Intracellular trafficking of hybrid gene delivery vectors

Abstract

Viral and non-viral gene delivery vectors are in development for human gene therapy, but both exhibit disadvantages such as inadequate efficiency, lack of cell-specific targeting or safety concerns. We have recently reported the design of hybrid delivery vectors combining retrovirus-like particles with synthetic polymers or lipids that are efficient, provide sustained gene expression and are more stable compared to native retroviruses. To guide further development of this promising class of gene delivery vectors, we have investigated their mechanisms of intracellular trafficking. Moloney murine leukemia virus-like particles (M-VLPs) were complexed with chitosan (Chi) or liposomes (Lip) comprising DOTAP, DOPE and cholesterol to form the hybrid vectors (Chi/M-VLPs and Lip/M-VLPs, respectively). Transfection efficiency and cellular internalization of the vectors were quantified in the presence of a panel of inhibitors of various endocytic pathways. Intracellular transport and trafficking kinetics of the hybrid vectors were dependent on the synthetic component and used a combination of clathrin- and caveolar-dependent endocytosis and macropinocytosis. Chi/M-VLPs were slower to transfect compared to Lip/M-VLPs due to the delayed detachment of the synthetic component. The synthetic component of hybrid gene delivery vectors plays a significant role in their cellular interactions and processing and is a key parameter for the design of more efficient gene delivery vehicles.

Endocytosis and intracellular traffic of cholesterol-PDMAEMA liposome complexes in human epithelial-like cells

“…However, conventional liposome formulations generally suffer from poor cell penetration and low stability in bloodstream; therefore, they need to be modified to improve their performance in vivo [5,6]. Different endocytic pathways could be involved in the incorporation of liposomes into eukaryotic cells [7,8], and the direct fusion of liposomes with plasma membrane can occur as well [9]. Endocytosis process can be classified in two broad categories: phagocytosis (the uptake of large particles and microorganisms) and pinocytosis (the uptake of fluid and solutes) [10].…”

Intracellular Delivery of Glucose Oxidase for Enhanced Cytotoxicity toward PSMA‐Expressing Prostate Cancer Cells

“…To better understand how anti‐PSMA‐PLL‐ g ‐PEG/GOX complexes are internalized by LNCaP cells, the mechanism of uptake by the complexes was evaluated using pharmacological inhibitors with methods described by Keswani et al LNCaP cells were grown to ≈70% confluency in RPMI 1640 supplemented with 10% FBS before being seeded on 96‐well plates at a density of 1 × 10 4 cells per well. Prior to uptake studies, the cytotoxicity of each inhibitor toward LNCaP cells was determined in order to treat the cells with the maximum amount of inhibitor without reducing cell viability lower than 90% . The maximum concentration of inhibitor tolerable to the cells was then used for uptake studies.…”

Lipid rafts-mediated endocytosis and physiology-based cell membrane traffic models of doxorubicin liposomes

“…It is well known that the fusogenic lipids DOPE may promote endosomal escape [1,15,18]. As mentioned in the cell membrane traffic studies, ≥ 20 mol% of DOPE can improve the internalization of liposomes.…”

“…However, the design of effective liposomes of anti-tumor drugs and gene therapeutics remains a major obstacle [1]. The liposomes comprising 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-DSPE) and cholesterol have shown promising utility [2,3], but the mechanisms of their cell process including cell membrane binding, internalization and intracellular trafficking remain unclear.…”