Cholesterol domains in cationic lipid/DNA complexes improve transfection

The interaction between the cationic lipid DOTAP and cholesterol is examined in high cholesterol formulations by differential scanning calorimetry (DSC). Preparation of liposomes above 66 mol% cholesterol results in formulations that exhibit a calorimetric transition for anhydrous cholesterol at 38-40 °C. The enthalpy of this transition progressively increases at higher cholesterol contents, and is not detected below 66 mol% cholesterol. Furthermore, the enthalpy changes indicate that the composition of the non-domain forming portion containing DOTAP saturated with cholesterol is relatively constant above 66 mol% cholesterol. Greater transfection efficiency in the presence of 50% serum is observed at the formulations with high cholesterol contents where anhydrous cholesterol domains are detected by DSC. Although formulations possessing higher cholesterol exhibited a greater resistance to serum-induced aggregation, maintenance of small particle size does not appear to be responsible for the enhanced transfection efficiency. Additional studies quantifying albumin binding suggest that cholesterol domains in the lipid/DNA complex do not bind protein, and this may enable these moieties to enhance transfection by facilitating membrane fusion.     

The role of lipid charge density in the serum stability of cationic lipid/DNA complexes

To evaluate the role of lipid charge density in the serum stability of DOTAP-Chol/DNA complexes (lipoplexes), lipid-DNA interactions, extent of aggregation, supercoil content, and in vitro transfection efficiency of lipoplexes were investigated. In general, higher serum concentration destabilized, and increasing molar charge ratio of DOTAP to negatively charged phosphates in the DNA (DOTAP(+)/DNA(-)) stabilized lipoplexes in serum as assessed by the criteria used in this study. The increase of cholesterol content led to increased serum stability, and DOTAP:Chol (mol/mol 1:4)/DNA lipoplex with DOTAP(+)/DNA(-) ratio 4 was the most serum stable formulation of all the formulations examined, and maintained lipid-DNA interactions, did not aggregate and exhibited high in vitro transfection efficiency in 50% (v/v) serum. The increased stability of this formulation could not be explained by the decreased charge density of the lipid component. Furthermore, no single parameter examined in the study could be used to consistently predict the in vitro transfection efficiency of lipoplexes in serum. Surprisingly, no correlation between the maintenance of supercoiled DNA content and in vitro transfection efficiency was found in the study.     

Human serum albumin enhances DNA transfection by lipoplexes and confers resistance to inhibition by serum

Cationic liposome-DNA complexes ('lipoplexes') are used as gene delivery vehicles and may overcome some of the limitations of viral vectors for gene therapy applications. The interaction of highly positively charged lipoplexes with biological macromolecules in blood and tissues is one of the drawbacks of this system. We examined whether coating cationic liposomes with human serum albumin (HSA) could generate complexes that maintained transfection activity. The association of HSA with liposomes composed of 1, 2-dioleoyl-3-(trimethylammonium) propane and dioleoylphosphatidylethanolamine, and subsequent complexation with the plasmid pCMVluc greatly increased luciferase expression in epithelial and lymphocytic cell lines above that obtained with plain lipoplexes. The percentage of cells transfected also increased by an order of magnitude. The zeta potential of the ternary complexes was lower than that of the lipoplexes. Transfection activity by HSA-lipoplexes was not inhibited by up to 30% serum. The combined use of HSA and a pH-sensitive peptide resulted in significant gene expression in human primary macrophages. HSA-lipoplexes mediated significantly higher gene expression than plain lipoplexes or naked DNA in the lungs and spleen of mice. Our results indicate that negatively charged HSA-lipoplexes can facilitate efficient transfection of cultured cells, and that they may overcome some of the problems associated with the use of highly positively charged complexes for gene delivery in vivo.     

Herpes simplex virus-enhanced cationic lipid/DNA-mediated transfection

Liposome plasmid DNA complexes (lipoplexes) are often inefficient in mediating gene transfer and expression because of DNA degradation in lysosomal vesicles. Because herpes simplex virus (HSV) enters cells by fusion of the virus envelope with the plasma membranes, thereby overriding the endosomal pathway, HSV/lipoplex mixtures could be useful for improving gene transfer particularly when the mixture uses highly defective HSV particles that fail to express cytotoxic viral gene products. To evaluate this possibility, lipoplexes composed of cationic liposomes and lacZ reporter plasmids were compared for their ability to transduce cells in culture in the presence and absence of infectious HSV particles. The results showed that HSV increased the efficiency of cell transduction by approximately 4-100-fold compared with lipoplex vector alone, depending on the cell type targeted for gene delivery. The increased efficiency of transduction was virus dose dependent and required virus entry.     

Improvement of DNA transfection with cationic liposomes

The increasing use of cationic liposomes as vectors for DNA transfection of eukaryotic cells is due to its high efficiency and reproducibility. After the interaction of the DNA cationic-liposome complexes (DNA-CLC) with the plasma membrane, the entry into the cells delivers the DNA-CLC to the endosome-lysosome pathway where some of the DNA-CLC are degraded. The non-degraded DNA that escapes to the cytoplasm, still has to transverse the nuclear membrane to be transcribed and then translated. To improve the efficiency of the whole process, we can manipulate the DNA (sequences, promoters, enhancers, nuclear localisation signals, etc), the DNA-CLC (lipids) or the plasmatic, endosomal and/or nuclear cellular membranes (ultrasound, electroporation, Ca++, pH of the endosomes, mitosis, fusogenic peptides, nuclear localisation signals, etc). Most of these methods have been generally used individually but in combination, may greatly improve the efficiency and reproducibility of in vitro transfection. While much of this work remains yet to be done and present results further explored, the application of these efforts is essential to the future development of new gene therapy strategies.     

Synthetic cationic amphiphiles for liposome-mediated DNA transfection

The compounds with efficient DNA transfection ability into eukaryotic cells were searched from various synthetic amphiphiles which have cationic heads and long saturated hydrocarbon tails. The efficiency of amphiphiles in gene transfer was examined by the transient expression of cytochrome b5 from its cDNA in COS cells. Among various synthetic amphiphiles, including N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride which is commercially available lipid, O,O'-didodecyl-N-[p-(2-trimethylammonioethyloxy)benzoyl]-(L) -glutamate bromide was highest in efficiency. The optimum condition for the amount of the amphiphile and DNA, and the incubation time were established to be 7.5-15 micrograms/22 mm dish and 1-10 micrograms/22 mm dish, and 48-72 h, respectively.     

Counterion effects on transfection activity of cationic lipid emulsion

Cationic lipid emulsion systems consisting of 1,2-dioleoyl-sn-glycero-3-trimethyl-ammonium-propane (DOTAP) and plasmid DNA with various counterions in the lipid headgroups were prepared. The transfection activity of the cationic lipid emulsion systems was then investigatedin vitro andin vivo. The complex formation of plasmid DNA and lipid emulsion was affected by the counterions through charged headgroup repulsion and also by the salt concentration in the media. As such, the transfection activity of the DOTAP emulsion system can be controlled by changing the counterions.     

Cellular uptake of cationic lipid/DNA complexes by cultured myoblasts and myotubes

Several cationic lipids which are highly efficient for delivering genes in vitro do not increase gene delivery in vivo after an intramuscular injection. In order to elucidate the origin of this phenomenon, we have studied the cellular uptake and intracellular fate of cationic lipid/DNA complexes in vitro on myogenic mouse cells (myoblasts and myotubes) of the C2 cell line and of primary cultures. We used a cationic lipid with a spermine head group and its fluorescent analog, and a fluorescent plasmid obtained by nick-translation. In myoblasts, transgene expression was obtained and lipoplexes were internalized in cytoplasmic vesicles. In myotubes, no transgene expression could be detected and we observed an absence of lipoplex internalization. The in vitro uptake of cationic lipid was inversely correlated with the degree of fusion of C2 cell myotubes cultures.     

Neomycin improves cationic lipid-mediated transfection of DNA in human cells

Delivery of oligonucleotides has been a major impediment in the development of nucleic acid based drugs. In this report, we show that neomycin, an aminoglycoside antibiotic, when combined with a cationic lipid preparation such as DOTAP, enhances transfection efficiency of both reporter plasmids and oligonucleotides and results in a significant increase in transgene expression. The results described here open a new lead in ongoing efforts for oligonucleotide delivery.     

The role of the helper lipid dioleoylphosphatidylethanolamine (DOPE) for DNA transfection cooperating with a cationic lipid bearing ethylenediamine

Gene therapy is expected to treat various incurable diseases including viral infections, autoimmune disorders, and cancers. Cationic lipids (CL) have been used as carriers of therapeutic DNAs for gene therapy because they can form a complex with DNA and such a complex can be incorporated into cells and transport the bound DNA to cytosol. The CL/DNA complexes are called lipoplexes and categorized as a non-viral vector. Lipoplexes are often prepared by adding a neutral phospholipid dioleoylphosphatidylethanolamine (DOPE) to CL in order to enhance transfection. However, the role of DOPE is not fully understood. We synthesized a new CL having an ethylenediamine cationic head group, denoted by DA, and found that addition of DOPE to DA achieved a good efficiency, almost in the similar level of commonly used transfection reagent Lipofectamine 2000 (Invitrogen). The composition of DA:DOPE = 1:1 showed the highest efficiency. This lipoplex showed structural transition when pH was changed from 7 to 4, corresponding pH lowering in late endosome, while DOPE itself showed structural transition at more basic pH around 8. The present data showed that the DOPE/DA composition determines the structural transition pH and choosing a suitable pH, i.e., a suitable composition, is essential to increase the transfection efficiency.     

Insulin receptor activation and down-regulation by cationic lipid transfection reagents

Background  

Transfection agents comprised of cationic lipid preparations are widely used to transfect cell lines in culture with specific recombinant complementary DNA molecules. We have found that cells in culture are often resistant to stimulation with insulin subsequent to treatment with transfection agents such as LipofectAMINE 2000™ and FuGENE-6™. This is seen with a variety of different readouts, including insulin receptor signalling, glucose uptake into muscle cells, phosphorylation of protein kinase B and reporter gene activity in a variety of different cell types     

Characterization of DNA/lipid complexes by fluorescence resonance energy transfer

Fluorescence resonance energy transfer (FRET) is a potential method for the characterization of DNA-cationic lipid complexes (lipoplexes). In this work, we used FRET models assuming a multilamellar lipoplex arrangement. The application of these models allows the determination of the distance between the fluorescent intercalator on the DNA and a membrane dye on the lipid, and/or the evaluation of encapsulation efficiencies of this liposomal vehicle. The experiments were carried out in 1,2-dioleoyl-3-trimethylammonium-propane/pUC19 complexes with different charge ratios. We used 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and 2-(4,4-difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-PC) as membrane dyes, and ethidium bromide (EtBr) and BOBO-1 as DNA intercalators. In cationic complexes (charge ratios (+/-) >or= 2), we verified that BOBO-1 remains bound to DNA, and FRET occurs to the membrane dye. This was also confirmed by anisotropy and lifetime measurements. In complexes with all DNA bound to the lipid (charge ratio (+/-) = 4), we determined 27 A as the distance between the donor and acceptor planes (half the repeat distance for a multilamellar arrangement). In complexes with DNA unbound to the lipids (charge ratio (+/-) = 0.5 and 2), we calculated the encapsulation efficiencies. The presented FRET methodology is, to our knowledge, the first procedure allowing quantification of lipid-DNA contact.     

Systemic administration of cationic phosphonolipids/DNA complexes and the relationship between formulation and lung transfection efficiency

Performances of cationic lipid formulations for intravenous gene delivery to mouse lungs have been previously reported. We report in this study that cationic phosphonolipids, when appropriately formulated, can be good synthetic vectors for gene delivery to lung after intravenous administration. One of our reagents, GLB43, was capable of mediating a 500-fold higher expression in the lungs of mice than could be obtained with free pDNA alone (P=0.018). We demonstrate that the most important parameters for cationic phosphonolipid transfection activity after systemic administration are the chemical structure of the cationic phosphonolipid, the lipid to DNA charge ratio and the inclusion of co-lipid in the formulation. We report using a luciferase reporter gene that transfection activity in vivo 24 h after cationic phosphonolipid systemic administration could not be predicted from in vitro analysis. In contrast to in vitro studies, cationic phosphonolipids including the oleyl acyl chains (GLB43) were more effective than its analogue with the myristyl acyl chains (GLB73). Using pathological analysis of animal livers, we demonstrate that the toxicity level was correlated with the lipoplex formulation and the lipid to DNA ratio.     

Gene transfection into HeLa cells by vesicles containing cationic peptide lipid

Lipid vesicles are potentially useful as microcapsules for drug and/or gene delivery. We developed cationic lipid vesicles consisting mainly of sorbitan monooleate (Span 80) and cationic peptide lipid (CPL), and evaluated the CPL vesicles as gene transfection vectors. The optimum CPL concentration for gene transfection into HeLa cells was found to be 20 wt % of total lipid, and such CPL vesicles did not exhibit significant cytotoxicity. Co-culture of Poly-L-lysine and plasmids prior to making CPL vesicle-plasmid complexes was effective. Lipofection using LipofectAMINE was suppressed in 10% serum-supplemented medium. The transfection efficiency of 20 wt % CPL vesicles, however, was not affected by serum in the medium when plasmids were treated with poly-L-lysine.     

The counterion influence on cationic lipid-mediated transfection of plasmid DNA

A panel of DOTAP analogs was prepared by altering the anionic counterion that accompanies the trimethylammonium polar domain. The transfection of plasmid DNA into NIH3T3 cells and mouse lung was examined using the counterion analogs. The in vitro transfection activity decreased as follows: DOTAP · bisulfate > trifluoromethanesulfonate ∼ iodide ∼ bromide > dihydrogenphosphate ∼ chloride ∼ acetate > sulfate. A similar activity trend was observed in vivo.     

Physicochemical and transfection properties of cationic Hydroxyethylcellulose/DNA nanoparticles

In this study the physicochemical and transfection properties of cationic hydroxyethylcellulose/plasmid DNA (pDNA) nanoparticles were investigated and compared with the properties of DNA nanoparticles based on polyethylene imine (PEI), which is widely investigated as a gene carrier. The two types of cationic hydroxyethylcelluloses studied, polyquaternium-4 (PQ-4) and polyquaternium-10 (PQ-10), are already commonly used in cosmetic and topical drug delivery devices. Both PQ-4 and PQ-10 spontaneously interact with pDNA with the formation of nanoparticles approximately 200 nm in size. Gel electrophoresis and fluorescence dequenching experiments indicated that the interactions between pDNA and the cationic celluloses were stronger than those between pDNA and PEI. The cationic cellulose/pDNA nanoparticles transfected cells to a much lesser extent than the PEI-based pDNA nanoparticles. The low transfection property of the PQ-4/pDNA nanoparticles was attributed to their neutrally charged surface, which does not allow an optimal binding of PQ-4/pDNA nanoparticles to cellular membranes. Although the PQ-10/pDNA nanoparticles were positively charged and thus expected to be taken up by cells, they were also much less efficient in transfecting cells than were PEI/pDNA nanoparticles. Agents known to enhance the endosomal escape were not able to improve the transfection properties of PQ-10/pDNA nanoparticles, indicating that a poor endosomal escape is, most likely, not the major reason for the low transfection activity of PQ-10/pDNA nanoparticles. We hypothesized that the strong binding of pDNA to PQ-10 prohibits the release of pDNA from PQ-10 once the PQ-10/pDNA nanoparticles arrive in the cytosol of the cells. Tailoring the nature and extent of the cationic side chains on this type of cationic hydroxyethylcellulose may be promising to further enhance their DNA delivery properties.     

Linear DNA low efficiency transfection by liposome can be improved by the use of cationic lipid as charge neutralizer

A plasmid expressing the beta-galactosidase enzyme was used to transfect Vero cells in order to evaluate the efficiency of a liposome-mediated transfection by circular and linear DNA. The results obtained showed a low rate of transfection by linear DNA:liposome complexes. To explore whether the structure of the complexes was interfering with the transfection, atomic force microscopy (AFM) was used. It has confirmed the difference between the linear and circular condensates: whereas the circular DNA:liposome complexes presented compact spherical or cylindrical structures of about 100-800 nm, the linear DNA showed pearl necklace-like structures, with pearls varying from 250 to 400 nm. On the basis of the theory proposed by Kuhn et al. (1999), low concentrations of cationic amphihile were used to neutralize or reverse the DNA charge in order to improve the transfection efficiency of the linear DNA. Using this method, we were able to obtain the expression of the transgene without an associated toxicity observed with the linear DNA liposome delivery.     

Cholesterol enhances cationic liposome-mediated DNA transfection of human respiratory epithelial cells

Cationic liposome transfection reagents are useful for transferring polynucleotides into cells, and have been proposed for human pulmonary gene therapy. The effect of adding cholesterol to cationic lipid preparations has been tested by first formulating the cationic lipid N-[1-(2,3-dioleoyloxy)propyl-N-[1-(2-hydroxy)ethyl]-N,N-dimethyl ammonium iodide (DORI) with varying amounts of dioleoylphos-phatidylethanolamine (DOPE) and cholesterol. Cholesterol was found to enhance lipid-mediated transfection in both the respiratory epithelial cells and mouse fibroblasts. These findings will facilitate nucleic acid transfection of many cell types including differentiated epithelial cell monolayers, and therefore may be useful for examining gene regulation in various cell types and for developing pulmonary gene therapy.Abbreviations (DORI) N-[1-(2,3-dioleoyloxy)propyl]-N-[1-(2-hydroxy)ethyl]-N,N-dimethyl ammonium iodide - (DOPE) dioleoylphosphatidylethanolamine - (DOTMA) N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethyl ammonium chloride - (Mem) Eagle's modified essential medium - (DMEM) Dulbecco's Modified Eagle's Medium     

Deposition gene transfection using bioconjugates of DNA and thermoresponsive cationic homopolymer

A poly(N,N-dimethylaminoethylmethacrylate) (PDMAEMA) homopolymer with both thermoresponsive and cationic characteristics was applied to a vector for use in deposition transfection. PDMAEMA with a molecular weight of 2.5 × 10(5) g mol(-1) was synthesized by photoinduced radical polymerization. Polyplexes approximately 750 nm in size were formed by mixing PDMAEMA with luciferase-encoding plasmid DNA. The polyplexes had a lower critical solution temperature (LCST) of approximately 30 °C. In addition, they exhibited excellent adsorption and durability on a polystyrene surface, as confirmed by a surface chemical compositional analysis. When HeLa cells and primary cells were cultured on a substrate coated with the polyplexes, high transgene expression and cell viability of more than 90% were obtained at low charge ratios (PDMAEMA/plasmid DNA ratio) ranging from 2 to 8. In addition, transgene expression was sustained for over 2 weeks post-transfection whereas decreased expression was observed 5 days post-transfection when the conventional solution-mediated transfection method was used. Thus, high and sustained transgene expression as well as high cell viability can be realized by using small amounts of PDMAEMA as a deposition transfection material.