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1 Application of filamentous phages In nanobiotechnology.

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Presentation on theme: "1 Application of filamentous phages In nanobiotechnology."— Presentation transcript:

1 1 Application of filamentous phages In nanobiotechnology

2 2 1) 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.

3 3 2) Use the isolated phages as nucleation centers for the fabrication of nanoparticles and nanowires

4 4 Phages as vehicles for gene and vaccine delivery Application of filamentous phages As nanomedicines Phages as tool for imaging Phages as vehicles for gene and drug delivery

5 5 Combine: 1) Gene delivery 2) Specific targeting 3) Imaging Pasqualiny and Arap group

6 6

7 7 Components of a targeted drug carrying platform Drug Targeting moiety Specificity Affinity Chemical tolerance Drug Carrier High capacity biocompatibility Labile linker Proper kinetics Temporal Spatial Potency

8 8 The filamentous bacteriophage as a targeted drug-carrying nanomedicine - g3 protein - g6 protein - g8 protein - g9 protein - g7 protein XII I g3 g8 g5 g2 g10 IG g4 g1 g9 g6 800-2000 nm (dependent on the size of the packaged genome) 6nm Scheme 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).

9 9 IgG complexed to fUSE5-ZZ phage through a p3-displayed ZZ domain - IgG - ZZ domain fUSE5-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. 10 12 fUSE5-ZZ phages were complexed with 0.6  g (*3 dil) or 0.2  g (*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. M13 KO7 pCANTAB5- ZZ fUSE5 - ZZ A B C

10 10 Exposed amine residues : 2 P8 coat protein monomer (of ~ 3000) DNA interacting zone

11 11 Preparation of drug-linker adduct Chloramphenicol was modified in two steps to create an ester bond between CAM and a linker (originated in glutatic anhydride) The linker CAM complex is activated for lysine conjugation by the NHS procedure.

12 12 Kinetics of drug release by serum esterases (here: 10% horse serum analysis by HPLC)

13 13 Partial growth inhibition of staphylococcus aureus by antibody-targeted drug-carrying phages (3000 Cam/phage) Similar Partial growth inhibition was obtained by peptide-targeted phages

14 14 The limitations: 1)Limited arming efficiency due to drug hydrophobicity 2)Solubility of the platform also affected 3)Vulnerability of the targeting moiety (ZZ domain) to amine chemistry

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