Intracellular Distribution and Mechanism of Delivery of Oligonucleotides Mediated by Cationic Lipids

Purpose. To study the parameters influencing the intracellular trafficking of oligonucleotides delivered by cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) lipids and to elucidate the mechanism of uptake. Methods. We have studied the intracellular localization of fluorescently labeled oligonucleotide (F-ODN) delivered by DOTAP using confocal microscopy and measured inhibition of luciferase synthesis. The delivery mechanism of ODN/DOTAP complexes was investigated using inhibitors of the endocytosis pathway. Results. F-ODN delivered by DOTAP liposomes redistribute from punctate cytoplasmic regions into the nucleus. The nuclear uptake of F-ODN depends on: charge ratio (+/–), time of incubation, temperature and presence of serum. A positively charged complex is required for enhanced uptake. The association of neutral lipids with DOTAP reduced the optimum charge ratio without altering the delivery efficiency. DOTAP lipids increased >100 fold the antisense activity of a specific anti-luciferase ODN. Inhibitors of the endocytosis pathway show that the majority of F-ODN are introduced through an endocytic pathway mainly involving uncoated vesicles. Nuclear accumulation of oligonucleotides can be decreased by inhibitors of actin microfilaments, energy metabolism and proteins implicated in the fusion of endosomes. Nuclear uptake is independent of acidification of the endosomal vesicles and unaffected by inhibitors of microtubules. Conclusions. Oligonucleotides are delivered by cationic lipids into the cytoplasm at an early stage of the endocytotic pathway which leads to a marked increase in antisense activity and oligonucleotide nuclear uptake.     

Magnetically enhanced nucleic acid delivery. Ten years of magnetofection-progress and prospects

Nucleic acids carry the building plans of living systems. As such, they can be exploited to make cells produce a desired protein, or to shut down the expression of endogenous genes or even to repair defective genes. Hence, nucleic acids are unique substances for research and therapy. To exploit their potential, they need to be delivered into cells which can be a challenging task in many respects. During the last decade, nanomagnetic methods for delivering and targeting nucleic acids have been developed, methods which are often referred to as magnetofection. In this review we summarize the progress and achievements in this field of research. We discuss magnetic formulations of vectors for nucleic acid delivery and their characterization, mechanisms of magnetofection, and the application of magnetofection in viral and nonviral nucleic acid delivery in cell culture and in animal models. We summarize results that have been obtained with using magnetofection in basic research and in preclinical animal models. Finally, we describe some of our recent work and end with some conclusions and perspectives.     

Nucleic acid delivery using magnetic nanoparticles: the Magnetofection technology

In recent years, gene therapy has received considerable interest as a potential method for the treatment of numerous inherited and acquired diseases. However, successes have so far been hampered by several limitations, including safety issues of viral-based nucleic acid vectors and poor in vivo efficiency of nonviral vectors. Magnetofection has been introduced as a novel and powerful tool to deliver genetic material into cells. This technology is defined as the delivery of nucleic acids, either 'naked' or packaged (as complexes with lipids or polymers, and viruses) using magnetic nanoparticles under the guidance of an external magnetic field. This article first discusses the principles of the Magnetofection technology and its benefits as compared with standard transfection methods. A number of relevant examples of its use, both in vitro and in vivo, will then be highlighted. Future trends in the development of new magnetic nanoparticle formulations will also be outlined.     

Mechanism of oligonucleotide release from cationic liposomes.

We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/ fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone did not release ODN, even at 100-fold charge excess (-/+). Based upon these results, we propose that the complex, after internalization by endocytosis, induces flip-flop of anionic lipids from the cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm.     

Magnetic Nanoparticles Enhance Adenovirus Transduction <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis>

Purpose  

Adenoviruses are among the most powerful gene delivery systems. Even if they present low potential for oncogenesis, there is still a need for minimizing widespread delivery to avoid deleterious reactions. In this study, we investigated Magnetofection efficiency to concentrate and guide vectors for an improved targeted delivery.     

Gene chemistry: functionally and conformationally intact fluorescent plasmid DNA

We describe an effective approach using a peptide nucleic acid (PNA) "clamp" to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. To demonstrate this approach a highly fluorescent preparation of plasmid DNA was generated by hybridizing a fluorescently labeled PNA to the plasmid. Fluorescent plasmid prepared in this way was neither functionally nor conformationally altered. PNA binding was sequence specific, saturable, extremely stable, and did not influence the nucleic acid intracellular distribution. This method was utilized for the first time to study the biodistribution of conformationally and functionally intact plasmid DNA in living cells after cationic lipid-mediated transfection. A fluorescent plasmid expressing green fluorescent protein (GFP) enabled simultaneous colocalization of both plasmid and expressed protein in living cells and in real time. GFP was shown to be expressed in cells containing detectable nuclear fluorescent plasmid. The fluorescent PNA-labeled plasmid revealed a marked difference in the nuclear uptake between oligonucleotide and plasmid, suggesting that nuclear entry of plasmid may require cell division. This detection method provides a way to simultaneously monitor the intracellular localization and expression of plasmid DNA in living cells, and to elucidate the mechanism of plasmid delivery and its nuclear import with synthetic gene delivery systems.