By 2030 there could be 27 million incident cases of cancer worldwide, 17 million cancer deaths annually and 75 million persons alive with cancer within five years of diagnosis. Active targeting of an adhesion molecule that is expressed on the surface of the tumor cells, is a promising area of research and targeted nanoparticle delivery to cancer is an area of interest that may offer hope in improving the treatment of therapy-resistant cancers. Unfortunately, cancer-specificity by selective recognition of individual cellular receptors has rarely been achieved adequately to date, suffering from unintended delivery to healthy organs as some target disease markers are also expressed in areas other than tumors. The focus of this work is on the problem of specific delivery of non-viral gene delivery vehicles to cancer cells versus healthy cells. Proposed is the development of a multi-targeted therapeutic system, that will make the recognition of cancer cells more specific.
The hypothesis of this work is that a higher degree of specificity for cancer cells could be achieved by designing a modular multi-targeted non-viral system that: i) introduces simultaneous targeting of the overexpressed cancer surface receptors alpha(5)beta(1) and alpha(6)beta(4) integrins as the first level of targeting at the extracellular level, and ii) targets the upregulated transcriptional activity of NF-kB that is inactive in healthy cells while it is highly upregulated in a variety of diseases including cancer, thereby introducing a new form of transcriptional targeting as the second level of specific targeting. To test this hypothesis the following tasks have been set:
Research Task 1: Evaluate the effect of peptide concentration on the miscibility of peptide-amphiphiles and polyethylene glycol (PEG) molecules. Langmuir-Blodgett membranes will be designed that will mimic the interfaces of the dual-ligand stealth liposomes by mixing lipids, lipidated PEG and the peptide-amphiphiles that bind specifically to the alpha(5)beta(1) and alpha(6)beta(4) integrins. The effect of peptide concentration on the mixing behavior between the peptides and PEG will be investigated by atomic force microscopy.
Research Task 2: To engineer multi-targeted stealth liposomes, functionalized with peptide-amphiphiles that bind specifically to the alpha(5)beta(1) and alpha(6)beta(4) integrins, and deliver them to cells with different integrin expression levels. Peptide-functionalized stealth liposomes will be prepared with different concentrations of the two peptides. The liposomes will be delivered to different cancer cells with varying levels of integrin expression. Fluorescent dyes will be encapsulated initially, and the binding and internalization of the liposomes will be evaluated with plate assays and confocal microscopy studies. The liposomes will be evaluated further with respect to promoting specific transcriptional targeting as they will deliver the diphtheria toxin fragment A (DTA) gene under the control of the NF-kB promoter. The toxicity of different cells with varying levels of integrin expression will be evaluated with plate assays.
Research Task 3: To evaluate the multi-targeted stealth liposomes in vivo. Dual-ligand formulations from Research Task 2 that will be shown to bind best to DLD-1 colon cancer cells will be evaluated in this task for their ability to reach the colon tumor site in vivo, with microPET/CT imaging and biodistribution studies. Non-targeted and single-ligand stealth liposomes will be used as controls. The single- and dual-ligand optimal formulations will further be evaluated for their efficacy and toxicity. For these studies the DTA gene under the control of the NF-kB promoter will be employed.
