Cationic liposome-DNA Complexes Research Articles

Liposome-induced DNA compaction and reentrant condensation investigated by dielectric relaxation spectroscopy and dynamic light scattering techniques

Interaction of DNA with oppositely charged objects, such as multivalent ions, cationic surfactants, cationic liposomes, basic proteins, and alcohols, up to nano- or mesoscopic particles, gives rise to a very interesting and fascinating phenomenology, where the shape, size, and stability of the resulting aggregates depend on a delicate balance between different driving forces, mainly of electrostatic origin. We have studied the cationic liposome-DNA complexes during the whole complexation process, below, close to, and above the isoelectric condition, where the number of cationic lipids equals the number of phosphate groups on the DNA chain. We took advantage of the combined use of dynamic light scattering, laser Doppler electrophoretic mobility, and radio-wave dielectric relaxation measurements in order to characterize both the structural parameters (hydrodynamic radius) and the electrical parameters (charge and counterion concentration) of the resulting structures. These structures are fundamentally of two types, clusters of liposomes stuck together by DNA chains (cluster phase in low-density colloidal suspension) and coexisting DNA coils and DNA globules, according to the procedure through which interactions occur (liposomes in excess DNA solution or DNA in excess liposome suspension).

Transfection efficiency boost by designer multicomponent lipoplexes

Cationic liposome–DNA complexes (lipoplexes) have emerged as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. We investigated the correlation between structural evolution of multicomponent lipoplexes when interacting with cellular lipids, the extent of DNA release and the efficiency in transfecting mouse fibroblast (NIH 3T3), ovarian (CHO) and tumoral myofibroblast-like (A17) cell lines. We show, for the first time, that the transfection pattern increases monotonically with the number of lipid components and further demonstrate by means of synchrotron small angle X- ray scattering (SAXS) that structural changes of lipoplexes induced by cellular lipids correlate with the transfection efficiency. Specifically, inefficient lipoplexes either fused too rapidly upon interaction with anionic lipids or, alternatively, are found to be extremely resistant to solubilization. The most efficient lipoplex formulations exhibited an intermediate behaviour. The extent of DNA unbinding (measured by electrophoresis on agarose gel) correlates with structural evolution of the lipoplexes but DNA-release does not scale with the extent of transfection. The general meaning of our results is of broad interest in the field of non-viral gene delivery: rational adjusting of lipoplex composition to generate the proper interaction between lipoplexes and cellular lipids may be the most appropriate strategy in optimizing synthetic lipid transfection agents.

Systemic and Specific Delivery of Small Interfering RNAs to the Liver Mediated by Apolipoprotein A-I

Tissue-targeted delivery of small interfering RNA (siRNA) must be achieved before RNA interference (RNAi) technology can be used in practical therapeutic approaches. In this study, the potential of apolipoprotein A-I (apo A-I) for the systemic delivery of nucleic acids to the liver is demonstrated using real-time in vivo imaging. As a proof of concept, synthetic siRNAs against hepatitis B virus (HBV) were formulated into complexes of apo A-I and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (DTC-Apo) and injected intravenously (i.v.) into a mouse model carrying replicating HBV. We show that administration of these nanoparticles can significantly reduce viral protein expression by receptor-mediated endocytosis. The advantages of the apo A-I-mediated siRNA delivery method are its liver specificity, its effectiveness at low doses (< or = 2 mg/kg) in only a single treatment, and its persistent antiviral effect up to 8 days. The liver-targeted gene silencing was also shown by in vivo images, in which bioluminescent signals emitted from the liver were efficiently reduced after i.v. administration of luciferase-specific siRNA and DTC-Apo lipoplex. Thus, our unique approach to siRNA delivery creates a foundation for the development of a new class of promising therapeutics against hepatitis viruses or hepatocyte genes related to tumor growth.

DNA release from cationic liposome/DNA complexes by anionic lipids

The authors found that recently developed multicomponent cationic liposome DNA complexes (lipoplexes) exhibit higher transfection efficiency with respect to usually employed binary lipoplexes in NIH 3T3 and A17 cell lines. Interaction of lipoplexes with anionic liposomes (model of cellular membranes) was investigated by synchrotron small angle x-ray diffraction. The authors used one-dimensional DNA packing density to estimate the molar fraction of DNA released from lipoplexes by anionic lipids.

Dynamic light scattering as a screening tool to monitor the maturation time of cationic liposome–DNA complexes

Dynamic light scattering as a screening tool to monitor the maturation time of cationic liposome–DNA complexes

Gene delivery using cationic liposomes

The development of cationic liposomes for gene delivery has been ongoing for almost 20 years; however, despite extensive efforts to develop a successful therapeutic agent, there has been limited progress towards generating an effective pharmaceutical product. Since the introduction of N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride, an immense number of different cationic lipids have been synthesised and used to formulate cationic liposome–DNA complexes. Structural modification of the cationic lipids and the addition of components within the delivery system that can facilitate the fusion, cellular uptake and targeting of liposome–DNA complexes have all been used in a bid to enhance their transfection efficiency. Unfortunately, the overall impact of these improvements is still nominal, with the vast majority of clinical trials (∼ 85%) continuing to rely on more potent viral delivery of DNA despite their associated toxicity issues. Key characteristics of the most effective cationic liposomes for the delivery of plasmid DNA (from a consensus of the literature) is identified here and the problems of converting these attributes into an effective pharmaceutical product are outlined.

Cationic liposome–DNA complexes: from liquid crystal science to gene delivery applications

At present, there is an unprecedented level of interest in the properties and structures of complexes consisting of DNA mixed with oppositely charged cationic liposomes (CLs). The interest arises because the complexes mimic natural viruses as chemical carriers of DNA into cells in worldwide human gene therapy clinical trials. However, since our understanding of the mechanisms of action of CL-DNA complexes interacting with cells remains poor, significant additional insights and discoveries will be required before the development of efficient chemical carriers suitable for long-term therapeutic applications. Recent studies describe synchrotron X-ray diffraction, which has revealed the liquid crystalline nature of CL-DNA complexes, and three-dimensional laser-scanning confocal microscopy, which reveals CL-DNA pathways and interactions with cells. The importance of the liquid crystalline structures in biological function is revealed in the application of these modern techniques in combination with functional transfection efficiency measurements, which shows that the mechanism of gene release from complexes in the cell cytoplasm is dependent on their precise liquid crystalline nature and the physical and chemical parameters (for example, the membrane charge density) of the complexes. In [section sign] 5, we describe some recent new results aimed at developing bionanotube vectors for gene delivery.

Protective immunity against acute phleboviral infection elicited through immunostimulatory cationic liposome-DNA complexes

Cationic liposome-DNA complexes (CLDC) have been demonstrated to induce potent antitumor activities. The ability of these complexes to elicit protective immunity against viral infections has not been fully explored. Here we report findings on the use of CLDC as an antiviral agent in a mouse model of acute phleboviral (Punta Toro virus) disease. CLDC treatment of mice challenged with Punta Toro virus (PTV) resulted in dramatic increases in survival and reduced viral burden and other parameters indicative of protection against disease. CLDC were effective when administered by intraperitoneal and intravenous routes and elicited protective immunity when given within 1 day of virus challenge. Treatments administered 36 h or longer after challenge, however, were not effective in preventing mortality or disease. CLDC treatment induced release of a number of potential antiviral cytokines including IFN-γ, IL-12, and IFN-α. Taken together, our findings indicate that non-specific immunotherapy with CLDC appears to be an effective treatment for blocking PTV-induced disease and suggests that further exploration in other viral disease models may be warranted.

60. Radiation Improves Local Delivery of a Virus-Mimicking Nanostructure Complex in Tumors

Efficiency and specificity of gene delivery to tumors are major concerns in cancer gene therapy. We have previously used regional ionizing radiation to improve adenovirus uptake and to enhance gene expression in intrahepatic tumors (Zhang, M, et al. Mol Ther. 2003, 8:21-28; Qian, J, et al. Cancer Res. 2005, 65:5493-5497). However, non-specific gene expression and cytotoxicity in normal liver is still inevitable. Here, we explore the potentials of radiation in improving gene delivery by a virus-mimicking nanostructure complex, transferrin (Tf)-cationic liposome-DNA complex (Tf- lipoplex) (Liang, Xu, et al.

Lipoplex and LPD Nanoparticles for In Vivo Gene Delivery

INTRODUCTION Lipoplex (cationic liposome-DNA complex) is formed via electrostatic interaction of anionic nucleic acids with cationic liposomes. A thin film of lipids is dried on the bottom of a glass tube and rehydrated in an aqueous solution. The resulting liposome suspension is passed through polycarbonate filters of desired pore size. This protocol also describes the preparation, physical properties, and biological activity of liposome-polycation-DNA (LPD) nanoparticles. The LPD nanoparticles contain a highly condensed DNA core surrounded by lipid bilayers with an average size of ~100 nm. The nanoparticle complex is injected into mice, and expression of the transfected DNA is monitored with an appropriate assay.

In Vivo Studies of Dialkynoyl Analogues of DOTAP Demonstrate Improved Gene Transfer Efficiency of Cationic Liposomes in Mouse Lung

A novel set of dialkynoyl analogues of the cationic, gene delivery lipid DOTAP (1) was synthesized. Structure-activity studies demonstrate that replacement of the cis-double bonds of DOTAP with triple bonds in varying positions alters both the physical properties of the resultant cationic liposome-DNA complexes and their biological functionalities, both in vitro and in vivo. Particularly, in vivo studies demonstrate that pDNA transfection of mouse lung endothelial cells with lead analogue DS(14-yne)TAP (4):cholesterol lipoplexes exhibits double the transfection level with less associated toxicity relative to the well-established DOTAP:cholesterol system. In fact, 4:cholesterol delivers up to 3 times the dose of pDNA in mice than can be tolerated by DOTAP, leading to nearly 3 times greater marker-gene expression. X-ray diffraction studies suggest that lipoplexes containing analogue 4 display increased stability at physiological temperatures. Our results thus suggest that analogue 4 is a potentially strong candidate for the gene therapy of lung tumors.

Type I Interferons potently suppress gene expression following gene delivery using liposome–DNA complexes

Gene delivery by intravenous injection of cationic liposome-DNA complexes (LDC) can generate efficient transgene expression in the lungs and other organs, but the duration of expression is typically short. Previous studies have suggested a major role for interferon-gamma (IFN-gamma) and TNF in this process. However, plasmid DNA is also capable of eliciting production of type I IFNs. Therefore, we assessed the ability of LDC to elicit production of type I IFNs in vivo and assessed the effects of type I IFNs on suppression of transgene expression following in vivo gene delivery with LDC. Injection of LDC was found to induce production of high levels of both IFN-alpha and IFN-beta in vivo. Moreover, the levels of transgene expression following in vivo gene delivery were markedly increased in mice lacking functional type I IFN receptor genes, compared to wild-type mice or mice lacking IFN-gamma or TNF receptors. Addition of recombinant IFN-alpha and IFN-beta inhibited transgene expression by in vitro-transfected endothelial cells, and incubation of macrophages with LDC in vitro triggered production of both IFN-alpha and IFN-beta. Therefore, type I IFNs appear to play a key role in suppressing transgene expression in vivo following systemic nonviral gene delivery using LDC.

590. Efficient, Serum-Resistant Transfection of Murine Squamous Cell Carcinoma Cells by Metafectene and GeneJammer: Application to HSV-tk/Ganciclovir Gene Therapy

Current treatments of head and neck squamous cell carcinoma (HNSCC) are largely unsatisfactory, and the five-year survival rate has not improved over the last two decades. The genetic approach to the treatment of HNSCC is based on the hypothesis that expression of therapeutic genes in target cells will cause a cytotoxic effect or mediate apoptosis. Oral cancer is a particularly appropriate target for gene therapy, since direct injection of genes into most primary and recurrent lesions is possible. Although generally efficient in transducing cells, viral vectors suffer from problems of immunogenicity, toxicity, limits in the size of exogenous DNA, and the risk of inducing tumorigenic mutations and generating active viral particles through recombination. Synthetic cationic liposome-DNA (lipoplexes) or cationic polymer-DNA complexes (polyplexes) constitute a promising alternative to the use of viral vectors and provide a simple means of transferring DNA into target cells. The inhibitory effect of serum on transfection mediated by non-viral vectors represents a serious limitation for their use in vivo. The presence of serum proteins, however, is unavoidable in vivo, and is advantageous in vitro because it increases cell viability and reduces transfection-associated cytotoxicity. In this study, we examined the effect of high concentrations of fetal bovine serum (FBS) on the delivery of luciferase, |[szlig]|-galactosidase and Herpes Simplex Virus thymidine kinase (HSV-tk) gene to murine squamous cell carcinoma cells, SCC-7, by the polycationic liposome, Metafectene, and the polyamine reagent, GeneJammer. Transfection was optimized using a luciferase-expressing plasmid. The level of gene expression in cell lysates was evaluated by measuring enzyme activity after a 48-h incubation. The optimal ratios for delivering the luciferase gene were 2 |[mu]|l Metafectene:1 |[mu]|g DNA and 3 |[mu]|l GeneJammer:0.5 |[mu]|g DNA. When transfection was performed in the presence of 20-60% FBS, both Metafectene- and GeneJammer-mediated luciferase expression were reduced only by about 20%. SCC-7 cells were also transfected with the |[szlig]|-galactosidase expressing plasmid in the absence or presence of 60% FBS. Approximately 60-70% of the cells were positive for |[szlig]|-gal staining. The expression of |[szlig]|-galactosidase was essentially not affected by serum. Cells transfected with the HSV-tk plasmid were incubated in the absence or the presence of ganciclovir (GCV; 20 |[mu]|g/ml) for the indicated periods of time. The Alamar Blue assay was used to determine GCV-mediated cytotoxicity. Mock-transfected cells served as controls. The delivery of the HSV-tk gene by Metafectene in the absence and presence of 60% FBS, followed by GCV treatment for 3 days, resulted in 68% and 36% cytotoxicity, respectively. With GeneJammer, transfection in 0% and 60% FBS resulted in 60% cytotoxicity. After 6 days, 90-100% cytotoxicity was observed in the presence of 0% or 60% FBS. Our observations suggest that both GeneJammer and Metafectene may be useful for the gene therapy of OSCC in animal models.

Use of immunostimulatory liposome‐nucleic acid complexes in allergen‐specific immunotherapy of dogs with refractory atopic dermatitis – a pilot study

This pilot study evaluated the effects of immunostimulatory liposome-plasmid-DNA complexes combined with specific allergens for immunotherapy of refractory canine atopic dermatitis. Seven dogs with previously diagnosed atopic dermatitis and unsatisfactory response to at least 12 months of conventional allergen-specific immunotherapy underwent a series of six intradermal injections (weeks 0, 2, 4, 6, 10 and 14), with patient-specific allergen extracts contained in cationic liposome-DNA complexes. Degree of pruritus was assessed on a visual analogue scale. Lesion scores were determined using the Canine Atopic Dermatitis Extent and Severity Index (CADESI) and medication usage was recorded at weeks 0 and 14. Canine cytokine mRNA expression in peripheral blood mononuclear cells collected prior to treatment and at the completion of the study was determined for IFN-gamma, IL-4, TNF and IL-10 genes using quantitative reverse transcription competitive polymerase chain reaction. Repeated intradermal injections of specific allergens incorporated into liposome-nucleic acid complexes were well tolerated in all seven dogs. There was a significant improvement in pruritus scores (P = 0.0277) and concurrent significant decrease in IL-4 production (P = 0.0428) at the completion of the trial compared to pretreatment values. Medication scores, CADESI and production of other cytokines did not change significantly with treatment. These early results suggest that antigen-specific immunotherapy using a novel liposome-nucleic acid complex vaccine may be beneficial for treatment of established atopic dermatitis in dogs using lower antigen doses. Further investigations in larger numbers of dogs with earlier stages of disease are warranted.

New multivalent cationic lipids reveal bell curve for transfection efficiency versus membrane charge density: lipid–DNA complexes for gene delivery

Gene carriers based on lipids or polymers-rather than on engineered viruses-constitute the latest technique for delivering genes into cells for gene therapy. Cationic liposome-DNA (CL-DNA) complexes have emerged as leading nonviral vectors in worldwide gene therapy clinical trials. To arrive at therapeutic dosages, however, their efficiency requires substantial further improvement. Newly synthesized multivalent lipids (MVLs) enable control of headgroup charge and size. Complexes comprised of MVLs and DNA have been characterized by X-ray diffraction and ethidium bromide displacement assays. Their transfection efficiency (TE) in L-cells was measured with a luciferase assay. Plots of TE versus the membrane charge density (sigmaM, average charge/unit area of membrane) for the MVLs and monovalent 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP) merge onto a universal, bell-shaped curve. This bell curve leads to the identification of three distinct regimes, related to interactions between complexes and cells: at low sigmaM, TE increases with increasing sigmaM; at intermediate sigmaM, TE exhibits saturated behavior; and unexpectedly, at high sigmaM, TE decreases with increasing sigmaM. Complexes with low sigmaM remain trapped in the endosome. In the high sigmaM regime, accessible for the first time with the new MVLs, complexes escape by overcoming a kinetic barrier to fusion with the endosomal membrane (activated fusion), yet they exhibit a reduced level of efficiency, presumably due to the inability of the DNA to dissociate from the highly charged membranes in the cytosol. The intermediate, optimal regime reflects a compromise between the opposing demands on sigmaM for endosomal escape and dissociation in the cytosol.

Membrane-permeant, DNA-binding agents alter intracellular trafficking and increase the transfection efficiency of complexed plasmid DNA

Nuclear delivery of extracellular DNA by nonviral vectors is inhibited by a series of cell membrane and compartmental barriers. Certain cationic amphiphiles that partition through cellular membranes to bind genomic DNA can enhance nuclear delivery of plasmid DNA. Specifically, delivering plasmid DNA complexed to the DNA-binding dye Hoechst 33258 produces cellular transfection levels similar to those achieved by cationic liposome:DNA complexes (CLDC), with less toxicity. Incorporating Hoechst into CLDC or polyethyleneimine:DNA complexes significantly increased reporter gene expression, as well as the percentage of cells transfected. Hoechst:CLDC significantly improved transfection of nondividing cells and efficiently transfected cells in the presence of anionic molecules that block cellular uptake of and transfection by CLDC alone. Hoechst:CLDC also increased gene expression in mouse tissues following intravenous delivery. Delivery of fluorescently labeled plasmid DNA via Hoechst altered its intracellular trafficking by both minimizing lysosomal sequestration and accelerating delivery into the nucleus. Agents such as Hoechst constitute a novel class of nonviral carriers that can confer their membrane-permeant properties on complexed DNA, thus redirecting its intracellular trafficking. In addition, binding of Hoechst 33258 to specific chromosomal DNA target sequences and its ability to modulate transcription may further enhance the expression of delivered genes.

Increased Resistance of DNA Lipoplexes to Protein Binding In Vitro by Surface-modification with a Multivalent Hydrophilic Polymer

The potential of cationic liposomes as DNA delivery vehicles for gene therapy is significantly limited by their instability upon systemic administration. Their strong positive charge induces non-specific binding of serum proteins and subsequent clearance from the circulation. This work investigates the ability of the multivalent reactive copolymer of poly[N-(2-hydroxypropopyl) methacrylamide], pHPMA (MA–GG–ONp) to shield lipoplexes from non-specific protein binding. The polymer was found to react with cationic liposome–DNA complexes (lipoplexes) in both an electrostatic and covalent manner to form an external polymer coat. Polymer coating resulted in an increase in lipoplex diameter (by up to 100 nm) that was proportional to the amount of polymer used, with a concomitant reduction in surface charge from strongly positive to neutral (from 30 to 0 mV). Polymer-coated lipoplexes exhibited increased stability to protein binding compared to untreated liposomes and reduced non-specific uptake into cells in vitro.

Adsorption and Desorption Behaviors of Cationic Liposome-DNA Complexes upon Lipofection in Inside and Outside Biomembrane Models Using a Dynamic Quasi-Elastic Laser Scattering Method

The dynamic behaviors of cationic liposome-DNA complexes in inside and outside biomembrane models upon lipofection were investigated using the time-resolved quasi-elastic laser scattering (QELS) method. Inside and outside biomembrane models with similar phospholipid compositions to those in living cells were formed at a tetradecane/phosphate buffered saline (TD/PBS) interface. Cationic liposome-DNA complexes were injected into the buffer subphase, and their adsorption/desorption behaviors at the biomembrane models were monitored through changes in the interfacial tension. We found that the adsorption rate of the complexes increased 2.6 times more in the outside model than in the inside one. The adsorption rate of DNA alone did not show a remarkable difference from one side to the other; however, the adsorption rate of the cationic liposome alone showed a similar tendency to that of the liposome-DNA complex. These results indicated that the difference in lipid composition induced a different dynamic behavior of exogenous biomolecules and that the cationic liposomes played an important role in the faster incorporation of DNA into cells upon lipofection.

Cationic liposomes for gene delivery: novel cationic lipids and enhancement by proteins and peptides.

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, the barriers to efficient gene expression, and novel cationic lipids used for transfection. We also describe 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 to emulate in vivo conditions.

1143. Biological Efficacy and Toxicity of Naked Plasmid DNA and Cationic Liposome-DNA Complexes in Ovine Lung

1143. Biological Efficacy and Toxicity of Naked Plasmid DNA and Cationic Liposome-DNA Complexes in Ovine Lung