Research Article
Delivery of siRNA into breast cancer cells via phage fusion protein-targeted liposomes

https://doi.org/10.1016/j.nano.2010.10.004Get rights and content

Abstract

Efficacy of siRNAs as potential anticancer therapeutics can be increased by their targeted delivery into cancer cells via tumor-specific ligands. Phage display offers a unique approach to identify highly specific and selective ligands that can deliver nanocarriers to the site of disease. In this study, we proved a novel approach for intracellular delivery of siRNAs into breast cancer cells through their encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins. The targeted siRNA liposomes were obtained by a fusion of two parental liposomes containing spontaneously inserted siRNA and fusion phage proteins. The presence of pVIII coat protein fused to a MCF-7 cell-targeting peptide DMPGTVLP in the liposomes was confirmed by Western blotting. The novel phage-targeted siRNA-nanopharmaceuticals demonstrate significant down-regulation of PRDM14 gene expression and PRDM14 protein synthesis in the target MCF-7 cells. This approach offers the potential for development of new anticancer siRNA-based targeted nanomedicines.

From the Clinical Editor

In this study, the authors report a novel approach for targeted intracellular delivery of siRNAs into breast cancer cells through encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins.

Graphical Abstract

siRNA-loaded liposome targeted by phage protein fused with a MCF-7 cell-specific peptide DMPGTVLP. The hydrophobic helix of the protein is anchored in the lipid bilayer, whereas the N-terminal fusion peptide DMPGTVLP is displayed on the surface of the liposome. The siRNA molecules are pictured as strands inside the liposomes.

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Section snippets

Reagents

L-α-phosphatidylcholine (ePC); 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt; DPPG); 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt; DOTAP); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)2000] (ammonium salt; PEG2000-PE ); and cholesterol (CHOL) were from Avanti Polar Lipids Inc. (Alabaster, Alabama).

Sodium cholate, 2.5% CHAPS: 3-[(3-cholamidopropyl)dimethylammonio]-1-proanesulfonate, bovine serum albumin (BSA), phenylmethanesulfonyl

Selection of breast cancer cell-specific phages

To obtain MCF-7 breast cancer cell-binding phages, the billion-clone library of phages harboring 8- mer peptides on all 4,000 copies of major coat proteins27 was used for in vitro selection. A portion of the library containing 100 billion phage particles was depleted first for phages binding a cell culture flask and then was applied to MCF-7 cells. Unbound phages were removed, whereas bound phages were eluted with mild acid. The eluted phages were amplified, purified and used in subsequent

Discussion

To enhance a potential anticancer efficiency of liposome-encapsulated siRNAs, we specifically targeted them via fusion with preselected phage protein specific for cancer cells MCF-7. To simplify the procedure and exclude any chemical conjugation reactions, we used spontaneous insertion of the phage proteins and siRNA into parental liposomes, followed by their fusion in mild conditions. The tumor-specific protein was isolated from the phage that was affinity selected from multibillion-clone

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    This work was supported by the NIH grant 1 R01 CA125063-01 and Calvert Research Institute LLC grants to Valery A. Petrenko. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of National Cancer Institute of NIH and Calvert Research Institute.

    There are no disclosures or any conflicts of interest with regard to this publication.

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