Liposomal formulations achieved 7.7% penetration of the injected dose into the brain of mice.
Liposomal formulations protected encapsulated plasmid DNA from enzymatic degradation.
The formulations exhibited low hemolytic potential and low cytotoxicity at a concentration of 100 nM phospholipid.
Cellular uptake of the nanoparticles was facilitated through multiple endocytosis pathways.
CPP-Tf-conjugated demonstrated effective transfection in brain endothelial, primary glial, and primary neuronal cells.
Tf and TAT modifications on liposomes enhanced their ability to cross the and transfect neuronal cells.
Simplified
BACKGROUND: The potential of gene therapy for treatment of neurological disorders can be explored using designed lipid-based nanoparticles such as , which have demonstrated ability to deliver nucleic acid to brain cells. We synthesized liposomes conjugated to (CPPs) (vascular endothelial-cadherin-derived peptide [pVec], pentapeptide QLPVM and HIV-1 trans-activating protein [TAT]) and transferrin (Tf) ligand, and examined the influence of surface modifications on the liposome delivery capacity and transfection efficiency of encapsulated plasmid DNA. The design of liposomes was based on targeting molecular recognition of transferrin receptor overexpressed on the (BBB) with enhanced internalization ability of CPPs.
METHODS: CPP-Tf-liposomes were characterized by particle size distribution, zeta potential, protection of encapsulated plasmid DNA, uptake mechanisms and transfection efficiencies. An in vitro triple co-culture BBB model selected the liposomal formulations that were able to cross the in vitro BBB and subsequently, transfect primary neuronal cells. The in vivo biodistribution and biocompatibility of selected formulations were also investigated in mice.
RESULTS: Liposomal formulations were able to protect the encapsulated plasmid DNA against enzymatic degradation and presented low hemolytic potential and low cytotoxicity at 100 nM phospholipid concentration. Cellular internalization of nanoparticles occurred via multiple endocytosis pathways. CPP-Tf-conjugated liposomes mediated robust transfection of brain endothelial (bEnd.3), primary glial and primary neuronal cells. Liposomes modified with Tf and TAT demonstrated superior ability to cross the barrier layer and subsequently, transfect neuronal cells compared to other formulations. Quantification of fluorescently labeled liposomes and in vivo imaging demonstrated that this system could efficiently overcome the BBB and penetrate the brain of mice (7.7% penetration of injected dose).
CONCLUSION: In vitro screening platforms are important tools to enhance the success of brain-targeted gene delivery systems. The potential of TAT-Tf-liposomes as efficient brain-targeted gene carriers in vitro and in vivo was suggested to be related to the presence of selected moieties on the nanoparticle surface.
Key numbers
62.3%
GFP Expression in bEnd.3 Cells
Percentage of bEnd.3 cells expressing GFP after treatment with TAT-Tf-.
7.7%
Brain Penetration Rate
Percentage of injected dose that penetrated the brain after administration of TAT-Tf-.
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B.S.R. is supported by a doctoral fellowship from The Brazilian National Council for Scientific and Technological Development (CNPq, Brazil) with a scholarship for B.S.R (Full Doctorate Fellowship (GDE): 221327/2014-2). The authors report no other conflicts of interest in this work.
PubMed
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