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.     

Effect of lipid composition on the structure and theoretical phase diagrams of DC-Chol/DOPE-DNA lipoplexes

Lipoplexes constituted by calf-thymus DNA (CT-DNA) and mixed cationic liposomes consisting of varying proportions of the cationic lipid 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and the zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphoetanolamine (DOPE) have been analyzed by means of electrophoretic mobility, SAXS, and fluorescence anisotropy experiments, as well as by theoretically calculated phase diagrams. Both experimental and theoretical studies have been run at several liposome and lipoplex compositions, defined in terms of cationic lipid molar fraction, α, and either the mass or charge ratios of the lipoplex, respectively. The experimental electrochemical results indicate that DC-Chol/DOPE liposomes, with a mean hydrodynamic diameter of around (120 ± 10) nm, compact and condense DNA fragments at their cationic surfaces by means of a strong entropically driven electrostatic interaction. Furthermore, the positive charges of cationic liposomes are compensated by the negative charges of DNA phosphate groups at the isoneutrality L/D ratio, (L/D)(?), which decreases with the cationic lipid content of the mixed liposome, for a given DNA concentration. This inversion of sign process has been also studied by means of the phase diagrams calculated with the theoretical model, which confirms all the experimental results. SAXS diffractograms, run at several lipoplex compositions, reveal that, irrespectively of the lipoplex charge ratio, DC-Chol/DOPE-DNA lipoplexes show a lamellar structure, L(α), when the cationic lipid content on the mixed liposomes α ≥ 0.4, while for a lower content (α = 0.2) the lipoplexes show an inverted hexagonal structure, H(II), usually related with improved cell transfection efficiency. A similar conclusion is reached from fluorescence anisotropy results, which indicate that the fluidity on liposome and lipoplexes membrane, also related with better transfection results, increases as long as the cationic lipid content decreases.     

Factors affecting DNA binding and stability of association to cationic liposomes

Lipoplexes are complexes formed between cationic liposomes (L(+)) and polyanionic nucleic acids (P(-)). They are commonly used in vitro and in vivo as a nucleic acid delivery system. Our study aims are to investigate how DOTAP-based cationic liposomes, which vary in their helper lipid (cholesterol or DOPE) and in media of different ionic strengths affect the degree, mode of association and degree of condensation of pDNA. This was determined by ultracentrifugation and gel electrophoresis, methods based on different physical principles. In addition, the degree of pDNA condensation was also determined using the ethidium bromide (EtBr) intercalation assay. The results suggest that for cationic lipid compositions (DOTAP/DOPE and DOTAP/cholesterol), 1.5 M NaCl, but not 0.15 M NaCl, both prevent lipoplex formation and/or induce partial dissociation between lipid and DNA of preformed lipoplexes. The higher the salt concentration the greater is the similarity of DNA condensation (monitored by EtBr intercalation) between lipoplex DNA and free DNA. As determined by ultracentrifugation and agarose gel electrophoresis, 30-90% of the DNA is uncondensed. SDS below its critical micellar concentration (CMC) induced "de-condensation" of DNA without its physical release (assessed by ultracentrifugation) for both DOTAP/DOPE and DOTAP/cholesterol lipoplexes. As was assessed by agarose gel electrophoresis SDS induced release of 50-60% of DNA from the DOTAP/cholesterol lipoplex but not from the DOTAP/DOPE lipoplex. This study shows that there are conditions under which DNA is still physically associated with the cationic lipids but undergoes unwinding to become less condensed. We also proved that the helper lipid affects level and strength of the L(+) and DNA(-) electrostatic association; these interactions are weaker for DOTAP/cholesterol than for DOTAP/DOPE, despite the fact that the positive charge and surface pH of DOTAP/cholesterol and DOTAP/DOPE are similar.     

Characterization of the interplay between the main factors contributing to lipoplex-mediated transfection in cell cultures

Transfection efficiency of lipoplex-mediated gene delivery is multifactorial. However, the mode of interaction between the factors which affect transfection is not fully understood. To help fill this deficiency we evaluated the effect of the interplay between several variables that affect transfection efficiency in cell cultures. For this, we applied the Analysis of Variance Model with Fixed Effects and Repeated Measures to assess the data. The variables studied include: two different genes, Luc, and human growth hormone (hGH), in three different plasmids (two of which contain the luciferase (Luc) gene, but different promoter-enhancer regions (CMV and H19) and one plasmid coding hGH with a S16 promoter); three topoisoforms of pDNA (supercoiled (SC), open circular (OC), and closed circular (CC)); three cationic lipid compositions, all based on the monocationic lipid DOTAP (100% DOTAP, DOTAP/DOPE 1 : 1, and DOTAP/cholesterol 1 : 1, all ratios are mole ratios); two DNA-/L+ charge ratios (0.2 and 0.5); and two cell lines (NIH 3T3 and MBT-2). Our statistical analysis confirmed that the cell type, the gene used for transfection, the promoter type, the type of helper lipid, and DNA-/DOTAP+ charge ratio, all affect transfection efficiency in a statistically significant manner. The most efficient lipoplex formulation in both cell lines was that based on DOTAP (without helper lipid), having CC plasmid DNA. We suggest that for obtaining the most transfection-efficient lipoplex one should select the best topoisoform of pDNA for each particular cell type, and complex it with cationic liposomes having optimal lipid composition.     

Effect of "helper lipid" on lipoplex electrostatics

Lipoplexes, which are complexes between cationic liposomes (L+) and nucleic acids, are commonly used as a nucleic acid delivery system in vitro and in vivo. This study aimed to better characterize cationic liposome and lipoplex electrostatics, which seems to play a major role in the formation and the performance of lipoplexes in vitro and in vivo. We characterized lipoplexes based on two commonly used monocationic lipids, DOTAP and DMRIE, and one polycationic lipid, DOSPA--each with and without helper lipid (cholesterol or DOPE). Electrical surface potential (Psi0) and surface pH were determined using several surface pH-sensitive fluorophores attached either to a one-chain lipid (4-heptadecyl hydroxycoumarin (C17HC)) or to the primary amino group of the two-chain lipids (1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-carboxyfluorescein (CFPE) and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-7-hydroxycoumarin) (HC-DOPE). Zeta potentials of the DOTAP-based cationic liposomes and lipoplexes were compared with Psi0 determined using C17HC. The location and relatively low sensitivity of fluorescein to pH changes explains why CFPE is the least efficient in quantifying the differences between the various cationic liposomes and lipoplexes used in this study. The fact that, for all cationic liposomes studied, those containing DOPE as helper lipid have the least positive Psi0 indicates neutralization of the cationic charge by the negatively-charged phosphodiester of the DOPE. Zeta potential is much less positively charged than Psi0 determined by C17HC. The electrostatics affects size changes that occurred to the cationic liposomes upon lipoplex formation. The largest size increase (based on static light scattering measurements) for all formulations occurred at DNA-/L+ charge ratios 0.5-1. Comparing the use of the one-chain C17HC and the two-chain HC-DOPE for monitoring lipoplex electrostatics reveals that both are suitable, as long as there is no serum (or other lipidic assemblies) present in the medium; in the latter case, only the two-chain HC-DOPE gives reliable results. Increasing NaCl concentrations decrease surface potential. Neutralization by DNA is reduced in a NaCl-concentration-dependent manner.     

Hydration of lipoplexes commonly used in gene delivery: follow-up by laurdan fluorescence changes and quantification by differential scanning calorimetry.

Lipoplexes, which are formed spontaneously between cationic liposomes and negatively charged nucleic acids, are commonly used for gene and oligonucleotide delivery in vitro and in vivo. Being assemblies, lipoplexes can be characterized by various physicochemical parameters, including size distribution, shape, physical state (lamellar, hexagonal type II and/or other phases), sign and magnitude of electrical surface potential, and level of hydration at the lipid-DNA interface. Only after all these variables will be characterized for lipoplexes with a broad spectrum of lipid compositions and DNA/cationic lipid (L(+)) mole (or charge) ratios can their relevance to transfection efficiency be understood. Of all these physicochemical parameters, hydration is the most neglected, and therefore the focus of this study. Cationic liposomes composed of DOTAP without and with helper lipids (DOPC, DOPE, or cholesterol) or of DC-Chol/DOPE were complexed with pDNA (S16 human growth hormone) at various DNA(-)/L(+) charge ratios (0.1-3.2). (DOTAP=N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DC-Chol=(3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholester ol; DOPC=1, 2-dioleoyl-sn-glycero-3-phosphocholine; DOPE=1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine). The hydration levels of the different cationic liposomes and the DNA separately are compared with the hydration levels of the lipoplexes. Two independent approaches were applied to study hydration. First, we used a semi-quantitative approach of determining changes in the 'generalized polarization' (GP) of laurdan (6-dodecanoyl-2-dimethylaminonaphthalene). This method was recently used extensively and successfully to characterize changes of hydration at lipid-water interfaces. Laurdan excitation GP at 340 nm (GP(340)DOTAP. The GP(340) of lipoplexes of all lipid compositions (except those based on DC-Chol/DOPE) was higher than the GP(340) of the cationic liposomes alone and increased with increasing DNA(-)/L(+) charge ratio, reaching a plateau at a charge ratio of 1. 0, suggesting an increase in dehydration at the lipid-water interface with increasing DNA(-)/L(+) charge ratio. Confirmation was obtained from the second method, differential scanning calorimetry (DSC). DOTAP/DOPE lipoplexes with charge ratio 0.44 had 16.5% dehydration and with charge ratio 1.5, 46.4% dehydration. For DOTAP/Chol lipoplexes with these charge ratios, there was 17.9% and 49% dehydration, respectively. These data are in good agreement with the laurdan data described above. They suggest that the dehydration occurs during lipoplex formation and that this is a prerequisite for the intimate contact between cationic lipids and DNA.     

The use of fluorescence resonance energy transfer to monitor dynamic changes of lipid-DNA interactions during lipoplex formation

Fluorescence resonance energy transfer (FRET) was used to monitor interactions between Cy3-labeled plasmid DNA and NBD-labeled cationic liposomes. FRET data show that binding of cationic liposomes to DNA occurs immediately upon mixing (within 1 min), but FRET efficiencies do not stabilize for 1-5 h. The time allowed for complex formation has effects on in vitro luciferase transfection efficiencies of DOPE-based lipoplexes; i.e., lipoplexes prepared with a 1-h incubation have much higher transfection efficiencies than samples with 1-min or 5-h incubations. The molar charge ratio of DOTAP to negatively charged phosphates in the DNA (DOTAP+/DNA-) also affected the interaction between liposomes and plasmid DNA, and interactions stabilized more rapidly at higher charge ratios. Lipoplexes formulated with DOPE were more resistant to high ionic strength than complexes formulated with cholesterol. Taken together, our data demonstrate that lipid-DNA interactions and in vitro transfection efficiencies are strongly affected by the time allowed for complex formation. This effect is especially evident in DOPE-based lipoplexes, and suggests that the time allowed for lipoplex formation is a parameter that should be carefully controlled in future studies.     

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.     

In vitro cationic lipid-mediated gene delivery with fluorinated glycerophosphoethanolamine helper lipids.

There is a need for the development of nonviral gene transfer systems with improved and original properties. "Fluorinated" lipoplexes are such candidates, as supported by the remarkably higher in vitro and in vivo transfection potency found for such fluorinated lipoplexes as compared with conventional ones or even with PEI-based polyplexes (Boussif, O., Gaucheron, J., Boulanger, C., Santaella, C., Kolbe, H. V. J., Vierling, P. (2001) Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophosphoethanolamine helper lipid. J. Gene Med. 3, 109-114). Here, we describe the synthesis of fluorinated glycerophosphoethanolamines (F-PEs), close analogues of dioleoylphosphatidylethanolamine (DOPE), and report on their lipid helper properties vs that of DOPE, as in vitro gene transfer components of fluorinated lipoplexes based on pcTG90, DOGS (Transfectam), or DOTAP. To evaluate the contribution of the F-PEs to in vitro lipoplex-mediated gene transfer, we examined the effect of including the F-PEs in lipoplexes formulated with these cationic lipids (CL) for various CL:DOPE:F-PE molar ratios [1:(1 - x):x with x = 0, 0.5 and 1; 1:(2 - y):y with y = 0, 1, 1.5, and 2], and various N/P ratios (from 10 to 0.8, N = number of CL amines, P = number of DNA phosphates). Irrespective of the F-PE chemical structure, of the colipid F-PE:DOPE composition, and of the N/P ratio, comparable transfection levels to those of their respective control DOPE lipoplexes were most frequently obtained when using one of the F-PEs as colipid of DOGS, pcTG90, or DOTAP in place of part of or of all DOPE. However, a large proportion of DOGS-based lipoplexes were found to display a higher transfection efficiency when formulated with the F-PEs rather than with DOPE alone while the opposite tendency was evidenced for the DOTAP-based lipoplexes. The present work indicates that "fluorinated" lipoplexes formulated with fluorinated helper lipids and conventional cationic lipids are very attractive candidates for gene delivery. It confirms further that lipophobicity and restricted miscibility of the lipoplex lipids with the endogenous lipids does not preclude efficient gene transfer and expression. Their transfection potency is rather attributable to their unique lipophobic and hydrophobic character (resulting from the formulation of DNA with fluorinated lipids), thus preventing to some extent DNA from interactions with lipophilic and hydrophilic biocompounds, and from degradation.     

Phase behavior of cationic amphiphiles and their mixtures with helper lipid influences lipoplex shape,DNA translocation,and transfection efficiency

Cationic lipids are widely used for gene transfection, but their mechanism of action is still poorly understood. To improve this knowledge, a structure-function study was carried out with two pyridinium-based lipid analogs with identical headgroups but differing in alkyl chain (un)saturation, i.e., SAINT-2 (diC18:1) and SAINT-5 (diC18:0). Although both amphiphiles display transfection activity per se, DOPE strongly promotes SAINT-2-mediated transfection, but not that of SAINT-5, despite the fact that DOPE effectively facilitates plasmid dissociation from either lipoplex. This difference appears to correlate with membrane stiffness, dictated by the cationic lipid packing in the donor liposomes, which governs the kinetics of lipid recruitment by the plasmid upon lipoplex assembly. Because of its interaction with the relatively rigid SAINT-5 membranes, the plasmid becomes inappropriately condensed, which results in formation of structurally deformed lipoplexes. This structural deformation does not affect its cellular uptake but, rather, hampers plasmid translocation across endosomal and/or nuclear membranes. This is inferred from the observation that both lipoplexes effectively translocate much smaller oligonucleotides into cells. In fact, SAINT-5/DOPE-mediated transfection is greatly improved when, before lipoplex assembly, the plasmid is stabilized by condensation with polylysine. The results emphasize a role of the structural shape of the plasmid in gaining cytosolic/nuclear access. Moreover, it has been proposed that such a translocation is promoted when the lipoplex adopts the hexagonal phase, and data are presented that demonstrate that the lamellar SAINT-5/DOPE lipoplex adopts such a phase after its interaction with acidic phospholipid-containing membranes.     

Multilamellar Cationic Liposomes are Efficient Vectors for in Vitro Gene Transfer in Serum

Abstract

Multilamellar vesicles (MLVs) containing the cationic lipid DOTAP were used as vectors to lipofect a number of culture cell lines in the presence of serum. The lipofection efficiency of lipoplexes made of MLVs and the plasmid pSV-β galactosidase are much less sensitive to the lipofection-inhibitory effect of serum than the conventionally used lipoplexes made of sonicated small unilamellar vesicles (SUVs). In order to determine the factors favoring the lipofection efficiency of MLVs, we measured the size, as well as the cellular association and uptake of MLV and SUV lipoplexes containing DOTAP alone or DOTAP:DOPE (1:1). Electron microscope images of these complexes were taken to confirm their structure and size. The single most important factor that correlates with transfection efficiency in serum is the size of the lipoplex. SUV lipoplexes remain smaller than 300 nm in the presence of serum, and the lipofection efficiencies are low. MLV lipoplexes are larger (>300 nm) and the lipofection efficiency, as well as cellular association and uptake, are much higher than those of SUV lipoplexes. Exceptions are those lipoplexes made of MLVs of DOTAP and DOPE (1:1) combined with DNA at higher charge ratios, which form hexagonal structures and show poor lipofection as well as cellular association and uptake, even if their lipoplex size exceeds 300 nm. This finding lends credence to our theory of the serum inhibition effect upon lipofection, and suggests ways to improve the transfection efficiency in the presence of serum, by fabricating lipoplexes of defined sizes.     

Lamellarity of cationic liposomes and mode of preparation of lipoplexes affect transfection efficiency.

Transfection of NIH-3T3 cells by a human growth hormone expression vector complexed with liposomes composed of N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) with or without helper lipids was studied. The transfection efficiency was dependent on the lamellarity of the liposomes used to prepare the lipoplexes. Multilamellar vesicles (MLV) were more effective than large unilamellar vesicles (LUV) of approximately 100 nm, irrespective of lipid composition. The optimal DNA/DOTAP mole ratio for transfection was 相似文献   

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.     

The use of fluorescence resonance energy transfer to monitor dynamic changes of lipid-DNA interactions during lipoplex formation

Fluorescence resonance energy transfer (FRET) was used to monitor interactions between Cy3-labeled plasmid DNA and NBD-labeled cationic liposomes. FRET data show that binding of cationic liposomes to DNA occurs immediately upon mixing (within 1 min), but FRET efficiencies do not stabilize for 1-5 h. The time allowed for complex formation has effects on in vitro luciferase transfection efficiencies of DOPE-based lipoplexes; i.e., lipoplexes prepared with a 1-h incubation have much higher transfection efficiencies than samples with 1-min or 5-h incubations. The molar charge ratio of DOTAP to negatively charged phosphates in the DNA (DOTAP⁺/DNA⁻) also affected the interaction between liposomes and plasmid DNA, and interactions stabilized more rapidly at higher charge ratios. Lipoplexes formulated with DOPE were more resistant to high ionic strength than complexes formulated with cholesterol. Taken together, our data demonstrate that lipid-DNA interactions and in vitro transfection efficiencies are strongly affected by the time allowed for complex formation. This effect is especially evident in DOPE-based lipoplexes, and suggests that the time allowed for lipoplex formation is a parameter that should be carefully controlled in future studies.     

Development and Characterization of siRNA Lipoplexes: Effect of Different Lipids,In Vitro Evaluation in Cancerous Cell Lines and In Vivo Toxicity Study

Cationic liposomes have long been used as non-viral vectors for small interfering RNA (siRNA) delivery but are associated with high toxicity, less transfection efficiency, and in vivo instability. In this investigation, we have developed siRNA targeted to RRM1 that is responsible for development of resistance to gemcitabine in cancer cells. Effect of different lipid compositions has been evaluated on formation of stable and less toxic lipoplexes. Optimized cationic lipoplex (D₂CH) system was comprised of dioleoyl-trimethylammoniumpropane (DOTAP), dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), hydrogenated soya phosphocholine (HSPC), cholesterol, and methoxy(polyethyleneglycol)₂₀₀₀–1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG₂₀₀₀–DSPE). D₂CH lipoplexes have shown particle size (147.5 ± 2.89 nm) and zeta potential (12.26 ± 0.54 mV) characteristics essential for their in vivo use. In vitro cytotoxicity study has shown low toxicity of developed lipoplexes as compared with lipofectamine-2000 up to N/P ratio as high as 7.5. Cell uptake studies and gene expression studies have confirmed intracellular availability of siRNA. In addition, developed lipoplexes also showed ~3 times less hemolytic potential as compared with DOTAP/DOPE lipoplexes at lipid concentration of 5 mg/mL. Lipoplexes also maintained particle size less than 200 nm on exposure to high electrolyte concentration and showed >70% siRNA retention in presence of serum showing siRNA protection conferred by lipoplexes. Furthermore, in vivo acute toxicity studies in mice showed that formulation was non-toxic up to a dosage of 0.75 mg of siRNA/kg as lipoplexes and 300 mg lipid/kg as blank liposomes indicating tolerability of lipoplexes at a dose much higher than required for therapeutic use. Promising results of this study warrant further investigation of developed siRNA lipoplexes for cancer treatment.KEY WORDS: cancer, gene expression, lipoplex, siRNA, toxicity     

Interaction of oligonucleotides with cationic lipids: the relationship between electrostatics, hydration and state of aggregation

Lipoplexes, which are spontaneously formed complexes between oligonucleotide (ODN) and cationic lipid, can be used to deliver ODNs into cells, both in vitro and in vivo. The present study was aimed at characterizing the interactions associated with the formation of lipoplexes, specifically in terms of electrostatics, hydration and particle size. Large unilamellar vesicles (approximately 100 nm diameter), composed of either DOTAP, DOTAP/cholesterol (mole ratio 1:1) or DOTAP/DOPE (mole ratio 1:1) were employed as a model of cationic liposomes. Neutral vesicles ( approximately 100 nm diameter), composed of DOPC/DOPE (mole ratio 1:1), were employed as control liposomes. After ODN addition to vesicles, at different mole ratios, changes in pH and electrical surface potential at the lipid-water interface were analyzed by using the fluorophore heptadecyl-7-hydroxycoumarin. In separate 'mirror image' experiments, liposomes were added at different mole ratios to fluorescein isothiocyanate-labeled ODNs, thus yielding data about changes in the pH near the ODN molecules induced by the complexation with the cationic lipid. Particle size distribution and turbidity fluctuations were analyzed by the use of photon correlation spectroscopy and static light-scattering, respectively. In additional fluorescent probe studies, TMADPH was used to quantify membrane defects while laurdan was used to measure the level of hydration at the water-lipid interface. The results indicate that mutual neutralization of cationic lipids by ODNs and vice versa is a spontaneous reaction and that this neutralization is the main driving force for lipoplex generation. When lipid neutralization is partial, induced membrane defects cause the lipoplexes to exhibit increased size instability.     

New effects in polynucleotide release from cationic lipid carriers revealed by confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking

We report on new insights into the mechanisms of short single and double stranded oligonucleotide release from cationic lipid complexes (lipoplexes), used in gene therapy. Specifically, we modeled endosomal membranes using giant unilamellar vesicles and investigated the roles of various individual cellular phospholipids in interaction with lipoplexes. Our approach uses a combination of confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking, revealing several new aspects of the release: (a) phosphatidylserine and phosphatidylethanolamine are equally active in disassembling lipoplexes, while phosphatidylcholine and sphingomyelin are inert; (b) in contrast to earlier findings, phosphatidylethanolamine alone, in the absence of anionic phosphatidylserine triggers extensive release; (c) a double-stranded DNA structure remains well preserved after release; (d) lipoplexes exhibited preferential binding to transient lipid domains, which appear at the onset of lipoplex attachment to originally uniform membranes and vanish after initiation of polynucleotide release. The latter effect is likely related to phosphatidyleserine redistribution in membranes due to lipoplex binding. Real time tracking of single DOTAP/DOPE and DOTAP/DOPC lipoplexes showed that both particles remained compact and associated with membranes up to 1-2 min before fusion, indicating that a more complex mechanism, different from suggested earlier rapid fusion, promotes more efficient transfection by DOTAP/DOPE complexes.     

New effects in polynucleotide release from cationic lipid carriers revealed by confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking

We report on new insights into the mechanisms of short single and double stranded oligonucleotide release from cationic lipid complexes (lipoplexes), used in gene therapy. Specifically, we modeled endosomal membranes using giant unilamellar vesicles and investigated the roles of various individual cellular phospholipids in interaction with lipoplexes. Our approach uses a combination of confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking, revealing several new aspects of the release: (a) phosphatidylserine and phosphatidylethanolamine are equally active in disassembling lipoplexes, while phosphatidylcholine and sphingomyelin are inert; (b) in contrast to earlier findings, phosphatidylethanolamine alone, in the absence of anionic phosphatidylserine triggers extensive release; (c) a double-stranded DNA structure remains well preserved after release; (d) lipoplexes exhibited preferential binding to transient lipid domains, which appear at the onset of lipoplex attachment to originally uniform membranes and vanish after initiation of polynucleotide release. The latter effect is likely related to phosphatidyleserine redistribution in membranes due to lipoplex binding. Real time tracking of single DOTAP/DOPE and DOTAP/DOPC lipoplexes showed that both particles remained compact and associated with membranes up to 1-2 min before fusion, indicating that a more complex mechanism, different from suggested earlier rapid fusion, promotes more efficient transfection by DOTAP/DOPE complexes.     

Effect of “helper lipid” on lipoplex electrostatics

Lipoplexes, which are complexes between cationic liposomes (L⁺) and nucleic acids, are commonly used as a nucleic acid delivery system in vitro and in vivo. This study aimed to better characterize cationic liposome and lipoplex electrostatics, which seems to play a major role in the formation and the performance of lipoplexes in vitro and in vivo. We characterized lipoplexes based on two commonly used monocationic lipids, DOTAP and DMRIE, and one polycationic lipid, DOSPA—each with and without helper lipid (cholesterol or DOPE). Electrical surface potential (Ψ₀) and surface pH were determined using several surface pH-sensitive fluorophores attached either to a one-chain lipid (4-heptadecyl hydroxycoumarin (C17HC)) or to the primary amino group of the two-chain lipids (1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-carboxyfluorescein (CFPE) and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-7-hydroxycoumarin) (HC-DOPE). Zeta potentials of the DOTAP-based cationic liposomes and lipoplexes were compared with Ψ₀ determined using C17HC. The location and relatively low sensitivity of fluorescein to pH changes explains why CFPE is the least efficient in quantifying the differences between the various cationic liposomes and lipoplexes used in this study. The fact that, for all cationic liposomes studied, those containing DOPE as helper lipid have the least positive Ψ₀ indicates neutralization of the cationic charge by the negatively-charged phosphodiester of the DOPE. Zeta potential is much less positively charged than Ψ₀ determined by C17HC. The electrostatics affects size changes that occurred to the cationic liposomes upon lipoplex formation. The largest size increase (based on static light scattering measurements) for all formulations occurred at DNA⁻/L⁺ charge ratios 0.5-1. Comparing the use of the one-chain C17HC and the two-chain HC-DOPE for monitoring lipoplex electrostatics reveals that both are suitable, as long as there is no serum (or other lipidic assemblies) present in the medium; in the latter case, only the two-chain HC-DOPE gives reliable results. Increasing NaCl concentrations decrease surface potential. Neutralization by DNA is reduced in a NaCl-concentration-dependent manner.     

In vitro lipofection with novel asymmetric series of 1,2-dialkoylamidopropane-based cytofectins containing single symmetric bis-(2-dimethylaminoethane) polar headgroups

Novel N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl[bis-(2-dimethylaminoethane)] bivalent cationic lipids were synthesized and evaluated for in vitro transfection activity against a murine melanoma cell line. In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), only the dioleoyl derivative 22 (1,2lb5) elicited transfection activity. The transfection activity of this lipid was reduced when formulated with DOPE. Contrary to that, the dimyristoyl derivative 19 (1,2lb2) mediated no activity when used alone but induced the highest levels of marker gene expression in the presence of DOPE. In an effort to correlate the transfection activity with cationic lipid structures, the physicochemical properties of cationic lipids in isolation and of lipoplexes were studied with surface tensiometry, photon correlation spectroscopy, gel electrophoresis mobility shift assay, and fluorescence techniques. In regard to the lipoplex properties, gel electrophoresis mobility shift assay and EtBr exclusion fluorescence assay revealed that the 1,2lb5 was the only lipid to associate and condense plasmid DNA, respectively. Photon correlation spectroscopy analysis found that 1,2lb5/DNA complexes were of relatively small size compared to all other lipoplexes. With respect to the properties of isolated lipids, Langmuir monolayer studies and fluorescence anisotropy on cationic lipid dispersions verified high two-plane elasticity and increased fluidity of the transfection competent dioleoyl derivative 1,2lb5, respectively. The results indicate that high transfection activity is mediated by cationic lipids characterized by an expanded mean molecular area, high molecular elasticity, and increased fluidity.