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Advances in RNA technology during the past two decades have led to the construction of replication-competent RNA, termed replicons, RepRNA, or self-amplifying mRNA, with high potential for vaccine applications. Cytosolic delivery is essential for their translation and self-replication, without infectious progeny generation, providing high levels of antigen expression for inducing humoral and cellular immunity. Synthetic nanoparticle-based delivery vehicles can both protect the RNA molecules and facilitate targeting of dendritic cells—critical for immune defense development. Several cationic lipids were assessed, with RepRNA generated from classical swine fever virus encoding nucleoprotein genes of influenza A virus. The non-cytopathogenic nature of the RNA allowed targeting to dendritic cells without destroying the cells—important for prolonged antigen production and presentation. Certain lipids were more effective at delivery and at promoting translation of RepRNA than others. Selection of particular lipids provided delivery to dendritic cells that resulted in translation, demonstrating that delivery efficiency could not guarantee translation. The observed translation in vitro was reproduced in vivo by inducing immune responses against the encoded influenza virus antigens. Cationic lipid-mediated delivery shows potential for promoting RepRNA vaccine delivery to dendritic cells, particularly when combined with additional delivery elements. Keywords: replicon, RepRNA, self-amplifying, cationic lipids, dendritic cells, nanoparticle delivery, influenza vaccines, humoral immunity, cellular immunity

International audience; Background: Gene delivery within the central nervous system at postnatal age is one of the most challenging tasks in neuroscience and currently only a few effective methods are available. Comparison with existing methods: For postnatal central nervous system cells, viral approaches are commonly used for genetic engineering but they face several biosafety requirements for production and use making them less accessible to the community. Conversely, lipid-based methods are widely used in cell culture but face limitation in vivo mainly due to the inflammatory responses they induce. To this aspect, the use of a transgenic mouse line can represent a credible answer to the community working on rat models still requires an effective and successful solution to circumvent these difficulties. New method: We describe a new polymer-based gene delivery system allowing persistent and robust in vivo transfection with low DNA amount, reduced inflammation and high diffusion. The expression profile along the brain, the stability, the diffusion of the DNA together with the quantity of cells transfected were evaluated through in vivo approaches. Results: With a single low-volume injection, we targeted different cell types within the rat brain. We measured the diffusion rate ranging from 1 to 5 mm based on the injected volume, in the three-dimensions axis. Finally, we modified brain susceptibility to epileptic seizures using a specific knock-down of the neuronal specific potassium chloride transporter 2. Conclusions: This safe and easy system opens perspectives for non viral gene delivery in the rat brain with perspectives to study brain function in vivo.

Microglia, the resident phagocytic cells of the brain, have recently been the subject of intense investigation given their role in pathology and normal brain physiology. In general, phagocytic cells are hard to transfect with plasmid DNA. The BV2 cell line is a murine cell line of microglial origin which is often used to study this cell type in vitro. Unfortunately, this microglial cell line is, like other phagocytic cells, resistant to transfection.Magnetofection is a well-established transfection method that combines DNA with magnetic particles which, under the influence of a magnetic field, ensures a high concentration of particles in proximity of cultured cells. Only recently, Glial-Mag was specifically developed for efficient transfection of microglia and microglial cell lines.Magnetofection with Glial-Mag yielded a transfection efficiency of 34.95% in BV2 cells, 24h after transfection with an eGFP-expressing plasmid. Efficient gene delivery caused a modest and short-lived cell activation (as measured by IL6 secretion) that ceased by 24h after transfection.Here we show that Glial-Mag magnetofection of BV2 cells yielded a significantly higher transfection efficiency (34.95%) compared to other chemical transfection methods including calcium-phoshate precipication (0.34%), X-tremeGENE (3.30%) and Lipofectamine 2000 (12.51%).Transfection of BV2 cells using Glial-Mag magnetofection is superior compared to other chemical transfection methods and could be considered as the method of choice to chemically transfect microglial cell lines.

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.

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., Graphical abstract

An approach is described for making transcriptionally active PCR (TAP) fragments that were used directly in in vitro and in vivo expression experiments. TAP fragments encoding reporter genes were amplified in 1 day using typical PCR methodology and were expressed in cultured cells and in mice at levels comparable with a widely used cytomegalovirus promoter-based plasmid expression vector. Following intramuscular injection, a TAP fragment encoding hepatitis B surface antigen (HBsAg) induced anti-HBsAg antibody titers comparable with those induced by supercoiled plasmid encoding the same antigen. Epitope-tagged TAP fragments were generated and transfected into cells for rapid, high throughput immunocytochemical analysis of the tagged gene products. TAP fragments were also transferred directly into expression vectors by in vivo homologous recombination without conventional cloning, affording a high throughput cloning approach that does not require restriction enzyme digestion, ligations, or thymidine adenine complementation cloning. The methodology has been adapted to a robotic work station enabling the high throughput generation of transcriptionally active genes at the rate of more than 400 different genes per day. This technology offers a practical approach to directly utilize genome sequence data to generate functional proteomes.

There are many very effective methods to introduce transcriptionally active DNA into viable cells but approaches to deliver functional proteins are limited. We have developed a lipid-mediated delivery system that can deliver functional proteins or other bioactive molecules into living cells. This delivery system is composed of a new trifluoroacetylated lipopolyamine (TFA-DODAPL) and dioleoyl phosphatidylethanolamine (DOPE). This cationic formulation successfully delivered antibodies, dextran sulfates, phycobiliproteins, albumin, and enzymes (beta-galactosidase and proteases) into the cytoplasm of numerous adherent and suspension cells. Two systems were used to demonstrate that the proteins were delivered in a functionally active form. First, intracellular beta-galactosidase activity was clearly demonstrated within X-gal-stained cells after TFA-DODAPL:DOPE-mediated delivery of the enzyme. Second, the delivery system mediated delivery of several caspases (caspase 3, caspase 8, and granzyme B) into cultured cell lines and primary cells triggering apoptosis. Mechanistic studies showed that up to 100% of the protein mixed with the lipid formulation was captured into a lipid-protein complex, and up to 50% of the input protein associated with cells. This lipid-mediated transport system makes protein delivery into cultured cells as convenient, effective, and reliable as DNA transfection.

Five analytical assays are described that provide a platform for systematically evaluating the effect of formulation variables on the physical properties of cationic lipid-DNA complexes (lipoplexes). The assays are for (i) lipid recovery, (ii) total DNA, (iii) free DNA, (iv) nuclease sensitivity, and (v) physical stability by filtration. Lipid recovery was determined by measuring lipid primary amino groups labeled with the fluorescamine reagent in the presence of the detergent Zwittergent. Zwittergent was effective at disrupting lipoplexes, making the primary amine accessible to the fluorescamine reagent. Total DNA was determined with the PicoGreen reagent, also in the presence of Zwittergent. The PicoGreen assay in the absence of Zwittergent gave the percentage of the total DNA that was not complexed with cationic lipid. The results of this assay for free DNA agreed well with the amount of DNA that could be separated from complexes by centrifugation as well as with the amount of DNA that was accessible to DNase I digestion. Monitoring the lipid and DNA recoveries after filtration through polycarbonate membranes provided a quantitative method for assessing changes in lipoplex physical characteristics. Together, these assays provide a convenient high-throughput approach to assess physical properties of lipoplexes, allowing systematic evaluation of different formulations.

Cationic 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...

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.

Cationic 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...

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.

Protocol Summary Primary neural stem cells (NSCs) can be cultivated and differentiated in vitro but are difficult to transfect using conventional methods. We describe a simple and rapid magnetofection-based method suitable for the lab bench as well as for high-throughput projects. Our method yields high transfection efficiency and can be used for deciphering the genetic control of neural cell differentiation.

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.

Encapsulation of oligonucleotides in antibody-targeted liposomes (immunoliposomes) which bind to target cells permits intracellular delivery of the oligonucleotides. This approach circumvents problems of extracellular degradation by nucleases and poor membrane permeability which free phosphodiester oligonucleotides are subject to, but leaves unresolved the inefficiency of encapsulation of oligonucleotides in liposomes. We have coupled oligonucleotides to cholesterol via a reversible disulfide bond. This modification of oligonucleotides improved their association with immunoliposomes by a factor of about 10 in comparison to unmodified oligonucleotides. The presence of cholesteryl-modified oligonucleotides incorporated in the bilayer of liposomes did not interfere with the coupling of the targeting protein to the liposome surface. Free or cholesterol coupled oligonucleotides associated with liposomes and directed against the tat gene of HIV-1 were tested for inhibition of HIV-1 proliferation in acutely infected cells. We demonstrate that the cholesteryl-modified as well as unmodified oligonucleotides acquire the target specificity of the antibody on the liposome. Their antiviral activity when delivered into cells is sequence-specific. The activity of these modified or unmodified oligonucleotides to inhibit the replication of HIV was the same on an equimolar basis (EC50 around 0.1 microM). Cholesterol coupled oligonucleotides thus offer increased liposome association without loss of antiviral activity.

Experiments performed in our laboratory or in collaboration with other groups demonstrating the delivery to cells of mono-, oligo- and polynucleotides from antibody-targeted small liposomes are reviewed. Biologically-active molecules delivered into cells via these liposomes include: phosphorylated derivatives of dideoxyuridine, which have activity against the human immunodeficiency virus; the oligonucleotide (2'-5') (A)n and various analogues, which have anti-viral and antiproliferative activity; and antisense phosphodiester and phosphorothioate oligodeoxynucleotides, which may have both gene-specific and nonspecific effects. Polynucleotides include: the RNA duplex poly (rI:rC), and related molecules, which are inducers of interferon and other cytokines; long RNA antisense molecules and plasmids. Advantages for delivery by liposomes, as compared to use of the same molecules free in solution include: protection against degradation, reduction of toxicity, improved pharmacokinetics and the possibilit...

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. Magnetic 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. Magnetic 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. The 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.

Antisense oligonucleotides inhibit HIV replication in vitro, but their activity is limited by their sensitivity to nucleases and low cellular uptake. To see whether these problems could be circumvented, we compared effects of HIV-1 rev and tat gene-specific antisense phosphodiester or phosphorothioate oligonucleotides, either free in solution or encapsulated in antibody-targeted liposomes (immunoliposomes), on acutely or chronically infected cells. Phosphodiester antisense oligonucleotides were inactive in their free form in acutely and chronically infected cells (up to a concentration of 50 microM). When encapsulated in immunoliposomes directed to HLA class I molecules expressed by targeted cells, they inhibited viral replication (at a concentration of 0.5 microM) in acutely infected cells in a sequence-specific manner. The same phosphodiester antisense oligonucleotides in liposomes had no antiviral activity in chronically infected cells. In acutely infected cells, phosphorothioate oligonucleotides free in solution inhibited the replication of HIV without sequence specificity and had slightly greater activity, also nonspecific, when encapsulated in liposomes. Phosphorothioate antisense (anti-rev) oligonucleotides specifically blocked HIV replication in chronically infected cells. When encapsulated in targeted liposomes the efficiency of inhibition for these cells was increased by at least 60-fold relative to the same oligonucleotide free in solution.

Primary neural stem cells (NSCs) can be cultivated and differentiated in vitro but are difficult to transfect using conventional methods. We describe a simple and rapid magnetofection-based method suitable for the lab bench as well as for high-throughput projects. Our method yields high transfection efficiency and can be used for deciphering the genetic control of neural cell differentiation.

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.Magnetofectins were formed by noncovalent interaction between LV particles and polycation-coated iron oxide nanoparticles. Efficiency of LV-mediated transduction (as evaluated through green fluorescent protein (GFP) expression by cytofluorimetric analysis) was measured in bronchial epithelial cells in the presence or absence of a magnetic field. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) release; cell monolayer integrity by measurement of transepithelial resistance (TER) and evaluation of correct zonula occludens-1 (ZO-1) localization at tight junctions (TJs) by immunofluorescence and confocal microscopy.In nonpolarized cells, magnetofectins enhanced LV-mediated transduction at multiplicity of infection (MOI) of 50 up to 3.9-fold upon a 24-h incubation, to levels that approached those achieved at MOI of 200 for LV alone, in the presence or absence of the magnetic field. Magnetofection significantly increased the percentage of transduced cells up to 186-fold already after 15 min of incubation. In polarized cells, magnetofection increased GFP+ cells up to 24-fold compared to LV alone. Magnetofection did not enhance LDH release and slightly altered TER but not ZO-1 localization at the TJs.We conclude that magnetofection can facilitate in vitro LV-mediated transduction of airway epithelial cells, in the absence of overt cytotoxicity and maintaining epithelial integrity, by lowering the necessary vector dose and reducing the incubation time required to achieve efficient transduction.

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.

A stable single vial lipoplex formulation has been developed that can be stored frozen without losing either biological activity or physical stability. This formulation was identified by systematically controlling several formulation variables and without introducing either stabilizers or surfactants. Analytical assays were used to unambiguously characterize the formulations. The critical formulation parameters were: (1) the size of the cationic liposomes; (2) the rate and method of DNA and cationic liposome mixing; and (3) the ionic strength of the suspending vehicle. The mixing conditions were precisely controlled by using a novel, specially designed continuous flow pumping system in which the DNA and liposome solutions were mixed at the junction of a T-connector. Homogenous cationic liposome preparations were prepared by extrusion in two different size ranges of either 400 or 100 nm. Extruded liposomes produced more monodisperse and physically stable lipoplex formulations than unextruded liposomes, but the formulations prepared with 100 nm liposomes were less active in in vitro transfection assays than either the 400 nm or unextruded liposomes. Low ionic strength and 5% sorbitol were required for the lipoplex formulations to survive freezing and thawing. A frozen lipoplex formulation stored for more than a year maintained its biological activity. These results have broad implications for the pharmaceutical development of lipoplex formulations for gene delivery.

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.

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.

Phosphodiester and phosphorothioate oligonucleotides in alpha and beta configurations directed against the initiation codon region of the HIV-1 rev gene were evaluated for their ability to inhibit HIV-1 replication in acutely and chronically infected human CEM cells. Encapsulation in antibody-targeted liposomes (immunoliposomes) permitted intracellular delivery and distinction between oligonucleotide-mediated inhibition of viral entry and intracellular effects on viral RNA. Our results are consistent with four mechanisms of antiviral activity for these antisense oligonucleotides: (i) interference with virus-mediated cell fusion by free but not liposome-encapsulated phosphorothioate oligonucleotides of any sequence; (ii) interference with reverse transcription in a sequence non-specific manner by phosphorothioate oligonucleotides in alpha and beta configurations; (iii) interference with viral reverse transcription in a sequence-specific and RNase-H-independent manner by alpha and beta phosphodiester oligonucleotides; (iv) interference with viral mRNA in a sequence-specific and RNase-H-dependent manner by beta-phosphorothioate oligonucleotides.

Among the 2′,3′-dideoxynucleoside 5′-triphosphates containing a physiological base, 2′,3′-dideoxyuridine 5′-triphosphate (ddUTP) has been reported to be among the most powerful inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (RT) in cell-free systems. However, in contrast to other dideoxynucleosides, 2′,3′-dideoxyuridine (ddU) is inactive in treatment of HIV-infected cells in culture, since it is a poor substrate for cellular nucleoside kinases. This problem cannot be overcome by the use of phosphorylated ddU because such compounds are unable to cross cell membranes. To promote entry and thus bypass the limiting steps of intracellular phosphorylation, we have encapsulated mono- and tri-phosphorylated ddU in liposomes coupled to monoclonal antibodies (immunoliposomes). We investigated antiviral effects in two human T cell lines (MT-4, CEM). We observed that ddU nucleotides remain phosphorylated for several weeks after encapsulation in immunoliposomes, and potent antiviral activity is obtained when these drugs are delivered into infected cells by cell-specific antibodies (ED50 ⩽1 μM on CEM). In contrast, no inhibition was observed with non-targeted liposomes containing phosphorylated ddU, or with empty liposomes, whether targeted or not.

We have examined two T lymphocyte cell surface molecules, CD4 and CD7, as targets for specific delivery of drugs from antibody-directed liposomes. The efficiency of uptake by peripheral lymphocytes, thymocytes, and two CEM sublines (CEM.MRS and CEM-T4) of anti-CD4 and anti-CD7 liposomes containing methotrexate was evaluated by the methotrexate-mediated inhibition of the incorporation of d -[ 3 H]Urd into DNA. This was compared with similar liposomes targeted to MHC-encoded HLA class I molecules, which are known to be efficiently taken up by T cells. Despite the lower expression of CD7 molecules relative to HLA class I on most cell lines, CD7 was shown to be a good target for drug delivery. The results of an internalization study using radiolabeled Protein A showed that a higher proportion of CD7 molecules was internalized than HLA class I molecules. CD4-targeted liposomes, in contrast, were relatively ineffective for drug delivery for lymphoid cells, and only partially inhibited CEM-T4 cells. The lack of toxicity correlated with poor internalization of the target molecule on most cell lines. The drug effect of anti-CD4 liposomes was more pronounced on HeLa-T4, which is an epithelial cell line transfected with the CD4 gene. In contrast to lymphoid cells, these cells efficiently internalized CD4 molecules. PMA is known to down-regulate surface expression of CD4 molecules on various T cells. Internalization of CD4 was induced by PMA, but PMA failed to induce cytotoxicity of CD4-targeted liposomes for CEM.MRS. The internalized drug was probably degraded rapidly because internalized anti-CD4 antibody-bound Protein A was degraded very rapidly.