Presentation on theme: "Application of filamentous phages"— Presentation transcript:
1Application of filamentous phages In nanobiotechnology
21) Peptide phage display: isolate binders of semiconductors Phage structure and phage display selection process. (a) Schematic diagram of phage and its genome and (b) phage-display process to identify specific binding peptide motifs against desired targets.
32) Use the isolated phages as nucleation centers for the fabrication of nanoparticles and nanowires
4Application of filamentous phages As nanomedicines Phages as vehicles for gene and vaccine deliveryPhages as tool for imagingPhages as vehicles for gene and drug delivery
5Combine: 1) Gene delivery 2) Specific targeting 3) Imaging Pasqualiny and Arap group
7Components of a targeted drug carrying platform CarrierHigh capacitybiocompatibilityDrugLabile linkerProper kineticsTemporalSpatialTargetingmoietySpecificityAffinityChemical tolerancePotency
8The filamentous bacteriophage as a targeted drug-carrying nanomedicineXIIIg3g8g5g2g10IGg4g1g9g66nmnm (dependent on the size of the packaged genome)- g3 protein- g6 protein- g8 protein- g9 protein- g7 proteinScheme of the filamentous Fd phage. In out system, the minor coat protein, p3 (g3p) carries the targeting moiety which the drug, and the engineered release mechanism are on p8 (g8p).
9IgG complexed to fUSE5-ZZ phage through a p3-displayed ZZ domain A BCM13 KO7pCANTAB5-ZZfUSE5 - ZZ- IgG- ZZ domainfUSE5-ZZ phage used for targeting. A Scheme of the filamentous fUSE5-ZZ phage. B. Evaluation of ZZ domain display by an immunoblot. Phage particles (each lane is identified with the corresponding phage name below it) were separated by SDS/PAGE and electro-botted onto nitrocellulose, and the p3 minor coat protein or the derived ZZ domain-p3 fused derivative was detected with an anti-p3 antibody. The upper arrow marks the position of the ZZ-p3 fusion, while the lower arrow marks the position of the wild-type p3 coat protein. C. Evaluation of antibody binding capacity by competitive ELISA fUSE5-ZZ phages were complexed with 0.6 mg (*3 dil) or 0.2 mg (*9 dil) of HRP-conjugated rabbit anti mouse IgG as tracer, in the presence of varying concentrations of protein-A purified human IgG. The residual HRP on the phages was detected using the substrate TMB.
10P8 coat protein monomer (of ~ 3000) Exposed amine residues : 2P8 coat protein monomer (of ~ 3000)DNA interacting zone
11Preparation of drug-linker adduct Chloramphenicol was modified in two steps to create an ester bond between CAMand a linker (originated in glutatic anhydride)The linker CAM complex is activated for lysine conjugation by the NHS procedure.
12Kinetics of drug release by serum esterases (here: 10% horse serum analysis by HPLC)
13Partial growth inhibition of staphylococcus aureus by antibody-targeted drug-carrying phages (3000 Cam/phage)Similar Partial growth inhibition was obtainedby peptide-targeted phages
14The limitations: Limited arming efficiency due to drug hydrophobicity Solubility of the platform also affectedVulnerability of the targeting moiety (ZZ domain) to amine chemistry