Functionalized liposomal nanoparticles for efficient gene delivery system to neuronal cell transfection

Abstract

Liposome based delivery systems provide a promising strategy for treatment of neurodegenerative diseases. A rational design of brain-targeted liposomes can support the development of more efficient treatments with drugs and gene materials. Here, we characterized surface modified liposomes with transferrin (Tf) protein and penetratin (Pen), a cell-penetrating peptide, for efficient and targeted gene delivery to brain cells. PenTf-liposomes efficiently encapsulated plasmid DNA, protected them against enzymatic degradation and exhibited a sustained in vitro release kinetics. The formulation demonstrated low cytotoxicity and was non-hemolytic. Liposomes were internalized into cells mainly through energy-dependent pathways especially clathrin-mediated endocytosis. Reporter gene transfection and consequent protein expression in different cell lines were significantly higher using PenTf-liposomes compared to unmodified liposomes. The ability of these liposome to escape from endosomes can be an important factor which may have likely contributed to the high transfection efficiency observed. Rationally designed bifunctional targeted-liposomes provide an efficient tool for improving the targetability and efficacy of synthesized delivery systems. This investigation of liposomal properties attempted to address cell differences, as well as, vector differences, in gene transfectability. The findings indicate that PenTf-liposomes can be a safe and non-invasive approach to transfect neuronal cells through multiple endocytosis pathways.

<p>Development and screening of brain-targeted lipid-based nanoparticles with enhanced cell penetration and gene delivery properties</p>

“…For liposome preparation, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/2,3-dioleoyl oxy-propyl-trimethylammonium chloride (DOTAP)/cholesterol/CPP-PEG-lipid (Avanti Polar Lipids, Alabaster, AL, USA) were mixed in chloroform:methanol (2:1, v/v) at a molar ratio of 45:45:2:4 mol%, respectively 36–38. The solvents were evaporated, and the thin lipid film was hydrated with HEPES buffer (pH 7.4) yielding CPP-liposomes.…”

“…The hydrodynamic size, polydispersity index (PDI) and zeta potential of the liposomal formulations were determined using a Zetasizer Nano ZS 90 (Malvern Instruments, Malvern, UK) at 25°C 36–38. Morphological examination of liposomes was performed via transmission electron microscopy (TEM) (JEM-2100; JEOL, Peabody, MA, USA).…”

“…Fluorescence intensity was measured using a SpectraMax M5 spectrophotometer (Molecular Devices, San Jose, CA, USA) at λ ex 354 nm and λ em 458 nm. Percent encapsulation was calculated following the equation: Encapsulation efficiency (EE) = (F Total – F NE )/F Toatl ×100%, where F Total is the fluorescence intensity of total amount of plasmid (addition of Hoechst 33342 followed by addition of Triton X-100) and F NE is the fluorescence intensity of non-encapsulated plasmid into liposomes (addition of Hoechst 33342) 36–38. To investigate the stability of liposomes, TAT-Tf-liposomes were added to 1 mL culture medium containing 10% FBS.…”

Breaking Barriers: Bioinspired Strategies for Targeted Neuronal Delivery to the Central Nervous System

“…Rodrigues et al modified liposomes with TAT, a cationic peptide derived from the transactivating protein of HIV-1. TAT, known as an enhancer of NP passage through BBB, is one of many cell-penetrating peptides that can be used to functionalize the nanosystem's surface to improve its internalization capabilities [53]. For further reading on cell penetrating peptides, please refer to the review by Guidotti et al [54].…”

Nanoscopy for endosomal escape quantification

“…Numerous strategies to enhance the escape of NP have also been suggested, including: endosomal buffering agents, membrane fusogenic peptides, lysosomotropic chemical agents (20,23,(37)(38)(39)(40)(41)(42)(43)(44), morphological-dependent changes (45) or external stimuli such as photochemical internalization (PCI) (46,47).…”