonly used driving force to induce the self-

assembly of protein cages into larger three-dimensional (3-D) structures, because these

are easy to introduce and most of the time reversible. Highly ordered assembly of

protein cages are constructed to introduce a variety of applications such as biosensors,

cascade catalysis and bio-imaging. To realize the long-ranged orientation of protein

cages, there are two main factors: a, the optimal binding affinity of the building blocks;

b, the valency of multivalent interactions of the component used for the supramolecular

Chapter 2

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interactions. This section will mainly focus on the assembly of protein cages into

higher-ordered structures via non-covalent interactions.

2.4.1 Hierarchical lattices of protein cages via electrostatic

interactions

Electrostatic interaction induced assembly of protein cages into hierarchical

structure is of great interest. There are three main advantages of applying electrostatic

interaction: a) most natural protein cages carry either negative or positive surface

charge near neutral pH, taking TMV84 and CCMV53 as example, they are negatively

charged on their outer surface, and the charges on the surface are distributed evenly due

to the intrinsic symmetrical structure of these protein cages; b) electrostatic interaction

can be controlled through pH and salt concentration; c) both synthetic and natural

components can be introduced as the contrary building blocks to provide vary

functions.

To achieve ordered assembly, salt concentration is vital to control that electrostatic

interactions at the optimal range. Stronger interactions will result in irregular structures,

while weaker interaction is unable to bring building blocks together. Kostiainen et al.

developed a temporary platform to induce assembly of virus protein cages (CCMV and

Ferritin) into higher-ordered structure and disassemble through irradiation (Figure 2.7

A, B).85, 86 The assembly of CCMV-dendron complexes can be controlled in size with

different concentrations and generations of the dendron as well as salt concentration

(Figure 2.7 B). Similarly, super lattices were produced by using recombinant magneto-

ferritin particles (RMPs) in a comparable way, allowing the magnetic properties of

nanoparticle to be tuned through the process of assembly and disassembly of

superstructures (Figure 2.7 A). Further study of these protein-cage and dendrimer

system were carried out to gain insight in the assembly process of CCMV and

PAMAM (Figure 2.7 F),87 as well as Ferritin ((Figure 2.7 D).88 With the understanding

of the principle of tuning assembly conditions, Beyeh et al. constructed cyclophane-

protein cage frameworks which bridge the gap between molecular frameworks and

colloidal nanoparticle crystals30 (Figure 2.7 C). Such host-guest supramolecular

structure are interesting for various applications, for example, creating biohybrid

system with functional organic dyes (Figure 2.7 H).89


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Figure 2.7. A: strategy for the assembly and optically triggered disassembly of RMP-dendron

complexes.86 Copyright© 2011 with permission from American Chemical Society. B: schematic illustration

of the assembly and disassembly of CCMV-dendron superstructures.85 Copyright© 2010, with permission

from Springer Nature. C: schematic illustration of the assembly of cyclophane-protein cage framework.30

Copyright© 2018 with permission from American Chemical Society

(https://pubs.acs.org/doi/full/10.1021/acsnano.8b02856, further permissions related to the material excerpted

should be directed to the ACS). D: graphic abstract of the tunable assembly of superlattices with different

generation of PAMAM dendrimers.88 Copyright© 2015, with permission from American Chemical Society.

E: graphic abstract of higher-ordered assembly of P22 VLP into protein cage lattices for multistep catalysis.90

Copyright© 2018, with permission from American Chemical Society. F: effect of size on the clustering of

CCMV with PAMAM dendrimers.87 Copyright© 2014, with permission from Wiley. G: carton of stimuli

responsive hierarchical assembly of P22 VLPs.83 Copyright© 2018, with permission from American

Chemical Society. H: hierarchical organization of organic dyes and Apoferritin into photoactive crystals

through electrostatic interaction.89 Copyright© 2015, with permission from American Chemical Society

(https://pubs.acs.org/doi/abs/10.1021/acsnano.5b07167, further permissions related to the material excerpted

should be directed to the ACS). I: cartoon representation of the formation of a light-emitting metal-organic

biohybrid complex via self-assembly of organoplatinum (Ⅱ) metallacycle and TMV.84 Copyright© 2016,

with permission from American Chemical Society.

In addition to CCVM and Ferritin, P22 VLPs are also capable of assembling into

long-ranged ordered structures via complementary charge interaction by modifying the

exterior surface of these VLPs90, 91 (Figure 2.7 E, G). Tian et al.84, reported negatively

charged TMV rod-like virus can be assembled into 3-D micrometer-sized bundles

through the electrostatic interactions with a positively charged TPE derivative TPE-Pt

https://pubs.acs.org/doi/full/10.1021/acsnano.8b02856

https://pubs.acs.org/doi/abs/10.1021/acsnano.5b07167

Chapter 2

19

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(TMV/TPE-Pt-MC) (Figure 2.7 I). This hierarchical assembly resulted in enhanced

fluorescence represented not only metal-organic biohybrid materials but also turn-on

fluorescence. Oppositely charged soft matter mediated hierarchical assembly of protein

cages provide experimental understanding of the critical factors for the production of

long-range ordered structures. It’s worthwhile to obtain insight into the assembly

between protein cages and rigid nanoparticles like gold nanoparticles, Kostiainen et

al..92 managed to obtain binary superlattices by using CCMV and Ferritin via

electrostatic interactions. AB-type superlattices obtained with a Ferritin derivative

indicated that the crystal structure is defined by protein cages rather than the

encapsulation of nanoparticles. This protein cage directed construction of nanoparticle

superlattices, will facilitate biocompatible delivery and sensing applications in

biological systems.

2.4.2 Assembly of virus-like-particles into superlattices through other

supramolecular interactions

Despite its effectiveness for non-covalent self-assembly, controlling the

electrostatic interaction as a way to obtain desired properties is still challenging, as it is

electrolyte concentration dependent. Other non-covalent interactions such as transition

metal coordination, hydrophobic interaction and protein-protein interaction have been

utilized to obtain hierarchical structures. Metal coordination is widely used to construct

metal-organic frameworks (MOF) for their application in separation, storage, and

catalysis93-96. Inspired by the assembly of synthetic organic hybrid crystals, Ferritin

was applied as building blocks for the construction of metal-protein cage crystals due

to its feasibility for genetically engineered symmetrical metal-ion binding site (Figure

2.8 A).97 A bcc crystal structure was produced by vapor diffusion in the presence of

excess ZnCl2 to saturate all the binding sites of the Ferritin cage. Another attempt to

induce ordered assembly of nanoparticles is by using tobacco mosaic virus (TMV) coat

protein.98 Zhang et al. mutated the outer rim of TMV disks with 4 histidine for metal

coordination to construct well-organized 2D monolayer (Figure 2.8 C-F).

Protein-protein specific interaction can be used to construct a protein

macromolecular framework99 (Figure 2.8 B), McCoy et al. managed to construct long-

range ordered functional protein network which is called protein macromolecular

framework (PMF) by P22 VLPs and its specific protein linker. This entire process is


20

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templated by an amine terminated dendrimer, where the preassembly was performed

via electrostatic interactions, followed by the substitution with the specific protein

linker. The assembly formed directly by P22 VLPs and the ditopic cementing protein

linker are rather loose compared to the latter methods.

Figure 2.8. A: scheme for the assembly of ferritin into 3D crystals.97 Copyright © 2015 with permission

from American Chemical Society (https://pubs.acs.org/doi/full/10.1021/jacs.5b07463, further permissions

related to the material excerpted should be directed to the ACS). B: schematic illustration for the assembly of

protein macromolecular framework from P22 VLPs.99 Copyright © 2018 with permission from American

Chemical Society. C: The schematic of assembly of T103C‐TMV‐4his disks into monolayer sheets via

Cu2+–histidine interactions. Three different functional sites contained in TMV monolayer sheet are shown

with different geometric figures (circle, rectangle, and triangle), resulting in three possibilities for binding

fNPs.98 D and E: TEM and AFM characterization of TMV monolayer sheet. F: SAXS data of the 2D

honeycomb AuNP lattice derived from TMV. Copyright © 2019 with permission from Wiley. G: cartoon of

hierarchical assembly of P22 VLPs via aromatic interactions. H: Fast Fourier transform (FFT) of 4FF lattices.

I: real map from invert FFT of H, J, structural model based on the real map in I.100 Copyright © 2018 with

permission from American Chemical Society.

Despite the novo method of constructing PMF, the linker for the protein cages

should be specific to restrain the generic application for the production of hierarchical

assemblies. As one of the non-covalent interactions, π – π stacking between aromatic

amino acids play an important role in protein-protein recognition, ligand binding,

structural stabilization and protein folding. Inspired by this, Zhou et al.100 mutated Glu

162 in native recombinant human H-chain ferritin (rHuHF) with Phe, Tyr and Trp to

induce the formation of 2D array or 3D simple cubic superlattices, respectively (Figure

https://pubs.acs.org/doi/full/10.1021/jacs.5b07463

Chapter 2

21

2

2.8 G-J). Due to the aromatic interaction, the assembly is reversible by tune salt

concentration and pH. Further applications were carried out for the production of

nanoparticle assay. This unconventional strategy only requires small and simple

modifications of the protein cage surface, which is easy to realize. Secondly, the

modification method can be generalized since a large variety of protein assemblies

have symmetry axes.

2.5 Summary and future perspectives

In summary, supramolecular interactions provide a rich and fulfilling methodology

in designing ordered assembly systems of virus like particle for the encapsulation of

therapeutics,101 inorganic functional cargos, nucleonic acids, organic dyes for potential

bio applications.27, 102, 103 With the development of supramolecular chemistry, a better

understanding of thermodynamics and the behavior of multivalent systems has been

made; allowing for a major step towards the understanding of the assembly pathways

and mechanisms of virus-like particles. A more detailed study is still required for a

thorough understanding of VLPs assembly which will facilitate the rational design of

de novo protein cages and provide insight into virus infection pathways for biomedical

purposes.

The fabrication of protein cage super lattices by supramolecular interaction saw

enormous progress in last decade.28 More work in the years ahead will enormously

enhance our knowledge of the fundamental aspects of protein based hierarchical

assemblies. This will in turn reward for the application of these systems in the biomedical

field.

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29

Chapter 3

Construction of Viral Protein-based Hybrid

Nanomaterials Mediated by Molecular

Glues

Herein we report a generic strategy to construct advanced viral protein -

based hybrid nanomaterials by using molecular glue inspired from mussels.

A dopamine modified commercial ly available poly (isobutylene-alt-maleic

anhydride) (PiBMAD) was designed as molecular glue, whi ch served as a

universal adhesive material for construction of multicomponent hybrid

nanomaterials. As a proof of concept, gold nanorods (AuNRs) and single

walled carbon nanotubes (SWCNTs) were first coated with PiBMAD, and

subsequently, the on-surface PiBMAD templated the coating of viral capsid

proteins around the nanoobjects. The resulted protein coated hybrid

nanomaterials showed an improved biocompatibility than raw

nanomaterials. It is anticipated that this line of research suggests a

general and facile approach for constructing sophisticated hybrid

nanomaterials without the need for tedious surface modification.


Construction of Viral Protein-based Hybrid Nanomaterials Mediated by

Molecular Glues

30

3

3.1 Introduction

Nanomaterials find numerous applications in catalysis,1, 2 biosensors,3 drug

delivery4 and bioimaging5, 6 with their controlled chemical compositions and structures,

large surface-to-volume ratios, various functional groups and surfaces. Among them,

protein cages especially virus based hybrid nanomaterials, show extraordinary

advantages in electronics,7 catalysis,8 drug/gene delivery,9 imaging and

immunotherapy,10 on account of their reversible assembly, uniform size, facile

modification, and capability of functional cargo encapsulation.11, 12

By far, functional protein-organic/inorganic hybrid nanomaterials are mainly

prepared via self-assembly of proteins,11, 13, 14 chemical conjugation,5, 15 and protein

immobilization.16 Take the Cowpea Chlorotic Mottle Virus (CCMV) for instance,

protein cages derived from this virus have been widely used as nanocarriers with various

cargos such as polystyrene sulfonate (PSS),17 oligonucleotides,18 functionalized

enzymes19, gold nanoparticles20, and luminescent materials etc.13, 12 For efficient loading,

negatively charged cargo surfaces are generally required, to guarantee strong

electrostatic interactions between the capsid proteins and the desired cargos. Therefore,

for lots of nanomaterial cargo candidates such as silicon quantum dots, gold nano-objects,

and carbon nanotubes with neutral surfaces, a surface modification either by coupling

oligonucleotides6, or PSS, or thiol-based ligands is a prerequisite.21 However, the surface

modification usually involves tedious organic synthesis, meanwhile, it suffers from

aggregation of these nanoobjects that leads to instability and the loss of intrinsic

properties. To tackle this problem, we propose a generic adhesive material that can serve

as an interfacial molecular glue to facilitate the coating of nano cargos with CCMV

capsid proteins, towards protein-organic/inorganic hybrid nanomaterials.

The structure design and core capabilities of this molecular glue should meet two

requirements: (i) as adhesive materials, they can intimately interact with a wide range of

nanomaterial surfaces; and (ii) as templates to induce the coating of capsid proteins on

nanomaterials, they should incorporate negatively charged functional groups (i.e,

carboxylic acids, sulfates, phosphates). Catechol functionalized polymers inspired by

natural mussels has been developed as tissue adhesives for bio-applications22-24 since the

catechol group is capable to have strong noncovalent interactions with metal ions,

Chapter 3

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3

polarized organic groups, or covalent crosslinking via oxidation.23, 25, 26, 27 Hence, the

catechol group is an ideal building block to meet the first requirement for constructing

the molecular glue. Meanwhile, commercially available poly (isobutylene-alt-maleic

anhydride) that inherently contains multiple carboxyl groups can serve as the template

building block to meet the second requirement. Therefore, we prepared catechol

modified poly (isobutylene-alt-maleic anhydride) as the molecular glue for constructing

protein-organic/inorganic hybrid nanomaterials.

The molecular glue was synthesized by animation of poly(isobutylene-alt-maleic

anhydride) with dopamine (PiBMAD).28 Dopamine was used to introduce the catechol

group for the enhanced adhesiveness of this generic molecular glue. As a proof of

concept, two representative nanomaterials, gold nanorods (AuNRs) and single walled

carbon nanotubes (SWCNTs), have been selected as cargo candidates to verify the

capability of this molecular glue (Scheme 3.1).

Scheme 3.1. Schematic representation of mussel inspired molecular glue to induce the coating of two

nanoobjects, AuNRs and SWCNTs, by CCMV capsid proteins. Both of AuNRs and SWCNTs were first

coated with PiBMAD, then CCMV capsid proteins were coated on their surface through electrostatic

interactions.


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3.2 Results and discussion

3.2.1 Synthesis of molecular glue

PiBMAD was synthesized as described in the supporting information (Figure 3.2

A). To maintain water solubility of the polymer, around 10 dopamine groups were

attached onto PiBMA according to 1H-NMR spectrum of PiBMAD (Figure 3.2 B).

Figure 3.2. A: synthesis of PiBMAD, B: 1H-NMR spectrum of PiBMAD in which the signals used to

determine the degree of functionalization are marked.

The capability of PiBMAD to induce the assembly of CP was first checked. The

formation of virus-like particles (VLPs) was evident by fast protein liquid

chromatography (FPLC) equipped with a UV-vis detector (Figure 3.3C). The absorption

data showed overlapping signals at two detection wavelengths ( = 260 nm and 280 nm)

at an elution volume of V ≈ 11 mL, indicating the formation of VLPs. The fraction was

isolated and analyzed by transmission electron microscopy (TEM) and dynamic light

scattering (DLS) (Figure 3.3B, D). TEM analysis revealed the formation of spherical

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nanoparticles with an average diameter of 18.9 nm. DLS showed that the isolated fraction

contained nanoparticles with diameter of 18.0 ± 3.6 nm, which is consistent with the

TEM results. These data are in line with the previously confirmed T = 1 icosahedral

symmetry for CCMV VLPs formed at neutral pH on a polyanionic template.29

Figure 3.3. Preparation of PiBMAD scaffolded VLPs. A: scheme of PiBMAD cargo induced formation

of VLPs, B: TEM images of formed VLPs, C: FPLC purification of PiBMAD formed VLPs, D: DLS data

shows well distributed VLPs sized around 18 nm

3.2.2 Ligand exchange and encapsulation of gold nanorods and

single-walled carbon nanotubes

Gold nanorods (AuNRs) belong to a highly interesting class of nanosized objects

for a plethora of biomedical and biotechnological applications such as sensing, imaging,

and others. However, these applications rely on their stability in biological fluids,

avoiding deleterious aggregation and precipitation. Surface coating of gold nanorods

with biocompatible proteins may facilitate the bio-applications of these nanomaterials.

To this end, surface coating of AuNRs by CCMV proteins was successfully realized

mediated by PiBMAD (Figure 3.4A), detailed experimental methods are described in the

supporting information. The coated AuNRs were purified with FPLC (Figure 3.4B), the

UV-Vis spectra showed signals at elution volume of V ≈ 7.5 mL by monitoring the


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characteristic absorption of capsid proteins (i.e. = 260 nm and 280 nm). The elution

volume is smaller than the typical elution volume of VLPs (11 mL), suggesting the

formation of larger particles. Other than that, the transverse plasmon resonance peak of

AuNRs at = 515 nm was also and only detected in the first fraction, confirming that

this fraction consists of coated gold nanorods (CCMV-AuNRs).30

Figure 3.4. A: schematic presentation of CCMV-AuNRs mediated by molecular glue PiBMAD, B: FPLC

purification of CCMV-AuNRs, C: changes of zeta potential of AuNRs at different coating steps, D: UV-Vis

spectra of original CTAB-AuNRs, PiBMAD-AuNRs, CCMV-AuNRs, and PiBMAD-VLPs.

Chapter 3

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Figure 3.5. TEM and AFM analysis of PiBMAD-AuNRs and CCMV-AuNRs. A: TEM image of

PiBMAD-AuNRs indicates a size around 10*40 nm, B: Negative stained TEM image of CCMV-AuNRs

shows a well-defined protein layer (thickness around 5 nm), C: AFM image of PiBMAD-AuNRs, D: AFM

image of CCMV-AuNRs, E and F: height analysis of PiBMAD-AuNRs and CCMV-AuNRs respectively, for

the PiBMAD-AuNRs, the height is around 15±2.3 nm which demonstrate around 2.5 nm coating of

PiBMAD; while the height of CCMV-AuNRs is around 25 nm, which indicate around 5 nm coating of CP,

this again in line with the natural capsid thickness.

Zeta potential measurements were carried out to check the change of surface charge

of AuNRs after coating (Figure 3.4C). When the AuNRs were first coated by PiBMAD,

the zeta potential of AuNRs reversed from positive to negative, due to the abundant

carboxyl groups of polymer chains. When finally coated by capsid proteins, the zeta

potential of CCMV-AuNRs increased from -75 mV to -10 mV. The longitudinal plasmon

resonance peak (LPRW) of the original AuNRs was located at = 808 nm, after coating


Molecular Glues

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by PiBMAD, the LPRW of PiBMAD-AuNRs was slightly shifted to = 805 nm, while

after coating by capsid proteins, a 50 nm red shift of the longitudinal plasmon resonance

(Figure 3.4D) was observed.

The morphology of PiBMAD-AuNRs and CCMV-AuNRs were studied by

transmission electronic microscopy (TEM) and atomic force microscopy (AFM)

micrographs. Both TEM and AFM analysis showed that the morphology of AuNRs was

unchanged after coating with PiBMAD (Figure 3.5A, C). The original height of AuNRs

was 10 nm, while overall height of PiBMAD-AuNRs was around 15 nm (Figure 4e),

suggesting a ~ 2.5 nm thickness of polymer layer. The thickness of capsid protein layer

on CCMV-AuNRs is estimated to be ~5 nm (Figure 3.5 B-F), which is consistent with

the thickness of the native CCMV capsid shell, suggesting a monolayer coating of capsid

protein on the AuNRs surface. AFM micrograph and height analysis on single PiBMAD-

AuNRs and CCMV-AuNRs further supports this conclusion (Figure 3.6).31

To avoid the aggregation of AuNRs during coating, an optimized concentration of

PiBMAD is required (Figure 3.7). The strongest LPRW absorption peak of PiBAMD-

AuNRs suggest minimal aggregation when the PiBMAD concentration is 3 mg/mL. This

might because less catechol is attached to the surface of AuNRs at lower concentrations

of PiBMAD. When the concentration of PiBMAD is too high, catechol groups of

PiBMAD might cause inter-nanoparticle crosslinking 32, 33. To note that, buffer solution

(i.e, salt concentrations, pH) also plays an important role in coating CP onto the AuNRs

(Figure 3.8). FPLC data (Figure 3.8A) indicates a thicker layer of CP is coated on AuNRs

at pH 6.8 compared to pH 7.2, which in line with a higher UV absorption at 280 nm by

CP (Figure 3.8B).

Chapter 3

37

3 Figure 3.6. A and C: AFM morphology and height measurement of PiBMAD-AuNRs, B and D: CCMV-

AuNRs .

Figure 3.7. UV-vis data of PiBMAD-AuNRs formed at various polymer concentrations, showing an

increased absorption of PiBMAD at = 280 nm at higher polymer concentrations.

Based on the large surface area, excellent chemical stability, and rich polyaromatic

structure, carbon nanotubes (CNTs) is an emerging nanomaterial as scaffold for

biosensors34-36, anticancer drug delivery cargos37. The main challenges of CNTs being


Molecular Glues

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3

biomaterials is their poor aqueous solubility due to the high hydrophobicity. To improve

the aqueous solubility, functionalization of CNTs with biocompatible and hydrophilic

functional organic species are general approaches.38-40 Herein, we demonstrate a facile

protein coating strategy on single walled carbon nanotubes (SWCNTs) by employing the

molecular glue PiBMAD without modification of SWCNTs. The detailed coating

process is described in the supporting information.

Figure 3.8. A: FPLC results CCMV-AuNRs formed in various encapsulation buffers, B: UV-vis data of

CCMV-AuNRs formed in various encapsulation buffers, which suggests a thicker coating of CP on the surface

of AuNRs at pH 6.8 encapsulation buffer (50 mM tris, 200 mM NaCl).

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SWCNTs dispersed with sodium dodecyl sulfate (SDS) showed few bundles (Figure

3.9A). After ligand exchange with PiBMAD, the size and distribution of SWCNTs were

almost unchanged (Figure 3.9B). While after coating with CPs, the average diameter of

CCMV-SWCNTs was around 20 nm (Figure 3.9C), suggesting a protein monolayer with

~5 nm thickness on the surface of SWCNTs. Raman spectroscopy (Figure 3.10) also

confirmed the presence SWCNT before and after coating with PiBMAD, based on the

unchanged characteristic peaks associated with CNT.

Figure 3.9. TEM images of A: SDS-SWCNTs, B: PiBMAD-SWCNTs, C: CCMV-SWCNTs.


Molecular Glues

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3

Figure 3.10. Raman shift of SWCNTS dispersed in water by (a) SDS and (b) PiBMAD.

Figure 3.11. Alternative ligands were used as templates for coating of AuNRs (A-C), and SWCNTs (D-

F).

In control experiments, other ligands such as bis(p-

sulfonatophenyl)phenylphosphine dihydrate dipotassium salt (BSPP) and Poly-Dopa

Chapter 3

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(PDA) were used as templates for CCMV capsid protein coating (Figure 3.11). For the

AuNRs, both BSPP and PiBMAD templated the formation of CCMV-AuNRs and an

apparent single layer of CP coating was observed, while PDA templated coating induced

aggregation of AuNRs which might be caused by crosslinking of AuNRs by the PDA

(Figure 3.11A-C). Regarding to SWCNTs, only PiBMAD induced the formation of well-

dispersed CCMV-SWCNTs (Figure .11D-F). These results again convinced the concept

of using PiBMAD as a generic molecular glue for the construction of viral protein coated

hybrid nanomaterials.

3.2.3 Cell viability study

To further demonstrate the biocompatibility of CCMV-AuNRs and CCMV-

SWCNTs for potential bio-applications, cytotoxicity studies were carried out at various

concentrations of the biohybrid materials. As shown in Figure 3.12A, the cells incubated

with CCMV-AuNRs revealed significant higher viability compared with CTAB-AuNRs

and PiBMAD-AuNRs over the tested concentration range (from 1.25 to 5 nM), in spite

of a concentration dependent cytotoxicity shown for all three samples. The SWCNTs

with diverse surface coating all demonstrate cell viability at the various concentrations

studied (range from 5-50 µg/mL) (Figure 3.12B).

Figure 3.12. cell cytotoxicity of AuNRs and SWCNTs at various concentration. A: cell viability of

AuNRs at various concentration (1.25 nM - 5 nM), B: cell viability of SWCNTs at various concentration

(5µg/mL - 50 µg/mL).

3.3 Conclusions

In summary, we have demonstrated a generic strategy to prepare viral protein-based

hybrid biomaterials mediated by an elaborate molecular glue inspired by mussels.


Molecular Glues

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3

AuNRs and SWCNTs were selected as typical nanomaterials to verify the concept of this

molecular glue. Catechol modified negatively charged polymers showed the capability

to mediate the co-assembly of these nano cargos and CCMV capsid proteins. This

strategy provides a facile and general coating process compared with traditional methods.

In contrast to other commonly used ligands such as thiolate polymer,21 single strand

short DNA fragments,41 the feasibility of preparing PiBMAD in a wide diversity makes

PiBMAD a promising interface molecular glue for the construction of viral protein-based

hybrid nanomaterials. Moreover, it is anticipated that through elaborate molecular design,

the concept of this molecular glue can be further expanded for the preparation of hybrid

nanomaterials.

3.4 Acknowledgments

The authors are grateful to Dr. E. G. Keim and Dr. Jun Wang (MESA+ Institute for

Nanotechnology, University of Twente) for assistance with TEM and AFM respectively.

Dr. Liulin Yang is gratefully acknowledged for his help in images and manuscript

preparation. Many thanks to Naomi Hamelmann for the cell experiments and fruitful

discussions.

3.5 Materials and methods

3.5.1 General

All chemicals were purchased from Sigma-Aldrich and used as received unless

stated. Deionized water used for buffers, reactions and dialysis media was of ultrapure

quality (Milli-Q, 18.2 MΩ·cm). Membrane filters (Spectra/Por® regenerated cellulose

tubing) used for dialysis were purchased from Spectrum Laboratories, Inc., USA.

3.5.2 Characterizations

Ultra-Centrifugation

Fiberlight F14S-6x250 and Step Saver 70V6 rotors (Thermo Fisher Scientific), have

been used.

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UV-vis Spectroscopy

UV-Vis absorption spectra were acquired using a PerkinElmer Lambda 850 UV-Vis

Spectrometer. Samples are prepared in a 1cm quartz glass cuvette. Each recording cycle

was performed within 44 s.

Transmission electron microscopy (TEM)

TEM analysis was performed using Philips CM300 microscope operating at 300 kV.

A droplet of the samples was casted on a 200-mesh copper grid for 2 min before the

excess solvent was blotted away using a sterile paper. Samples were negatively stained

by applying 5 µL 1 % (w/v) uranyl acetate in MilliQ water onto the grid for 30 s and

removed afterwards.

Fast protein liquid chromatography (FPLC)

FPLC analysis were performed on a GE Healthcare ÄKTApurifierTM system

equipped with a Superose 610/300 GL column from GE Healthcare and a fractionating

device. Injection of 500 µL pre-filtered samples which are injected on a 24 mL

superpose-6 column. Compound elution is monitored using a UV-Vis spectrometer at

260 nm, 280 nm and 520 nm. Fractionation are collected separately.

Dynamic light scattering (DLS)

DLS analysis was performed using a Nanotrac (Anaspec) instrument and Microtrac

FLEX Operating software with laser wavelength of 780 nm and a scattering angle of 90°

at 25°C. The observed size and standard deviation of nanoparticles were calculated by

taking an average of 5 measurements.

Nuclear magnetic resonance (NMR)

Carbon-13 and proton NMR spectra of polymers were recorded using a Bruker 400

MHz NMR. Polymers were dissolved in dimethyl sulfoxide-d6 in concentrations of 30

mg/mL and 0.5-1.0 mg/mL for 13C- and 1H-NMR respectively.

Gel permeation chromatography (GPC)

The polydispersity of PiBMA was analyzed using a Waters e2695 Separations

Module equipped with Agilent PLgel 5 μm MIXED-D 300x7.5 mm column. DMF was

used as eluent, with molecular weights calibrated against linear standards of polyethylene

glycol (PEG).

Inductively coupled plasma optical emission spectroscopy (ICP-OES)


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3

The gold concentration of commercial nanorod stock solution was determined using

a PerkinElmer dual view 8300 ICP-OES. Calibrations were made against dilutions of a

commercial gold standard of 1000 mg/L, ranging from 0.001 to 100 ppm. Emissions

from gold atoms and ions in excited state were measured at software recommended

wavelengths for Au: 267.6, 242.8 and 208.2 nm.

Raman spectroscopy

Raman spectra of CNT samples were acquired using a custom-built Raman setup

containing a 647 nm laser (30 mW). Calibrations were performed against an argon

mercury light source as well as a white light source for further corrections. 50 μL of

sample was deposited on a glass slide and measured. From each measurement the

average of 100 spectra was used.

Zeta potential

The zeta potential of gold nanorods with different ligands was measured at neutral

pH, 25 °C with a Malvern Zetasizer Nano ZS ZEN3600, using a 633 nm laser.

3.5.3 Experimental section

Synthesis of PiBMAD

1 g of PiBMA was dissolved in 50 mL aqueous solution of 2 M NaOH. The solution

was then hydrolyzed (Figure S1a) at 80 °C for 3 h, afterwards the solution turned clear.

After cooling to room temperature, 15 mL of HCl (10 M) was added to precipitate

PiBSA. The white, sticky polymer was then collected by short centrifugation and

dissolved in 15 mL deionized water. In order to remove residual reagents, the solution

was dialyzed against deionized water overnight using a 1 kD MWCO dialysis tube. The

solution was then freeze dried (Labconco FreeZone 4.5) for 48 h to obtain 250-500 mg

poly (isobutylene-alt-maleic acid) (PiBSA).

100 mg PiBSA was dissolved in 5 mL dimethylformamide (DMF) containing 1-

ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl, 0.1 M) and N-

hydroxysuccinimide (NHS, 0.1 M). This mixture was stirred for 20 min. Meanwhile, a

second solution was prepared of 47.4 mg dopamine·HCl (0.5 M) in 0.5 mL aqueous

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NaHCO3 (0.5 M) and Na2S2O3 (reducing agent, 0.1 M). The mixture was then added to

the organic PiBSA solution and left to react overnight under mild magnetic stirring. The

mixture was diluted 10X using deionized water and purified by dialysis against deionized

water (1 kD MWCO dialysis tubing), refreshing the medium every 3 h for at least five

times. The solution was then freeze dried (Labconco FreeZone 4.5) for 48 h, after which

the PiBMAD powder is collected.

Encapsulation of PiBMAD by CCMV VLPs

PiBMAD was dissolved (6.0 mg/mL) in pH 7.2 encapsulation buffer (50 mM Tris,

200 mM NaCl, 5 mM MgCl2). 50 μL of this PiBMAD solution was added to 200 μL CP

solution (6.0 mg/mL, also in pH 7.2 encapsulation buffer). The mixture was gently

shaken overnight at 4 °C.

Preparation of CCMV-AuNRs

When extracting AuNRs from solution by means of centrifugation, to maximize the

yield of AuNRs, samples were centrifuged as follows: A gold nanorod solution is

centrifuged at 6,000 g for 10 min. Then the pellet is collected, and the supernatant is

centrifuged again at 8,000 g for 10 min. The resulting pellet is collected, and the

supernatant is again centrifuged at 10,000 g for 10 min. This is repeated once more at

14,000 g for 10 min, resulting in a total of four pellets after one round of centrifugation.

All the centrifugation of AuNRs was performed in this manner, unless stated differently.

The ligand exchange of CTAB-coated gold nanorods with PiBMAD was based on

general methods found in literature,1 where (poly)dopamine coatings are applied to gold

nanorods. However, since oxidation of dopamine is undesired in our case, pH was

lowered to 7.5 and small amount of reducing agent (Na2S2O3) was included. Briefly, 1

mL CTAB-AuNRs (optical density = 0.95) from stock solution was centrifuged at 20 °C.

The pellet was suspended in 2 mL Tris buffer (10 mM, pH 7.5) containing PiBMAD (0.5


Molecular Glues

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3

- 4.0 mg/mL or 50-400 equiv. by weight conc.) and 5 mM Na2S2O3. The nanorod

suspension was then ultrasonicated for 30 min to accelerate displacement of the CTAB

bilayer from the surface and subsequently incubated overnight at 4 °C. PiBMAD coated

AuNRs were pelleted by centrifugation at 20 °C and resuspended in 1 mL deionized

water. UV-Vis-NIR absorption spectra and zeta potentials were recorded to confirm the

presence of gold (SPR peaks) and polymer respectively.

Subsequently 1 mL PiBMAD-AuNRs were centrifuged once at 20 °C. The pellets

were suspended and concentrated to OD ≈ 2-2.5 in 200 μL pH 6.8 cold encapsulation

buffer (50 mM Tris, 200 mM NaCl and 5 mM MgCl2). CP solution from storage, was

freshly dialyzed to cold pH 6.8 encapsulation buffer, and diluted to a concentration of

6.0 mg/mL. The PiBMAD-AuNRs and CP solution were then mixed in 1:1 volume ratio

and gently stirred overnight at 4 °C. Note that due to the difficult purification of the

nanorods after ligand exchange, there is still free PiBMAD in the nanorod solution,

which is able to induce the formation of spherical VLPs. Hence an excess amount of CP

is applied. After encapsulation CCMV-AuNRs were purified by FPLC. The collected

fractions could be further purified by centrifugation at 6000 g for 10 min and were

analyzed using UV-Vis-NIR and TEM.

Preparation of CCMV-SWCNTs

The dispersion of carbon nanotubes in aqueous media is a challenging process.

Commonly used (ionic) surfactants, stated in decreasing order of dispersion quality,2 are:

sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), sodium dodecylbenzene

sulfonate (SBDS), tetradecyl trimethyl ammounium bromide (TTAB) and sodium

cholate (SC). Dispersion generally occurs during long periods of ultrasonication, when

more and more surfactant molecules are between bundled CNTs, ultimately breaking

them free.3 Dispersion results seemed very inconsistent and the concentration ratio

Chapter 3

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3

between SDS and dispersed CNT seem vary between 1.25 and 2,000.4 In this research,

amide functionalized SWCNT were used, with bundle dimensions of 4-6 x 1000 nm

(commercial specifications). 0.5 mg of black SWCNT powder was added to 1 mL

aqueous SDS (10 mg/mL). The solution was then ultrasonicated in an ice bath for 3 h.

After sonication, the SWCNT solution was centrifuged at 16,000 g for 1 h, precipitating

all undispersed and amorphous carbon. The supernatant, containing SDS-SWCNTs was

collected and the presence of carbon nanotubes was investigated using Raman

spectroscopy and TEM. Then 2 mg/mL PiBMAD solution in pH 7.5 Tris (20 mM, 10

mM Na2S2O3) was prepared and added to aqueous SDS-SWCNT in a 1:1 vol. ratio. The

mixture was then ultrasonicated for 2-3 h and centrifuged at 16,000 g for 30 min to

remove aggregated SWCNT. The supernatant was collected and dialyzed (12-14 kD

MWCO) against two to five volumes of 300 mL pH 6.8 encapsulation buffer (50 mM

Tris, 200 mM NaCl, and 5 mM MgCl2) to remove free polymer. After dialysis,

aggregated carbon nanotubes were sonicated for 30 min for potential redispersion, and

then centrifuged for 30 mins at 16,000 g. Due to insufficient removal of polymer during

dialysis, the solution was washed at least six times using a 100 kD spinl filter with pH

6.8 encapsulation buffer. The carbon nanotubes, mostly sticking to the filter (around

90%), are entered back in solution in aggregated form by thoroughly flushing the filter

with 100 μL encapsulation buffer using a pipette and could be redispersed by 5-20 min

ultrasonication. Then PiBMAD-SWCNTs (100 μL pH 6.8 encapsulation buffer) were

added to 10 μL CP (6 mg/mL, pH 6.8 encapsulation buffer) and gently stirred overnight

at 4 °C. Excess CP was removed by washing at least three times with deionized water

using a 100 kD spin filter.


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Alternative ligands

Besides our designed polymer glue, two alternatives are proposed as templates for

the encapsulation of gold nanorods: polyDOPA (PDA)5,6 and bis(p-

sulfonatophenyl)phenylphosphine (BSPP).7

PolyDOPA, for the growth of PDA films on gold nanorods, 1 mL CTAB-AuNRs

from stock solution was centrifuged and suspended in 2 mL Tris pH 8.5 buffer containing

0.5 mg/mL L-DOPA. The solution was ultrasonicated for 30 min to allow for film

growth, where the thickness depends on the sonication time and concentration of L-

DOPA. After sonication, the PDA-AuNRs could not be precipitated into pellet anymore,

unless the pH of the solution was significantly lower than the pKa of the carboxylic group

(≈ 2.3) of L-DOPA. Centrifugation at pH = 1.8 resulted in pellet which was suspended

into 300 μL pH 6.8 encapsulation buffer (50 mM Tris, 200 mM NaCL, 5 mM MgCl2).

The PDA-AuNRs were then added (1:1 vol.) to 6.0 mg/mL CP (pH 6.8 enc. buffer) and

mildly stirred overnight at 4 °C. Samples were inspected using TEM.

Bis(p-sulfonatophenyl)phenylphosphine (BSPP), the capping of AuNRs by BSPP

was performed according to the method of capping spherical gold nanoparticles (AuNPs)

described by A. Liu et al. Briefly,7 1 mL AuNRs from stock solution was centrifuged

and the pellet was suspended into 2 mL aqueous BSPP (0.5 - 3.0 mg/mL). The solution

was mildly stirred overnight at room temperature. BSPP-AuNRs were then centrifuged

and concentrated in 200 μL pH 6.8 encapsulation buffer (50 mM Tris, 200 mM NaCl, 5

mM MgCl2). A solution of freshly dialyzed CCMV CP into pH 6.8 encapsulation buffer

was then diluted to 6 mg/mL and added to the BSPP-AuNR solution in a 1:1 volume

ratio. The solution was then mildly stirred overnight at 4 °C. CCMV-BSPP-AuNRs were

then purified by size exclusion chromatography (FPLC), the collected gold fractions

were analyzed with TEM and UV-Vis-NIR. The CCMV coated nanorods could be

Chapter 3

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concentrated by centrifugation at 6,000 g for 10 mins at 4°C and resuspension of the

pellet.

Attempts to coat SWCNTs with BSPP and PDA were made. The coating of SWCNTs

with BSPP and PDA were performed nearly identical to the method described previously

for AuNRs. BSPP was added in deionized water instead of tris buffer. For L-DOPA the

following modifications were applied: a slightly elevated pH of 8.5 and the exclusion of

Na2S2O3 to facilitate oxidation and a shorter ultrasonic treatment (1 h instead of 3 h) to

limit film growth.

3.

3.6 References

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H. Schoenherr, W. J. Zhang and X. Jiang, Langmuir, 2014, 30, 1089-1099.

4. M. Malmsten, Curr. Opin. Colloid Interface Sci., 2013, 18, 468-480.

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16. W. Zhang, K. Patel, A. Schexnider, S. Banu and A. D. Radadia, ACS Nano,

2014, 8, 1419-1428.

17. M. Comellas-Aragones, A. de la Escosura, A. J. Dirks, A. van der Ham, A.

Fuste-Cune, J. J. L. M. Cornelissen and R. J. M. Nolte, Biomacromolecules,

2009, 10, 3141-3147.

18. M. V. De Ruiter, N. J. Overeem, G. Singhai and J. J. L. M. Cornelissen, Journal

of Physics Condensed Matter, 2018, 30.

19. L. Schoonen, S. Maassen, R. J. M. Nolte and J. C. M. van Hest,

Biomacromolecules, 2017, 18, 3492-3497.



21. S. Basiruddin, A. Saha, N. Pradhan and N. R. Jana, Langmuir, 2010, 26, 7475-

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E. Richardson and N. G. Tassi, Nat Mater, 2003, 2, 338-342.

53

Chapter 4

Functionalization of Fluorescent

Nanodiamonds with Capsid Protein for

Bioimaging

Functional hybrid nanoparticles are constructed by the coating of

nanodiamonds with virus capsid proteins that show potential capability for

cell imaging.


.

Encapsulation of Fluorescent Nanodiamonds with Capsid Protein for

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4.1 Introduction

Nanodiamonds (NDs) have attracted increasing attention in the past decad due

to their intrinsic optical properties with the conbination of excellent

biocompatibility,1 Nanodiamonds containing nitrogen-vacancy (NV) defect

centers possess excellent physical properties like bright fluorescence with superior

photostability and nonblinking. When excited by a laser, the NV centers within

the nanodiamond emit photons which extend farther into the near-infrared

(wavelength = >800nm) and are capable of being used in vivo imaging ( =700-

900nm),2 making nanodiamonds well suited as fluorescent probes for biological

imaging.

The properties of NDs maninly depend on the defect center and are

independent of their size and shape, therefore NDs have been used for different

applications. More specifically, the NV centers with their electron spin and

magnetic spin resonance, are sensitive to environmental changes, which can be

detected by optical approaches and can be used for quantum sensing. These

intrinsic properties of NDs make them promising nanoscale quantum sensors to

detect magnetic3 and electronic properties,4 temperature,5 pressure and strain.

These defects are also used as fluorescent lables due to their photostability.

To facilitate practical application of fluorescent nanodiamonds in biology,6-10

numerous studies have been done on chemically conjugating functional groups

such as bioactive ligands and stimuli responsive molecules on the surface of the

nanodiamonds.11 Progress has been made in the development of advanced

methods with improved ability of bioconjugation.12, 13 The major concern of these

functionalized nanodiamonds is their stability in physiological medium, such as

phosphate buffered saline (PBS).14 Tzeng et al15 found that bovine serum albumin

(BSA) could be served as an stabilizing agent for nanodiamonds in PBS.

Furthermore, Moscariello et al16 successfully utilized human serum albumin-

based biopolymer (polyethylene glycol) (dcHSA-PEG) coated fluorescent

nanodimaonds (NDs) for blood-brain barrier (BBB) targeting and cell-cell

transportation, which envisioned systemic application of dcHSA-NDs as traceable

transporters for in vivo brain imaging, sensing, and drug delivery.

Chapter 4

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Herein, we managed to coat fluorescent nanodiamonds with the capsid protien

(CP) of the cowpea chlorotic mottle virus (CCMV). Viral hybrid materials have

attracted tremendous attention for applications such as nanoreactors,17

bioimaging,18 biosensing,19 etc. The RNA of CCMV can be removed by

disassembly and the proteincs can be reassembled into virus like particles around

negatively charged templates through (mostly) electrostatic interactions between

the CP and the cargo.20 Furthermore, feasible methods for the surface modification

of CCMV have been developed for various biological modification.18 This chapter

aimed to construct a platform of CCMV coated nanodiamonds (CP-NDs) for

bioimaging and tracking. Therefore, we coated carboxilic acid terminated

nanodiamonds with CP and studied their uptake in living cells.


4.2.1 Nanodiamond encapsulation

As a proof of concept, we first tried to coat NDs in Tris buffer (50 mM Tris, 50 mM

NaCl, 10 mM KCl, pH 7.2) with CP directly. Due to the low density of carboxylic acid

group on the surface of NDs, aggregates were formed while mixing CP together with

NDs as shown in Figure 4.1; dynamic light scattering measurements illustrate that the

size of the NDs increased to approximately 400 nm in diameter after incubation with CP

overnight (Figure 4.1 A). This is further confirmed by transmission electron microscopy

(TEM) images (Figure 4.1 B).

Figure 4.1. Coating of nanodiamonds (NDs) in Tris buffer (50 mM Tris, 50 mM NaCl, 10 mM

KCl, pH 7.2), A: dynamic light scattering measurement of NDs and CP-NDs, B: TEM images of

CCMV coated NDs (CP-NDs).


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Poly(vinylpyrrolidone) (PVP) was introduced to prevent aggregation of NDs

(NDs PVP)) in buffer solution,21 so a similar coating process was carried out to

construct CP coated NDs. Fast protein liquid chromatography (FPLC) was used

to remove uncoated CP from the assembly mixture (Figure 4.2 A). The fraction

eluting at V ≈ 8 mL indicated that NDs were successfully coated with CP both in

PBS buffer and Tris buffer, while NDs with PVP formed aggregates in Tris buffer

at the same salt concentration as CP-NDs. To further prove the coating of CP on

the surface of NDs, sodium dodecyl sulfate–polyacrylamide gel electrophoresis

(SDS-PAGE) was used and confirm that CP monomers are present (Figure 4.2 B).

Figure 4.2. Coating of PVP stabilized nanodiamonds (NDs (PVP)) in Tris buffer (50 mM Tris, 50

mM NaCl, 10 mM KCl, pH 7.2) and 20 mM PBS (150 mM NaCl, pH 7.2), A: FPLC chromatograms

of NDs (PVP) and CP-NDs, B: SDS-PAGE images of CP-NDs, 1, standard protein ladder; 2, CP-NDs

in Tris; 3, CP-NDs in PBS; 4, CP in Tris.

4.2.2 Characterization of capsid protein coated nano-diamonds

To further evaluate CP-NDs from the elution fractions of the FPLC system,

zeta potential and dynamical light scattering (DLS) were used (Figure 4.3). As

shown in Figure 4.3 A, a change from the uncoated NDs to CP-NDs is obvious

(zeta potentials: NDs ( -50 mV), NDs (PVP) (-10 mV), CP-NDs (-15 mV)). To

address the impact of CP and PVP on the size of NDs, DLS was used to measure

the size of uncoated NDs and CP-NDs (Figure 4.3 B). The average hydrodynamic

diameters changed from 35 ± 2 nm before coating to 46 ± 3 nm for NDs (PVP)

and 52 ± 5 nm for CP-NDs respectively. The morphology of the NDs after coating

was characterized by transmission electron microscopy (TEM) (Figure 4.4).

Discrete nanoparticles of CP-NDs were observed indicating the coating and

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stabilization of NDs by CP, negatively stained TEM of CP-NDs (Figure 4.4 A),

furthermore, points to the presence of protein layer on the surface of NDs (Figure

4.4 B).

Figure 4.3. Characterization of coated NDs, A: Zeta potential measurement of uncoated NDs, NDs

(PVP) and CP-NDs, B: DLS evaluation of uncoated NDs, NDs (PVP) and CP-NDs.

Figure 4.4. Characterization of coated NDs, A: TEM images of negatively stained (uranyl acetate)

CP-NDs, B: TEM images of unstained CP-NDs.


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To confirm that the coating of NDs with CP is successful, Atomic Force

Microscopy (AFM) was used to study the deformation of CP-NDs which revealed

the core-shell structure (Figure 4.5). The measurement was conducted in liquid

state with a Bruker Dimension FastScan BioTM atomic force microscope, which

was operated in PeakForce mode. AFM probes with a nominal spring constant of

0.25 Nm-1 (FastScan-D, Bruker) were used. As showed in Figure 4.5A, it was

found that the CP-NDs have pretty good distribution in the height sensor. To better

illustrate the core-shell structure of CP-NDs, the deformation sensor was adapted

due to different softness between CP shell and ND core, it is revealed in figure

4.5B and C, all of CP-NDs showed homogenous shell around core. Moreover, as

illustrated in the insert of figure 4.5C, the high magnification picture, a higher

resolution of core-shell structure is observed.

Figure 4.5. Deformation of coated NDs, A: height AFM images of CP-NDs, B: deformation AFM

images of CP-NDs, C: deformation AFM images of CP-NDs.

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4.2.3 Cell uptake and single nanoparticle tracking

To evaluate the use of CP-NDs for in vivo applications, we coated fluorescent

nanodiamonds (fND) with CP and investigated their uptake by Primary Neuronal cells

and Hela cells with a single nanoparticle tracking system. As shown in Figure 4.6, the

properties were evaluated by fluorescence spectroscopy. Similar emission peaks of CP-

fNDs and fNDs (PVP) were obtained when excited at λ = 560 nm (Figure 4.6 A), which

showes that the coating of fNDs with CP minimally affects the fluorescent properties

(Figure 4.6 B).

Figure 4.6. Fluorescence properties of coated NDs, A: fluorescent emission spectrum of CP-fNDs and

fNDs (PVP), B: fluorescent intensity of CP-fNDs and fNDs (PVP).


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Figure 4.7. Three-dimensional tracking of a single fND in Primary Neuronal cells, A: confocal

images of a live cell after uptake of CP-fNDs in XY dimensions, B: confocal images of a live cell after

uptake of CP-fNDs in XZ dimensions.

Furthermore, CP-fNDs were added to cell culture of both Primary Neuronal cells and

HeLa cells and incubated for 4 h. A single nanoparticle tracking system was applied to

check the uptake.22, 23 For Primary Neuronal cells, no obvious uptake were found (Figure

4.7), CP-fNDs are prone to stick to the cell membrane (Figure 4.7 A) while no signals

was obtained inside the filament of these neuronal cells (Figure 4.7 B). The weak

negative charge of CP-fNDs might prevent uptake by these cells.

On the contrary, a clear uptake of single nanoparticles was observed in HeLa

cells according to the confocal images (Figure 4.8). To further investigate the

movement of nanoparticles inside HeLa cells, confocal images were taken at

different exposure time intervals: 0 s (Figure 4.8 A), 5 mins (Figure 4.8 B), 10

mins (Figure 4.8 C), respectively. It can be found that there are a vast number of

fluorescent ND spots present in the living HeLa cells, which are mainly located

near the cell membrane. The size of fluorescent ND spots is roughly comparable

to endosomes, which suggests that the uptake pathway of CP-fNDs might be a

process of endocytosis. As marked with the red circle, the upper left two

fluorescent spots move away gradually with time and eventually disappear.

Moreover, the intrinsic fluorescent property of NDs, i.e, long fluorescent lifetime,

shows a great potential of CP-NDs as long-term tracking agent. This can be used

to track the motion of nanoparticles at subcellular level. In addition, compared to

the uptake behaviour of CP-fNDs in primary neuronal cells with that of Hela cells,

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no uptake was observed in the former, the reason might be the surface charge of

CP-fNDs.

Figure 4.8. Three-dimensional tracking of a single fND in HeLa cells, A: confocal images of a

live cell after uptake of CP-fNDs in XY dimensions after 0 second’s tracking, B: confocal images of a

live cell after uptake of CP-fNDs after 5 mins’ tracking, C: confocal images of a live cell after uptake

of CP-fNDs in after 10 mins’ tracking.

4.3 Conclusions

We have demonstrated a protein hybrid system of nanodiamonds coated with

virus coat protein. The fluorescent nanodiamonds retain their fluorescent

properties. The CP coated nanodiamonds can be used for cell imaging and in vivo

tracking inside HeLa cells. 3D tracking of single nanoparticles in a live HeLa cell

was carried out and motion of nanodimaonds were observed under confocal

microscopy. This method provides a platform for the functionalization of

nanodiamonds towards advanced hybrid functional materials.

4.4 Acknowledgments

Yingke Wu is gratefully acknowledged for the help of production of nanodiamonds

and fruitful discussion, Dr. Jana Hedrich is gratefully acknowledged for the cell


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experiments. Prof. Dr. Tanja Weil is grateful acknowledged for the fruitful discussion.

Many thanks to Regine van der Hee MSc, Ing. Bianca Ruël and Marcel de Bruine for

their scientific and technical support.


4.5.1 General

All reagent and reactants have been purchased by Sigma Aldrich. Nanodiamonds and

fluorescent nanodiamonds were purchased fromFND BIOTECH Water used for buffer

solutions and reaction process was of MilliQ quality (Millipore, R=18.2 MΩCM-1).

Citric acid stabilized gold nanoparticles have been purchased from NanoComposix (7nm

gold core, 0.05 mg/ml) and were dispersed in MilliQ water. Quartz glass cuvette were

purchased from Hellma-analytics, Quartz SUPRASIL (QS). Diced borofloat wafers

(Schott) and polished silicon wafers (p++) were used for studies on flat substrates.

Amicon Ultra centrifuge filter with 10K MWCO from Pall MicrosepTM Advance

Centrifuge Device with Omega Membrane were used for particle preparation.

4.5.2 Characterizations

Hybrid particle formation

In a typical experiment NDs, NDs (PVP) or fNDs (PVP) solution (400 μL, 0.2

mg/mL; H2O) is added to a solution of CCMV coat protein (100 μL, 15 mg/mL; pH 7.2;

250 mM Tris, 500 mM NaCl) and allowed to incubate overnight at 4 ⁰C. The reaction

mixture is subsequently resulting CP-NDs are purified using preparative FPLC.

FPLC

FPLC size exclusion chromatography samples, ranging from 100 μL up to 500 μL

dialyzed overnight to the coat protein buffer (pH 7.5; 50 mM Tris, 500 mM NaCl). The

UV absorption 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.

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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 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 core.

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 Au NPs with different ligands at pH 7.2 was characterized by

a Zetasizer Nano ZS ZEN3600 instrument (Malvern Instruments) at 25 C̊ with 633 nm

laser.

Single nanoparticle tracking experiment

Thirty thousand of Hela cells or primary neuronal cells were plated in a µ-Slide 8-

well chambered coverslip (ibidi GmbH, Germany) in 300 µL medium. The cells were

incubated overnight to allow adhesion. Then, 3 µL of CP-fND (2 mg/mL) were mixed

with 297 µL cell culture medium to reach media concentrations of CP-fND for 0.02

mg/mL. and then the mixture was added to the cells to replace the old cell culture

medium. The cells were further incubated for 4 hours, before imaging, the cells were

washed with PBS buffer for 3 times. Imaging was then performed using a homemade

confocal laser scanning microscopy, the key hardware of the setup consists of an oil-

immersion (Olympus, 1.35, 60×), a 532 nm continuous-wave laser, a spectrometer

(Princeton Instruments, Acton SP 300i), an avalanche photo-diode (APD, Excelitas,

SPCM-AQRH), optical filter for confocal imaging is a bandpass started from 640nm.

For compatibility with the Ibidi cell-chamber, the recommended Ibidi immersion oil by

Ibidi was used for the objective of the confocal microscope. On the detection arm, a


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motorized flip-mirror was used to conduct fluorescence to the APD or the spectrometer,

for confocal imaging and tracking, or for spectral measurements respectively.

AFM

Atomic force microscopy was conducted in liquid state with a Bruker Dimension

FastScan BioTM atomic force microscope, which was operated in PeakForce mode. AFM

probes with a nominal spring constant of 0.25 Nm-1 (FastScan-D, Bruker) were used.

The samples were diluted with TRIS buffer (5 mM TRIS, 5 mM NaCl, 1 mM EDTA,

12 mM MgCl2) to a concentration of 0.05 mg/mL – 0.1 mg/mL. Sample solution (30 µL)

was added onto a freshly cleaved mica substrate (circular, 15 mm) and incubated for at

least 15 min to allow deposition of the structures. Remaining solution was removed and

300 µL TRIS buffer was applied onto the mica surface, forming a droplet for measuring

in liquid. Samples were scanned with scan rates between 1 and 2 Hz and scan sizes

between 5 and 1 µm. Images were processed with Nano-Scope Analysis 1.8.

Fluorescence analysis

To evaluate the effect of protein coating on the fluorescent property of fNDs,

fluorescent emission scans were performed with λ ex = 560 nm by using a PerkinElmer

LS55 fluorescence spectrometer.

4.6 References

1. Y. Wu, F. Jelezko, M. B. Plenio and T. Weil, Angewandte Chemie International

Edition, 2016, 55, 6586-6598.

2. Y. T. Lim, S. Kim, A. Nakayama, N. E. Stott, M. G. Bawendi and J. V.

Frangioni, Molecular Imaging, 2003, 2, 15353500200302163.

3. G. Waldherr, J. Beck, P. Neumann, R. S. Said, M. Nitsche, M. L. Markham, D.

J. Twitchen, J. Twamley, F. Jelezko and J. Wrachtrup, Nature Nanotechnology,

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4. E. Van Oort and M. Glasbeek, Chemical Physics Letters, 1990, 168, 529-532.

5. V. M. Acosta, E. Bauch, M. P. Ledbetter, A. Waxman, L. S. Bouchard and D.

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6. H.-C. Chang, in Nanodiamonds: Applications in Biology and Nanoscale

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7. J. I. Chao, E. Perevedentseva, C. C. Chang, C. Y. Cheng, K. K. Liu, P. H.

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Boston, MA, 2010, DOI: 10.1007/978-1-4419-0531-4_9, ch. 9, pp. 189-224.

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Boston, MA, 2010, DOI: 10.1007/978-1-4419-0531-4_7, pp. 151-174.

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Y. C. Lee, A. L. Yu and H.-C. Chang, Adv. Funct. Mater., 2013, 23, 5737-5745.

12. Y. Kuo, T.-Y. Hsu, Y.-C. Wu and H.-C. Chang, Biomaterials, 2013, 34, 8352-

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13. A. Krueger and D. Lang, Advanced Functional Materials, 2012, 22, 890-906.

14. J. K. Lim, S. A. Majetich and R. D. Tilton, Langmuir, 2009, 25, 13384-13393.

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Angewandte Chemie International Edition, 2011, 50, 2262-2265.

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67

Chapter 5

Host-guest Interactions Inside Virus-Like

Particles

Host-guest interactions play a vital role in molecular recognition and

transportation. Most of these processes happen in microenvironments such

as organelles, cells or even in nano-compartments like protein cages. To

study these interactions inside biological nano-compartments remains a

challenge. To address this challenge, we report a facile strategy to create

nano-compartments containing specific host or guest molecules. To

increase the understanding of host -guest interactions in confined spaces,

attempts were carried out using 19F-NMR or fluorescence spectrometry.

Furthermore, host-guest interactions were used to induce assembly or

disassembly of Azo-CP VLPs, which provided a new perspective of

constructing responsive protein cages.

Part of this chapter will be submitted.

Host-guest Interaction Inside Virus-like Particles

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5.1 Introduction

Non-covalent interactions provide multiple advantages in constructing functional

materials as they are cost effective and environmentally friendly.1, 2 Interactions such as

electrostatic interactions, π- π stacking, van der Waals forces, and hydrophobic effects

have been widely used by many researchers during the last decades to fabricate

functional materials for biomedical applications.3-5 Host-guest chemistry is an area that

is very well understood both experimentally and theoretically. In particular, a host-guest

system is a chemical system that involves two or more chemical compounds that interact

with each other and ultimately attach to each other non-covalently in a controlled

manner. Often, more than one type of supramolecular interactions is involved in host-

guest systems. A detailed understanding of such systems has been obtained by studying

the binding energies6 and models7, 8 to fully understand the assembly mechanism for

potential applications in producing functional materials.

The development of organic chemistry allows for the construction of a plethora of

desired host molecules, which can specifically interact with guest molecules. Both the

binding constant and affinity in these complexes can be determined experimentally.6

There are few factors that affect their thermodynamics and self-assembly structure, such

as temperature, concentration, chirality and solvents. In Nature, host-guest interactions

play a vital role in molecular recognition and transportation, while most of these

processes happen in microenvironments such as organelles, cells or even in nano-

compartments like protein cages. In this Chapter we study the influence of the local

environment on the binding processes, if this local environment affects the binding

affinity and whether or not the confinement environment interferes with the local binding

structure.

Here we use protein cages as a model system to study host-guest interaction in

confined spaces which also provide a specific microenvironment. Cowpea chlorotic

mottle virus (CCMV) is a widely studied plant virus that infects black eyed peas.9 The

wild type CCMV has an icosahedral T=3 structure sized in diameter around 28 nm. The

genome of CCMV can be removed and the capsid proteins (CP) of CCMV are able to

reassemble into virus-like particles (VLP).10 More interesting is that the nanosized pore

of the CCMV shell allows a variety of guest molecules to diffuse in and out of CCMV

protein cages.

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In this chapter, we choose β-cyclodextrin (β-CD) as a model host molecule since it is

easy to be chemically modified and widely used as a building block for supramolecular

assemblies.11-14 Different methods and guest molecules were used to study their host-

guest interactions inside CCMV VLPs. We first constructed VLPs by using β-CD

modified negatively charged polymer poly(isobutylene-alt-maleic acid) (PiBMA) as a

template. Afterwards, two different guest molecules, i.e. hexafluoro phosphate and

naphthol, were used to study their host-guest interactions inside VLPs,19F-NMR and

fluorescence spectrometry were applied respectively to analyze the different properties

of these two guest molecules. Furthermore, azobenzene (Azo) was covalently attached

to the N-terminus of the CP (Azo-CP) which resulted in the formation of VLPs because

of the hydrophobic or π - π stacking interactions of this azobenzene group. These self-

assembled VLPs can be disassembled by adding host molecules to block the interaction

between aromatic groups. While adding another competitive guest, for example

adamantane (ADA), the disassembled Azo-CP reassembled into VLPs. These results

provide another novel approach to construct VLPs which is also stimuli responsive.


5.2.1 CD-PiBMA modification and encapsulation

To successfully encapsulate β-CD, we first synthesized CD-RhB-PiBMA (fCDP)

(Figure 5.1A) by a stepwise amidation of poly(isobutylene-alt-maleic anhydride) (MW

6 kDa) with lissamineTM rhodamine B ethylenediamine (RhB) followed by 6-

monodeoxy-6-monoamino-β cyclodextrin.15-17 and the subsequent ring-opening of the

unreacted anhydrides with a sodium hydroxide solution.18, 19 The crude material was

dialyzed and freeze dried to obtain the final product. The modification degree of RhB

and β-CD was determined by UV-vis and 1H-NMR, indicating that approximately 5 β-

CD units are attached to each polymer and on average 1.5 RhB units per polymer

backbone.


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Figure 5.1. Schematic illustration of cyclodextrin encapsulated protein cages and the analytical data of

fCDP loaded VLPs (CPfCDP). A: structure illustration of CD-RhB-PiBMA, B: cartoon of CDP encapsulated

VLPs based on electrostatic interaction, C: fast protein liquid chromatography (FPLC) data with UV-vis

detection, where two main fractions eluted, the left one with smaller elution volume was assigned to VLPs,

while the other is unencapsulated CD-RhB-PiBMA, D: dynamic light scattering of CPfCDP, E: UV-vis

spectrum of the collected CPfCDP fraction after FPLC, F: transmission electron microscopy (TEM) image of

CPfCDP.

VLPs were obtained by using fCDP as the negatively charged template (Figure 5.1B),

since the positively charged N-terminus of the CP is the main part of the capsid protein

that interacts with its original genome through electrostatic interactions.20-23 After

overnight incubation, fast protein liquid chromatography (FPLC) analysis revealed

successful formation of VLPs (Figure 5.1C), the first fraction eluted at V=11 mL, this

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fraction exhibit absorption bands at λ=560 nm resulting from the UV absorbance of RhB

together with bands at λ=260 nm and λ=280 nm which are assigned to CP of CCMV.

The size of fCDP templated VLPs was determined by dynamic light scattering (DLS)

(Figure 5.1D), which indicated a diameter of around 22 nm. Further proof was provided

by negatively stained transmission electron microscopy (TEM) (Figure 5.1F), where

spherical particles were found with a diameter of 18 ± 4 nm, indicating particles with a

triangulation number T=1. The UV-vis spectrum is also in line with the FPLC result,

since it shows the fraction contains both capsid protein and cyclodextrin modified

polymer (CD-RhB-PiBMA).

5.2.2 Host-guest analysis via 19F-NMR

Figure 5.2. Schematic representation of the host-guest interaction of PF6 -anion and β-cyclodextrin.


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There are multiple approaches to obtain the binding constant and binding affinity in

supramolecular systems, such as NMR,24-26 fluorescence microscopy,27 isothermal

titration calorimetry,28 UV-vis,29 etc. 19F-NMR spectroscopy is a powerful alternative to

1H-NMR because of its high sensitivity (83% with respect to 1H). Furthermore, 19F-NMR

spectroscopy has its unique potential in structural identification of compounds because

of its much larger dispersion in chemical shifts than 1H, 15N, or 13C nuclei.

Figure 5.3. 19F-NMR spectroscopy analysis of the host-guest interaction of PF6- anion and β-cyclodextrin

in 50 mM Tris buffer (supplemented with 300 mM NaCl, pH 7.2), A: 19F-NMR spectra of NaPF6 and β-CD at

various concentration ratios (total concentration of host and guest compounds: 10 mM), B: Job’s plot data

analysis to determine the complex stoichiometry of the NaPF6- β-CD host-guest interaction, C: 19F-NMR

spectra of NaPF6 with varying β-CD concentration (6, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0 mM from top to bottom),

D: 19F-NMR chemical shift displacement versus c(β-CD)/c(NaPF6) (NaPF6 concentration is 2 mM). the

chemical shift values are downfield peak value of the 19F doublet signals shown in A and C.

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Figure 5.4. 19F-NMR spectroscopy analysis of the host-guest interaction of PF6- anion and fCDP in 50

mM Tris Buffer (supplemented with 300 mM NaCl, pH 7.2), A: 19F-NMR spectra of NaPF6 with varying fCDP

concentration (4.5, 4, 2, 1, 0.5, 0.25, 0.125 mM from top to bottom), B: 19F-NMR chemical shift displacement

versus c(β-CD)/c(NaPF6) (NaPF6 concentration is 0.5 mM). the chemical shift values are upfield peak values

of the 19F doublet signals shown in A.

Here, we used a small fluorine anion, hexafluorophosphate anion (NaPF6), as a

fluorine guest molecule, since they have been studied by calorimetric experiments.30 As

a proof of principle, we investigate the interaction between β-CD and NaPF6 (Figure 5.2).

To gain an insight of NaPF6- β-CD interaction, the stoichiometry of NaPF6- β-CD

complex has been determined by Job’s plot experiment.31, 32 19F-NMR spectra were

obtained for a series of solutions in which the total concentration of the two components

was maintained constant (10 mM) while the ratio (CNaPF6 )/(CNaPF6 + Cβ-cyclodextrin) was

varied between 0 and 1 (Figure 5.3A and B). The maximum at 0.5 in the Job’s plot

indicates that the stoichiometry for the NaPF6- β-CD interaction is 1:1 (Figure 5.3B). To

further investigate the binding of NaPF6-β-CD, the chemical shift of NaPF6 was

measured (Figure 5.3C and D). The fluorine peaks showed an upfield shift with

increasing concentration of β-CD from 0 to 6 mM (the concentration of NaPF6 was kept

as constant at 2 mM). The shift of NaPF6-β-CD is different from NaPF6-α-CD according

to Gómez et al,26 who indicated that different binding structures were formed. To study

the interaction of NaPF6-β-CD complex with β-CD modified polymer, the chemical shift

of NaPF6 was measured (Figure 5.4A and B). Similarly, the fluorine peaks showed an

upfield shift while increasing the concentration of β-CD from 0 to 4.5 mM (the

concentration of NaPF6 was kept as constant at 0.5 mM).


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Figure 5.5. 19F-NMR spectroscopy analysis of the host-guest interaction of NaPF6 (1 mM) and VLPs in

50 mM Tris buffer (supplemented with 300 mM NaCl, pH 7.2), A: 19F-NMR spectra of NaPF6 with varying

CPfCDP concentration (4, 3, 1, 0.5, 0.25, 0 mM from top to bottom), B: 19F-NMR spectra of NaPF6 with

varying CPfP concentration (1.2, 0.9, 0.6, 0.3, 0.15, 0 mM from top to bottom), C: dynamic light scattering of

CPfP, D: fast protein liquid chromatography (FPLC) data with UV-vis detection of CPfP VLPs.

With a better understanding of NaPF6-β-CD complex, further investigation of NaPF6

CPfCDP complex was carried out (Figure 5.5A), by titration of NaPF6 (1 mM) into

CPfCDP in different concentrations of β-CD (4, 3, 1, 0,5, 0.25, 0 mM from top to

bottom). Unexpectedly, a downfield shift rather than an upfield one was obtained, and

the fluorine peaks of NaPF6 became wider with increasing concentrations of CPfCDP.

As a control sample, VLPs without β-CD were prepared in a similar way as discussed in

5.2.1. In short, PiBMA-RhB (fP) was used as negatively charged template to induce the

formation of CPfP VLPs (CPfP), FPLC (Figure 5.5C) was used to analyze and purify the

mixture after overnight incubation, where the fraction at 11 mL elution volume was

collected and analyzed with DLS (Figure 5.5D), which showed that similar sized VLPs

were formed. The titration of NaPF6 (1 mM) into CPfP at different concentrations was

carried out in a similar way as CPfCDP (Figure 5.5 B). The fluorine shift showed a

similar trend as CPfCDP where a downfield shift and wider peaks were found.

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Although it shows that 19F-NMR spectroscopy is a powerful technique to study host-

guest interactions in biological systems, it appears that the host-guest interactions in

CCMV VLPs are more complicated than simple NaPF6-β-CD systems. Since

electrostatic interactions between NaPF6 and capsid protein, as well as hydrophobic

interactions of NaPF6 with the interface of CCMV VLPs shell can compete with the

envisioned host-guest interaction of NaPF6-β-CD we assume that the competition

prohibits a detailed study of the PF6- binding to the encased CDs. For this reason no

binding affinities were calculated and to reach the original goal of this chapter, another

system or technique is required.

5.2.3 Host-guest analysis by fluorescence spectrometry

Figure 5.6. Cartoon of β-CD-PiBMA (CDP) and β-naphthol (β-NP)

Fluorescence spectrometer is a widely used technique to study host-guest interactions

between β-CD and its fluorescence guest molecules.33, 34 Here we used β-naphthol, a

fluorescent colorless crystalline solid with the formula C10H7OH, as a fluorescent guest

molecule to study host-guest interactions inside VLPs. β-naphthol (β-NP) is well known

as starting material to synthesize dyes and drugs in organic chemistry, but it is also


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suspected to be harmful as it would cause different pathological changes when penetrated

into skin.35, 36

Figure 5.7. Fluorescence emission data of β-NP (5 µM) titrated with the different host systems studied. A:

β-CD, B: PiBMA, C: CDP, D: CPP, E: CPCDP, F: CCMV native virus (concentration of D, E and F were

taken as the concentration of capsid protein).

To encapsulate β-CD and avoid overlap of fluorescence signals, β-CD-PiBMA

(CDP) (Figure 5.6) was used instead of fCDP. Both CDP and CPCDP VLPs were

produced in similar ways as fCDP and CPfCDP. Then, fluorescent emission spectra were

recorded by titrating various concentrations of host systems (Figure 5.7) into a β-NP

solution. Figure 5.7A shows the increased emission of β-NP at 360 nm with increasing

concentrations of β-CD, which is in line with literature; it indicates the formation of a

host-guest complex.33 The fluorescence intensity of β-NP decreased due to quenching

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when titrating other complexes or compounds with β-NP (Figure 5.7 B-F). To analyze

the quenching effect and shift of emission when β-NP interacts with different compounds

or systems, the ratio of the fluorescence intensity at = 360 nm and 410 nm were

compared at different concentrations of host system (Figure 5.8A). The results indicate

that there is no binding when mixing β-NP with PiBMA, while all the other systems do

form host-guest complex systems. Interestingly, both CCMV native virus and CPP

interact with β-NP; CCMV showed a similar binding affinity as CPCDP, while CPP has

an even higher affinity to β-NP. Certainly, further investigation needs to be done in order

to understand this β-NP binding behavior. In most cases quenching is caused by the

formation of aggregates or by the change of the physical environment such as pH (the

interaction with protons) or solvents. As for β-NP, researchers have claimed that the

presence of H+ in solution would quench the fluorescence of β-NP.37, 38 We analyzed the

quenching of the β-NP fluorescence in the presence of the different host systems studied

(Figure 5.8A), which showed that CPCDP has the largest quenching effect compared to

the other host systems, CCMV and CDP display similar quenching and neither CD or

PiBMA has any quenching effect on β-NP’s fluorescence. From previous work in our

group we have concluded that the inner pH of VLPs based on CCMV is slightly lower

than bulk solution,39 which might cause the quenched fluorescence. Another hypothesis

is that after binding to β-NP on CDP, smaller sized aggregates might form due to the

hydrophobic interactions of β-NP, while the confined space of CPCDP would again

facilitate this process and eventually influence the fluorescence of β-NP.

To conclude, fluorescence spectrometry was applied to study the host-guest

interactions inside CCMV based VLPs. Unfortunately, due to the complex chemical

environment of the protein cage, we failed to obtain conclusive data to compare the β-

CD- β-NP interaction in bulk solution and in the confinement of a protein cage.


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Figure 5.8. Data analysis of resulted fluorescence spectra from figure 5.7 while the concentrations here

represent β-CD. A: fluorescence intensity ratio (I360/I410) at λ=360 nm and λ=410 nm of different complex, B:

quenching factor (I0/I) of different host systems while mixing with β-NP (here we used the emission at λ=360

nm).

5.2.4 Host-guest interaction induced reversible assembly of

azobenzene modified capsid proteins

In the last two parts, we aimed to construct VLPs with non-covalent host molecules

by encapsulating β-CD conjugated polymers via electrostatic interactions. However, no

conclusive results were obtained by using either 19F-NMR or fluorescence spectrometry.

The interaction between host and guest molecule should be stronger than the electrostatic

or hydrophobic interactions between the guest compound and capsid protein.

Herein, we managed to covalently attach azobenzene (Azo) to the N-terminal part of

the CP, since azobenzene is one of the best characterized photo-swiches and it is well

known to interact with β-CD and form a host-guest complex. Additionally, this host-

guest interaction can be swiched on and off by switching Azo from the trans isomer to

the cis isomer.40, 41 We used sortase A (SrtA) mediated conjugation to attach Azo to the

N-terminus of CP.42-44 SrtA is widely used to functionalize proteins by cleaving between

threonine and glycine at an LPXTG recongnition motif and subsequentely reacting with

an N-terminal glycine. Similar methods have been used to modify the N-termini of CP

with varying functional motifs. In this part, we synthesized C-terminal Azo-ALPETGG

(AzoPep) and subsequently conjugated Azo to CP to obtain Azo-CP. This Azo-CP is

capable of forming VLPs when dialyzed into pH 5 Tris buffer. It stays stable while

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switching pH from 5 to 7.5. Azobenzene conjugated VLPs disassociated after adding β-

CD at neutral pH while showing reversibility by adding another competing guest having

a stronger binding affinity (Figure 5.9).

Figure 5.9. Schematic illustration for the construction and formation of light responsive VLPs.

His-CP and SrtA were expressed in bacteria similar to the method used by Schoonen

et al.44. After production and Ni2+-NTA column purification, FPLC was used for further

purification (Figure 5.10A and B). The chromatogram (Figure 5.10B) shows mainly one

peak at elution volume (V) around V = 18 mL, which corresponds to capsid protein

dimers. For the SrtA chromatogram (Figure 5.10A), one major peak was found around

V = 19 mL, which is expected since the molecular weight of SrtA is around 27 kDa

(smaller than CP dimers, that have a molecular weight of ~40 kDa).


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Figure 5.10. Data analysis of SrtA and SrtA catalyzed Azo-CP conjugation. A: FPLC data of SrtA, B:

FPLC data of His-CP, C: FPLC of Azo-CP formed at pH 5, D: UV-vis absorption of Azo-CP collected from

first fraction obtained by FPLC purification, E: SDS-PAGE of 1- protein standard & ladder, 2-AzoPep, 3-His-

CP, 4-SrtA, 5-reaction mixture, 6-purified Azo-CP, F: TEM image of Azo-VLPs formed at pH 5.

SrtA and His-CP are mixed with AzoPep to conjugate Azo on the N-terminal of His-

CP. After 24 hours incubation, the reaction mixture was dialyzed in pH 5 buffer to obtain

VLPs, after which FPLC was used to remove SrtA and other small compounds (Figure

5.10C) and two peaks were observed. One peak eluted around V = 12 mL, which is in

line with the T=1 VLPs, which is confirmed by TEM where VLPs were visualized

around 18 ± 2 nm in diameter (Figure 5.10F). The second peak around V = 19 mL is

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assigned to SrtA or His-CP. The first fraction was collected and characterized by UV-

vis (Figure 5.10D), which showed around 25% of His-CP was successfully modified

with Azo. The SDS-PAGE result indicated that pure SrtA and His-CP were obtained

after careful purification, and successful removal of SrtA after the reaction (Figure

5.10E).

Figure 5.11. Analysis of the stability and reversibility of Azo-CP, A: DLS of Azo-CP VLPs, B: FPLC of

reassembled Azo-CP VLPs by removing the interaction of CD-Azo through guest competition, C: TEM image

of Azo-CP VLPs at pH 7.5, D: TEM image of reassembled Azo-CP VLPs.

After functionalizing CP with azobenzene, the resulting VLPs were dialyzed into

neutral pH (pH 7.5). DLS showed that VLPs are stable at neutral pH, likely because of

the hydrophobic interactions between the azobenzene moieties within the capsids. To

further prove this the hydrophilic host molecule β-CD-SO3, which is expected to form an

Azo-CD complex, was added to block this hydrophobic interaction. As shown in Figure

5.11A, DLS showed the disassembly of Azo-CP VLPs. Furthermore, another stronger

competing guest molecule with a higher binding affinity (ADA) was added to the

disassembled Azo-CP mixture. The disassembled VLPs reassembled into VLPs

according to DLS data. The resulting mixture was analyzed by FPLC (Figure 5.11B), the


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fraction at elution volume V = 12 mL proved our hypothesis and the UV absorption at λ

= 325 nm (the typical absorption of Azo) illustrated the existence of Azobenzene. To

study the morphology, VLPs at pH 7.5 (Figure 5.11C) and after reassembly (Figure

5.11D) were characterized by TEM and in both images VLPs around 18 nm in diameter

were observed. Future studies are planned to investigate the photo responsiveness of the

obtained materials in detail.

5.3 Conclusions

To obtain a better understanding of host-guest interactions in biologically relevant

nanocompartments, we used CCMV capsids as our model system and cyclodextrin as

our model host molecule. We encapsulated cyclodextrin into CCMV VLPs and two

different host-guest systems were studied by 19F-NMR and fluorescence microscopy.

Due to the complexity of the local environment of CCMV VLPs, both the choice of

methods and guest molecules were constrained. Unfortunately, we were unable to

compare the binding constant in bulk solution with host-guest complex in VLPs.

However, a better understanding of the chemical environment inside CCMV VLPs was

obtained. When studying host-guest interaction in such kind of biological nano-

compartments, the affinity of the host-guest complex should be strong, and the pore size

of VLPs should demonstrate size and charge selectivity. This approach provides a new

way to study the confinement effect and the molecular diffusion in and out of biological

nano-compartments.

Furthermore, Azo-CP VLPs were constructed and are stable at neutral pH. They are

also chemically responsive, demonstrating the capability of reversible assembly by

adding host molecules or guest competitors. This responsive system provides new

possibilities to construct smart carriers for biomedical applications such as biosensors

and controlled drug delivery.

5.4 Acknowledgments

Dr. Alberto Juan Ruiz Del Valle is gratefully acknowledged for the NMR

experiments and fruitful discussion, Dr. Mark de Ruiter is gratefully acknowledged for

the help of production of protein in bacteria lab. Many thanks to Regine van der Hee,

MSc, Ing. Bianca Ruël and Marcel de Bruine for their scientific and technical support.

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5.5.1 General

All reagents and reactants were purchased from Sigma Aldrich and used without

further purification. Water used for buffers and reactions was of MilliQ quality

(Millipore, R=18.2 MΩcm-1). Quartz glass cuvettes were purchased from Hellma-

analytics, Quartz SUPRASIL (QS), for UV-Vis analysis.

5.5.2 Materials preparation

Native CCMV and coat protein isolation

The production and isolation of native CCMV and coat proteins is described in

Chapter 3. The coat protein with a concentration of 18 mg/mL was prepared for further

use.

Synthesis of CDP (β-CD -PiBMA)

To a solution of PiBMA (40 mg, 6.66 μmol) in dry DMSO (8 mL), a solution of 6

monodeoxy-6-monoamino-β-cyclodextrin (121 mg, 0.1067 mmol) and DIPEA (35 μL,

0.213 mmol) in DMSO (2 mL) was added. The mixture was reacted overnight at 80 ˚C.

To the crude reaction mixture, water (10 mL) was added and the unreacted anhydride

rings were opened with NaOHaq (0.26 mL 1 M NaOH). Afterwards, the mixture was

purified by dialysis (SpectraPor membrane, MWCO 6-8 kD) for 1 week. Pure PiBMA-

CD was obtained by freeze-drying (45 mg, 42% yield). The degree of grafting was

calculated from the ratio between the CH3 protons of the polymer and the C1H of β-CD.

1H-NMR (400 MHz, D2O, pH 11): δ 0.25-1.25 (br, 234 H, CH3), 1.25-3.0 (br m, 136 H,

PiBMA backbone), 3.01-4.18 (m, 337 H, C2-6H of CD), 4.88-5.1 (br, 60 H, C1H of CD)

19.

Synthesis of fCDP (β-CD-RhB-PiBMA)

25 mg of PiBMA was dissolved in 5 mL dry DMSO containing 2.5 mg lissamine

RhB ethylene diamine and allowed to react at 45 ⁰C for one day, after which a solution

of 6-monoamino-β-cyclodextrin (60.5 mg) and DIPEA 81 µL in 1.5 mL DMSO was

added. The mixture was allowed to react for 2 days at 60 ⁰C. 10 mL MilliQ with 1.3 mL

NaOH (0.1 M) was added to the reaction mixture to open unreacted anhydride rings.

Subsequently, the mixture was purified through dialysis (Spectra Pro membrane, MWCO

6-8 kDa) for one week, and freeze dried. 1H-NMR was used to characterize the final


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product, the degree of grafting of β-CD was calculated from the ratio between the CH3

protons of the polymer and the C1H of β-CD, which showed around 5 CD were grafted.

The degree of modification with RhB was calculated with a UV-vis calibration curve,

which resulted in 1.5 RhB conjugated per polymer backbone. This result is in line with

literature19.

Synthesis of β-CD-SO3

To modify β-cyclodextrin, per-6-iodo- β-cyclodextrin (β-CD-I) were synthesized as

described in literature11. Briefly, 15 mmol of Ph3P was dissolved into 16 mL dry DMF,16

mmol of I2 was carefully added into this solution over 10 mins and the mixture was

gradually heated up to 50 ⁰C. Then, 1 mmol of dry β-cyclodextrin (β-CD) was added into

the solution, the temperature was heated to 70 ⁰C while stirring under N2 atmosphere for

18 h. Next, the whole reaction was stopped and DMF was removed under reduced

pressure. NaOMe in MeOH was then prepared by adding Na (0.42 g) to MeOH (6 mL)

in an ice bath. The NaOMe was added into the residues and stirred for 30 mins. The

reaction mixture was then precipitated into MeOH and then washed with MeOH 3 times

and dried under vacuum. The crude powder was Soxhlet extracted with MeOH for 20 h

until there was no more discoloration of the solvent. Subsequently, the product was first

air dried and dried under high vacuum which resulted in a white powder. 1H-NMR (400

MHz, CD3SOCD3) was carried out, the results of which were in line with the literature11.

Heptakis-[6-deoxy-6-(2-sulfanylethanesulfonic acid)]- β-CD (β-CD-SO3) was

prepared by reacting β-CD-I with 2-mercaptoethanesulfonate as described in literature7,

in short, 0.2 mmol of CD-I was dissolved into 5 ml DMSO with 3.5 mmol of both

triethylamine (TEA) and 2-mercaptoethanesulfonate, then the reaction mixture was

stirred for 3 days at 60 ⁰C under N2 atmosphere. The reaction was precipitated into

acetone and methanol, filtered and dried under vacuum. 1H-NMR (400 MHz, D2O) was

carried out. The NMR spectrum showed the successful preparation of β-CD-SO3.

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Encapsulation of CDP and fCDP

CDP and fCDP and free coat protein (in 50 mM Tris, 300 mM NaCl, 5 mM MgCl2,

pH 7.2) were mixed in a final weight ratio of polymer to coat protein of 1:1. The mixture

was incubated for at least 8 h at 4 ⁰C. Afterwards, purification was carried out by FPLC

with elution buffer (50 mM Tris, 300 mM NaCl, 5 mM MgCl2, pH 7.2).

Figure 5.12. 1H-NMR spectrum of β-CD-I

Figure 5.13. 1H-NMR spectrum of β-CD-SO3


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SrtA production

E. coli BL21 AI bacteria were transformed with a pQE30 plasmid carrying the Sortase

gene, followed by incubation in LB medium (1 mL) for 1 h at 37 °C. After this short

incubation phase, the cells were transferred into fresh LB medium (4 mL) with ampicillin

(100 mg/L) and were incubated at 37 °C for 4 h. This preculture was then transferred

into LB medium (500 mL) with ampicillin (100 mg/L) and the bacteria were incubated

for 24 h at 37 °C. The bacteria were pelleted and resuspended in lysis buffer (50 mM

NaH2PO4, 300 mM NaCl, 5 mM imidazole and supplemented with 1 mM

phenylmethanesulfonyl fluoride, pH 8.0) and lysed by sonication. The lysate was

centrifuged (14,000 x g, 30 min, 4 °C) and the supernatant was incubated with Ni-NTA

beads for 2 h at 4 °C. Ni-NTA beads were washed with wash buffer (50 mM NaH2PO4,

300 mM NaCl, 10 mM imidazole, pH 8.0) and the purified protein was eluted from the

beads with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, pH 8.0).

For storage the protein was dialyzed against Sortase buffer (50 mM Tris, 150 mM NaCl,

10 mM CaCl2, pH 7.5). The purity was verified by SDS-PAGE.

His-CP production

His-CP is expressed according to the procedure described by Minten et al45. Briefly,

His-CP is expressed in a colony of BL21(DE3) pLysS (E. coli) cells. One colony is used

to inoculate 10 mL of Lysogeny Broth (LB) with ampicillin (0.050 g·L-1) and

chloramphenicol (0.025 g·L-1). After 16 h of growth at 37 °C, the culture is transferred

to 800 mL of LB and grown until an optical density of 0.4-0.6 is reached. Protein

expression is induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final

concentration of 1 mM. After 5 hours expression at 32 °C the bacteria are pelleted by

centrifugation at 10,000 rpm and 4 °C for 15 minutes. The supernatant is discarded, and

the pelleted cells are stored at -20 °C.

The cells are lysed using BugBuster® Protein Extraction Reagent according to the

manufacturers protocol (Novagen). His-CP is purified by incubating the BugBuster

solution with 2 mL of Ni-NTA agarose beads for 1 h at 4 ° C. After settling in a column,

the flow-through is collected and the column is washed with 40 mL of wash buffer (50

mM Tris-HCl, 25 mM imidazole, and 1.5 M sodium chloride, pH 8.0). The capsid protein

is then eluted from the column using approximately 10 mL of elution buffer (50 mM

Tris-HCl, 250 mM imidazole, and 1.5 M sodium chloride, pH 8.0). The outflow was

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collected in small fractions and the presence of protein in these fractions was checked

using UV/Vis spectroscopy. The fractions containing His-CP were dialyzed to cleaning

buffer (50 mM Tris-HCl, 0.5 M sodium chloride, pH 7.5) for 16 hours, to remove the

excess of imidazole. Further purification was performed using SEC, after which the

purified His-CP was concentrated using spin filtration, and dialyzed to capsid storage

buffer (50 mM sodium acetate, 1 M sodium chloride, 1 mM sodium azide, pH 5) and

stored at 4 °C until further use.

AzoPep preparation and purification

ALPETGG was synthesized using automatic Fmoc solid-phase peptide synthesis

(SPPS) (50 mg resin per reactor *5). The resin was swollen in NMP for 60 min prior to

use. 65.61 mg Azo-COOH in 1 mL NMP, HOBT (40.53 mg in 1 mL NMP) and DIPEA

(91 µL) were added to each reactor. After 12 hours stirring at rt, the peptide was cleaved

from the resin by treatment with a mixture of 1,2-ethanedithiol: H2O: TFA (2.5:2.5:95)

for 3 h. The peptide was precipitated in Et2O and separated from the solvent by

centrifugation yielding a yellow solid. The mixture was dissolved in MilliQ and freeze

dried. The crude peptide was purified by reversed phase HPLC. The fractions containing

the peptide were combined and lyophilized yielding a yellow powder. Liquid

Chromatography–mass Spectrometry (LC-MS): m/z for AzoPep (M + H+) calculated

852, found 851.7169.

AZO-CP conjugation and VLPs formation

For a typical SrtA-mediated coupling experiment, stock solutions of SrtA, CCMV

and the LPETG-containing reactant were prepared in Sortase buffer. The components

were added together to final concentrations of 50 µM SrtA and 50 µM CCMV and Azo-

ALPETGG. The solutions were shaken at 21 °C for 24 h. The reaction progress was

followed by SDS-PAGE analysis.

After 24 hours, the reaction mixture was purified using zeba column (MWCO: 7000

kDa), then dialyzed into pH 5 capsid storage buffer (50 mM NaOAc, 500 mM NaCl, 5

mM MgCl2, pH 5) overnight and purified by FPLC.

5.5.3 Characteristics

Nuclear magnetic resonance (NMR)


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19F-NMR titration and proton NMR spectra were recorded using a Bruker 400 MHz

NMR. Polymers or virus-like particles were dissolved in 10% D2O + 90 % H2O at various

concentrations.

Fluorescence analysis

For the fluorescence study of host-gust systems, all the samples were prepared in Tris

buffer, pH 7.2 (300 mM NaCl, 50 mM Tris). To evaluate the effect of host-guest

interactions on the fluorescent property of β-NP, fluorescent emission scans were

performed with λ ex = 220 nm by using a PerkinElmer LS55 fluorescence spectrometer.

5.6 References

1. G. Thangavel, M. W. M. Tan and P. S. Lee, Nano Convergence, 2019, 6.

2. J. L. Mann, A. C. Yu, G. Agmon and E. A. Appel, Biomaterials Science, 2018,

6, 10-37.

3. X. Ma and Y. Zhao, Chemical Reviews, 2015, 115, 7794-7839.

4. M. Valldeperas, A. Salis, J. Barauskas, F. Tiberg, T. Arnebrant, V. Razumas,

M. Monduzzi and T. Nylander, Current Opinion in Colloid and Interface

Science, 2019, 44, 130-142.

5. D. P. Goronzy, M. Ebrahimi, F. Rosei, Arramel, Y. Fang, S. De Feyter, S. L.

Tait, C. Wang, P. H. Beton, A. T. S. Wee, P. S. Weiss and D. F. Perepichka,

ACS Nano, 2018, 12, 7445-7481.

6. F. Biedermann and H. J. Schneider, Chemical Reviews, 2016, 116, 5216-5300.

7. A. Steffen, C. Thiele, S. Tietze, C. Strassnig, A. Kamper, T. Lengauer, G. Wenz

and J. Apostolakis, Chemistry, 2007, 13, 6801-6809.

8. D. J. White, N. J. Reiter, R. A. Sikkink, L. Yu and F. Rusnak, Biochemistry,

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9. L. O. Liepold, J. Revis, M. Allen, L. Oltrogge, M. Young and T. Douglas,

Physical Biology, 2005, 2, S166-S172.

10. L. Lavelle, J.-P. Michel and M. Gingery, J. Virol. Methods, 2007, 146, 311-316.

11. R. K. Peter R. Ashton, and J.Fraser Stoddart*, J. Org. Chem, 1996, 61, 903-

908.

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13. S. Chelli, M. Majdoub, M. Jouini, S. Aeiyach, F. Maurel, K. I. Chane-Ching

and P.-C. Lacaze, Journal of Physical Organic Chemistry, 2007, 20, 30-43.

14. Y. M. Zhang, Y. H. Liu and Y. Liu, Advanced Materials, 2019, DOI:

10.1002/adma.201806158.

15. H. Law, J. M. Benito, J. M. García Fernández, L. Jicsinszky, S. Crouzy and J.

Defaye, The Journal of Physical Chemistry B, 2011, 115, 7524-7532.

16. X. Li, Z. Zhou, W. Zhou, L. Dai and Z. Li, Analyst, 2011, 136, 5017-5024.

17. A. Kulkarni, K. DeFrees, S.-H. Hyun and D. H. Thompson, Journal of the


18. M. Weickenmeier, G. Wenz and J. Huff, Macromolecular Rapid

Communications, 1997, 18, 1117-1123.

19. L. Graña Suárez, W. Verboom and J. Huskens, Chemical Communications,

2014, 50, 7280-7282.

20. M. Comas-Garcia, R. F. Garmann, S. W. Singaram, A. Ben-Shaul, C. M.

Knobler and W. M. Gelbart, J. Phys. Chem. B, 2014, 118, 7510-7519.

21. R. F. Garmann, M. Comas-Garcia, A. Gopal, C. M. Knobler and W. M. Gelbart,

J. Mol. Biol., 2014, 426, 1050-1060.

22. R. F. Garmann, R. Sportsman, C. Beren, V. N. Manoharan, C. M. Knobler and

W. M. Gelbart, J. Am. Chem. Soc., 2015, 137, 7584-7587.

23. M. Verwegen and J. J. L. M. Cornelissen, Macromol. Biosci., 2015, 15, 98-110.

24. M. Scherer, D. L. Caulder, D. W. Johnson and K. N. Raymond, Angewandte

Chemie International Edition, 1999, 38, 1587-1592.

25. M. M. Becker and B. J. Ravoo, Chemical Communications, 2010, 46, 4369-

4371.

26. M. V. Gómez, D. N. Reinhoudt and A. H. Velders, Small, 2008, 4, 1293-1295.

27. D. Granadero, J. Bordello, M. J. Pérez-Alvite, M. Novo and W. Al-Soufi,


28. D.-Z. Sun, L. Li, X.-M. Qiu, F. Liu and B.-L. Yin, International Journal of

Pharmaceutics, 2006, 316, 7-13.

29. R. Fang, Y. Liu, Z. Wang and X. Zhang, Polymer Chemistry, 2013, 4, 900-903.

30. L. A. Godínez, B. G. Schulze-fiehn, S. Patel, C. M. Criss, J. D. Evanseck and

A. E. Kaifer, Supramolecular Chemistry, 1996, 8, 17-22.


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31. E. J. Olson and P. Bühlmann, The Journal of Organic Chemistry, 2011, 76,

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32. C. Y. Huang, in Methods in Enzymology, ed. D. L. Purich, Academic Press,

1982, vol. 87, pp. 509-525.

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Surfaces A: Physicochemical and Engineering Aspects, 2015, 481, 244-251.

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Alvarado and J. C. Menendez, Luminescence, 2005, 20, 162-169.

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Aravindakumar, The Journal of Physical Chemistry A, 2013, 117, 11261-

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Photobiology A: Chemistry, 1993, 71, 115-118.

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802.

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Akira, Chemistry Letters, 2004, 33, 890-891.

42. R. Parthasarathy, S. Subramanian and E. T. Boder, Bioconjugate Chemistry,

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43. D. A. Levary, R. Parthasarathy, E. T. Boder and M. E. Ackerman, PLOS ONE,

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Bioconjugate Chemistry, 2015, 26, 2429-2434.



91

Chapter 6

Controlling the Self-organization of Protein

Cages via Supramolecular Interactions

In nature, protein cages are used as protective shells for organelles,

allowing survival in harsh environments and preventing the degradation of

enzymes. Protein cages are promising candidates for targeted drug

delivery, bio-catalysis as well as constraint polymer synthesis. In this study

we report the use of supramolecular interactions to form higher order

complexes with protein cages derived from the cowpea chlorotic mottle

virus or encapsulins. These high order structures exhibit collective

properties which individual nanoparticles do not possess. Inclusion of the

photolabile o-nitrobenzyl linker allows for the controlled dissociation of

the positively charged groups by UV-irradiation, resulting in the

dissociation of the supramolecular complexes. Such co mplexes are

deposited on a surface and patterns are fabricated with the aid of

lithography. Moreover, metal coordination is used to induce the a ssembly

of encapsulin protein cages and introduces a means to control the assembly

of protein cages.

Part of this chapter will be submitted.

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6.1 Introduction

Organelles are subunits within a cell with specific functions and are often enclosed

within their own lipid bilayers. Some organelle microcompartments however are made

from protein cages and can be found in several organisms, for example bacteria.1 A shell

around an enzyme can protect it from harsh environments and degradation. Protein cages

are ideal building blocks in bio-nanotechnology due to their mono-dispersity combined

with well-defined symmetries, their non-toxicity and their biodegradability.

Additionally, the cargo and the in- and exterior can be modified.2-4 An advantage of

protein cages over synthetic nano-capsules is that the protein capsids can be modified at

precisely known locations by the means of genetic engineering or chemical

modification.5

Hierarchical two- and three-dimensional assemblies have also been of great interest in

the field of protein cages, as these higher order structures are sometimes able to exhibit

collective properties of which individual nanoparticles lack.6-10 General strategies for the

construction of these 2D and 3D-architectures is the use of linkers which can bind through

covalent and non-covalent interactions. Where traditional chemistry typically relies on

covalent bonds, supramolecular chemistry focuses on the weaker, reversible non-

covalent bonds11-15. These non-covalent interactions include hydrogen bonding, π-π

stacking, hydrophobic interactions and electrostatic interactions. The approach using

non-covalent interactions is more promising in the construction of biomaterials for biological

applications since they implicate more versatile parameters, such as flexibility and

geometry, that can be adjusted with relative ease. In this chapter, we report the

construction of the ordered assembly of the CCMV virus and protein cages by

supramolecular interactions.

The CCMV virus was be assembled into higher order supramolecular complexes with

the help of dendrons.7, 16, 17 The dendron acts as a single molecular scaffold containing

multiple positively charged ligands. Inclusion of a photolabile linker results in controlled

release of the covalently attached spermine group. Upon UV-irradiation at a λ = 365 nm,

photoisomerization takes place and the linker is cleaved from the multivalent scaffold,

resulting in individual spermine groups with relatively weak electrostatic interactions. It

has been reported previously that a certain degree of multi-valency is necessary for the

complexation of positively charged dendrons with CCMV to be efficient. These high

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order supramolecular complexes of CCMV and the dendron were deposited onto silicon

wafers and lithography was used in order to create patterns on the surface (Figure 6.1).

In this way, bottom-up and top-down approaches are combined into the fabrication of a

functional surface. This approach should also be valid for the use for other VLPs or

negatively charged particles.

Figure 6.1. Graphical abstract: Supramolecular complexes formed by the co-assembly of dendrons and

protein cages are deposited on a surface and patterned by lithography

Figure 6.2. Graphical abstract: Supramolecular complexes assembled by metal ion-protein cage

coordination.

As well as using electrostatic interactions to construct higher ordered assemblies of

protein cages, we tried to utilize metal ion coordination18-20 to control the organization

of protein cages. We genetically engineered the bacterial nano-compartment encapsulin

from T. maritima (Tm). Two mutations at E64-N65 and E127-K128 of Tm with a 10-

histidine loop were constructed. These two mutant encapsulins are referred to as Tm 64


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and Tm 127, respectively. The mutated Tm protein cages with histidine on the outer face

were used as building blocks to assemble into ordered structures by the addition of

divalent metal ions. Furthermore, these metal-ion coordinated assemblies can be tuned

by a chelating interaction, increasing amounts of imidazole were added to the incubation

buffer (Figure 6.2).27 Imidazole serves as a ligand competing with the protein for the

coordination of nickel.


6.2.1 Controlling the organization of CCMV-dendron complexes on

surfaces

Figure 6. 3. NMR characterization of pll-G1.

The first generation, dendron was synthesized according to the reported literature

procedure21 and the following generations were synthesized by a modified version of the

procedure outlined by Kostiainen et al.16 The NMR spectrum of first generation

Newkome-type dendron (pll-G1) can be found in Figure 6.3. The purity of the compound

is estimated by the ratio of integrals, although the margin of error is significant due to

the overcrowded spectrum. The protons adjacent to the amines were normalized to 30

protons (peaks 1, 3, 4, 7 and 8), which is the expected amount. Peak 23 which

corresponds to the Cbz group has an integral of 4.77 (expected 5H) and peaks 20 & 21

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have an integral of 14.45 (expected 12H), which suggests that the synthesis of the first

generation dendron was successful, albeit with some minor impurities, which is in good

agreement with data found by Kostiainen et al.16

Figure 6. 4. Dynamic light scattering of 40 mg/L CCMV with varying pll-G1 concentrations.

The isoelectric point (pI) of CCMV is approximately 3.7, which is independent of the

ionic strength of the solution. Therefore, if the pH is higher than the isoelectric point, the

exterior of the protein cages will be negatively charged. Thus, in order to assemble these

protein cages into larger aggregates by electrostatic interactions, positively charged

groups are required. In this study, a Newkome-type dendron containing polyamine

groups was synthesized in order to fulfill this function. The structure of the first

generation dendron can be found in Figure 6.3. The polyamine spermine groups

(highlighted in red) are positively charged when the pH is below 8. Attractive

electrostatic interactions between CCMV and pll-G1 are therefore expected in the pH

range of 4-8. CCMV was mixed with different concentrations of pll-G1 to observe co-

assembly, as seen in Figure 6.4. Similar results have been reported previously by

Kostiainen et al., wherein dendron concentrations of 10mg/L and above are sufficient to

complex CCMV efficiently, such that no free CCMV is available in solution17. Increased


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dendron concentrations results in the formation of larger aggregates. Irradiation with

UV-light at = 365 nm dissociates the large supramolecular complexes into free CCMV.

Figure 6. 5. Cross section of substrate made by LbL-assembly.

Initially, two different approaches for the deposition of protein cages were applied.

Layer-by-Layer (LbL) assembly as well as a direct dendron-CCMV complex deposition

method. For direct deposition, CCMV and the dendron are mixed before deposition,

guaranteeing a well-ordered packing. Polyelectrolyte LbL films are constructed by

alternating positively and negatively charged species, more specifically the dendron and

the protein cages. An advantage of LbL assembly is the relatively simple and versatile

method to achieve higher ordered structures and the possibility of the inclusion of various

functionalities into the surface.22, 23 However, as visualized in Figure 6.5, the film

thickness after 12 alternating layers is very small, even within the dimensions of a

monolayer. It is possible that the cation chains of the dendron are pointing down towards

the negatively charged layer which hinders the attachment of the consecutive layer,

which would highlight the major flaw in the use of a dendron opposed to a dendrimer.

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Figure 6. 6. A: AFM image of LbL assembled dendron-CCMV complex, B: AFM image of direct

complex deposition of dendron-CCMV complex, C: SEM image of LbL assembled dendron-CCMV complex,

D: SEM image of direct complex deposition of dendron-CCMV complex.

The manner of deposition clearly influences the packing of the CCMV on the

substrate, as can be seen in the atomic force microscopy (AFM) and scanning electron

microscopy (SEM) images as shown in Figure 6.6. When the dendron and CCMV were

mixed before deposition, one can clearly observe a very condensed packing, whereas

using LbL assembly results in significant gaps in the packing of the film.

A dendron-CCMV complex was deposited by LbL and selectively irradiated with

UV light with a dose of 1840 mJ/cm2 by use of a photomask. The irradiation dose was

chosen as Kostiainen et al. reported a plateau in the molar absorption coefficient

subsequent to this dosage, indicating the full release of the surface groups of pll-G1. The

photomask pattern could faintly be observed using HR-SEM, which can be seen in Figure

6.7. The dimensions of the mask correspond to the lines observed by SEM as the line


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width is 2.5 µm and the line spacing is 5 µm. A control experiment was performed using

a commercially available dendrimer (PAMAM-G2) which does not contain the o-

nitrobenzyl linkers, in order to confirm that the UV exposure did not just damage the

protein structure. No pattern was visible, suggesting that such high UV dose does not

strongly influence the structure of the protein cages. AFM measurements did not show

any sign of the pattern, which suggests that the protein cages remained attached to the

surface after irradiation and a consecutive washing step. A plausible cause could be that

the layer is compressed after contact with the mask and the non-specific adsorption of

the CCMV on the surface prevents the removal of individual protein cages. The pattern

observed in SEM could originate from the change in charge/electronic landscape of the

dendron.

Figure 6. 7. SEM characterization of CCMV layers on substrate after irradiation via a photo maks, using

the cross-linkers; A: photo labile pll-G1, B: photo stable PAMAM G4.

6.2.2 Self-assembly of Encapsulins into supramolecular structures

via metal coordination

Tm 64 and Tm 127 were genetically engineered and expressed in E. coli, with

mutated amino acid sequences at E64-N65 and E127-K128, respectively. Both Tm 64

and Tm 127 are genetically fused with monomeric teal fluorescent protein (mTFP) to

improve their stability and add the option of fluorescent analysis. Purification processes

were monitored by SDS-PAGE (Figure 6.8A and 6.8B), which indicate that Tm 127

(Figure 6.8A) encapsulated a larger quantity of mTFP than Tm 64 (Figure 6.8B). Tm

encapsulins are spherical protein cages, 20-25 nm in diameter. To characterize the

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morphology of Tm 127 and Tm 64, TEM analysis was carried out. The TEM images

reveal proteins cages with diameters of around 22 nm for both the Tm 127 (Figure 6.8C)

and Tm 64 (Figure 6.8D) samples. This is in line with the non-mutated encapsulin

particles which suggests that the mutation of Tm does not change its morphology. Further

analysis was done by fast protein liquid chromatography (FPLC). The chromatogram of

Tm 127 reveals a protein peak at V = 11-13 mL which is characteristic for encapsulin

particles (Figure 6.8E). Chromatogram of Tm 64 is similar to that of Tm 127 (Figure

6.8F). The characteristic absorption of mTFP at λ = 350 nm in the FPLC traces at the

expected elution volume of Tm 127 (Figure 6.8E) and Tm 64 (Figure 6.8 F) which

verifies the presence of mTFP in the Tm protein cages.


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Figure 6.8. Production of Tm 64 and Tm127. L-mixture in lysis buffer, FT-, W1-first wash, W2-second

wash, E1 to E5-eluted fractions. A: SDS-PAGE analysis of Tm 127, B: SDS-PAGE of Tm 64, C: transmission

electron microscopy (TEM) characterization of Tm 127, D: TEM images of Tm 64, E: FPLC chromatogram

of Tm 127, F: FPLC chromatogram of Tm 64.

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Figure 6.9. Metal ion induced assembly of Tm protein cages. A: DLS results of Ni2+ (2 µM) induced

assembly of Tm 64 (20 µM) at various concentrations of imidazole, B: DLS results of Ni2+ (2 µM) induced

assembly of Tm 127 (20 µM) at various concentrations of imidazole.

His-tags are widely used for efficient protein purification when linked to the N- or C-

terminal part of a recombinant protein to allow a high affinity interaction with Ni2+-NTA

resins24. In recent years, His-tag linked proteins have been used as building blocks for

the construction of 2-D or 3-D organized structures.10, 25, 26 To study the assembly,

dynamic light scattering (DLS) was used to monitor the size of Tm 127 and Tm 64 when

adding Ni2+ at different concentrations of imidazole (Figure 6.9). Figure 6.9A illustrates

that when the concentration of imidazole is at 10 mM or 50 mM, Tm 64 assembled into

aggregates larger than the individual protein cages. At 100 mM imidazole, the observed

particles had a diameter around 22 nm, which corresponds to individual TM 64

encapsulin. In the case of Tm 127, large assemblies were found only when the

concentration of imidazole is 10 mM (Figure 6.9 B) at higher imidazole concentrations

only free encapsulin was observed.

To further study the assembly of encapsulin with different metal ions in the presence

of different concentrations of imidazole, a titration of Tm 127 and Tm 64 with Ni2+ and

Co2+ was performed. Figure 6.10 shows that a lower Ni2+ concentration than Co2+ is

needed to assemble 50% of the free Tm 127 and Tm 64 in nonlinear manner (as observed

by the scattering intensity in DLS). Tm64 has a stronger propensity to assembly under

the influence of the metals studied, compared to Tm127.


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Figure 6.10. DLS data for the assembly of Tm encapsulin nanoparticles with Ni2+ and Co2+ in the presence

of 10 mM and 50 mM imidazole. A: titration of metal ions to Tm 127 (5 µM), B: titration of metal ions to Tm

64 (5 µM).

Negative-stained TEM was used to characterize the metal ion induced assembly of

Tm 64 (Figure 6.11) and Tm 127 (Figure 6.12). Figure 6.11 indicates the formation of

large size assemblies of Tm 64 in the range of micrometers when mixed with Ni2+ and

Co2+, even in the presence of 50 mM imidazole. However, no individual proteincage

could be detected. We anticipate that the His10 loop on Tm 64 is facing outward, which

facilitates the coordination of the His-tag to the metal ions and aggregates form under all

studied conditions. On the other hand, the influence of imidazole on the assembly of

Tm127 nanoparticles is significant. According to Figure 6.12, the formation of defined

superstructures is observed without imidazole (Figure 6.12A), while only small clusters

of assembled Tm 127 nanoparticles are observed in the presence of 10 mM imidazole

along with a large number of free particles (Figure 6.12B). Furthermore, no defined

superstructures are observed when the concentration of imidazole was increased to 50

mM ((Figure 6.12C). Our results show that His10 mutated Tm encapsulins can form large

supramolecular assemblies through metal ion coordination.

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Figure 6.11. TEM characterization of the assembly of Tm 64 particles (40 µM) mixed with Ni2+ (20 µM)

and Co2+ (20 µM) in the presence of 0 mM imidazole (A), 10 mM imidazole (B) and 50 mM imidazole (C).

Aggregates were observed, however, no individual protein nanocages could be detected.


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Figure 6.12. TEM characterization for the assembly of Tm 127 particles (60 µM) mixed with Ni2+ (30

µM) and Co2+ (30 µM) in the presence of 0 mM imidazole (A), 10 mM imidazole (B) and 50 mM imidazole

(C).

6.3 Conclusions

We have demonstrated supramolecular interaction induced assembly of protein cages

into higher order structures. A hierarchical assembly of CCMV is enabled by use of a

photocleavable dendron including an o-nitrobenzyl linker that allows for the controlled

dissociation of the positively charged groups by UV-irradiation, resulting in the

dissociation of the supramolecular complexes. The assembled complex of CCMV-

dendron was deposited on a surface by either LbL assembly or direct deposition.

Moreover, the photomask pattern was observed while using lithography to irradiate the

photocleavable dendron-CCMV complex.

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Metal ion coordination was used to induce the ordered assembly of His10 loop

mutated Tm encapsulins, which can be altered via varying the imidazole concentration.

More specifically, two types of mutant were obtained i.e. Tm 64 and Tm 127. Then nickel

and cobalt ions were utilized to interact with the His10 fragment to induce the assembly

of protein nanocages. Furthermore, imidazole was added to compete for the metal ions,

allowing the protein nanocage assembly to be tuned. Compared to the Tm 127, Tm 64

forms lager scale aggregates even at high concentrations of imidazole (50 mM), which

indicates stronger metal ion coordination of Tm 64 than that of Tm127. Interestingly,

lower concentration of nickel ion than cobalt ion is required to induce the assembly of

the mutated encapsulin materials. Our results provide new strategies to construct large

scale assemblies of protein nanocages, paving the way towards controlled assembly of

protein nanocages into superlattices through metal coordination.

6.4 Acknowledgements

We thank Dr. Sandra Michel-Souzy for the production and purification of Tm

encapsulins. We acknowledge Dr. Enrico G. Keim for assistance with TEM imaging and

Dr. Mark Smithers for the assistance with SEM imaging.


6.5.1 Synthesis of pll-G1

(i) Tris (1.21 g) was dissolved in DMSO (2 mL) and MilliQ (0.2 mL) water was

added. The solution was cooled to 15 ◦C under an argon atmosphere. NaOH (0.2 mL)

was added while stirring and 5 ml tert-butylacrylate was added dropwise. The reaction

mixture was stirred for 24h at room temperature. The product was purified using flash

column chromatography with 2:1 EtOAc: hexane with 0.05% v/v NH4OH as the elution

solvent to yield compound 1 (36% yield). The NMR spectra and mass spectra can be

found in Appendix S6.1 and S6.2.

(ii) Compound 1 (0.5 g) was dissolved in CH2Cl2 (7.3 mL) and 25% aqueous

Na2CO3 were added while stirring. Benzylchloroformate (0.44 mL) was added and the

reaction was stirred for 24h at room temperature. The aqueous layer was removed and

the CH2Cl2 dried over MgSO4. The compound was purified by flash column

chromatography using 5:1 heptane: EtOAc as the elution solvent to yield compound 2

(58% yield). NMR spectra and mass spectra can be found in Appendix S6.3 and S6.4.


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(iii) Compound 2 was dissolved in 96% formic acid (5 mL) and stirred for 18 hours

at room temperature. The solvent was removed by under reduced pressure to yield the

G1 precursor (92% yield). NMR spectra and mass spectra can be found in Appendix S6.5

and S6.6.

(i) BOCspermine (200 mg) and pll (Photolabile linker, 100 mg) were dissolved in

THF (5 mL). DCC (83 mg), HOBt (61 mg) and Et3N (41 ug) were added and the mixture

was left to stir for 24h at room temperature. The dicyclohexylurea precipitate was

removed by filtration and the crude product was purified by column chromatography

using 9:1 DCM:MeOH as the elution solvent to yield pll-BOCspermine (67 % yield)

(ii) pll-BOCspermine (145 mg), G1 precursor (23 mg), DCC (30 mg), HOBt (3

mg) and DMAP (2 mg) were dissolved in dry DCM (4 mL) under and argon atmosphere

and cooled on ice, after which pyridine (50 µL) was added to the mixture. The mixture

was stirred at 0 ◦C for 10 minutes then allowed to reach room temperature. The mixture

was heated to 65 ◦C and refluxed for 24 hours. The dicyclohexylurea precipitate was

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removed by filtration and the crude product was purified by flash column

chromatography using a 9:1 DCM:MeOH elution solvent (38% yield).

(iii) pll-G1-BOCspermine was dissolved in DCM. HCl gas was bubbled through the

solution. The reaction was stirred for ca. 45 minutes. The solvent was removed to yield

pll-G1.


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6.5.2 Deposition of protein cages on surface

Prior to the deposition, each substrate was activated by either oxygen plasma (5 min,

40 mA) or UV Ozone (> 30 min). Layer-by-Layer assembly was done immersion of the

substrate in a solution of CCMV (100 mg/L in for 5 min, rinsing with Milli-Q water,

immersion of the substrate in a solution of pll-G1 dendron (100 mg/L, in pH = 5, 10 mM

NaCl) for 5 min then rinsing with Milli-Q water.


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For direct deposition, a solution of pll-G1 (40 mg/L in pH = 5, 10 mM NaCl) was

mixed in a ratio with CCMV (50 mg/L in pH = 5, 10 mM NaCl). The substrate was

immersed overnight in the solution then rinsed with Milli-Q water before drying.

6.5.3 Production of Encapsulin

Competent cells of strain Rosetta were transformed with pCDFDuet-tmenc127-H-

128 or pCDFDuet-tmenc64-H-65 and pETDuet-mTFPEflp. The bacteria were grown

until 0,5 ODU (Optical Density Units) at 600nm at 37°C on LB medium with

Streptomycin (Sm) (30 µg/ml) and Ampicillin (Ap) (50 µg/ml). The induction was

performed with 1mM of IPTG during 12h at 25°C. Bacteria were collected by

centrifugation and broken by sonication (2 x 1min) in cold buffer HEPES pH8 50mM,

NaCl 150mM, EDTA 1mM, MgCl2 20mM, 1 protease inhibitor tablet/7mL

(cOmpleteTM), Lysozyme 0,5mg/mL, DNAse 20µg/ml, Imidazole 30mM, Beta-

mercaptoethanol (βme) 15mM. The lysate was cleared by ultracentrifugation (20000 x

g) to remove unbroken debris and membranes. The cleared lysate containing Tm127H-

mTFP or Tm64H-mTFP were loaded onto 5-ml Ni-NTA agarose beads (Protino® Ni-

NTA Agarose) using Biorad gravity column and the immobilized proteins were washed

in washing buffer (50mM HEPES pH 8.0, 150mM NaCl, βme 15mM, 30mM imidazole)

and eluted in elution buffer (50mM HEPES pH 8.0, 150mM NaCl, βme 15mM, 500mM

imidazole). Then the imidazole was removed using PD10 Desalting column (GE

Healthcare) with the buffer 50mM HEPES pH 8.0, 150mM NaCl, βme 15mM.

6.5.4 General methods

Lithography

Irradiation with UV light was done with the EVG 620. Hg light bulb intensity was

set at 12 mW/cm2 and a constant dose of 1840 mJ/cm2. A mask with lines of width of

2.5 µm and a spacing of 5 µm was used. Alignment of the mask with the substrate was

done in soft contact mode.

HR-Scanning Electron Microscopy

Zeiss MERLIN HR-SEM. Acceleration voltages of 0.8-1.2 kV were used. Samples

were stained with a 1 wt% uranyl acetate solution in MQ Excess stain was removed with

a filter paper after 45 seconds and the sample was dried with nitrogen gas.

Atomic Force Microscopy

AFM Bruker Fast Scan in ScanAsyst mode, with PeakForce Tapping mode. Tip

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diameters are approximately 4 nm.

Dynamic Light Scattering

Anaspec nanotrac wave dynamic light scattering instrument was used. Each sample

was measured 3-5 times. A refractive index of 1.54 is used for spherical VLPs in

combination with the viscosity of water.

6.6 References

1. C. P. Satori, M. M. Henderson, E. A. Krautkramer, V. Kostal, M. M. Distefano

and E. A. Arriaga, Chemical Reviews, 2013, 113, 2733-2811.

2. J. Lucon, S. Qazi, M. Uchida, G. J. Bedwell, B. LaFrance, P. E. Prevelige and

T. Douglas, Nature Chemistry, 2012, 4, 781-788.

3. G. Destito, R. Yeh, C. S. Rae, M. G. Finn and M. Manchester, Chemistry &

Biology, 2007, 14, 1152-1162.

4. Y. Ma, R. J. M. Nolte and J. J. L. M. Cornelissen, Advanced Drug Delivery

Reviews, 2012, 64, 811-825.

5. S. D. Brown, J. D. Fiedler and M. G. Finn, Biochemistry, 2009, 48, 11155-

11157.

6. Y. Bai, Q. Luo, W. Zhang, L. Miao, J. Xu, H. Li and J. Liu, J. Am. Chem. Soc.,

2013, 135, 10966-10969.

7. S. Välimäki, J. Mikkilä, V. Liljeström, H. Rosilo, A. Ora and M. A. Kostiainen,


8. N. K. Beyeh, Nonappa, V. Liljeström, J. Mikkilä, A. Korpi, D. Bochicchio, G.

M. Pavan, O. Ikkala, R. H. A. Ras and M. A. Kostiainen, ACS Nano, 2018, 12,

8029-8036.

9. A. Korpi, E. Anaya-Plaza, S. Välimäki and M. Kostiainen, Wiley

Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 2019, 0,

e1578.

10. J. Zhang, K. Zhou, Y. Zhang, M. Du and Q. Wang, Advanced Materials, 2019,

31, 1901485.

11. S. Zhang, Nature Biotechnology, 2003, 21, 1171-1178.

12. G. V. Oshovsky, D. N. Reinhoudt and W. Verboom, Angewandte Chemie

International Edition, 2007, 46, 2366-2393.


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13. M. A. Kostiainen, C. Pietsch, R. Hoogenboom, R. J. M. Nolte and J. J. L. M.

Cornelissen, Adv. Funct. Mater., 2011, 21, 2012-2019.

14. V. Liljeström, J. Seitsonen and M. A. Kostiainen, ACS Nano, 2015, 9, 11278-

11285.

15. X. Ma and Y. Zhao, Chemical Reviews, 2015, 115, 7794-7839.

16. M. A. Kostiainen, O. Kasyutich, J. J. L. M. Cornelissen and R. J. M. Nolte,

Nature Chemistry, 2010, 2, 394-399.

17. M. A. Kostiainen, P. Ceci, M. Fornara, P. Hiekkataipale, O. Kasyutich, R. J. M.

Nolte, J. J. L. M. Cornelissen, R. D. Desautels and J. Van Lierop, ACS Nano,

2011, 5, 6394-6402.



19. T. Tosha, H. L. Ng, O. Bhattasali, T. Alber and E. C. Theil, Journal of the


20. P. A. Sontz, J. B. Bailey, S. Ahn and F. A. Tezcan, Journal of the American


21. C. M. Cardona and R. E. Gawley, The Journal of Organic Chemistry, 2002, 67,

1411-1413.

22. N. F. Steinmetz, K. C. Findlay, T. R. Noel, R. Parker, G. P. Lomonossoff and

D. J. Evans, ChemBioChem, 2008, 9, 1662-1670.

23. P. A. Suci, M. T. Klem, F. T. Arce, T. Douglas and M. Young, Langmuir, 2006,

22, 8891-8896.

24. J. Schmitt, H. Hess and H. G. Stunnenberg, Molecular Biology Reports, 1993,

18, 223-230.

25. S. P. Qiao, C. Lang, R. D. Wang, X. M. Li, T. F. Yan, T. Z. Pan, L. L. Zhao, X.

T. Fan, X. Zhang, C. X. Hou, Q. Luo, J. Y. Xu and J. Q. Liu, Nanoscale, 2016,

8, 333-341.

26. H. Nakashima, K. Furukawa, Y. Kashimura, K. Sumitomo, Y. Shinozaki and

K. Torimitsu, Langmuir, 2010, 26, 12716-12721.

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6.7 Appendix

Figure S6.1. NMR Spectrum of Compound 1.

Figure S6.2. Mass Spectrum of Compound 1, C25H49O9 (505.64) MS : 506 m/z.


Interactions

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6

Figure S6.3. NMR Spectrum of Compound 2 NMR ratios : 27 (p1), 6 (p2), 12 (p3+4), 2 (p5) and 5 (p6).

Figure S6.4. Mass Spectrum of Compound 2, C33H53NO11 (639.4) MS : 640 m/z, 662 (+ Na).

Figure S6.5. NMR Spectrum of G1 Precursor NMR ratios : 6 (p1), 12 (p2+3), 2 (p4) and 5 (p5).

Chapter 6

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Figure S6.5. Mass Spectrum of G1 Precursor C21H29NO11 (471.5) MS : 472 m/z, 494 (+ Na).

116

117

Chapter 7

Perspectives of Protein-cage based

Supramolecular Functional Materials

In this chapter, we aim to reflect on what have been achieved and the

challenges that remain for the construction of protein-cage based

supramolecular functional materials. Furthermore, future research

directions based on this thesis are discussed.

Perspectives of Protein-cage based Supramolecular Functional Materials

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7.1 Introduction

Protein-cage based supramolecular functional materials have attracted significant

attention due to the intrinsic uniformed size, feasibility of functionalization and

biocompatibility.1-6 The Cowpea Chlorotic Mottle Virus (CCMV), for example, has been

utilized for drug and gene delivery,7, 8 construction of inorganic/protein hybrid

materials,9, 10 nanoreactors,11 etc. Functional materials based on CCMV protein cages are

constructed by electrostatic interaction of the positively charged N-terminal capsid

proteins and the negatively charged cargoes, however, there are limitations for the

encapsulation of functional cargos, such as charge density, size, shape and stability. To

tackle these limitations, researchers have made efforts to understand the assembly

pathway and mechanism of CCMV,12-16 which provided vital information on the design

principles for the fabrication of CCMV-based functional materials. Apart from CCMV,

bacteria produced protein cages, such as T. maritima (TM) encapsulin, have

demonstrated their great potential for the construction of functional materials due to their

thermo stability and availability for genetical modification.17 Here, we reflect on the

preparation and applications of protein cage based supramolecular functional materials

that are composed of complex biological moieties such as CCMV and TM encapsulin.

7.2 Achievement and challenges

In the work described in this thesis, we made an attempt to elaborate on the

application of CCMV from building blocks for functional materials to models for the

understanding of host-guest interaction in confined space. More specifically, we faced

with the challenge of coating non-spherical inorganic functional materials such as gold

nanorods (GNRs), single walled carbon nanotubes (SWCNTs) and fluorescent

nanodiamonds (fNDs) with capsid proteins (CP) of CCMV, a generic template was

developed and used to fabricate CCMV-GNRs and CCMV-SWCNTs virus like particles.

Additionally, fNDs were successfully coated with CP for the first time and applied to

cell imaging and single nanoparticle tracking in living cells. At the same time, we were

faced with new challenges. One of the major concerns when preparing these non-

conventional hybrid functional materials was the purification efficiency. Particularly, for

the purification of CCMV-SWCNTs, multiple washing steps were carried out after

ligand exchange and coating of CP to remove free polymers and free CP, which resulted

Chapter 7

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in a rather low yield of coated CCMV-SWCNTs. Furthermore, more studies need to be

carried out to understand the arrangement of CP that is coated on the surface of these

inorganic nanomaterials as well as their stabilities in biological environments such as

cell culture medium.

Other than the construction of protein-cage based functional materials, attempts to

understand host-guest interactions inside virus-like particles (VLPs) was carried out,

multiple systems and methods were utilized. Due to the complexity of the interior

chemical-physical environment of VLPs, no conclusive results were obtained, albeit only

cyclodextrin based host-guest systems were studied. For further study, a better

methodology and system should be applied to overcome the complex chemical

environment of VLPs, furthermore, the system should be kept simple to allow

comparison with computational models.

Additionally, both CCMV and TM encapsulin were used as building blocks for long-

range ordered assemblies of 2D and 3D functional materials. Techniques such as

lithography were employed to create pattern by photo cleavable CCMV-dendron

complexes. Nonspecific interaction appears to be one of the obstacles when ordered

CCMV-dendron complexes are deposited on a substrate. Ordered assembly of TM

encapsulin was achieved through metal ion coordination. Weak supramolecular

interactions are the key to control assembly of protein cages for hierarchically ordered

structures, which require more study to understand design parameters, the extent of

supramolecular interactions between building blocks and the optimal conditions such as

concentration, buffer, temperature, etc.

Overall, we made progress in the development of supramolecular functional materials

based on CCMV and TM encapsulin. Stimuli-responsive, higher ordered assemblies are

formed via incorporation of photo-responsive dendrons, metal ion coordination

interactions. The presented research provides a new approach to fabricate protein based

functional materials and supports novel strategies towards the development of

biocompatible and smart materials.


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7.3 Outlook

In this thesis we have developed and constructed capsid protein coated gold nanorods,

carbon nanotubes and nanodiamonds as biohybrid nanomaterials for biomedical

applications. We envision that similar strategies could be extended to systems with other

non-spherical inorganic nanomaterials such as nano-oxides, nanocomposite oxides and

nanometallic alloys. These capsid protein coated hybrid nanomaterials would ultimately

provide new insights and perspectives for biomaterials. Nevertheless, there are still some

challenges left to overcome, such as the stabilities of capsid proteins, the limited

strategies applicable for the coating of the nanomaterials with these proteins and

incorporation of stimuli responsive functionalities. If we use other supramolecular

interactions such as host-guest interaction, metal ion coordination as new methods to

construct capsid protein-inorganic biohybrid functional materials, we could overcome

these challenges. Chemically or genetically modified capsid protein with azobenzene

(Azo-CP), for example, can be used to prepare capsid protein coated nanomaterials based

on host guest interactions between the azobenzene and cyclodextrins. Additionally, this

system is intrinsically dynamic and can rearrange, since the host-guest interaction is

reversible and responds to the addition of chemicals or irradiation with light. Similarly,

other stimuli responsive guest molecules can be conjugated to capsid proteins, which

opens the door for enhanced control over the molecular and assembly, towards the

construction of protein-(in)organic biohybrid nanomaterials.

Furthermore, the great potential of using protein nanocages as building blocks to

fabricate ordered 2D or 3D functional materials has been explored in the last decade.

Through optimization and investigation of the details in the electrostatically driven

assembly of synthetic polymers and protein nanocages, protein based superlattices can

be designed and fabricated that have the potential for biomedical applications. On the

other hand, with the combination of microbiology and supramolecular chemistry,

genetically engineered protein cages can be used as a multivalent platform to study

multivalent interactions with biomimetic system such as vesicles, supported lipid

bilayers and nanoparticles. Taking Tm encapsulin as an example, a Histidine loop can

be incorporated to study multivalent interactions between Tm encapsulin and Nickel-

NTA (Nitrilotriacetic acid) functionalized supported lipid bilayers, these systems might

be responsive to external stimuli such as chemicals and pH. This will enormously expand

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our knowledge about selectivity and dynamics of biologic systems. Furthermore,

investigation of the interactions between histidine mutated Tm encapsulins and Nickel-

NTA vesicles as may yield improved insight into nanoparticle-cell interactions and

communication. In addition, the multivalent interactions of protein nanocages can be

used as a driving force for fabricating stimuli responsive hydrogels. Simple networks can

be constructed and used as model systems to develop artificial smart system that could

read and process signals in local environment.

7.4 References





2019, DOI: 10.1038/s41586-019-1185-4.

2. T. L. Li, Z. Wang, H. You, Q. Ong, V. J. Varanasi, M. Dong, B. Lu, S. P. Paşca

and B. Cui, Nano Letters, 2019, 19, 6955-6963.



2362.

4. B. S. Sandanaraj, M. M. Reddy, P. J. Bhandari, S. Kumar and V. K. Aswal,

Chemistry - A European Journal, 2018, 24, 16085-16096.

5. M. V. De Ruiter, N. J. Overeem, G. Singhai and J. J. L. M. Cornelissen, Journal

of Physics Condensed Matter, 2018, 30.

6. M. B. Van Eldijk, L. Schoonen, J. J. L. M. Cornelissen, R. J. M. Nolte and J. C.

M. Van Hest, Small, 2016, 12, 2476-2483.

7. I. Barwal, R. Kumar, S. Kateriya, A. K. Dinda and S. C. Yadav, Sci. Rep., 2016,

6, 37096.

8. P. Lam and N. F. Steinmetz, Biomater. Sci., 2019, 7, 3138-3142.




Cornelissen, Adv. Funct. Mater., 2018, 28, n/a.


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3084-3091.

12. Y. Miao, J. E. Johnson and P. J. Ortoleva, J. Phys. Chem. B, 2010, 114, 11181-

11195.

13. C. Globisch, V. Krishnamani, M. Deserno and C. Peter, PLoS One, 2013, 8,

e60582.

14. M. Comas-Garcia, R. D. Cadena-Nava, A. L. N. Rao, C. M. Knobler and W. M.

Gelbart, J. Virol., 2012, 86, 12271-12282.

15. M. Comas-Garcia, R. F. Garmann, S. W. Singaram, A. Ben-Shaul, C. M.

Knobler and W. M. Gelbart, J. Phys. Chem. B, 2014, 118, 7510-7519.

16. S. J. Maassen, M. V. de Ruiter, S. Lindhoud and J. J. L. M. Cornelissen, Chem.

- Eur. J., 2018, 24, 7456-7463.

17. L. Schoonen, R. J. M. Nolte and J. C. M. van Hest, Nanoscale, 2016, 8, 14467-

14472.

123

Summary

In Nature, supramolecular interactions can be found everywhere, for example,

protein-protein and protein-DNA interactions. These noncovalent interactions are

reversible, stimuli responsive and dynamic. Viruses are a perfect example of natural

assemblies via supramolecular interactions; viruses envelop their genome inside protein

cages via both electrostatic interactions and hydrophobic interactions. Furthermore,

viruses are uniform sized building blocks for the development of new advanced

functional materials. More specifically, capsids of viruses can be produced by removing

their genome in a disassembly step and thereafter reassemble into virus-like particles

either by controlling salt concentration or introducing foreign cargos. The focus of the

research reported in this thesis is to design and synthesize materials based on the protein

of the Cowpea Chlorotic Mottle Virus, with the aim to control the assembly behavior by

combining aspects from polymer and supramolecular chemistry. Furthermore, we

studied the encasement of functional materials with these proteins in order to modify

their properties, in particular the biocompatibility. Finally, the 3D arrangement of protein

cages by non-covalent interactions is investigated.

The work described in this thesis combined supramolecular chemistry and physical

virology. Chapter 2 provides an overview of the relevant literature. A generic bio-

inspired molecular glue (PiBMAD) to facilitate encapsulation of non-spherical inorganic

materials into CCMV protein cages was developed (Chapter 3). Both gold nanorods and

single-walled carbon nanotubes were successfully coated with capsid protein by using

PiBMAD. Their ease of preparation and versatility makes PiBMAD a promising

interface molecular glue for construction of viral protein-based hybrid nanomaterials. By

taking advantage of the functional groups on the interior surface of capsids, the coating

of nano-diamonds with capsid protein through electrostatic interactions was achieved,

stabilizing these nano-diamonds in solution while keeping their intrinsic physical

properties for biological application (Chapter 4). Chapter 4 illustrates that the potential

of coating various inorganic nanomaterials with capsid protein goes far beyond spherical

nanomaterials.

Summary

124

To better understand the host-guest interactions in a confined space, cyclodextrin

modified polymers were encapsulated inside CCMV, the host-guest interactions were

studied via 19F-NMR and fluorescence spectrometry, which showed the complexity of

the CCMV system. More specifically, weak interactions between capsid proteins and

guest molecules prevented getting better insight into the host-guest interactions inside

CCMV VLPs. Nevertheless, after the chemical modification of the N-terminal of the

capsid protein with azobenzene via Sortase-A enzyme, Azo-CP self-assembled into

virus-like particles (VLPs) through hydrophobic interactions at neutral pH. The

assembled VLPs are reversible by adding different chemical reagents and modification

of CP provides a new strategy to study host-guest interactions in the cavity of CCMV

VLPs (Chapter 5).

Furthermore, supramolecular interactions were utilized to fabricate 2D and 3D

assemblies with protein cages. Using both CCMV and Thermotoga maritima (Tm)

encapsulin as compartmentalized nanocarriers, stimuli responsive assembly was

obtained through the supramolecular interactions of CCMV and an optically responsive

dendron. The photomask pattern was observed while using lithography to irradiate the

photocleavable dendron-CCMV complex. Metal ion coordination was used to induce

assembly of His10 loop mutated Tm encapsulin, which can be altered by varying the

imidazole concentration (Chapter 6).

Overall, there is great potential for the application of viruses in various fields,

however, because of the intrinsic properties of capsid proteins, further functionalization

should be carried out. The study presented in this thesis highlights the enormous

possibilities of using supramolecular interactions to obtain different dimensional

biomaterials for various applications, and more knowledge on the structure and

interactions between each capsid protein was obtained.

125

Samenvatting

In de natuur zijn supramoleculaire interacties, zoals eiwit-eiwit en eiwit-DNA

interacties, alom vertegenwoordigd. Deze niet-covalente interacties zijn omkeerbaar,

beïnvloedbaar door stimuli en dynamisch. Virussen zijn een perfect voorbeeld van

natuurlijke assemblages die worden gevormd via supramoleculaire interacties; virussen

omvatten hun genetische materiaal in eiwitkooien via zowel elektrostatische als

hydrofobe interacties. Virussen zijn bovendien bouwstenen van uniforme grootte voor

de ontwikkeling van nieuwe, geavanceerde functionele materialen. Capsiden van

virussen kunnen verkregen worden door het genetische materiaal tijdens een opbreek-

fase te verwijderen en naderhand kan dit opnieuw tot een virus-achtig deeltje herbouwd

worden door de zoutconcentratie aan te passen of nieuwe lading te introduceren. De

focus van het in deze thesis beschreven onderzoek is het ontwerpen en maken van

materialen gebaseerd op de eiwitten van de Cowpea Chlorotic Mottle Virus (CCMV),

met als doel om de constructie te controleren door aspecten van polymeer en

supramoleculaire chemie te combineren. We bestuderen de encapsulering van

functionele materialen met deze eiwitten om vervolgens hun eigenschappen, met name

biocompatibiliteit, aan te passen. Ook wordt de 3D organisatie van de eiwitkooien door

niet-covalente interacties onderzocht.

Het werk dat is beschreven in deze thesis combineert supramoleculaire chemie en

fysische virologie. In Hoofdstuk 2 staat een overzicht van de relevante literatuur. Een

generieke, bio-geïnspireerde, moleculaire lijm (PiBMAD) was ontwikkeld om de

omhulling van niet-bolvormige, anorganische materialen in CCMV-eiwitkooien te

faciliteren (Hoofdstuk 3). Zowel gouden nanostaafjes als koolstof nanobuisjes zijn

succesvol gecoat met capside eiwitten door het gebruik van PiBMAD. De eenvoudige

bereiding en het brede toepassingsveld maken PiBMAD een veelbelovende moleculaire

lijm voor het maken van virale, eiwit-gebaseerde, hybride nanomaterialen. Door gebruik

te maken van de functionele groepen aan de binnenkant van de capsiden kon het coaten

van nanodiamanten met capside eiwitten worden bewerkstelligd middels elektrostatische

interacties, waardoor deze nanodiamanten gestabiliseerd werden in oplossing terwijl hun

intrinsieke, fysische eigenschappen voor biologische toepassingen behouden bleef

Samenvatting

126

(Hoofdstuk 4). Hoofdstuk 4 laat zien dat de potentie om verschillende anorganische

nanomaterialen te coaten met capside eiwitten verder reikt dan alleen bolvormige

nanomaterialen.

Om meer inzicht te krijgen in host-gast interacties in een besloten ruimte werden

polymeren met cyclodextrines gemodificeerd en deze werden omhuld in CCMV. De

host-gast interacties werden bestudeerd met 19F-NMR en fluorescentie spectrometrie,

hetgeen de complexiteit van het CCMVsysteem aantoonde. Zwakke interacties tussen de

capside eiwitten en de gast-moleculen verhinderden verdere diepgaande analyse van de

host-gast interacties in CCMV. Na de chemische modificatie van de N-terminus van de

capside eiwitten met azobenzeen middels het Sortase A enzym, vormde Azo-CP

virusachtige nanodeeltjes (VLPs) via hydrofobe interacties bij neutrale pH. Deze VLPs

zijn reversibel door verschillende reagentia toe te voegen en de modificatie van capside

eiwitten vormt een strategie om de host-gast interacties in de holte van CCMV VLPs te

bestuderen (Hoofdstuk 5).

Supramoleculaire interacties werden ook gebruikt om 2D en 3D assemblages te

maken met de eiwitkooien. Middels zowel CCMV als Thermotoga maritima (Tm)

encapsulines als gecompartimentaliseerde nanodragers, werd stimuli responsieve

assemblages verkregen door de supramoleculaire interacties van CCMV en een optisch

responsief dendron. Het fotomasker patroon werd geobserveerd tijdens de lithografie om

het foto-splitsbare dendron-CCMV-complex te bestralen. Metaal-ion coördinatie werd

gebruikt om de vorming van His10 loop gemuteerde Tm encapsulin te induceren, welke

veranderd kan worden door de imidazool concentratie te variëren (Hoofdstuk 6).

Er is grote potentie voor het gebruik van virussen in verschillende velden, maar

verdere functionalisatie moet worden uitgevoerd vanwege de intrinsieke eigenschappen

van de capside eiwitten. De in deze thesis beschreven studie verduidelijkt de potentie

van het gebruik van supramoleculaire interacties om verschillende dimensionale

biomaterialen te verkrijgen voor diverse toepassingen en vergroot de kennis van de

structuur en interacties tussen elk capside eiwit.

127

Acknowledgments

With the completion of my thesis I would like to express my gratitude and

appreciation to all those who have helped and supported me through my PhD study and

my stay in The Netherlands.

Foremost, I would like to express my sincere gratitude to my promotor Prof. Jeroen

Cornelissen. Thank you for offering me the opportunity to join this group; for providing

facilities and scientific guidance to carry out scientific works and to develop my research

skills. Thank you for your support, advices, and immense knowledge. It was a great

pleasure to have been part of BNT group in these past four and half years. I am grateful

for all these times I have been given freedom and opportunity to develop my own ideas,

which undoubtedly shaped my scientific life and helped me to become a better

researcher.

I would like to express my gratitude to Prof. Tanja Weil from Max Planck Institute

for Polymer Research, Dr. Renko de Vries from Wageningen University, Dr. Saskia

Lindhoud, Prof. Jurriaan Huskens and Prof. Nathalie Katsonis from University of

Twente. I am grateful for the time you spent for reading my thesis and for being part of

my graduation committee members. Tanja, thank you for adjusting your schedule for

my defense. I am also grateful for the opportunity to collaborate with you and your group,

for the fruitful discussion and meetings we had during the last few months. Saskia, thank

you for your help and advice for the SAXS experiments. Jurriaan, I am very thankful

for the insightful discussions during the colloquia and the meetings. Nathalie, Tibor and

Jos, I enjoyed a lot the daily conversations we had, and I appreciate very much being

invited as an external group member to your group activities. Wim and Pascal, thank

you for your feedback and input during the colloquia.

I would like to thank all the staff members: Marcel, Bianca, Richard, and Regine.

Thank you for providing the technical support that research requires, for offering help

whenever I needed it. I also enjoyed the conversations we had during coffee breaks.

Nicole H. and Izabel, thank you for your help regarding the arrangement of schedules,

conferences and the administration. I also would like to thank the support staff of the

Acknowledgments

128

MESA+ institute: Rico, Ine and Mark for their help with the TEM, SEM and other

shared equipment.

The accomplishment of this dissertation would have not been possible without the

fruitful collaborations with Yinke Wu from the research group of Prof. Tanja Weil in

Mainz, and with Minmin Zhang and Prof. Serge Lemay from MESA+ institute at the

University of Twente. It was a great honor to have the opportunity to work with you.

Furthermore, I would like to thank fellow postdocs and PhD students in the BNT & MnF

clusters who have worked and collaborated with me on my projects: Alberto, Liulin,

Sandra, Sara, Melissa, Naomi and Pia, thank you for your help, suggestions and

discussions. I wish you all the best for your future. I would like to thank my master and

bachelor students, Sandro Peeters, Wouter v.d. A, Sander and Andrea, not only

because I have learned a lot from you but also because you guys contributed a lot to my

thesis. I feel so lucky and I had lots of fun working with you guys.

I would also like to thank the virus-lunch group: Mark, Stan, Robin, Aijie, Gaurav,

Liulin, Aref, Melissa, Rindia and Sandra, thank you for the nice discussions and

suggestions. I wish you all the best for your bright future. Robin, we started our PhD on

the same day. Thank you for the translation of my summary and the correction of my

thesis. I am looking forward to meeting you again in China. Melissa, many thanks for

your help with my thesis, good luck for your job hunting. Sandra, you are always

cheerful and energetic. Working with you in the lab was incredibly fun. Thank you for

the energy and suggestions. I wish you, Mathieu and your two adorable boys all the best.

Being far away from home can be difficult at times, fortunately I have been

surrounded with ridiculously wonderful people along the way, who have made my life

in Enschede exciting. There will always be a place in my heart and thoughts to cherish

these unforgettable moments with whom I have met over the past five years. Aijie and

Liulin, thank you for picking me up at the airport when I arrived in The Netherlands for

the first time in 2015, for your hospitality and help, which made me feel at home. I had

lots of fun during the trips we had together to Belgium, Austria, Italy and Budapest.

Aijie, you are the first person I contacted before I came to Enschede. I am grateful for

your arrangement of my trip and apartment. You are a nice friend and a hard-working

scientist. Looking back, you gave me a lot of suggestions for my projects. With all the

knowledge and experience you got over last six years, I am truly confident about your

Acknowledgments

129

bright future. Liulin, you are more than a friend for me, thank you for all the daily

supervision and encouragement you gave to me in the very beginning of my PhD. I have

learned a lot from you, you are hard-working and smart. Thank you for motivating me

to challenge myself to independently tackle sophisticated scientific questions and

improve my communication skills. I wish you all the best for your career in Xiamen

University.

Rindia, Supa, He, Yao, Xiu, Qin, Min, Jianfeng, Jinfeng and Yi, thank you for

your help, suggestions, funny and nice chats, laughter and jokes, delicious food and all

the wonderful games we played. I will cherish the moments we shared together in

Enschede, and I am quite sure that we will meet again someday. Wish you all the best

with your study and career.

Sports became very important to relax and recharge myself in the past three years.

Luckily, I was surrounded by these wonderful friends with whom I went to sport

together. First, I would like to thank Xiu and Yao, we started to do strength training

together in 2017. Since then going to the gym became a routine, it was fun to workout

with you and I benefited a lot from doing sports. Thank you for your support and

suggestions when I was frustrated. I feel so much privileged to have you girls have my

back.

Furthermore, I would like to thank the group lesson buddies, Jacopo, Lucia and Pia,

I enjoyed a lot in the power pump group lesson and team beats. Jacopo, thank you for

your support and suggestions, I have no doubt that you will be very successful in

academia, wish you and Lucia all the best. Pia, it was fun to meet you in this wonderful

group, thank you for your suggestions and help, I am very grateful for the moments we

spent together, with your passion and motivation for sure you will be successful in your

career. Daniele (Danniiii), Cande (Dramalaria), and Luca, we used to go to X-core

and body pump group lessons together. Thank you for the company and encouragement,

you guys are awesome and cheerful. Danniiii, you are a very considerate and trustable

friend, thank you for your support and company. Moving to the same office with you

was definitely the right choice. Thank you for sharing snacks and driving me home from

time to time; we managed to expand our “territory” from our own table to the whole

office during last few months . Wish you and Simon all the best for your next

adventure in Munster. Dramalaria, the drama queen of our sports club, it was fun to be

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130

with you, wish you all the best in Argentina. Luca, thank you for your time and help in

designing my cover. You are so kind, inspiring, warmhearted, and talented in various

aspects (movie editing, drawing, linguistics). With all these weapons, for sure you will

rock your PhD. Julien, it was fun to go climbing together, you are one of the coolest

people I’ve ever met. Thank you for the encouragement to challenge myself to reach my

limit, wish you all the best for your future, keep rocking and being chill.

I would like to thank the tennis club, Qirong, Remi, Dhanya and Ruben, it was a

great pleasure to spend few hours together playing tennis every now and then. Qirong,

you are very hard-working and determined, your trust and honesty flattered me a lot. I

wish you a successful career in South Korea. Remi and Dhanya, I am very grateful for

you guys being my paranymphs, for your ultimate friendship and support. Remi, it is

always nice to be with you, you are such an amazing friend. Thank you for all the jokes

and laughter you brought to us. You are also a great father, wish you, Manuela and little

Arthur a bright future, don’t forget to visit us in China someday. Dhanya, I am so grateful

for your support and help, you are a very determined and smart girl. It is always relaxing

and cheerful being with you. Your dedication of doing sports together with Deepak

inspired me a lot. I have no doubt that you will have a bright future together. If you would

like to travel to China in the future, don’t forget to come to Chendu :).

I would like to thank my office mate Federico (Fede) and the neighbor Pramod.

Fede, you are probably one of the most organized persons I’ve met. Without your

encouragement, I would have never finished my thesis. Thank you for being my personal

‘therapist’, I wish you and your family all the best in U.S.A. Hopefully we will have

some collaborations soon. Pramod, it was nice to meet you in this group, wish you and

Isa all the best for your future. I would also like to thank the ‘female club’, Maike

Wiemann, Gigi, Naomi, Pia, Muhabbat, Maaike, Sandra, Dodo, Haasna,

Almudena, Dhanya and Anamarija. You girls are awesome, thank you for the jokes

and laughter in the lab. Gigi and Naomi (laboratory wives), thank you for your help and

support, it was fun to have you girls around. I am very grateful for your empathy and

sympathy, wish you all the best for your PhD. Muhabbat and Maaike thank you for

your support in the conferences, wish you all the best for your future. Maike Wiemann,

thank you for your effort to improve my alcohol tolerance even though it was not that

successful. It was a great pleasure to be with you after work. We have similar weird sense

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131

of humor, but I love all the jokes we had. Thank you for sharing your experience with

me. I am so happy that you are enjoying your life in Veldhoven.

I would also like to express my gratitude to the other group members I met here,

Nicolas, Manee, Nicole, Jan Willem, Gulistan, Siyu and Alexander, spending time

with you guys was always fun.

I would like to thank my friends who strongly supported me during my PhD. I have

been fortunate to meet Chuan and Jun: being with you was always relaxing and

inspiring. You guys helped me a lot to put myself together in the last few months of my

PhD. Chuan, you are very passionate and motivated. Thank you for always asking the

right question; every lunch break was inspiring and joyful; every discussion was

valuable. Thank you for shaping my way of thinking; thank you for always being so

supportive and warm to my frustrations and negative thoughts. Unfortunately, I will not

really pay you back, but for sure you will be very successful as a rising star in your field.

Wish you and Kui all the best in The Netherlands. Jun, it was a pity that we only met in

the last year of my PhD. I am lucky to have had the synergy with you, you are very

considerate and warmhearted, and very talented in cooking and singing . Thank you

for all the support and delicious food. I am so happy (jealous) that you will work with

Chuan in the following years, there is no doubt that you will enjoy this adventure. I

would also like to thank my Chinese friends from all over The Netherlands, Zhijun,

Dingding and Xiangzhen, we came to The Netherlands to do our PhD in the same year,

I am happy that you guys are progressing as planned, it was fun to have these annual

gatherings with you.

To my family, 王冲，谢谢你一直以来的理解和支持，这些年来有很多不容易，

希望我们能携手一起面对生活的风风雨雨，相信我们也会越来越好。感谢我的妈

妈，弟弟，妹妹，妹夫，姐姐，姐夫及家人们对我的关心和支持。

曹叔琴

Shuqin Cao (Jenny)

Enschede, 2020

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About the author

Shuqin Cao was born in Chongqing, China on August

30th in 1989. She pursued her bachelor’s degree in Polymer

Materials and Engineering at Sichuan University of China

from 2008 to 2012. During her bachelor research project,

she constructed a glucose responsive LbL system as a

controlled delivery system of insulin under the supervision

of Prof Dr. Jianshu Li. She received her Master degree in

biomaterials in 2015 at Sichuan University, China, her

thesis was entitled “Dipeptide-assisted self-assembly of

Salmon Calcitonin into supramolecular nanoparticles for long-last therapy” and was

carried out under the supervision of professor Jianshu Li at the polymer science and

engineering institute of biomaterials.

In September 2015 Shuqin started her PhD in the Biomolecular Nanotechnology

group under the supervision of Prof. Dr. Jeroen J.L.M. Cornelissen. Combining aspects

from polymer and supramolecular chemistry she aimed to control the assembly behavior

of virus-like particles. Furthermore, she studied the encasement of functional materials

with these proteins in order to modify their properties, more specifically, the

biocompatibility. Finally, the 3D arrangement of protein cages by non-covalent

interactions in investigated.

133

Publications

Published

1. Shuqin Cao#, Yanpeng Liu#, Hui Shang#, Sheyu Li, Jian Jiang, Xiaofeng Zhu,

Peng Zhang, Xianlong Wang, Jianshu Li*; Supramolecular nanoparticles of

calcitonin and dipeptide for long-term controlled release; Journal of

Controlled Release; 256 (2017), 182–192.

2. Liulin Yang#, Aijie Liu, Shuqin Cao, RindiaM.Putri, Pascal Jonkheijm,* and

Jeroen J. L. M. Cornelissen*; Self-Assembly of

Proteins:TowardsSupramolecular Materials; Chem. Eur.J; (2016), 22,

15570–15582.

3. Zaifu Lin#, Shuqin Cao, Xingyu Chen, Wei Wu, and Jianshu Li*;

Thermoresponsive Hydrogels from Phosphorylated ABA Triblock Copolymers:

A Potential Scaffold for Bone Tissue Engineering; Biomacromolecules; 2013,

14, 2206−2214.

4. Jiaojiao Yang#, Shuqin Cao, Jiahui Li, Jianyu Xin, Xingyu Chen, Wei Wu,

Fujian Xu and Jianshu Li; Staged self-assembly of PAMAM dendrimers into

macroscopic aggregates with a microribbon structure similar to that of

amelogenin; Soft Matter; 2013, 9, 7553.

5. Jiaojiao Yang#, Shuqin Cao, Jianyu Xin, Xingyu Chen & Wei Wu & Jianshu Li

；Calcium carbonate deposition on layer-by-layer systems assembled from star

polymers; J Polym Res; (2013), 20, 157.

6. Jun Luo#, Shuqin Cao, Xingyu Chen, Shuning Liu, Hong Tan, Wei Wu, Jianshu

Li*; Super long-term glycemic control in diabetic rats by glucose-sensitive LbL

films constructed of supramolecular insulin assembly; Biomaterials; (2012),

33, 8733-8742.

7. Wei Wu#, Jing Liu, Shuqin Cao, Hong Tan, Jianshu Li, Fujian Xu, Xiao Zhang;

Drug release behaviors of a pH sensitive semi-interpenetrating polymer

network hydrogel composed of poly (vinyl alcohol) and star poly[2-

(dimethylamino) ethyl methacrylate; International Journal of Pharmaceutics;

416 (2011) 104-109.

134

Manuscripts in preparation

8. Shuqin Cao#, Sandro Peeters, Naomi Hamelmann, Liulin Yang*, Jeroen J. L.

M. Cornelissen*; Construction of Viral Protein-based Hybrid Nanomaterials

Mediated by Molecular Glues; under preparation.

9. Shuqin Cao#, Jeroen J. L. M. Cornelissen*; Self-assembly of virus-like-

particles induced by supramolecular interactions; RSC book "Supramolecular

Protein Chemistry" Chapter (2020), under preparation.

10. Minmin Zhang#, Shuqin Cao#，Aijie Liu, Jeroen J. L. M. Cornelissen, Serge

G. Lemay*; Self-assembly of viral capsid proteins driven by compressible

nanobubbles; under preparation.

11. Shuqin Cao#, Yingke Wu# et. al, Encapsulation of Nano-diamonds with Capsid

Protein of CCMV for in vivo imaging; under preparation.

12. Shuqin Cao# et. al, Self-organization of protein cages mediated by metal

coordination, under preparation.

13. Shuqin Cao# et. al, Chemical and light responsive virus-like particles based on

azobenzene modified capsid proteins, under preparation.

Documents

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