Olivier Zelphati

Effect of serum components on the physico-chemical properties of cationic lipid/oligonucleotide complexes and on their interactions with cells

The interactions among serum components and cationic lipid-nucleic acid complexes are central to the understanding of how serum inhibits cellular delivery of oligonucleotides in vitro and in vivo. In this study, we show that several serum proteins, in particular bovine serum albumin (BSA), lipoproteins (HDL and LDL) and macroglobulin, interact with cationic lipid/oligonucleotide complexes, alter the complex diameter and zeta potential (from positive to negative values), and significantly interfere with the ability of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) to deliver phosphorothioate oligonucleotides (ODN) into cells. Serum and BSA do not dissociate the ODN and lipid components, therefore inhibition of delivery cannot be attributed to a displacement of cationic lipid from the ODN. Rather BSA at 2.5 mg/ml, comparable to the amount found in 10% serum, decreases the cell association of ODN by about 5-fold and nuclear uptake of ODN by greater than 20-fold. In contrast, immunoglobulin G, the other major serum component, alters the zeta potential from positive to near neutral, has a modest effect on the diameter of the complex but does not affect cell association or nuclear delivery of the ODN at amounts found in 10% serum. Other molecules found in serum, specifically oleic acid and heparin, displace the ODN from the complex and thus interfere with delivery. This displacement is attenuated by first incubating the complex with BSA. Another manifestation of serum-complex interactions is that ODN significantly and cationic liposomes slightly, activate complement. However, formation of the complex markedly reduces the complement activation of the ODN. Finally, the effect of serum can be partially counteracted by the selection of the helper lipid (DOPE or cholesterol). Inclusion of a helper lipid reduces the effective charge ratio (cationic groups/anionic thioates) required to deliver ODN into cells and permits delivery in the presence of greater percentages of serum in the culture medium. These results support the current view that the binding of serum proteins to the complex is a significant factor in modulating the activity of cationic lipid-ODN complexes in culture and after intravenous administration.

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

AbstractPurpose. 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

Abstract 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.

Liposomes as a carrier for intracellular delivery of antisense oligonucleotides: a real or magic bullet?

Abstract Antisense oligonucleotides are specific inhibitors of gene expression. They represent a promising tool in fighting viral, malignant and inflammatory diseases. In many cases their activity is limited by their low cellular uptake and lack of target cell recognition. One approach to circumvent these problems is the use liposomes as an oligonucleotide carrier. The encapsulation of oligonucleotides in liposomes is useful for several reasons: (1) protection of oligonucleotides from nuclease degradation; (2) enhancement of cellular uptake in several cell types; (3) improvement of oligonucleotide potency, especially in vitro; (4) modification of their intracellular distribution (this is particularly true for cationic liposomes); (5) increased retention of the oligonucleotides in cells; (6) potential for slow release depots for modified oligonucleotides. However, encapsulation of oligonucleotides in liposomes may decrease the access of the oligonucleotide to tissues outside of the vascular system which may restrict the use of oligonucleotides encapsulated in liposomes or other particulate carriers to accessible cells or tissues. In this review results obtained in vitro and in vivo, using liposome encapsulated oligonucleotides are described and the benefits of liposomes as an oligonucleotide carrier are analyzed.

Recent Advances in Magnetofection and Its Potential to Deliver siRNAs In Vitro

This chapter describes how to design and conduct experiments to deliver siRNA to adherent mammalian cells in vitro by magnetic force-assisted transfection using self-assembled complexes of small interfering RNA (siRNA) and cationic lipids or polymers that are associated with magnetic nanoparticles. These magnetic complexes are targeted to the cell surface by the application of a magnetic gradient field. In this chapter, first we describe the synthesis of magnetic nanoparticles for magnetofection and the association of siRNA with the magnetic components of the transfection complex. Second, a simple protocol is described in order to evaluate magnetic responsiveness of the magnetic siRNA transfection complexes and estimate the complex loading with magnetic nanoparticles. Third, protocols are provided for the preparation of magnetic lipoplexes and polyplexes of siRNA, magnetofection, downregulation of gene expression, and the determination of cell viability. The addition of INF-7 peptide, a fusogenic peptide, to the magnetic transfection triplexes improved gene silencing in HeLa cells. The described protocols are also valuable for screening vector compositions and novel magnetic nanoparticle preparations to optimize siRNA transfection by magnetofection in every cell type.

How are Nucleic Acids Released in Cells from Cationic Lipid-Nucleic Acid Complexes?

AbstractCationic lipid-nucleic acid complexes are widely used to deliver oligonucleotides, RNA and DNA into cells. Although much has been learned about the structure and forces that hold the complex together, an understanding of the mechanism of release of the nucleic acids from the complex into cells has been lacking. Recent studies have shown that anionic liposomes with compositions similar to the cytoplasmic face of the endosomal membrane are potent agents for inducing the rapid release of oligonucleotides and DNA from cationic lipid-nucleic acid complexes. Based upon these results, we propose that after the cationic lipid/nucleic complex is internalized by endocytosis it destabilizes the endosomal membrane. This destabilization induces flip-flop of anionic lipids from the cytoplasmic facing monolayer, which laterally diffuse into the complex and form a charge neutral ion-pair with the cationic lipids. This results in displacement of the nucleic acid from the cationic lipid and subsequent release of th...

Magnetic Nanoparticles Enhance Adenovirus Transduction In Vitro and In Vivo

ABSTRACTPurposeAdenoviruses 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.MethodMagnetic nanoparticles formulations were complexed to a replication defective Adenovirus and were used to transduce cells both in vitro and in vivo. A new integrated magnetic procedure for cell sorting and genetic modification (i-MICST) was also investigated.ResultsMagnetic nanoparticles enhanced viral transduction efficiency and protein expression in a dose-dependent manner. They accelerated the transduction kinetics and allowed non-permissive cells infection. Magnetofection greatly improved adenovirus-mediated DNA delivery in vivo and provided a magnetic targeting. The i-MICST results established the efficiency of magnetic nanoparticles assisted viral transduction within cell sorting columns.ConclusionThe results showed that the combination of Magnetofection and Adenoviruses represents a promising strategy for gene therapy. Recently, a new integrated method to combine clinically approved magnetic cell isolation devices and genetic modification was developed. In this study, we validated that magnetic cell separation and adenoviral transduction can be accomplished in one reliable integrated and safe system.

CATIONIC LIPOSOMES AS AN OLIGONUCLEOTIDE CARRIER: MECHANISM OF ACTION

AbstractCationic liposomes are a useful in vitro but as yet unproven in vivo delivery system for oligonucleotides. An understanding of the mechanism of delivery mediated by cationic lipid/oligonucleotide complexes has been lacking. In this review, we describe recent results concerning several steps of the delivery process, including the formation of complexes, the intracellular distribution of the oligonucleotide and the lipid, as well as the uptake pathway and site of intracellular release. Cationic liposomes form a polyelectrolyte complex with the oligonucleotides, protect them from nuclease degradation, enhance their cellular uptake and improve the oligonucleotide potency. In the majority of cell types studied, cationic lipids deliver oligonucleotides into the cell predominately via an endocytotic pathway rather then by fusion with the plasma membrane. We propose that the oligonucleotide is released from the complex when anionic lipids from the cytoplasmic facing lipid monolayer of the cell flip into c...

Magnetically guided lentiviral‐mediated transduction of airway epithelial cells

Lentiviral (LV) vectors are able to only slowly and inefficiently transduce nondividing cells such as those of the airway epithelium. To address this issue, we have exploited the magnetofection technique in in vitro models of airway epithelium.

3D-fection: cell transfection within 3D scaffolds and hydrogels

BACKGROUND 3D matrices are widely used as cell growth supports in basic research, regenerative medicine or cell-based drug assays. In order to genetically manipulate cells cultured within 3D matrices, two novel non-viral transfection reagents allowing preparation of matrices for in situ cell transfection were evaluated. RESULTS Two lipidic formulations, 3D-Fect™ and 3D-FectIN™, were assessed for their ability to transfect cells cultured within 3D solid scaffolds and 3D hydrogels, respectively. These reagents showed good compatibility with the most widespread types of matrices and enabled transfection of a wide range of mammalian cells of various origins. Classical cell lines, primary cells and stem cells were thus genetically modified while colonizing their growth support. Importantly, this in situ strategy alleviated the need to manipulate cells before seeding them. CONCLUSION Results presented here demonstrated that 3D-Fect and 3D-FectIN reagents for 3D transfection are totally compatible with cells and do not impair matrix properties. 3D-Fect and 3D-FectIN, therefore, provide valuable tools for achieving localized and sustained transgene expression and should find versatile applications in fundamental research, regenerative medicine and cell-based drug assays.