Abstract
Peptide ligands can enhance delivery of nucleic acid-loaded nanoparticles to tumors by promoting their cell binding and internalization. Lung tumor lesions accessible from the alveolar side can be transfected, in principle, using gene vectors delivered as an aerosol. The cell surface marker CD49f (Integrin α6) is frequently upregulated in metastasizing, highly aggressive tumors. In this study, we utilize a CD49f binding peptide coupled to linear polyethylenimine (LPEI) promoting gene delivery into CD49f-overexpressing tumor cells in vitro and into lung lesions in vivo. We have synthesized a molecular conjugate based on LPEI covalently attached to the CD49f binding peptide CYESIKVAVS via a polyethylene glycol (PEG) spacer. Particles formed with plasmid DNA were small (<200 nm) and could be aerosolized without causing major aggregation or particle loss. In vitro, CD49f targeting significantly improved plasmid uptake and reporter gene expression on both human and murine tumor cell lines. For evaluation in vivo, localization and morphology of 4T1 murine triple-negative breast cancer tumor lesions in the lung of syngeneic BALB/c mice were identified by MRI. Polyplexes applied via intratracheal aerosolization were well tolerated and resulted in measurable transgene activity of the reporter gene firefly luciferase in tumor areas by bioluminescence imaging (BLI). Transfectability of tumors correlated with their accessibility for the aerosol. With CD49f-targeted polyplexes, luciferase activity was considerably increased and was restricted to the tumor area.
Highlights
Receptor-mediated delivery of nucleic acids to tumor cells is an important issue in the development of viral and synthetic gene delivery systems
It is associated with phosphatidylinositol 3-kinase (PI3K)/Akt signaling and is responsible for maintaining stemness properties in other stem cells by interaction with OCT4 and SOX2.10 Both benign and malignant cells derived from the prostate capable of spheroid formation show a high CD49f expression, whereas other stem cell markers such as CD133 seem to be less important.[11]
Synthesis of linear polyethylenimine (LPEI)-polyethylene glycol (PEG)-Peptide Conjugate LPEI was synthesized by acidic hydrolysis of poly(2-ethyl-2-oxazoline) with HCl (6 M)
Summary
Receptor-mediated delivery of nucleic acids to tumor cells is an important issue in the development of viral and synthetic gene delivery systems. We and others have developed targeted delivery systems based on polycations for nucleic acid condensation, PEG for shielding purposes, and peptidic ligands for receptor targeting.[1,2,3,4] the affinity of a rather small peptide can be considerably lower when compared with the whole protein, the high number of targeting peptides per transfection particle induces a cooperative binding mode.[5] When compared with their protein counterparts, such peptide ligands are advantageous by several means, i.e., being fully synthetic, less immunogenic, and preferred for the nucleic acid condensation process due to their lower molecular weight.[1] Most of the potentially targetable antigens on tumor cells are not unique de novo antigens, but rather overexpressed compared with the surrounding non-malignant tissue.[6] Integrin a6 (ITGA6, CD49f) is a glycosylated transmembrane protein that dimerizes preferentially with integrin b1 and b4 forming laminin binding heterodimers.
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