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Multi-component Polymeric System for Tumour Cell-Specific Gene Delivery Using a Universal Bungarotoxin Linker

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ABSTRACT

Purpose

A new universal tool for specific, non-covalent and non-destructive attachment of a recombinant antibody fragment to a polymer-modified adenovirus has been utilised to regulate the tropism of adenoviral gene delivery vector.

Methods

We have prepared a multivalent reactive N-(2-hydroxypropyl)methacrylamide-based copolymer (PHPMA) bearing an α-bungarotoxin-binding peptide (BTXbp). The copolymer was used for covalent surface modification of adenoviral vectors (Ad). The α-bungarotoxin protein (BTX) has a nanomolar binding affinity for BTXbp, allowing non-covalent linkage of BTX fusion proteins. A single chain variable fragment of anti-PSMA antibody bearing BTX (scFv-BTX) binding to the prostate-specific membrane antigen (PSMA) was conjugated with the copolymer-coated adenovirus to enable specific infection of prostate cancer cells via PSMA receptors.

Results

As shown by ELISA, the copolymer-coated virus exhibited much reduced binding to anti-Ad antibodies. Infection of PC-3 and LNCaP prostate cancer cells was ∼100-fold less efficient with copolymer-coated Ad than with un-modified Ad. Conjugation of scFv-BTX with Ad-PHPMA-BTXbp led to 5–10-fold restoration of infection in PSMA-positive LNCaP cells. In PSMA-negative PC-3 cells, the conjugation of scFv-BTX with Ad-PHPMA-BTXbp gave no enhancement of infection.

Conclusions

We have shown that the presented Ad-PHPMA-BTXbp/scFv-BTX system can be used as a universal tool for a receptor-specific virotherapy.

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Abbreviations

Ad:

adenovirus

BCA:

bicinchoninic acid

BTX:

α-bungarotoxin

BTXbp:

α-bungarotoxin-binding peptide

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HINGS:

heat-inactivated normal goat serum

HPMA:

N-(2-hydroxypropyl)methacrylamide

IPTG:

isopropyl β-D-1-thiogalactopyranoside

Ma-GG-OH:

N-methacryloylglycylglycine

Ma-GG-TT:

3-(N-methacryloylglycylglycyl) thiazolidine-2-thione

PEG:

poly(ethylene glycol)

PHPMA:

N-(2-hydroxypropyl)methacrylamide-based copolymer

PBS:

phosphate buffered saline

PSMA:

prostate specific membrane antigen

scFv-BTX:

single chain variable fragment of anti-PSMA antibody bearing BTX

TMB:

3,3′,5,5′-tetramethylbenzidine

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ACKNOWLEDGEMENTS

Financial support of this project by EU grant GIANT No. 512087, the Grant Agency of the Czech Republic, grant No. 203/08/0543 and the Academy of Sciences of the Czech Republic, grant No. 200200651 is gratefully acknowledged.

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Correspondence to Michal Pechar.

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Ralph A. Willemsen, Michal Pechar and Robert C. Carlisle contributed equally to the manuscript

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Willemsen, R.A., Pechar, M., Carlisle, R.C. et al. Multi-component Polymeric System for Tumour Cell-Specific Gene Delivery Using a Universal Bungarotoxin Linker. Pharm Res 27, 2274–2282 (2010). https://doi.org/10.1007/s11095-010-0088-8

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  • DOI: https://doi.org/10.1007/s11095-010-0088-8

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