Development of High-Functionality and -Quality Lipids with RGD Peptide Ligands: Application for PEGylated Liposomes and Analysis of Intratumoral Distribution in a Murine Colon Cancer Model

High-functionality and -quality (HFQ) lipids have a discrete molecular weight and good water dispersibility and can be produced by solid-phase peptide synthesis. Therefore, HFQ lipids are a promising material for the preparation of ligand-grafted PEGylated liposomes. Recently, we have reported serine–glycine repeated peptides ((SG) n ) as a spacer of HFQ lipids and to substitute a conventional PEG spacer. We demonstrated the advantage of using (SG) n spacers for peptide ligand presentation on the liposomal surface in vitro; however, the use of (SG) n spacers in ligand-grafted PEGylated liposomes in vivo has not been validated. The aim of this study was to validate the in vivo targeting ability of HFQ lipid-grafted PEGylated liposomes. We synthesized lipids containing GRGDS (RGD–(SG) n –lipid) to target integrin α v β 3 and prepared RGD–(SG) n /PEGylated liposomes. Subsequently, their cellular uptake characteristics in murine colon carcinoma (Colon-26) cells were evaluated. Two-color imaging of liposomes and tumor blood vessels following tissue clearing was performed to examine the spatial intratumoral distribution of liposomes. RGD–(SG) 5 /PEGylated liposomes were selectively associated with the cells in vitro. In vivo analysis of intratumoral distribution following tissue clearing revealed the superior targeting ability of RGD–(SG) 5 /PEGylated liposomes compared with that of conventional RGD–PEG 2000 /PEGylated liposomes for both tumor tissues and tumor blood vessels. We successfully synthesized RGD-HFQ lipids to prepare RGD-grafted PEGylated liposomes for the efficient targeting of integrin α v β 3 -expressing cells. To the best of our knowledge, this is the first report of the intratumoral distribution of ligand-grafted PEGylated liposomes by two-color imaging following tissue clearing.