Phage as a Genetically Modifiable Supramacromolecule in Chemistry, Materials and Medicine

Abstract

Filamentous bacteriophage (phage) is a genetically modifiable supramacromolecule. It can be pictured as a semiflexible nanofiber (∼900 nm long and ∼8 nm wide) made of a DNA core and a protein shell with the former genetically encoding the latter. Although phage bioengineering and phage display techniques were developed before the 1990s, these techniques have not been widely used for chemistry, materials, and biomedical research from the perspective of supramolecular chemistry until recently. Powered by our expertise in displaying a foreign peptide on its surface through engineering phage DNA, we have employed phage to identify target-specific peptides, construct novel organic-inorganic nanohybrids, develop biomaterials for disease treatment, and generate bioanalytical methods for disease diagnosis. Compared with conventional biomimetic chemistry, phage-based supramolecular chemistry represents a new frontier in chemistry, materials science, and medicine. In this Account, we introduce our recent successful efforts in phage-based supramolecular chemistry, by integrating the unique nanofiber-like phage structure and powerful peptide display techniques into the fields of chemistry, materials science, and medicine: (1) successfully synthesized and assembled silica, hydroxyapatite, and gold nanoparticles using phage templates to form novel functional materials; (2) chemically introduced azo units onto the phage to form photoresponsive functional azo-phage nanofibers via a diazotization reaction between aromatic amino groups and the tyrosine residues genetically displayed on phage surfaces; (3) assembled phage into 2D films for studying the effects of both biochemical (the peptide sequences displayed on the phages) and biophysical (the topographies of the phage films) cues on the proliferation and differentiation of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) and identified peptides and topographies that can induce their osteogenic differentiation; (4) discovered that phage could induce angiogenesis and osteogenesis for MSC-based vascularized bone regeneration; (5) identified novel breast cancer cell-targeting and MSC-targeting peptides and used them to significantly improve the efficiency of targeted cancer therapy and MSC-based gene delivery, respectively; (6) employed engineered phage as a probe to achieve ultrasensitive detection of biomarkers from serum of human patients for disease diagnosis; and (7) constructed centimeter-scale 3D multilayered phage assemblies with the potential application as scaffolds for bone regeneration and functional device fabrication. Our findings demonstrated that phage is indeed a very powerful supramacromolecule suitable for not only developing novel nanostructures and biomaterials but also advancing important fields in biomedicine, including molecular targeting, cancer diagnosis and treatment, drug and gene delivery, stem cell fate direction, and tissue regeneration. Our successes in exploiting phage in chemistry, materials, and medicine suggest that phage itself is nontoxic at the cell level and can be safely used for detecting biomarkers in vitro. Moreover, although we have demonstrated successful in vivo tissue regeneration induced by phage, we believe future studies are needed to evaluate the in vivo biodistribution and potential risks of the phage-based biomaterials.

Figure 1

Scheme of phage structure and phage display technique. Filamentous phage (~900 nm long and ~8 nm wide) is a nanofiber-like virus composed of ~3000 highly ordered copies of major coat protein (pVIII) and 5 copies of the minor coat proteins (pIII, pVI, pVII, and pIX) surrounding a circular ssDNA, which encodes all the coat proteins. By insertion of DNA encoding foreign peptides into the genes of specific coat proteins, the peptides can be displayed at the tip by genetic fusion to the N-terminus of pIII (pIII display) or along the length by genetic fusion to the N-terminus of the pVIII (pVIII display).

Figure 2

Scheme of the selection of mesenchymal stem cell (MSC)-targeting peptides by screening a phage-displayed random peptide library against target cells. The process is called biopanning. Reproduced with permission from ref . Copyright 2010 American Chemical Society.

Figure 3

(A) Scheme of the phage-directed formation of hexagonal mesoporous silica; (B) TEM images of phage–silica composites before (B-1) and after (B-2) calcination; (C) cross-sectional TEM images of phage–silica composites before (C-1) and after (C-2) calcination. Reproduced with permission from ref . Copyright 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 4

Schematic representation of photoresponsive azo-phage and reversible photoisomerization under UV exposure and dark conditions. Reproduced with permission from ref . Copyright 2013 Nature Publishing Group.

Figure 5

(A) Fabrication of phage films by a layer-by-layer self-assembly method for inducing the osteoblastic differentiation of iPSCs. Real-time PCR (B) and immunofluorescence staining (C) assays for evaluating the expression levels of the osteogenic marker proteins, including osteoclacin (OCN) and osteopontin (OPN). Collagen (COL) was used as a reference marker, and plates without phage films were the control (CON). Reproduced with permission from ref . Copyright 2014 American Chemical Society.

Figure 6

(A) Scheme of the integration of RGD–phage and rMSCs into a 3D printed bioceramic scaffold for bone regeneration. (B) Micro-CT images showing bone regeneration using the scaffold filled with wild-type phage (negative control, B-1), with RGD–phage (VAM, B-2), and with both RGD–phage and VEGF (positive control, B-3). (C) Quantification of bone volume density (bone volume/tissue volume) within the scaffolds. The bone volume density within the VAM scaffold is higher than that in the blank and negative control groups. (D) Number of newly formed blood vessels within the scaffolds. There are significantly more blood vessels formed in VAM scaffold than negative control. Reproduced with permission from ref . Copyright 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 7

(A) ELISA results for the evaluation of selected rMSC-binding peptide by biopanning, showing that the peptide VTAMEPGQ has the highest rMSC-binding affinity. (B) Affinity and specificity tests of FITC-labeled VTAMEPGQ with rMSCs. Fluorescence was detected from 1 μM (B-1) and 10 μM (B-2) FITC-VTAMEPGQ in rMSCs. But no specific fluorescence was observed from either 1 μM (B-3) or 10 μM (B-4) FITC-labeled control peptide in rMSCs. No specific fluorescence was observed from 10 μM FITC-VTAMEPGQ in either rat fibroblastic cells (B-5) or rat smooth muscle cells (B-6). Scale bar = 100 μm. Reproduced with permission from ref . Copyright 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 8

(A) Schematic of photosensitizer conjugation onto phage for developing targeted PDT. (B) In vitro evaluation of PDT on control MCF-7 cells (B-1, B-2, B-3) and target SKBR-3 cells (B-4, B5, B-6): (B-1, B-4) light microscopy images of MCF-7 and SKBR-3 cells; (B-2, B-5) fluorescent images of MCF-7 and SKBR-3 cells using live/dead cell viability kit upon incubation with photosensitizer and laser irradiation; (B-3, B-6) fluorescent images of MCF-7 and SKBR-3 cells using live/dead cell viability kit upon incubation with phage–photosensitizer complex and laser irradiation. All images were captured at 10× objectives. Reproduced with permission from ref . Copyright 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 9

(A) Scheme for using VMSNCs for gene delivery to MSCs; (B) Mechanism of the magnetic/silica nanocluster formation and cleavage with intracellular glutathione (GSH); (C) The EGFP-VEGF (vascular endothelial growth factor tagged with green fluorescence protein) gene transfection efficiency by VMSNCs and control vectors. MSNCs: magnetic silica nanoclusters, WT: wild-type, MPTS: (3-mercaptopropyl)-trimethoxysilane, CTAB: cetyltrimethylammonium bromide, PEI: polyethylenimine. Reproduced with permission from ref . Copyright 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 10

(A) Schematic of using phage displaying anti-Sap2-IgG targeting (ASIT) peptide and decorated with magnetic nanoparticles (MNP) (ASIT-MNP-phage) (A-1) for the ultrasensitive detection of anti-Sap2-IgG from human serum (A-2, A-3). (B) The detection of anti-Sap2 IgG in serum of Candida albicans-infected cancer patients: (B-1) percentage of anti-Sap2 IgG positive population among all cancer patients (1, Candida albicans-infected patients; 2, healthy control); (B-2) percentage of anti-Sap2 IgG positive population among patients of each specific cancer type (1, lung cancer; 2, breast cancer; 3, intestinal cancer; 4, others). WT: wild-type, HRP: horseradish peroxidase, TMB: 3, 3’, 5, 5’-tetramethylbenzidine, PK: MNP-binding peptide (PTYSLVPRLATQPFK). Reproduced with permission from ref . Copyright 2015 American Chemical Society.

Figure 11

(A) Schematic for using a magnetic field to guide the alignment of MNP-decorated phage (MNP-phage); (B) TEM image of the MNP-phage; (C) SEM image of the single-orientation horizontally aligned MNP-phage; (D) centimeter-scale single-orientation horizontally aligned MNP-phage; (E, F) optical images of double-orientation multilayered MNP-phage arrays. Reproduced with permission from ref . Copyright 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.