Phage-Enabled Nanomedicine: From Probes to Therapeutics in Precision Medicine.

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Angew Chem Int Ed Engl. Author manuscript; available in PMC 2017 February 16. Published in final edited form as: Angew Chem Int Ed Engl. 2017 February 13; 56(8): 1964–1992. doi:10.1002/anie.201606181.

Phage-Enabled Nanomedicine: From Probes to Therapeutics in Precision Medicine Kegan S. Sunderland, Department of Chemistry and Biochemistry Stephenson Life Sciences Research Center, University of Oklahoma 101 Stephenson Parkway, Norman, Oklahoma 73019 (USA)

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Prof. Mingying Yang, and Institute of Applied Bioresource Research College of Animal Science, Zhejiang University Yuhangtang Road 866, Hangzhou, Zhejiang 310058 (China) Prof. Chuanbin Mao Department of Chemistry and Biochemistry Stephenson Life Sciences Research Center, University of Oklahoma 101 Stephenson Parkway, Norman, Oklahoma 73019 (USA). School of Materials Science and Engineering, Zhejiang University Hangzhou, Zhejiang 310027 (China)

Abstract

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Both lytic and temperate bacteriophages (phages) can be applied in nanomedicine, in particular, as nanoprobes for precise disease diagnosis and nanotherapeutics for targeted disease treatment. Since phages are bacteria-specific viruses, they do not naturally infect eukaryotic cells and are not toxic to them. They can be genetically engineered to target nanoparticles, cells, tissues, and organs, and can also be modified with functional abiotic nanomaterials for disease diagnosis and treatment. This Review will summarize the current use of phage structures in many aspects of precision nanomedicine, including ultrasensitive biomarker detection, enhanced bioimaging for disease diagnosis, targeted drug and gene delivery, directed stem cell differentiation, accelerated tissue formation, effective vaccination, and nanotherapeutics for targeted disease treatment. We will also propose future directions in the area of phage-based nanomedicines, and discuss the state of phage-based clinical trials.

To eat is to treat

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Bacteriophages (phages) are bacteria-specific viruses, which makes them promising antibacterial agents. They can additionally be engineered to target particular cells or tissues, and can also be modified with drugs or nanomaterials. This Review will summarize the current use of phage structures in precision nanomedicine, in particular, their use as nanoprobes for precise disease diagnosis and nanotherapeutics for targeted disease treatment.

Correspondence to: Mingying Yang; Chuanbin Mao.

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Keywords biosensing; nanomedicine; phages; probes; therapeutics

1. Introduction 1.1. History of Phages A century ago, on September 3, 1917, Félix O’Hérelle announced he had “isolated an


invisible microbe endowed with an antagonistic property against the bacillus of Shiga”.[1] He called these antagonists “bacteriophages” (phages). Since this discovery, phages have been employed for several uses. For example, lytic phages were widely used as an antibacterial agent until the invention of antibiotics seemingly appeared to make them obsolete.[2] However, with the development of antibiotic-resistant bacteria, medical professionals around the world have resorted to employing lytic phages to treat antibioticresistant bacterial infections.[3] In spite of their immunogenicity, phages have been reported to elicit only a mild immune response, suggests the potential for use in humans.[3] In fact, the field of phage therapeutics is currently a very vibrant area, especially in the context of the new clusters of regularly interspaced short palindromic repeat and associated genes (CRISPR/Cas) system and its ability to provide selective pressure for antibiotic-sensitive bacteria.[4] Indeed, even large-scale human clinical trials have been completed or are underway, such as the phagoburn (phase I–II clinical trials) project funded by the European Union under the 7th Framework Program for Research and Development.[2b, 5] The phagoburn project focuses on treating infected burn wounds with lytic phages, and it represents a large-scale clinical trial for the use of phages in medicine. With the developments in nanobiotechnology since 2000, both lytic and temperate phages as biological nanoparticles have begun to fulfil a new role in nanomedicine. Precision medicine, that is, ultrasensitive disease diagnosis and targeted disease treatment, in particular has been greatly advanced by emerging research with phages. At the forefront of these advancements are intricate systems in which the natural biology of phages is utilized, in addition to enhancements through genetic engineering. Such integrated systems make it possible, for example, to invent new highly sensitive ways to detect diseases, as well as Angew Chem Int Ed Engl. Author manuscript; available in PMC 2017 February 16.

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create biomaterials that are a hybrid of genetically engineered phages and inorganic materials for targeted disease treatment. They also allow precision targeting in drug and gene delivery because they can be genetically engineered to bear targeting molecules or be used as a platform for identifying targeting molecules. This precision targeting is particularly useful for diseases such as cancer, in which standard methods of treatment affect far more than their intended target, thereby causing severe side effects.

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The lack of medicinal use of phages in the US and UK to date has been attributed to a lack of acceptance by the mainstream scientific and medical authorities, owing to a lack of studies from Eastern European practitioners that meet western standards for medical efficacy and safety.[3] However, with increasing resistance to antibiotics and the pressing need for precision medicine, phages have become a versatile and extraordinary tool in nanomedicine. With promising new therapies available and the completion of several clinical trials, phage therapy has now been developed into a commercially viable technology. With this development, companies such as Pherecydes Pharma (based in the United Kingdom and France) have been leading the way to a new era of phage therapeutics. 1.2. Classification of Phages

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The versatility of phages in nanomedicine with regard to precision targeting, self-assembled biomaterials, and many more techniques is in large part due to the natural biology and structure of phages. From a biological perspective, phages are viruses that specifically infect bacteria. They are classified taxonomically into families, but due to rapid changes in the established taxonomies from year to year, the most current existing taxonomical families should be referenced according to the international committee on taxonomy of viruses.[3,6] Each family is sorted according to the structure of the capsids encompassing their genomes, the chemical and structural composition of their genomes (linear, circular, double or singlestranded, DNA or RNA), and the mechanism of mRNA production.[3] In terms of making use of phage biology, there are several reviews encompassing phage display[7] and bacterial biosensing.[8] Phage display is useful for finding peptide ligands that can specifically bind to disease-associated receptors and microenvironments.[3, 9] Additionally, nanotechnology often employs the use of phage morphologies, such as the naturally occurring filamentous structure of M13 phages. The use of phages in several nanomaterials has also been previously reviewed.[10] However, there has yet to be a review primarily dedicated to the overall use of phages in nanomedicine (Figure 1). 1.3. Focus of this Review: Use of Phages in Nanomedicine

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Nanomedicine is the application of nanotechnology in medicine.[3] This review is focused on the use of phages, which are biological nanostructures, in nanomedicine. Natural phage structures have been utilized alone or with abiotic materials to develop applications in nanomedicine, including nanotherapeutics, bioimaging probes, biomimetic biomaterials, targeted gene and drug carriers, tissue regenerative scaffolds, matrices for directing stem-cell fate, probes for detecting disease biomarkers, and numerous others. Previous reviews dealing with phages have not encompassed the recent advances in nanomedicine, which will constitute the main topic for this review.

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2. Lytic and Temperate Phages as Nanoparticles: Chemistry and Biology Phages exist in various shapes and sizes, from long rod-like filaments to more compact shapes with much shorter tails. A few representative phages are shown in Table 1. Despite their many differences, phages can be divided into two broad categories, namely lytic and temperate phages (Table 1).[11] In essence, lytic phages break open and kill the host bacteria after infection to bring about release from the bacteria. However, temperate phages do not kill the host bacteria; instead, they only use the host bacteria as a “factory” for amplification and are finally secreted from the host. Additionally, it should be noted that some phages share qualities from both categories. For example, the temperate phage lambda can follow a lytic or lysogenic lifecycle.

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Lambda phage has a head measuring around 30 nm in radius and a tail measuring 150 nm in length.[12] The head consists of 830 protein subunits and functions as a capsid containing the viral genome, which is made up of 48 502 base pairs of double-stranded linear DNA.[13] The DNA itself makes up about half the weight of the entire phage. The tail of lambda phages consists of a 150 nm hollow tube with a conical cap measuring around 15 nm. While the inside of the tail measures around 3 nm in diameter, the outside diameter is 9–18 nm and may contain rough, knob-like structures. Infection begins when the phage attaches to the host cell and injects its DNA into the cell. From here, the phage enters either the lytic or lysogenic cycle.[14]

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If followed, the lytic cycle involves immediate replication of the phage DNA by the bacterial replication machinery. The DNA templates are then used for the production of phage proteins, which are assembled into phage particles. When phage particles build up, the cell undergoes lysis and is broken into fragments.[12b The phages are then released and begin infecting additional bacteria.

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Upon viral DNA entry into the bacteria cell, the phage may also enter a lysogenic pathway, in which the viral DNA integrates into the host genome through recombination to produce a prophage. Although less common, the lysogenic pathway can be followed when conditions are unfavorable for bacterial cell replication. Thus, this phage can also have temperate qualities. The prophage is then replicated each time the bacterial cell replicates. In lambda phage, to prevent a phage from being cut out of the genome and entering the lytic cycle, the lambda repressor is synthesized from the transcribed cI gene product and occupies the OR1 and OR2 sites.[15] This represses the cro gene. If conditions make cell death likely, the repressor protein is cleaved in half by recA (from the cell’s distress response to DNA damage) and the cro gene is derepressed.[15a, 16] The cro protein is then synthesized and occupies the OR3 site, turning off transcription of the cI gene.[15a This allows the prophage to be cut out of the host genome and enter the lytic cycle. 2.1. Professionally Lytic Phages Professionally lytic phage types are generally composed of a head and flexible tail, but lack the long filamentous shape shared by most temperate phages. These phages primarily resort to lysing the host cells during the infection cycle, and some key examples of professionally lytic phages include the T1–T7 phages.[11] These phages are often used in phage-based


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antibacterial strategies. An example of a professionally lytic phage is T7 phage. T7 phage is a member of the Podoviridae family and has an icosahedral head with a short tail. When T7 phages infect cells, several internal capsid proteins are ejected into the host cell wall and assemble an ejectosome that enables translocation of DNA from the T7 viral capsid into the cell.[17] The viral DNA immediately begins a process in which a set of proteins necessary for replicating the phage DNA are transcribed and translated from the viral DNA template. This results in several copies of the viral DNA being made.[17] Next, a second set of proteins are made and assemble into the various structural components of the phages. Finally, while the assembly process is taking place, lysozyme is produced. The lysozyme will lyse the host’s cell wall to allow the release of the mature viruses.[18] This whole process is actually quite fast, and depending on the fitness of the phages, it can take just a matter of minutes to complete.[19] Therefore, professionally lytic phages have gained a lot of attention for antibacterial applications.

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2.2. Temperate Phages

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The temperate inoviruses are a group of filamentous phages that includes the Ff, M13, f1, fd, Ike, and N1 phages.[3] So far, filamentous phages have found more applications in templating nanomaterials synthesis than professionally lytic phages, probably because filamentous phages tend to be used to template nanowire synthesis and can self-assemble into higher-order structures. Filamentous temperate phages can be pictured as a long flexible nanofiber composed of five structural capsid proteins, which encase a relatively large circular single strand of DNA.[10a, 20] One example includes wild-type M13 (Table 1), which measures approximately 9300 Å in length but has a diameter of only 65 Å.[21] The M13 virion contains of approximately 6407 bases of circular single-stranded DNA (ssDNA) in its genome, but longer genomes generate longer phages.[10d, 21, 22] Although there are five different proteins, the majority of a filamentous phage made up of around 2700 copies of a major coat protein (pVIII) encoded by a single gene called gene VIII,[20] which are helically arranged to form a filamentous tube.[10a pVIII is the protein that is often modified for desirable characteristics in the laboratory, and filamentous temperate phages can serve as a highly structured nanomaterial. The ends of filamentous phages are composed of minor coat proteins. One end is composed of five copies each of the proteins pIII and pVI, while the opposite end displays five copies each of the proteins pVII and pIX.[20, 22, 23] The filamentous structure and major coat proteins are what gives these phages the potential for creating many self-assembled nanostructures and developing excellent targeting abilities.

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Unlike lytic phages, filamentous phages are secreted from bacteria without bacterial cell lysis.[10a, 20] Filamentous phages infecting Escherichia coli are among the most productive phages, and can produce titers of up to 1013 phages per milliliter of culture.[10a The infection cycle begins when the minor coat protein at the tip, pIII, utilizes its N2 domain to bind to the F-pilus on the surface of a bacterial cell.[24] The F-pilus will then retract, pulling the phage closer to the surface of the bacterium.[21, 24, 25] Next, it is thought that the N1 domain of pIII binds to the TolAIII domain on the highly conserved inner membrane complex, TolQRA.[10a, 24] Interaction of the C domain of pIII with the TolQRA complex or some conformational rearrangement of the C domain of pIII then exposes the hydrophobic Cterminal helix of pIII, thus allowing insertion of the helix into the inner membrane.[10a


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Finally, the phage ssDNA is injected into the cytoplasm, where it can either begin replicating (episomal) or integrate into the host’s chromosome (temperate).[10a If the ssDNA takes the temperate route, it becomes a substrate for the XerCD recombinase, which will bind to the forked hairpin loop termed AttP[10a and integrate the viral DNA into the bacterial genome through site-specific recombination at the dif site located between the chromosomal DNA and a previously inserted satellite phage such as RS1φ or TLCφ.[26] This process is called lysogenization. After integration into the chromosome, there may come a time when it is beneficial for the virus to undergo induction. Such an event could occur if the cell undergoes damage as shown in experiments using UV light or mitomycin C, which will induce a cellular SOS response in which LexA is degraded.[10a This results in PA promoter transcription, which will eventually lead to release of the recirularized old ssDNA, which is a combination of the satellite and prophage between the two origins of replication. It is important to note that the resulting replicon is not the same as the one initially integrated into the chromosome.[10a The ssDNA can then replicate by the episomal path.

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Negative-strand synthesis of the ssDNA is carried out by bacterial RNA polymerase, which will first generate an RNA primer that is subsequently released from the template ssDNA.[27] Bacterial DNA polymerase III then creates a replica of the negative strand based on the RNA primer sequence. Synthesis of the positive-strand is then initiated by the nonstructural phage protein pII. Supercoiling occurs on the double-stranded DNA (dsDNA), which forms a stem-loop structure that allows a nick in the positive strand of the dsDNA to be created at the origin of replication.[10a Rolling-circle replication then completes a new copy of the viral DNA.[28] Copies of the replicative form of the viral DNA then serve as a template to produce the non-structural phage proteins pII, pV, and pX to help with packaging of the substrate, and genome replication.[10a, 21] The non-structural proteins pI, pIV, and pXI are also produced from the replicative form by bacterial cell transcriptional and translational machinery and form a transport complex spanning the outer and inner membranes of the bacteria cell. The phage structural proteins pVII, pIX, pVIII, pVI, and pIII are then inserted into the membrane for latter assembly into the phage virion.[10a, 23] The positive strands of viral DNA are then coated by dimers of the ssDNA-binding protein pV, and then transported to the cell membrane for packaging by the transport complex and assembled into the exported virion form of the phages.[10a, 21]

3. Phage-Directed Nanomaterials Synthesis and Assembly for Biomedical Applications

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Phages are a powerful building material and can be assembled into highly organized nanomaterials with various desirable characteristics.[29] In addition to using the natural structure of phage itself as a nanomaterial, phages can easily be genetically engineered to display peptides that provide affinity characteristics and serve as a biointeractive peptide motif, or even to form self-assembled nanomaterials.[30, 44, 48–51] Phages have even been used in patterned molecular inks.[31]


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3.1. Magnetic Nanomaterials


Phages have been utilized in the synthesis and assembly of magnetic nanomaterials.[32] Typically filamentous phages are used because of their natural fibrous structure encased by the major coat protein pVIII. This coat protein is often modified to interact with magnetic nanoparticles or serve as a template for magnetic nanoparticle synthesis.[32c Complexes of phages and magnetic nanoparticles have been employed in several cases, such as in tumor detection[33] or 3D scaffolds for tissue regeneration,[32d,f] among others.[34] In particular, these magnetic-phage complexes have shown great promise in precision detection methods.[35] For example, a T7 phage-conjugated magnetic probe has recently been developed for the rapid detection of E. Coli in drinking water supplies.[35a In this technique, a T7 phage conjugated to iron oxide magnetic beads (Dynabeads MyOne) is used to capture E. coli BL21 and separate it from drinking water. Since T7 phages are professionally lytic, the E. coli cells are then lysed by the phages and an endogenous β-galactosidase is released from the bound bacterial cells. The released β-galactosidase is then detected using chlorophenol red-β-D-galactopyranoside. This is a colorimetric substrate, which produces a color change from yellow to red in the presence of β-galactosidase. Through this strategy, E. coli at a concentration of 1 × 104 colony forming units per milliliter can be detected in as little as 2.5 h. In addition, high specificity was demonstrated against a background of competing bacteria.[35a A schematic overview of the technique is shown in Figure 2. 3.2. Metallic Nanoparticles

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Phages can also be utilized in the self-assembly of nanomaterials involving inorganic molecules such as gold,[32e,53b, 58] silver,[59] titanium dioxide,[60] copper,[61] aluminum,[62] and many others. Of these metals, it has been demonstrated that filamentous phages can form networks with gold (enhanced by the presence of imidazole).[58a Importantly, the phages still maintained cell targeting, thus suggesting that phages can be used for both targeting and imaging owing to their ability to maintain specificity for cells while associating with inorganic gold nanoparticles.[58a Such properties are very promising for disease diagnosis and biosensing. Self-assembled phage–gold networks have even been used to create other types of nanomaterials, including sensors for hydrogen sulfide[63] and ammonia gas.[64] Additionally, Capehart et al. have demonstrated that MS2 phage capsids can be assembled around gold nanoparticles to form a metal-enhanced fluorophore.[53b The resulting metal-enhanced fluorophore can serve as an imaging agent. Modifications to the surface of the MS2 phage capsid have also been used to demonstrate that the system can be fine-tuned to create complex systems, which could perhaps be further developed to create a precision delivery system for the imaging agent.

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3.3. Semiconducting Nanocrystals Phages can serve as a template for the synthesis of quantum dots.[47, 65] Quantum dots such as CdS self-assemble into hexameric and pentameric patterns on the shells of P22 phages, possibly through interaction with protein pockets.[47] These assemblies may provide enhanced photoactivity important for tissue imaging, although studies have not yet been carried out on the imaging of tissues with these assemblies.[47]


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3.4. Nanotubes

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Fluorescence imaging is a highly desirable technique in terms of the detection of diseases and monitoring of treatment response, and has been widely applied.[66] Single-walled carbon nanotubes (SWNTs) show photoluminescence in the second near-infrared window (λ= 950– 1499 nm), which gives a high tissue penetration depth and avoiding problems with autofluorescence. The fact that the excitation wavelength is far from the emission wavelength makes them ideal for medical diagnostics.[49, 67] However, SWNTs lack stability in biocompatible environments, as well as targeting ability. To solve this problem, Yi et al. engineered the major coat protein (pVIII) of M13 phages to bind and evenly distribute SWNTs along the surface of phages.[49] Conjugation to the phages pVIII coat protein still allowed cell targeting and also stabilized the SWNTs in a biocompatible environment.[49] Phage assembled SWNTs may thus serve as powerful imaging agents in vitro. A schematic representation of M13–SWNT and its biomedical application is shown in Figure 3.

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3.5. Bone Minerals

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Bioinspired mineralization has recently gained momentum in bone regeneration. In natural bone, the mineral hydroxylapatite (HAP) is preferentially oriented with the c-axis parallel to the collagen fibers.[68] Such organization and orientation is difficult to achieve in artificial bone materials. Genetically engineered filamentous phages have been successfully employed to mimic collagen fibers in bone to template the nucleation and assembly of HAP.[68, 69] Phage-mediated HAP nucleation has also been enhanced by genetically fusing proteinderived peptides, such as Glu8 (E8) derived from the non-collagenous protein bone sialoprotein (BSP)[69a or dentin matrix protein-1 (DMP1),[68] to M13 phage’s pVIII coat protein. The resultant HAP assemblies are potential building blocks for the fabrication of a bone-like matrix for inducing bone formation. These materials could potentially be used in bone regenerative implant scaffolds in the near future. 3.6. 3D Nanostructured Scaffolds

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Scaffolds often require very particular characteristics in terms of order and interactions with substrates. Phages have been shown to be a viable material for creating several types of 3D scaffolds for use in tissue regeneration and biosensing. The long rod shape and monodispersity of filamentous phages make it a natural nanofiber for self-assembled 2D and 3D structures. In addition, its major coat protein can be genetically modified to serve as a promoter of cellular differentiation with regards to osteogenesis.[70] Phages can be used in 3D printed mineral scaffolds to induce vascularization and osteogenesis.[71] Filamentous phages can also form directionally organized liquid-crystal-like materials for promoting nerve tissue regeneration.[52a In addition, various hydrogel scaffolds have been created by utilizing phage technology.[58b,c, 72] For example, Balc.o et al. optimized the conditions of phage-based hydrogels for the biosensing of bacteria.[72a Sawada et al. were also able to create highly regular hybrid hydrogel structures by utilizing filamentous phage displaying tag-peptides (antigens) and antibody-immobilized gold nanoparticles, thereby opening up new opportunities in soft materials science.[58b These hydrogels could potentially be used in molecular sensing devices.[58b Molecular sensing devices have a wide variety of applications in disease detection and monitoring for imbalances of molecules in the human body.


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4. Targeting Phage Nanoparticles for Precision Medicine Through the use of phage libraries and natural selection methods, phages can be tailored for several targeted affinity characteristics. Genetically engineered phages can be created through a simple biopanning method that has been proven to be useful for targeting inorganic structures, cells, tumors, and organs. 4.1. Phage Libraries

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Generically, a library of polypeptides displayed on the surface of phages (such as M13 filamentous phage) is referred to as a phage library. More specifically, there are three systems: phage, hybrid, and phagemid. The variants and techniques in phage library display have already been widely reviewed.[7g, 20, 21, 73] For example, a phage system using M13 phages makes use of the single copy of gene III in the phage genome by ligating the sequence for a fusion peptide to pIII. Consequently, every copy of pIII in the assembled phages will then display the corresponding fusion protein. In addition to temperate phage libraries, there are also libraries of professionally lytic phages.[113] For example, the T7 12-mer library (X12) and the T7 7-mer disulfideconstrained library (CX7C) are lytic phage libraries. When compared to non-lytic phage libraries such as the M13 Ph.D.-12™ and Ph.D.-C7C™ libraries from New England BioLabs, there is actually some benefit to using the lytic phage libraries. The T7 phage library shows fewer amino acid biases, more normal distributions of the net charge of peptides, an increased peptide diversity, and a more normal distribution of the hydrophobicity of peptides when compared to the standard M13 temperate phage libraries.[74]

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Fusion libraries created from phage display techniques can become extremely diverse. For example, we have reported using a landscape phage library to target SKBR-3 breast cancer cells, and such a library consists of a multibillion-member collection of phages.[75] In this case, each phage had random octamers fused to each of their, roughly 3900 copies of the major coat protein pVIII.[50a With such diverse surface possibilities, phage libraries such as this become powerful tools for searching for targeting peptides through biopanning. 4.2. Biopanning

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Biopanning is a common technique for naturally selecting phages. Compared to other methods such as Kunkel muta-genesis and PCR-driven library selection,[20] biopanning has several advantages. Biopanning does not require extensive knowledge of the target material, and with titers of up to 1013 phages per milliliter of culture, the scope and power of selective binding is immense.[10a, 76] During biopanning, a receptor is immobilized on a solid support. A diverse phage library is then added. The immobilized receptors capture genetically fit phages while the unbound phages are washed away. In this way, the resultant phage population is enriched for binding to the specific receptor. The bound phages are then amplified and subsequent rounds of selection are carried out to improve the binding properties.[20] A schematic of the biopanning process is shown in Figure 4. More details on biopanning can be found in numerous reviews on phage selection.[20, 21, 73, 76a, 77]


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4.3. Binding to Inorganic Species and Nanostructures

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Phages are not limited to binding to biological molecules. The coat proteins of phages are often selected to bind inorganic species such as gold nanoparticles,[58a, 78] single-walled carbon nanotubes,[49] Pb2+ cations for use in nanowires,[79] and CaO·MoO3 (powellite),[80] among numerous others (e.g., ZnO, Cr2O3, GeO2, Ag, and GaAs).[80] Although this list is far from complete, this serves as compelling evidence that phages are a powerful tool for building diverse self-assembled nanostructures, which may find potential applications in disease diagnosis and treatment. 4.4. Cell Targeting

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Phages are an exceptionally good tool for cell targeting since their natural function is to target and replicate within cells, and they are less hindered by the site of injection and blood flow than other vectors.[81] Their major coat proteins can easily be modified for a high specificity for certain cell types. Such targeting is usually accomplished by carrying out in vitro selection using cells as the immobilized targets.[21] The phages are selected through the well-known biopanning method. Virtually any cell type can be selected for by this mechanism, and current selection techniques are able to rapidly discover and identify cellsurface markers.[82]

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Phages can be a powerful tool for cancer-cell targeting. Biopanning with phages has an enormous advantage when it comes to selectively binding to cancer cells because of the diverse peptide libraries available and the ability to easily select and amplify phages displaying good binding peptides for further rounds of selection. For example, filamentous phages capable of targeting SKBR-3 breast cancer cells were obtained through biopanning. These genetically selected phages were also shown to cause membrane ruffling and rearrangements of the cellular actin cytoskeletons during phage entry.[75] 4.5. Tissue and Organ Targeting

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It can be difficult to specifically target tissues and organs for drug delivery or imaging. For example, the relative impermeability of the blood–brain barrier (BBB) can make drug delivery to the central nervous system (CNS) difficult. In order to enhance brain-specific drug delivery, Li et al. used a 12-mer phage-display peptide library to isolate peptides capable of guiding drugs to the brain.[83] This led to the isolation of a 12-mer peptide (Pep TGN), which was selected through phage display combined with in vivo screening.[83] After identification, the Pep TGN peptide was covalently conjugated onto poly(ethylene glycol)– poly(lactic-co-glycolic acid) based nanoparticles, which were shown to have significantly higher cellular uptake than uncoated nanoparticles.[83] In nude mice, the peptide coated nanoparticles showed enhanced brain accumulation efficiency and lower accumulation in the liver or spleen.[83] A fluorescent probe (coumarin 6) was also used to evaluate brain delivery and showed a significantly higher accumulation of peptide coated nanoparticles in the brain than unmodified nanoparticles.[83] The consensus sequence for the brain-targeting peptide was found to be TGNYKALHPHNG.[83] In addition to the newer brain-targeting peptides, phages have long been a source for other organ-targeting peptides. These organs include but are not limited to the skin, lung,


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pancreas, intestine, uterus, adrenal gland, and retina.[76b, 84] There have even been efforts to map the entire human vascular system by using phage-display techniques.[76b Finally, there have also been a tremendous number of tissue-targeting peptides discovered through phage-display techniques. Some of these motif sequences are for breast cancer, hepatocarcinoma, melanoma, prostate cancer, gastric cancer, colon cancer, head and neck cancer, cervical cancer, ovarian cancer, rhabdomyosarcoma, osteosarcoma, lymphoma and leukemia, lung cancer, and barret’s esophagus cancers.[20] A detailed and comprehensive list and description of these cancer-targeting phage peptides have already been produced and reviewed, in addition to several other cancer-targeting peptide sequences.[20]

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Phages are an excellent candidate for drug/gene delivery. Phages naturally insert their genome into host bacterial cells, and filamentous phages have the ability to remain dormant without lysing the target cells. The phages can easily be genetically modified to display target-specific peptides. In addition, phages can have drugs conjugated to them and act as carriers, or their peptides can be incorporated into another delivery method such as a coating on nanoparticles. It follows that phages have the potential to become a widely applied tool in nanomedicine. 5.1. Phage Stability


If phages are to be a viable vessel for drug/gene delivery, then their stability is highly important. Phages must be stable enough to be administered in traditional methods, with oral administration being the most desired method. Jepson et al. investigated the stability of lambda phage delivery system for a DNA vaccine, and the phages were found to be very robust.[85] Their stability was found to be unaffected by freezing or thawing over a six month period at 4 and −70 °C. In suspension, the half-life of the phages was 36 days at 20°C.[85] Freeze-drying without stabilizers led to 5–20% residual viability.[85] After desiccation, the half-lives ranged from 20 to 100 days at 20°C, although trehalose significantly increased the stability of the desiccated phages.[85] Perhaps most importantly, phage suspensions were stable over a 24-hour time period within a pH range of 3–11. This study shows that phages can be stored easily and can be stable under harsh pH conditions in vitro. It may even be possible to administer phage-based DNA vaccines through drinking water. In addition to pH stability, phages carry DNA in a protective protein matrix, which protects it from nuclease degradation.[85] These stability properties make phages an excellent delivery vessel for drug/gene delivery. 5.2. Phage-Inspired Antibiotics Professionally lytic phages naturally target, infect, and lyse bacterial cells.[81a, 86] Therefore, employing them as antibiotic agents could prove to be a natural solution to the increasing problem of antibiotic-resistant strains of bacteria.[4e, 87] For example, Zhang et al. reported that a combination of the antibiotic kanamycin and the professionally lytic phage SBW25Φ2 was able to dramatically decrease the survival rate of the bacterium Pseudomonas


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fluorescens.[88] The combined treatment also proved robust to immigration of bacteria from an untreated environment.[88] The key here is that bacteria may have evolved to survive kanamycin or phages, but not both at the same time. It is also possible that if bacteria do evolve to gain resistance to the phages, the phages will in turn evolve, thereby creating a coevolutionary arms race.[89] The therapeutic effects of such an event are unknown.[88]

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Bacterial antibiotic-resistance genes are generally carried by spread conjugative plasmids. Keeping this in mind, some phages have evolved to infect and kill plasmid-harboring cells, which gives rise to the possibility of using phages to remove bacterial plasmids and make them vulnerable to antibiotics.[57] Jalasvuori et al. showed that the use of plasmid-dependent phages (PRD1) on Escherichia coli and Salmonella enterica caused a dramatic reduction in antibiotic-resistant bacteria.[57] The loss of antibiotic resistance in bacteria harboring an RP4 plasmid was a result of them losing since this leads to the loss of suitable receptors for any PRD1 phages.[57] In this case, adapting to survive the PRD1 phages prevented the further spread of resistance through plasmids. Upon addition of PRD1 phages, the frequency of antibiotic-resistant bacteria dropped quickly to an average of five percent by day ten, regardless of phage addition frequency.[57] It should also be noted that at low frequency, the bacteria was able to keep their plasmids and become resistant to phages. However, these double mutants had a growth cost compared to the phage-resistant but antibiotic-susceptible mutants. They were additionally unable to conjugate. The plasmids presented in this study were the IncP-type plasmid RP4 and IncN-type plasmid RN3.[57]

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Phages have also been employed as an antibiotic by equipping them with a photosensitizer to target microbes. Embleton et al. were able to successfully conjugate the photosensitizer tin(IV) chlorin e6 (SnCe6) to phages and transport it with high specificity to their target microbe, Staphylococcus aureu. The strain of phage used was phage 75, which is a serogroup F staphylococcal phage that is used for typing staphylococci.[90] They found that red-light irradiation resulted in activation of the photosensitizer and thus killing of the microbe.[90] In principle, this strategy should kill even antibiotic-resistant strains of bacteria. This strategy is also often used in cancer treatments, as will be discussed in Section 6.2.

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In addition to the phage antibiotic strategies discussed, recent work has shown that lytic proteins expressed by phages may also be a therapeutic option.[91] Such proteins demonstrate bactericidal properties and these recombinant proteins are much simpler than the phage itself.[91] This leads to the potential for creating an antibacterial treatment with a high degree of control.[91] Some examples include the recombinant lyase gp17, which has been shown to inhibit the growth of E. coli 8401,[92] and the molecular dissection of the broad-spectrum antimicrobial endolysin PlySs2, which revealed unique binding and catalytic-domain properties against Staphylococci and Streptococci.[93] Avibrant new area of study in this field worth mentioning is the CRISPR/Cas system. This system enables direct editing of phage genomes. Phages modified with the CRISPR/Cas system can be used to sensitize antibiotic resistant bacteria by eliminating the plasmid that confers resistance.[4a, 94] Briefly, selective pressure can be applied to sensitize bacteria towards an antibiotic by providing a survival advantage in the presence of lytic phages. In the presence of lytic phages, those bacteria that did not undergo CRISPR/Cas treatment for


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sensitizing the bacteria to an antibiotic are at a survival disadvantage. Therefore, there is a selective pressure for the sensitive bacteria to survive.[4a–c] This gives a selection process that could help alleviate problems with antibiotic-resistant bacteria and could be of great interest in the future. The CRISPR/Cas system is traditionally a defense system against bacteriophages. However, the bacterium Campylobacter jejuni, which is responsible for Gullian–Barré syndrome,[95] was discovered to have both a type II CRISPR/Cas and a ganglioside-like sialyated lipooligosaccharade defense system against phages. The Campylobacter jejuni sialyltransferase Cst-II was found to be linked to Gullian–Barré syndrome, as well as to confer efficient phage resistance in the bacteria. CRISPR degeneration and mutations within the cas1, cas2, and csn1 genes was found to correlate with the presence of the sialytransferase. By inactivating the type II CRISPR/Cas marker gene csn1, virulence in cst-II-positive Campylobacter jejuni was reduced. This work demonstrates a novel link between bacterial defense against phages, virulence, Gullian– Barré syndrome, and a pathogenic bacterium that could have clinical relevance. 5.3. Phages as Drug Nanocarriers The emergence of covalent modification processes for viral capsids has laid the groundwork for converting viral capsids into modular carrier systems for drugs and imaging agents.[53a For example, Kovacs et al. were able to coat the surface of genome-free MS2 capsids with polyethylene glycol (PEG) chains and also incorporate 50–70 copies of a fluorescent drug cargo mimic into the capsid.[53a The capsid was shown to remain in the assembled state despite high levels of modification, thus making it a viable delivery vessel for drugs or imaging agents.[53a In a similar system developed by Tong et al, MS2 phage capsids are loaded with a cargo drug and the surface is coated with DNA for targeting (Figure 5).[53c

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There have also been recent advances in drug delivery by filamentous phages. Drugs can be attached to the phage surface without disrupting cell-targeting. For example, drug-loaded liposomes can deliver drugs to cells through a phage–liposome complex.[96] 5.4. Phage-Inspired Targeting Moeities for RNA Delivery

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Nucleic acids such as antisense oligonucleotides, small interfering RNA (siRNA), aptamers, and ribozymes are useful in medicine owing to their innate ability to interfere with protein expression in a predictable way.[97] However, the use of nucleic acid therapeutics has been limited by their instability under physiological conditions and lack of ability to migrate to the site of action.[97] For example, to deliver the siRNA, it can be packaged in several ways. Typically, the siRNA is at the center of the carrier particle and is surrounded by a biocompatible material such as a lipid bilayer, and the targeting peptide is left on the outside to allow greater interaction possibilities with cells. As previously mentioned, peptides from phages theoretically allow the targeting of any organ or cell type and will then carry the siRNA-containing particle to the site of action. These siRNA-containing particles have been shown to have potential in many applications. For instance, Bedi et al. have shown that pVIII-coated siRNA-containing liposomes could serve as a carrier to target breast cancer cells.[51] The engineered pVIII protein of


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filamentous phages was shown by western blotting to target MCF-7 cells, and the result was significant down-regulation of PRDM14 gene expression and PRDM14 protein synthesis.[51] Additionally, Bedi et al. evaluated the potential for treating cancer with siRNA particles coated by tumor-targeting peptides.[97] The tumor-targeting peptides were affinity selected from a landscape phage library against breast cancer cells and fused to pVIII. In this library, each phage clone displays a unique peptide as a fusion to each copy of pVIII, which constitutes the major coat of a filamentous fd-tet phage particle. The siRNA was encased by selected pVIII from filamentous phages and shown to specifically silence the model gene GAPDH in MCF-7 cells.[97] The siRNA-containing particles were also shown to be resistant to degradation by serum nucleases, thus making them a stable, highly selective potential nanomedicine.[97] This strategy is summarized in Figure 6.

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Chen et al. then extended this siRNA delivery approach to skin disorders.[98] siRNAcontaining particles coated with peptides derived from phage display gave enhanced penetration into porcine skin, an increase in epidermis accumulation of siRNA, and increased penetration into cells compared to delivery by aqueous solution.[98] The siRNAcarrying particle design is shown in Figure 7. Although many more applications exist, these studies give a sense of the broad applications of siRNA delivery by particles modified with the targeting peptides identified by phage display. 5.5. Phage-derived DNA carriers

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As discussed in Section 5.1, phages are an excellent carrier of DNA, owing to their stability over a range of pH values and ability to fend off nuclease degradation.[85] In terms of carrying a large amount of foreign DNA, phages possess a large cloning capacity. For example, lambda phages have a cloning capacity of around 20 kilobase pairs (kbp) of DNA, which is much higher than the 5 kbp maximum for plasmid DNA vaccines.[99] This large cloning capacity allows multiple inserts for many different vaccines or isotypes to be included on a single phage particle.[85] It also allows the insertion of large intron-containing genes, which is ideal for creating vaccines against eukaryotic parasites such as malaria or

Plasmodium falciparum.[85]

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One useful technique for the delivery of DNA into mammalian cells using phages involves the use of small cationic peptides called peptide/protein transduction domains. Wadia et al. were able to express the trans-activator of transcription domain (TAT) of human immunodeficiency virus type 1 (HIV-1) on lambda phages. TAT is encoded by the tat gene of the virus HIV-1. It is a regulatory protein that enhances viral transcription efficiency.[100] This method resulted in the efficient delivery of plasmid DNA into mammalian cells without cytotoxicity and in a concentration-dependent manner (Figure 8).[37] Tao et al. successfully used phages for DNA delivery by first allowing the DNA to be translocated into the empty head of the T4 phage.[42] The surface of each phage was decorated with fused outer-capsid proteins consisting of highly antigenic outer-capsid protein (Hoc) and small outer capsid protein (Soc).[42] The T4 vessels were shown to efficiently carry vaccine candidates, functional enzymes, and targeting ligands into cells.[42] Mice receiving a single dose of F1-V plague vaccine through delivery of both the gene and


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protein in the head of the T4 phages were shown to exhibit robust antibody activity and cellular immune responses.[42] In addition to the studies mentioned, there have been many successes in DNA delivery by phages for cancer treatments, as will be discussed in Section 6.[101] This wide success will likely lead to many new types of vaccines and gene therapies.[42]

6. Phage-Based Nanomedicines for Targeted Cancer Treatment

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In addition to siRNA treatment of cancer by means of peptide-coated siRNA-carrying particles, there have been several advances in phage-related cancer nanomedicine. Cancer is an example of a highly important disease that can be addressed by phage-based methods. Cancer and its accessibility to phage-derived approaches are on the cutting edge of potential cancer nanomedicine treatments. In order to better understand phage-based cancer treatment, it is first necessary to understand how the phages interact with a cancerous cell, before moving on to drug conjugation methods. 6.1. Interaction between Phages and Cancer Cells

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If phages are to be involved in treating cancer, it is first important to understand how the phages themselves affect and enter cancer cells. Filamentous phages displaying the peptides VSSTQDFP and DGSIPWST were found to be selectively internalized by SKBR-3 breast cancer cells.[75] Upon entry of the phages, the SKBR-3 cell membranes showed ruffling and rearrangements of their actin cytoskeletons. Phage entry into the cells was found to occur by an energy-dependent mechanism.[75] The study also indicated that multivalent phage libraries could considerably increase the repertoire of cell-entry ligands for imaging, drug delivery, molecular monitoring, and profiling of breast cancer cells.[75] 6.2. Phage-Bound Photosensitizers in Photodynamic Therapy

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The use of photosensitizers in phage-based nanomedicine was briefly introduced in the context of antibiotics in Section 5.2, but they are also applicable to cancer treatment by photodynamic therapy (PDT). In PDT, a combination of endogenous oxygen, light, and photosensitizers are excited to produce singlet oxygen (1O2) that then induces cell death.[102] Recently, our group has explored the conjugation of the photosensitizer 9ethenyl-14-ethyl-4,6,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid, known as pyropheophorbida (PPa), to the N terminus of genetically modified phages specific for SKBR-3 cancer cells by using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) as a coupling reagent.[102a The SKBR-3 cancer cell targeting phages partially modified with PPa were shown to retain their targeting capabilities and the cells were killed by PDT upon excitation with a 658 nm laser.[102a 6.3. Inhibition of Cancer Cell Growth by Using Phage Vectors Targeted gene therapy has been applied via phage vectors to treat cancer.[101a, 103] Although phages naturally only infect bacteria, they can be modified for gene expression in eukaryotes.[101a, 104] For example, Kia et al. designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV).[101a The AAV/phage


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hybrid (AAVP) was able to deliver therapeutic genes to tumors with high specificity. The stress-inducible tumor-specific Grp78 promoter was also introduced to this system and the dual tumor-targeted AAVP provided persistent gene expression compared to silenced gene expression from the CMV promoter in the parental AAVP.[101a Histone deacetylation and DNA methylation were found to be associated with gene silencing in the CMV promoter of the parental AAVP. However, a higher expression from the Grp78 promoter was found in cancer cells when using the dual tumor-targeted AAVP.[101a Therefore, a combination of histone deacetylase inhibitor drugs coupled with the Grp78 promoter is a plausible improvement for AAVP-mediated gene expression in cancer cells.[101a 6.4. Direct Conjugation of Cancer Drugs to the Phage Surface


The anthracyline antibiotic doxorubicin is a well-known cancer drug that was originally isolated from the fungus Streptomyces peucetius. Doxorubicin acts through several mechanisms. It kills cells through the release of free radicals[106] and also interacts with DNA to inhibit the progression of topoisomerase II (which is responsible for relaxing supercoils in DNA during transcription).[107] In clinical trials, it has been shown to be effective but may also be associated with a slight risk of cardiac dysfunction (4% higher than controls), which increases with age or antihypertensive medications. According to the original paper, after administration of the drug, the ejection fraction from the left ventricle was measured as an indicator of heart function when used to treat breast cancer.[108] In addition, the side effects of common chemotherapeutic treatment such as nausea, vomiting, hematopoietic suppression, and alopecia are also associated with doxorubicin.[106b Doxorubicin has also been shown to have potential for treating ovarian cancer,[106b, 109] prostate cancer,[106b, 110] leukemia,[106b, 111] non-Hodgkin lymphomas,[106b, 112] Hodgkin’s disease,[106b, 113] and sarcomas.[106b, 114] As with any drug, and particularly chemotherapeutic drugs, the issue lies in targeting cancerous cells with high specificity, while sparing healthy cells. Several targeting strategies have been employed, such as the use of aptamer-conjugated and doxorubicin-loaded unimolecular micelles,[110a or tumor-targeted quantum-dot–mucin 1 aptamer–doxorubicin conjugates.[109a Perhaps a more effective targeting approach would be the use of phages for delivering doxorubicin to cancerous cells.

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Filamentous phages have already been utilized as a carrier of doxorubicin for cancer treatment.[50b, 105] Filamentous phages can be genetically modified to display a specificityconferring ligands and conjugated to drugs such as doxorubicin or hygromycin.[50b Drugcarrying phages are able to bind to antibody receptors on the cell membranes of cancer cells and cause endocytosis, intracellular degradation, and drug release. This technique produced growth inhibition of target cells in vitro, with a potential targeting advantage of greater than 1000 times that of corresponding free drugs.[50b An overview of a treatment method using MCF-7-targeted phage–Doxil nanoparticles to treat breast cancer is shown in Figure 9.

7. Nanostructured Phage Matrix for Inducing Tissue Regeneration Phage-based scaffolds for tissue regeneration have gained attention recently owing to their ability to display peptides and create well-defined self-assembled nanostructures. These self-


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assembled biomimetic structures have been employed in neural regeneration and bone-tissue regeneration.[52a, 70] 7.1. Phage-Induced Stem Cell Differentiation

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Cell differentiation can involve a variety of factors. Genetically modified phages have the advantage of displaying specific peptides or carrying differentiation-inducing factors to stem cells.[115] For example, displaying the peptides Arg-Gly-Asp (RGD, which makes up a celladhesion integrin-binding motif[52a, 71]) and PHSRN on M13 phages has been shown to induce osteoblastic differentiation of mesenchymal stem cells (MSCs) without the addition of other osteogenic supplements.[115b In addition, RGD and IKVAV (a laminin motif known to promote neural cell adhesion and neurite extension[116]) have been displayed on M13 phages and shown to promote the proliferation and differentiation of neural progenitor cells when self-assembled into a 3D scaffold.[52a In addition, phage technology has been implemented to differentiate induced pluripotent stem cells (iPSCs). The reprogramming of somatic cells to be pluripotent has opened up new possibilities in tissue regeneration by providing an alternative to the ethical issue of using embryonic stem cells and overcoming serious immune system rejection issues.[117] M13 phage-based matrices have recently been shown to be capable of directing iPSC fate first into mesenchymal progenitor cells (MPCs) and then to osteoblast cells to generate a safe and efficient cell source for tissue regeneration.[118] 7.2. Phage-Induced Tissue Formation

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Although phages have been shown to direct cell behavior in vitro, the use of the intact phages to induce tissue formation had never been reported until it was recently found that phages could be used to induce the formation of vascularized bone tissue in vivo in a rat model.[71] Previously developed bone tissue engineering strategies had either failed to produce high blood vessel densities in new bones or met with limited success.[71, 119] Partial success had been achieved through biomimetic 3D scaffolds,[120] the use of growth factors like vascular endothelial growth factor (VEGF),[121] or potent cell sources such as stem cells or mature vascular cells.[122, 71] Recently, we developed a process in which phage nanofibers induced vascularized osteogenesis in 3D-printed bone scaffolds to heal bone defects in a rat radial-defect model.[71]

8. Phage-Based Nanoprobes for Precise Disease Diagnosis

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The ability of a phage to target diverse biologically active materials and carry imaging agents provides opportunities for their use in biosensing and disease diagnosis. Ideally, phages could act as a sort of magic bullet for targeting diseased cells and may greatly improve disease detection methods. 8.1. Phages as a Molecular Imaging Agent in Disease Detection Genes can produce biologically active peptides for which the natural targets are proteins or protein-coupled receptors. These peptides play crucial regulatory roles, which are often mediated by binding to receptors such as G-protein-coupled receptors.[123] Their binding can regulate G proteins, tyrosine kinases, and transcription processes among others.[124] In


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terms of disease imaging and diagnosis, several of these receptors are significantly overexpressed in certain diseases and tumors.[125] For example, the integrin binding motif Arg-Gly-Asp (RGD) is widely used for tumor targeting[126] and can be incorporated into filamentous phage coat proteins to target overexpressed integrins in cancerous tissues.[127] Phages expressing the RGD peptide motif may even have potential in treating inflammatory arthritis.[128] In addition, after phages are genetically selected for peptides that bind overexpressed receptors, the phages can be designed to carry an imaging agent to the site of the disease, thereby enabling imaging and disease diagnosis.

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Magnetic resonance imaging (MRI) can be of great use for in vivo imaging as it provides highly resolved anatomical and functional information in a non-invasive manner. Recently, a system was developed that utilizes P22 viral capsids and ferritin protein cages conjugated to GdIII chelators for use in vascular imaging.[129] The system was validated by visualizing the intravascular system of a mouse through MRI. This included visualizing the mammary arteries, jugular vein, and superficial vessels of the head to an isotropic resolution of 250 μm. Such a system is promising for applications to provide critical information on vascular diseases and physiology. The system used is summarized in Figure 10.[129] 8.2. Phage-Inspired Targeted Imaging for Cancer Diagnosis

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Overexpressed receptors that are often found in diseases like cancers can be targeted by phages carrying an imaging agent. For example, Kelly et al. were able to directly label phage clones with far-red fluorochromes and image them in vivo by multichannel fluorescence ratio imaging.[131] The fluorescently labeled phages also retained tumor-targeting specificity upon labeling and could be quantitated throughout the entire depth of the tumor by fluorescent tomographic imaging.[131] In addition to fluorescence imaging, it is possible to perform targeted single-photon emission computerized tomography (SPECT). BT-474 breast cancer cells can be targeted and imaged with a 1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid (DOTA)-conjugated peptide radio-labeled with 111In.[130] Images generated from the SPECT method are shown in Figure 11. DePorter and McNaughton have also recently developed a method of diagnosing prostate cancer by delivering protein cargo to cancer cells.[132] The cargo is carried by M13 phages to the diseased cells and consists of a protein that can serve as an imaging probe. An overview of this technique is shown in Figure 12. 8.3. Phage-Based Detection of Bacterial and Fungal Infection

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Identifying bacterial contamination is an important step for improving health, and it is very important in the context of the food industry,[133] detection of biological threats,[77c, 134] and diagnosis of bacterial infections. Phages can be useful bacterial diagnosis tools since they specifically recognize and lyse bacterial isolates, which in turn, provides evidence of viable cells.[72a, 135] Several methods have been employed to detect bacteria. One approach used the DSM JG004 phage strain in phage-based hydrogels to measure the diffusion of the bacteria P. aeruginosa into the hydrogels and produce a chromogenic bacterial biosensing response.[72a Another approach, which has a limit of detection from 104 to 103 cfumL−1, involves measuring the fluorescence by flow cytometry of bacteria flowing over T4 phage


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coated Dyna beads, which selectively capture and concentrate E. coli K12 cells (cfu=colonyforming units).[32b Yet another approach involves the use of phage receptor binding proteins coupled to magnetic separation with real-time PCR, enables the detection of Campylobacter jejuni cells at concentrations as low as 100 cfumL−1.[32a] Recently, we have also developed an ultrasensitive method for the rapid detection of human serum antibody biomarkers using biomarker-capturing fd phage.[136] This method provides a faster, more sensitive way to detect the fungus Candida albicans, which contributes to high mortality rates in cancer patients who become infected.[136] While the previous gold standard method for detecting Candida albicans takes about a week, this method is capable of providing a diagnosis in only 6 h at approximately 1.1 pgml−1, which is roughly two orders of magnitude lower than the traditional antigen-based technique.[136] These studies clearly show the power of phages as a tool for detecting bacterial and fungal infections. For further reference, several additional phage-based biosensing techniques are also summarized by Peltomaa et al.[8b

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8.4. Phage-Mediated Detection of Biomarkers at the Molecular Level

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In molecular diagnostics, the ability to count a disease biomarker molecule with the naked eye seemed to be impossible. However, such a technique has been developed by employing T7 phages (Figure 13). Briefly, the phages are genetically engineered to give off fluorescence as well as to bind an miRNA-capturing gold nanoparticle in a one-to-one manner.[35b The target miRNAs can then crosslink the coupled T7 phage and gold nanoparticle complex with miRNA-capturing magnetic microparticles. This forms a sandwich complex of equimolar phage and miRNA. The T7 phages can then be released from the complex and titered to give a macroscopic fluorescent plaque in a petri dish with its host bacterial medium. The plaques can then be counted with the naked eye with detection limits of approximately 3 and 5 aM for single- and two-target miRNAs, respectively.[35b In this way, disease biomarkers can be quantified by the naked eye at the single-molecule level.

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Detecting biomarkers for viral disease diagnosis has also undergone recent advances. After exposure to an illness-causing virus, the human body produces antibodies for targeting that virus in case of future exposures. This immune response can be taken advantage of by using a blood test to detect all present and past exposures to viruses. Typically, such a test was done for one virus at a time. However, Xu et al. have developed a blood test capable of identifying antibodies against all known human viruses at the same time with less than 1 μL of blood.[137] The process, termed VirScan, uses phage immunoprecipitation sequencing technology. The process uses a programmable DNA microarray to synthesize 93904 200mer oligonucleotides, which encode 56-residue peptide tiles with 28-residue overlaps. These cover the sequences of all viruses annotated to have human tropism in the UniProt database. A T7 phage library including displayed peptide sequences from 206 species of virus and over 1000 strains is then created for screening. Incubation of the library with blood serum (which already contains past exposure antibodies) allows the recovery of present antibodies for each virus that a person has previously or is currently infected with. Non-binding phages are washed away and PCR along with massively parallel DNA sequencing on the phage DNA are carried out to quantify the enrichment of each library member and later provide a readout of all previous viral infections a person has had. This technique is described further in Figure 14.


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To further demonstrate the versatility of phage-inspired techniques for detecting biomarkers, Ghoshal et al. were able to use phage display for the identification of serum biomarkers of traumatic brain injury.[138] Peptides that could preferentially bind the serum biomarkers for brain injury were selected using an M13 phage Ph.D.-12 phage library. The presence of injured brain serum biomarkers could then be determined through enzyme-linked immunosorbent assay (ELISA). The test compared non-injured brain serum to injured brain serum. In the ELISA, the phages bind the biomarkers and are detected using a horseradish peroxidase (HRP)-conjugated anti-M13 antibody in the presence of the substrate 3,3′,5,5′tetramethyl benzidine by measuring the absorbance at 450 nm. The peptides were found to work through preferential targeting of glial fibrillary acidic protein. This protein has been previously shown to be a promising biomarker of traumatic brain injury.[138] The animal model used was C57BL/6 mice. This technology has implications for helping to determine the extent and severity of traumatic brain injuries or distinguish them from more mild concussions.

9. Phage-Based Vaccination 9.1. A Phage-Based Type A Influenza Vaccine An exciting field of phage-based nanomedicine deals in vaccinations.[139] In general, phages can be used to display a conserved peptide that is otherwise present on dangerous viruses. After injection of the virus-mimicking phage, an antibody response is safely generated, which confers immunity to the actual virus.

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Type A influenza viruses are a major problem in human populations. Seasonal vaccinations are available, but need to be continually developed to keep up with the variability of influenza. The matrix protein 2 of type A influenza is currently of great interest since it is conserved in human and avian influenza. Therefore, it may be a potential target for a universal type A influenza vaccine. Deng et al. were able to fuse the extracellular domain of matrix protein 2 to the N terminus of the major coat protein (pVIII) of M13 phages. The recombinant phages were then tested in BALB/c mice for their ability to induce an antibody response. Immunization of mice with the recombinant phages in the presence of Freund’s adjuvant was shown to induce a robust matrix protein 2 immune response and protected BALB/c mice from infection by the human and avian type A influenza viruses.[139b 9.2. A Phage-Based Vaccine against the Fungus Candida albicans

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Phage-based vaccines have also been created for certain fungal infections. The fungus Candida albicans is responsible for high morbidity and mortality rates among immunocompromised patients. While there are several virulence factors associated with Candida albicans, the secretory aspartyl proteinase (SAP) family proteins are considered to confer notable virulence attributes. These attributes include phenotypic switching, adhesion, hyphal formation, secretion of hydrolytic enzymes, and dimorphism. SAP2 is the most abundant of these SAPs. Therefore, a phage-based vaccine was created by displaying a short peptide epitope from this target.[140] Filamentous fd phages displaying the epitope (the peptide Val-Lys-Tyr-Thr-Ser) were shown to produce a strong immune response in mice. Phage-immunized mice showed dramatically increased survival after exposure to/infection


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with Candida albicans compared to non-immunized mice. Therefore, this phage-based vaccine holds great promise for further clinical trials in humans.[140] Figure 15 demonstrates this concept. 9.3. A Phage-Based Plague Vaccine

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In addition to viral and fungal infections, phages have been used to make vaccines for bacterial infections, for example, against Yersinia pestis, which causes plague. Although ancient, the disease still exists in some parts of the world today. Plague was responsible for wiping out a third of Europe’s population in the 14th century. For this reason, it has been a priority to stock FDA-approved plague vaccines. However, vaccines such as that produced from killed whole cells have been discontinued due to the requirement for multiple immunizations, demonstration of high reactogenicity (a property of a vaccine capable of causing undesired reactions), and lack of complete protection. For these reasons, work has been completed in developing a T4 phage based vaccine for plague that utilizes T4 phage as a nanoparticle delivery system. A potential target protein, the major antigen Caf 1, was modified and displayed on T4 phage. The new vaccine was then evaluated in mice.[141] The vaccine conferred 100% protection against an intranasal administration of Y. pestis CO92 (which is responsible for pneumonic plague), while all control animals died by day 4.[141b 9.4. A Vaccine for Phage-Based Hormonal Fertility Control


Extraordinarily, phage-based vaccines are not limited to the typical disease prevention concepts discussed. They have also been implemented in controlling the release of hormones. For example, gonadotropin-releasing hormone is a 10-mer peptide that acts as a master reproductive hormone and regulates the release of major gonadotrophic hormones.[139c Antigenic preparations based on gonadotropin-releasing hormone can stimulate the production of antibodies, which will inactivate endogenous gonatropinreleasing hormones. Thus there is potential for regulating sex hormones as well as creating a fertility control tool. In addition, gonadotropin-releasing hormone vaccines are thought to be possible candidates for the treatment of prostate and hormone-sensitive reproductive cancers. Hence, a potential gonadotropin-releasing hormone vaccine was created by utilizing filamentous phage display.[139c Potential immune-response-generating peptides were developed through selection of a phage display library against several types of major gonadotrophic hormones. Five of the selected peptides were then tested in mice as a single dose of 5 × 1011 virions displaying the peptides and were found to stimulate the production of major gonadotrophic hormone antibodies. However, they did not lead to the suppression of testosterone levels, which is an indirect indicator of the ability of the antibodies to neutralize the major gonadotrophic hormone. One of these peptides was additionally tested at a higher dosage of 2 × 1012 virions per mouse, combined with a poly(-lactide-coglycolide) based adjuvant. After this adjustment, there was a multifold increase in major gonadotrophic hormone antibodies as well as a significant reduction in serum testosterone. This indicated that the phage-based vaccine could induce an antibody response capable of suppressing these hormones.[139c


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10. Phage Structure Mimetics and Phage-Derived Particle Variants The development of biocompatible non-viral gene delivery vectors is highly desirable to avoid immune and inflammatory responses. However, non-viral vectors often lack homing and internalization capabilities.[143] To overcome this issue, phage-mimicking nanoparticles have been created.[143b For instance, particles were coated with filamentous phage pVIII protein to mimic phages and improve homing and internalization into MSCs, which led to significantly enhanced transfection efficiency. These MSC-targeting phage-like nanoparticles have desirable applications in treating health problems associated with the heart,[144] brain, bone, spinal cord, and blood vessels.[143b

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Additionally, phage-like particles have been developed as nanocarriers for the transport of cargo across the blood–brain barrier. One such example involved the development of a bifunctional viral nanocontainer utilizing the Salmonella typhimurium phage P22 capsid.[142] The capsid was genetically engineered to incorporate ziconotide in the interior cavity. Next, HIV Tat peptide was chemically attached to the exterior of the capsid for better cell penetration. The phage-like particles were then able to transport across the blood–brain barrier in models of rat and human brain microvascular endothelial cells. Such a system has great potential for transporting drugs across the blood–brain barrier, which is normally difficult to cross. An illustration of the technique is given in Figure 16.[142]

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Phage-like particles can carry compounds with antibacterial properties as well. For example, Douglas et al. were able to construct catalytic antimicrobial nanoparticles with phage-like particles. Briefly, a hydrogen peroxide producing enzyme was encapsulated into a phage P22 phage-like particle. The hydrogen peroxide product could then serve to inhibit bacterial growth. To be more general, such systems suggest that other enzymes may also be encapsulated into the phage-like particles as well, thus suggesting great opportunities in nanomedicine in the future.[145]

11. Status of Phage Use in Humans: Onward to Clinical Trials

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The field finally has progressed to the point where phage therapy can be tested in human clinical trials. Much of the early attention for clinical phage therapies can be attributed to antibiotic therapies.[146] For example, the bacterial strains Pseudomonas aeruginosa, Staphylococcus aureus, and Clostridium difficile are three prominent strains that are difficult to treat with conventional antibiotics.[5b Phage therapies are now being pursued commercially with a few products in phase 1 and 2 clinical trials.[5b, 147] Worth mentioning is the current phagoburn (phase I–II clinical trials) project funded by the European Union under the 7th Framework Program for Research and Development.[2b, 5] This project was launched on June 1st, 2013 and has continued for 36 months, and is a comparative study on treating infection of burn wounds with phages. The study is currently being carried out primarily through a collaboration between France, Switzerland, and Belgium, although the project involves both private and public partners. The main project coordinator for phagoburn is the French Ministry of Defense. However, the private company Pherecydes Pharma is a clinical study sponsor, the company Clean Cells is in charge of biosafety testing, Statitec is in charge of clinical trial data management, and France Europe Innovation is in


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charge of financial and administrative management. The study involves treating burns on each arm of 110 patients with phage cocktails. However, with two different phage cocktails to test, the entire study will necessarily be doubled to 220 patients, 110 for each cocktail tested. For the study, one burned arm is infected with Escherichia coli and the other with Pseudomonas aeruginosa.

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The reference antibiotic treatment used as a comparison is silver sulfadiazine.[5a The recent clinical trials in France, Switzerland, and Belgium are not isolated examples. Phage therapies are also being tested in Georgia, Poland, Germany, and Russia.[4e Of these, Germany has even recently (2015) opened up a therapeutic phage bank at the German Leibniz Institute—Deutsche Sammlung von Microorganism and Zellkulturen GmbH.[4e In the past, it has been more difficult to get approval for phage therapy clinical trials in the United States. However, even the United States now acknowledges several phage therapy clinical trials planned, underway, or completed, according to the U.S. National Institutes of Health.[148] Indeed, after the landmark 2015 phage-based therapeutics meeting hosted by the U.S. National Institute of Allergy and Infectious Diseases, it is clear that the biotechnology industry is in clear support of the use of phage therapies. However, it should be noted that there is still some skepticism among physicians, which will need to be overcome to gain wider spread approval of phage therapies in the United States. Even so, a large amount of clinical trials has been completed or are underway. Table 2 lists a sample of completed phage-based clinical trials listed by the U.S. National Institutes of Health.[148]

12. Outlook and Perspective

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Phage-based nanomedicine is an incredibly diverse and powerful field. This field will undoubtedly continue to grow. As new selections of phages continue, more methods for disease diagnosis and treatment are becoming a reality. Phage-based nanomedicine is at the forefront of precision nanomedicine. By genetically manipulating phages to bind specific targets, phages may hold the key to solving many of the problems caused by the lack of specificity of current approaches. Lack of specificity is known to be one of the major problems in medicine today. This is clearly evident in cases such as cancer treatments. Undoubtedly, precision-based nanomedicine is sure to gain more attention in the coming years. However, despite advances, the use of phages in humans has still not gained widespread acceptability. There exists a gap between animal trials and use in humans that must be addressed. Approval from the US Food and Drug Administration is necessary, and will likely come with increasingly promising research on phage-based nanomedicine.

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There is a lack of knowledge as to the fate of phages in the human body. More research is necessary to address immune response and cellular interactions. Few studies have been completed on the internalization of phages into cancer cells,[50a the stability of phages under various pH conditions,[85] or the complex relationships that phage communities have in humans.[149] This is especially important for phages proposed for use in medical treatments and will be essential to the acceptance of phage-based nanomedicine in humans. Additionally, while the assembly of phages into 3D scaffolds is promising for tissue regeneration, it is highly difficult to reproducibly produce such scaffolds in large sizes.


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Strategies such as 3D printing and the use of magnetic fields to align phage materials hold great promise and may prove to be invaluable in this respect.[32f, 71]

13. Concluding Remarks Phages offer vast and powerful new techniques in nanomedicine. Their ability to display genetically selected peptides and form self-assembled nanostructures has allowed them to be used in fields as diverse as disease diagnosis and treatment, tissue regeneration, bacterial and fungal infection detection, vaccination, and gene therapy. With the increasing research and growing applications of phage-based nanomedicine, this field will undoubtedly continue to grow and produce much needed techniques in nanomedicine.

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One area of particular interest in the future will be precision medicine. Through methods such as biopanning, many more binding motifs are discovered each year. Each binding sequence can act as a type of magic bullet to a particular tissue, cell type, or disease marker, or endless varieties of other targets. Through precision-based nanomedicine, unwanted side effects or diseases that cannot currently be cured, such as the late stages of cancer, will have potential solutions. One thing in particular is certain. This is an exciting and booming field.

Acknowledgments

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We gratefully acknowledge financial support from National Institutes of Health (CA200504, CA195607, and EB021339), National Science Foundation (CMMI-1234957 and CBET-1512664), Department of Defense office of the Congressionally Directed Medical Research Programs (W81XWH-15-1-0180), Oklahoma Center for Adult Stem Cell Research (434003) and Oklahoma Center for the Advancement of Science and Technology (HR14-160). We would also like to acknowledge generous support from National Natural Science Foundation of China (51673168), Zhejiang Provincial Natural Science Foundation of China (LZ16E030001), the State of Sericulture Industry Technology System (CARS-22-ZJ0402), and National High Technology Research and Development Program 863 (2013AA102507).

Abbreviations

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AAV

adeno-associated virus

AAVP

AAV/phage hybrid

APTES

aminopropyltriethoxysilane

BBB

blood–brain barrier

BSP

bone sialoprotein

CNS

central nervous system

CRISPR/Cas

Clusters of Regularly Interspaced Short Palindromic Repeat and associated genes

DOTA

1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

dsDNA

double stranded DNA

E8

Glu8 peptides


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EDC

1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride

FD

freeze dried

GFP

green fluorescent protein

HAP

hydroxyapatite

HIV-1

human immunodeficiency virus type 1

HAART

highly active anti-retroviral therapy

iPSC

induced pluripotent stem cell

MSC

mesenchymal stem cell

MR

magnetic resonance

ND

not determined

PDT

photodynamic therapy

PPa

9-ethenyl-14-ethyl-4,6,13,18-tetramethyl-20-oxo-3phorbinepropanoic acid

RGD

Arg-Gly-Asp peptide

SEM

scanning electron microscopy

Sac2

secretory aspartyl proteinase 2

SPECT

single-photon emission computerized tomography

ssDNA

single-stranded DNA

SWNTs

single-walled carbon nanotubes

TEM

transmission electron microscopy

VEGF

vascular endothelial growth factor

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Author Manuscript

Biographies

Author Manuscript

Kegan Sunderland completed his bachelor of science at Hamline University in biochemistry and mathematics in 2013. In 2015 he completed his Master’s and became a PhD candidate in biochemistry at the University of Oklahoma in the group of Chuanbin Mao. He is a recipient of the Provost Distinction in Teaching award at the University of Oklahoma. His research is focused on tissue engineering, virus-enabled nanomedicine, and bionanomaterials.

Author Manuscript

Mingying Yang received her PhD in 2005 from Tokyo University of Agriculture and Technology in the group of Asakura Tetsuo. She then carried out postdoctoral studies in the same group, before taking a faculty position at Zhejiang University in 2007. Her research is focused on engineering and biomedical applications of natural protein nanostructures.


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Chuanbin Mao received his PhD in 1997 from Northeastern University in China in the group of Lian Zhou. He completed postdoctoral studies at Tsinghua University, followed by a faculty position in Tsinghua University. He then became a visiting scholar at the University of Tennessee at Knoxville. He carried out postdoctoral studies at the University of Texas at Austin and then took a faculty position at the University of Oklahoma in 2005. His research is now focused on phage nanobiotechnology, biomaterials, and nanomedicine.

Author Manuscript Author Manuscript Angew Chem Int Ed Engl. Author manuscript; available in PMC 2017 February 16.

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Author Manuscript Author Manuscript Figure 1.


Overview of phage-based nanomedicine strategies. (Cancer Treatment) Strategies involving precision targeting by phages, which can also have cancer-treating drugs attached to the surface or encapsulated in the interior. Cell/Tissue Targeting: Biopanning on phage libraries has produced a large variety of precision-targeted peptides for specific cell and tissue types. Gene Therapy: Improved phage vectors have been created by incorporating cis genetic elements of adeno-associated virus to allow gene therapy in eukaryotic cells, which phage cannot otherwise infect. Tissue Regeneration: Phages have been incorporated into 2D and 3D scaffolds including hydrogels. Displayed peptides on phages can be tuned to help control stem cell fate and improve tissue regeneration. In addition, the physical structure of the phage itself can promote tissue regeneration by forming structures that mimic the 3D niche of the cells. Imaging: A variety of imaging techniques have been developed in which phages are utilized for precision targeting. Strategies often involve transport of an imaging agent to the desired location by attaching it to or encapsulating it in the phage structure. Stem Cell Fate: Phage-displayed peptides and self-assembled phage scaffolds can be used to control stem cell fate. Drug Delivery: Precision drug delivery methods have been developed by utilizing phages. Drugs can be conjugated onto the surface of phages or encapsulated within a phage such as MS2. The phage itself or attached targeting ligands can then serve as a precision transport system to a specific target. Vaccines: Genetically engineered phages can display peptides that safely mimic dangerous pathogens and generate an immune response for future attacks by that pathogen.


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Author Manuscript Figure 2.

Author Manuscript

Detecting E. coli in drinking water utilizing a T7 phage conjugated magnetic probe. 1) Bacteria are separated from drinking water with a magnet after T7 phages are allowed to interact with the bacteria. 2) T7 phage infects the E. coli and causes the release of βgalactosidase. 3) Chlorophenol red-β-D-galactopyranoside hydrolysis catalyzed by βgalactosidase produces a colorimetric readout.[35a


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Figure 3.

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A) Depiction of an M13–SWNT imaging probe. The pVIII coat protein of the phages are genetically modified to disperse SWNTs along its surface. The pIII protein of the phage is genetically engineered for precision targeting. B) Circulation of M13–SWNT in the blood. Circulation time is defined as the time point at which the %IDg−1 of SWNT in the blood falls to 5%. The blood circulation time for M13–SWNT was around 60 minutes. Each listed data point is the mean±standard deviation (SD) for n=3 animals. (C) M13–SWNT fluorescent images of a mouse injected with 2 μgmL−1 (200 μL, 0.022 mgkg−1 of M13– SWNTs) probe solution. Of the labelled tissues, the liver and spleen can be seen very clearly on the dorsal side. The images were taken at 2 h post injection with an acquisition time of 0.5 seconds at varying time points as shown.[49]

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Summary of the process of biopanning. Briefly, a target ligand is immobilized on a surface. A bacteriophage library is then applied to the surface (a negative selection for the surface without ligand can also be done first), followed by washing, after which only those phages displaying peptides that bind the target will stay bound. The surviving phages are then released by disrupting the interaction. The released phages are amplified and the selection is repeated, usually 3 to 4 times. At the end, phages can be titered and individual plaques can be picked to be sent for sequencing. The most frequently occurring peptides identified from sequencing are then presumed to be the best binders for the target ligand.


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Figure 5.

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A) The synthetic strategy for developing MS2 phage capsids for cargo delivery is shown. An N87C mutation on the MS2 coat protein allows site-specific alkylation. Exterior surface modification of the aptamer with a phenylene diamine group is then completed. A T19paF mutation on the capsid then enables the attachment of modified DNA to the surface of the MS2 through a NaIO4-mediated oxidative coupling reaction. B, C) Images of cellular targeting and uptake with aptamer-labeled capsids. The scale bars: 3 μm. (D) Flow cytometry confirmation of cell targeting. Only those MS2 capsids modified with strand B were bound to the Jurkat cells (blue trace). Capsids not modified on the exterior and capsids modified with strand C (green and yellow traces, respectively) showed only background autofluorescence (red trace). The live-cell confocal images show colocalization of B-labeled capsids with LDL-labeled endosomes as shown in (B), but not with transferrin-labeled endosomes as shown in (C).[53c


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Figure 6.

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A) Depiction of siRNA-coated phage-like nanoparticles created by mixing cholatesolubilized phage fusion protein with siRNA. The cholate was then gradually removed. The N terminus of the phage protein is a targeting ligand, and the positively charged C terminus interacts with the siRNA. B–D) Images showing the binding of the phage protein/siRNA complex (80:1) to MCF-7 (B and C) or MCF-10A (D) cells.[97] The incubation time with the siRNA-coated phage-like nanoparticles was 24 hours for (B) and 4 hours for (C).


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Figure 7.

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A) A diagram of the SPACE peptide (discovered utilizing phage display) bound to cationic ethosomes for the topical delivery of siRNA. The SPACE peptide is directly conjugated to the siRNA or the ethosomal particle surface, and also exists in free form. B) The SPACE peptide directs the internalization of the model drug, fluorescein isothiocyanate (FITC), in a concentration-dependent manner. Representative images showing FITC (green) internalization into skin cells after 120 minutes of incubation at (i) 1 mgmL−1, (ii) 4 mgmL−1, (iii) 7 mgmL−1, or (iv) 10 mgmL−1 FITC–SPACE peptide. The cell nuclei were stained with Hoechst 33342 (blue) and dead cells are indicated by ethidium bromide staining (red). Dead cells were ignored during analysis. (C) The free SPACE peptide increases internalization of the model drug FITC. It also shifts the intracellular distribution from endocytotic structures into the cytoplasm. Internalization of FITC into human adult epidermal keratinocytes when incubated for 15 min with 0.1 mgmL−1 FITC-SPACE peptide with or without 10 mgmL−1 free SPACE peptide are shown. Values represent the mean±SD for n=3. D, E) Representative images of internalization and intracellular localization when the cells are incubated with 0 mgmL−1 (D) or 10 mgmL−1 (E) free SPACE peptide.[98]


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Figure 8.

In situ detection of trans-activator of transcription (TAT)-driven phage-mediated green fluorescent protein (GFP) expression in cultured cells. COS-1 cells (2.5×104) were incubated for 6 hours at 37°C, with 500 μL of medium containing 2.5×1010 pfu of TAT GFP phages (A,B), 2.5×109 pfu of TAT GFP phages (C,D), or 2.5×1010 pfu of wild-type GFP phages (E,F). pfu=plaque-forming units. After 48 h, the cells were counterstained with 4′,6diamidine-2-phenylindole HCl and examined by fluorescence microscopy, with a GFPA cube (A,C,E) or a WU cube (B,D,F), as described in the report. G) Preparation of phage λ particles displaying foreign peptides. DNA is packaged through recognition of the COS site into the phage λ prohead, which consists of E protein. Next, the chimeric D protein with the foreign peptide is assembled onto the prohead, followed by binding of the head to the tail.[37]


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Author Manuscript Author Manuscript Author Manuscript Figure 9.

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Overview of tumor treatment with M13 phage–Doxil nanoparticles modified with the MCF-7-targeted 8-mer landscape library f8/8. Significant tumor remission was accompanied by enhanced apoptosis. There was extensive necrosis and low toxicity. This treatment is ideal for breast cancer therapy.[105]


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Author Manuscript Figure 10.

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Magnetic resonance imaging strategy utilizing P22 phages. GdIII-chelating agents are attached to either the interior or exterior surface of P22 viral capsids. This system allows non-invasive imaging of the intravascular system as shown in the magnetic resonance (MR) image.[129]


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A) Selected phage clones from biopanning for binding to BT-474 breast cancer cells. B) Individual phages were incubated with either target BT-474 cells or normal 184A.1 breast epithelial cells. Total bound phages were quantified and normalized with respect to wildtype phage. The shaded bars represent a mean of three replicate experiments. The error bars give the standard deviation. C) Peptide 51 in Vitro cell binding assays. Biotinylated peptide 51 and a control peptide were incubated with BT-474 human breast cancer and 184A.1 normal breast epithelial cells fixed onto microscope slides. After washing, bound peptides were detected by addition of an anti-biotin AlexaFluor488-conjugated antibody. Strong binding was observed for 51 with the target BT-474 cells. Strong binding was not shown for normal breast epithelial cells. The control peptide does not exhibit binding to either cell line. D) Imaging of BT-474 breast cancer in mice using a peptide developed by phage display. A tumor-bearing mouse was injected with 111In-DOTA-51 and peptide for imaging. T: tumor, K: kidney.[130]


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Scheme for the delivery of horseradish peroxidase to prostate cancer cells by utilizing phages as a carrier with a cancer-cell-penetrating peptide displayed on p3. Increased horseradish peroxidase delivery is indicated by a darker blue color in the colorimetric assay.[132]


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Figure 13.

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General strategy for counting miRNA with the naked eye. a) A T7–gold nanoparticle probe is created by conjugation with a thiolated miRNA-capturing oligonucleotide. The T7 phages are fluorescent. The magnetic microparticle probe is then prepared through conjugation of another biotinylated miRNA-capturing oligonucleotide onto a magnetic microparticle. The magnetic microparticles and T7–gold nanoparticles are then mixed in the presence of the target miRNA. A sandwich complex is formed following the recognition and capture of miRNA. The phages are then eluted through competitive binding followed by plating. The number of fluorescent phages is equal to the number of plaques formed, which is in turn equal to the number of miRNA target molecules. b) Diagram of a fluorescent T7 phage. c) Images of a Petri dish showing the presence of plaques when green fluorescence T7 phages are plated on the host bacteria media (right) and an absence of plaques when no phage is used to infect bacteria (left). d) Fluorescent images of the same Petri dish shown in (c) under the fluorescence scanner. The Petri dish on the right shows green plaques, whereas the one on the left does not. An excitation wavelength of λ=488 nm is used in detecting the green fluorescent plaques derived from the green fluorescent T7 phage–GNP probes.[35b


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General VirScan analysis of the human virome. A) Construction of the virome peptide library and VirScan screening procedure. a) The virome peptide library consists of 93904 peptide tiles, each consisting of 56 amino acids, with a 28 amino acid overlap, that covers the proteomes of all known human viruses. b) The 200-nt DNA sequences encoding the peptides were printed on a releasable DNA microarray. c) The released DNA was amplified and cloned into a T7 phage-display vector and packaged into virus particles displaying the encoded peptide on its surface. d) The library is mixed with a sample containing antibodies that bind to their cognate peptide antigen on the phage surface. e) The antibodies are immobilized, and unbound phages are washed away. f) Finally, amplification of the bound DNA and high-throughput sequencing of the insert DNA from bound phages reveals peptides targeted by sample antibodies. Ab: antibody, IP: immunoprecipitation. B) Antibody profile of a randomly chosen group of donors to show typical assay results. Each row represents a virus; each column a sample. The label above each chart indicates whether the donors are over or under 10 years of age. The color intensity of each cell indicates the number of peptides from the virus that were significantly enriched by antibodies in the sample. C) A scatter plot of the number of unique enriched peptides (after applying maximum-parsimony filtering) detected in each sample against the viral load in that sample. Data are shown for HCV-positive and HIV-positive samples for which we were able to obtain viral-load data. For the HIV-positive samples, red dots indicate samples from donors currently on highly active antiretroviral therapy (HAART) at the time the sample was taken, whereas blue dots indicate different donors before undergoing therapy. IU: international


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units. D) Overlap between enriched peptides detected by VirScan and human B-cell epitopes from viruses in immune epitope database. The entire pink circle represents the 1392 groups of nonredundant immune epitope database epitopes that are also present in the VirScan library (out of 1559 clusters total). The overlap region represents the number of groups with an epitope that is also contained in an enriched peptide detected by VirScan. The purple-only region represents the number of nonredundant enriched peptides detected by VirScan that do not contain an immune epitope database epitope. Data are shown for peptides enriched in at least one (left) or at least two (right) samples. E) Overlap between enriched peptides detected by VirScan and human B-cell epitopes in an immune epitope database from common human viruses. The regions represent the same values as in (D) except only epitopes corresponding to the indicated virus are considered, and only peptides from that virus that were enriched in at least two samples were considered. F) Distribution of number of viruses detected in each sample. The histogram depicts the frequency of samples binned by the number of virus species detected by VirScan. The mean and median of the distribution are both about 10 virus species.[137]


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The concept of displaying vaccine peptides on fd phages for Candida albicans. a) The epitope peptide of secretory aspartyl proteinase 2 (Sap2) was displayed on the phages. b) The engineered phages were injected into mice three times at 25 μg per mouse each time to create immunized mice. Next, two approaches were used to prove the success of the vaccine. The first approach (c) was to test whether Candida albicans could infect immunized mice, and this was successful evidenced by decreased fungal loading in the kidneys, fewer visceral lesions, and an increased survival rate (f). The second approach tested whether antibodies collected from immunized mice (c′) could cure infected mice (h′). This approach led to


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curing of the infection as evidenced by significantly lowered fungal loading in the kidneys (i ′).[140]

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A) An illustration of the concept viral nanocontainers encapsulating marine snail peptide MVIIA and the cell-penetrating peptide Tat(FAM) on the exterior to deliver cargo across the blood brain barrier. This process utilizes an endocytic pathway. B) Spinning-disc microscopy images of RMBVE cells after incubation (20 minutes) with conjugated viruslike particles using a DAPI filter (a,d) or a RhoB filter (b,e) for lysotracker (Excitation/ Emission=490/525 nm). c,f) Overlay of the DAPI, RhoB, and FITC channels, showing colocalization of conjugated virus-like particles with acidic organelles. Cells with hypertonic solution (0.4M sucrose) show reduced uptake of conjugated virus-like particles, but still the show the presence of lysosomes (e–f). Scale bar : 12 μm. g) Green fluorescence intensities plotted to show the reduced cellular uptake of the conjugated virus-like particles in cells incubated without hypertonic solution. Ten regions were selected for measuring the fluorescence intensities. C) Rab11 immunostaining indicating Tat(FAM)–P22-MVIIA virus-


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like particles in the recycling pathway. a–f) The distribution of Rab11, which is known to associate primarily with perinuclear recycling endosomes and regulate the recycling of endocytosed proteins. The immunoreactivity in cells treated with fluorescent virus-like particles and stained for Rab11 are demonstrated through by overlays images for DAPI and FITC (a,d), RhoB and DAPI (b,e), or all three (c,f). The arrows in (c) and (f) show the concentrated virus-like particles inside recycling endosomes as a result of to colocalization with Rab11. g) Quantitation of the images for green (FITC) and red (RhoB) filters shown in (a–f). *p =0.55 or p >0.05. Scale bar: 12 μm.[142]


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Table 1

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Various structures and applications of phages in nanomedicine. Type of Phage

Electron micrograph image

Schematic

Common Applications

Delivery of plasmid DNA into cells without cytotoxicity and in a concentration-dependent manner to mammalian cells.[37]

λ Phage (temperate)

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Electron micrograph of a lambda phage negatively stained with 2% uranyl acetate.[36] Food preservation[39] and antibiotics.[40] Magnetically-assisted impedimetric detection of bacteria.[32b Phage-based DNA and protein packaging, expression, and display systems.[41] T4 phage is also implemented in DNA delivery for potential vaccines.[42]

T4 Phage (professionally lytic)

TEM image of a T4 phage.[38]

Detection of protein markers through real-time immuno-PCR.[44] Detection of viral infections.[45] Rapid detection of E. Coli in drinking water supplies.[35a Naked-eye counting of miRNA for disease diagnosis.[35b

T7 Phage (professionally lytic)

Author Manuscript

Electron micrograph of negatively stained T7 phages.[43]

Podovirus P22 (professionally lytic)

Similar to T7.

Podovirus P22 of Salmonella typhimurium at 297000× magnification.[46]


Assembly of CdS nanocrystals for enhanced photoactivity in tissue imaging.[47]

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Type of Phage


Schematic

Common Applications

Author Manuscript

Used for the identification of B. anthracis, the etiologic agent of anthrax.[46]

Siphovirus γ (professionally lytic)

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Siphovirus γ of B. anthracis at 297000× magnification.[46] Widely used for selfassembled nanostructures such as nanowires[32c and functionalized SWNTs.[49] Also prominent in targeted drug delivery, cancer treatments, and imaging.[50] Filamentous phages have also been implemented in siRNA delivery for gene therapy[51] and tissue regeneration[52]

M13 and fd Phages (temperate)

TEM image of M13 phage[48]

Author Manuscript

Multifunctional drug delivery vessels and tissue imaging.[53]

Levivirus MS2 (professionally lytic)

Levivirus MS2 of E. coli at 297000× magnification.[46] Genome free schematic of MS2 phage particle.

Author Manuscript

Φ29 Phages (professionally lytic)


Development of a DNA packaging nanomotor associated with developing hybrid bionanostructures.[55]

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Type of Phage


Schematic

Common Applications

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CryoEM map of a Φ29 phage. High densities are white and low densities are black. The various proteins and DNA are labeled.[54]

PRD1 Phages (professionally lytic) Cryo-electron micrographs of wild-type PRD1 phages mixed with a preparation of susI packaging mutant at 2 μm underfocus. The arrow points to a DNA filled phage particle and the inset shows a particle which has lost its DNA and forms a tail from the membrane.[56]


Antibiotic studies involving the loss of antibiotic resistance in bacteria harboring an RP4 plasmid.[57]

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Table 2

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U.S. National Institute of Allergy and Infectious Diseases phage usage in completed clinical trials.[148]

Author Manuscript

Year Completed

Study

Condition(s)

2012

Phage Effects on Pseudomonas Aeruginosa

Cystic Fibrosis

2010

Prospective Study Into the Performance of the MicroPhage S. Aureus/ MSSA/MRSATest Directly From Blood Culture Positives

Bacteremia

2008

A Prospective, Randomized, Double-Blind Controlled Study of WPP-201 for the Safety and Efficacy of Treatment of Venous Leg Ulcers

Venous Leg Ulcers

2008

MicroPhage S. Aureus/MSSA/MRSA Blood Culture Beta Trial

Bacteremia; Staphylococcal Infection; Sepsis Infection

2007

T4N5 Liposomal Lotion in Preventing the Recurrence of Nonmelanoma Skin Cancer in Patients Who Have Undergone a Kidney Transplant

Actinic Keratosis ; Basal Cell Carcinoma of the Skin ;Recurrent Skin Cancer ;Squamous Cell Carcinoma of the Skin

2006

Interleukin-2 Plus Anti-HIV Therapy in HIV-Infected Children With Weakened Immune Systems

HIV Infections

2000

The Use of Phage Phi X174 to Assess the Immune Competence of HIVInfected Patients In Vivo

HIV Infections

2003

Study of Immune Responses and Safety of Recombinant Human CD40 Ligand in Patients With X-Linked Hyper-IgM Syndrome

Immunoproliferative Disorder

1997

Phase IStudy of BMS-188667 (CTLA4Ig) in Patients With Psoriasis Vulgaris

Psoriasis Vulgaris


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