Using disaccharides to enhance in vitro and in vivo transgene expression mediated by a lipid-based gene delivery system

BACKGROUND: Lipid-based vectors have been widely applied to in vivo and in vitro gene delivery. Disaccharides can effectively stabilize lipid membranes. This study examined whether disaccharides could enhance the transgene expression mediated by lipid-based vectors. METHODS: Different disaccharides were incorporated into the vectors prepared with DOTAP/protamine/DNA (LPD) or with DNA/cationic liposomes containing DOTAP, DOTAP/Chol, DOTAP/DOPE, or DC-Chol/DOPE. The levels of transgene expression and internalized plasmid of CHO cells were represented by the percentages of GFP-positive cells and the fluorescence intensity of ethidium-monoazide covalently labeled plasmid, respectively. The vectors containing either cellobiose or trehalose were also intravenously injected into mouse tail vein to investigate the potentials of in vivo applications. RESULTS: For enhancing the transgene expression, cellobiose was found to be effective for all the vectors whereas maltose decreased the effectiveness of DOTAP/Chol liposomes and LPD. For the internalization of plasmid, most disaccharides were able to increase the cellular delivery of DOTAP, DOTAP/Chol, and DOTAP/DOPE liposomes, but caused decreases in the cellular entry of DC-Chol/DOPE liposomes. An approximately linear correlation between the internalized plasmid and the transgene expression was observed for all the treatments in this study. When the vectors were administered to mouse by intravenous injection, 10-fold and 3-fold increases in the luciferase expression of lung were observed for DOTAP liposomes containing 330 mM cellobiose and trehalose, respectively. CONCLUSIONS: This study showed that using trehalose and cellobiose with a lipid-based delivery system provides a straightforward approach to effectively enhance both in vitro and in vivo transgene expression.     

Cationic Liposomes Containing Disulfide Bonds in Delivery of Plasmid DNA

Abstract

Cationic liposomes are non-viral gene transfer vectors for in vitro and in vivo experiments. In the present studies, we investigated whether a disulfide linkage in a cationic lipid was reducible by cell lysate resulting in the release of plasmid DNA and enhanced gene transfection. We also investigated if the differences in transgene production were from differences in total amount of cellular associated plasmid DNA. We systematically compared the gene transfection of disulfide bond containing-cationic lipid, 1', 2'-dioleoyl-sn-glycero-3'-succinyl-2-hydroxyethyl disulfide ornithine conjugate (DOGSDSO), its non-disulfide-containing analog, 1', 2'-dioleyl-sn-glycero-3'-succinyl-1, 6-hexanediol ornithine conjugate (DOGSHDO), 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP). Two transgene reporter systems (i.e., luciferase and green fluorescent protein (GFP)) were used to address transgene transgene expression and transgene efficiency. Experiments with the luciferase expression plasmid resulted in transgene activity up to 11 times greater transgene production for the disulfide containing lipid in at least two different cell lines, COS 1 and CHO cells. When transgene expression was determined by GFP activity, DOGSDSO liposomes were four times greater than the non-disulfide lipid or positive control (DOTAP) liposomes. By quantifying nucleic acid uptake by flow cytometry it was also demonstrated that increase expression was not solely from an increase in cellular plasmid DNA accumulation. These results demonstrate that cationic lipids containing a disulfide linkage are a promising method for gene transfer.     

Cationic Liposome-Mediated Gene Delivery Via Systemic Administration

Abstract

Cationic liposomes have been studied as a potential carrier for delivering genes to cells for the purpose of gene therapy. This report summarizes our efforts to characterize the in vivo expression of transgene delivered by cationic liposomes via intravenous administrtion. Using a CMV driven gene expression system containing cDNA of luciferase or green fluorescence protein gene as a reporter and two commonly used cationic lipids, 2, 3-dioleoyloxypropyl-1-trimethyl ammonium chloride (DOTMA) and 2, 3-dioleoyloxyl-1-trimethylammonium propanyl chloride (DOTAP), we demonstrate that a significant level of gene expression can be obtained in different organs including the lung, heart, spleen, liver and kidneys following intravenous administration in the mouse. Our finding show that the transfection efficiency of cationic liposomes is determined by the structure of the cationic lipids, the lipid composition of liposomes and cationic lipid to DNA ratio. Furthermore, gene expression was short in duration, peaked between 4-24 hours post injection, and dropped to less than 1% of the peak level within a 4 day period. Experiments with repeated injections revealed that cells initially transfected by the first transfection were not fully responsive to the subsequent second transfection for approximately 14 days.     

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.     

FACTORS INFLUENCING THE EFFICIENCY OF LIPOPLEXES MEDIATED GENE TRANSFER IN LUNG AFTER INTRAVENOUS ADMINISTRATION*

The objectives of this study were to test the influence of different parameters on the in vivo cationic lipid mediated gene transfer in lung after intravenous administration. Luciferase activity was evaluated in lung tissue 24 hours after intravenous administration of different types of lipoplexes. These included lipoplexes prepared using cationic phosphonolipids or DOTAP and various amounts of plasmid DNA. Using two different plasmids we tested the influence of plasmid size on transfection efficiency in vivo. In a last series of experiments, lipoplexes were prepared using different excipients (water, NaCl or 5% glucose solution) and three injection volumes were tested. We demonstrate that chemical structure modifications such as cation substitution and increment of the aliphatic chain length significantly improve transfection efficiency. High luciferase levels are obtained by increasing lipid to DNA charge ratio and plasmid DNA dose and decreasing plasmid size. Lipoplexes prepared in physiological NaCl solution and injected using a volume of 800μl are significantly the most effective.

Cationic lipid mediated gene transfer in lung tissue after intravenous administration is influenced by factors including cationic lipid chemical structure, lipid to DNA ratio and plasmid dose. Nevertheless, plasmid size, injection volume and the excipient, used for the lipoplexes preparation, are also important factors and must be considered for an optimization of in vivo gene delivery using intravenous administration.     

Characterization of cationic lipid DNA transfection complexes differing in susceptability to serum inhibition

Background

Cationic lipid DNA complexes based on DOTAP (1,2-dioleoyl-3-(trimethyammonium) propane) and mixtures of DOTAP and cholesterol (DC) have been previously optimized for transfection efficiency in the absence of serum and used as a non-viral gene delivery system. To determine whether DOTAP and DC lipid DNA complexes could be obtained with increased transfection effciency in the presence of high serum concentrations, the composition of the complexes was varied systematically and a total of 162 different complexes were analyzed for transfection efficiency in the presence and absence of high serum concentrations.

Results

Increasing the ratio of DOTAP or DC to DNA led to a dose dependent enhancement of transfection efficiency in the presence of high serum concentrations up to a ratio of approximately 128 nmol lipid/μg DNA. Transfection efficiency could be further increased for all ratios of DOTAP and DC to DNA by addition of the DNA condensing agent protamine sulfate (PS). For DOTAP DNA complexes with ratios of ≤ 32 nmol/μg DNA, peak transfection efficiencies were obtained with 4 μg PS/μg DNA. In contrast, increasing the amount of PS of DC complexes above 0.5 μg PS /μg DNA did not lead to significant further increases in transfection efficiency in the presence of high serum concentrations. Four complexes, which had a similar high transfection efficiency in cell culture in the presence of low serum concentrations but which differed largely in the lipid to DNA ratio and the amount of PS were selected for further analysis. Intravenous injection of the selected complexes led to 22-fold differences in transduction efficiency, which correlated with transfection efficiency in the presence of high serum concentrations. The complex with the highest transfection efficiency in vivo consisted of 64 nmol DC/ 16 μg PS/ μg DNA. Physical analysis revealed a predicted size of 440 nm and the highest zeta potential of the complexes analyzed.

Conclusions

Optimization of cationic lipid DNA complexes for transfection efficiency in the presence of high concentrations of serum led to the identification of a DC complex with high transduction efficiency in mice. This complex differs from previously described ones by higher lipid to DNA and PS to DNA ratios. The stability of this complex in the presence of high concentrations of serum and its high transduction efficiency in mice suggests that it is a promising candidate vehicle for in vivo gene delivery.     

Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: Interplay between nanostructure and composition

Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid–protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP–DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP–DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol–DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.     

Structure and internal organization of overcharged cationic-lipid/peptide/DNA self-assembly complexes

The combination of cationic lipids with cationic peptides and DNA vectors can produce synergistic effects in gene delivery to eukaryotic cells. Binary complexes of cationic lipids with DNA are well-studied whereas little information is available about the structure of the ternary lipid/peptide/DNA (LPD) complexes and mechanisms defining DNA protection and delivery. Here we use synchrotron small angle X-ray scattering and dynamic light scattering zeta-potential measurements to determine structure and the net charge of supramolecular aggregates of complexes in mixtures of plasmid DNA, cationic liposomes formed from DOTAP, plus a linear cationic ε-oligolysine with the pendant α-amino acids Leu-Tyr-Arg (LYR), ε-(LYR)K10. These ternary complexes display multilamellar structures with relatively constant separation between DOTAP bilayers, accommodating a hydrated monolayer of parallel DNA rods. The DNA-DNA distance in the complexes varies as a function of the net positive to negative (lipid+peptide)/DNA charge ratio. An explanation for the observed dependence of DNA-DNA distance on charge ratio was proposed based on general polyelectrolyte properties of non-stoichiometric polycation-DNA mixtures.     

Cationic lipids and cationic ligands induce DNA helix denaturation: detection of single stranded regions by KMnO4 probing

Cationic lipids and cationic polymers are widely used in gene delivery. Using 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid, we have investigated the stability of the DNA in DOTAP:DNA complexes by probing with potassium permanganate (KMnO4). Interestingly, thymidines followed by a purine showed higher susceptibility to cationic ligand-mediated melting. Similar studies performed with other water-soluble cationic ligands such as polylysine, protamine sulfate and polyethyleneimine also demonstrated melting of the DNA but with variations. Small cations such as spermine and spermidine and a cationic detergent, cetyl trimethylammonium bromide, also rendered the DNA susceptible to modification by KMnO4. The data presented here provide direct proof for melting of DNA upon interaction with cationic lipids. Structural changes subsequent to binding of cationic lipids/ligands to DNA may lead to instability and formation of DNA bubbles in double-stranded DNA.     

A simple but effective cancer vaccine consisting of an antigen and a cationic lipid

Developing a cancer vaccine with a potent adjuvant, which is safe for human use, remains to be an unmet need. In this study, we developed a simple, safe, yet efficient, peptide-based therapeutic cancer vaccine, DOTAP/E7 complex, which comprises only two molecules: a DOTAP cationic lipid and a peptide antigen derived from E7 oncoprotein of human papillomavirus (HPV) type 16. The anti-cancer activity of DOTAP/E7 against existing HPV positive TC-1 tumor was compared to that of our previous LPD/E7 formulation, which contains bacterial DNA CpG motifs. Tumor-bearing mice showed significant tumor inhibition following a single vaccination of either formulation at the optimal lipid dose, suggesting that DOTAP liposome alone can provide a potent adjuvant activity without plasmid DNA. E7 peptide formulated with DOTAP induced migration of activated dendritic cells (DC) to the draining lymph node (DLN) and efficiently generated functional antigen-specific CD8⁺ T lymphocyte responses. Accumulation of CD8⁺ tumor infiltrating T cells and apoptosis at tumor sites were observed after treatment with DOTAP/E7 complexes, which was also associated with a decreased amount of CD25⁺Foxp3⁺ regulatory T cells in treated animals. Reactive oxygen species (ROS) induced by DOTAP cationic lipid in DLN revealed a plausible mechanism of the initial interaction between DC and DOTAP. An adequate amount of ROS generation was apparently required for the initiation of the vaccine mechanism; however, an overdose of DOTAP induced massive ROS production and apoptosis of DC in DLN, which led to diminished anti-cancer immunity. Overall, these results indicate that cationic lipid DOTAP alone serves as an efficient vaccine adjuvant for the induction of a therapeutic, antigen-specific anti-cancer activity.     

Nanoparticles evading the reticuloendothelial system: Role of the supported bilayer

We have previously shown that the PEGylated LPD (liposome-polycation-DNA) nanoparticles were highly efficient in delivering siRNA to the tumor with low liver uptake. Its mechanism of evading the reticuloendothelial system (RES) is reported here. In LPD, nucleic acids were condensed with protamine into a compact core, which was then coated by two cationic lipid bilayers with the inner bilayer stabilized by charge-charge interaction (also called the supported bilayer). Finally, a detergent-like molecule, polyethylene glycol (PEG)-phospholipid is post-inserted into the lipid bilayer to modify the surface of LPD. The dynamic light scattering (DLS) data showed that LPD had improved stability compared to cationic liposomes after incubation with a high concentration of DSPE-PEG₂₀₀₀, which is known to disrupt the bilayer. LPD prepared with a multivalent cationic lipid, DSGLA, had enhanced stability compared to those containing DOTAP, a monovalent cationic lipid, suggesting that stronger charge-charge interaction in the supported bilayer contributed to a higher stability. Distinct nanoparticle structure was found in the PEGylated LPD by transmission electron microscopy, while the cationic liposomes were transformed into tubular micelles. Size exclusion chromatography data showed that approximately 60% of the total cationic lipids, which were located in the outer bilayer of LPD, were stripped off during the PEGylation; and about 20% of the input DSPE-PEG₂₀₀₀ was incorporated into the inner bilayer with about 10.6 mol% of DSPE-PEG₂₀₀₀ presented on the particle surface. This led to complete charge shielding, low liver sinusoidal uptake, and 32.5% injected dose delivered to the NCI-H460 tumor in a xenograft model.     

Enhancement of transfection efficiency by protamine in DDAB lipid vesicle-mediated gene transfer.

We have previously developed a simple gene transfection procedure mediated by cationic lipid vesicles for animal cells, in which a commercially available cationic surfactant, dimethyldioctadecyl ammonium bromide (DDAB), was used for making lipid vesicles. In the present study, we examined enhancement of transfection efficiency for this method by adding protamine to plasmid DNA solution before the formation of DNA/lipid vesicle complexes. Both free-base protamine and protamine sulfate provided enhanced transfection efficiency and expression level, but the optimal amount of the two protamines was different. The enhancement in transfection efficiency and expression level by protamines was observed in all the cell lines (COS-7, Hela, NIH3T3, MDCK, and BHK-21C13) and all the plasmids (pCMVbeta, pmiwZ, and pCH110) tested. The enhancement in both transfection efficiency and expression level was at most 20-fold compared with that using only DDAB lipid vesicles. Protamines seemed to protect DNA from degradation by DNase and promote DNA delivery into a nucleus.     

Systemic administration of LPD prepared with CpG oligonucleotides inhibits the growth of established pulmonary metastases by stimulating innate and acquired antitumor immune responses

Intravenous (i.v.) administration of a lipopolyplex consisting of a ternary complex of DOTAP:cholesterol cationic liposomes, protamine sulfate, and noncoding plasmid DNA (LPD-pDNA) is capable of stimulating a potent Th-1 cytokine response and inhibiting the growth of established tumors in mice. Both activities are mainly elicited by unmethylated CpG motifs in the plasmid DNA (pDNA) component, which are bacterial in origin. Since oligodeoxynucleotides (ODN) that possess a consensus immunostimulatory CpG motif of RRCpGYY (R is purine and Y is pyrimidine) can mimic the immunostimulatory actions of bacterial DNA, we hypothesized that i.v. administration of LPD prepared with GpG-ODN would mimic the ability of LPD-pDNA to stimulate Th-1 cytokines and antitumor activity and provide an improved vector for probing the immune mechanisms underlying the observed antitumor effects. These hypotheses were tested for the treatment of established 24JK experimental pulmonary metastases that are syngeneic in C57BL/6 mice. Mice treated with LPD containing 25 microg of the prototypical phosphodiester (PO) CpG-ODN 1668 (tccatGACGTTcctgatgct, motif capitalized) demonstrated a dramatic reduction in lung tumor burden (>80% inhibition, P<0.01) compared to dextrose-treated controls. The antitumor effect was dependent on the CpG dinulceotide and correlated with the ability to stimulate serum Th-1 cytokines (TNF-alpha, IL-12, and IFN-gamma). Both activities required assembly of CpG-ODN in a cationic liposome/DNA complex (lipoplex) or the LPD lipopolyplex. LPD delivery of both PO-1668 and phosphorothioated (PS)-1668 stimulated a greater cytokine response compared to delivery of free ODN. Furthermore, within the LPD complex, both PO- and PS-1668 had similar ability to stimulate Th-1 cytokines with respect to potency and duration of response, thus eliminating the need for the PS modification. In tumor cell lysis assays, LPD-CpG DNA stimulated development of an acquired, tumor-specific CD8+ cytotoxic T-lymphocyte (CTL) activity that was dependent on CpG DNA. LPD was also capable of stimulating NK activity; however, this was not dependent on CpG DNA. Only formulations that concomitantly stimulated NK activity and CpG-specific, Th-1 cytokine were capable of stimulating the development of tumor-specific CTL activity and significant inhibition of tumor growth. Thus, we propose a model where CpG DNA in complex with cationic liposome-based lipoplexes or lipopolyplexes stimulates antitumor NK activity and CpG-stimulated Th-1 cytokine production. The combination of these two activities of the innate immune system subsequently direct the development of an acquired, tumor-specific CTL response that in total are effective for inhibiting the growth of established tumors in mice.     

Proteolipidic vectors for gene transfer to the lung

In order to develop improved synthetic gene transfer vectors, we have synthesized bifunctional peptides composed of a DNA binding peptide (P2) and ligand peptides selected by the phage display technique on tracheal epithelial cells. We have evaluated the capacity of these peptides to enhance the gene transfer efficiency of the cationic lipid DOTAP to the mouse lung. To optimize the in vivo transfection efficiency, we first compared the efficiency of DOTAP to transfect the lung by either intravenous injection or aerosolization. We then tested DNA/Peptide/DOTAP complexes formed at different Peptide/DNA and DOTAP/DNA charge ratios. Under optimal conditions, precompaction of DNA by peptide P2 gave a higher expression in the mouse lung using the luciferase reporter gene than DOTAP/DNA complexes. A further increase of transfection efficiency was obtained with the bifunctional peptide P2-9. Experiments performed with the GFP reporter gene showed expression in the alveolar parenchyme.     

Immunostimulation of dendritic cells by cationic liposomes

A nano-aggregate liposome-polycation-DNA (LPD), composed of a cationic lipid, protamine and plasmid DNA was found to effectively deliver a human papillomavirus (HPV)-E7 epitope antigen to the antigen presenting cells of the immune system, eliciting enhanced anti-tumor immune responses in mouse models of cervical carcinoma. Both the cationic liposome and plasmid DNA were essential for the full immunostimulation activity of LPD. Interestingly, cationic liposomes alone could stimulate the antigen presenting dendritic cells (DC) leading to the expression of co-stimulatory molecules, CD80 and CD86. However, cationic lipids could not stimulate DC for the expression of pro-inflammatory cytokines. Moreover, they were unable to enhance the expression of NF-κB, suggesting that dendritic cells stimulation by cationic lipids is signaled through an NF-κB independent mechanism. DC stimulation was specific to cationic lipids, the zwitterionic and anionic lipids showed little or no activity. The ability of different cationic lipids to stimulate the expression of co-stimulatory molecules on DC varied significantly. In general, the cationic lipids bearing ethyl phosphocholine head groups were better stimulants than their trimethylammonium counterparts. In case of the cationic lipids bearing trimethyl ammonium head groups, the ones bearing unsaturated or shorter saturated hydrophobic chains exhibited enhanced immunostimulatory activity. The LPS-induced TNF-α expression by dendritic cells was inhibited by active cationic lipids but not the inactive ones, suggesting the possible involvement of lipopolysaccharide binding protein (LBP) in cationic lipid mediated DC stimulation. Based on the structure-specific activation of dendritic cells by cationic lipids, a model for the immunostimulation of DC by such lipids is proposed.     

Immunostimulation of dendritic cells by cationic liposomes

A nano-aggregate liposome-polycation-DNA (LPD), composed of a cationic lipid, protamine and plasmid DNA was found to effectively deliver a human papillomavirus (HPV)-E7 epitope antigen to the antigen presenting cells of the immune system, eliciting enhanced anti-tumor immune responses in mouse models of cervical carcinoma. Both the cationic liposome and plasmid DNA were essential for the full immunostimulation activity of LPD. Interestingly, cationic liposomes alone could stimulate the antigen presenting dendritic cells (DC) leading to the expression of co-stimulatory molecules, CD80 and CD86. However, cationic lipids could not stimulate DC for the expression of pro-inflammatory cytokines. Moreover, they were unable to enhance the expression of NF-kappaB, suggesting that dendritic cells stimulation by cationic lipids is signaled through an NF-kappaB independent mechanism. DC stimulation was specific to cationic lipids, the zwitterionic and anionic lipids showed little or no activity. The ability of different cationic lipids to stimulate the expression of co-stimulatory molecules on DC varied significantly. In general, the cationic lipids bearing ethyl phosphocholine head groups were better stimulants than their trimethylammonium counterparts. In case of the cationic lipids bearing trimethyl ammonium head groups, the ones bearing unsaturated or shorter saturated hydrophobic chains exhibited enhanced immunostimulatory activity. The LPS-induced TNF-alpha expression by dendritic cells was inhibited by active cationic lipids but not the inactive ones, suggesting the possible involvement of lipopolysaccharide binding protein (LBP) in cationic lipid mediated DC stimulation. Based on the structure-specific activation of dendritic cells by cationic lipids, a model for the immunostimulation of DC by such lipids is proposed.     

Intracellular accumulation of secreted proteoglycans inhibits cationic lipid-mediated gene transfer. Co-transfer of glycosaminoglycans to the nucleus.

Molecules secreted by potential target cells may interfere with cationic lipid-mediated gene transfer. This has been studied using human lung fibroblasts and human epidermoid lung cancer cells. Secreted cell medium components caused a substantial decrease both in the uptake of cationic lipid-DNA complexes (2-4-fold) and in reporter gene expression (100-1000-fold). Metabolic labeling of the cell medium showed that especially [35S]sulfate-labeled macromolecules competed with DNA for binding to the cationic lipid. Release of DNA from the cationic lipid by cell medium components was demonstrated by an ethidium bromide intercalation assay. In the presence of the cationic lipid, the secreted macromolecules were internalized by the cells. By enzymatic digestions, it was shown that the competing macromolecules consist of chondroitin/dermatan sulfate and heparan sulfate proteoglycans and that the effects on transfection were mediated by the polyanionic glycosaminoglycan portion of the proteoglycan. Accordingly, pretreatment of cell medium with the polycationic peptide protamine sulfate abrogated the inhibitory effects on gene transfer. Fluorescence microscopy studies revealed that heparan sulfate, internalized as a complex with cationic lipids, accumulated in the cell nuclei. These results support the view that the lack of specificity of this type of gene transfer vehicle is a major hindrance to efficient and safe in vivo administration.     

DOTAP cationic liposomes prefer relaxed over supercoiled plasmids

Cationic liposomes and DNA interact electrostatically to form complexes called lipoplexes. The amounts of unbound (free) DNA in a mixture of cationic liposomes and DNA at different cationic lipid:DNA molar ratios can be used to describe DNA binding isotherms; these provide a measure of the binding efficiency of DNA to different cationic lipid formulations at various medium conditions. In order to quantify the ratio between the various forms of naked DNA and supercoiled, relaxed and single-stranded DNA, and the ratio between cationic lipid bound and unbound DNA of various forms we developed a simple, sensitive quantitative assay using agarose gel electrophoresis, followed by staining with the fluorescent cyanine DNA dyes SYBR Green I or SYBR Gold. This assay was compared with that based on the use of ethidium bromide (the most commonly used nucleic acid stain). Unlike ethidium bromide, SYBR Green I DNA sensitivity and concentration-dependent fluorescence intensity were identical for supercoiled and nicked-relaxed forms. DNA detection by SYBR Green I in solution is approximately 40-fold more sensitive than by ethidium bromide for double-stranded DNA and approximately 10-fold for single-stranded DNA, and in agarose gel it is 16-fold more sensitive for double-stranded DNA compared with ethidium bromide. SYBR Gold performs similarly to SYBR Green I. This study shows that: (a) there is no significant difference in DNA binding isotherms to the monocationic DOTAP (DOTAP/DOPE) liposomes and to the polycationic DOSPA (DOSPA/DOPE) liposomes, even when four DOSPA positive charges are involved in the electrostatic interaction with DNA; (b) the helper lipids affect DNA binding, as DOTAP/DOPE liposomes bind more DNA than DOTAP/cholesterol; (c) in the process of lipoplex formation, when the DNA is a mixture of two forms, supercoiled and nicked-relaxed (open circular), there is a preference for the binding to the cationic liposomes of plasmid DNA in the nicked-relaxed over the supercoiled form. This preference is much more pronounced when the cationic liposome formulation is based on the monocationic lipid DOTAP than on the polycationic lipid DOSPA. The preference of DOTAP formulations to bind to the relaxed DNA plasmid suggests that the binding of supercoiled DNA is weaker and easier to dissociate from the complex.     

Novel ortho ester-based,pH-sensitive cationic lipid for gene delivery in vitro and in vivo

Context: Cationic lipoplexes are less toxic than viral gene vectors and more convenient to prepare but their efficiencies of gene delivery are generally lower.

Objective: To develop ortho ester-based, pH-sensitive lipoplexes for efficient gene delivery both in cultured cells and in vivo.

Materials and methods: A novel cationic and acid-labile lipid (DOC) containing a cationic headgroup and a cholesterol-derived lipid tail joined together by an acid-labile ortho ester linker was designed and synthesized. DOC was formulated into liposomes with the conical helper lipid DOPE, and then into lipoplexes with plasmid DNA encoding a luciferase reporter gene. The physicochemical properties of the lipoplexes (size, surface charge and pH-sensitivity) were characterized. Gene delivery by DOC/DOPE/DNA lipoplexes was also evaluated in CV-1 cells and in CD-1 mice following intratracheal injection. Lipoplexes consisting of the acid-stable cationic lipid DC-Chol were characterized as a control.

Results: DOC formed cationic lipoplexes with DOPE and DNA. After incubation at acidic pH 4.6, DOC/DOPE/DNA lipoplexes lost their positive charges and aggregated with one another as a result of DOC hydrolysis. Both in CV-1 cell culture and in CD-1 mice, DOC/DOPE/DNA lipoplexes increased the luciferase gene expression by 5- to 10-fold compared with the analogous but acid-stable DC-Chol/DOPE/DNA lipoplexes.

Discussion and conclusion: Incorporation of an acid-labile ortho ester linker into a cationic lipid is a viable approach to enhance gene delivery by the corresponding lipoplexes both in cultured cells and in vivo.     

Interplay in lipoplexes between type of pDNA promoter and lipid composition determines transfection efficiency of human growth hormone in NIH3T3 cells in culture.

This study was aimed to investigate if and to what extent there is an interplay between lipoplex physicochemical properties and plasmid promoter type affecting transfection efficiency in vitro. To reduce the number of variables only one cell type (NIH3T3 cells), one gene (human growth hormone), one cationic lipid (DOTAP) in a plasmid >85% in supercoiled form, and the same medium conditions were used. The variables of the physicochemical properties included presence and type of helper lipid (DOPE, DOPC, or cholesterol, all in 1:1 mole ratio with DOTAP), size and lamellarity of the liposomes used for lipoplex preparation (large unilamellar vesicles, LUV, versus multilamellar vesicles, MLV), and DNA(-)/cationic lipid(+) charge ratio, all containing the same human growth hormone but differing in their promoter enhancer region. Two of the promoters were of viral origin: (a) SV40 promoter (simian virus early promoter) and (b) CMV promoter (cytomegalovirus early promoter); two were of mammalian cell origin: (c) PABP promoter (human poly(A)-binding protein promoter) and (d) S16 promoter (mouse ribosomal protein (rp) S16 promoter). Transfection studies showed that, irrespective of promoter type, large (> or =500 nm) MLV were superior to approximately 100 nm LUV; the extent of superiority was dependent on liposome lipid composition (larger for 100% DOTAP and DOTAP/DOPE than for DOTAP/DOPC and DOTAP/cholesterol). The optimal DNA(-)/DOTAP(+) charge ratio for all types of lipoplexes used was 0.2 or 0.5 (namely, when the lipoplexes were positively charged). Scoring the six best lipoplex formulations (out of 128 studied) revealed the following order: pCMV (DOTAP/DOPE) > pSV (DOTAP/DOPE)=pCMV(DOTAP/cholesterol)=pS16 (100% DOTAP)=pS16 DOTAP/DOPE > pCMV (DOTAP/DOPC). The lack of trivial consistency in the transfection efficiency score, the pattern of transfection efficiency, and statistical analysis of the data suggest that there is cross-talk between promoter type and lipoplex lipid composition, which may be related to the way the promoter is associated with the lipids.