Viruses and their potential in bioimaging and biosensing applications

Abstract

Successful development of ultrasensitive constructs for bioimaging and biosensing is a challenging task. Recently, viruses have drawn increasing attention due to their exquisite three-dimensional structures and unique properties, including multivalency, orthogonal reactivities, and responsiveness to genetic modifications. With such well-characterized structures, functional units, such as imaging and binding motifs, can be engineered on the surface of viruses in a programmable, polyvalent manner, which leads to novel nanosized sensing/imaging systems with enhanced signaling and targeting performance. This review highlights some recent progress in the applications of viruses in bioimaging and biosensing.

Kai Li

Kai Li is a chemistry PhD candidate in the group of Professor Xiaobing Lu of the State Key Laboratory of Fine Chemicals at Dalian University of Technology, P. R. China. He joined the Wang lab in September, 2007. His research focuses on the modification of filamentous virus.

Huong Giang Nguyen

Huong Giang Nguyen is currently a senior undergraduate student at the University of South Carolina pursuing her BS in Chemistry. She joined the Wang lab in the spring of 2007, and her research focus is the bioconjugation of plant viruses. She hopes to attend graduate school in fall 2010.

Xiaobing Lu

Professor Xiaobing Lu was born in 1970. He received a PhD in organic chemistry from Dalian University of Technology in 2002. In 1997 he joined the faculty of Dalian University of Technology and obtained a Professor position in State Key Laboratory of Fine Chemicals in 2005. He has received awards from the Chinese Chemical Society (Young Chemist Award), Fok Ying Tung Education Foundation (Young Teacher Prize) and NSFC (National Outstanding Young Investigator). His research interests focuses on catalytic transformations of carbon dioxide, asymmetric catalysis, coordination polymerization with an emphasis on polymer stereochemistry.

Qian Wang

Professor Qian Wang received a BS degree in chemistry in 1992 from Tsinghua University. He obtained a PhD in Organic Chemistry from Tsinghua University in 1997 (supervisor: Prof. Yufen Zhao). After postdoctoral experiences with Prof. Manfred Schlosser at University of Lausanne (1997–1999) and Prof. M. G. Finn at the Scripps Research Institute (1999–2003), he started as an Assistant Professor at University of South Carolina in 2003, where he is currently the Robert L. Sumwalt Professor of Chemistry. His research interest focuses on creating 3D programmable scaffolds to probe the cellular activities. He is also interested in new bioconjugation chemistry and development of protein markers or enzyme inhibitors by combinatorial synthesis. See also: http://www.chem.sc.edu/people/facultyStaffDetails.asp?SID=245.

Introduction

In the past three decades, a wide selection of nanoscale, multifunctional devices and systems have been developed for the applications in biosensing and bioimaging.^1–3 Compared to conventional methods, these nanosystems are characterized with better sensitivity and specificity, and possibly quicker response in biological and medical analyses.⁴ Nanoparticles, including nanogold and other metallic nanoparticles, carbon nanotubes, silica nanoparticles, quantum dots, magnetic nanoparticles, dendrimers and polymeric nanoparticles, are among the best studied nanosystems for biosensing and imaging, primarily due to the recent progress in their syntheses and structural characterizations.^3,5–8 As a comparison, biological nanoparticles, including viruses, ferritins, heat shock proteins and enzyme complexes, can offer well-defined three-dimensional structures that are malleable for genetic and/or chemical modifications with near-atomic precision,⁹ which is very attractive for many applications in bioimaging and biosensing.

As a kind of biogenic species, viruses have emerged as platforms for broad applications ranging from materials to medicine.^10–12 Viruses are self-assembled architectures that occur in a wide range of shapes and sizes (Fig. 1). These naturally occurring nanoscaffolds have many unique properties in comparison to synthetic nanoparticles. For instance, for each kind of virus, all the particles are nearly identical; therefore, they are mono-disperse in shape and size, which is difficult to achieve by laboratory synthesis. In addition, viruses are water soluble and stable in aqueous buffer, which are features more attractive for biological applications. Furthermore, the multicopies of the viral coat proteins provide a large surface area that enables the multivalent display of functional groups. Moreover, they can be purified in large quantity inexpensively. In this review, we highlight how these combined advantages of viruses can promote their applications in biosensing and bioimaging.

Fig. 1 Three-dimensional structures of some representative viruses used in sensing and imaging applications. CPV: canine parvovirus; CCMV: cowpea chlorotic mottle virus; MS2 bacteriophage; CPMV: cowpea mosaic virus; M13 bacteriophage; TMV: tobacco mosaic virus; TYMV: turnip yellow mosaic virus. All the models are generated using PyMol (http://www.pymol.org) with coordinates obtained from RCSB Protein Data Bank (http://www.pdb.org).

Multivalency and defined local environment

To effectively and economically carry and protect genomic information, most of the simple viruses are composed of multicopies of identical protein subunits to form an icosahedral or helical structure.¹³ The development of structural biology, especially X-ray crystallography and high resolution electron microscopy, has uncovered the major capsid proteins of plant viruses and bacteriophages as fairly rigid and ordered 3D structures (Fig. 1). Very often, the coat protein (capsid) structure of a virus obtained from X-ray crystallography study can reveal its solution structural features under the same pH and ionic strength. Therefore, despite its big size, a simple plant virus (and some insect viruses and bacteriophages) can be considered as a molecule where regioselective modification is achievable and predictable.¹⁴ Thus, viruses can function as nanoscale scaffolds where the multivalent attachment of functional ligands at defined positions is possible, which is proven to be important for sensing and imaging applications.

For example, using traditional bioconjugation methods, chemists can ‘fix’ fluorescent molecules at specific, designated positions on the virion surface, where the spatial separation of the dye molecules helps to prevent fluorescence quenching, resulting in ‘super bright’ viral particles. In one study, cowpea mosaic virus (CPMV), an icosahedral plant virus about 30 nm in size, was labeled with more than 40 Cy5 dyes with no significant fluorescence quenching detected due to the large intermolecular distance between the dye molecules.¹⁵ Similarly, turnip yellow mosaic virus (TYMV), a spherical plant virus with an average diameter of 28 nm, was loaded with up to 40 fluorescein or N,N,N′,N′-tetramethylrhodamine molecules via an amidation reaction (Fig. 2a), which is equal to a local concentration of 4.6 mM of dyes surrounding the virus with no observed fluorescence quenching (Fig. 2b).¹⁶

Fig. 2 (a) Bioconjugation of TYMV with fluorescein (FL) and N,N,N′,N′-tetramethylrhodamine (TMR) dyes. (b) Fluorescence intensity vs. dye loading for modified-TYMV with FL () and TMR (). (Reprinted with permission from ref. 16. Copyright 2007, American Chemical Society.)

On the other hand, clustering of the targeting molecules on the surface of viruses allows for multivalent target–receptor interactions, a principle of polyvalency, which can be applied to improve the binding affinity.¹⁷ The multivalent display of tumor targeting molecules, such as folic acid, on a virus surface has been shown to enhance their uptake by cancer cells.¹⁸ Therefore, a viral scaffold offers a unique multivalent system to display targeting or signaling motifs, which can potentially enhance the sensitivity of recognition and signaling.

Orthogonal reactivities: targeting and signaling

For every single sensing/imaging application, both the targeting and signaling motifs should be displayed. To achieve this goal, traditional orthogonal bioconjugation techniques have permeated fundamental virus chemistry, decorating addressable amino acids, such as glutamic/aspartic acids, lysines, and cysteines (Fig. 3).^9,12 In addition, Francis and co-workers have recently reported a highly efficient reaction that can specifically target tyrosine residues.¹⁹ A detailed analysis of advantages and shortcomings of all available conjugation methods for viruses has recently been reviewed by Lee et al.⁹

Fig. 3 Orthogonal bioconjugation strategies targeting the endogenous amino acids on the viral capsid.

As an example, CPMV mutants were functionalized by orthogonal chemical modification with fluorescent dyes on the lysine residues and immunoglobulins (chicken or mouse IgGs) on the cysteine residues (Fig. 4).²⁰ Such an entity, with both the targeting and signal motifs, could be used as a tracer in immunoassays with the hope of an improved detection limit due to the high dye loadings.¹⁹ The sandwich immunoassay result clearly demonstrated that once immobilized to the surface of the virus, the antibodies still maintained functionalities. The dual-modified virus produced a stronger signal than the molar equivalent of dye-modified antibody. The lower limit of detection of dual-modified virus can reach 1 ng/mL, which is ideal for future medical diagnostic applications.²⁰ It was shown that such a virus-based tracer could be stored for up to 7 weeks whilst still maintaining structural integrity.²⁰ Additionally, the functionalities of the attached dyes and antibodies were preserved if the conjugated viral particles were lyophilized in the presence of a cryoprotectant.²¹

Fig. 4 (a) Schematic illustration of the procedure for virus-based microarray analysis. Mutant CPMV particles (EF-CPMV) are conjugated with AlexaFluor^® 647 (Alexa) and chicken or mouse IgG before usage.²⁰ (b) Charge-coupled-device image of the direct immunoassay, with Alexa–EF-CPMV–IgG, taken using the Naval Research Laboratory (NRL) array biosensor. (Reprinted with permission from ref. 20. Copyright 2006, Elsevier.)

In another study, the orthogonal reactivities of cowpea chlorotic mottle virus (CCMV) were obtained via the coupling of fluorophores to surface-exposed carboxylic acid (glutamate and aspartate), amine (lysine), and genetically-inserted thiol (cysteine) residues.²² The degree and spatial distribution of the modifications showed that up to 540 FL dyes could be decorated on the amine groups, 100 FL dyes on the thiol groups, and 560 cadaverine dyes on the carboxyl groups. Tobacco mosaic virus (TMV) and MS2 bacteriophage have also been reported to afford dual-modified viruses using orthogonal bioconjugation chemistries,^19,23 indicating that high-level modified viruses provide a promising platform for potential biosensing and bioimaging applications.

Genetic modification and phage display

In order to enhance the selective modification on the viral particles, genetic tools have been employed to generate mutations that express new reactive residues on the exterior surface of the viral capsids, with precise regio-control. For instance, while the crystal structure of wild-type CPMV is known to display reactive lysine residues on its outer surface, no cysteine residues are assessable, which is further confirmed by the virus's inertness toward thiol-reactive reagents. Hence, site-directed genetic insertion of cysteine residues to the virus, which results in the symmetrical display of 60 thiol groups around the 30 nm-diameter capsid, has generated a mutant virus enhanced with new and higher (cysteine) reactivity, in addition to its already present and still active lysine reactivity.²⁴ The EF-CPMV of Fig. 4 was one of the exemplary, cysteine-added CPMV particles generated by Johnson, Finn and co-workers.^24,25 Similar cysteine-addition strategies have been widely used in the genetic modification of CCMV,²⁶ TMV,^27–29 and other viral systems and protein cages.^30–32

In other cases, reactive groups that are native to the viral coat protein can be replaced by non-reactive groups to control and, thus, boost the modification selectivity of the viral particles. For example, although native CPMV has five reactive lysines on the exterior surface, Chatterji et al. were able to create mutant CPMV with a single reactive lysine site per subunit by mutating the other reactive lysine residues into arginines, which, when coupled with site-directed cysteine mutations, could offer the possibility of dual modifications that are tailored and predictable for both groups.³³ A more elegant approach to introduce orthogonal reactive residues has recently been developed by incorporating either azide- or alkyne-containing unnatural amino acids onto the capsid proteins of bacteriophage³⁴ and virus-like particles composed of hepatitis B virus or bacteriophage Qβ (Fig. 5).³⁵ The exogenous azide and alkyne groups could be readily addressed by Cu(I)-catalyzed cycloaddition reaction, which provided the capability to display a wide variety of functional units with precise spacing control.³⁵ Such spacing control could be further expanded to the insertion of peptide sequences, e.g. RGD peptide, to target specific cells for the purpose of medical imaging and therapy.^36,37

Fig. 5 (a) Hepatitis B virus (HBV) dimer. The locations of the methionine residues allowed for the genetic incorporation of the azide group. (b) Three-dimensional structure of HBV. (c) Bacteriophage Qβ (mutants K16M and T93M) dimer. Genetic incorporation of azide and alkyne groups at the locations of the methionine residues. (d) Three-dimensional structure of bacteriophage Qβ. (Reprinted with permission from ref. 35. Copyright 2008, American Chemical Society.)

Phage display is another powerful tool to genetically engineer the capsid proteins of filamentous bacteriophages. Unlike the genetic mutagenesis technology mentioned above, the power of phage display relies on multicopy expression and selection, rather than precise design. A phage library may contain billions of different phages, thus making it possible to obtain, via affinity selection, a small collection of phages with recognition peptides for essentially any target analyte.³⁸ The guest peptide may be fused to either the minor coat protein pIII, totaling five copies, or the major coat protein pVIII.^39–41 Phage display has gained much attention in recent years in biosensing applications due to its tremendous potential, particularly the advantages it offers over the conventional use of antibodies.^39,40,42 In many cases, the selected phage clone offers selectivity and sensitivity to the target molecule comparable to those of antibodies. However, unlike antibodies, it is not limited to only certain target molecules due to its large library size.³⁸ Moreover, its production is easy through propagation in host cells and is cheaper and more homogeneous when compared to monoclonal and polyclonal antibodies, respectively.⁴⁰ Another advantage is its stability under a variety of environmental conditions, whereas antibodies tend to lose their binding function under less favorable conditions, such as at higher temperatures, for example.^40,43 Its stability and promptness are particularly important for field-use detectors, where real-time sensing would be beneficial.

A variety of methods and instrumentations, such as fluoroimmunoassays,²³ quartz crystal microbalance (QCM),^25,28 electrical resistance,²⁷ electrochemical impedance spectroscopy,²⁹ opto-fluidic ring resonator,³⁰ and surface plasmon resonator³¹ have been successfully applied to phage-based sensing of a diverse class of analytes, ranging from single molecules to whole cells,³¹ testifying to the versatility of phage display. For instance, virus-based electrodes have been constructed, whose resistance increased when the electrically resistive covalent virus layer composed of bacteriophage M13 selected for anti-M13 monoclonal antibody and prostate-specific membrane antigen (PSMA) specifically bound the antibody or antigen.⁴⁴ The bacteriophage was covalently linked to the electrode via a self-assembled monolayer of N-hydroxysuccinimide thioctic ester activated on the gold electrodes (Fig. 6). It was shown that the change in resistance was more sensitive and provided a better signal/noise ratio (in high frequency domain) than the capacitive impedance.⁴⁴ This method was recently expanded to a QCM detector for mass-based biosensing.^45,46

Fig. 6 Schematic diagram depicting the stepwise assembly of the covalent virus layer for biosensing.^44–46

Interior encapsulation vs. exterior modification

Most viruses have well-defined inner cavities, which encapsulate the viral RNAs or DNAs. After removal of the genome, the capsids of viruses provide a uniquely defined inside environment to entrap inorganic nanoparticles, polymers and enzymes.^47–53 Furthermore, the interior cavities of viruses, in addition to the exterior surfaces, can also be chemically manipulated. While it is clear that exterior modifications offer advantages such as polyvalent display and orthogonal reactivities important to biosensing and bioimaging, interior encapsulation or modification proves useful in many cases as well. For example, anchoring ligands to the interior surface of a viral capsid has minimal impact on the virus's receptor targeting since this leaves the exterior surface available for derivatization with the targeting molecules, fostering the use of viruses as imaging reagents.⁵⁴ An intriguing study has been reported by Francis and co-workers.⁵⁵ Using bacteriophage MS2 as a scaffold, either the exterior or interior surface was used to display ligands to chelate Gd(III) as contrast agents for magnetic resonance imaging (MRI) (Fig. 7). The interior-modified MS2 particles showed much higher water solubility and better stability than their exterior-modified counterparts did. More significantly, the ionic relaxivity for MS2 capsids decorated with internal Gd-chelates was much higher than for those with Gd-chelates on the exterior surface, likely due to different rigidities between linkers.^55,56 Although the in vivo imaging application has yet to be determined, the study opens the door for the future clinical use.

Fig. 7 Exterior (top) and interior (bottom) modifications of MS2 bacteriophage to display ligands to chelate Gd(III). (Reprinted with permission from ref. 56. Copyright 2008, American Chemical Society.)

In vivo imaging

Fluorescent virus probes have been reported for in vivo imaging.¹⁷ In one study, Oregon Green-488 anchored CPMV particles were generated to study the possible biodistribution of viral particles in mice with three delivery routes: oral, intramucosal, and intravenous injection.⁵⁷Via confocal fluorescence microscopy and viral RNA detection, the orally-dosed CPMV particles were found in a wide variety of tissues, including the spleen, kidneys, liver, lungs, stomach, duodenum, jejunum, ileum, lymph nodes, and brain after several days of administration of viral particles.⁵⁷ The CPMV particles appeared to be intact after recovery from mouse tissues. This result supports the remarkable stability of the virus even after undergoing a series of harsh conditions, such as the degradative and acidic environment of the stomach, pancreatic lipases, and bilary digestive enzymes of the duodenum.⁵⁷

Fluorescently-labeled CPMV particles have also been used in imaging the vascular system in living animals.⁵⁸ CPMV was modified in high levels with AlexaFluor and fluorescein dyes, showing no detectable quenching. Unlike a similar-sized fluorescent nanosphere, the super bright viral particle remained monodisperse in the vascular endothelium of a living mouse, allowing for a higher resolution of macro- and micro-vasculature in vivo and in fixed tissues using fluorescence microscopy (Fig. 8a). Similarly, in the chick embryo, dye-loaded CPMV particles quickly labeled the embryonic and extraembryonic vasculature, and provided good signals for visualizing the vasculature to a depth of 500 µm.⁵⁸ Angiogenesis and the human fibrosarcoma-mediated tumor vasculature also could be visualized using fluorescently-labeled CPMV. As a result of the internalization of the particles by endothelial cells, the dye-labeled CPMV could be used to identify arterial and venous vessels and monitor the neovascularization of the tumor microenvironment (Fig. 8b).⁵⁸ In another study, dye-labeled murine polyoma virus-like particles (VLPs) were generated and used for analyzing the lateral movement of incoming VLPs bound to living cells and artificial lipid bilayers via a combination of total internal reflection fluorescence microscopy and single-particle tracking technology.⁵⁹

Fig. 8 (a) Comparison of intravital imaging with CPMV–AlexaFluor 555 (left) and fluorescent nanospheres (right) in Ell.5 mouse embryo. Scale bar, 1.1 mm. Other than the nanosphere, no aggregates were observed using dye-labeled CPMV. (b) Intravital vascular mapping of tumor angiogenesis using CPMV–AlexaFluor 555 (left) and CPMV–AlexaFluor 488 (right). Arrows indicate a site of angiogenesis. Sprout and arrowheads indicate newly formed vessels. Scale bar, 100 µm. (Reprinted with permission from ref. 58. Copyright 2006, Macmillan Publishers Ltd.)

One of the natural properties of viruses is the specific binding between the viral capsid proteins and the receptors on the host cell surface. Benefiting from this characteristic, canine parvovirus (CPV), a viral pathogen of dog that has a natural affinity toward transferrin receptors (TfRs) highly expressed on some tumor cells, was the basis for the expression and generation of non-infectious canine parvovirus virus-like particles (CPV-VLPs) for attachment of fluorescent reagents.⁶⁰ The fluorescence confocal microscopy analysis showed that the dye-modified CPV-VLPs retained their specificity for TfR-expressing tumor cells while they did not interact with TfR-negative cells.⁶⁰

As mentioned previously, phage display technology can generate a bifunctional entity which can target tumors while delivering agents. Chen et al. engineered a chimera phage vector containing a PIII-displayed α_v integrin-targeting moiety and a PVIII-displayed streptavidin-binding adaptor moiety. The special phage not only could guide luminescent quantum dots to KS1767 cells, but also to the in vivo tumor cells.⁶¹

Conclusion and future directions

Thanks to their well-organized 3D structures, orthogonal reactivities, and possibilities of being genetically modified, viruses have emerged as ideal platforms for sensing and imaging applications. Virus-based probes can be modified with a variety of signaling agents (e.g. near-infrared fluorescent dyes, magnetic contrast imaging agents) at high local concentrations to lower the detection limit. Furthermore, viruses can be covered with biocompatible polymers to make them stable under long-term storage or boost their half-life in the in vivo imaging study.⁶² Despite the many advantages of the virus-based sensing probes, they exhibit limited stability under harsher conditions or in organic solutions. There are two ways to cope with such problems: one is to search for special viruses that can be isolated from extreme environments, and the other way is to use genetic tools to generate temperature- and solvent-tolerant virus mutants.^63,64 Further study of the toxicity of and the immune response to viruses in the in vivo environment is crucial. In addition, although it is theoretically possible to generate genetic libraries for all viruses, only phages (M13, fd) have been broadly used so far. Additional efforts in the genetic manipulations of viruses to generate mutants for cell targeting should be further pursued.

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