1 application of filamentous phages in nanobiotechnology

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1 Application of filamentous phages In nanobiotechnology

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Application of filamentous phagesIn nanobiotechnology

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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.

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

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Phages as vehicles for gene and vaccine delivery

Application of filamentous phagesAs nanomedicines

Phages as tool for imaging

Phages as vehicles for gene and drug delivery

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Combine:1) Gene delivery2) Specific targeting3) Imaging

Pasqualiny and Arap group

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Components of a targeted drug carrying platform

Drug

DrugTargeting

moiety

Specificity

Affinity

Chemical tolerance

Drug Carrier

High capacitybiocompatibility

Labile linker

Proper kineticsTemporalSpatial

Potency

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The filamentous bacteriophageas 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).

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IgG complexed to fUSE5-ZZ phage through a p3-displayed ZZ domain

- IgG

- ZZ domain

0

10

20

30

40

50

60

70

80

90

100

110

0.01 0.10 1.00 10.00 100.00 1000.00competing human IgG (μg/ml)

% o

f m

axim

al s

ign

al

*3 dil *9 dil

fUSE5-ZZ phage used for targeting. A Scheme of the filamentous fUSE5-ZZ phage. B. Evaluationof 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

KO

7

pCA

NT

AB

5-Z

ZfUSE

5 -

ZZ

A B

C

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Exposed amine residues : 2

P8 coat protein monomer (of ~ 3000)

DNA interacting zone

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Preparation of drug-linker adductChloramphenicol 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.

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

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Partial growth inhibition of staphylococcus aureusby antibody-targeted drug-carrying phages (3000 Cam/phage)

Similar Partial growth inhibition was obtainedby peptide-targeted phages

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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