A Mathematical Treatment of Multiple Intermittent Intravenous Infusions in a One-Compartment Model.

BACKGROUND AND OBJECTIVE: Compartment models in pharmacokinetics are generally described for a single dose and can only simulate drug concentration as a function of the first dosing interval. Our objective was to create a one-compartment model for constant rate repetitive intravenous intermittent infusions where drug concentration can be simulated as a function of real time. METHODS: The analytical solutions to differential equations that were set for the time periods of drug infusion and elimination after an intermittent intravenous infusion were used to derive sequences patterns and determine the partial sums of mathematical series for multiple intermittent infusion doses. RESULTS: The model's original theory was supplemented with explicit solutions to the concentration and AUC, at non steady state conditions, with and without a loading dose, for both the infusion and elimination time periods after repetitive intermittent intravenous infusions. The validity and accuracy of these formulas in calculating drug concentration and AUC was verified by mathematical proofs, numerical methods and the principle of superposition. CONCLUSIONS: Drug concentration and AUC can be simulated using the newly described one-compartment pharmacokinetic model in the entire time-domain of therapy after multiple and repetitive intermittent intravenous infusions and not just within the first dosing interval.

Physicochemical and biological characterization of 1,2-dialkoylamidopropane-based lipoplexes for gene delivery.

Elucidation of the molecular and formulation requirements for efficient lipofection is a prerequisite to enhance the biological activity of cationic lipid-mediated gene delivery systems. To this end, the in vitro lipofection activity of the ionizable asymmetric 1,2-dialkoylamidopropane-based derivatives bearing a single primary amine group as the cationic head group was evaluated. The electrostatic interactions of these cationic lipids with plasmid DNA in serum-free medium were investigated by means of gel electrophoresis retardation and Eth-Br quenching assays. The effect of the inclusion of the helper lipid DOPE in the formulation on these interactions was also considered. The physicochemical properties of these lipids in terms of bilayer fluidity and extent of ionization were investigated using fluorescence anisotropy and surface potential techniques, respectively. The results showed that only the active lipid, 1,2lmp[5], existed in a liquid crystalline state at physiological temperature. Moreover, the extent of ionization of this lipid in assemblies was significantly higher that it's saturated analogues. Inclusion of the helper lipid DOPE improved the encapsulation and association between 1,2lmp[5] and plasmid DNA, which was reflected by the significant boost of lipofection activity of the 1,2lmp[5]/DOPE formulation as compared to the lipid alone. In conclusion, membrane fluidity and sufficient protonation of ionizable cationic lipid are required for efficient association and encapsulation of plasmid DNA and elicit of improved in vitro lipofection activity.

Physicochemical characterization of 9-aminocamptothecin in aqueous solutions.

The present manuscript provides a detailed physicochemical and thermodynamic characterization of 9-aminocamptothecin (9AC) which can be used as a tool to develop novel formulation strategies for optimum pharmacological activity. The pKa of 9AC was determined to be 2.43 at 37°C, while the basicity of quinoline nitrogen of 9AC was found to decrease with increasing temperature due to a positive enthalpy of deprotonation of 10.36 kJ mol(-1). The equilibrium solubility as well as the intrinsic solubility of the drug was found to increase with increasing temperature and decreasing pH. The enthalpies of solution of unionized and ionized forms of 9AC obtained from isothermal and iso-pH equilibrium solubility measurements were found to be 36.01 and 24.72 kJ mol(-1), respectively. Equilibrium hydrolysis studies revealed the hydrolytic susceptibility of 9AC with only 14% of active lactone species remaining at physiological pH 7.4. The intrinsic partition coefficient log P of the free base, 9AC-lactone, was estimated to be 1.28 (a characteristic of molecules suitable for oral absorption). The estimated pKa and log P values of 9AC, combined with its increased solubility at lower pH, are features that can be utilized to develop novel drug delivery systems to optimize the antitumor activity of 9AC.

Physicochemical characterization, solubilization, and stabilization of 9-nitrocamptothecin using pluronic block copolymers.

Solid-state properties and physicochemical characteristics of 9-nitrocamptothecin (9NC) were investigated with a view of molecular and bulk level understanding of its poor aqueous solubility and hydrolytic instability that prevent efficient drug delivery and pharmacological activity. 9NC bulk drug substance was found to be a nonhygroscopic, yellowish crystalline solid with long rectangular prism-shaped particle morphology and a sharp melting point at 264°C. Hydrolysis of 9NC-lactone occurs above pH 4, whereas complete conversion of lactone to carboxylate was recorded above pH 8. At saturated conditions, appreciable concentrations of 9NC-lactone were detected at pH as high as 11. 9NC undergoes oxidation in the presence of dimethyl sulfoxide with formation of 9NC-N-oxide. The total solubility of lactone and carboxylate forms of 9NC in deionized water was found to be less than 5 µg/mL, whereas the solubility of 9NC-lactone in aqueous acidic media was determined to be approximately 2.5 µg/mL. Incorporation of 10% pluronic copolymers P123, F127, and F68 in 10 mM HCl increased 9NC solubility by 13-fold, eightfold, and fivefold, respectively. The thermodynamic stability of drug-loaded pluronic micelles was evaluated under isothermal variable volume conditions and found F127, among all poloxamers, to offer the best hydrolytic protection efficacy for 9NC.

Kinetic and thermodynamic studies of 9-aminocamptothecin hydrolysis at physiological pH in the presence of human serum albumin.

As a first step towards improving the aqueous stability of 9-aminocamptothecin (9AC), a detailed kinetic and thermodynamic investigation of the hydrolysis reaction of 9AC was carried out, using a first derivative absorption spectrophotometry technique. It was found that 9AC-lactone decayed with a half-life of 25 min in PBS at pH 7.4 and 310.15K. The activation energy (Ea) associated with the hydrolysis of 9AC-lactone was 87.3 ± 3.8 kJmol(-1), whereas the positive enthalpy and entropy values of the 9AC-lactone hydrolysis reaction indicated that the reaction is endothermic and entropically driven. Similarly to other camptothecin analogs, except for SN38, the activation energy for 9AC-lactone hydrolysis in the presence of Human Serum Albumin (HSA) was about 10 kJmol(-1) lower than that determined in plain PBS, whereas the equilibrium 9AC-lactone concentration was decreased in the presence of HSA as compared to that in plain PBS. The lower Ea for 9AC hydrolysis in presence of HSA fully explained the shift of lactone-carboxylate equilibrium towards the carboxylate form with only 4% of active 9AC-lactone remaining in the presence of HSA under physiological conditions. Finally, affinity studies of several camptothecin analogs with HSA, showed that the association constants of the lactone species with HSA are similar and pointed out that the superior stability of the SN38 over the other two analogs (9AC and 9-nitrocamptothecin) is not due to the higher affinity of lactone toward HSA, but it is rather due to the lower affinity of the SN38-carboxylate toward HSA.

Physicochemical characteristics associated with transfection of cationic cholesterol-based gene delivery vectors in the presence of DOPE.

The physicochemical properties of a novel series of cholesterol-based cationic lipids in the presence of DOPE were studied by various techniques in an effort to correlate cationic lipid structure with transfection efficacy. It was found that while DOPE improves the ß-gal activity of the active AC and MC derivatives, the overall zeta potential of the particles, pDNA complexation and condensation is not improved. This is in stark contrast with the tertiary amine derivative DC whose dispersion properties were improved and its monolayer surface potential is restored at high molecular surface density in the presence of DOPE. Overall the transfection activity mediated by DC and the quaternary ammonium TC derivative was greatly improved in the presence of DOPE and is attributed to decreased cytotoxicity, improved fusogenicity and cellular association.

From gene delivery to gene silencing: plasmid DNA-transfecting cationic lipid 1,3-dimyristoylamidopropane-2-[bis(2-dimethylaminoethane)] carbamate efficiently promotes small interfering RNA-induced RNA interference.

The cationic lipid 1,3-dimyristoylamidopropane-2-[bis(2-dimethylaminoethane)] carbamate (1,3lb2) was applied as a delivery system for small interfering RNA (siRNA) to inhibit the production of vascular endothelial growth factor (VEGF) in vitro in human prostate carcinoma cell line PC-3. VEGF protein silencing peaked at 94% when cationic lipid-nucleic acid complexes (lipoplexes) were formulated at a nitrogen:phosphorothioate ratio (N:P) of 2 with a dose concentration of 53.7 nM, and the performance of these lipoplexes was not impeded by serum. Knockdown efficiency was maintained for at least 72 h, and an IC(50) of 12 nM lasted for 48 h. Only 20% of the total siRNA became cell-associated at this N:P, at a rate of 25 ng/h. Lipoplexes of the optimal formulation were relatively monodisperse, having an average diameter of 634 nm and a zeta potential of -21.3 mV. Formation of the 1,3lb2-siRNA complex reached 94% at an N:P of 2 and was positively cooperative; the binding constant was calculated in the range of 10(5) M(-1), and a Hill coefficient of 3 was determined. 1,3lb2 was found to be a nontoxic and potent carrier of siRNA that binds to the nucleic acid effectively and whose lipoplexes promote long-lasting inhibition, have high biological activity at low N:Ps, and are functional in the presence of serum.

Thermodynamic studies and loading of 7-ethyl-10-hydroxycamptothecin into mesoporous silica particles MCM-41 in strongly acidic solutions.

A major limitation of the clinical efficacy of the 7-ethyl-10-hydroxycamptothecin (SN38) therapy is its facile conversion of the active lactone form into a less active carboxylate species at physiological pH and limited aqueous solubility. The present manuscript embodies a detailed description of several physicochemical properties of SN38 and further details the thermodynamic basis for its poor aqueous solubility. The ionization and increased solubility of the drug in highly acidic media were subsequently employed to efficiently load the positively charged drug in its biologically active lactone form into mesoporous silica material of type MCM-41, achieving a maximum loading of 250 mg of SN38 per gram of silicate. It was also found that the equilibrium association constant K(A) varies with the extent of drug adsorption. At low and high drug load, corresponding to one SN38 molecule bound for every 70 and 13 -SiO(2)-, K(A) was determined to be 1253.5 and 127.39 M(-1), respectively.

The interaction energies of cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine in spread mixed monolayers at the air-water interface.

The interaction of cholesterol with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was investigated in insoluble miscible mixed monolayers at the air-water interface using a Langmuir balance technique. The strong condensation effects observed at all compositions were quantified on the basis of excess thermodynamic properties of the system. It was found that partial molar areas and work of compression of cholesterol in the mixed monolayers were greatly reduced and increased, respectively, at xDOPE of 0.8, while, in accord with the "umbrella model", the character of cholesterol monolayers was drastically affected even at mole fractions of DOPE as low as 0.2. Calculated Gibbs free energies of mixing were shown to be symmetric about equimolar lipid quantities and considerably decreased at high surface pressures. Interaction energy parameters calculated from values of excess Gibbs energy are found to decrease linearly with surface pressure at a rate of 100 kT m.N(-1), regardless of composition. All evidence points out that cholesterol-DOPE molecular interactions can be adequately simulated using a simple regular mixture model.

Physicochemical characterization and membrane binding properties of camptothecin.

The intrinsic solubility of the CPT-lactone free base in acidic pH was determined to be 3.44 and 5.11 microM at 22 and 37 degrees C, respectively. The equilibrium solubility of the drug was found to increase with increasing temperature and decreasing pH. The enhanced solubility of the drug at very low pH is attributed to the protonation of the nitrogen atom in the ring B and the increased solvency of the highly concentrated acidic media. The logarithmic value of the intrinsic partition coefficient P of the free base CPT-lactone form was estimated to be 1.65, characteristic of a molecule suitable for absorption. Association constants K(f) of the drug for bilayers composed of the zwitterionic (DOPC) and the negatively charged (DOPG) were determined at acidic pH by fluorescence anisotropy to be 35.4 +/- 4.5 M(-1) and 93.1 +/- 11.0 M(-1), respectively, indicative of the CPT tendency to preferentially bind to negatively charged membranes. The results indicate that at highly acidic media, CPT is positively charged and exists at its stable lactone form of increased solubility and capacity to bind to negatively charged membranes. These features could be used to invent novel formulation strategies to optimize the antitumor activity of CPT.

Structure-transfection activity studies of novel cationic cholesterol-based amphiphiles.

Inclusion of DOPE in lipoplex formulations has hampered the establishment of a correlation between cationic lipid structure, biological specificity, and transfection activity, simply because the presence of a helper lipid not only alters the physicochemical properties of the lipoplex but also modifies cell surface specific interactions during the process of transfection. To this end, four cationic cholesterol-based derivatives were synthesized by systematically varying the methylation of the polar headgroup, after which the physicochemical properties, in the absence of DOPE and serum, were correlated with their transfection activity and interaction with cell membranes. It was found that only the primary and secondary amine derivatives, AC-Chol and MC-Chol, respectively, are able to mediate in vitro cell transfection. These results were consistent with fusion experiments and cell internalization studies which illustrated that although cell surface binding occurs for all of the cationic lipids, only the active analogues were able to gain entry into the cytosol. Given the minute differences in the physical properties of these cationic derivatives, we speculate that the biological specificity of the active cationic derivatives either triggers endocytotic pathways leading to eventual endosomal fusion allowing cytoplasmic access to the packaged DNA or other endocytotic pathways that avoid lysosomal degradation.

Uncertainty analysis of drug concentration in pharmaceutical mixtures.

Using a Taylor expansion to first order, a novel method was developed to calculate the uncertainty of drug concentration in pharmaceutical dosage forms. The method allows, in principle, calculation of the maximum potential error in drug concentration in a mixture composed of an infinite number of ingredients that are measured on multiple balances of variable sensitivity requirements.

The Conventional Langmuir Trough Technique as a Convenient Means to Determine the Solubility of Sparingly Soluble Surface Active Molecules: Case Study Cholesterol.

The feasibility of a method based on mass preservation [G. Schwarz, J. Zhang, Chem. Phys. Lipids, 110 (2001) 35-45] to determine the solubility of Cholesterol in water from monomolecular films on air/water interface was investigated. Using a mass balance equation, it was found that Cholesterol undergoes an exponential desorption at very low surface pressures followed by an almost linear desorption into the subphase at higher surface pressures until monolayer collapse. Processing of the surface pressure measurements as a function of trough area in accord with the theory, enabled the accurate determination of the molecular dimensions of Cholesterol as a function of surface pressure. Slight modification of the theory enabled accurate quantification of the surface pressure-independent apparent solubility of Cholesterol and the amount of Cholesterol desorbed into the subphase as a function of surface pressure, in the nanomolar range.

Novel N,N '-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids for gene delivery--synthesis, in vitro transfection activity, and physicochemical characterization.

Novel N,N'-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids were synthesized and their physicochemical properties in lamellar assemblies with and without plasmid DNA were evaluated to elucidate the structural requirements of these double-chained pH-sensitive surfactants for potent non-viral gene delivery and expression. The highest in vitro transfection efficacies were induced at +/-4:1 by the dimyristoyl, dipalmitoyl and dioleoyl derivatives 1,3lb2, 1,3lb3 and 1,3lb5, respectively, without inclusion of helper lipids. Transfection activities were reduced in the presence of either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine alone or in combination with cholesterol for all derivatives except 1,3lb5, which maintained reporter gene expression levels at +/-4:1 and yielded increased lipofection activity at a lower charge ratio of +/-2:1. Ethidium bromide displacement indicated efficient plasmid DNA binding and compaction by the transfection-competent analogs. Dynamic light-scattering and electrophoretic mobility studies revealed lipoplexes of the active lipids with large particle sizes (mean diameter>or=500 nm) and zeta potentials with positive values (low ionic strength) or below neutrality (high ionic strength). Langmuir film balance studies showed high in-plane elasticity of these derivatives in isolation. In agreement with the monolayer experiments, fluorescence polarization studies verified the fluid nature of the highly transfection-efficient amphiphiles, with gel-to-liquid crystalline phase transitions below physiological temperature. The active compounds also interacted with endosome-mimicking vesicles to a greater extent than the poorly active derivative 1,3lb4, as revealed by fluorescence resonance energy transfer experiments. Taken together, the results suggest that well-hydrated and highly elastic cationic lipids with increased acyl chain fluidity and minimal cytotoxicity elicit high transfection activity.

Effect of spacer attachment sites and pH-sensitive headgroup expansion on cationic lipid-mediated gene delivery of three novel myristoyl derivatives.

The transfection activity and physicochemical properties of the dimyristoyl derivatives from three novel series of double-chained tertiary cationic lipids were compared. Two of the derivatives were constructed as isomers with different linkages of the same bis-(2-dimethylaminoethane) polar headgroup and hydrophobic chains to the diaminopropanol backbone, while the third was designed with a hydrophilic region containing only a single ionizable amine group. Such systematic molecular changes offer a great opportunity to delineate factors critical for transfection activity, which in this work include the intramolecular distance between the hydrophobic chains and pH-expandability of the polar headgroup. The physical studies comprised a variety of techniques, including pKa determination, Langmuir monolayer studies, fluorescence anisotropy, gel electrophoresis mobility shift assay, ethidium bromide displacement assay, particle size distribution, and zeta potential. These studies are crucial in the development of lipid-based gene delivery systems with improved efficacy. Physicochemical characterization revealed that a symmetric bivalent pH-expandable polar headgroup in combination with greater intramolecular space between the hydrophobic chains provide for high transfection activity through efficient binding and compaction of pDNA, increased acyl chain fluidity, and high molecular elasticity.

Physicochemical properties affecting lipofection potency of a new series of 1,2-dialkoylamidopropane-based cationic lipids.

The in vitro transfection activity of a novel series of N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl-(aminoethane) derivatives was evaluated against a mouse melanoma cell line at different +/- charge ratios, in the presence and absence of helper lipids. Only the unsaturated derivative N,N'-dioleoyl-1,2-diaminopropyl-3-carbamoyl-(aminoethane), (1,2lmp[5]) mediated significant increase in the reporter gene level which was significantly boosted in the presence of DOPE peaking at +/- charge ratio of 2. The electrostatic interactions between the cationic liposomes and plasmid DNA were investigated by gel electrophoresis, fluorescence spectroscopy, dynamic light scattering and electrophoretic mobility techniques. In agreement with the transfection results, 1,2lmp[5]/DOPE formulation was most efficient in associating with and retarding DNA migration. The improved association between the dioleoyl derivative and DNA was further confirmed by ethidium bromide displacement assay and particle size distribution analysis of the lipoplexes. Differential scanning calorimetry studies showed that 1,2lmp[5] was the only lipid that exhibited a main phase transition below 37 degrees C. Likewise, 1,2lmp[5] was the only lipid found to form all liquid expanded monolayers at 23 degrees C. In conclusion, the current findings suggest that high in vitro transfection activity is mediated by cationic lipids characterized by increased acyl chain fluidity and high interfacial elasticity.

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.

Correlation of the physicochemical properties of symmetric 1,3-dialkoylamidopropane-based cationic lipids containing single primary and tertiary amine polar head groups with in vitro transfection activity.

The physicochemical properties of a novel series of symmetric 1,3-dialkylamidopropane-based cationic amphiphiles [M. Sheikh, J. Feig, B. Gee, S. Li, M. Savva, In vitro lipofection with novel series of symmetric 1,3-dialkoylamidopropane-based cationic surfactants containing single primary and tertiary amine polar head groups, Chem. Phys. Lipids 124 (2003) 49-61] were studied by several techniques, in an effort to correlate cationic lipid structure with transfection efficacy. It was found that only the unsubstituted amine and tertiary amine dioleoyl derivatives 1,3lmp5 and 1,3lmt5, respectively, mediated in vitro transfection activity in the absence of helper lipids. This activity pattern was consistent with ethidium bromide fluorescence quenching studies, which indicated that only these two derivatives bound to and efficiently condense plasmid DNA at physiological pH. Dynamic light scattering indicated that lipoplexes made by these two cationic lipids were relatively small particles below 1 microm, in sharp contrast to lipoplexes bigger than 3 microm composed of saturated cationic derivatives. Transmission electron microscopy studies clearly indicated that cationic lipid dispersions made by saturated derivatives form multilamellar tubules at physiological pH. Calorimetric studies showed that cationic amphiphiles with saturated acyl chains longer than 12 carbons exhibit solid-to-liquid crystalline phase transitions above 37 degrees C. In agreement with the microscopy and calorimetry studies, Langmuir film balance experiments indicated that saturated derivatives with hydrophobic chains longer that 12 carbons are not well hydrated and exist at a chain-ordered state at ambient temperature. Calculation of compressibility moduli from monolayer compression isotherms at 23 degrees C suggested that monolayers made by cationic lipids bearing saturated acyl chains are less compressible relative to those of the dioleoyl derivatives 1,3lmp5 and 1,3lmt5. In conclusion, high hydration, increased fluidity and high elasticity of cationic lipid assemblies in isolation, all correlate with high in vitro transfection activity.

Synthesis, in vitro transfection activity and physicochemical characterization of novel N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) amphiphilic derivatives.

A novel series of N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) cationic derivatives was synthesized and screened for in vitro transfection activity at different charge ratios in the presence and absence of the helper lipids DOPE and cholesterol. Physicochemical properties of lipid-DNA complexes were studied by gel electrophoresis, fluorescence spectroscopy and dynamic light scattering. The interfacial properties of the lipids in isolation were studied using the Langmuir film balance technique at 23 degrees C. It was found that only lipoplexes formulated with the dioleoyl derivative, 1,2lmt[5], mediated significant in vitro transfection activity. Optimum activity was obtained with 1,2lmt[5]/DOPE mixture at a +/-charge ratio of 2. In agreement with the transfection results, 1,2lmt[5] was the only lipid found to complex and retard DNA migration as verified by gel electrophoresis. Despite the efficient complexation, no significant condensation of plasmid DNA was observed as indicated by fluorescence spectroscopy measurements. Monolayer studies showed that the dioleoyl derivative 1,2lmt[5] was the only lipid that existed in an all liquid-expanded state with a collapse area and collapse pressure of 59.5 A2 and 38.7 mN/m, respectively. This lipid was also found to have the highest elasticity with a compressibility modulus at monolayer collapse of 80.4 mN/m. In conclusion, increased acyl chain fluidity and high molecular elasticity of cationic lipids were found to correlate with improved transfection activity.

In vitro lipofection with novel series of symmetric 1,3-dialkoylamidopropane-based cationic surfactants containing single primary and tertiary amine polar head groups.

A novel series of symmetric double-chained primary and tertiary 1,3-dialkoylamido monovalent cationic lipids were synthesized and evaluated for their transfection activities. In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), only the primary and tertiary dioleoyl derivatives 1,3lmp5 and 1,3lmt5, respectively elicited transfection activity. This is a striking difference between symmetrical 1,2-diacyl glycerol-based monovalent cationic lipids that always found both dioleoyl and dimyristoyl analogues being efficient transfection reagents. In the presence of helper lipid, all cationic derivatives induced marker gene expression, except the dilauroyl analogues 1,3lmp1 and 1,3lmt1 that elicited no transfection activity. Combining electrophoretic mobility data of the lipoplexes at different charge ratios with transfection activity suggested two requirements for high transfection activity with monovalent double-chained cationic lipids, that is, binding/association of the lipid to the plasmid DNA and membrane fusion properties of the lipid layers surrounding the DNA.