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

Abstract

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) ) as a spacer of HFQ lipids and to substitute a conventional PEG spacer. We demonstrated the advantage of using (SG) spacers for peptide ligand presentation on the liposomal surface in vitro; however, the use of (SG) 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)-lipid) to target integrin αβ and prepared RGD-(SG) /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)/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)/PEGylated liposomes compared with that of conventional RGD-PEG/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 αβ-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.

Targeted co-delivery of protein and drug to a tumor in vivo by sophisticated RGD-modified lipid-calcium carbonate nanoparticles

“…The RGD-(SG)5-lipid was synthesized based on a solid phase peptide synthesis method [25]. The other inorganic chemicals were obtained from Nacalai Tesque (Kyoto, Japan).…”

“…The encapsulation efficiency (EE) was calculated in accordance with the following equation: For the quantitation of the cellular association of DiD, a fluorescence-activated flow cytometer (LSRfortessa X-20, Becton Dickinson, USA) was used to detect the fluorescence. To study the mechanism of cellular association, RGD-modified liposomes (RLipo) were prepared in accordance with a previous report [25]. The cells were seeded in a 24-well plate at a density of intravenously injected via the tail vein every other day for five times.…”

“…GRGDS (RGD) peptides are known to be selective ligands toward the αvβ3 integrin that is overexpressed on tumor neovasculature and tumor cells including those derived from melanoma, prostate, breast, and colon cancers [23,24]. We developed HFQ lipids containing the GRGDS (RGD) peptide (RGD-(SG)5-lipid) and succeeded in the more efficient delivery of RGD-(SG)5/PEGylated liposomes to a colon tumor in mice compared with conventional RGD− PEG2000/PEGylated liposomes [25]. In this study, the RGD-(SG)5-lipid was inserted into the lipid-based CaCO3 nanoparticles to provide them with a targeting capability.…”

Development of a DNA Vaccine for Melanoma Metastasis by Inhalation Based on an Analysis of Transgene Expression Characteristics of Naked pDNA and a Ternary Complex in Mouse Lung Tissues

“…Although the intrapulmonary distribution of gene expression after inhalation is important information for therapeutic applications, it has not yet been elucidated in detail. Our group developed a multicolor deep imaging system using a tissue-clearing method and confocal microscopy in several organs [18][19][20][21]. We herein applied the tissue-clearing method to evaluate the intrapulmonary distribution of transgene expression using a multicolor deep imaging system for the first time.…”

Effects of Tissue Pressure on Transgene Expression Characteristics via Renal Local Administration Routes from Ureter or Renal Artery in the Rat Kidney

“…More recently, we have optimized a multicolor deep imaging system, by a tissue clearing method and confocal microscopy, in various organs [10][11][12][13]. Using this imaging system, we have revealed that pDNA can be delivered to peritubular fibroblast of renal cortex in suctioned kidney, after systemic injection in mice [10].…”