RESUMEN
Cancer is the second leading cause of death globally after heart diseases. Currently used highly cytotoxic anti-cancer drugs not only kill cancer cells but also often kill non-cancerous healthy body cells, causing adverse side effects. Efforts are now being directed towards developing tumor-selective chemotherapy. Tumor/tumor endothelial cell selective peptide ligands are being covalently grafted onto the exo-surfaces of drug carriers such as liposomes, polymers, etc. A number of prior studies used conjugation of tumor/tumor endothelial cell-selective RGDK- or CGKRK-peptide ligands on the outer surfaces of liposomes, metal-based nanoparticles, single walled carbon nanotubes (SWNTs), etc. However, studies aimed at examining the relative cell membrane fusogenicities and the relative degrees of cellular uptake for the RGDK- and CGKRK-ligand-grafted nanometric drug carriers have not yet been undertaken. Herein, using the widely used liposomes of DOPC, DOPE, DOPS and cholesterol (45 : 25 : 20 : 15, w/w ratio) as the model biomembranes and the fluorescence resonance energy transfer (FRET) assay for measuring membrane fusogenicities, we show that the liposomes of the RGDK-lipopeptide are more biomembrane fusogenic than the liposomes of the CGKRK-lipopeptide. Notably, such FRET assay-derived relative biomembrane fusogenicities of the liposomes of RGDK- and CGKRK-lipopeptides were found to be consistent with their relative degrees of cellular uptake in cultured cancer cells. The present findings open the door for undertaking in-depth in vivo studies aimed at evaluating the relative therapeutic potential of different nanocarriers of drugs/genes/siRNA having tumor-targeting RGDK- and CGKRK-peptides on their exo-surfaces.
Asunto(s)
Liposomas , Liposomas/química , Humanos , Lipopéptidos/química , Lipopéptidos/farmacología , Oligopéptidos/química , Membrana Celular/metabolismo , Membrana Celular/química , Transferencia Resonante de Energía de Fluorescencia , Portadores de Fármacos/química , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Colesterol/química , Colesterol/metabolismo , Fosfatidilcolinas/química , Antineoplásicos/química , Antineoplásicos/farmacologíaRESUMEN
To ensure effective immune response in genetic immunizations, DNA/mRNA vaccines need to be delivered to body's antigen presenting cells (APCs) which is a challenging task. This is primarily due to presence of high concentrations of various degradative enzymes inside them. To this end, mannose receptor (over expressed in APCs) selective cationic liposomes have been used in the past for delivering antigen-encoded plasmid DNA to APCs. APCs also express integrin receptors on their cell surfaces. However, studies aimed at delivering DNA vaccines into APCs via integrin receptors have not yet been undertaken. Herein, we report on the use of cationic liposomes of a priorly disclosed α5ß1 integrin receptor selective RGDK-lipopeptide for macrophage transfection. In this study, we have used pCMV-GFP (as model DNA vaccine) and RAW 264.7 cells (mouse macrophages cells) as model APC. We show that the liposomes of RGDK-lipopeptide containing a previously reported endosome-disrupting histidinylated lipid and DOPE (as co-lipid) in 0.5:0.5:1.0 mole ratio are the most competent in transfecting macrophage cells (44%). Findings in the fluorescence resonance energy transfer based membrane fusogencity assay revealed that the enhanced macrophage transfection efficiency of the liposomes containing RGDK-lipopeptide, endosome-disrupting histidinylated and DOPE may originate from its higher membrane fusogenicity than that for liposomes containing only RGDK-lipopeptide and DOPE. The presently described biologically safe liposomal formulations of RGDK-lipopeptide are expected to find biomedical applications in future for combating cancer and infectious diseases through genetic immunizations.
RESUMEN
Molecular level understanding of liposome-gene interaction is immensely important for the research progress and technological advancement of gene delivery, which is highly significant due to a wide range of applications of gene therapy. The liposomal gene delivery method is one of the most promising techniques due to its efficacy to easily fuse with the cell membrane and its lower toxicity. In vivo gene delivery using liposomes is reported to be extremely successful. However, the success of gene delivery depends on various factors including the chemical nature of the structural unit of the liposome. To explore the regulative factor(s) for liposomal gene delivery, we systematically analyze the linker orientation effect on the gene delivery efficiency of liposomes through a density functional theory (DFT) study. Interestingly, it is observed that the liposome-gene interaction is not the regulating factor for successful gene delivery. The success depends on the gel to liquid melting temperature of the liposome.