Phage protein-targeted cancer nanomedicines

Abstract

Nanoencapsulation of anticancer drugs improves their therapeutic indices by virtue of the enhanced permeation and retention effect which achieves passive targeting of nanoparticles in tumors. This effect can be significantly enhanced by active targeting of nanovehicles to tumors. Numerous ligands have been proposed and used in various studies with peptides being considered attractive alternatives to antibodies. This is further reinforced by the availability of peptide phage display libraries which offer an unlimited reservoir of target-specific probes. In particular landscape phages with multivalent display of target-specific peptides which enable the phage particle itself to become a nanoplatform creates a paradigm for high throughput selection of nanoprobes setting the stage for personalized cancer management. Despite its promise, this conjugate of combinatorial chemistry and nanotechnology has not made a significant clinical impact in cancer management due to a lack of using robust processes that facilitate scale-up and manufacturing. To this end we proposed the use of phage fusion protein as the navigating modules of novel targeted nanomedicine platforms which are described in this review.

1. Introduction

Nano-encapsulated drugs (nanomedicines) offer a less toxic alternative for patients with cancer diseases. They reduce non-specific drug delivery to normal tissues and deliver drugs to tumors [1–3]. It is commonly believed that efficiency of nanomedicines can be significantly increased by their ligand-mediated targeting (LMT) to cancer cells. While the potential benefit of LMT was shown in numerous experiments with animals [4–7], this technology has not made a significant clinical impact on cancer treatment [8,9]. The major challenge in translation of LMT into clinical development has been the difficulty in developing targeted nanomedicines with optimal biophysicochemical properties, biological behaviors, and pharmacological profiles, while using robust processes that facilitate scale-up and manufacturing.

Though most studies of nanomedicine targeting have used antibodies, these therapies show limited efficacy in solid tumors mainly due to their large size (~160 kDa) which precludes efficient penetration, a problem aggravated by the elevated tumor interstitial pressure [10]. Furthermore, optimal tumor penetration may be hindered by a high affinity interaction between the antibody and the first few antigen molecules it comes in contact with thereby preventing uniform diffusion, a phenomenon first described as “binding site barrier” [11] and later demonstrated conclusively [12]. Also, immunogenicity and non-specific uptake by the reticulo-endothelial system contribute to a less than favorable clinical outcome. Research has indicated that pharmacokinetic properties are improved with the use of smaller ligands underscoring the need for smaller molecules for tumor directed therapies [13]. Peptides being in the range of 1–2 kDa and demonstrating acceptable affinity and specificity to their targets represent an attractive alternative to antibodies [14]. Much of the problems attendant with the larger sized antibodies appears to be resolved with peptides. They possess impressive tumor penetrating capacity, are generally not recognized by the MPS and are less likely to instigate immune responses. In addition, they exhibit a higher activity per mass and greater stability [15]. The availability of combinatorial peptide libraries from which potential tumor-specific ligands can be extracted adds another dimension to the concept of peptide-targeted nanomedicines. The use of phage displayed peptide libraries for obtaining tumor-specific peptides and their application in tumor-targeting has been illustrated with several examples in recent reviews [14,16–18]. Liposomal targeting mediated by phage display-derived peptides has been amply demonstrated [19–26]. A critical factor within this scenario is that of the conjugation chemistry required to attach the ligand to the liposomal surface. Numerous covalent coupling techniques including the formation of a disulfide bond, cross-linking between primary amines, reactions between a carboxylic acid and primary amine, between maleimide and thiol, between hydrazide and aldehyde or between a primary amine and free aldehyde can be used (reviewed in [27,28]). The conjugation procedures adapted from the armamentarium of organic chemistry are quite efficient for preparation of various targeted liposomes in small scale – for their preliminary laboratory and clinical study. However, the cost and reproducibility of these derivatives in quality and quantity sufficient for pharmaceutical applications are challenging problems. The use of the conjugates may be less efficient at the scaling step, when very standardized and pharmaceutically acceptable preparations are required. For example, 2085 mg of lipid conjugated with single chain antibody was obtained through a laborious procedure involving high volume propagation of bacteria, several chromatographic steps and sophisticated conjugation procedures yielding a preparation which had 93% purity [29]. Also, preparative conditions for the addressed vesicles differ idiosyncratically from one targeted particle to another.

To facilitate the development and screening of targeted nanomedicines, significant efforts has been made to develop methods of high-throughput selection of cancer cell-binding phage proteins and their self-assembly into nanovehicles [30]. These efforts resulted in the development of several new targeted nanomedicine platforms, which have been patented and proven using human breast, prostate, pancreatic and lung cancer cells [30–43]. This review describes these platforms, the key targeting components of which are the tumor-specific major coat proteins preselected using phage display technique.

2. Phage display

Phage display technology emerged as a synergy of two fundamental concepts: combinatorial peptide libraries and fusion phage [44]. The first concept replaced the traditional collections of natural or individually synthesized compounds for libraries of peptides obtained in parallel synthesis as grouped mixtures [45,46] (reviewed in [47]); the second—allowed displaying foreign peptides on the surface of bacterial viruses (bacteriophages) as part of their minor or major coat proteins [48,49] (reviewed in [44,50]). The merge of these two concepts resulted in development of phage display libraries—multibillion clone compositions of self-amplified and self-assembled biological particles [44].

Fusion phage, the fount of phage display technology, was first described by Smith [49] and involves modifying the phage genome to achieve surface display of a peptide on the virion while maintaining its viability and infectivity. Phage display has since then evolved into a widely used screening resource for molecular targets supporting a burgeoning repertoire of applications [51–54].

The potential of phage display libraries is founded on phage biology. Bacteriophages are viruses that infect bacteria and in particular the phage vectors employed in phage display infect the bacterium Escherichia coli. The filamentous phage of class Ff (M13, fd, and f1) widely exploited to develop phage display systems possess single stranded circular DNA enclosed in a tubular capsid composed predominantly of pVIII major coat protein with few copies of minor coat proteins at the ends of the virion (Fig. 1). In-frame oligonucleotide inserts into one of the coat protein genes results in the expression of the foreign amino acids as part of the corresponding coat protein, creating a hybrid fusion protein displayed on the surface of the phage particles. Thus, the extraneous peptide is now linked to the phage genotype and this forms the cornerstone for the creation of phage display libraries. Phage display libraries are created by splicing unique randomized oligonucleotide inserts into individual phages so that each phage displays a unique peptide.

Fig. 1.

Electron micrograph of filamentous phage. Adapted from [105].

Availability and amenability of numerous phage display vectors has permitted the development of numerous phage display systems [50]. One such system in particular, type 8, involves the expression of a guest peptide on each copy of the major coat protein pVIII of the phage particle. The intense iterative display of peptides on the surface of the phage creates defined organic landscapes leading to the nomenclature, landscape phage (Fig 2). A landscape phage is considered not just as a genetic carrier for foreign peptides, like in the traditional phage display approach, but rather as a nanoparticle (nanotube) with emergent physicochemical characteristics determined by the specific landscapes formed by the thousands of random peptides fused to the major coat protein pVIII [55]. Despite the extra burden, such particles could retain their infectivity and progeny-forming ability making them a rich source of selectable nanomaterials.

Fig. 2.

Segment of landscape phage. Foreign peptides are pictured with white and yellow atoms; their arrangement corresponds to the model of Marvin, 1994. Adapted from [105].

3. Landscape phage libraries

A landscape library is a multibillion-clone population of landscape phages with different structures. For example, landscape libraries f8/8 and f8/9 used in studies in our laboratory [56], were constructed by splicing a degenerate coding sequence into the beginning of the coat-protein gene in the vectors f8-1 and f8-6 [50], replacing wild-type codons 2–4 or 2–5, as shown below;

DNA

GCAGNKNNKNNKNNKNNKNNKNNKNNGGATCCCGCAAAAGCGGCCTTTGACTCC…

pVIII

A

X

X

X

X

X

X

X

X

D

P

A

K

A

A

F

D

S…

1

5

6

7

8

9

10

11

12

13…

where N is any nucleotide, and K is G or T. As a result, every protein subunit in the landscape phage is five amino acids longer than in the wild-type phage, and displays a “random” sequence of eight or nine amino acids [55,56]. In any single clone, the random peptide is the same in every particle, but almost every clone displays a different random peptide. Landscape phages and their fusion proteins have been shown to serve as substitute antibodies against antigens and receptors, including live cancer cells [33,34,57–60]. The targeting protein can be obtained from the phage in a very pure form in one step – using size exclusion chromatography, or by extraction of the protein from phage by isopropanol–chloroform mixture.

The guest peptides in a landscape phage constitute up to 25% by weight of the phage particle and overlays 50% of its surface. The multivalent display of peptides, apart from contributing to avidity allows for a greater freedom in manipulating the phage filament as a scaffold for biospecific interactions. In fact, landscape phages have been proposed as substitute antibodies with nanomolar and subnanomolar affinities and high specificity for protein and glycoprotein antigens. When phage particles mimic antibodies many of the intrinsic limitations of antibodies can be overcome. A culture of cells secreting phages is an efficient protein production system. Phages are secreted from the cell nearly free of intracellular components and can be purified by routine procedures. The phage structure is robust and purified phage can be stored indefinitely without losing infectivity. These characteristics make landscape phage libraries an ideal source for peptide ligands.

Landscape phage probes have been identified for a variety of targets belonging to both the organic and inorganic realms and encompassing a wide spectrum of complexity [30]. The process of identifying phage probes is termed affinity selection and a generic protocol for this procedure has been described in detail elsewhere [33].

As a pertinent example, we successfully managed to isolate highly specific and selective landscape phage probes for PC3 prostate tumor cells. Further, we demonstrated that these probes could be fluorescently labeled and labeling does not adversely affect the target association properties of the phage particles as evidenced by fluorescence microscopy and flow cytometry [34]. This study provided the proof of concept of using the landscape phage particle itself as target-specific nanoparticles with diagnostic applications, a fact that has already been proved with pIII display constructs [61–65]. In addition, landscape phage have been used as diagnostic probes for bacteria [66–68], spores [69], as components of gene delivery systems [70], biospecific adsorbents [71] and molecular recognition interfaces in detection systems [72,73]. Phage derived probes allow the fabrication of bioselective materials by self-assemblage on metal, mineral or plastic surfaces [74–77]. These observations led us to surmise the utility of derivatized phage components as targeting moieties of drug carriers. In particular, the pVIII major coat protein with the N-terminal guest peptide has unique properties which qualify its use as a targeting ligand for clinically important nanomedicines, such as PEGylated liposomes, micelles and complexes with nucleic acids.

4. Phage major coat protein pVIII

The ability of the major coat protein pVIII to associate with micelles and liposomes emerges from its intrinsic function as a membrane protein judged by its biological, chemical, and structural properties. During infection of the host E. coli, the phage coat is dissolved in the bacterial cytoplasmic membrane, while viral DNA enters the cytoplasm [78]. The protein is synthesized in infected cell as a water-soluble cytoplasmic precursor, which contains an additional leader sequence of 23 residues at its N-terminus. Prior to assembly of viral progeny, precursor coat protein pVIII integrates into inner membrane independently of the SEC translocation machinery of the host bacterium. When this protein is inserted into the membrane, the leader sequence is cleaved off by a leader peptidase. Later, during the phage assembly, the newly synthesized proteins are transferred from the membrane into the coat of the emerging phage. Thus, the major coat protein can change its conformation to accommodate various distinctly different forms of the phage and its precursors: phage filament, intermediate particle and membrane-bound form. Despite this remarkable adaptability, the coat protein contains only 50 amino acid residues. It is very hydrophobic and insoluble in water when separated from virus particles or membranes [79] (Fig. 3A). In virus particles it forms a single, somewhat distorted α-helix with only the first four to five residues mobile and unstructured [80] (Fig. 3B). It is arranged in layers with a fivefold rotational symmetry and approximate twofold screw symmetry around the filament axis, as shown in Fig. 3C.

Fig. 3.

pVIII phage major coat protein. (A) Schematic showing the primary structure and domain organization of the pVIII major coat protein in phage M13. (B) Three-dimensional structure of pVIII coat protein showing its helical structure. White area corresponds to the position of the guest peptides. Yellow area shows segment that can be randomized [106]. (C) Fragment of three-dimensional structure of landscape phage showing the arrangement of pVIII units. Adapted from [107].

The structure of the major coat protein in the phage virions, micelles and bilayer membranes is well resolved [81–83]. A variety of structural models for the protein in the membrane-bound state have been proposed, with dominating I-shaped and L-shaped structures, depending on the lipid model studied [81]. In dehydrated lipid bilayers and micelles, the 16-Å-long amphipathic helix (residues 8–18) rests on the membrane surface in L-form, while in hydrated lipids—a natural ‘stress-free’ environment it extends from the lipid bilayer of liposomes as an I-form α-helix. In liposomes, the 35-Å-long trans-membrane (TM) helix (residues 21–45) crosses the membrane at an angle of 26° up to residue Lys40, where the helix tilt changes (Fig. 4). The helix tilt accommodates the thickness of the phospholipid bilayer, which is 31 Å for the palmitoyl–oleoyl–phosphatidylcholine and palmitoyl–oleoyl–phosphatidylglycerol—typical lipids of E. coli membrane components. Tyr 21 and Phe 45 at the lipid–water interfaces delimit the TM helix, while a half of N-terminal and the last C-terminal amino acids, including the charged lysine side chains, emerge from the membrane interior. The transmembrane and amphipathic helices are connected by a short turn (Thr 19–Glu 20). In micelles having a curved surface, N-terminal domain of the protein is forced to bend back on this surface, thus providing a variety of protein shapes including L- and U-shapes in addition to extended structures.

Fig. 4.

The model of the pVIII protein in the lipid bilayer environment. Adapted from [108].

Spontaneous insertion of the major coat protein into lipid membranes is believed to be mediated by interplay of electrophoretic influences (membrane potential), electrostatic forces (charges on membrane and protein) and hydrophobic interactions. The hydrophobicity of the transmembrane domain is chiefly responsible for driving the insertion of the coat protein to allow for thermodynamic equilibrium whereas the membrane potential and charges on the protein in question are the major determinants of the topology of the membrane associated protein [84,85]. The spontaneous insertion of a membrane protein can be envisioned as a two-step process. The first step involves binding of the protein to the membrane parallel to the plane of the membrane accompanied with simultaneous insertion of the hydrophobic region into the hydrocarbon core of the lipid bilayer (Fig. 5A). The second step would involve the release of the hydrophilic tail into the trans side (Fig. 5B), following probably the mechanisms characteristic for cell-penetrating peptides [86]. The structural characteristics of the membrane-bound coat protein after spontaneous insertion was shown to be predominantly α-helical [87]. Association of coat protein with lipid vesicles has been determined by fluorescence energy transfer (FET) between the coat protein’s tyrosine and tryptophan residues (donors) and a diphenylhexatriene (DPH) in the lipids (acceptor). The membrane penetration of coat proteins depends on the concentration of lipids available and almost complete association is seen at high lipid to protein ratios [84]. Furthermore, lipid membranes with negative charges demonstrate higher amenability to spontaneous insertion whereas rigid lipid membranes preclude spontaneous insertion. What is important to note, coat protein insertion into liposomes follows an almost exclusive N terminus_out topological preference. A mutant of coat protein Pf3 of the bacteriophage Pf3 having three additional leucine residues in the transmembrane region and a complete replacement of charged amino acids by asparagine (3L–4N) was shown to spontaneously insert into lipid vesicles [88]. In an extension of this research, hybrid peptides comprising a fusion of a single IgG binding unit of Staphylococcal protein A attached to the N-terminus of the coat protein mutant was shown to spontaneously insert into liposomes and resulted in an emergent liposomal property that of antibody binding [89]. These observations formed the basis for our hypothesis that target-specific phage fusion proteins would function as liposomal and micelle ligands and targeting would improve the performance of anticancer agents. The next two sections provide a summary of studies undertaken to demonstrate the feasibility of using tumor-specific phage fusion protein as navigating modules for anticancer nanoparticles.

Fig. 5.

Spontaneous insertion of the major coat protein into lipid membranes. (A) In the first step, the coat protein binds to the membrane parallel to the plane of the membrane with the insertion of the hydrophobic region into the hydrocarbon core of the lipid bilayer. (B) In the second step, the hydrophilic tail is released into the trans side, a process probably approximating the mode adopted by cell-penetrating peptides.

5. Phage protein-mediated delivery of anticancer nanomedicines

The unique characteristics of phage major coat protein and its recombinant fusion variants allows their use for development of targeted anticancer nanomedicines. This novel approach exploits the intrinsic membrane properties of the phage major coat protein, i.e., its propensity to reside in the bacterial internal membrane during phage infection and ability to spontaneously integrate into the lipid bilayer in vitro. The phage specific for the target organ, tissue or cell is selected from the multibillion clone landscape phage libraries [34,57,59,60,70,90], its pVIII coat protein isolated and inserted into liposomes or micelles, as exemplified below.

Individual proteins or a mixture of targeted phage proteins can be introduced into the preformed PEGylated doxorubicin-loaded liposomes, such as commercially available Doxil^® (Caelyx^®, Lipodox^®) (Fig. 6) by their incubation with the cholate-stabilized phage fusion protein. The crude formulation is dialyzed and the cholate-free liposomes are purified by size-exclusion chromatography. The following properties of the targeted liposomes are controlled: (a) liposome morphology, size and size distribution; (b) content of encapsulated drug; (c) presence and orientation of inserted fusion proteins; (d) liposome ability to keep entrapped prototype drugs and reporter protein moieties both in water solutions and in blood plasma; (e) specific binding activity of the phage protein-bearing liposomes; (f) cytotoxic activity of the targeted nanomedicines towards the target cancer cells in vitro and in vivo.

Fig. 6.

Phage fusion protein-targeted nanoparticles. Target-specific phage fusion protein selected from phage libraries can be introduced into drug-loaded PEGylated liposomes exploiting their intrinsic propensity to spontaneously integrate into the lipid bilayers. Explanation in the text. Adapted from CNRS international magazine, © F. Caillaud/CNRS Photothèque/SAGASCIENCE.

A major concern, in initial studies, related to the ability of the fusion protein to penetrate through the polyethylene glycol (PEG) crown surrounding the liposomes. PEG as an inert, biocompatible polymer forms a protective layer over the liposome surface that masks surface charge, increase the hydrophilicity of liposomes, slows down liposome clearance by opsonins, inhibits destruction of liposomes by lipoproteins and increases their bioavailability [2,91].

Reiterating the earlier discussion, the hydrophobic C-terminal domain of the major coat protein and its fusion forms accommodate perfectly well in the micelles and liposomes formed by natural phospholipids. Existing structural data suggest that the amphiphilic and hinge domains of the major coat protein may also accommodate well in the random coiled corona of the PEGylated vesicles, displaying the fusion peptides on their surface. Since data on the exact random coil conformation of the PEG moieties are quite sparse [26,92], we elucidated a true conformation of the phage proteins in PEGylated vesicles in a model experiment using streptavidin-coated gold beads as a model target and the major coat protein with a fused streptavidin binding peptide as a targeting ligand [36]. The translation of the peptide specificity for streptavidin to the liposomes was evidenced by a functional test utilizing streptavidin-conjugated colloidal gold nanoparticles. As Fig. 7 aptly demonstrates, targeted liposomes show a clear propensity to have the streptavidin-conjugated colloidal gold particles studded on their shell as against control liposomes. To determine protein topology in the lipid membrane and prove the display of the binding peptides on the crown of the PEGylated nanoparticles, the protein-modified liposomes were treated first with proteinase K and then analyzed by Western blot probed for the presence of the N-terminal and C-terminal protein sequences, using N- and C-terminus-specific antibodies. Based on these model experiments it was concluded that the target-specific peptide fused to the major coat protein is exposed on the PEG crown of the drug-loaded vesicle. In following experiments it was demonstrated that the specificity of phage proteins towards cancer cells translates to the protein-modified nanomedicines, increasing their specific binding and cytotoxic activity towards the target cancer cells. It was demonstrated that the phage major coat proteins fused to cancer cell-targeting peptides can serve as an anchor for association of these peptides with drug-loaded liposomes and micelles without destruction of their integrity. Targeting peptides are exposed and allow for specific binding of drug-loaded phage-nanoparticles to cancer cells [35,38,43].

Fig. 7.

Functional test for targeted liposomes with streptavidin conjugated colloidal gold nano-particles. Targeted liposomes prepared by co-incubation of purified major coat protein specific for streptavidin and DOXIL^® were tested using streptavidin conjugated colloidal gold particles for the specificity and functionality of the incorporated phage coat protein. (A) Targeted liposomes studded with streptavidin conjugated colloidal gold particles with a particle to liposome ratio of 1.8. (B) Non-targeted liposomes subjected to the same assay with a particle to liposome ratio of 0.09. Adapted from [36].

Following conclusive proof of concept studies for using phage fusion protein as a simple and cost-effective means for preparing tumor-targeted liposomes, Wang et al. undertook studies to optimize phage liposome preparative conditions that would maximize their targeting efficacy. Accordingly, a factorial design approach was used to determine the role of preparative protocol, phage protein content and lipid composition of the liposomal membrane on the targeting efficiency of the phage liposomes. Results indicated that a specific lipid cocktail of ePC:CHOL:DPPG:DOTAP:-PEG2K-PE:45:30:20:2:3 with 0.5% phage fusion protein had the most positive effect on the targeting specificity of the phage liposomes. Furthermore, the post-insertion protocol wherein the fusion protein is inserted into fully formed liposomes produced formulations which were more uniform and stable than the direct incorporation approach wherein the fusion protein is added at the time of liposome formation [39]. In another study using liposomes modified with MCF-7-specific phage fusion protein [57], Wang et al. demonstrated that the targeting specificity of modified liposomes was solely due to the presence of the tumor-specific peptide on the N-terminus of the phage fusion protein in the liposomal bi-layer. Removal of this binding peptide by proteolytic treatment, substitution of this peptide with a random-peptide or the presence of free peptide in the assay abolished the specific interaction between the modified liposomes and their target MCF-7 cells [40]. Results obtained from liposomal studies were also translatable to other nanoparticles like micelles. Thus, paclitaxel containing micelles targeted with phage fusion protein specific for MCF-7 cells showed preferential binding to their cognate target cells as compared to non-target cells resulting in a significantly higher cytotoxicity towards target MCF-7 cells than free drug or non-targeted micelle formulations. This differential toxicity was not seen in non-target C166 cells leading the authors to surmise that cancer cell-specific phage proteins identified from phage display peptide libraries can serve as a “substitute antibody” for polymeric micelle-based pharmaceutical preparations [41,42].

In contrast to the sophisticated and poorly controllable conjugation procedures used for coupling of peptides and antibodies to the targeted vesicles, the new landscape phage-based approach relies on and mimics the extremely precise natural mechanisms of biosynthesis and self-assembly of phage particles. When landscape phage libraries serve as a reservoir of the targeted membrane proteins one of the most troublesome steps of the conjugation technology is bypassed. Furthermore, it does not require idiosyncratic reactions with any new shell-decorating polymer or targeting ligand and may be easily adapted to a new liposome or micelle composition and a new addressed target. No re-engineering of the selected phage is required at all: the phages themselves serve as the source of the final product—coat protein genetically fused to the targeting peptide.

6. Phage fusion protein-mediated delivery of anticancer siRNA

Synthetic small fragments of RNA are known to inhibit specific protein expression by suppressing target genes at the mRNA level, a mechanism described as RNA interference (RNAi). High specificity, high efficiency, and low toxicity have made these small interfering RNA (siRNA) popular as potential anticancer agents. However, delivery of siRNA into the cells remains challenging due to their instability in physiological fluids as well as their inability to cross cell membranes to achieve intracellular accumulation at the site of action [93]. For these reasons, other mechanisms to deliver siRNA to target cells have been devised that include viral and bacterial delivery, the use of liposomes or nanoparticles, and stabilization of siRNA by chemical modification [94]. Despite some improvement in the bioavailability, many siRNA-carrier systems, including cationic polymers and lipids, suffer from inherent deficiencies associated with poor cellular delivery mainly due to rapid uptake into tissues of phagocytic systems [95–97]. PEGylated (‘stealth’) liposomes remain the ideal choice of carrier for siRNA delivery mainly due to their biological inertness, non-toxicity and protection from nuclease degradation [98]. Further improvements to the efficacy of delivery can be achieved if the liposomal vehicles of siRNA can be specifically targeted to tumor cells by conjugation with targeting ligands [99]. For example, siRNA-loaded immunoliposomes targeted with anti-transferrin antibody produced specific inhibition of Her-2 expression in breast cancer animal model and tumor growth inhibition in pancreatic cancer animal model [100]. Attachment of cell-penetrating peptides (CPP), a family of peptides able to translocate across the cell membrane, have also been used to deliver siRNA into cancer cells [101]. It was shown that liposomes bearing a synthetic arginine-rich CPP are stable and can efficiently transfect lung tumor cells in vitro. Recent study has shown that systemic delivery of siRNA encapsulated in targeted liposomes can efficiently silence oncogenes in metastatic tumor cells [102]. Taken together, siRNA-containing targeted liposomes represent a promising treatment avenue for cancer. However, the drawbacks in the production of targeted liposomal preparations as outlined in the previous section led us to consider phage proteins as easily available targeting components of the siRNA-loaded liposomal vehicles.

Bedi et al. described a new strategy for targeted siRNA delivery using phage fusion proteins as navigating modules for liposomes. The targeted siRNA liposomes were produced by a novel approach in which two different parental liposomes, one containing spontaneously inserted siRNA and the other containing fusion phage proteins are fused together to obtain the final therapeutic formulation [32]. For conceptual studies, the PRDM14 gene – a member of the family of genes that encode proline rich domain proteins (PRDM), was chosen as a target for siRNA-mediated silencing because it has been suggested to play an important role in carcinogenesis [103]. MCF-7 specific phage protein DMPGTVLP (here and later fusion phages and their proteins will be designated by the sequence of inserted foreign peptides) was derived from landscape phage libraries and has already been described in previous sections and publications as targeting ligands for drug-loaded liposomes [38,57]. For the production of siRNA-loaded liposomes, the authors had to adapt a novel three-step approach in lieu of the afore-mentioned simplistic approach as the spontaneous insertion of RNA and phage fusion protein into pre-formed liposome follow different mechanistic principles. First, siRNAs was inserted into a liposome formed by a mixture of neutral and positively charged lipids (plus-liposome). This liposome has a positively charged interface that attracts the negatively charged siRNAs and drives their internalization [104]. Second, the fusion phage protein (DMPGTVLP) was inserted into a liposome formed by neutral and negatively charged lipids (minus-liposome). This liposome has a negatively charged interface that attracts C termini of the major coat protein, drives their translocation through the lipid bilayer and allows their anchoring at the internal liposomal interface, as was studied before [84]. Third, the plus liposomes loaded with siRNAs and minus liposome loaded with phage protein were fused together to integrate into the protein-targeted particles containing siRNA. MCF-7 cells treated with siRNA–protein liposomes showed a significant down-regulation of the PRDM14 gene expression as compared to the cells treated with control liposomal preparations that included the plus- and minus-liposomes by themselves as well as plain liposomes. The level of down-regulation was comparable to that achieved by a complex of siRNA with lipofectamine, a ‘gold standard’, although toxic carrier for siRNAs. These results were further supported by western blot analysis that revealed that PRDM14 protein expression was diminished in cells treated with siRNA–DMPGTVLP phage protein liposomes.

In an elegant extension of this concept, Bedi et al. undertook studies to evaluate the direct use of fusion phage proteins as nanocarriers of siRNA [31]. The basis of their approach was the premise that phage coat proteins act as natural vehicles for phage genetic material during phage life cycle; encapsulating the nucleic acid after its production, conveying the same to the host cells and then releasing the genetic payload at the host cell membrane for further propagation. Thus, phage fusion proteins could and would act as natural vehicles for small fragments of nucleic acids like siRNA (Fig. 8). Accordingly, MCF-7-specific phage fusion proteins were incubated with GAPDH siRNA to form particles, called “phagelike nanoparticles” or “nanophages”. These entities, seen as small particles of diameter 11 nm, protected the siRNA from degradation by serum nucleases, retained the target-binding ability of the fusion phage protein and were able to deliver a siRNA payload to the target cells to achieve knockdown of the GAPDH gene. Thus, nanophages may represent a very attractive formulation for delivery of therapeutic siRNA into tumors.

Fig. 8.

Phage like nanoparticles or nanophage formed by mixing of cholate solubilized phage fusion protein with siRNA with the gradual removal of cholate. N-terminus of phage protein is the targeting ligand whereas positively charged C-terminal end of the major coat protein interacts with negatively charged phosphate groups of siRNA. Adapted from [31].

7. Conclusions

Filamentous bacteriophages, viruses that infect bacteria, are in many ways ideal nanomaterials. Their structure and infection cycle are encoded in small genomes that can be readily manipulated using simple recombinant DNA techniques. They have well-defined geometry and uniformity favorable for nano-fabrication. Moreover, they have turned out to be remarkably tolerant of structural alteration. The atomic structures of filamentous phages and their component proteins have been determined, allowing precise engineering of their modified forms with predetermined shapes and functions and a precise spatial distribution of fused functional peptides at a nanoscale level.

Phage nanobiotechnology has evolved from phage display, a suite of techniques for surveying vast populations of peptides for rare structures with some desired target behavior. The surveys are accomplished by selective strategies that exploit the ability to display phage-encoded peptides on the outer virion surface by genetic fusion to phage coat proteins. The phage libraries that are the initial input to selection can comprise billions of distinct phage clones, displaying billions of distinct peptides. The most common of survey strategy is affinity selection, which enriches for phage whose displayed peptides bind an immobilized selector. The selector can be a conventional biomolecule such as an antibody or receptor, or it can be a complex biological structure such as whole cells in culture or whole tissues in living animals. In most phage display applications, the desired end-product is the displayed peptide; the virion serves only a vehicle for discovery. In phage nanotechnology, in contrast, it is the entire phage proteins or ensembles of proteins and virion that is the goal of discovery. In summary, it is in the ability to select nanomaterials with favorable behavior from vast initial nanomaterial libraries that phage nanotechnology differs most dramatically from other modes of nanotechnology.

New modes of peptide display designed to modify the properties of the entire virion have come recently to the fore. In “landscape” libraries, described in this review, almost a quarter of the virion’s surface area differs from one phage clone to another. These creative developments have already paid off in contributions to multiple areas of medicine and technology, including medical diagnostics and monitoring, molecular imaging, targeted drug and gene delivery, vaccine development, and bone and tissue repair. We hope that the assembling in this review recent achievements in targeted drug delivery using phage nanotechnology will help the members of the growing community of phage bioengineers stay abreast of recent trends and inspire them to create new ones. Further advances in these areas will require the collective efforts of specialists in diverse fields—medical doctors, microbiologists, structural biologists, chemists, pharmacologists, and many others.

Abbreviations

LMT

ligand-mediated targeting

PEG

polyethylene glycol

TM

trans-membrane

FET

fluorescence energy transfer

DPH

diphenylhexatriene

ePC

phosphatidylcholine (egg)

CHOL

cholesterol

DPPG

1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]

DOTAP

1,2-dioleoyl-3-trimethylammonium-propane

PEG2K-PE

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)2000]

siRNA

small interfering RNA

CPP

cell-penetrating peptides