Novel N,N ¢-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids for gene delivery – synthesis, in vitro transfection activity, and physicochemical characterization Michael Spelios and Michalakis Savva Division of Pharmaceutical Sciences, Arnold & Marie Schwartz College of Pharmacy & Health Sciences, Long Island University, Brooklyn, NY, USA Keywords cationic lipid; elasticity; FRET; gene delivery; lipoplex Correspondence M Savva, Division of Pharmaceutical Sciences, Arnold & Marie Schwartz College of Pharmacy & Health Sciences, Long Island University, 75 DeKalb Avenue, Brooklyn, NY 11201, USA Fax: +1 718 780 4586 Tel: +1 718 488 1471 E-mail: msavva@liu.edu (Received 23 September 2007, revised November 2007, accepted November 2007) doi:10.1111/j.1742-4658.2007.06185.x 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 + ⁄ ) : 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 + ⁄ ) : and yielded increased lipofection activity at a lower charge ratio of + ⁄ ) : 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 ‡ 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 endosomemimicking 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 The development of highly potent and minimally toxic cationic lipids for nucleic acid delivery depends on generation of meaningful structure–activity relation- ships The rational design of efficacious transfection amphiphiles is based on understanding the impact of each of the lipid structural components on gene Abbreviations DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOTAP, 1,2-dioleoyl-3trimethylammonium-propane; DPH, 1,6-diphenyl-1,3,5-hexatriene; EGFP, enhanced green fluorescent protein; EtBr, ethidium bromide; FITC, fluoroscein isothiocyanate; FRET, fluorescence resonance energy transfer; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NBD-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl); ONPG, 2-nitrophenyl b-D-galacto pyranoside; PA, 1,2-dipalmitoyl-sn-glycero-3-phosphate; PC, L-a-phosphatidylcholine (egg, chicken); pDNA, plasmid DNA; Rh-PE, 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-(lissamine rhodamine B sulfonyl); SFM, serum-free medium; TNS, 2-(p-toluidino)naphthalene-6-sulfonic acid 148 FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS M Spelios and M Savva transfer, namely the polar headgroup, the nonpolar tail (either a cholesterol moiety or a pair of aliphatic hydrocarbon chains), and the linker tethering both regions together Since the advent of lipofection 20 years ago [1], cationic lipids with new molecular architectures have been developed and analyzed as gene-delivery vectors, as seen in numerous recent publications Liu et al [2] synthesized a series of 16 carbamate-linked cationic lipids, differing in their hydrocarbon chain length, quaternary ammonium head and counter-ion species, and examined their biological performance Spacer modifications were studied in cholesterol-based and aliphatic gemini cationic lipids to determine their effects on the transfecting abilities of these dimeric surfactants [3–5] Rajesh et al [6] were the first to report the influence of linker orientation reversal on the transfection efficiencies and physicochemical properties of two cationic amphiphiles with identical hydrophilic and hydrophobic constituents Other groups have also recently described the design, syntheses, physicochemical characterization and transfection properties of novel cationic amphiphiles [7–11] In an effort to further delineate the structural properties of these lipofection reagents that confer superior transfection activity, a novel series of N,N¢-diacyl-1,3diaminopropyl-2-carbamoyl bivalent cationic lipids was synthesized containing a symmetric bis-[2-dimethylamino-ethyl]-amine polar headgroup at the 2-position and hydrophobic chains at the 1- and 3- positions of the 1,3-diamino-2-propanol backbone (Fig 1) The series, designated 1,3lb, consists of four saturated lipids, ranging in chain length from 12 to 18 carbons, and a single monounsaturated derivative with a double bond between the 9th and 10th carbons of each 18-carbon chain Physicochemical characterization of the cationic lipids in lamellar assemblies with and without plasmid DNA and in media of various ionic strengths comprised a variety of studies and techniques, including pKa determination, isothermal monolayer compression, fluorescence anisotropy, ethidium bromide displacement, dynamic light scattering, determination of zeta potential, and fluorescence resonance energy transfer (FRET), and is indispensable for elucidating the structural properties of these amphiphiles that induce high transfection activity This work is a continuation of a recent study highlighting the superior gene delivery mediated by the dimyristoyl derivative 1,3lb2 from the aforementioned series as compared to two other cationic lipid vectors, a conformational isomer and a monovalent analog [12] It was determined that a symmetrical bivalent pH-expandable polar headgroup, in combination with greater intramolecular space between the hydrophobic Novel cytofectins for gene delivery H3C H3C N CH3 N H3C N O O O NH O NH R R The R group varies with the derivative: C11H23 for dilauroyl (1,3lb1) C13H27 for dimyristoyl (1,3lb2) C15H31 for dipalmitoyl (1,3lb3) C17H35 for distearoyl (1,3lb4) C17H31 for dioleoyl (1,3lb5) Fig Structure of the 1,3lb derivatives chains, promotes highly efficacious in vitro lipofection through efficient binding and compaction of pDNA, increased acyl chain fluidity and high molecular elasticity The current study is a further examination of the 1,3lb cytofectin involving systematic molecular changes; specifically, determination of the effects of hydrophobic chain length and degree of unsaturation on target gene expression Results Biological analysis Lipoplexes of 1,3lb cationic lipids with and without helper lipid(s) were examined at various + ⁄ ) charge ratios for transfection activity The shortest saturated chain derivative 1,3lb1 was completely inefficient at promoting lipofection at all charge ratios, both in the absence and presence of neutral colipid(s) Formulations lacking either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or phospholipid and cholesterol induced the highest levels of reporter gene expression at + ⁄ ) : 1, the exception being 1,3lb4 which exhibited low activity throughout the range of charge ratios tested (Fig 2A) The dipalmitoyl derivative 1,3lb3 elicited higher in vitro transfection activity than 1,3lb5, contrary to findings that unsaturated derivatives are typically the FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS 149 Novel cytofectins for gene delivery M Spelios and M Savva β-gal concentration (mU/well) A 1100 1,3lb2 1000 900 800 700 600 500 400 300 200 100 1,3lb3 1,3lb4 1,3lb5 DOTAP +/– charge ratio β-gal concentration (mU/well) B 1100 1,3lb2/D 1000 900 800 700 600 500 400 300 200 100 1,3lb3/D 1,3lb4/D 1,3lb5/D DOTAP +/– charge ratio β-gal concentration (mU/well) C 1100 1,3lb2/D/c 1000 900 800 700 600 500 400 300 200 100 1,3lb3/D/c 1,3lb4/D/c 1,3lb5/D/c DOTAP +/– charge ratio Fig In vitro transfection activity of cationic lipids in the absence of helper lipid(s) (A) and in the presence of DOPE (B) or DOPE and cholesterol (C), as measured in a murine skin cell line (B16-F0 melanoma cells) by ONPG assay (n = 3) most transfection-competent [13–15] The activity of b-galactosidase was approximately two- to threefold greater than with 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP)-mediated gene delivery Qualitative analysis of transfection efficiency by detection of enhance green fluorescent protein (EGFP), as seen in 150 Fig 3, revealed the same activity trends as quantitatively determined by the 2-nitrophenyl b-d-galacto pyranoside (ONPG) assay: 1,3lb3 > 1,3lb2 $ 1,3lb5 > DOTAP > 1,3lb4 Fluorescein covalently attached to plasmid allowed visual tracking of cellular uptake Internalization of exogenous nucleic acid occurred to the greatest extent with the aid of 1,3lb2, as indicated by the higher fluoroscein isothiocyanate (FITC)-plasmid DNA (pDNA) intensity (green) when compared to the fluorescence yields of labeled plasmid transported via other derivatives (Fig 4A) Lipoplexes formed with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl (Rh-PE)-labeled dispersions (red) of 1,3lb2 were visualized as distinctly yellow spots, suggesting well-associated complexes of nucleic acid and lipid (Fig 4B); overlaid FITC and rhodamine images of cells transfected using the other transfection-active analogs showed colocalization of plasmid and lipid to a similar degree (not shown) In accordance with these results, the dimyristoyl derivative was the most efficient of the transfection-active compounds at condensing pDNA as monitored by ethidium bromide (EtBr) displacement Images obtained after transfection with 1,3lb1 (results not shown) showed significantly fewer cells, and fluorescent patches where no cells were present, indicating large aggregates with a high affinity for the plate surface that were not internalized and are probably responsible for the elevated cytotoxicity In fact, except along the edges of the wells where cells were densely packed and multilayered, accounting for the 46% survival (data not shown), no viable cells were detected after exposure to 1,3lb1 Dynamic lightscattering studies revealed 1,3lb1-containing lipoplexes (+ ⁄ ) : 1) of the largest size with a mean diameter around lm Use of DOPE and cholesterol to enhance the genedelivery properties of cationic lipids has been extensively documented [16–21] For 1,3lb2 and 1,3lb3, transfection activity was appreciably reduced at the highest tested charge ratio by the incorporation of DOPE, falling below levels reported for 1,3lb5; a similar result was observed when cholesterol was added (Fig 2B,C) The lipofection efficiency of 1,3lb5 increased significantly at + ⁄ ) : 1, climbing above that of commercially available DOTAP, and rose moderately with the inclusion of cholesterol The distearoyl derivative continued to mediate low levels of transgene expression at all charge ratios, even after the addition of DOPE alone or in combination with cholesterol Increasing the + ⁄ ) charge ratio beyond : resulted in decreased transfection activity for the most active derivatives in all formulations (data not shown) FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS M Spelios and M Savva Novel cytofectins for gene delivery A D B E C F Fig Fluorescence of EGFP in B16-F0 cells transfected with lipoplexes of (A) 1,3lb1, (B) 1,3lb2, (C) 1,3lb3, (D) 1,3lb4 and (E) 1,3lb5 at ± : in the absence of helper lipid(s), and with (F) DOTAP at ± : Images were acquired at 10 · magnification MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction analysis revealed a low cytotoxicity of lipoplexes at all charge ratios and compositions, with cell viability greater than 60%, except for formulations containing 1,3lb1, which were poorly tolerated (data not shown) FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS 151 Novel cytofectins for gene delivery A 1,3lb2 M Spelios and M Savva B 1,3lb3 1,3lb4 1,3lb5 152 FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS M Spelios and M Savva Novel cytofectins for gene delivery Fig (A) Fluorescence images of B16-F0 cells treated with lipoplexes of FITC–pDNA (B) FITC (top), rhodamine (center) and overlaid fluorescence and brightfield (bottom) images of cells transfected using Rh-PE-labeled (1 mol%) dispersions of 1,3lb2 Lipoplexes were prepared at a charge ratio of ± : 1, and images were captured h after transfection at 20 and 10 · magnification for (A) and (B), respectively Changes in the membrane surface charge of pH-stable 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) ⁄ cholesterol vesicles containing mol% 1,3lb amphiphile were monitored by measuring the fluorescence intensity of 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS) in the lipid bilayers as a function of pH As expected, the pH titration curves (not shown) of the derivatives overlapped in accordance with their identical polar headgroup Curve fitting of the experimental data revealed a pKa between 7.1 and 7.4, indicating that only about 30–50% of the tertiary amine groups are charged at physiological pH (Table 1) Higher values were found for the isolated triamine (pKa1 = 8.959, pKa2 = 9.592) [22], and may be attributed to reduced hydration of the cationic lipids compared to the free amine, as well as tight packing of the hydrophobic chains, which promotes charge distribution over adjacent lipid molecules The pH titration curves were fitted to a modified version of the Henderson–Hasselbach equation, which contains an adjustable parameter C that affects the slope of the transition region In the original equation, this parameter is equal to for a univalent base (and -1 for a monoprotic acid) The pKa values listed in Table for the bivalent lipids are the mean of two acid dissociation constants, one for each of the ionizable amino groups, resulting in pH titration curves with slopes of lower steepness (C less than unity) The TNS assay was used to ascertain the pKa values of the cationic lipids in assemblies where they were Table Acid dissociation constants of cationic lipids as determined by nonlinear fitting of TNS fluorescence intensity–pH plots Lipid pKa Ca Coefficient of determinationb 1,3lb1 1,3lb2 1,3lb3 1,3lb4 1,3lb5 7.24 7.09 7.37 7.36 7.42 0.40 0.52 0.40 0.58 0.53 0.991 0.994 0.984 0.996 0.995 % ionization at pH 7.2 41 33 48 48 51 a C is an adjustable parameter affecting the slope of the transition region of the fitted pH titration curves, as calculated from Eqn (1) b Goodness-of-fit statistics for pKa were assessed within a 95% confidence interval 40 mM Tris, pH7.2 100 EtBr fluorescence (% of max.) pKa studies well separated from one another, precluding influences of the hydrophobic anchors of the derivatives with respect to the number of carbon atoms and double bonds in the aliphatic chains These pKa values deviate from those obtained using vesicles where the derivatives are in greater contact with each other, such as the dispersions under investigation, and van der Waals forces between adjacent cationic lipid molecules, as dictated by their hydrophobic chain length and degree of unsaturation, are a major contribution to the extent of the bis-[2-dimethylamino-ethyl]-amine polar headgroup hydration, and, subsequently, protonation Thus, the acid dissociation constants in Table are molecular descriptors of the derivatives and not necessarily offer insight into the differences in transfection activities 80 1,3lb1 1,3lb2 60 1,3lb3 1,3lb4 40 1,3lb5 20 0 0.2 0.4 0.6 0.8 1.2 1.4 1.6 1.8 2.2 2.4 +/– charge ratio SFM 100 EtBr fluorescence (% of max.) Physicochemical characterization 80 60 1,3lb1 1,3lb2 40 1,3lb3 1,3lb4 20 1,3lb5 0 0.4 0.8 1.2 1.6 2.4 +/– charge ratio 2.8 3.2 3.6 Fig Percentage ethidium bromide displacement against the charge ratio of lipoplexes in Tris buffer or SFM Parabolic curve fits of the experimental data (solid points) are shown as dashed lines FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS 153 Novel cytofectins for gene delivery M Spelios and M Savva Cationic lipid–pDNA binding studies Binding curves of EtBr-intercalated pDNA titrated with aliquots of cationic lipid dispersions are shown in Fig With respect to the saturated derivatives, there was a reduction in plasmid compaction efficiency with increasing hydrophobic chain length Interestingly, the transfection-inactive lipid 1,3lb1 was the most efficient at condensing pDNA, with complete charge neutralization of the negative charges of pDNA at about + ⁄ ) 2.3 in low-ionic-strength medium This finding is contradictory to previous work suggesting inefficient DNA condensation and dehydration by 12-carbon fatty acid chains [23] However, 1,3lb1 was also the most toxic to the cells, accounting for its transfection inactivity The monounsaturated derivative 1,3lb5 displayed intermediate nucleic acid condensation efficacy, with full EtBr exclusion at approximately + ⁄ ) 3.1 Increasing the ionic strength resulted in complete probe displacement at higher charge ratios for all lipids, but there was no effect on the binding trends Langmuir monolayer studies The surface properties of the cationic lipids were investigated using the Langmuir film balance technique Monolayers of the cationic lipids, with the exception of the distearoyl derivative, exist in an all liquidexpanded state at 23 °C Monolayer collapse occurred at lower mean molecular areas and higher surface pressures as the acyl chain length increased from 1,3lb1 to 1,3lb14 (Table 2) Tighter lipid packing associated with the additional van der Waals forces of longer hydrophobic chains more effectively excludes interstitial water and reduces surface tension, allowing a greater reduction in the available surface area of the monolayer prior to its collapse The dimyristoyl and dioleoyl derivatives 1,3lb2 and 1,3lb5, respectively, shared similar collapse parameters and comparable transfection activities (Fig 2A) The mixed-phase state of monolayers composed of the poorly transfection-efficient derivative 1,3lb4 is evidenced by an L1-to-L2 transition in the compression isotherm Specifically, liquid-expanded behavior was observed up to a surface pressure and ˚ mean molecular area of 24 mNỈm)1 and 70 A2, respectively, at which point a transition occurred to a chainordered phase The pressure continued to rise upon further surface area reduction until the monolayer ˚ finally collapsed at 49 mNỈm)1 and 39 A2 At 37 °C, the two-dimensional transition was absent and only a liquid-expanded state was evident For all derivatives, molecular dimensions increased and compression forces decreased at monolayer collapse in response to elevated temperature Differences between the monolayer collapse parameters of the cationic lipids were not as apparent at 37 °C; the derivatives possessed similar mean molecular areas at monolayer collapse, and their collapse pressures were nearly identical Molecular elasticity is correlated with transfection activity, and was assessed by first-derivative analysis of the p–A isotherm; the smaller the slope at monolayer collapse (dp ⁄ dAc), the higher the compressibility The value of dp ⁄ dAc was highest for 1,3lb4 at both 23 °C and 37 °C, indicative of the lowest in-plane elasticity, and lipoplexes containing the distearoyl analog concomitantly generated minimum reporter gene expression (Fig 2A) The monounsaturated 1,3lb5 was found to be the most elastic at 23 °C, with a dp ⁄ dAc value of ˚ mNỈm)1ỈA)2 Phase-transition temperature studies DPH (1,6-diphenyl-1,3,5-hexatriene) was used to probe for cationic lipid bilayer phase changes by monitoring the depolarization of fluorescence of the extrinsic fluorophore in response to temperature The shortest saturated chain derivative 1,3lb1 and the monounsaturated analog 1,3lb5 displayed the most fluid behavior, Table Monolayer transitiona and collapse parameters of the 1,3lb series Measurements were performed using Tris buffer (40 mM, pH 7.2) as the subphase Values reported are the mean of n experiments ± standard deviation ˚ Mean molecular area (A2) 23 °C 1,3lb1 1,3lb2 1,3lb3 1,3lb4 (n (n (n (n = = = = 4,6) 4,3) 3,4) 11,6) 1,3lb5 (n = 7,7) a p (mNỈm)1) 23 °C 59.27 55.98 46.91 38.70 70.49 54.08 37 °C ± ± ± ± ± ± 4.89 2.55 1.39 3.03 3.43a 3.30 64.90 60.61 55.61 56.06 ± ± ± ± 3.22 2.29 3.41 3.81 57.92 ± 6.41 37.75 41.62 40.29 48.68 23.73 39.04 37 °C ± ± ± ± ± ± 2.01 0.94 0.72 6.71 1.09a 3.81 Phase stateb dp ⁄ dAc 36.59 36.31 38.62 39.89 23 °C ± ± ± ± 1.34 1.37 0.75 0.80 36.82 ± 2.54 1.27 1.55 1.32 2.29 ± ± ± ± 37 °C 0.07 0.10 0.04 0.51 1.00 ± 0.17 1.13 1.04 1.11 1.20 ± ± ± ± 23 °C 0.08 0.04 0.03 0.11 1.20 ± 0.14 Phase transition was determined by a discontinuity in the plot of dp ⁄ dA against mean molecular area (not shown) liquid-expanded and liquid-condensed states, respectively 154 b 37 °C L1 L1 L1 L2 L1 L1 L1 L1 L1 L1 L1 L1 and L2 indicate the FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS M Spelios and M Savva A 1,3lb1 0.4 Novel cytofectins for gene delivery 1,3lb2 1,3lb3 1,3lb4 1,3lb5 0.35 Anisotropy 0.3 0.25 0.2 0.15 0.1 0.05 0 10 20 30 40 50 60 T (°C) dr /dT B The behavior of the cationic lipids in two-dimensional monolayers and three-dimensional bilayers was compared As shown in Table 3, a gel-to-liquid crystalline transition temperature below 23 °C was found for 1,3lb1, 1,3lb2 and 1,3lb5, coinciding with the fluid state of these lipids as indicated by the p–A isotherm at 23 °C and 37 °C The three-dimensional phase transition exhibited by 1,3lb4 at 45 °C complies with monolayer compression data indicating the presence of a chain-ordered phase at 23 °C Taking into consideration the onset phase-transition temperature determined from the first-derivative profile of the r–T plot (Fig 6B) instead of the transition midpoint, the all liquid-expanded states at 23 °C and 37 °C of the dipalmitoyl and distearoyl derivatives, respectively, in monolayers complement the nature of these lipids in bilayer assemblies at these temperatures 1,3lb1 1,3lb2 Particle size and electrophoretic mobility studies 1,3lb3 1,3lb4 1,3lb5 10 20 30 T (°C) 40 50 60 Fig (A) Fluorescence anisotropy of DPH in cationic lipid bilayers as a function of temperature, and (B) first-derivative data of r–T plots Dispersions were prepared with 40 mM Tris, pH 7.2 existing in a liquid crystalline state within the range of temperatures scanned (Fig 6A) A gel-to-liquid crystalline phase transition was detected for all other derivatives (temperature span 6–7 °C), and increased in tandem with acyl chain length (Table 3) Only 1,3lb4 exhibited a three-dimensional phase transition above physiological temperature, signifying an ordered phase during transfection, with tight lipid packing, and induced low reporter gene expression Table Midpoint and onset phase-transition temperatures obtained from curve fits and first-derivative analysis, respectively, of the experimental data in Fig Lipid Tm (°C) Coefficient of determinationa Tonset (°C) Toffset (°C) Toffset ) onset 1,3lb1 1,3lb2 1,3lb3 1,3lb4 1,3lb5 1,3lb5 > 1,3lb3 > 1,3lb4 The dimyristoyl derivative exhibited the highest biomembrane fusogenicity of the active analogs, and lipoplexes of this cationic lipid were internalized to the greatest extent (Fig 4) For 1,3lb2, the percentage lipid mixing was approximately four times greater than for 1,3lb3, despite displaying an approximately 1.4-fold lower transfection activity than the most biologically active compound (Fig 2A) However, cell viability was compromised to a greater degree with formulations of 1,3lb2 (63% survival compared with 84% for 1,3lb3; data not shown) The same holds true for 1,3lb5 in comparison with 1,3lb3 Discussion The current project is part of a greater endeavor to understand the structural effects of double-chained amphiphilic molecules and their aggregates, in the presence and absence of pDNA, on cationic lipid-mediated gene delivery In particular, the + ⁄ ) charge ratio, neutral helper lipids, ionic strength, acyl chain length and degree of unsaturation, the number of ionizable amines in the polar headgroup, and the spatial arrangement of the hydrophobic and hydrophilic regions within the lipid molecule have been examined by our laboratory and correlated with transfection efficiency in an exploration of structure–function relationships that has spanned several papers [12,28–31] Generation of such relationships is essential to the development of lipofection reagents that are highly potent and minimally toxic Ewert et al [32] identified the membrane charge density (rM) of cationic lipid vectors that form lamellar complexes with DNA as a key universal parameter governing their transfection efficiency Our previous work with monovalent cationic lipids also shows this dependence on the average charge per unit area of the membrane Excluding helper lipids, only the dioleoyl derivatives from a series of primary and tertiary 1,3-dialkoylamido monovalent cationic lipids [31], differing in molecular structure from the 1,3lb series by only a single amine in the polar headgroup, elicited transfection activity Addition of a second amine group and the subsequent increase in rM increased the Novel cytofectins for gene delivery number of transfection-efficient derivatives to include the dimyristoyl and dipalmitoyl analogs Increased fluidity, or a low gel-to-liquid crystalline phase-transition temperature, of these lamellar assemblies under physiological conditions is another characteristic of the lipid vesicles in isolation that has been identified as critical for transfection activity [33–35] An investigation was recently completed regarding the transfection activity and physicochemical properties of a 1,2-diamino-3-propanol series containing an attachment of the same bivalent polar headgroup at the 3-position but with linkages of the acyl chains at the 1- and 2- positions of the 1,2-diamino-3-propanol backbone [30] The 1,3-dialkyl cationic amphiphiles reported herein feature hydrophobic chains of greater interchain distance than their 1,2-dialkyl counterparts, and the impact this has biologically and physicochemically on these vectors is remarkable Whereas only the dioleoyl derivative of the 1,2lb series generated transfection activity and efficiently bound and compacted pDNA in the absence of helper lipid(s), increasing the intramolecular space between the acyl chains activated the dimyristoyl and dipalmitoyl derivatives This structural modification also afforded these lipids higher two-plane elasticity and increased fluidity relative to their corresponding 1,2lb analogs, as indicated by the lower compressibility moduli and reduced gel-to-liquid crystalline phase-transition temperatures of the 1,3lb derivatives Remarkably, many of the physicochemical properties of the dilauroyl derivative, which was found to be a completely inefficient delivery system, are characteristic of an ideal cytofectin Dispersions of this lipid displaced EtBr from pDNA and condensed plasmid to the greatest extent, and were fusogenically superior in lipid-mixing studies with endosome-mimicking vesicles 1,3lb1 liposomes displayed the most fluid behavior out of all the saturated derivatives, as indicated by the absence of a gel-to-liquid crystalline phase transition In addition, lipoplexes containing 1,3lb1, due to better hydration, possessed the highest zeta potential at the + ⁄ ) charge ratio with the highest reporter gene expression However, 1,3lb1 solubilizes cell membranes and lyses cells in much the same way as strong micelleforming surfactants such as Triton X-100, and such high cytotoxicity rendered the dilauroyl derivative totally inactive Conclusion Five cationic lipid derivatives, differing in the length and degree of unsaturation of their hydrophobic chains, were analyzed with reference to their gene-delivery FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS 157 Novel cytofectins for gene delivery M Spelios and M Savva capabilities Lipofection mediated by the dimyristoyl, dipalmitoyl and dioleoyl derivatives 1,3lb2, 1,3lb3 and 1,3lb5, respectively, resulted in the highest b-galactosidase quantities at + ⁄ ) : in the absence of helper lipids Transfection activities were reduced in the presence of either DOPE alone or in combination with cholesterol for all derivatives except 1,3lb5, which maintained its reporter gene expression levels at + ⁄ ) : and yielded increased enzyme activity at a lower charge ratio of + ⁄ ) : EtBr displacement indicated efficient pDNA compaction by the transfection-competent analogs, irrespective of the ion concentration in the dispersion medium (Tris buffer or serum-free medium) Dynamic light-scattering and electrophoretic mobility studies revealed lipoplexes of active lipids with mean diameters of several hundred nanometers and positive zeta potentials at low ionic strength, or negative values in high-ionic-strength medium Langmuir film balance studies showed high hydration and in-plane elasticity of the active derivatives in isolation In agreement with the monolayer experiments, fluorescence polarization studies verified the fluid nature of the highly transfection-efficient lipids with gel-to-liquid crystalline phase transitions below physiological temperature FRET experiments revealed a greater degree, compared with the poorly active derivative 1,3lb4, of lipid mixing of the active compounds with anionic vesicles Lipids of the 1,3lb series were synthesized with the intent of enhancing the transport of exogenous genetic material into cells in vitro, and their design eliminates the need for additional components in the gene-delivery system (i.e DOPE and cholesterol), providing a simpler yet more potent formulation The evolution of synthetic cationic lipid carriers in our laboratory and the structure–activity data collected for the various series contribute to the long-term goals of understanding the mechanism of lipofection, and the rational design of pharmaceutically sound and therapeutically superior nonviral vectors for gene therapy in vivo Experimental procedures Bis-(2-dimethylamino-ethyl)-amine The synthesis was carried out as previously described [30] 1,3-Dimyristoylamidopropan-2-ol and 1,3-dimyristoylamidopropane-2-(p-nitrophenyl) carbonate The compounds were synthesized by a procedure similar to those previously described [31] 1,3-Dimyristoylamidopropane-2-[bis-(2-dimethylaminoethane)] carbamate (1,3lb2) To a solution of N,N¢-ditetradecanoyl-1,3-diaminopropane2-(p-nitrophenyl)-carbonate (0.0059 mol; 4.32 g) in 30 mL anhydrous CH2Cl2 were added bis-(2-dimethylaminoethyl)amine (0.0118 mol) and triethylamine (0.00118 mol) The reaction was stirred at room temperature for 3.5 h, after which time the solvent was evaporated under vacuum and the reaction mixture transferred to a separation funnel with 100 mL of water and 100 mL of ethyl acetate The aqueous layer was discarded, and the organic phase was washed three times with 100 mL saturated potassium bicarbonate solution to remove the p-NO2-phenol The oily residue that resulted after concentrating the organic layers was dissolved in a minimum amount of chloroform and loaded onto a silica gel column (26 · 2.8 cm) The column was eluted sequentially with 100 mL chloroform, and 1, 3, 5, 7, 8, 9, 10, 12 (300 mL) and 15% methanol ⁄ chloroform The 10% and 12% fractions were combined and concentrated to give 2.79 g (68%) of N,N¢-ditetradecanoyl-1,3-diaminopropyl-2carbamoyl-[bis-(2-dimethylaminoethane)] as a waxy material The calculated composition for C40H81N5O4 (relative molecular mass 695) was C, 69.06; H, 11.65; N, 10.07 That found was C, 68.54; H, 11.92; N, 9.96 MS (FAB) m ⁄ z 696.4 [M+H]+; 1H NMR (400 MHz, CDCl3, 20 °C, TMS) d 6.86–6.83 (t, 2H, HNCO), 4.72–4.70 (m, 1H, CH), 3.45– 3.30 [m, 8H, (CH2)2NC(O)O, CH2NHC(O)], 2.41–2.35 [m, 4H, (CH2)2N], 2.21–2.20 [d, coherent peak, 12H, N(CH3)2], 2.19–2.11 (t, 4H, CH2CO), 1.58–1.53 (m, 4H, CH2CH2CO), 1.23–1.20 [coherent peak, 40H, 10(CH2)2], 0.84–0.81 (t, 6H, CH3); 13C NMR (100 MHz, CDCl3, 20 °C, TMS) d 174.82 (NHCO), 156.37 [NC(O)O], 73.47 (CH), 59.28, 58.43, 46.98, 46.79, 40.66, 37.92, 33.10, 30.87, 30.84, 30.72, 30.59, 30.57, 30.55, 27.02, 23.89, 15.36 Materials Reagents and solvents were purchased from commercial suppliers and were used without further purification Synthesis N,N¢-diacyl-1,3-diaminopropyl-2-carbamoyl-[bis-(2-dimethylaminoethane)] derivatives were synthesized and identified to purity > 99%, as described below 158 1,3-Dilauroylamidopropane-2-[bis-(2-dimethylaminoethane)] carbamate (1,3lb1) The calculated composition for C36H73N5O4 (relative molecular mass 639) was C, 67.61; H, 11.42; N, 10.95 That found was C, 67.62; H, 11.57; N, 10.98 MS (EI) m ⁄ z 641.1 [M+H]+; 1H NMR (400 MHz, CDCl3, 20 °C, TMS) d 6.79–6.70 (t, 2H, HNCO), 4.73–4.70 (m, 1H, CH), 3.58–3.29 [m, 8H, (CH2)2NC(O)O, CH2NHC(O)], 2.41–2.35 FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS M Spelios and M Savva [m, 4H, (CH2)2N], 2.22–2.21 [d, coherent peak, 12H, N(CH3)2], 2.19–2.12 (t, 4H, CH2CO), 1.62–1.56 (m, 4H, CH2CH2CO), 1.24–1.21 [coherent peak, 32H, 10(CH2)2], 0.84–0.81 (t, 6H, CH3) 1,3-Dihexanoylamidopropane-2-[bis-(2-dimethylaminoethane)] carbamate (1,3lb3) The calculated composition for C44H89N5O4 (relative molecular mass 751) was C, 70.31; H, 11.85; N, 9.32 That found was C, 68.97; H, 11.60; N, 9.74 MS (FAB) m ⁄ z 752.7 [M+H]+; 1H NMR (400 MHz, CDCl3, 20 °C, TMS) d 6.80 (bs, 2H, HNCO), 4.73 (m, 1H, CH), 3.45–3.31 [m, 8H, (CH2)2NC(O)O, CH2NHC(O)], 2.41–2.38 [m, 4H, (CH2)2N], 2.23–2.21 [d, coherent peak, 12H, N(CH3)2], 2.17–2.13 (t, 4H, CH2CO), 1.60–1.57 (m, 4H, CH2CH2CO), 1.25–1.22 [coherent peak, 48H, 12(CH2)2], 0.86–0.83 (t, 6H, CH3); 13C NMR (100 MHz, CDCl3, 20 °C, TMS) d 174.82 (NHCO), 156.41 [NC(O)O], 73.51 (CH), 59.41, 58.50, 47.11, 46.87, 40.70, 38.00, 33.14, 30.93, 30.89, 30.75, 30.62, 30.59, 27.05, 23.93, 15.39 1,3-Distearoylamidopropane-2-[bis-(2-dimethylaminoethane)] carbamate (1,3lb4) The calculated composition for C48H97N5O4 (relative molecular mass 807) was C, 71.37; H, 12.02; N, 8.67 That found was C, 70.62; H, 12.18; N, 8.35 MS (FAB) m ⁄ z 808.6 [M+H]+,780.6 [M+-(CH3)2]; 1H NMR (400 MHz, CDCl3, 20 °C, TMS) d 6.90–6.88 (bs, 2H, HNCO), 4.72– 4.70 (m, 1H, CH), 3.47–3.29 [m, 8H, (CH2)2NC(O)O, CH2NHC(O)], 2.40–2.36 [m, 4H, (CH2)2N], 2.21–2.20 [d, coherent peak, 12H, N(CH3)2], 2.15–2.11 (t, 4H, CH2CO), 1.58–1.55 (m, 4H, CH2CH2CO), 1.20 [coherent peak, 56H, 14(CH2)2], 0.84–0.81 (t, 6H, CH3); 13C NMR (100 MHz, CDCl3, 20 °C, TMS) d 174.83 (NHCO), 156.40 [NC(O)O], 73.48 (CH), 59.38 [N(CH3)2], 58.49 [N(CH3)2], 47.07 [(CH2)2N], 46.87 [(CH2)2N], 40.67, 37.96, 33.13, 30.91, 30.87, 30.74, 30.62, 30.59, 27.03, 23.92, 15.33 1,3-Dioleoylamidopropane-2-[bis-(2-dimethylaminoethane)] carbamate (1,3lb5) The calculated composition for C48H93N5O4 (relative molecular mass 803) was C, 71.73; H, 11.58; N, 8.72 That found was C, 70.45; H, 11.53; N, 8.38 MS (FAB) m ⁄ z 804.8 [M+H]+; 1H NMR (400 MHz, CDCl3, 20 °C, TMS) d 6.83 (bs, 2H, HNCO), 5.36–5.31 (m, 4H, CH=CH), 4.75–4.73 (m, 1H, CH), 3.56–3.52 [m, 4H, (CH2)2NC(O)], 3.37–3.29 [m, 4H, CH2NHC(O)], 2.44–2.41 [m, 4H, (CH2)2N], 2.26–2.24 [d, coherent peak, 12H, N(CH3)2], 2.19–2.15 (t, 4H, CH2CO), 2.00–1.95 (m, 8H, CH2CH=CHCH2), 1.61 (m, 4H, CH2CH2CO), 1.29–1.25 [coherent peak, 40H, 10(CH2)2], 0.88–0.84 (t, 6H, CH3); 13C NMR (100 MHz, CDCl3, 20 °C, Novel cytofectins for gene delivery TMS) d 174.81 (NHCO), 156.38 [NC(O)O], 131.40–130.52 (C=C), 73.44 (CH), 65.34, 59.28, 58.46, 46.96, 46.77, 40.58, 37.86, 33.86, 33.77, 33.70, 30.94, 30.90, 30.84, 30.79, 30.71, 30.66, 30.62, 30.53, 30.50, 30.40, 30.38, 30.32, 30.27, 30.12, 28.40, 28.38, 27.65, 23.87, 15.34 Plasmids pUC19-b-gal and pEGFP-N1 were propagated in DH5acompetent cells and collected according to standard protocols [36] Plasmid DNA was purified by gel permeation chromatography using a Sepharose 4B-packed column equilibrated with 2.5 m ammonium acetate Agarose gel electrophoresis and the spectrophotometrically determined A260 ⁄ A280 ratio verified that the pDNA was of high quality and purity Lipid dispersions and lipoplexes Cationic lipids, DOPE and cholesterol were dissolved separately in chloroform, and the appropriate volume of each solution was added to 12 · 75 mm borosilicate glass disposable culture tubes The concentration of cationic lipid and the molar ratio of cationic lipid : DOPE : cholesterol in the final lipid formulations were maintained at 0.3 mm and : : 1, respectively Complete evaporation of organic solvent, first under a stream of nitrogen gas and then by high vacuum desiccation, was followed by hydration of the dry lipid films in Tris buffer (40 mm, pH 7.2) at elevated temperature with periodic vortexing Lipoplexes were prepared at + ⁄ ) charge ratios of : 1, : and : by pipetting a constant volume of pDNA solution into an appropriate amount of diluted liposome preparation In vitro transfection studies Aliquots (250 lL) of lipoplexes in serum-free medium containing lg pUC19-b-gal plasmid DNA were added to approximately 50 000 B16-F0 cells seeded into each well of a 48-well tissue culture plate at least 12 h before transfection, and maintained in serum medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 50 unitsỈmL)1 penicillin and 50 lgỈmL)1 streptomycin) After incubation for h at 37 °C in a 5% CO2 in air atmosphere, the lipoplexes were removed and replaced with 0.5 mL fresh serum medium The cells were lysed after an additional 44 h, and lipofection activity in terms of reporter enzyme levels was quantified by a microplate colorimetric assay utilizing the b-galactosidase substrate ONPG Lipoplex cytotoxicity was assessed by MTT assay A similar procedure was followed for cells transfected with pEGFP-N1 pDNA Fluorescence images of intact cells were acquired 48 h after lipofection using a Zeiss Axiovert 200M inverted microscope (Carl Zeiss, Gottingen, ă FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS 159 Novel cytofectins for gene delivery M Spelios and M Savva Germany) For some experiments, a fluorescein label was covalently attached to the pUC19 plasmid, with a labeling efficiency of approximately one marker molecule every 20–60 nucleic acid base pairs (Mirus Label ITTM, Mirus Bio Corporation, Madison, WI, USA), and lipoplexes were formed with either unlabeled lipid dispersions or cationic vesicles labeled with mol% Rh-PE Images were captured after exchange of the lipoplexes for fresh serum medium pKa studies Studies were performed at excitation and emission wavelengths of 321 and 445 nm, respectively Excitation and emission slit widths were both nm Dry DOPC ⁄ cholesterol ⁄ cationic lipid films (0.95 ⁄ 0.95 ⁄ 0.1 molar ratio) were reconstituted with Tris buffer (40 mm, pH 7.2) to a final combined lipid concentration of mm The aqueous dispersions were dispensed as 100 lL aliquots into 4.5 mL plastic cuvettes with four optical windows, and diluted to mL with buffered solutions (40 mm Tris, 40 mm Mes) of varying pH containing TNS to give a lipid ⁄ probe molar ratio of 100 : Samples were stirred for 30 before measurement of TNS fluorescence intensity Nonlinear fitting of the pH titration curves was performed using psi-plot (version 7.01, Poly Software International, Pearl River, NY, USA) according to a modified version of the Henderson–Hasselbach equation F ¼ Aỵ B ỵ 10CpH Dị 1ị where A is the minimum fluorescence, B is the difference between the maximum and minimum emission intensities, C is a parameter affecting the slope of the transition region, and D is equal to the acid dissociation constant (pKa) of the cationic lipid [12] Goodness-of-fit statistics were assessed within a 95% confidence interval Langmuir monolayer studies Monomolecular cationic lipid films at the air–water interface were studied using a computer-controlled KSV Minitrough (KSV Instruments Ltd., Helsinki, Finland) equipped with a Wilhelmy plate electrobalance (KSV Instruments Ltd.) to measure surface pressure and two symmetrically moving hydrophilic Delrin barriers (Dupont, Wilmington, DE, USA) to reduce the available surface area Using a gas-tight microliter syringe, 20 lL of cationic lipid solution (0.75 mm in chloroform) were applied to the surface of 140 mL of the subphase (40 mm Tris, pH 7.2) within a thermoregulated Teflon trough (364 · 75 mm effective film area) After a time lag of 20 to ensure complete evaporation of organic solvent, the monolayer was compressed at a constant rate of 10 mmỈmin)1 Plots of surface pressure (p) against mean molecular area (A) were automatically generated Molecular compressibility was assessed from first-derivative analysis of the p–A isotherm Phase-transition temperature studies Cationic lipid dispersions (0.5 mm) were prepared with mol% DPH, ensuring minimal membrane perturbation by the fluorophore Studies were conducted at an excitation wavelength of 351 nm (slit width nm) using a Cary Eclipse spectrofluorometer (Varian Instruments, Walnut Creek, CA, USA) equipped with motorized polarizers and a temperature-controlled four-window cuvette holder Anisotropy (r) values of constantly stirred samples at various temperatures were calculated using the Cary Eclipse advanced reads application, software version 1.1 (Varian Instruments), from fluorescence intensities of emitted light at 430 nm (slit width nm) polarized parallel and perpendicular to the illuminating beam Nonlinear fitting of the r–T profiles was performed as described previously [30] Cationic lipid–pDNA binding studies Plasmid DNA (22.5 lg) and EtBr (0.8 lg) were combined in a quartz cuvette and diluted to 22.7 and 0.68 lm, respectively, using Tris buffer or serum-free medium (SFM) Cationic lipids (1 mm) were added in 6.8 lL aliquots with continuous stirring The enhanced fluorescence intensity of intercalated dye was measured at charge ratio increments of + ⁄ ) 0.2 : at an excitation wavelength of 515 nm (slit width nm) and an emission wavelength between 595 and 605 nm (slit width 2.5 nm) The emission readings were treated as described previously [30] to generate plots of percentage ethidium bromide displaced from cationic lipid-bound pDNA against the lipoplex charge ratio The data was not corrected for light-scattering effects, which caused a change in the fluorescence signal of less than 2%, as determined from samples lacking EtBr The binding profiles were modeled as parabolic functions of the form y = ax2 ± bx 160 Particle size and electrophoretic mobility studies Measurements were carried out at room temperature using a Malvern Zetasizer Nano ZS system (Malvern Instruments, Southboro, MA, USA) validated using polystyrene microspheres (Duke Scientific Corporation, Palo Alto, CA, USA) of 60 and 220 nm certified mean diameter Lipoplexes were prepared with either Tris buffer (40 mm, pH 7.2), filtered using a 0.2 lm filter, or with sterile SFM Liposomes were prepared with the same filtered buffer or SFM by diluting cationic lipid dispersions (0.3 mm) to 24 nm Mean diameters of samples in disposable polymethylmethacrylate cuvettes were obtained, using a 633 nm laser, from Gaussian analysis of the intensity-weighted particle size distributions using the instrument software (dispersion technology software 3.00, Malvern Instruments Ltd.) Electrophoretic mobility was measured in a folded capillary FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS M Spelios and M Savva cell (Malvern Instruments) using the laser Doppler velocimetry technique and converted by the software to zeta potential Lipid-mixing studies PC : PA vesicles (73 : 25) containing mol% each of NBD-PE and Rh-PE were prepared in phosphate buffer (pH 7.4) and diluted to mL (25 lm total lipid concentration) in a magnetically stirred quartz fluorescence cell, thermostatically controlled at 37 °C, with either the same buffer or acetate buffer pH 4.0 The ionic strength of buffers was adjusted to 154 mm using NaCl Negatively charged liposomes were treated with a twofold molar excess of cationic lipids, and emission scans (kex = 475 nm) were recorded at various times over a period of approximately 0.5 h The extent of fusion between labeled and unlabeled vesicles was calculated as   F t À F0 Â 100 2ị % lipid mixing ẳ F100 F0 where Ft, F0 and F100 are the NBD ⁄ Rh ratios of maximum probe fluorescence intensities at a particular time t, before addition of cationic lipids, and in the presence of 0.1% Triton X-100, respectively Acknowledgements This work was supported in part by a pre-doctoral fellowship in pharmaceutics from the PhRMA Foundation (Washington, DC) and by National Institutes of Health Grant EB004863 References Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM & Danielsen M (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure Proc Natl Acad Sci USA 84, 7413–7417 Liu D, Qiao W, Li Z, Cui X, Li K, Yu L, Yan K, Zhu L & Cheng L (2007) Carbamate-linked cationic lipids for gene delivery Bioorg Med Chem, doi:10.1016/ j.bmc.2007.10.009 Bajaj A, Kondaiah P & Bhattacharya S (2007) Synthesis and gene transfer activities of novel serum compatible cholesterol-based gemini lipids possessing oxyethylene-type spacers Bioconjug Chem 18, 1537– 1546 Bajaj A, Kondiah P & Bhattacharya S (2007) Design, synthesis, and in vitro gene delivery efficacies of novel cholesterol-based gemini cationic lipids and their serum compatibility: a structure–activity investigation J Med Chem 50, 2432–2442 Novel cytofectins for gene delivery Wettig SD, Badea I, Donkuru M, Verrall RE & Foldvari M (2007) Structural and transfection properties of amine-substituted gemini surfactant-based nanoparticles J Gene Med 9, 649–658 Rajesh M, Sen J, Srujan M, Mukherjee K, Sreedhar B & Chaudhuri A (2007) Dramatic influence of the orientation of linker between hydrophilic and hydrophobic lipid moiety in liposomal gene delivery J Am Chem Soc 129, 11408–11420 Antipina MN, Schulze I, Dobner B, Langner A & Brezesinski G (2007) Physicochemical investigation of a lipid with a new core structure for gene transfection: 2-amino-3-hexadecyloxy-2-(hexadecyloxymethyl)propan1-ol Langmuir 23, 3919–3926 Chen H, Zhang H, McCallum CM, Szoka FC & Guo X (2007) Unsaturated cationic ortho esters for endosome permeation in gene delivery J Med Chem 50, 4269–4278 Gardner RA, Belting M, Svensson K & Phanstiel O IV (2007) Synthesis and transfection efficiencies of new lipophilic polyamines J Med Chem 50, 308–318 10 Lamarche F, Mevel M, Montier T, Burel-Deschamps L, Giamarchi P, Tripier R, Delepine P, Le Gall T, Cartier D, Lehn P et al (2007) Lipophosphoramidates as lipidic part of lipospermines for gene delivery Bioconjug Chem 18, 1575–1582 11 Takahashi T, Kojima C, Harada A & Kono K (2007) Alkyl chain moieties of polyamidoamine dendron-bearing lipids influence their function as a nonviral gene vector Bioconjug Chem 18, 1349–1354 12 Spelios M, Nedd S, Matsunaga N & Savva M (2007) Effect of spacer attachment sites and pH-sensitive headgroup expansion on cationic lipid-mediated gene delivery of three novel myristoyl derivatives Biophys Chem 129, 137–147 13 Audouy S & Hoekstra D (2001) Cationic lipid-mediated transfection in vitro and in vivo (review) Mol Membr Biol 18, 129–143 14 Tranchant I, Thompson B, Nicolazzi C, Mignet N & Scherman D (2004) Physicochemical optimisation of plasmid delivery by cationic lipids J Gene Med 6, S24– S35 15 Wasungu L & Hoekstra D (2006) Cationic lipids, lipoplexes and intracellular delivery of genes J Control Release 116, 255–264 16 Banerjee R, Mahidhar YV, Chaudhuri A, Gopal V & Rao NM (2001) Design, synthesis, and transfection biology of novel cationic glycolipids for use in liposomal gene delivery J Med Chem 44, 4176–4185 17 Felgner JH, Kumar R, Sridhar CN, Wheeler CJ, Tsai YJ, Border R, Ramsey P, Martin M & Felgner PL (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations J Biol Chem 269, 2550–2561 FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS 161 Novel cytofectins for gene delivery M Spelios and M Savva 18 Ferrari ME, Rusalov D, Enas J & Wheeler CJ (2002) Synergy between cationic lipid and co-lipid determines the macroscopic structure and transfection activity of lipoplexes Nucleic Acids Res 30, 1808–1816 19 Liu L, Zern MA, Lizarzaburu ME, Nantz MH & Wu J (2003) Poly(cationic lipid)-mediated in vivo gene delivery to mouse liver Gene Ther 10, 180–187 20 Narang AS, Thoma L, Miller DD & Mahato RI (2005) Cationic lipids with increased DNA binding affinity for nonviral gene transfer in dividing and nondividing cells Bioconjug Chem 16, 156–168 21 Oudrhiri N, Vigneron JP, Peuchmaur M, Leclerc T, Lehn JM & Lehn P (1997) Gene transfer by guanidinium-cholesterol cationic lipids into airway epithelial cells in vitro and in vivo Proc Natl Acad Sci USA 94, 1651–1656 22 Yperman J, Mullens J, Francois JP & Van Poucke LC (1982) Calorimetric and potentiometric investigation of diethylenetriamine and its N-methyl-substituted derivatives in aqueous solution J Phys Chem 86, 298–303 23 Ghirlando R, Wachtel EJ, Arad T & Minsky A (1992) DNA packaging induced by micellar aggregates: a novel in vitro DNA condensation system Biochemistry 31, 7110–7119 24 Oberle V, Bakowsky U, Zuhorn IS & Hoekstra D (2000) Lipoplex formation under equilibrium conditions reveals a three-step mechanism Biophys J 79, 1447–1454 25 Ross PC & Hui SW (1999) Lipoplex size is a major determinant of in vitro lipofection efficiency Gene Ther 6, 651–659 26 Struck DK, Hoekstra D & Pagano RE (1981) Use of resonance energy transfer to monitor membrane fusion Biochemistry 20, 4093–4099 27 Asokan A & Cho MJ (2005) Cytosolic delivery of macromolecules Head group-dependent membrane permeabilization by pH-sensitive detergents J Control Release 106, 146–153 28 Aljaberi A, Chen P & Savva M (2005) Synthesis, in vitro transfection activity and physicochemical 162 29 30 31 32 33 34 35 36 characterization of novel N,N¢-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) amphiphilic derivatives Chem Phys Lipids 133, 135–149 Aljaberi A, Spelios M, Kearns M, Selvi B & Savva M (2007) Physicochemical properties affecting lipofection potency of a new series of 1,2-dialkoylamidopropanebased cationic lipids Colloids Surf B Biointerfaces 57, 108–117 Savva M, Chen P, Aljaberi A, Selvi B & Spelios M (2005) In vitro lipofection with novel asymmetric series of 1,2-dialkoylamidopropane-based cytofectins containing single symmetric bis-(2-dimethylaminoethane) polar headgroups Bioconjug Chem 16, 1411–1422 Sheikh M, Feig J, Gee B, Li S & Savva M (2003) 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, 49–61 Ewert K, Slack NL, Ahmad A, Evans HM, Lin AJ, Samuel CE & Safinya CR (2004) Cationic lipid–DNA complexes for gene therapy: understanding the relationship between complex structure and gene delivery pathways at the molecular level Curr Med Chem 11, 133– 149 Akao T, Nakayama T, Takeshia K & Ito A (1994) Design of a new cationic amphiphile with efficient DNA-transfection ability Biochem Mol Biol Int 34, 915–920 Boukhnikachvili T, Aguerre-Chariol O, Airiau M, Lesieur S, Ollivon M & Vacus J (1997) Structure of in-serum transfecting DNA–cationic lipid complexes FEBS Lett 409, 188–194 Gaucheron J, Wong T, Wong KF, Maurer N & Cullis PR (2002) Synthesis and properties of novel tetraalkyl cationic lipids Bioconjug Chem 13, 671–675 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY FEBS Journal 275 (2008) 148–162 ª 2007 The Authors Journal compilation ª 2007 FEBS ... 200 1,3lb1 20 1,3lb2 1,3lb3 –2 0 1,3lb4 1,3lb5 –4 0 –6 0 –8 0 + /– 1:1 + /– 2:1 + /– 4:1 + /– 1:1 liposomes + /– 2:1 + /– 4:1 liposomes Fig Particle size distribution (A,B) and zeta potential (C,D), as... – 13 28 42 – – 20 34 48 – – 6 – a Best-fit parameters were assessed within a 95% confidence interval In vitro transfection activity was found to be a function of the size of the cationic lipid–pDNA... diameter around lm Use of DOPE and cholesterol to enhance the genedelivery properties of cationic lipids has been extensively documented [1 6–2 1] For 1,3lb2 and 1,3lb3, transfection activity was appreciably