Cationic lipids are exploited as vectors ('lipoplexes') for delivering nucleic acids, including genes, into cells for both therapeutic and cell biological purposes. However, to meet therapeutic requirements, their efficacy needs major improvement, and better defining the mechanism of entry in relation to eventual transfection efficiency could be part of such a strategy. Endocytosis is the major pathway of entry, but the relative contribution of distinct endocytic pathways, including clathrin- and caveolae-mediated endocytosis and/or macropinocytosis is as yet poorly defined. Escape of DNA/RNA from endosomal compartments is thought to represent a major obstacle. Evidence is accumulating that non-lamellar phase changes of the lipoplexes, facilitated by intracellular lipids, which allow DNA to dissociate from the vector and destabilize endosomal membranes, are instrumental in plasmid translocation into the cytosol, a prerequisite for nuclear delivery. To further clarify molecular mechanisms and to appreciate and overcome intracellular hurdles in lipoplex-mediated gene delivery, quantification of distinct steps in overall transfection and proper model systems are required.
"Plasmid DNA and cationic liposomes self-assemble into complexes capable of binding to cell membranes. Intact lipoplexes have been shown to be internalised in large endocytic vesicles , from which they are proposed to escape into the cytoplasm at some point along the endosomal pathway , . Efficient transgene expression depends on nuclear entry of the plasmid DNA, in order to access the transcription machinery. "
[Show abstract][Hide abstract] ABSTRACT: Intracellular protein trafficking through secretory and endocytic pathways depends on the function of adaptor proteins that bind motifs on cargo proteins. The adaptor proteins then recruit coat proteins such as clathrin, enabling the formation of a transport vesicle. While studying the role of the clathrin adaptor proteins, AP-1, AP-2 and AP-3 in viral protein trafficking, we discovered that AP-1 and AP-3 potentially have a role in successful transfection of mammalian cells with DNA-liposome complexes (lipoplexes). We showed that AP-1, -2 and -3 are not required for lipoplexes to enter cells, but that lipoplexes and/or released DNA are unable to reach the nucleus in the absence of AP-1 or AP-3, leading to minimal exogenous gene expression. In contrast, gene expression from liposome-delivered mRNA, which does not require nuclear entry, was not impaired by the absence of AP-1 or AP-3. Despite the use of lipoplexes to mediate gene delivery being so widely used in cell biology and, more recently, gene therapy, the mechanism by which lipoplexes or DNA reach the nucleus is poorly characterised. This work sheds light on the components involved in this process, and demonstrates a novel role for AP-1 and AP-3 in trafficking lipoplexes.
PLoS ONE 03/2014; 9(3):e91429. DOI:10.1371/journal.pone.0091429 · 3.23 Impact Factor
"Furthermore, it has been shown that cationic liposomes are superior to neutral and negatively charged liposomes in inducing antigen-specific cytotoxic T lymphocyte (CTL) responses and antibody production (Nakanishi et al. 1997, 1999) and use as delivery systems for nucleic acids in gene therapy (Karmali and Chaudhuri 2007; Wasungu and Hoekstra 2006). The ability of liposome–DNA complexes to enter the cell by endocytosis, escape from the endosome, and deliver nucleic acids into the cytoplasm has been well documented (Zabner et al. 1995; Hoekstra et al. 2007). We have already assessed the role of CpG ODNs in enhancement of immune response against two recombinant antigens including rgp63 or rLmSTI1 when entrapped into the liposomes (Badiee et al. 2008; Jaafari et al. 2007). "
[Show abstract][Hide abstract] ABSTRACT: Development of an effective vaccine against leishmaniasis is possible due to the fact that individuals cured from cutaneous leishmaniasis (CL) are protected from further infection. First generation Leishmania vaccines consisting of whole killed parasites reached to phase 3 clinical trials but failed to show enough efficacies mainly due to the lack of an appropriate adjuvant. In this study, an efficient liposomal protein-based vaccine against Leishmania major infection was developed using soluble Leishmania antigens (SLA) as a first generation vaccine and cytidine phosphate guanosine oligodeoxynucleotides (CpG ODNs) as an immunostimulatory adjuvant. 1, 2-Dioleoyl-3-trimethylammonium-propane was used as a cationic lipid to prepare the liposomes due to its intrinsic adjuvanticity. BALB/c mice were immunized subcutaneously (SC), three times in 2-week intervals, with Lip-SLA-CpG, Lip-SLA, SLA + CpG, SLA, or HEPES buffer. As criteria for protection, footpad swelling at the site of challenge and spleen parasite loads were assessed, and the immune responses were evaluated by determination of IFN-γ and IL-4 levels of cultured splenocytes, and IgG subtypes. The group of mice that received Lip-SLA-CpG showed a significantly smaller footpad swelling, lower spleen parasite burden, higher IgG2a antibody, and lower IL-4 level compared to the control groups. It is concluded that cationic liposomes containing SLA and CpG ODNs are appropriate to induce Th1 type of immune response and protection against leishmaniasis.
Parasitology Research 01/2012; 111(1):105-14. DOI:10.1007/s00436-011-2806-5 · 2.10 Impact Factor
"Lipoplexes within size range 200–300 nm have been previously reported . Although the size of the lipoplexes will determine the main route of entry with smaller lipoplexes (<300 nm) likely to enter via clathrin mediated endocytosis, and larger particles (>500 nm) entering cells via caveoli mediated endocytosis [28,29], one recent report shows that the actual entry route for functional siRNA mediated gene silencing might possibly be fusion with the plasma membrane rather than the endocytosis pathway . The ζ-potentials measurements showed that all the lipoplexes had positive values within the range 28-50 mV. "
[Show abstract][Hide abstract] ABSTRACT: Four guanidine derivatives of N4,N9-diacylated spermine have been designed, synthesized, and characterized. These guanidine-containing cationic lipids bound siRNA and formed nanoparticles. Two cationic lipids with C18 unsaturated chains, N1,N12-diamidino-N4,N9-dioleoylspermine and N1,N12-diamidino-N4-linoleoyl-N9-oleoylspermine, were more efficient in terms of GFP expression reduction compared to the other cationic lipids with shorter C12 (12:0) and very long C22 (22:1) chains. N1,N12-Diamidino-N4-linoleoyl-N9-oleoylspermine siRNA lipoplexes resulted in GFP reduction (26%) in the presence of serum, and cell viability (64%). These data are comparable to those obtained with TransIT TKO. Thus, cationic lipid guanidines based on N4,N9-diacylated spermines are good candidates for non-viral delivery of siRNA to HeLa cells using self-assembled lipoplexes.
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