Effect of Polyethyleneglycol Spacer on the Binding Properties of Nuclear Localization Signal-Modified Liposomes to Isolated Nucleus

Abstract

The successful control of intracellular trafficking is prerequisite for the development of a gene delivery system. To date, efforts to overcome the rate-limiting processes have been extensive. To improve endosomal escape, pH-dependently membrane-fusogenic lipids 1) and/or peptides, 2,3) or polycations with proton-sponge activity 4,5) have been integrated into carriers. In non-dividing cells, it is generally thought that the nuclear membrane is the ultimate barrier to be overcome, since it is composed of a double lipid bilayer structure, and mutual interactions between the cytoplasm and the intranuclear compartment are limited exclusively to transport through the nuclear pore complex (NPC). Small viruses (i.e. including Parvovirus 6) or Hepatitis B virus 7) ), which are able to pass through the threshold size of the NPC (ca. 39 nm 8) ) directly enter to the nucleus through the NPC, presumably aided by nuclear localization signals that are located on their capsid proteins. In contrast, adenovirus with a large genomic DNA (36 kDa) is approximately 90 nm in diameter, which exceeds the threshold size of the NPC. Nevertheless, it can achieve a high level of gene expression activity due to its highly efficient nuclear delivery. In the process of delivering adenovirus genome DNA to the nucleus, two ratelimiting steps are possible. First, adenoviral particles dock to the NPC. The subsequent binding of histone H1 to the adenovirus capsid, and import factors (i.e. importin 7) induce the disassembly of the adenovirus capsid structure, thus triggering the nuclear transfer of genomic DNA. 9)In a previous study, we reported on a nuclear gene delivery system that mimics the mechanism of adenoviral nuclear entry. In this carrier, pDNA was condensed with a polycation, followed by encapsulation with envelopes composed of lipid bilayers. For the targeting the NPC, the surface of the nuclear envelope was modified with nuclear localization signals derived from SV40 (NLS) or nuclear targeting sugars by incorporating stearylated NLS (STR-NLS) 10) or cholesterolconjugated sugars 11) into the lipid film. As a result, gene expression activity was drastically enhanced, compared to that of the NLS-or sugar-unmodified carriers. To further enhance the nuclear targeting activity of the preparation and gene expression efficiency, the binding activity of the lipid envelope to the nucleus needs to be further otimized.In the present study, we evaluated the nuclear binding activity of liposomes modified with a NLS with and without a PEG spacer, in order to investigate the effect of the topology of the NLS on the liposome surface on its ability to bind to the nucleus. MATERIALS AND METHODSEgg york phosphatidylcholine (EPC), cholesterol (Cho), distearoyl-sn-glycero-3-phoshoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG), N-[(3-maleimide-1-oxopropyl)aminopropylpolyethyleneglycol-carbamyl]distearoylphosphatidyl-ethanolamine (DSPE-PEG-Mal) and 7-nitrobenz-2-oxa-1,3-diazole labeled DOPE (NBD-DOPE) were purchased from Avanti Polar lipids (A...

Delivery of nanomedicines to extracellular and intracellular compartments of a solid tumor

“…In cultured cells, binding of NLS-liposomes to the nucleus increases in a NLS density-dependent manner. Interestingly, this process appears to be enhanced by pegylation, such that the efficiency of NLS-PEG-liposomes binding with the nucleus is greater than the binding of unpegylated liposomes even at low NLS density [156]. Combination of NLS and pH sensitive fusogenic lipid enables the transfer of plasmid DNA into the nucleus in an energy-dependent manner [157].…”

Advancing Nonviral Gene Delivery: Lipid- and Surfactant-Based Nanoparticle Design Strategies

“…Unfortunately, these peptides are more efficient in linear DNA delivery and electrostatic interaction between them results in shielding of the peptide by the DNA, hindering its function [133]. Grafting a PEG spacer onto the surface of the lipoplexes bypasses this deficiency, improving nuclear transport [134]. Another shuttle peptide is the 38-amino acid M9 sequence of the human mRNA-binding protein hn-RNPA1, a hydrophobic peptide having a transport mechanism distinct from that of nuclear localization signals [135,136].…”