The role of the helper lipid on the DNA transfection efficiency of lipopolyplex formulations (original) (raw)
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Cationic Liposomes for Gene Delivery: Novel Cationic Lipids and Enhancement by Proteins and Peptides
Current Medicinal Chemistry, 2003
Cationic liposome-DNA complexes, also called "lipoplexes", constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. Here we review the mechanisms of lipoplex-mediated gene delivery and barriers to efficient gene expression, novel cationic lipids used for transfection, and methods for enhancing gene transfer via the use of proteins, including transferrin, albumin and asialofetuin, and synthetic peptides, including GALA and nuclear localization signal peptides. We underscore the importance of understanding the mechanisms of cytoplasmic and nuclear entry of DNA and its dissociation from lipoplexes. We emphasize that the in vitro transfection activity of new lipoplex constructs should be tested in the presence of high serum concentrations. MECHANISMS OF LIPOPLEX-MEDIATED GENE DELIVERY Successful gene delivery via lipoplexes is thought to require several conditions to be met: (i) condensation of DNA into the lipoplex structure and its protection from
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2014
Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid-protamine/ deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP-DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP-DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol-DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically:
Journal of peptide science : an official publication of the European Peptide Society, 2018
The design, synthesis and formulation of non-viral gene delivery vectors is an area of renewed research interest. Amongst the most efficient non-viral gene delivery systems are lipopolyplexes, in which cationic peptides are co-formulated with plasmid DNA and lipids. One advantage of lipopolyplex vectors is that they have the potential to be targeted to specific cell types by attaching peptide targeting ligands on the surface, thus increasing both the transfection efficiency and selectivity for disease targets such as cancer cells. In this paper, we have investigated two different modes of displaying cell-specific peptide targeting ligands at the surface of lipopolyplexes. Lipopolyplexes formulated with bimodal peptides, with both receptor binding and DNA condensing sequences, were compared with lipopolyplexes with the peptide targeting ligand directly conjugated to one of the lipids. Three EGFR targeting peptide sequences were studied, together with a range of lipid formulations and...
Solid lipid nanoparticles (SLNs) are a promising system for the delivery of lipophilic and hydrophilic drugs. They consist of a solid lipid core that is stabilized by a layer of surfactants. By the incorporation of cationic lipids in the formulation, positively charged SLNs can be generated, that are suitable carriers for nucleic acids (DNA, siRNA). Considering the beneficial effect of hDelepleivrelirpeidsboynPthueblitsrahninsfgecTtieocnhenfoficloiegnycytow: iSthUcNaYtioUnipcsltipaotesoMmeds,ictahleUenffievcetrsoiftythe helper lipid 1,2- IP: 218.4.248.194 On: Sun, 11 Oct 2015 10:41:22 dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) on transfection with cationic lipid-containing solid lipid nanoparticles Copyright: American Scientific Publishers was investigated in PC3 prostate cancer cells. The inclusion of DOPE in SLN formulations, instead of promoted, strongly inhibited SLN transfection efficiency, by frustrating the accommodation of DNA by the particles, as was revealed by biochemical analysis. SLNs devoid of DOPE maintained a homogenous size distribution of ∼150 nm following lipoplex assembly and cellular delivery, and showed transfection efficiency comparable to that of Lipofectamine 2000® (LF2k). Moreover, the SLNs maintain their high transfection efficiency after lyophilization and long-term storage (1–2 years), an important asset for biomedical applications. There is even the possibility to lyophilize the SLN carrier together with its DNA cargo, which represents an interesting pharmaceutical advantage of the SLN formulations over LF2k. These results reflect marked differences between the physicochemical properties of cationic liposomes and SLNs, the latter requiring more critical lipid-depending properties for effective ‘packaging’ of DNA but displaying a higher storage stability than cationic lipid based carriers like LF2k.
Lipopolyplexes as Nanomedicines for Therapeutic Gene Delivery
Methods in Enzymology, 2012
We describe an efficient, nonviral gene transfer system that has been developed by employing polyethylenimine (PEI 800, 25, 22 kDa), and DOTAP and cholesterol (Chol) as lipids (lipopolyplex), at three different lipid/DNA molar ratios (2/1, 5/1, and 17/1) by using five different protocols of formulation. Condensation assays revealed that PEIs of 800, 25, and 22 kDa were very effective in condensing plasmid DNA, leading to a complete condensation at N/P ratios above 4. Addition of DOTAP/Chol liposomes did not further condense DNA. Increasing the molar ratio lipid/DNA in the complex resulted in higher positive values of the zeta potential, while the particle size increased in some
Cationic liposomes for gene delivery
Expert Opinion on Drug Delivery, 2005
Cationic liposome-DNA complexes (lipoplexes) constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. This review will focus on various parameters governing lipoplex biological activity, from their mode of formation to in vivo behaviour. Particular emphasis is given to the mechanism of interaction of lipoplexes with cells, in an attempt to dissect the different barriers that need to be surpassed for efficient gene expression to occur. Aspects related to new trends in the formulation of lipid-based gene delivery systems aiming at overcoming some of their limitations will be covered. Finally, examples illustrating the potential of cationic liposomes in clinical applications will be provided.
Development of a DNA-liposome complex for gene delivery applications
2017
The association structures formed by cationic liposomes and DNA (Deoxyribonucleic acid)-liposome have been effectively utilized as gene carriers in transfection assays. In this research study, cationic liposomes were prepared using a modified lipid film hydration method consisting of a lyophilization step for gene delivery applications. The obtained results demonstrated that the mean particle size had no significant change while the polydispersity (PDI) increased after lyophilization. The mean particle size slightly reduced after lyophilization (520 ± 12 nm to 464 ± 25 nm) while the PDI increased after lyophilization (0.094 ± 0.017 to 0.220 ± 0.004). In addition. The mean particle size of vesicles increases when DNA is incorporated to the liposomes (673 ± 27 nm). According to the Scanning Electron Microscopy (SEM) and transmission electron microscopy (TEM) images, the spherical shape of liposomes confirmed their successful preservation and reconstitution from the powder. It was found that liposomal formulation has enhanced transfection considerably compared to the naked DNA as negative control. Finally, liposomal formulation in this research had a better function than Lipofectamine® 2000 as a commercialized product because the cellular activity (cellular protein) was higher in the prepared lipoplex than Lipofectamine® 2000.
Colloids and Surfaces B-biointerfaces, 2009
Among non-viral vectors, cationic liposomes are the most promising carriers in gene delivery. But the most critical issue about their application is their low transfection efficiency compared to viral vectors. In this study, we tried to make a comparison between transfection efficiency of different liposomal formulations and to investigate the effect of membrane fluidity and other physical properties of liposomes and lipoplexes such as size and charge ratio on the transfection efficiency in in vitro environment. Different gene delivery systems were developed by using liposomes composed of 1,2-dioleoyl-3trimethylammonium-propane (DOTAP) or 3--[N-(N N -dimethylaminoethane)-carbamoyl] cholesterol (DC-CHOL) in combination with other lipids including 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), egg L-␣-phosphatidylcholine (EPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). These multilamellar vesicle (MLV) liposomes were extruded through 100 nm polycarbonate filters to produce small unilamellar vesicles (SUVs). Transfection activity of these lipoplexes in Neuro2A cells was tested using pRL-CMV encoding Renilla luciferase. We could not establish any direct correlation between high fluid membranes and high transfection efficiency because DOTAP:DPPE had a better result than DOTAP:DOPE while DC-CHOL:DOPE was more successful in gene transfer than DC-CHOL:DPPE. It was revealed that the use of these two helper lipids with different Tm (DPPE: 64 • C and DOPE: −11 • C) along with DOTAP increased transfection efficiency but formulation of these phospholipids with DC-CHOL was led to a significant reduction in transfection activity. Generally, DOTAP:DPPE, DC-CHOL:DOPE and DOTAP:DOPE:DPPE formulations showed the highest transfection activity. The results of this study showed that, in designing of liposome based non-viral vectors, different parameters such as size, lipid composition and the use of helper lipid should be considered.
Bioreducible Liposomes for Gene Delivery: From the Formulation to the Mechanism of Action
PLOS One, 2010
Background: A promising strategy to create stimuli-responsive gene delivery systems is to exploit the redox gradient between the oxidizing extracellular milieu and the reducing cytoplasm in order to disassemble DNA/cationic lipid complexes (lipoplexes). On these premises, we previously described the synthesis of SS14 redox-sensitive gemini surfactant for gene delivery. Although others have attributed the beneficial effects of intracellular reducing environment to reduced glutathione (GSH), these observations cannot rule out the possible implication of the redox milieu in its whole on transfection efficiency of bioreducible transfectants leaving the determinants of DNA release largely undefined.