From Infection to Healing: the Use of Plant Viruses in Bioactive Hydrogels

Received: 4 February 2020 Revised: 8 June 2020 Accepted: 23 June 2020 DOI: 10.1002/wnan.1662

ADVANCED REVIEW

From infection to healing: The use of plant viruses in bioactive hydrogels

Christina Dickmeis | Louisa Kauth | Ulrich Commandeur

Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Abstract Germany Plant viruses show great diversity in shape and size, but each species forms unique nucleoprotein particles that are symmetrical and monodisperse. The Correspondence Christina Dickmeis, Institute for genetically programed structure of plant viruses allows them to be modified by Molecular Biotechnology, RWTH Aachen genetic engineering, bioconjugation, or encapsulation to form virus University, Aachen, Germany. nanoparticles (VNPs) that are suitable for a broad range of applications. Plant Email: christina.dickmeis@molbiotech. rwth-aachen.de VNPs can be used to present foreign proteins or epitopes, to construct inorganic hybrid materials, or to carry molecular cargos, allowing their utilization Funding information as imaging reagents, immunomodulators, therapeutics, nanoreactors, and bio- Deutsche Forschungsgemeinschaft, Grant/ Award Number: 654127; RWTH Aachen sensors. The medical applications of plant viruses benefit from their inability University to infect and replicate in human cells. The structural properties of plant viruses also make them useful as components of hydrogels for tissue engineering. Hydrogels are three-dimensional networks composed of hydrophilic polymers that can absorb large amounts of water. They are used as supports for tissue regeneration, as reservoirs for controlled drug release, and are found in contact lenses, many wound healing materials, and hygiene products. They are also useful in ecological applications such as wastewater treatment. Hydrogel-based matrices are structurally similar to the native extracellular matrix (ECM) and provide a scaffold for the attachment of cells. To fully replicate the functions of the ECM it is necessary to augment hydrogels with biological cues that regu- late cellular interactions. This can be achieved by incorporating functionalized VNPs displaying ligands that influence the mechanical characteristics of hydrogels and their biological properties, promoting the survival, proliferation, migration, and differentiation of embedded cells.

This article is categorized under: Implantable Materials and Surgical Technologies > Nanomaterials and Implants Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals LLC.

WIREs Nanomed Nanobiotechnol. 2020;e1662. wires.wiley.com/nanomed 1of32 https://doi.org/10.1002/wnan.1662 2of32 DICKMEIS ET AL.

KEYWORDS hydrogels, nanoparticles, plant viruses, tissue engineering

1 | INTRODUCTION

Virus nanoparticles (VNPs) are nanomaterials based on augmented natural viruses. They have many advantages for applications in the biomedical and material sciences, including their uniformity and versatility. VNPs are uniform because they are genetically encoded, therefore the particles are identical, symmetrical, and monodisperse. They are versatile because they can be functionalized by genetic engineering, bioconjugation, and/or encapsulation. VNPs based on plant viruses have the added advantages of safety and inexpensive production. Most plant-based food products contain plant viruses (Latham & Wilson, 2008; Lesemann & Winter, 2002; Rochow, 1972) and some species are even propa- gated in fog and clouds (Castello et al., 1995) and in cigarettes (Liu, Vaishnav, Roberts, & Friedland, 2013; Wetter, 1975). People are exposed to plant viruses constantly but are never infected. The corresponding VNPs are also noninfectious, and because they are nucleoprotein structures they are also biocompatible and biodegradable (Czapar et al., 2016; van Kan-Davelaar, van Hest, Cornelissen, & Koay, 2014). Plant VNPs are inexpensive to produce because, like the native virus, they replicate naturally in plants. VNPs produced in plants may carry plant glycans (Lomonossoff & D'Aoust, 2016; Sack, Hofbauer, Fischer, & Stoger, 2015). If this is undesirable, specialized plant lines are available that have been engineered to eliminate plant glycans or produce oligosaccharides similar to those synthesized in human cells (Schoberer & Strasser, 2018). Based on these advantages, plant VNPs have been used to develop inorganic hybrid materials (Lee, Lim, & Harris, 2012; Wen & Steinmetz, 2016) and modified to carry drugs, contrast and imaging reagents, immunomodulators, enzymes, and biosensors (Chen, Butler, Chen, & Suh, 2019; Cormode, Jarzyna, Mulder, & Fayad, 2010; Koudelka, Pitek, Manchester, & Steinmetz, 2015; Lam & Steinmetz, 2018; Manchester & Singh, 2006; Wen & Steinmetz, 2016; Wu, Wu, Nakagawa, & Gao, 2019; Young, Debbie, Uchida, & Douglas, 2008). The programma- bility of viruses allows their use as targeted drug delivery vehicles. Some plant VNPs have a natural tendency to accumulate in tumors, a property influenced by the particle shape and distribution of surface charges, which affect the enhanced permeability and retention effect (Lee et al., 2013; Shukla et al., 2015). Targeting can also be engineered by modifying the surface charge and chemistry or by adding specific targeting peptides or shielding molecules such as polyethylene glycol (PEG) (Pitek, Wen, Shukla, & Steinmetz, 2016). For example, new forms of cancer therapy have been developed based on potato virus X (PVX) modified to deliver doxorubicin (Le, Lee, Shukla, Commandeur, & Stein- metz, 2017) and tobacco mosaic virus (TMV) modified to deliver phenanthriplatin (Czapar et al., 2016). The capsids provide a scaffold that can simultaneously encapsulate drugs while presenting targeting ligands on the external surface (Hovlid et al., 2014). Capsids can also be used to encapsulate imaging reagents or to display them externally. For example, PVX particles displaying fluorescent markers such as mCherry or green fluorescent protein have been used for imaging in plants, preclinical animal models, and human cells (Shukla et al., 2014). Many of the advantages of plant viruses are shared by bacteriophages, but the latter are contaminated with lipopolysaccharides when produced in bacteria, and additional purification steps are thus needed during downstream processing (Szermer-Olearnik & Boratynski, 2015). The well-defined structures of plant VNPs make them particularly suitable for tissue-engineering applications, providing an alternative strategy where there is a lack of donor tissue or a risk of rejection after implantation. Hydrogels are colloidal gels in which a liquid is dispersed in a three-dimensional (3D) network of hydrophilic polymer chains, allowing the adsorption of large quantities of water or biological fluids (El-Sherbiny & Yacoub, 2013). This contrasts with other colloidal gels in which the liquid is dispersed among solid particles. Hydrogels can be used to support organs or tissue implants, or as a matrix to embed cells for the regrowth of tissues de novo (El-Sherbiny & Yacoub, 2013). They can also be developed as controlled drug-release systems (Kim, Lee, et al., 2015; Merino, Martín, Kostarelos, Prato, & Vázquez, 2015), wound healing materials (Gupta, Agarwal, & Alam, 2011), superabsorbent materials for hygiene products such as diapers (Zohuriaan-Mehr, Omidian, Doroudiani, & Kabiri, 2010), and the filling of contact lenses (Michalek, Hobzova, Pradny, & Duskova, 2010). They have also been used in agriculture (Abobatta, 2018) and for wastewater treatment (Ali Shah & Ali Khan, 2019). The construction of hydrogels has been comprehensively reviewed by Mantha et al. (2019). In tissue engineering, hydrogels provide a support scaffold for implanted cells and therefore mimic the mechanical properties of the extracellular matrix (ECM). However, to support tissue regeneration they must also provide the appropriate biological signals that control cell behavior (Luckanagul et al., 2012). Bioactive scaffolds DICKMEIS ET AL. 3of32 can be developed by incorporating plant VNPs into hydrogels, allowing the presentation of peptides that promote cell proliferation, adhesion, migration, or differentiation. A recent review covering plant virus-based materials for biomedical applications included a brief description of VNP-based hydrogels (Eiben et al., 2019). Here we expand on this specific theme by describing the use of plant viruses for the creation of composite hydrogels in more detail, focusing on the strategies used to produce VNP-augmented hydrogels and the novel functions conferred by plant VNPs in the context of tissue engineering.

2 | PLANT VIRUSES

Plant viruses are natural supramolecular structures ranging in size from tens to hundreds of nanometers. They are composed of multiple copies of one or more identical coat protein subunits that self-assemble to form a capsid enclosing the virus genome, allowing them to be adapted into nanocarriers. VNPs derived from plant viruses remain stable when exposed to high temperatures, extreme pH and many solvents (Pokorski & Steinmetz, 2011). Structurally, there are two major classes of plant viruses—those with icosahedral capsids and those with rod-shaped or filamentous capsids—and we describe each in turn below.

2.1 | Virus structures

2.1.1 | Icosahedral viruses

Icosahedral viruses (often described superficially as spherical viruses) form a capsid that shows icosahedral symmetry. An icosahedron is a polyhedron with 20 triangular faces and 60 asymmetric units, characterized by overall symmetry involving different axes of rotation (Caspar & Klug, 1962). Icosahedral viruses are therefore built from multiples of 60 coat protein subunits, with the size and complexity of the capsid represented by the triangulation number T (Caspar & Klug, 1962). The simplest plant viruses assemble from 60 subunits (T = 1) and the most complex icosahedral plant viruses discovered thus far assemble from 180 subunits (T = 3), although this limits the packaging capacity by restricting the internal volume (Solovyev & Makarov, 2016). The largest RNA known to be packaged into icosahedral plant virus capsids is ~7.5 kb in length (Solovyev & Makarov, 2016). Icosahedral particles range from 18 to 500 nm in diameter (Pokorski & Steinmetz, 2011). Assembly may require multiple lower-order steps and cannot occur as a single higher-order reaction. The family Bromoviridae (bromoviruses), particularly cowpea chlorotic mottle virus (CCMV), has been used as model system to study the assembly of icosahedral plant viruses because infectious virions can be produced easily from purified RNA and coat protein (Bancroft & Hiebert, 1967; Hiebert & Bancroft, 1969). The CCMV genome is made up of three unique single-stranded RNA molecules encapsidated independently in separate capsids each featuring 180 identical coat protein subunits (Narayanan & Han, 2017). In contrast, the capsids of cowpea mosaic virus (CPMV, family Sec- oviridae, subfamily Comovirinae) feature 120 coat proteins, 60 copies each of the small (S) and large (L) subunits (Hesketh, Meshcheriakova, Thompson, Lomonossoff, & Ranson, 2017). However, CPMV is structurally similar to CCMV because the 120 coat protein subunits form 180 domains, and for this reason CPMV is often described as having pseudo T = 3 symmetry (Lin et al., 1999). CPMV has often been used for the display of amino acids or small peptides, and the modified coat proteins can be used as nanoscale building blocks for the design of new materials (Chatterji et al., 2002; Chatterji, Ochoa, Ueno, Lin, & Johnson, 2005; Cho et al., 2014; Dickmeis, Altintoprak, van Rijn, Wege, & Commandeur, 2018; Steinmetz, Cho, Ablack, Lewis, & Manchester, 2011; Wang, Lin, Johnson, & Finn, 2002).

2.1.2 | Rigid rod-shaped viruses

Almost half of all known species of plant virus with a positive-sense single-stranded RNA genome have capsids that display helical symmetry (Stubbs & Kendall, 2012). Helical capsids typically have a central channel along the virion axis which contains the genomic RNA, and the coat proteins are assembled along it. Unlike icosahedral viruses, which have a maximum capacity for nucleic acid based on the internal volume of the capsid, helical viruses build the capsid around 4of32 DICKMEIS ET AL. the genome and can therefore accommodate much longer RNAs (Solovyev & Makarov, 2016). Viruses with helical symmetry can have stiff, rod-shaped capsids or flexuous, filamentous capsids. Members of the family Virgaviridae have rod-shaped capsids, including the genera Furovirus, Hordeivirus, Pecluvirus, Pomovirus, Tobamovirus, and Tobravirus (Adams, Antoniw, & Kreuze, 2009). Such viruses have alpha-like replication proteins that form a distinct phylogenetic group (Koonin, Dolja, & Morris, 1993). The particles are 20– 25 nm in diameter made up of coat proteins with a molecular weight of 19–24 kDa. Their RNA genome has a 30 tRNA- like structure (Adams et al., 2009). Some members of the family Virgaviridae encode a cell-to-cell movement protein (Melcher, 2000) whereas others contain a triple gene block (Morozov & Solovyev, 2003). TMV is a well-studied rod-shaped plant virus and, as the type member of the genus Tobamovirus, it was the first virus structure to be resolved (Holmes, Stubbs, Mandelkow, & Gallwitz, 1975). TMV features 2,130 coat proteins encapsidating an RNA strand of 6,395 nucleotides. The resulting virion is 300 nm in length and 18 nm in diameter (Stubbs, 1999; Stubbs & Kendall, 2012). The virus is particularly interesting in terms of materials science because it offers a highly versatile scaffold that provides surfaces with distinct amino acid compositions (Namba & Stubbs, 1986; Pokorski & Steinmetz, 2011). TMV has been functionalized with the amino acids cysteine (Yi et al., 2005) and lysine (Smith et al., 2006) to present reactive groups for bioconjugation, resulting in the VNPs named TMV1cys and TMV1lys, respectively.

2.1.3 | Flexuous filamentous viruses

Flexuous filamentous viruses also show helical symmetry. Filamentous virions are formed by viruses of the families Alphaflexiviridae, Betaflexiviridae, Closteroviridae, and Potyviridae, which include at least 19 recognized genera (Faquet, Mayo, Maniloff, Dessselberg, & Ball, 2005; Kendall et al., 2008). More filamentous viruses than rigid rod-shaped viruses have been described (King, Adams, Carstens, & Lefkowitz, 2011), possibly reflecting the higher fitness of these viruses in flowering plants (Solovyev & Makarov, 2016). However, whereas a large body of structural data now exists for rigid rod-shaped and icosahedral viruses, few filamentous viral structures have been solved. Very recently, the structure of PVX was solved to a resolution of 2.2 Å by high-resolution cryogenic electron microscopy, providing the first details of protein–protein and protein–RNA interactions essential to the integrity of potexviruses (Grinzato et al., 2020). Due to their flexibility, instability and in many cases limited abundance, filamentous viruses are difficult to study using methods successfully applied to TMV and other viruses (Kendall et al., 2008; Solovyev & Makarov, 2016). There may be structural and evolutionary relationships among flexuous viruses (Aiken & Gill, 1966; Dolja, Boyko, Agranovsky, & Koonin, 1991; Morozov, Lukasheva, Chernov, Skryabin, & Atabekov, 1987; Shukla, Strike, Tracy, Gough, & Ward, 1988) but there is little sequence conservation among the coat proteins of different species. PVX, the type member of the genus Potexvirus (family Alphaflexiviridae), features 1,270 coat protein subunits arranged in a helical architecture around the 6.4 kb genomic RNA strand, forming a high-aspect-ratio particle with dimensions of 515 × 15 nm (Huisman, Linthorst, Bol, & Cornelissen, 1988; Kendall et al., 2008; Sober et al., 1988). The C-terminus is located inside the assembled particle whereas the N-terminus is exposed and therefore accessible for genetic modifications, allowing the display of amino acids or small peptides (Kendall et al., 2008; Lee, Uhde-Holzem, Fischer, Commandeur, & Steinmetz, 2014; Lico et al., 2009; Nemykh et al., 2008).

2.2 | Modification of plant viruses

Multiple techniques can be used to modify plant virus coat proteins and capsids, including genetic engineering, bioconjugation, encapsulation, and biomineralization (Figure 1) (Koudelka et al., 2015; Vignali et al., 2019). Genetic engineering allows the insertion, removal, or replacement of specific amino acids (Klem, Willits, Young, & Douglas, 2003; Miller, Presley, & Francis, 2007; Peabody, 2003; Wang, Lin, Johnson, et al., 2002) and is the preferred method for the display of functional groups or small peptides on the virus surface. These changes may include the extension of the N- terminus or C-terminus of the coat protein, the insertion of internal loops, or the insertion or replacement of single amino acids to enable functionalization (Chatterji et al., 2005; Medintz et al., 2005) or to alter the surface properties of the virus (Douglas et al., 2002). For example, this modification strategy can be used for the introduction of purification/ immunodetection tags or targeting sequences (Yildiz, Shukla, & Steinmetz, 2011). DICKMEIS ET AL. 5of32

FIGURE 1 Modifications and applications of plant virus nanoparticles (VNPs) and virus like particles (VLPs). Plant virus capsids can be modified by genetic engineering, bioconjugation, biomineralization or used for the encapsulation of small molecules. These modifications can be used for applications such as biomaterial design, molecular imaging, and drug delivery

Bioconjugation uses classical chemistry to modify virus particles. Carboxylate groups on the side chains of acidic amino acids, amine groups on arginine and lysine residues, phenol groups on tyrosine residues, and sulfhydryl groups on cysteine residues can all be functionalized by direct conjugation or modified to display a new functional group as a ligation handle. For example, functional groups can be added specifically to amine or carboxylate groups using selective conjugation methods based on N-hydroxysuccinimide chemistry (Evans, 2008). Native TMV particles offer accessible tyrosine residues on the external surface and glutamic acid residues on the internal surface that can be functionalized with different molecules simultaneously (Schlick, Ding, Kovacs, & Francis, 2005). The self-assembly of viruses can also be exploited for the encapsulation of cargo molecules. This can be triggered by surface charge, electrostatic interactions, or binding interactions (Daniel et al., 2010; Dixit et al., 2006; Huang et al., 2007; Sun et al., 2007). In terms of virus modification, biomineralization is the ability of capsid protein to assemble around a mineral core or act as a nucleation center for mineralization (Koudelka et al., 2015). Encapsulated minerals can be used as contrast agents, for example, CCMV capsids were used to carry an Fe(II) cargo for magnetic resonance imaging and the treatment of hyperthermia (Douglas et al., 2002).

2.3 | Production of nanoparticles

2.3.1 | Plant VNPs

Plant VNPs include infectious particles carrying a functional genome as well as noninfectious particles that carry either a defective genome or no genome at all, the latter also being defined as virus-like particles (VLPs). We therefore use VNPs and VLPs hereafter to refer to genome-containing replicating particles and genome-free non-replicating particles, respectively. The ability of VNPs to replicate means that gram quantities can be produced by molecular farming in plant hosts (Yildiz et al., 2011). Molecular farming is especially efficient in tobacco species, which can be used as VNP production factories (Figure 2). Plants can be inoculated with the virus, its genomic RNA or a cDNA copy thereof. For inoculation, three or four leaves of 4-week-old plants are rubbed with an abrasive such as Celite 545 and then with a virus suspension of 10 μg of DNA/RNA (Lee et al., 2014). Leaves are harvested 14–21 days post-infection for the extraction of VNPs. Alternatively, plants can be infected with Agrobacterium tumefaciens carrying a cDNA copy of the virus genome. The expression of this cDNA then yields many copies of the virus genomic RNA, mirroring the strategy used for the transient expression of recombinant proteins (Peyret & Lomonossoff, 2015). More VNPs can be produced by 6of32 DICKMEIS ET AL.

FIGURE 2 Schematic summary of the production options for virus nanoparticles (VNPs)/virus like particles (VLPs). Plant VNPs can be produced by molecular farming in plants following inoculation with intact particles, genomic RNA or cDNA, or infection with Agrobacterium tumefaciens carrying a cDNA copy of the virus genome. After infection, plant material is harvested and the tissue is disrupted. Cell debris is removed and the extract is used for particle purification by precipitation and gradient separation. Plant VLPs can be produced by expressing coat proteins in suitable hosts such as plants, bacteria or yeast. After cell lysis, subunits can be isolated by precipitation and chromatography. The particles are assembled in vitro and can be purified as described above for VNPs. Alternatively, VLPs can be produced directly from VNPs by partial or complete disassembly followed by the degradation or capture of the nucleic acids and subsequent capsid reassembly

inoculating uninfected plants with either the crude extract of infected plants or with purified VNPs, allowing large-scale production (Uhde-Holzem, Fischer, & Commandeur, 2007).

2.3.2 | Plant virus-like particles

VLPs self-assemble in the same manner as replicating particles, but the absence of the viral genome means they cannot replicate (Pumpens & Grens, 2002). VLPs retain the ability to enter target cells (because this depends on surface structures) but are noninfectious and therefore present a low risk to the environment. In contrast to VNPs, which can be produced by infecting host plants, VLPs are produced by the expression of proteins necessary for particle formation (Figure 2). VLPs can be produced in heterologous expression systems such as plants (Saunders, Sainsbury, & Lomonossoff, 2009), bacteria (usually Escherichia coli) (Brown, Naves, Wang, Ghodssi, & Culver, 2013), or yeast (Brumfield et al., 2004; Mueller, Kadri, Jeske, & Wege, 2010). Alternatively, VLPs can be produced directly from VNPs by pH-induced swelling followed by alkaline hydrolysis of the released nucleic acids (Yildiz et al., 2011) or the disassembly of intact VNPs and reassembly into VLPs following the removal of nucleic acids (Mueller et al., 2010). When using animal viruses as the basis for vaccine development, VLPs are preferred to VNPs because they are noninfectious but they retain the advantages of immunogenicity and antigen stability (Chen & Lai, 2013). They combine the best attributes of whole-virus and subunit antigens for vaccine development. The success of the hepatitis B virus surface antigen (Pumpens et al., 2008) and human papillomavirus capsid protein L1 (Frazer, 2004) as commercial vaccines against hepatitis B and cervical cancer has promoted new interest in VLPs. Accordingly, plant-derived VLPs have emerged in several fields of biotechnology because they can carry and display heterologous molecules or serve as building blocks for new materials. VLPs can also be used for biomedical imaging (Cormode et al., 2010; Manchester & DICKMEIS ET AL. 7of32

Singh, 2006), the synthesis of inorganic materials (Bode, Minten, Nolte, & Cornelissen, 2011), gene therapy (Campos & Barry, 2007; Parker, Nicklin, & Baker, 2008), and the development of films and arrays suitable for engineering and elec- tronics (Flynn, Lee, Peelle, & Belcher, 2003; Lee, Muhammad, et al., 2012; Rong, Niu, Lee, & Wang, 2011). For example, CPMV particles have been used to present fluorescent dyes or gold clusters (Wang, Lin, Tang, Johnson, & Finn, 2002) whereas TMV has been used as a scaffold for the assembly of nanoscale biopolymers (Gleba, Klimyuk, & Marillonnet, 2007).

3 | HYDROGELS

Tissue engineering aims to create biomimetic tissues with high biochemical and structural similarity to native tissue counterparts in order to augment or replace damaged tissues in vivo. Scaffolds are therefore needed to provide suitable architectural, biological and chemical characteristics that support cell growth. Native tissues and organs are composites of different materials and structures, including multiple cell populations and ECM that interact in a synchronous manner. The connective junctions are particularly prone to injury and degeneration, and often fail to regenerate after surgical repair due to inferior scar tissue development (Galatz, Ball, Teefey, Middleton, & Yamaguchi, 2004; Lee, Robinson, & Lu, 2016). These junctions are characterized by an ECM with spatial differences in composition, affecting cell phenotypes, cell organization, and region-specific mechanical properties (Lee et al., 2016). The ECM is a natural hydrogel, containing various polymers such as collagen, elastin, mucopolysaccharides, gelatin, and hyaluronan, combined with various cell types and a pervasive fluid medium (Kyburz & Anseth, 2015). Synthetic hydrogels are crosslinked networks with a high content of water and tissue-like elastic properties. Hydro- gels are extremely hydrophilic due to the presence of many carboxyl, amide, amino, and hydroxyl groups distributed along the backbone of polymer chains. In the swollen state, hydrogels resemble living tissue because they are soft and rubbery (El-Sherbiny & Yacoub, 2013). Hydrogels are used as 3D scaffolds to support cell growth and new tissue development in grafts and organ replacements. Cells are either encapsulated during hydrogel production or seeded onto preformed cytocompatible scaffolds. The polymerization conditions of certain types of hydrogels during encapsulation can be too harsh for the survival of some cells, and in such cases the preformed scaffold is the better option (Nicodemus & Bryant, 2008). However, cells encapsulated in hydrogels are advantageous because the composite can be injected directly into target sites in vivo and will diffuse into small spaces in adjacent tissues, improving adhesion (Peretti et al., 2006). Strategies to produce hydrogel scaffolds encompassing living cells are therefore preferred, and these must include the biological cues that encourage cells to interact with their microenvironment and show spatiotemporal dynamic properties relevant for complex tissue engineering processes (Burdick & Murphy, 2012). The first biobased hydrogels were prepared from collagen and/or fibrin (Lee & Mooney, 2001), but other substrates have been tested for biocompatibility including chitosan and alginate (Ju, Kim, Kim, & Lee, 2002; Matsukuma, Sambai, & Otsuka, 2017). Biobased hydrogels are often produced from elastin, collagen, insect-derived silk, and resilin (Desai & Lee, 2015). These protein-based polypeptide materials can be modified by genetic engineering to refine their structure or bioactivity, and the expression of recombinant proteins allows the production of monodisperse ECM-like materials with no risk of rejection or disease transmission, unlike conventional allografts and xenografts (Desai & Lee, 2015). Protein-based systems are also superior for biomedical applications due to their intrinsic biocompatibility and biodegradability (Nettles, Chilkoti, & Setton, 2010; Werkmeister & Ramshaw, 2012).

3.1 | Formation of hydrogels

Hydrogels containing embedded cells are produced by mixing living cells with a gelling solution and allowing it to set. The gelling solution and setting process must therefore be cytocompatible, as must any degradation products that arise during gelation. Several gelation mechanisms can be exploited, including covalent crosslinking or electrostatic interactions between polymer chains. Although most natural hydrogels form without covalent crosslinks, the latter are preferred because they allow much finer control of the mechanical and degradation properties of the final gel (Nicodemus & Bryant, 2008). Such hydrogels are typically produced from biocompatible macromolecular monomers with molecular weights ≥3 kDa, with water-soluble components and appropriate buffers to prevent cell lysis (Nicodemus & Bry- ant, 2008). Polymerization is typically induced by radical groups or chemical crosslinking, each of which we now con- sider in turn. 8of32 DICKMEIS ET AL.

Radical chain polymerization involves the use of biocompatible polymers modified with vinyl groups (e.g., fumarate) that produce radicals in response to triggers such as illumination, a temperature shift or a small-molecule inducer. The resulting signal propagates through carbon–carbon double bonds to form high-molecular-weight chains. The chemical initiators used for these reactions tend to be cytotoxic, which restricts their use in hydrogels containing embedded cells. Even so, radical chain polymerization is useful for cell encapsulation because it promotes rapid polymerization (in the order of seconds). For example, redox systems are two-component initiators typically consisting of a peroxide- based oxidizing agent and an amine reducing agent (Hong et al., 2007). The toxicity of these systems is primarily driven by the pH of the initiators and to a lesser extent by the active centers produced during the reaction (Temenoff, Shin, Conway, Engel, & Mikos, 2003). Photo-initiators can also be cytotoxic, mainly reflecting the initiator chemistry and concentration, but toxicity was also found to depend on the proliferation rate of the encapsulated cells (Bryant, Nuttelman, & Anseth, 2000; Williams, Malik, Kim, Manson, & Elisseeff, 2005). Photo-initiators are usually triggered by UV light, thus the exposure time has a major impact on cell viability. Chemical crosslinking (or step-growth polymerization) is spontaneous, so there is no requirement for an initiator, but the process is typically slower than radical chain polymerization. Thiol-containing proteins, peptides, and polysac- charides have been used for cell encapsulation via this method (Park, Lutolf, Hubbell, Hunziker, & Wong, 2004; Shu, Liu, Palumbo, Luo, & Prestwich, 2004). In Michael-type addition reactions, a nucleophilic thiolate and electrophile unsaturated ester form a thioester linkage. Thiolates can form under slightly basic conditions, and cysteine-containing proteins can be used as a reaction component (Park et al., 2004; Shu et al., 2004; Xiao, Ahmad, Liu, & Prestwich, 2006).

3.2 | Hydrogel design

A major advantage of hydrogels is that the polymers can be modified to display molecules that promote cell attachment (Huebsch et al., 2010), cell growth (Sakiyama-Elbert & Hubbell, 2000; Shah et al., 2010; Sierra, Cordova, Chen, & Rajadhyaksha, 2015), or other functionalities (Lee, Muhammad, et al., 2012). Proteins that bind particular substrates can be aligned in hydrogels to confer tissue structure, or enzymes can be used to transform these substrates into nano- structured materials. This allows the development of “smart” biomaterials that react to cellular processes or environmental changes such as pH or temperature (Anderson, Burdick, & Langer, 2004). Controlling the structural and biochemical characteristics of hydrogels using protein-based biomaterials allows the production of tissue replacements with tailored mechanical and therapeutic properties. In this context, hydrogels should be designed to simulate the ECM appropriate for the embedded cells (Box 1), promoting cell survival, growth, and the maintenance of cell functions, and if necessary inducing differentiation (Figure 3). Scaffold design allows the fine tuning of the hydrogel properties and can influence the differentiation of cells by providing mechanical signals (Drury & Mooney, 2003; Hollister, 2005; Lee et al., 2016). The diffusion of proteins and other molecules through the hydrogel, as well as its stiffness, degradability and propensity for swelling, must be considered as

BOX 1 THE EXTRACELLULAR MATRIX The extracellular matrix (ECM) consists of noncellular components such as collagens, proteoglycans/glycosami- noglycans, elastin, fibronectin, laminins, and several other glycoproteins, which build a three-dimensional (3D) network present in all tissues and organs (Frantz, Stewart, & Weaver, 2010). All of the components are synthesized in and secreted by cells. The ECM provides cells with the structural and biochemical support required for attachment, growth, differentiation, tissue morphogenesis and homeostasis. The ECM is tissue-specific, reflecting its unique composition and topology in each microenvironment due to the particular cell types present and the interactions between them. However, the ECM in all tissues shares common functions such as the promotion of cell adhesion, cell-to-cell communication and differentiation. It also provides compressive strength and elasticity (Frantz et al., 2010). The ECM is a dynamic structure that remodels continuously via enzymatic or nonenzymatic processes in response to the embedded cells and the wider environment. The formation of the ECM is necessary for proper development, growth and wound healing, and the properties of the ECM must therefore be understood and replicated for tissue engineering to be successful. DICKMEIS ET AL. 9of32

FIGURE 3 The favorable traits of hydrogels for tissue engineering applications

design elements. These characteristics can be influenced by the degree of crosslinking or may depend on the addition of certain proteins or chemicals. Hydrogels with high mechanical strength can be produced by increasing the crosslinking density, but this limits the swelling capacity and reduces the mesh size, thus inhibiting diffusion. The distribution of newly synthesized matrix molecules within the hydrogel is strongly influenced by the hydrogel structure (Bryant & Anseth, 2003; Bryant, Durand, & Anseth, 2003; Söntjens, Nettles, Carnahan, Setton, & Grinstaff, 2006). Furthermore, different cell types require different mechanical properties and biological signals. For example, chondrocytes and osteo- blasts can be encapsulated in PEG hydrogels without biological signals (Benoit, Durney, & Anseth, 2006; Bryant et al., 2003), whereas human mesenchymal stem cells (hMSCs) require the presence of components such as RGD peptides that promote cell adhesion (Nuttelman, Tripodi, & Anseth, 2005; Salinas, Cole, Kasko, & Anseth, 2007). Further- more, the porosity of the gels can influence cell motility and the transport of soluble molecules (Mendes, Baran, Reis, & Azevedo, 2013). A fibrous architecture is needed to support neuronal cells and promote neurite outgrowth. This is because peripheral nerve regeneration requires the longitudinal alignment of Schwann cells to promote proliferation, which is followed by the formation of Büngner bands around fractured axons. The aligned Schwann cells secrete and deposit highly anisotropic ECM components providing pathways to direct axonal regeneration, thus bridging the gap of the nerve injury (Ribeiro-Resende, Koenig, Nichterwitz, Oberhoffner, & Schlosshauer, 2009). Aligned fibrous materials are thought to mimic the native architecture to guide axonal and neurite extension, promote cell migration, and enhance axon regeneration (Cooper, Bhattarai, & Zhang, 2011; Yang, Murugan, Wang, & Ramakrishna, 2005). Hydrogels can also be designed to respond to stimuli such as a pH or temperature shift, exposure to a specific wave- length of light, or exposure to electric or magnetic fields (Chen et al., 2018). The ability to fine tune the properties of hydrogels also makes them promising in membrane applications (Yang, Adrus, Tomicki, & Ulbricht, 2011). For example, thin-layer hydrogels are often fabricated on porous mechanical supports to control membrane selectivity by altering the mesh size. Such hydrogel layers are particularly suitable for wastewater treatment, where membrane fouling can be avoided by using hydrophilic hydrogels with high fouling resistance (Le-Clech, Chen, & Fane, 2006; Sadeghi, Yi, & Asatekin, 2018).

4 | PLANT VIRUSES FOR THE MODIFCATION OF HYDROGELS

There is increasing interest in the development of high-aspect-ratio nanotubes (Butcher, Offeddu, & Oyen, 2014) that convert a soft polymer matrix into a stiff filler under mechanical stress by absorbing energy (Schadler, Giannaris, & Ajayan, 1998). Synthetic nanoparticles are mainly used to create composite materials that modify the physical 10 of 32 DICKMEIS ET AL. properties of hydrogels, including conductivity, tensile strength and tensile modulus. Nanoparticles can also be used to make gels responsive to stimuli such as light (Wang et al., 2015; Wu et al., 2018), changes in pH (Shen et al., 2012), the imposition of a magnetic (Reddy et al., 2011), or electric field (Servant et al., 2014), and changes in temperature (Kim, Liu, et al., 2015). The design and application of hydrogels containing synthetic nanoparticles have been comprehensively reviewed by Chen et al. (2018). Silver nanoparticles can also be used to confer antimicrobial activity upon hydrogels for wound dressing applications (Varaprasad, Mohan, Vimala, & Mohana Raju, 2011). The best-characterized nanotubes are carbon nanotubes and cellulose nanocrystals. The former are hydrophobic so they are not well dispersed in hydrogels, tending instead to form clusters and bundles that destabilize the matrix (Lu, 1997; Thostenson, Ren, & Chou, 2001). The properties of carbon nanotubes can be improved, but the methods are laborious and can influence particle stability. In contrast, cellulose nanocrystals are hydrophilic, biocompatible and thus good candidates for hydrogels (Habibi, Lucia, & Rojas, 2010). However, their structure is highly variable and influenced by the processing and purification method, making it difficult to produce monodisperse preparations (Eyholzer, Lopez-Suevos, Tingaut, Zimmermann, & Oksman, 2010). Plant viruses overcome the disadvantages of both of these structures because they are hydrophilic and self-assembling, resulting in monodisperse preparations of molecularly identical subunits (Desai, 2012; França, Zhang, Veres, Yahia, & Sacher, 2013; Mendes et al., 2013). Plant viruses are therefore robust building blocks for nanomaterials, and a promising alternative for the development of high-aspect-ratio protein-based nanostructures for use in aqueous solutions (Zheng, Dougherty, Konkolewicz, Steinmetz, & Pokorski, 2018). Plant viruses are suitable scaffolds for the development of innovative nanomaterials (Lee, Muhammad, et al., 2012) because they provide dimensional and organizational uniformity, in contrast to many artificial nanomaterials (Liu, Alim, Balandin, Mathews, & Dodds, 2005; Singh, Gonzalez, & Manchester, 2006). Plant viruses occupy the same size range as synthetic nanomaterials, that is, ranging from a few tens of nanometers to several hundred nanometers (Doug- las & Young, 2006). The diversity of plant virus structures allows the development of a library of architectures to meet the needs of different applications. Rod-shaped VNPs can be functionalized to construct high-aspect-ratio nanomaterials, whereas icosahedral VNPs are used as templates for the production of spherical nanoparticles (Wen & Steinmetz, 2016). For the construction of biomimetic structures for tissue engineering applications, filamentous particles allow the dense presentation of biological signals to promote cell attachment and proliferation (Wen & Stein- metz, 2016). To promote cell survival and proliferation, hydrogels should have a mesh size of 100–300 μm and interconnecting channels of ~40 μm (Barbetta, Barigelli, & Dentini, 2009). The minimal pore size determines the rate of nutrient and oxygen diffusion as well as waste removal and influences cell adhesion, cell interactions and migration (Bružauskaitė, Bironaitė, Bagdonas, & Bernotienė, 2016). However, pore sizes below 300 μm are preferred to enhance vascularization (Karageorgiou & Kaplan, 2005). The most extensively studied plant virus (TMV) has been use to modify porous alginate hydrogels to introduce well-defined pores in the size range 100–500 μm and this did not require an additional crosslinking step (Luckanagul et al., 2012).

4.1 | Strategies to incorporate plant virions into hydrogels

Several strategies can be used to functionalize hydrogels with VNPs, depending on which polymer forms the base material of the gel and which functional groups are required. Hydrogels can be constructed from synthetic or natural polymers or directly from the VNPs (Table 1).

4.1.1 | Crosslinking by nucleic acid hybridization

The icosahedral cucumber mosaic virus (CMV) was shown to assemble with nonspecific DNA in vitro into rod-shaped structures (Xu et al., 2008), which allowed the DNA-templated assembly of CMV coat proteins into a protein-based hydrogel (Xu et al., 2014). The CMV coat protein was expressed in E. coli BL21(DE3) Rosetta cells, purified from the resulting inclusion bodies, and then mixed with X-shaped or Y-shaped DNA molecules at a ratio of one coat protein per five base pairs of DNA at room temperature overnight. The resulting gel contained nanoscale holes, highlighting the hybridization DNA molecules. Gels prepared at lower coat protein/DNA ratios were softer, and the lowest ratio that preserved the hydrogel structure was 1:2. Lower temperatures slowed the hydrogel formation process and took up to 2 days. TMV could also be functionalized in an ordered manner on chitosan surfaces, which can be used as the basis for further functionalization strategies in gels (Yi et al., 2005). For the patterned assembly of TMV on the surface, DICKMEIS ET AL. 11 of 32

TABLE 1 Plant viruses used for hydrogel production

Hydrogel/crosslinking Virus Modification method pH Temperature Time Reference CMV Recombinant CP Assembled with branched 7.5 4C to RT 2 days to O/N Xu, Tao, and DNA Xu (2014) PVX WT, PVX-RGD, PVX-MIP No crosslinking N/D RT 15 min Lauria et al. (2017) TMGMV WT PPEGMEA, PEGDMA, O- 7.0 45C 3 hr Zheng Am-CTA and VA-044 et al. (2018) TMGMV WT PVA 7.0 RT + 40C 48 hr + 6 hr Zheng et al. (2018) TMV TMV1cys 50-RNA hybridization on 7.0 30C O/N Yi et al. (2005) DNA-linked chitosan TMV WT Aniline, poly(sulfonated 4–5 RT 24 hr Niu et al. (2007) styrene) and ammonium persulfate

TMV WT BaCl2 Neutral RT 24 hr Wu et al. (2011) TMV WT, TMV-RGD, TMV-AN Electrospinning N/D N/A N/A Wu et al. (2011) TMV WT + TMV-RGD No crosslinking to porous N/D; acidic RT, flash frozen in 5 min Luckanagul

alginate N2 and freeze- et al. (2012) dried TMV TMV-β-CD βCD to azo-hyaluronan under 7.4 RT N/D Chen, Zhao, Lin, UV light Su, and Wang (2014) TMV TMV1cys DTT coupling to 7.4 RT 15 min to 2 hr Maturavongsadit methacrylated hyaluronic et al. (2016) acid TMV TMV1cys PEGDA 7.4 RT 1 hr Southan et al. (2018) TMV WT Dual-reactive diazonium 7.0–8.5 37–50C5–30 min Ma et al. (2018) TMV TMV1cys: Pd–TMV PEG200 and PEGDA N/D RT N/D Sadeghi, Liu, Yi, and Asatekin (2019)

Abbreviations: AN, fluorescent anthracene motif; CP, coat protein; DTT, dithiotreitol; N/A, not applicable; N/D, not determined/reported; O-Am-CTA, oligoacrylamide-chain-transfer agent; O/N, overnight; PEGDMA, polyethylene glycol dimethacrylate ether acrylate; PPEGMEA, poly (polyethylene glycol) methyl ether acrylate; PVA, polyvinyl alcohol; RT, room temperature, VA-044, 2,20-azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride; WT, wild-type.

TMV1cys virions (see Section 2.1.2) were partially disassembled to reveal the 50-end, which was then hybridized with DNA linked to the chitosan. Using this method, fluorescence-labeled TMV was linked to electro-patterned chitosan- coated silicon chips and laid the foundation for the construction of TMV-based gels for applications such as corrosion prevention, biosensor development, antimicrobial coatings, drug release, and cell encapsulation (Maerten, Jierry, Schaaf, & Boulmedais, 2017). TMV1cys was also incorporated into PEG-based hydrogel microparticles containing amino-chitosan (Jung & Yi, 2014). These TMV-assembled microparticles were functionalized by biorthogonal tetrazine trans-cyclooctene cycloaddition with the red fluorescent protein R-phycoerythrin and an anti-R-phycoerythrin antibody.

4.1.2 | Crosslinking with chemical agents

Plant viruses can be used for direct crosslinking with chemicals if appropriate side chains are (a) already available on surface-exposed amino acid residues or (b) suitable side chains can be introduced by genetic modification. Two further 12 of 32 DICKMEIS ET AL. criteria must be satisfied for these methods to work: (c) the virus particles must remain stable under the reaction conditions needed for crosslinking and (d) a biocompatible crosslinking agent must be used. For example, TMV has been modified with β-cyclodextrin (βCD) to create multifunctional VNPs (Chen, Zhao, Lin, Huang, & Wang, 2013) that were later used for the production of hydrogels based on hyaluronan (Chen et al., 2014). The cyclic oligosaccharide βCD is advantageous for supramolecular chemistry due to its low cost, high solubility and biocompatibility (Szejtli, 1998). It has a hydrophilic external surface and a hydrophobic internal cavity that can accommodate molecules such as azobenzene (Chen & Liu, 2010). The TMV-βCD particle was therefore crosslinked to azo-modified hyaluronan to generate the hydrogel. However, even large quantities of the VNP (up to 3% [wt/wt] in the gelation mix) were insufficient to form solid gels, whereas the flexible bacteriophage M13 was able to produce solid hydrogels at a concentration of only 0.5% (wt/wt). TMV1cys was crosslinked into methacrylated hyaluronan hydrogels using a thiolene coupling reaction (Maturavongsadit et al., 2016). The same reaction was used to crosslink TMV1cys to PEG diacrylate (PEG-DA) hydrogels (Southan et al., 2018). The thiols can be coupled in aqueous solution via a thiol-Michael reaction under neutral to slightly alkaline conditions without a catalyst. TMV was first coupled to PEG-DA and in a second radical crosslinking step to the photo-initiator Irgacure 2959 to form the hydrogel. PEG-DA was used at a concentration of 20% (wt/wt), and 0.1% (wt/wt) Irgacure 2959 was used to start the gelation process in response to UV irradiation. TMV was added at concentrations of 0.1–2.0% (wt/wt) but did not influence the radical polymerization of the acrylate groups. A dual-reactive diazonium crosslinking reagent was recently described for the functionalization of exposed tyrosine residues (Ma et al., 2018). It was possible to crosslink wild-type TMV with this linker under mild conditions (37C, pH 7.0) and 1.25 mM of the linker was sufficient to form hydrogels containing 0.25% (wt/wt) TMV. The gelation was complete after 30 min at 37C or 5 min at 50C, whereas only semi-fluid gels were produced at 25C. Gelation also required a pH ≥7.0.

4.1.3 | Hydrogel formation without crosslinking

Biomimetic matrices can be created by mixing VNPs/VLPs in a polymer gel without crosslinking, as long as the particles are not washed out of the hydrogel and do not influence the gelling properties in a negative manner. The mesh size of the hydrogel is important in this regard. For example, ~20% (wt/wt) PEG-DA hydrogels have an average mesh size of 2 nm (Canal & Peppas, 1989; Southan et al., 2018). Thus, most plant VNPs/VLPs should be retained in the hydrogel. TMV has been used to modify porous alginate hydrogels and was added during the gelation process (Luckanagul et al., 2012). The addition of TMV did not influence pore formation but the architecture of the pores was less compact compared to unmodified hydrogels and facilitated the formation of larger, well-defined pores (Luckanagul et al., 2012). Furthermore, TMV did not change the swelling properties of the gel and was stably integrated without crosslinking. TMV has also been mixed with polyvinyl alcohol (PVA) to produce composite fibers by electrospinning (Wu et al., 2011). The resulting fibers were 200–400 nm in diameter, and the addition of TMV or TMV-RGD did not change the nanofiber diameters but allowed the robust attachment of baby hamster kidney fibroblasts (BHK cells). Most of the TMV particles were aligned along the long axis of the fiber and improved the stability of the fiber in water. Such fibers can be used to produce biomimetic ECMs by adding biomimetic cues to the particle surface. Wu, Jiang, Zan, Lin, and Wang (2017) were able to control the alignment of TMV particles in alginate hydrogels via shear flow induced by the unidirectional flow of the precursor solution and the orientation was quickly fixed during the rapid sol–gel transition in situ. The orientation and distance between the TMV particles could be regulated by adjusting the concentration of alginate and TMV. The introduction of TMV reduced the degree of swelling in the hydrogel and improved the mechanical strength of the gel (Wu et al., 2017). TMV was also homogeneously integrated into PEG-DA hydrogels without crosslinking (Southan et al., 2018). Washout analysis revealed that TMV was present only in the first elution fraction and that the total quantity of virus washed from the gel was 1–3% of the initial amount. This indicated that only TMV particles adsorbed on the hydrogel surface were washed out whereas internal particles remained stable.

4.2 | Hydrogels based on a plant virus matrix

As an alternative to embedding plant viruses into polymer matrices, hydrogels can be constructed directly from plant VNPs/VLPs or coat protein solutions. The particles or coat proteins must be crosslinked by chemical agents or per- suaded to interact by introducing functional sites that favor the assembly of higher-order protein structures. Several DICKMEIS ET AL. 13 of 32 plant viruses have been used in this manner as a basis for hydrogel formation (Table 1). Native TMV has been crosslinked with a dual-reactive diazonium crosslinker (Ma et al., 2018). The resulting gels were not digested by naturally occurring enzymes such as caseins and lysozymes suggesting they are likely to remain stable and degrade only slowly following their incorporation into tissue. The hydrogels were loaded with camptothecin and the antitumor drug did not change the gelation characteristics. During dialysis, 72.1% of the drug was released after 72 hr. The functionality and biosafety of the gels were tested by spraying or smearing them onto tobacco plants. None of the plants were infected, indicating that crosslinking prevents the release of individual virions. As discussed earlier, CMV coat proteins assembled with non-specific DNA in vitro into rod-shaped structures (Xu et al., 2008) which allowed the DNA- templated assembly of CMV coat proteins into a protein-based hydrogel using X-shaped or Y-shaped DNA (Xu et al., 2014). It would be interesting to determine whether different combinations or ratios of branched DNA affect the characteristics of the gel and allow the fine-tuning of properties such as stiffness or stability. TMV has also been incorporated into porous alginate hydrogels to determine its influence on gel properties (Luckanagul et al., 2012). Approximately 13% of the particles were released from the gel during the first 24 hr and the release rate plateaued thereafter. The response of the gels to compression changed from linear without the virus to a nonlinear response with the virus, indicating that the virus-containing gels were stiffer in the lower strain regions but more pliable at higher strains. No mass losses were observed in 4 weeks, which allowed the completion of cell culture experiments. Tobacco mild green mosaic virus (TMGMV) has been used as a high-aspect-ratio nanotube to modify the characteristics of a hydrogel (Zheng et al., 2018). TMGMV was chosen because it resembles TMV in many ways but has a much narrower host range, which improves the biosafety of the final product. TMGMV was crosslinked to a PEG hydrogel which was produced by reversible addition-fragmentation chain-transfer polymerization. The virus was stable under the gelation conditions (3 hr, 45C) and increased the toughness of poly-PEGMEA (PPEGMEA) hydrogels. Increasing concentrations of the virus (0.1–5% TMGMV) reduced the swelling capacity from sixfold to fourfold as anticipated because the virus was physically crosslinked in the gel. Gels containing up to 2% TMGMV were smooth matrices with homogenously dispersed virions, achieving a consistent morphology. In contrast, those containing 5% TMGMV showed additional effects, such as defects and voids within the matrix caused by aggregation, which allowed more water to enter and improved the swelling of the hydrogels. The compressive modulus of the gels containing 2% TMGMV was approximately twofold higher than the pure polymer control. Shear rheology testing showed stable elastic properties, and the storage modulus (G0) of gels containing 1% TMGMV was sevenfold higher than that of pure polymer gels. TMGMV has also been used to strengthen PVA hydrogels, which show a high degree of hydrogen bonding. The gels were formed by solvent casting, with TMGMV included at concentrations ranging from 0.8% to 3.2%. PVA films adopt a crystalline structure due to hydrogen bonding, but this was modified in the presence of the virus. The tensile modulus and strength of the gels increased in the presence of up to 1.6% TMGMV due to the greater stiffness, but dropped at higher concentrations because the virus showed an increasing tendency to aggregate. Whereas TMGMV increased the mechanical strength of PPEGMEA and PVA hydrogels, TMV reduced the mechanical strength of hyaluronan hydrogels (Maturavongsadit et al., 2016). Hyaluronan is naturally abundant in the synovial fluid of cartilage and is therefore useful for the preparation of cartilage-mimicking hydrogels (Tibbitt & Anseth, 2009). TMV1cys has been crosslinked into hyaluronan hydrogels by mixing the virus with methyl hyaluronan (Maturavongsadit et al., 2016). The gels remained stable for 6 months at room temperature and showed a similar structure to hyaluronan hydrogels. The TMV1cys-hyaluronan hydrogels formed abundant interconnected pores of 20– 100 μm. Although chondrocytes prefer pore sizes of 10–15 μm (Grad, Zhou, Gogolewski, & Alini, 2003), the gels helped to maintain the phenotypes of these cells. The swelling rate of the virus-modified hydrogel was slightly higher than that of the pure polymer, with a 3.5-fold increase in weight and no weight loss during a period of 21 days (Maturavongsadit et al., 2016). The addition of TMV accelerated gelation, which is advantageous for injectable hydrogels. Furthermore, the modified hydrogels supported the survival of bone marrow-derived stem cells (BMSCs) and promoted cho- ndrogenesis without supplements, which may account for the lower metabolic activity of the cells compared to those in unmodified gels. BMSCs encapsulated in TMV1cys-hyaluronan gels expressed higher levels of bone morphogenetic protein 2 (BMP2) and accumulated more collagen. The deposition of collagen added to the mechanical strength of the gel, thus outperforming the unmodified gels during later culture states. TMV1cys has also been crosslinked to PEG-DA hydrogels by the same method (Figure 4a), and increasing the concentration of TMV1cys led to a significant increase in G0, which was not the case for gels containing unlinked wild-type TMV (Southan et al., 2018). The trend of hydrogel 0 0 0 stiffness was: G TMV1cys > G PEG-DA > G wt-TMV. This was not significant when the solid content of the gel was 20.1– 20.3% (wt/wt) but became more significant at higher concentrations: p < .05 at 21.0% (wt/wt) and p < .01 at 22.0% (wt/ 14 of 32 DICKMEIS ET AL. wt). The equilibrium water content was consistent in all formulations. Interestingly, the mechanical properties of PEG- DA and porous alginate hydrogels were influenced by TMV in different ways, with the virus increasing the compressive modulus of the alginate gels during unconfined uniaxial compression (Luckanagul et al., 2012). This shows that the type of hydrogel (and thus the non-covalent interactions between the polymer and virus particle) has a significant effect, and must be taken into account during hydrogel design. Alginate forms more noncovalent interactions with VNPs than the PEG backbone, which explains the difference between PEG-DA and alginate gels in the presence of TMV. The absence of larger TMV aggregates in the hydrogels containing TMV1cys indicates that PEG preferentially forms bonds between coat proteins on the same TMV particle rather than linking coat proteins from different particles during the thiol-Michael reaction prior to crosslinking. In contrast, the presence of 1.5% (wt/wt) TMV in alginate gels resulted in immediate clouding, indicating aggregate formation, whereas TMV-PEG-DA solutions stayed clear upon mixing (Wu et al., 2017). To support neural cells, TMV has been aligned in polyaniline films (TMV/PANi) and electroactive TMV nanofibers with polyaniline and polystyrene (TMV/PANi/PSS) (Wu et al., 2015). TMV can form conductive nanofibers several micrometers in length following incubation with aniline, polysulfonated styrene and ammonium persulfate at room temperature (Niu et al., 2007). The films remained stable when incubated in PBS for 3 days. At pH 4, TMV/PANi/PSS − materials showed a conductivity of 10 5 S/cm. Rat PC12 cells seeded on surfaces modified with TMV proliferated more efficiently in the conductive formulation and extended neurites on these surfaces. The alignment of TMV-electroactive nanofibers by high pressure shear forces (Zan, Feng, Balizan, Lin, & Wang, 2013) guided neurite growth along the fibers and produced longer neurites. PVX has also been tested as a flexible nanoparticle for hydrogel production in tissue engineering applications (Lauria et al., 2017). Virus particles were added during the gelation of agarose hydrogels and formed filamentous, network-like nanostructures within the hydrogels and on coated surfaces, which remained stable for more than 14 days. The morphology observed following the incorporation of high concentrations of particles during film formation possibly reflects liquid crystalline phase transition (Wang, Wang, Li, & Mao, 2013). The higher aspect ratio and flexuous morphology of PVX can influence hydrogels in a different manner to rigid particles such as TMV, due to the less constrained but more extensive interaction with polymers. Similarly, flexible bacteriophages modified with βCD interact more efficiently with azo-modified hyaluronan to produce functional hydrogels, whereas rigid TMV particles were unable to produce stable gels (Chen et al., 2014).

4.3 | Plant viruses for the addition of biomimetic cues to artificial environments

Ligand density and ligand concentration in the microenvironment influence the behavior of cells and therefore control proliferation and differentiation. The direct modification of hydrogels to display peptide ligands cannot achieve precise control over ligand distribution (Destito, Yeh, Rae, Finn, & Manchester, 2007). In contrast, plant VNPs or VLPs allow the dense local concentration of peptide ligands, which can significantly enhance their effects. For example, the spacing of the integrin-binding peptide RGD has a huge impact on cell responses and differentiation (Cavalcanti-Adam et al., 2006; Deeg et al., 2011; Le Saux, Magenau, Böcking, Gaus, & Gooding, 2011; Wang, Yan, et al., 2013). VNPs based on bacteriophage M13 (Chung et al., 2011), CPMV (Hovlid et al., 2012), turnip yellow mosaic virus (TYMV) (Zan, Sitasuwan, Powell, Dreher, & Wang, 2012), TMV (Lee, Nguyen, et al., 2012; Luckanagul, Lee, You, Yang, & Wang, 2015; Luckanagul et al., 2016; Wu et al., 2011), and PVX (Lauria et al., 2017) have already been used to display clustered RGD peptides. Table 2 shows peptide sequences that can be used as biomimetic cues to modify cell responses. The augmenta- tion of viruses with such peptides, and the resulting cell responses, are summarized in Table 3.

4.3.1 | Plant virus coatings

Several studies have addressed the effects of TMV and other plant viruses carrying functional peptides when applied as coatings to tissue culture vessels (Kaur, Valarmathi, et al., 2010; Lee, Muhammad, et al., 2012; Maturavongsadit et al., 2016; Sitasuwan et al., 2012; Sitasuwan et al., 2014) or when incorporated into porous alginate hydrogels and implanted in mice and rats (Luckanagul et al., 2012; Luckanagul et al., 2016; Maturavongsadit et al., 2016). Plant viruses were first tested for their effect on cultured cells by coating them onto cell culture plates before seeding cells onto the surface, and observing the impact on properties such as cell adhesion, survival and differentiation. TMV DICKMEIS ET AL. 15 of 32

FIGURE 4 Plant virus functionalization can modify cell behavior on 2D and 3D scaffolds. (a) Production of TMV-PEG-DA hydrogels. (i) Schematic representation of the coupling of PEG-DA to TMV1cys via a thiol-Michael reaction resulting in TMV particles functionalized with a double bond (TMV-PEG-DA). (ii) Subsequent photo-polymerization of the attached acrylate functionalities with more PEG-DA to hydrogels (Reprinted with permission from Southan et al. (2018). Copyright 2018 The Royal Society of Chemistry (RSC) on behalf of the European Society for Photobiology, the European Photochemistry Association, and RSC). (b) Hydrogel films and fibers support neurite outgrowth. (i) Production of 2D-TMV/PANi/PSS films to support neurite growth. (ii) AFM images of aligned TMV/PANi/PSS nanofibers in capillaries coated with PDDA/PSS2.5 (white arrow shows the direction of the flow; scale bar = 1 μm). (iii) Representative fluorescence (FDA staining) images of PC12 cells cultured on aligned TMV/PANi/PSS for 3 days (Reprinted with permission from Wu, Feng, Zan, Lin, and Wang (2015). Copyright (2015) American Chemical Society). (c) Porous alginate hydrogels can support bone regeneration. (i) Generation of virus-functionalized porous composite hydrogels. (ii) TMV was added 5 min before the foamy mixture was frozen and lyophilized and the lyophilized sample was cross-linked with CaCl2. (iii) Scanning electron microscopy (SEM) images of hydrogels and TMV particles, TMV- PAH at different magnifications. (iv) SEM images of a single cell inside TMV-PAH, scale bar = 10 μm (Reprinted with permission from Luckanagul et al. (2012). Copyright (2012) American Chemical Society). (d) PVX functionalized with biomimetic cues for the functionalization of agarose hydrogels. (i) Schematic representation of genetically engineered PVX for the presentation of MIP and RGD and a combination thereof. (ii) SEM image of hMSC pseudopodia interconnected with PVX-RGD-MIP on TCPS coated with 0.05 mg/cm2 PVX after 8 hr (scale bar = 2 μm). (iii) Human MSCs embedded in 0.5% agarose with 0.2% PVX-RGD incubated for 14 days, scale bar = 100 μm (Reprinted with permission from Lauria et al. (2017). Copyright 2017 Elsevier) 16 of 32 DICKMEIS ET AL.

TABLE 2 Peptides that can be used as biomimetic cues to modify cell responses

Motif Peptide sequence Function in hydrogel Reference RGD GRGDSPG, AVTGRGDSPASS Cell adhesion Lee, Nguyen, et al. (2012) IKVAV GGIKVAVGGG Promote neurite growth, and Jung, Moyano, and selective differentiation of Collier (2011) neural progenitor cells MIP3 MSESDSSDSDSKS Mineralization-inducing peptide Lauria et al. (2017) BMP2-binding peptide YPVHPST BMP2 binding for bone Behanna, Donners, Gordon, regeneration and Stupp (2005) TGFβ1-binding peptide LPLGNSH TGFβ1 binding for cartilage Behanna et al. (2005) regeneration DGEA DGEA ECM-mimicking biomaterial Anderson et al. (2011) VEGF mimetic peptide GKLTWQELYQLKYKGI Ischemic tissue repair Webber et al. (2011) YIGSR GGYIGSRGGG Laminin cell adhesion motif Jung et al. (2011) REDV GGREDVGGG Fibronectin cell adhesion motif Jung et al. (2011) RYVVLPR RYVVLPR Laminin cell adhesion motif Genové, Shen, Zhang, and Semino (2005) TAGSCLRKFSTM TAGSCLRKFSTM Collagen IV cell adhesion motif Genové et al. (2005) ALKRQGRTLYGF ALKRQGRTLYGF Osteogenic growth and Horii, Wang, Gelain, and osteopontin cell adhesion motif Zhang (2007) DGRGDSVAYG DGRGDSVAYG SKPPGTSS SKPPGTSS Bone marrow homing peptide, Gelain, Bottai, Vescovi, and 3D cell culture Zhang (2006) PFSSTKT PFSSTKT P15 GTPGPQGIAGQRGVV Collagen I mimetic Lee, Nguyen, et al. (2012) PHSRN3 EDRVPHSRNSIT Fibronectin cell adhesion motif

(Kaur, Valarmathi, et al., 2010) and TYMV (Kaur et al., 2008) were shown to promote the differentiation of rat BMSCs more efficiently than uncoated surfaces. TMV promoted the upregulation of osteospecific genes soon after the induction of osteogenesis, with BMP2 expression reaching a peak after 8 hr (Sitasuwan et al., 2012). TMV mineralized with phos- phate triggered the upregulation of osteospecific genes more efficiently than native TMV (Kaur, Wang, Sun, & Wang, 2010) but TMV functionalized with the cell-binding motif GRGDSPS (TMV-RGD) induced even faster differentiation and calcium sequestration, and osteospecific gene expression was observed as soon as 2 days after induction (Lee, Muhammad, et al., 2012). Chemically coupled RGD with a spacing of 2–4 nm on the TMV surface showed greater stability but only a slight increase in cell attachment compared to recombinant TMV-RGD particles (Sitasuwan et al., 2014). Wild-type TMV, PVX, TYMV, and TVMV supported osteogenesis and caused the differentiating BMSCs to cluster on plates coated with poly-D-lysine, whereas CPMV did not promote clustering and there was no enhancement of osteogenesis (Metavarayuth et al., 2015). The authors hypothesized that BMSCs attached weakly to TMV, PVX, TYMV, and TVMV, permitting a certain amount of motility and allowing the formation of cell aggregates that facilitated osteogenesis, whereas CPMV has a vimentin-binding ligand that binds cells tightly and reduces motility (Koudelka et al., 2009). BHK and Chinese hamster ovary (CHO) cells did not attach strongly to wild-type TMV or bovine serum albumin (BSA) control coatings (Lee, Nguyen, et al., 2012) but showed improved adhesion to modified TMV particles displaying cell adhesion motifs, such as TMV-RGD1 (-S155GPATG-RGD-) and TMV-RGD7 (-S155AVTG-RGD-). The strength of attachment differed according to the displayed motif, possibly reflecting the tertiary conformation adopted by the flanking sequences. For example, the fibronectin-binding site (PHSRN) was weaker than the RGD sequences and the collagen- derived peptide DGEA was only slightly more effective than native TMV. Overall, TMV-P15 achieved the strongest attachment to BHK and CHO cells. Neuronal cells tend to cluster on unfavorable surfaces (Saha, Irwin, Kozhukh, Schaffer, & Healy, 2007) and hence need suitable cell-binding motifs on artificial surfaces to encourage cell DICKMEIS ET AL. 17 of 32

TABLE 3 Effect of plant viruses on different cell types

Plant virus Modification Cell type Material Effect Reference 2D coatings and films CPMV WT Rat BMSCs PDL-coated plates No effect on Metavarayuth, osteogenesis Sitasuwan, Luckanagul, Feng, and Wang (2015) PVX WT Rat BMSCs PDL-coated plates Early osteogenesis 6 hr Metavarayuth after osteoinduction et al. (2015)

TMV TMV-PO4 Rat BMSCs APTES-coated glass Higher upregulation of Kaur, Wang, slides osteospecific genes et al. (2010) compared to wt virions TMV WT Rat BMSCs APTES-coated glass Promotion of Kaur, Valarmathi, slides osteogenic et al. (2010) differentiation inducing early-onset mineralization TMV WT, TMV-RGD, BHK cells Co-electrospun Improved cell Wu et al. (2011) TMV-AN PVA and TMV attachment on TMV- fibrous substrates RGD in a 12-well plate TMV TMV-RGD Rat BMSCs Polyelectrolyte Promotion of cell Lee, Muhammad, membrane differentiation and et al. (2012) calcium sequestration within 2 days of osteoinduction TMV WT Rat BMSCs APTES-coated glass BMP2 mRNA and Sitasuwan slides protein levels highest et al. (2012) after 8 hr of osteoinduction TMV WT, TMV-RGD, BHK and CHO cells High-binding 96- No cytotoxicity, Lee, Nguyen, TMV-P15, TMV- well plates improved et al. (2012) DGEA, TMV- attachment on TMV PHSRN3 coated surfaces TMV TMV-RGDa Rat BMSCs APTES-coated glass Improved cell adhesion Sitasuwan, Lee, Li, slides Nguyen, and Wang (2014) TMV WT Rat BMSCs PDL-coated plates Early osteogenesis 6 hr Metavarayuth after osteoinduction et al. (2015) TMV WT Rat PC12 neural cells TMV/PANi and Lower viability on Wu et al. (2015) TMV/PANi/PSS particles compared coated glass slides to glass, but significant higher cell proliferation TMV WT, TMV-RGD Mouse adult PPCs Inkjet printing on Cell adhesion and Yang et al. (2015) BSA-coated or formation of tailored PS-coated silicon islets wafers TMV WT, TMV-RGD, Mouse N2a neural cells Added to culture No cytotoxicity, Feng et al. (2015) TMV-P15, TMV- medium, PDL- improved DGEA, TMV- coated plates proliferation on PHSRN3 TMV coated surfaces (Continues) 18 of 32 DICKMEIS ET AL.

TABLE 3 (Continued)

Plant virus Modification Cell type Material Effect Reference TMV TMV, TMV1lys Mouse NIH-3T3 fibroblasts PDADMA and PSS Supported high cell Tiu et al. (2017) bilayers on QCM density, healthy cells crystals TVCV WT Rat BMSCs PDL-coated plates Early osteogenesis 6 hr Metavarayuth after osteoinduction et al. (2015) TYMV WT Rat BMSCs Silane-coated glass BMSCs adhered, Kaur, Valarmathi, slides proliferated and Potts, and differentiated into Wang (2008) osteoblast-like cells and showed an early maturation and mineralization of cells TYMV WT Rat BMSCs PDL-coated plates Early osteogenesis 6 hr Metavarayuth after osteoinduction et al. (2015) 3D scaffolds PVX WT, PVX-MIP, Human MSC Agarose hydrogels No cytoxicity, Lauria et al. (2017) PVX-RGD improved cell attachment on PVX- RGD, improved mineralization with PVX-MIP and-RGD TMV WT, TMV-RGD Rat BMSCs Porous alginate TMV and TMV-RGD Luckanagul hydrogels promoted calcium et al. (2012) deposition TMV WT, TMV-RGD implanted in mice Porous alginate No toxicity or chronic Luckanagul hydrogels inflammation et al. (2015) detected TMV TMV1cys Rat BMSCs Methacrylate No cytotoxicity, Maturavongsadit hyaluronate improved BMP2 et al. (2016) hydrogels expression and chondrogenic ECM formation TMV WT, TMV-RGD implanted in rats Porous alginate No systemic Luckanagul hydrogels inflammation or et al. (2016) severe immunogenic effects, TMV-RGD improved bone remodeling and maturation

Abbreviations: AN, fluorescent anthracene motif; ATPES, aminopropyltriethoxysilane; BMP2, bone morphogenetic protein 2; BMSC, bone marrow-derived stem cell; BHK, baby hamster kidney; CHO, Chinese hamster ovary; hMSC, human mesenchymal stem cell; PANi, polyaniline; PDL, poly-D-lysine; PPC, pancreatic progenitor cell; PPDADMAC, poly(diallyldimethyl ammonium chloride); PSS, polystyrene; QCM, quartz crystal microbalance; TVCV, turnip vein clearing virus; WT, wild-type. aRGD was added by CuAAC bioconjugation and was spaced at 2–4-nm intervals.

proliferation. Mouse Neuro2a cells tended to cluster on plates coated with wild-type TMV but formed other distribution patterns when plates were coated with TMV displaying cell-binding motifs, and extended longer neurites (Feng et al., 2015). DICKMEIS ET AL. 19 of 32

Thin TMV films have been constructed by the slow evaporation of a TMV suspension between two glass slides (Lin et al., 2011). When the concentration of the virus is too low, the evaporation process forms thin stripes rather than a continuous film. As the virus concentration increases the stripes broaden and eventually begin to overlap at a concentration of 0.1 mg/ml. Once the concentration reaches 0.7 mg/ml, a continuous TMV film is formed with particles mainly oriented parallel to the contact line. BMSCs cultured on TMV-coated glass slides spread on such templates and follow the orientation of the stripes. Furthermore, multilayer structures can be constructed by combining wild-type TMV and TMV1lys by electrostatically driven layer-by-layer assembly (Tiu et al., 2017). These TMV assemblies supported the adhesion of NIH-3T3 fibroblasts and cell attachment increased with increasing layer deposition. A cellulose acetate undercoat layer provided sufficient mechanical strength to create a free-standing multilayer membrane suitable as a 2D scaffold.

4.3.2 | Plant virus scaffolds (2D)

Films (2D scaffolds) can be used to create modified surfaces with novel properties and to design functional devices such as biosensors or biomaterials to study cell–surface interactions (Kehr, Atay, & Ergün, 2015). One example is the TMV/ PANi films and TMV/PANi/PSS fibers discussed above, which allow the directional growth of neurites (Figure 4b). However, other cells need a highly constrained environment for differentiation. For example, pancreatic progenitor cells (PPCs) are promising candidates for the production of β-cell mass during the treatment of diabetes (Noguchi, 2010). These cells perform better when configured as islet-like clusters (Ramiya et al., 2000) because larger clusters suffer nutrient and oxygen deprivation (Ramachandran, Williams, Huang, Novikova, & Stehno-Bittel, 2013). Therefore, it is advantageous to produce islets of 50–150 μm for a superior insulin response (Lehmann et al., 2007). Inkjet printing of TMV-RGD solutions in patterns allowed the fine-tuning of islet formation by adult mouse PPCs (Yang et al., 2015). This shows the suitability of plant viruses in inkjet printing techniques to produce bespoke patterns of biological signals. Superlattices can further augment the properties and potential applications of hydrogel films. TMV can be formu- lated into superlattices tens of micrometers in length by mixing with Ba2+ in a dilute solution (Li, Winans, & Lee, 2011). Surface-bound Ba2+ ions not only screen electrostatic repulsion between TMV particles but also induce mutual attraction when the molar ratio of Ba2+ to TMV reaches a critical value. Distances between TMV particles in the superlattice did not vary with the Ba2+/TMV ratio, TMV concentration or pH as long as the films were formed with a Ba2+/TMV molar ratio higher than the critical value. Analysis of the viscoelasticity of the superlattice showed that Ba2+TMV is rigid during initial contact but experiences deformation under constant pressure (Wang, Wang, Li, & Lee, 2014). Inter- estingly, other divalent ions such as Cd2+,Zn2+,Ca2+, and Mg2+ did not induce superlattice ordering (Li et al., 2011). However, TMV could be formed into superlattices by depletion interaction with carboxymethylcellulose and sodium dodecylsulfate (Li, Zan, Winans, Wang, & Lee, 2013). The mixture of TMV and methylcellulose can form physical gels when heated above a critical temperature, thus creating a temperature-responsive medium. Catalytically active VNPs in hydrogels can reduce heavy metals during the treatment of wastewater. For example, palladium-decorated TMV1cys (TMV1cys-Pd) can be used as a catalyst to reduce metals such as hexavalent chromium (Yang, Manocchi, Lee, & Yi, 2010). The incorporation of TMV1cys-Pd into hydrogels will allow the development of new wastewater treatment devices. For example, TMV-hydrogel microdroplets were created from a TMV PEG-DA solution using microfluidics followed by photo-polymerization of the droplets by exposure to UV light to create functional microparticles (Lewis et al., 2010). It was also possible to create Janus particles with different functionalities by combining TMV1cys-Pd with magnetic nanoparticles, allowing their separation from a bulk solution in a magnetic field. Photo-polymerization with PEG can be used to control the shape of the microparticles (Yang, Choi, Lee, & Yi, 2013; Yang, Kang, & Yi, 2018). The incorporation of catalytic VNPs in selective layers can prevent contact with foulants or macromolecules in wastewater, thus preserving their catalytic activity (Sadeghi et al., 2019). Interfacially initiated free radical polymerization (IIFRP) enables the fabrication of ultrathin hydrogel selective layers on porous supports (Sadeghi et al., 2018) so that TMV1cys-Pd nanoparticles can be integrated into PEG-based hydrogels. Ultrathin hydrogel layers achieved a high catalytic turnover for the reduction of hexavalent chromium, with up to 98% conversion in single-pass filtration experiments. The membranes were stable during long-term operation for at least 3 days. These results highlight the promising integration of plant virus nanomaterials to create advanced membranes with new and versatile functions. 20 of 32 DICKMEIS ET AL.

4.3.3 | Plant virus scaffolds (3D)

Plant virions can be used as building blocks for the construction of 3D hydrogels because they are monodisperse, polyvalent and biocompatible (Pokorski & Steinmetz, 2011). For example, TMV was incorporated into porous alginate hydrogels, which were seeded with BMSCs. The cells survived in hydrogels modified with TMV and TMV-RGD, and cells in the latter showed the highest metabolic activity (Luckanagul et al., 2012), penetrated deeply into the gel and adopted a consistent morphology (Figure 4c). The TMV and TMV-RGD gels induced the deposition of more calcium than unmodified gels, but only TMV-RGD induced early osteogenesis (although the process stalled after 7 days). The biocompatibility and potential toxicity of TMV-containing gels were tested by subcutaneous implantation into BALB/c mice (Luckanagul et al., 2015). The mice showed normal behavior after implantation and no weight loss was observed. The inflammatory response was monitored by counting total white blood cells, lymphocytes, monocytes and neutro- phils, and all counts remained within the normal range for the 4 weeks of the experiment, with no difference between the TMV gel group and controls. Macrophages and lymphocytes were observed in the tissue around the gel disks and mild inflammation in the center of the hydrogels was detected, but this resolved quickly in line with normal reactions during wound healing following the implantation of medical devices (Anderson, Rodriguez, & Chang, 2008). The appearance of pro-inflammatory cells 2 weeks after implantation indicated acute local inflammation, which is necessary to heal and reconstitute the implant site. The ultimate goal is the replacement of the implant through tissue regeneration, the migration of native parenchymal cells and the formation of fibroblastic scar tissue (Eiselt, Yeh, Latvala, Shea, & Mooney, 2000; Nunamaker, Purcell, & Kipke, 2007). TMV particles are cleared from the blood via the reticulo- endothelial system (RES) as described in Box 2, and the presence of many particles therefore has the potential to cause spleen enlargement and organ damage (Bruckman et al., 2014; Wu et al., 2013). However, no spleen damage was observed in mice implanted with the TMV-containing hydrogels, and the titer of TMV-specific antibodies was low, suggesting the VNPs are released slowly from the hydrogels into the circulation. The same porous alginate hydrogels were also tested as scaffolds for the regeneration of cranial defects generated in rats (Luckanagul et al., 2016). The total white blood cell count was generally lower in rats implanted with the porous alginate hydrogels compared to controls, but there were no significant differences between rats injected with different gel formulations. Furthermore, there was no major inflammatory response and no evidence of RES toxicity. Rats injected with gels carrying the TMV-RGD particles achieved a slightly higher bone volume and were more likely to regenerate denser bone structures. Given the different effects of rigid and flexible virions on the properties of hydrogels, PVX has also been tested as a hydrogel additive for tissue regeneration applications (Lauria et al., 2017). For example, PVX displaying the RGD peptide was mixed with agarose and the resulting gel was seeded with hMSCs (Figure 4d).The PVX virions formed a stable network in the gel, and the hMSCs interacted with the RGD peptide and associated with the fibers after 14 days. The hMSCs did not associate with the nonadhesive unmodified agarose gel, indicating that the PVX-RDG particles promote cell attachment. The hMSCs also grew normally on PVX-coated tissue culture plates and showed a comparable metabolic activity to cells on unmodified surfaces, but the RGD peptide promoted cell attachment, expansion and spreading.

BOX 2 CLEARANCE OF PLANT VIRUSES Plant viruses are regarded as foreign material by the human body and are targeted by natural clearance mechanisms. One of the most important is the reticulo-endothelial system (RES), which removes nanoscale foreign agents by phagocytosis and is therefore the main route for virus particle clearance (Bruckman et al., 2014; Wu et al., 2013). The primary cell types responsible for the RES are monocytes and macrophages, which accumulate in lymph nodes and the spleen, although Kupffer cells of the liver are also part of the RES (De Jong et al., 2013). Plant viruses with a negative surface charge tend to have short circulation times in vivo because they are rap- idly cleared by the RES (Wen & Steinmetz, 2016) whereas positively charged particles bind to the negative charges on the surface of mammalian cells and are retained for longer (Shukla et al., 2013). TMV and PVX particles are cleared from the circulation via the RES and the presence of large numbers of particles can therefore cause the enlargement of the spleen, leading to organ damage (Bruckman et al., 2014; Wu et al., 2013). DICKMEIS ET AL. 21 of 32

Interestingly, wild-type PVX mildly inhibited cell adhesion (as did the BSA control) reflecting the predominance of non-charged amino acid residues such as alanine, serine and threonine on the virion surface.

5 | CHALLENGES FOR THE UTILIZATION OF PLANT VIRUSES

Despite all the advantages of plant VNPs discussed above, several hurdles must be overcome before they can be used routinely for diagnostic, therapeutic or engineering applications. Plant VNPs do not replicate in humans and cannot therefore cause a human infectious disease, but they can still infect plants and must therefore be considered in terms of potential environmental damage. Although coat protein modifications tend to reduce the infectivity of VNPs in their natural hosts (Ma et al., 2018) they are nevertheless persistent, with some VNPs remaining infectious even after incubation in serum for 24 hr (Blandino et al., 2015). The potential for engineered VNPs to spread like viruses can be addressed by adopting simple precautions, such as using viruses with a narrow host range or a host range inconsistent with plants in the local environment of the clinic. The development of VLPs instead of VNPs eliminates the potential for infection, but not all plant viruses can be tailored to produce VLPs because many plant virions assemble around a nucleic acid template. One strategy that may overcome this hurdle is the use of deconstructed viruses, in which the genetic material is present but lacks key functions required for infection (Peyret & Lomonossoff, 2015). Alternatively, synthetic genetic material can be used that does not encode any genes but contains the necessary origin of assembly sequences (Shukla et al., 2015). Some viruses also assemble with nonspecific nucleic acids (Xu et al., 2008) or with RNAs from other plant viruses (Arkhipenko et al., 2011) and can be utilized to create new artificial structures (Wege & Koch, 2019; Xu et al., 2014). For icosahedral viruses such as CPMV, it is often possible to remove the genomic RNA after assembly using a simple chemical or enzymatic treatment (Ochoa, Chatterji, Lin, & Johnson, 2006) but this approach has yet to be demonstrated with filamentous viruses. Exposure to UV light can also lead to the production of non-infectious particles by generating lethal RNA damage without affecting the protein shell (Rae et al., 2008). In the case of barley stripe mosaic virus, damage to the virions was visible at the microscopic level after UV irradiation but the virions remained intact (Urban et al., 2011). The genomic RNA of cucumber green mottle mosaic virus and TMV can also be destroyed with gamma radiation, although this also compromises the integrity of the particles (Hong, Jeong, & Jeong, 2017; Jeong & Choi, 2017). Although plant viruses do not infect humans they are potentially immunogenic, and each VNP/VLP must be tested systematically to ensure safety. Biomaterials are generally regulated under the DIN EN ISO 10993 guidelines, and evaluation criteria vary depending on the nature and duration of contact (e.g., surface device vs. implant, temporary vs. long-term, and precise position). The evaluation involves extensive toxicity and reaction testing. Nanoparticles are rela- tively new as an innovation in biomedical devices, and safety evaluations have yet to be harmonized, particularly in the case of plant viruses (which are novel even within this emerging field). The clearance of plant viruses from the blood has been monitored and appears to follow the typical kinetics of nanoparticles (Bruckman et al., 2014; Pitek, Jameson, Veliz, Shukla, & Steinmetz, 2016; Wu et al., 2013) (see also Box 2). The most advanced plant virus hydrogels are those based on TMV, and these have been subject to the most extensive tests. In mice, TMV was released slowly from the hydrogel into the circulation and did not induce the significant production of antibodies (Luckanagul et al., 2015). Among seven rats implanted with TMV-RGD hydrogels, only four produced antibodies against TMV, whereas rats implanted with unmodified TMV hydrogels produced higher titers of TMV-specific antibodies (Luckanagul et al., 2016). This shows that the limited amount of antigen released from the hydrogels nevertheless leads to a humoral immune response lasting at least 10 weeks, indicating that the repeated application of hydrogels containing the same virus may trigger a stronger immune response. However, the persistence of this humoral response in animals already implanted with a virus-based hydrogel is unclear. A detailed analysis of cellular responses around the implant suggested that TMV induces cytokine production in migrating cells, thus recruiting inflammatory cells more efficiently to the implant site. This could accelerate the healing process but could also induce chronic inflammation ultimately leading to cancer (Hussain & Harris, 2007). However, this risk is expected to be low because TMV has a rather short biological half-life (Bruckman et al., 2014). Even if the modifications applied to VNPs render them inactive, as shown for a TMV-based hydrogel (Ma et al., 2018), antibodies can still be generated against the coat proteins. Humans already produce antibodies against some plant virus antigens because they are inhaled or ingested (Liu et al., 2013). However, no adverse effects have been observed thus far. Additionally, plant viruses can be shielded from immune detection to reduce their antigenicity by coating them with PEG (Bruckman et al., 2014; Lee et al., 2015) or serum albumin (Pitek, Jameson, et al., 2016). Eventually, it might be possible to produce artificial structures via 3D printing or synthetic pathways (Wu 22 of 32 DICKMEIS ET AL. et al., 2019). Furthermore, for hydrogel-based applications, anti-inflammatory signals could be added to the hydrogel in order to limit any immunostimulatory effects.

6 | CONCLUSION

The applications of hydrogels are diverse, ranging from biomaterials to sensors and from agricultural applications to wastewater treatment. Predictable modifications that confer physicochemical and biological properties such as cytocompatibility, tuneable internal structures, response to stimuli, injectability, and self-healing are therefore highly desirable (Zhi et al., 2018). However, it can be challenging to combine many desirable traits into a single hydrogel. Nanoparticles based on proteins or peptides are cytocompatible and allow the addition of biological cues for the construction of biomimetic ECMs to tailor cell responses (Halberstadt et al., 2002; Luckanagul et al., 2012). Proteins can be produced in hosts such as bacteria, yeast, mammalian or insect cells, and in plants (Sack et al., 2015). Plant virions offer the additional advantage of self-assembly into monodisperse nanoparticles adding further structural tuneability to hydrogels. Filamentous particles are especially promising for the modification of hydrogel mechanics, with different effects generated by particles structures (Chen et al., 2014; Wen & Steinmetz, 2016). VNPs allow the presentation of peptides or whole proteins to promote cell attachment and differentiation (Lauria et al., 2017; Luckanagul et al., 2016). VNPs directly crosslinked to the hydrogel matrix often change the mechanical behavior of the gel and can have different effects on different polymers (Maturavongsadit et al., 2016; Southan et al., 2018). VNPs can also be used directly for the production of hydrogels, providing a biodegradable cytocompatible matrix (Ma et al., 2018; Xu et al., 2014). VNPs are often added directly to the hydrogel production process without any specific crosslinking, and filamentous particles are well preserved in the matrix and not washed out due to their structure (Southan et al., 2018). They also contribute to pore formation and create more evenly distributed and well-defined pore structures in alginate hydrogels (Maturavongsadit et al., 2016). Plant VNPs have been used to modulate cell responses and the first studies have now evaluated their cytotoxicity and effect on different cell types. TMV is the best studied and its effects on a range on cell types have been investigated, including rat BMSCs (Kaur, Valarmathi, et al., 2010; Kaur, Wang, et al., 2010; Metavarayuth et al., 2015), rat neural cells (Wu et al., 2015), mouse neural cells (Feng et al., 2015), BHK, and CHO cells (Lee, Nguyen, et al., 2012), and mouse fibroblasts (Tiu et al., 2017). Most studies showed that the virus improved cell attachment, especially if the VNP was used to present cell-binding motifs. The cellular response depends on the type of virus. For example, TMV, PVX, TYMV and TVCV promote the early osteogenesis of rat BMSCs whereas CPMV has no effect on differentiation (Meta- varayuth et al., 2015). Human MSCs bound weakly to tissue culture slides coated with PVX because of the predominant display of noncharged and thus less-adhesive amino acid residues, whereas PVX presenting RGD or MIP sequences promoted cell attachment (Lauria et al., 2017). These results highlight the importance of surface-exposed amino acids for cell attachment and differentiation. The incorporation of VNPs into 3D-hydrogels may promote cell differentiation and integration without cytotoxic effects (Lauria et al., 2017; Luckanagul et al., 2015, 2016; Maturavongsadit et al., 2016). However, several challenges must be overcome before hydrogels containing plant viruses are widely adopted. Plant viruses are potentially immunogenic, and each VNP/VLP must therefore be tested systematically for safety. Initial studies with TMV-loaded hydrogels were promising because only a low level of antibody production was observed due to the slow release of TMV from the hydrogel, compared to the significant antibody titers induced by the direct adminis- tration of TMV particles. It is also necessary to evaluate the response of individuals already exposed to the VNPs or native viruses. VLPs provide an additional barrier of environmental safety because they do not replicate in plants, but it is not possible to generate VLPs for all plant viruses and this technical challenge must be overcome using strategies such as artificial virus genomes and deconstructed viruses lacking key virulence genes to avoid the unwanted infection of plants.

ACKNOWLEDGMENTS We thank Richard M. Twyman PhD for critically reading the manuscript. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – project number 654127. Louisa Kauth is funded by RWTH Aachen University Scholarship for Doctoral Students.

CONFLICT OF INTEREST The authors have declared no conflicts of interest for this article. DICKMEIS ET AL. 23 of 32

AUTHOR CONTRIBUTIONS Louisa Kauth: Writing-original draft. Christina Dickmeis: Writing-original draft. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ORCID Ulrich Commandeur https://orcid.org/0000-0002-8986-5224

RELATED WIREs ARTICLE From stars to stripes: RNA-directed shaping of plant viral protein templates-structural synthetic virology for smart biohybrid nanostructures

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How to cite this article: Dickmeis C, Kauth L, Commandeur U. From infection to healing: The use of plant viruses in bioactive hydrogels. WIREs Nanomed Nanobiotechnol. 2020;e1662. https://doi.org/10.1002/wnan.1662