Peptide microarray with ligands at high density based on symmetrical carrier landscape phage for detection of cellulase.

Peptide microarrays evolved recently as a routine analytical implementation in various research areas due to their unique characteristics. However, the immobilization of peptides with high density in each spot during the fabricating process remains a problem, which will affect the performance of the resultant microarray greatly. To respond to this challenge, a novel peptide immobilization method using symmetrical phage carrier was developed in this work. The cellulytic enzyme endoglucanase I (EG I) was used as a model for selection of its specific peptide ligands from the f8/8 landscape library. Three phage monoclones were selected and identified by the specificity array, of which one phage monoclone displaying the fusion peptide EGSDPRMV (phage EGSDPRMV) could bind EG I specifically with highest affinity. Subsequently, the phage EGSDPRMV was used directly to construct peptide microarray. For comparison, major coat protein pVIII fused EG I specific peptide EGSDPRMV (pVIII-fused EGSDPRMV) which was isolated from phage EGSDPRMV was also immobilized by traditional method to fabricate peptide microarray. The fluorescent signal of the phage EGSDPRMV-mediated peptide microarray was more reproducible and about four times higher than the value for pVIII-fused EGSDPRMV-based microarray, suggesting the high efficiency of the proposed phage EGSDPRMV-mediated peptide immobilization method. Further, the phage EGSDPRMV based microarray not only simplified the procedure of microarray construction but also exhibited significantly enhanced sensitivity due to the symmetrical carrier landscape phage, which dramatically increased the density and sterical regularity of immobilized peptides in each spot. Thus, the proposed strategy has the advantages that the immobilizing peptide ligands were not disturbed by their composition and the immobilized peptides were highly regular with free amino-terminal.

Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen.

Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825-165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.

Enhanced tumor delivery and antitumor activity in vivo of liposomal doxorubicin modified with MCF-7-specific phage fusion protein.

A novel strategy to improve the therapeutic index of chemotherapy has been developed by the integration of nanotechnology with phage technique. The objective of this study was to combine phage display, identifying tumor-targeting ligands, with a liposomal nanocarrier for targeted delivery of doxorubicin. Following the proof of concept in cell-based experiments, this study focused on in vivo assessment of antitumor activity and potential side-effects of phage fusion protein-modified liposomal doxorubicin. MCF-7-targeted phage-Doxil treatments led to greater tumor remission and faster onset of antitumor activity than the treatments with non-targeted formulations. The enhanced anticancer effect induced by the targeted phage-Doxil correlated with an improved tumor accumulation of doxorubicin. Tumor sections consistently revealed enhanced apoptosis, reduced proliferation activity and extensive necrosis. Phage-Doxil-treated mice did not show any sign of hepatotoxicity and maintained overall health. Therefore, MCF-7-targeted phage-Doxil seems to be an active and tolerable chemotherapy for breast cancer treatment. FROM THE CLINICAL EDITOR: The authors of this study successfully combined phage display with a liposomal nanocarrier for targeted delivery of doxorubicin using MCF-7-targeted phage-Doxil nanocarriers in a rodent model. The method demonstrated improved efficiency and reduced hepatotoxicity, paving the way to future clinical trials addressing breast cancer.

Phage Display's Prospects for Early Diagnosis of Prostate Cancer.

Prostate cancer (PC) is the second most diagnosed cancer among men. It was observed that early diagnosis of disease is highly beneficial for the survival of cancer patients. Therefore, the extension and increasing quality of life of PC patients can be achieved by broadening the cancer screening programs that are aimed at the identification of cancer manifestation in patients at earlier stages, before they demonstrate well-understood signs of the disease. Therefore, there is an urgent need for standard, sensitive, robust, and commonly available screening and diagnosis tools for the identification of early signs of cancer pathologies. In this respect, the "Holy Grail" of cancer researchers and bioengineers for decades has been molecular sensing probes that would allow for the diagnosis, prognosis, and monitoring of cancer diseases via their interaction with cell-secreted and cell-associated PC biomarkers, e.g., PSA and PSMA, respectively. At present, most PSA tests are performed at centralized laboratories using high-throughput total PSA immune analyzers, which are suitable for dedicated laboratories and are not readily available for broad health screenings. Therefore, the current trend in the detection of PC is the development of portable biosensors for mobile laboratories and individual use. Phage display, since its conception by George Smith in 1985, has emerged as a premier tool in molecular biology with widespread application. This review describes the role of the molecular evolution and phage display paradigm in revolutionizing the methods for the early diagnosis and monitoring of PC.

Phage Display in Cancer Research: Special Issue Editorial.

Soon after its birth in 1985, following a short lag period [...].

Affinity comparison of p3 and p8 peptide displaying bacteriophages using surface plasmon resonance.

Ever increasing demands in sensitivity and specificity of biosensors have recently established a trend toward the use of multivalent bioreceptors. This trend has also been introduced in the field of bacteriophage affinity peptides, where the entire phage is used as a receptor rather than the individual peptides. Although this approach is gaining in popularity due to the numerous advantages, binding kinetics of complete phage particles have never been studied in detail, notwithstanding being essential for the efficient design of such applications. In this paper we used an in house developed fiber-optic surface plasmon resonance (FO-SPR) biosensor to study the affinity and binding kinetics of phages, displaying peptide libraries. By using either peptide expression on the p3 or on the p8 coat proteins, a corresponding density of 5 up to more than 2000 peptides on a single virus particle was obtained. Binding parameters of 26 different filamentous phages, displaying peptides selective for enhanced Green Fluorescent Protein (eGFP), were characterized. This study revealed a broad affinity range of phages for the target eGFP, indicating their potential to be used for applications with different requirements in binding kinetics. Moreover, detailed analysis of koff and kon values of several selected p3 and p8 phages, using the FO-SPR biosensor, clearly showed the correlation between the binding parameters and the density at which eGFP-peptides are being expressed. Consequently, although p3 and p8-based phages both revealed exceptionally high affinities for eGFP, two p8 phages were found to have the highest affinity with dissociation constants (Kd) in the femtomolar range.

Synergetic Targeted Delivery of Sleeping-Beauty Transposon System to Mesenchymal Stem Cells Using LPD Nanoparticles Modified with a Phage-Displayed Targeting Peptide.

An important criterion for effective gene therapy is sufficient chromosomal integration activity. The Sleeping Beauty (SB) transposon system is a plasmid system allowing efficient insertion of transgenes into the host genome. However, such efficient insertion occurs only after the system is delivered to nuclei. Since transposons do not have the transducing abilities of viral vectors, efficient delivery of this system first into cells and then into cell nuclei is still a challenge. Here, a phage display technique using a major coat displayed phage library is employed to identify a peptide (VTAMEPGQ) that can home to rat mesenchymal stem cells (rMSCs). A nanoparticle, called liposome protamine/DNA lipoplex (LPD), is electrostatically assembled from cationic liposomes and an anionic complex of protamine, DNA and targeting peptides. Various peptides are enveloped inside the LPD to improve its targeting capability for rMSCs and nuclei. The rMSC-targeting peptide and nuclear localization signal (NLS) peptide can execute the synergetic effect to promote transfection action of LPD. The homing peptide directs the LPD to target the MSCs, whereas the NLS peptide directs transposon to accumulate into nuclei once LPD is internalized inside the cells, leading to increased gene expression. This suggests that rMSC-targeting peptide and NLS peptide within LPD can target to rMSCs and then guide transposon into nuclei. After entering the nuclei, SB transposon increase the insertion rates into cellular chromosomes. The targeting LPD does not show obvious cell toxicity and influence on the differentiation potential of rMSCs. Therefore, the integration of SB transposon and LPD system is a promising nonviral gene delivery vector in stem cell therapy.

Targeted delivery of siRNA into breast cancer cells via phage fusion proteins.

Nucleic acids, including antisense oligonucleotides, small interfering RNA (siRNA), aptamers, and rybozymes, emerged as versatile therapeutics due to their ability to interfere in a well-planned manner with the flow of genetic information from DNA to protein. However, a systemic use of NAs is hindered by their instability in physiological liquids and inability of intracellular accumulation in the site of action. We first evaluated the potential of cancer specific phage fusion proteins as targeting ligands that provide encapsulation, protection, and navigation of siRNA to the target cell. The tumor-specific proteins were isolated from phages that were affinity selected from a landscape phage library against target breast cancer cells. It was found that fusion phage coat protein fpVIII displaying cancer-targeting peptides can effectively encapsulate siRNAs and deliver them into the cells leading to specific silencing of the model gene GAPDH. Complexes of siRNA and phage protein form nanoparticles (nanophages), which were characterized by atomic force microscopy and ELISA, and their stability was demonstrated by resistance of encapsulated siRNA to degradation by serum nucleases. The phage protein/siRNA complexes can make a new type of highly selective, stable, active, and physiologically acceptable cancer nanomedicine.

Metastatic prostate cancer cell-specific phage-like particles as a targeted gene-delivery system.

BACKGROUND: One of the cardinal requirements for effective therapeutic management of tumors is the selective delivery of cancer drugs to the right site by ligand-decorated nanomedicines. Screening of 2 × 109 clone landscape phage library provides a reliable avenue for generating protein ligands specific for tumor cells. It was shown that selective phage proteins derived from landscape phage libraries against breast and prostate cancer cells are able to navigate drug or siRNA loaded liposomes to corresponding cancer cells with minimal toxicity to non-neoplastic cells. In an alternative platform, glioma cell-specific phage proteins were used for assembling in vivo cancer-specific phage-like particles, named 'phagemid infective particles' as targeted gene-delivery vehicles. METHODS: To extend the panel of anticancer cell phages, we have screened a 2 × 109 clone landscape phage library f8/8 to select phage clones specific for metastatic prostate cancer cell PC-3M. The phage clones were characterized for their selective interaction with PC-3M cells using phage capture assay, immunofluorescence microscopy and electron microscopy. A prostate cancer selective phage was converted to phage-like particles harboring emerald green fluorescent protein. RESULTS: Phage clone EPTHSWAT (designated by the sequence of inserted peptide) was found to be most selective for PC-3M cells and was observed to internalize PC-3M cells as revealed by immunofluorescence microscopy and electron microscopy. Conversion of this phage to phage-like particles harboring emerald green fluorescent protein and the expression of emerald green fluorescent protein in the phage-like particles treated PC-3M cells showed potential of adoption of this phage-like particle in prostate cancer therapeutic gene delivery. CONCLUSION: Successful employment of phage-like particles expressing emerald green fluorescent protein genes targeted to prostate cancer cells PC-3M confirms a prospect of their use for targeted delivery of therapeutic genes to cancer cells.

Specificity and promiscuity in human glutaminase interacting protein recognition: insight from the binding of the internal and C-terminal motif.

A large number of cellular processes are mediated by protein-protein interactions, often specified by particular protein binding modules. PDZ domains make up an important class of protein-protein interaction modules that typically bind to the C-terminus of target proteins. These domains act as a scaffold where signaling molecules are linked to a multiprotein complex. Human glutaminase interacting protein (GIP), also known as tax interacting protein 1, is unique among PDZ domain-containing proteins because it is composed almost exclusively of a single PDZ domain rather than one of many domains as part of a larger protein. GIP plays pivotal roles in cellular signaling, protein scaffolding, and cancer pathways via its interaction with the C-terminus of a growing list of partner proteins. We have identified novel internal motifs that are recognized by GIP through combinatorial phage library screening. Leu and Asp residues in the consensus sequence were identified to be critical for binding to GIP through site-directed mutagenesis studies. Structure-based models of GIP bound to two different surrogate peptides determined from nuclear magnetic resonance constraints revealed that the binding pocket is flexible enough to accommodate either the smaller carboxylate (COO(-)) group of a C-terminal recognition motif or the bulkier aspartate side chain (CH(2)COO(-)) of an internal motif. The noncanonical ILGF loop in GIP moves in for the C-terminal motif but moves out for the internal recognition motifs, allowing binding to different partner proteins. One of the peptides colocalizes with GIP within human glioma cells, indicating that GIP might be a potential target for anticancer therapeutics.

Phage-Displayed Mimotopes of SARS-CoV-2 Spike Protein Targeted to Authentic and Alternative Cellular Receptors.

The evolution of the SARS-CoV-2 virus during the COVID-19 pandemic was accompanied by the emergence of new heavily mutated viral variants with increased infectivity and/or resistance to detection by the human immune system. To respond to the urgent need for advanced methods and materials to empower a better understanding of the mechanisms of virus's adaptation to human host cells and to the immuno-resistant human population, we suggested using recombinant filamentous bacteriophages, displaying on their surface foreign peptides termed "mimotopes", which mimic the structure of viral receptor-binding sites on the viral spike protein and can serve as molecular probes in the evaluation of molecular mechanisms of virus infectivity. In opposition to spike-binding antibodies that are commonly used in studying the interaction of the ACE2 receptor with SARS-CoV-2 variants in vitro, phage spike mimotopes targeted to other cellular receptors would allow discovery of their role in viral infection in vivo using cell culture, tissue, organs, or the whole organism. Phage mimotopes of the SARS-CoV-2 Spike S1 protein have been developed using a combination of phage display and molecular mimicry concepts, termed here "phage mimicry", supported by bioinformatics methods. The key elements of the phage mimicry concept include: (1) preparation of a collection of p8-type (landscape) phages, which interact with authentic active receptors of live human cells, presumably mimicking the binding interactions of human coronaviruses such as SARS-CoV-2 and its variants; (2) discovery of closely related amino acid clusters with similar 3D structural motifs on the surface of natural ligands (FGF1 and NRP1), of the model receptor of interest FGFR and the S1 spike protein; and (3) an ELISA analysis of the interaction between candidate phage mimotopes with FGFR3 (a potential alternative receptor) in comparison with ACE2 (the authentic receptor).

On the mechanism of targeting of phage fusion protein-modified nanocarriers: only the binding peptide sequence matters.

The integration of pharmaceutical nanocarriers with phage display techniques is emerging as a new paradigm for targeted cancer nanomedicines. We explored the direct use of landscape phage fusion proteins for the self-assembly of phage-derived binding peptides to liposomes for cancer cell targeting. The primary purpose of this study was to elucidate the targeting mechanism with a particular emphasis on the relative contributions of the two motifs that make up the landscape phage fusion protein (a binding peptide and the phage pVIII coat protein) to the targeting efficiency. Using transmission electron microscopy and dynamic light scattering, we confirmed the formation of phage-liposomes. Using FACS analysis, fluorescence microscopy, and fluorescence photospectrometry, we found that liposomes modified with MCF-7-specific phage fusion proteins (MCF-7 binding peptide, DMPGTVLP, fused to the phage PVIII coat protein) provided a strong and specific association with target MCF-7 cancer cells but not with cocultured, nontarget cells including C166-GFP and NIH3T3. The substitution for the binding peptide fused to phage pVIII coat protein abolished the targeting specificity. The addition of free binding peptide, DMPGTVLP, competitively inhibited the interaction of MCF-7-specific phage-liposomes with target MCF-7 cells but showed no reduction of MCF-7-associated plain liposomes. The proteolysis of the binding peptide reduced MCF-7 cell-associated phage-liposomes in a proteinase K (PK) concentration-dependent manner with no effect on the binding of plain liposomes to MCF-7 cells. Overall, only the binding peptide motif was involved in the targeting specificity of phage-liposomes. The presence of phage pVIII coat protein did not interfere with the targeting efficiency.

Delivery of siRNA into breast cancer cells via phage fusion protein-targeted liposomes.

Efficacy of siRNAs as potential anticancer therapeutics can be increased by their targeted delivery into cancer cells via tumor-specific ligands. Phage display offers a unique approach to identify highly specific and selective ligands that can deliver nanocarriers to the site of disease. In this study, we proved a novel approach for intracellular delivery of siRNAs into breast cancer cells through their encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins. The targeted siRNA liposomes were obtained by a fusion of two parental liposomes containing spontaneously inserted siRNA and fusion phage proteins. The presence of pVIII coat protein fused to a MCF-7 cell-targeting peptide DMPGTVLP in the liposomes was confirmed by Western blotting. The novel phage-targeted siRNA-nanopharmaceuticals demonstrate significant down-regulation of PRDM14 gene expression and PRDM14 protein synthesis in the target MCF-7 cells. This approach offers the potential for development of new anticancer siRNA-based targeted nanomedicines. FROM THE CLINICAL EDITOR: In this study, the authors report a novel approach for targeted intracellular delivery of siRNAs into breast cancer cells through encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins.

Paclitaxel-loaded polymeric micelles modified with MCF-7 cell-specific phage protein: enhanced binding to target cancer cells and increased cytotoxicity.

Polymeric micelles are used as pharmaceutical carriers to increase solubility and bioavailability of poorly water-soluble drugs. Different ligands are used to prepare targeted polymeric micelles. Earlier, we developed the method for use of specific landscape phage fusion coat proteins as targeted delivery ligands and demonstrated the efficiency of this approach with doxorubicin-loaded PEGylated liposomes. Here, we describe a MCF-7 cell-specific micellar formulation self-assembled from the mixture of the micelle-forming amphiphilic polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugate, MCF-7-specific landscape phage fusion coat protein, and the hydrophobic drug paclitaxel. These micelles demonstrated a very low cmc value and specific binding to target cells. Using an in vitro coculture model, FACS analysis, and fluorescence microscopy we showed that MCF-7 targeted phage-micelles preferentially bound to target cells compared to nontarget cells. As a result, targeted paclitaxel-loaded phage-micelles demonstrated a significantly higher cytotoxicity toward target MCF-7 cells than free drug or nontargeted micelle formulations, but failed to show such a differential toxicity toward nontarget C166 cells. Overall, cancer cell-specific phage proteins identified from phage display peptide libraries can serve as targeting ligands ("substitute antibody") for polymeric micelle-based pharmaceutical preparations.

Cytoplasmic delivery of liposomes into MCF-7 breast cancer cells mediated by cell-specific phage fusion coat protein.

Earlier, we have shown that doxorubicin-loaded liposomes (Doxil) modified with a chimeric phage fusion coat protein specific toward MCF-7 breast cancer cells identified from a phage landscape library demonstrated a significantly enhanced association with target cells and an increased cytotoxicity. Based on some structural similarities between the N-terminus of the phage potein and known fusogenic peptides, we hypothesized that, in addition to the specific targeting, the phage protein may possess endosome-escaping potential and an increased cytotoxicity of drug-loaded phage protein-targeted liposomes may be explained by an advantageous combination of both, cell targeting and endosomal escape of drug-loaded nanocarrier. The use of the fluorescence resonance energy transfer (FRET) technique allowed us to clearly demonstrate the pH-dependent membrane fusion activity of the phage protein. Endosomal escape and cytosolic delivery of phage-liposomes was visualized with fluorescence microscopy. Endosome acidification inhibition by bafilomycin A 1 resulted in decreased cytotoxicity of the phage-Doxil, while the endosome disruption by chloroquine had a negligible effect on efficacy of phage-Doxil, confirming its endosomal escape. Our results demonstrated an endosome-escaping property of the phage protein and provided an insight on mechanism of the enhanced cytotoxicity of phage-Doxil.

Evolutionary selection of new breast cancer cell-targeting peptides and phages with the cell-targeting peptides fully displayed on the major coat and their effects on actin dynamics during cell internalization.

Filamentous phage as a bacteria-specific virus can be conjugated with an anticancer drug and has been proposed to serve as a carrier to deliver drugs to cancer cells for targeted therapy. However, how cell-targeting filamentous phage alone affects cancer cell biology is unclear. Phage libraries provide an inexhaustible reservoir of new ligands against tumor cells and tissues that have potential therapeutic and diagnostic applications in cancer treatment. Some of these identified ligands might stimulate various cell responses. Here we identified new cell internalizing peptides (and the phages with such peptides fused to each of ~3900 copies of their major coat protein) using landscape phage libraries and for the first time investigated the actin dynamics when selected phages are internalized into the SKBR-3 breast cancer cells. Our results show that phages harboring VSSTQDFP and DGSIPWST peptides could selectively internalize into the SKBR-3 breast cancer cells with high affinity, and also show rapid involvement of membrane ruffling and rearrangements of actin cytoskeleton during the phage entry. The actin dynamics was studied by using live cell and fluorescence imaging. The cell-targeting phages were found to enter breast cancer cells through energy dependent mechanism and phage entry interferes with actin dynamics, resulting in reorganization of actin filaments and increased membrane rufflings in SKBR-3 cells. These results suggest that, when phage enters epithelial cells, it triggers transient changes in the host cell actin cytoskeleton. This study also shows that using multivalent phage libraries considerably increases the repertoire of available cell-internalizing ligands with potential applications in targeted drug delivery, imaging, molecular monitoring and profiling of breast cancer cells.

Landscape phage fusion protein-mediated targeting of nanomedicines enhances their prostate tumor cell association and cytotoxic efficiency.

Tumor-specific cytotoxicity of drugs can be enhanced by targeting them to tumor receptors using tumor-specific ligands. Phage display offers a high-throughput approach to screen for the targeting ligands. We have successfully isolated phage fusion peptides selective and specific for PC3 prostate cancer cells. Also, we have demonstrated a novel approach of targeting liposomes through tumor-specific phage fusion coat proteins, exploiting the intrinsic properties of the phage coat protein as an integral membrane protein. Here we describe the production of Rhodamine-labeled liposomes as well as doxorubicin-loaded long-circulating liposomes targeted to PC3 prostate tumor cells via PC-specific phage peptides, as an extension of our previous studies. Targeting of labeled liposomes was demonstrated using fluorescence microscopy as well as flow cytometry. Targeting of doxorubicin-loaded liposomes enhanced their cytotoxic effect against PC3 cells in vitro, indicating a possible therapeutic advantage. The simplicity of the approach for generating targeted liposomes coupled with the ability to rapidly obtain tumor-specific phage fusion proteins via phage display may contribute to a combinatorial system for the production of targeted liposomal therapeutics for advanced stages of prostate tumor. From the clinical editor: This paper demonstrates targeting cytotoxic agents to tumor receptors using tumor-specific ligands. The authors describe the production of Rhodamine-labeled liposomes as well as doxorubicin loaded long circulating liposomes targeted to PC3 prostate tumor cells via PC-specific phage peptides. This approach may be especially relevant for advanced prostate tumors.

Colorimetric Assay of Bacterial Pathogens Based on Co3O4 Magnetic Nanozymes Conjugated with Specific Fusion Phage Proteins and Magnetophoretic Chromatography.

It is important to detect pathogens rapidly, sensitively, and selectively for clinical medicine, homeland security, food safety, and environmental control. We report here a specific and sensitive colorimetric assay that incorporated a bovine serum albumin-templated Co3O4 magnetic nanozyme (Co3O4 MNE) with a novel specific fusion phage protein and magnetophoretic chromatography to detect Staphylococcus aureus. The Co3O4 MNE was conjugated to S. aureus-specific fusion-pVIII (Co3O4 MNE@fusion-pVIII), screened from the S. aureus-specific phage AQTFLGEQD (the phage monoclone is denoted by the peptide sequence). The as-prepared triple-functional Co3O4 MNE@fusion-pVIII particles were capable of capturing S. aureus in sterile milk, which were then isolated from milk magnetically. Assisted by polyethylene glycol, the Co3O4 MNE@fusion-pVIII@S. aureus complex was separated from the free Co3O4 MNE@fusion-pVIII by magnetophoretic chromatography in an external magnetic field. After transferring the isolated Co3O4 MNE@fusion-pVIII@S. aureus complexes into a 96-well plate, diammonium salt of 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) and H2O2 were added to develop color because of the peroxidase mimetics activity of the Co3O4 MNE. A S. aureus concentration within 10-10,000 cfu/mL in milk can be detected (detection limit: 8 cfu/mL). The as-developed method is simple, cost-efficient, and sensitive, which is useful for rapidly diagnosing pathogenic bacteria and helpful to prevent disease outbreaks induced by pathogens in developing countries.

Liposomes targeted by fusion phage proteins.

Targeting of nanocarriers has long been sought after to improve the therapeutic indices of anticancer drugs. Here we provide the proof of principle for a novel approach of nanocarrier targeting through their fusion with target-specific phage coat proteins. The source of the targeted phage coat proteins are landscape phage libraries--collections of recombinant filamentous phages with foreign random peptides fused to all 4000 copies of the major coat protein. We exploit in our approach the intrinsic physicochemical properties of the phage major coat protein as a typical membrane protein. Landscape phage peptides specific for specific tumors can be obtained by affinity selection, and purified fusion coat proteins can be assimilated into liposomes to obtain specific drug-loaded nanocarriers. As a paradigm for inceptive experiments, a streptavidin-specific phage peptide selected from a landscape phage library was incorporated into approximately 100-nm liposomes. Targeting of liposomes was proved by their specific binding to streptavidin-coated beads.

Evolution of a Landscape Phage Library in a Mouse Xenograft Model of Human Breast Cancer.

Peptide-displayed phage libraries are billion-clone collections of diverse chimeric bacteriophage particles, decorated by genetically fused peptides built from a random combination of natural amino acids. Studying the molecular evolution of peptide-displayed libraries in mammalian model systems, using in vivo phage display techniques, can provide invaluable knowledge about the underlying physiology of the vasculature system, allow recognition of organ- and tissue-specific networks of protein-protein interactions, and provide ligands for targeted diagnostics and therapeutics. Recently, we discovered that landscape phage libraries, a specific type of multivalent peptide phage display library, expose on their surface comprehensive collections of elementary binding units (EBUs), which can form short linear motifs (SLiMs) that interact with functional domains of physiologically relevant proteins. Because of their unique structural and functional features, landscape phages can use an alternative mechanism of directed molecular evolution, i.e., combinatorial avidity selection. These discoveries fueled our interest in revisiting the in vivo evolution of phage displayed libraries using another format of display, i.e., landscape phages. In this study, we monitored the evolution of a landscape phage library in a mouse model with and without an implanted human breast cancer tumor xenograft. As expected, the multivalent architecture of landscape phage displayed proteins provided strong tissue selectivity and resulted in a huge diversity of tissue penetrating, chimeric phage particles. We identified several types of EBU interactions that evolved during the course of tissue distribution, which included interactions of EBUs with all tissue types, those EBUs that interacted selectively with specific organs or tissues with shared gene expression profiles or functionalities, and other EBUs that interacted in a tissue-selective manner. We demonstrated that landscape phage libraries are a rich collection of unique nanobioparticles that can be used to identify functional organ and tissue-binding elements after the evolution of a phage display library in vivo.