Cationic Amphiphiles with Fatty Acyl Chain Asymmetry of Coconut Oil

ARTICLE pubs.acs.org/bc

Cationic Amphiphiles with Fatty Acyl Chain Asymmetry of Coconut Oil Deliver Genes Selectively to Mouse Lung Voshavar Chandrashekhar,† Marepally Srujan,† Rairala Prabhakar,† Rakesh C. Reddy,† Bojja Sreedhar,‡ Kiran K. R. Rentam,§ Sanjit Kanjilal,† and Arabinda Chaudhuri*,† †

Division of Lipid Science and Technology, ‡Inorganic and Physical Chemistry Division and §Pharmacology Division, Indian Institute of Chemical Technology, Hyderabad 500 007, India

bS Supporting Information ABSTRACT: Recent structure-activity studies have revealed a dramatic influence of hydrophobic chain asymmetry in enhancing gene delivery efficacies of synthetic cationic amphiphiles (Nantz, M. H. et al. Mol. Pharmaceutics 2010, 7, 786-794; Koynova, R. et al. Mol. Pharmaceutics 2009, 6, 951-958). The present findings demonstrate for the first time that such a transfection enhancing influence of asymmetric hydrocarbon chains observed in pure synthetic cationic amphiphiles also works for cationic amphiphiles designed with natural, asymmetric fatty acyl chains of a food-grade oil. Herein, we demonstrate that cationic amphiphiles designed with the natural fatty acyl chain asymmetry of food-grade coconut oil are less cytotoxic and deliver genes selectively to mouse lung. Despite lauroyl chains being the major fatty acyl chains of coconut oil, both the in vitro and In vivo gene transfer efficiencies of such cationic amphiphiles were found to be remarkably superior (>4-fold) to those of their pure dilauroyl analogue. Mechanistic studies involving the technique of fluorescence resonance energy transfer (FRET) revealed higher biomembrane fusibility of the cationic liposomes of the coconut amphiphiles than that of the symmetric dilauroyl analogue. AFM study revealed pronounced fusogenic nonlamellar structures of the liposomes of coconut amphiphiles. Findings in the FRET and cellular uptake study, taken together, support the notion that the higher cellular uptake resulting from the more fusogenic nature of the liposomes of coconut amphiphiles 1 are likely to play a dominant role in making the coconut amphiphiles transfection competent.

’ INTRODUCTION Clinical success of gene therapy continues to remain critically dependent on the use of safe and efficient gene transfer reagents. Gene delivery reagents, popularly known as transfection vectors, are broadly classified into two types: viral and nonviral. Viral vectors are, in general, efficient in delivering genes into body cells. However, viral vectors suffer from numerous biosafetyrelated disadvantages including adverse inflammatory and immunogenic responses, insertional mutagenesis through random integration into the host genome, and so forth.1 On the other hand, because of their superior biosafety profiles, nonviral vectors such as cationic liposomes,2-7 cationic polymers,8 dendrimers,9,10 and so forth hold therapeutic promise. Among these arsenals of nonviral gene transfer reagents, the distinguishing features of cationic liposomes include their less immunogenic nature, robust manufacture, ability to deliver large pieces of DNA, and ease of handling and preparation techniques.11,12 A number of structure-activity studies have demonstrated in the past that the gene delivery efficiencies of cationic amphiphiles crucially depend upon molecular architectures of the hydrophobic alkyl chain lengths13-15 and the nature of polar headgroups,16-19 as well as on the nature of linker and spacer functionalities used in covalent tethering of the polar headgroups and the nonpolar tails r 2011 American Chemical Society

of cationic amphiphiles.20,21 In 2006, in a thought-provoking structure-activity study, Koynova et al. demonstrated dramatically superior (about 50-fold) in vitro gene transfer efficiency of a synthetic cationic amphiphile with asymmetric hydrocarbon chains, namely, oleoyldecanoyl-ethylphosphatidylcholine (C18:1/ C10-EPC) to that of its structurally very similar saturated asymmetric counterpart stearoyldecanoyl-ethylphosphatidylcholine (C18:0/C10-EPC).22 In this report, it was demonstrated that liposomal compositions of C18:1/C10-EPC containing two defined asymmetric hydrocarbon chains (oleoyl and decanoyl) formed a pronounced nonlamellar phase which, in turn, played a dominant role in imparting high membrane fusogenicity and thereby enhanced transfection properties to C18:1/C10-EPC.22 More recently, structure-activity studies by Koynova et al.23 and Nantz et al.24 have convincingly demonstrated the dramatic influence of hydrophobic domain asymmetry in modulating the gene transfer efficacies of synthetic cationic amphiphiles. These important recent structure-activity findings prompted us to design cationic amphiphiles possessing natural fatty acyl chain asymmetry of food-grade coconut oil. We reasoned that, if the mixture of fatty Received: December 1, 2010 Revised: January 21, 2011 Published: February 21, 2011 497

dx.doi.org/10.1021/bc100537r | Bioconjugate Chem. 2011, 22, 497–509

Bioconjugate Chemistry

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Scheme 1. Synthesis of Coconut Amphiphiles 1 (A) and Control Dilauroyl Amphiphile 2 (B)

spectrometer for ESI analysis. 1H NMR spectra were recorded on a Varian FT 200 MHz or AV 300 MHz NMR spectrometer. Column chromatography was performed with silica gel (Acme Synthetic Chemicals, India, 60-120 mesh). p-CMV-SPORT-βgal plasmid was a generous gift from Dr. Nalam Madhusudhana Rao of Centre for Cellular and Molecular Biology, Hyderabad, India. Lauric acid, cell culture media, fetal bovine serum, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), poly(ethylene glycol) 8000, o-nitrophenyl-β-D-galactopyranoside, N-methyl-N,N-diethanolamine, and cholesterol were purchased from Sigma-Aldrich, St. Louis, MO, USA. NP-40, antibiotics, and agarose were procured from Hi-media, India. Unless otherwise stated, all reagents were purchased from local commercial suppliers and were used without further purification. B16F10, HepG2, CHO, and COS-1 cells were procured from the National Centre for Cell Sciences (NCCS), Pune, India. Cells were grown at 37 °C either in Dulbecco’s modified Eagle’s medium (DMEM) or Minimum Essential Medium (MEM) with 10% FBS in a humidified atomosphere containing 5% CO2/95% air. Syntheses. The synthetic route used for preparing the coconut amphiphiles 1 and control dilauroyl amphiphile 2 were shown in parts A and B, Scheme 1, respectively. Structures of all the synthetic intermediates were confirmed by 1H NMR and ESI-MS. The purity of the control dilauroyl amphiphile 2 was confirmed by reverse-phase analytical HPLC in two different mobile phases. Synthesis of Coconut Oil Amphiphiles 1 (Scheme 1A). Steps a and b. Isolation of Coconut Oil Fatty Acids (I, Scheme 1A). 10 gm of coconut oil (Scheme 1A) was taken in a 250 mL rb flask, and 10% KOH in ethanol (25 mL) was added slowly to the coconut oil. This mixture was gradually heated to refluxing temperature with continuous stirring in oil bath until it was completely dissolved. Refluxing was continued for 4 h. After 4 h, the reaction mixture was

acids obtained from the hydrolysis of natural food-grade coconut oil (with high content of C12 lauroyl and significant amount of C18:1 oleoyl hydrocarbon chains) is used in preparing cationic amphiphiles, the resulting mixture of cationic amphiphiles will not only possess hydrophobic tails of asymmetric chain-lengths, but will also contain both saturated as well as transfection-enhancing unsaturated aliphatic hydrocarbon chains. Stated differently, liposomes prepared from such a mixture of cationic amphiphiles containing varying fatty acyl chains of coconut oil should, in principle, form a transfection-enhancing phase. Importantly, being derived from food-grade coconut oil, such cationic amphiphiles are also expected to be least cytotoxic. On the basis of this rationale, we synthesized cationic amphiphiles with asymmetric fatty acyl chains of coconut oil and evaluated their gene transfer efficiencies (both in vitro and In Vivo) and cell viabilities. Herein, we demonstrate for the first time that liposomes of cationic amphiphiles designed with natural fatty acyl chain asymmetry of food-grade coconut oil are less cytotoxic and selectively deliver genes to mouse lung. Despite lauroyl chains being the major fatty acyl chains of coconut oil, the in vitro and In Vivo gene transfer efficiencies of such cationic amphiphiles were found to be significantly superior (>4 fold) to those of their pure dilauroyl analogue. Findings in the FRET and cellular uptake study, taken together, support the notion that the higher cellular uptake resulting from the more fusogenic nature of the liposomes of coconut amphiphiles 1 is likely to play a dominant role in making the coconut amphiphiles transfection-competent.

’ EXPERIMENTAL PROCEDURES General Procedures and Reagents. Mass spectral data were acquired by using a commercial LCQ ion trap mass spectrometer (ThermoFinnigan, SanJose, CA, USA) equipped with an ESI source or micromass Quatro LC triple quadrapole mass 498

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taken out and allowed to cool to rt and then concentrated to half of its volume. This mixture was poured into a separatory funnel with the addition of water and hexane (25 mL each). The mixture was vigorously shaken to extract the potassium salt of the fatty acids into the aqueous layer. The aqueous layer was collected and acidified with 30% aqueous HCl to precipitate the fatty acids. The acidified white curdy fatty acid mixture was extracted with ethyl acetate (4 25 mL). The combined organic extract was washed with brine solution (2 25 mL), dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and vacuum-dried to give a white solid mixture of coconut fatty acids (I, Scheme 1A, 8.1 g, Rf = 0.4, 10:90 v/v ethylacetate/hexane). Step c. Preparation of Coconut Fatty Acyl Chlorides (II, Scheme 1A). The coconut fatty acid mixture (10 g, 49.9 mmol, I, Scheme 1A) prepared above in step b was taken in a 250 mL two-necked rb flask fitted with a calcium chloride guard tube and a dropping funnel. The fatty acid mixture was dissolved in a minimum amount of dry DCM (20 mL) with a catalytic amount of dry DMF, and oxalyl chloride (5.2 mL, 59.9 mmol) was added slowly drop by drop at room temperature with continuous stirring for 3 h. After 3 h, TLC was checked to confirm the complete conversion of acid to acid chloride. Excess oxalyl chloride was distilled off at 40-45 °C by downward distillation using an oil bath. The acid chloride mixture was then chased continuously with solvent DCM to ensure complete removal of excess oxalyl chloride. The residue left upon vacuum-drying afforded the mixture of coconut fatty acyl chlorides (10.3 g, 94% yield, Rf = 0.7, 5:95 methanol/chloroform, v/v). Step d. Synthesis of Coconut Oil Derived N-Methyl-N, Ndiethanolamine Fatty Acyl Esters (II, Scheme 1A). Coconut fatty acyl chloride mixture (5.14 g, 23.5 mmol) prepared above in step c was taken in a 250 mL rb flask and dissolved in 20 mL dry DCM by stirring at room temperature. N-Methyl-N,N-diethanolamine (1 g, 0.84 mmol) was added to the fatty acyl chloride mixture, followed by slow addition of triethylamine (3.4 mL, 23.5 mmol) at 0 °C. Stirring was continued for 12 h at room temperature, and then the reaction mixture was diluted with 20 mL DCM and washed with an excess volume of 5% aqueous HCl (6 15 mL to ensure complete removal of triethylamine). The DCM layer was washed with 5% aqueous sodium bicarbonate solution (3 15 mL) followed by a final wash with brine solution (2 20 mL). Combined organic layers were collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The residue upon column chromatographic purification using a 60-120 mesh size silica gel column and 2% methanol/chloroform, v/v, as eluent afforded the intermediate tertiary amine compound N-methyl-N,N-diethanolamine fatty acyl esters (II, Scheme 1A) as brown viscous liquid (3.5 g, 85% yield, Rf = 0.8, 5:95 methanol/ chloroform, v/v) which solidified upon storage at 4 °C. 1 H NMR of coconut oil derived N-methyl-N,N-diethanolamine difatty acyl ester II (300 MHz, CDCl3): δ/ppm = 0.9 [t, 6H, CH3-CH2-(CH2)5-15-]; 1.2-1.4 [m, 20-60H, -(CH2)5-15-]; 1.6 [t, 4H, CH3-(CH2)4-14-CH2-CH2]; 2.3 [t, 4H, CH3(CH2)4-14-CH2-CH2]; 2.4 [s, 3H, N-CH3]; 2.7 [t, 4H, N-CH2-CH2-O-]; 4.15 [t, 4H, N-CH2-CH2-O-]; 5.3 [t, 2H, CHdCH]. ESI-MS m/z: found 372 (Mþ1 for C8:C8); 400 (Mþ1 for C8:C10); 428 (Mþ1 for C8:C12 or C10:C10); 456 (Mþ1 for C8:C14 or C10:C12); 484 (Mþ1 for C8:C16 or C10: C14 or C12:C12); 512 (Mþ1 for C8:C18 or C10:C16 or C12: C14); 540 (Mþ1 for C10:C18 or C12:C16 or C14:C14); 566 (Mþ1 for C12:C18:1); 594 (Mþ1 for C14:C18:1); 595 (Mþ1

for C14:C18); 624 (Mþ1 for C16:C18); 622 (Mþ1 for C16: C18:1). Step e. Synthesis of Target Coconut Amphiphiles 1 (Scheme 1A). N-Methyl-N,N-diethanolamine fatty acyl esters (0.5 g, 1.01 mmol) (II, Scheme 1A, prepared above in step d) was taken in a 25 mL rb flask and added with 0.5 mL 2-iodoethanol (excess). This was stirred at 80 °C for 6 h. Reaction was diluted with 10 mL DCM, washed with water (3 10 mL), followed by brine solution (2 10 mL). Combined organic layers were collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The residue upon column chromatographic purification (using 60-120 mesh silica gel and 4% methanol/CHCl3, v/v, as eluent) and chloride ion exchange on Amberlite IRA 400 chloride resin in chloroform/methanol solvent mixture (30:70 v/v) to give a white solid of coconut amphiphiles 1. Crystallization by dissolving the compound in a few drops of methanol followed by addition of diethyl ether (20 mL) afforded the title compound coconut amphiphiles 1 as white solid (1, Scheme 1A, 0.25 g, 46% yield, Rf = 0.2, 5:95 methanol/ chloroform, v/v). 1 H NMR of coconut amphiphiles 1 (300 MHz, CDCl3): δ/ ppm = 0.9 [t, 6H, CH3-CH2-C9H18-]; 1.2-1.4 [m, 36H, -(CH2)9-]; 1.6 [t, 4H, CH3-(CH2)9-CH2-CH2]; 2.35 [t, 4H, CH3-(CH2)9-CH2-CH2]; 3.4 [s, 3H, N-CH3]; 3.8-3.9 [t, 6H, N-CH2-CH2-O]; 4.0-4.1 [t, 2H, N-CH2-CH2-OH]; 4.6 [t, 4H, -CO-O-CH2-]. ESI-MS m/z: found 472 (Mþ1 for C8:C14 or C10:C12); 500 (Mþ1 for C8:C16 or C10:C14 or C12:C12); 528 (Mþ1 for C8:C18 or C10:C16 or C12:C14); 556 (Mþ1 for C10:C18 or C12:C16 or C14:C14); 584 (Mþ1 for C12:C18:1); 612 (Mþ1 for C14:C18) or (Mþ1 for C14:C18:1); 640 (Mþ1 for C16:C18) or (Mþ1 for C16:C18:1). Synthesis of the Control Dilauroyl Amphiphile 2 (Scheme 1B). Synthesis of N,N-di-[O-dodecanoyl-2-hydroxyethyl]-N-hydroxyethyl-N-methyl ammonium chloride (control dilauroyl amphiphile 2, Scheme 1B). Step c. Preparation of Lauroyl Chloride (Scheme 1B). Lauric acid (5 g, 25 mmol) was taken in a 100 mL two-necked rb flask fitted with a calcium chloride guard tube and a dropping funnel. Lauric acid was dissolved in dry DCM (10 mL) with a catalytic amount of dry DMF, and oxalyl chloride (2.56 mL, 30 mmol) was added slowly drop by drop at room temperature with continuous stirring for 3 h. After 3 h, TLC was checked to confirm the complete conversion of acid to acid chloride. Excess oxalyl chloride was distilled off at 40-45 °C by downward distillation using an oil bath. The acid chloride was then chased continuously with solvent DCM to ensure complete removal of remaining oxalyl chloride. The residue left upon vacuum drying afforded the target lauroyl chloride (5.2 g, 95% yield, Rf = 0.7, 5:95 methanol/chloroform, v/v). Step d. Synthesis of N,N-Di-[O-dodecanoyl-2-hydroxyethyl]N-methyl Amine (Scheme 1B). Lauroyl chloride (5.12 g, 23.5 mmol, Scheme1B) prepared above in step c was taken in a 100 mL rb flask and dissolved in 20 mL dry DCM by stirring at room temperature. N-Methyl-N,N-diethanolamine (1 g, 8.4 mmol) was added followed by a slow addition of triethylamine (3.27 mL, 23.5 mmol) at 0 °C. Stirring was continued for 12 h at room temperature, and then the reaction mixture was diluted with 20 mL DCM and washed with 5% aqueous HCl (5 30 mL to ensure complete removal of triethylamine). The DCM layer was washed with 5% aqueous sodium bicarbonate solution (3 20 mL) followed by final wash with brine solution (2 20 mL). Combined organic layers were collected, dried over anhydrous 499



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sodium sulfate, and concentrated by rotary evaporation. The residue upon column chromatographic purification using a 60120 mesh size silica gel column and 2% methanol/chloroform, v/v, as eluent afforded the intermediate tertiary amine compound N,N-di-[O-dodecanoyl-2-hydroxyethyl]-N-methyl amine as yellow liquid (3.6 g, 89% yield, Rf = 0.8, 5:95 methanol/chloroform, v/v), which solidified upon storage at 4 °C. 1 H NMR of N,N-di-[O-dodecanoyl-2-hydroxyethyl]-N-methyl amine (300 MHz, CDCl3): δ/ppm = 0.9 [t, 6H, CH3-CH2C9H18-]; 1.2-1.4 [m, 36H, -O-CO-CH2-CH2-(CH2)9-]; 1.6 [t, 4H, CH3-(CH2)8-CH2-CH2-CO-]; 2.3 [t, 4H, CH3-(CH2)8CH2-CH2-CO-]; 2.4 [s, 3H, N-CH3]; 2.7 [t, 4H, N-CH2-CH2O-]; 4.2 [t, 4H, N-CH2-CH2-O-]. ESI-MS m/z: calcd 483.772 (for C29H57NO4), found 484 (Mþ). Steps e and f. Synthesis of Target N,N-Di-[O-dodecanoyl-2hydroxyethyl]-N-hydroxy ethyl-N-methyl Ammonium Chloride (Control Dilauroyl Amphiphile 2, Scheme 1B). A mixture of N, N-di-[O-dodecanoyl-2-hydroxyethyl]-N-methyl amine (0.5 g, 1.03 mmol) prepared above in step d and 2-iodoethanol (0.5 mL) was stirred at 80 °C for 6 h in a 25 mL rb flask. Reaction mixture was diluted with 10 mL DCM and washed with water (3 10 mL), followed by brine solution (2 10 mL). Combined organic layers were collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The residue upon vacuum drying afforded the iodide salt of control dilauroyl amphiphile as a whitish yellow solid, which upon chloride ion exchange over Amberlite IRA 400 chloride resin in chloroform/methanol solvent mixture (30:70 v/v) afforded the crude target amphiphile. The crude product upon recrystallization with a few drops of methanol and diethyl ether (20 mL) afforded the pure title compound control dilauroyl amphiphile 2 as a white solid (2, Scheme 1B, 0.23 g, 42% yield, Rf = 0.2, 5:95 methanol/chloroform, v/v). 1 H NMR of control dilauroyl amphiphile 2 (300 MHz, CDCl3): δ/ppm = 0.9 [t, 6H, CH3-CH2-C9H18-]; 1.2-1.4 [m, 36H, -(CH2)9-]; 1.5 [t, 4H, CH3-(CH2)9-CH2-CH2]; 2.3 [t, 4H, CH3-(CH2)9-CH2-CH2]; 3.3 [s, 3H, N-CH3]; 3.9 [t, 6H, N-CH2-CH2-O]; 3.9 [t, 2H N-CH2-CH2-OH]; 4.3 [t, 4H, -CO-O-CH2-]. ESI-MS m/z: calcd 528.786 (for C31H62NO5), found 528 (Mþ). Transfection Assay. Cells were seeded at a density of 10 000 cells (for B16F10) and 15 000 cells (for COS-1, CHO, HepG2) per well in a 96-well plate 12-18 h before the transfection. 0.3 μg of plasmid DNA was complexed with varying amounts of lipids (0.45-7.2 nmol) in plain DMEM/MEM medium (total volume made up to 100 μL) for 30 min. The lipid/DNA (() charge ratios were from 8:1 to 0.5:1 over these ranges of the lipids. The complexes were then added to the cells. After 3 h of incubation, DMEM/MEM was removed, and 10% complete medium was added to the cells. The reporter gene activity was estimated between 36 and 48 h. The cells were washed with PBS (2 100 μL) and lysed with 50 μL lysis buffer [0.25 M Tris-HCl, pH 8.0, 0.5% NP40]. Care was taken to ensure complete lysis. The β-galactosidase activity per well was estimated by adding 50 μL of 2 substrate solution [1.33 mg/mL of ONPG, 0.2 M sodium phosphate (pH 7.3), and 2 mM magnesium chloride] to the lysate in a 96-well plate. Absoption at 405 nm was converted to β-galactosidase units using a calibration curve constructed with pure commercial β-galactosidase enzyme. The values of β-galactosidase units in triplicate experiments assayed on the same day varied by less than 10%. The transfection experiment was carried in duplicate, and the transfection efficiency values

shown in Figure 1a-d are the average of triplicate experiments performed on the same day. The day-to-day variation in average transfection efficiency was found to be within 2-fold. The transfection profiles obtained on different days were identical. Cytotoxicity Assay. The cytotoxicities of coconut amphiphiles 1 and control dilauroyl amphiphile 2 were evaluated in representative CHO (Chinese hamster ovary) cells across the lipid:/DNA charge ratios of 8:1-0.5:1 using MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) based reduction assay as described earlier.19 The cytotoxicity assay was performed in 96-well plates by maintaining the same ratio of number of cells to amount of cationic lipid, as used in the previously described transfection experiments. Briefly, 4 h after the addition of lipoplexes, MTT (5 mg/mL in PBS) was added to cells and incubated for 4 h at 37 °C. Results were expressed as percent viability = [A540 (treated cells) - background/A540 (untreated cells) - background] 100. Fatty Acid Composition Profile by Gas Chromatography. 10 mg of coconut oil or cationic coconut amphiphiles 1 were taken separately in two 25 mL round-bottom flasks, dissolved in 5% H2SO4 in methanol, and the solutions refluxed for 4 h. Reaction mixtures were diluted with water (5 mL) and extracted with ethyl acetate (3 5 mL). Collected organic layers were further washed with saturated brine (2 5 mL) and dried over anhydrous sodium sulfate. Compositions of coconut oil fatty acid methyl esters were analyzed quantitatively by gas chromatography. The fatty acid methyl ester analysis was performed on Agilent 6890 series gas chromatograph equipped with a FID and the capillary column DB-23 (30 m 0.25 mm i.d. 0.5 μm film thickness). The injector and detector temperatures were maintained at 230 and 250 °C, respectively. The oven temperature was programmed for 2 min at 160-180 at 6 °C/ min, maintained for 2 min at 180 °C, increased further to 230 at 4 °C/min and finally maintained for 10 min at 230 °C. The carrier gas, nitrogen, was used at a flow rate of 1.5 mL/min. The injection volume was 1 μL, with a split ratio of 50:1. The fatty acid compositions of both coconut oil and coconut amphiphiles 1 are shown in the Table 1. Preparation of Liposomes and Plasmid DNA. 0.5 mM liposomes were prepared with 1:1 mol ratios of lipid and cholesterol. For lipid with a mixture of fatty acids, the average molecular weight was calculated based on the fatty acid composition percentages of cationic lipid as obtained by gas chromatographic analysis. The cationic lipids and cholesterol in the appropriate mole ratios were dissolved in chloroform (500 μL) in a glass vial. The solvent was removed with a thin flow of moisture-free nitrogen gas, and the dried lipid film was kept for drying under high vacuum for 6 h. 1 mL of sterile deionized water was added to the vacuum-dried lipid films, and the mixtures were allowed to swell overnight. The vials were then vortexed for 23 min at room temperature to produce multilamellar vesicles (MLVs). MLVs were then sonicated initially in a water bath (ULTRA SONIK 28X) followed by an ice bath until clarity using a Branson 450 sonifier at 100% duty cycle and 25 W output power to produce small unilamellar vesicles (SUVs). p-CMVSPORT-β-gal plasmid was amplified in DH5R-strain of Escherichia coli, isolated by an alkaline lysis procedure, and finally purified by PEG-8000 precipitation as described previously.17 The purity of plasmid was checked by A260/A280 ratio (around 1.9) and 1% agarose gel electrophoresis. Transfection Protocol for r5GFP Plasmid. Cells were seeded in 24-well plates (Corning Inc., Corning, NY, 4 104 500



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Figure 1. In vitro gene delivery efficiencies of coconut amphiphiles 1 (gray bars) and the control dilauroyl amphiphile 2 (open bars) in COS-1 (a), CHO (b), B16F10 (c), and HepG-2 (d) cells. Units of β-galactosidase activity were plotted against the varying lipid-to-DNA charge ratios (8:1-0.5:1). The black bars refer to transfection efficiencies of commercially available Lipofectamine 2000. The transfection values shown are the average of triplicate experiments performed on the same day.

cells per well) for 12 h in 500 μL of growth medium such that the well became 30-50% confluent at the time of transfection. Coconut amphiphiles 1 and control dilauroyl amphiphile 2 were complexed with R5GFP encoding pDNA (0.9 μg/well) at 2:1 lipid/DNA charge ratio in plain DMEM (total volume made up to 100 μL) for 15-20 min. The complexes were then diluted with 300 μL DMEM and added to the cells. After 4 h of incubation, DMEM was removed and cells were supplemented with complete medium. The cells were incubated for 24 h. Cells were washed with PBS (100 μL) and fixed with 3.8% paraformaldehyde in PBS at room temperature for 10 min. The green fluorescent cells expressing R5GFP were detected under an inverted fluorescence microscope (Nikon, Japan). DNA Binding Assay. The DNA bindig ability of the cationic coconut amphiphiles 1 and control dilauroyl amphiphile 2 were assessed by their gel retardation assay on a 1% agarose gel (prestained with ethidium bromide) across the varying lipid/ DNA charge ratios of 8:1 to 0.5:1. pCMV-β-gal (0.30 μg) was complexed with the varying amount of cationic lipids in a total volume of 30 μL in Hepes buffer (pH 7.4) and incubated at room temperature for 20-25 min. 4 μL of 6 loading buffer (0.25% bromophenol blue in 40% (w/v) sucrose with sterile H2O was added to it, and from the resulting solution, 30 μL was loaded on each well. The samples were electrophoresed at 80 V for 45 min, and the DNA bands were visualized in the gel documentation unit.

Table 1. Fatty Acid Composition Data of Coconut Oil and of Coconut Amphiphiles 1 fatty acid composition* (%) sample no methyl ester of fatty acid coconut oil coconut amphiphiles 1 1

methyl caproate (C6)

0.55

-

2

methyl caprylate (C8)

7.29

8.76

3 4

methyl caprate (C10) methyl laurate (C12)

5.61 45.8

6.36 39.5

5

methyl myristate (C14)

20.1

6

methyl palmitate (C16)

8.88

7

methyl stearate (C18:0)

2.99

8

methyl oleate (C18:1)

6.98

9

methyl linoleate (C18:2)

1.84

16.1 10.7 3.66 11.9 3.02

* According to gas chromatographic analysis of fatty acid methyl esters (C6; C8; C10; C12; C14; C16; C18:0; C18:1; C18:2) in coconut oil and in coconut amphiphiles 1.

DNase I Sensitivity Assay. Briefly, in a typical assay, 3 nmol of pCMV-β-gal (1 ug) was complexed with the varying amount of cationic coconut amphiphiles 1 and control dilauroyl amphiphile 2 (using indicated lipid/DNA charge ratios in a total volume of 30 μL in Hepes buffer (pH 7.4) and incubated at room temperature for 30 min on a rotary shaker. Subsequently, the 501


Bioconjugate Chemistry complexes were treated with 10 μL DNase I (at a final concentration of 1 μg/mL or 10 ng/3 nmol of pDNA) in the presence of 20 mM MgCl2 and incubated for 20 min at 37 °C. The reactions were then halted by adding EDTA (to a final concentration of 50 mM) and incubated at 60 °C for 10 min in a water bath. The aqueous layer was washed with 50 μL of phenol/chloroform (1:1 v/v) and centrifuged at 10 000 g for 5 min. The aqueous supernatants were separated, loaded (20 μL for coconut amphiphiles 1 and control dilauroyl amphiphile 2) on a 1% agarose gel (prestained with ethidium bromide), and electrophoresed at 80 V for 45 h. DNase-I treated and untreated naked DNA was also included in the same experiment. The binding was visualized after 45 min in the gel documentation unit. Zeta Potential (ζ) and Size Measurements. The sizes and the zeta potentials (surface charges) of neat liposomes and lipoplexes with varying charge ratios (8:1-0.5:1) were measured by photon correlation spectroscopy and electrophoretic mobility on a Zeta sizer 3000HSA (Malvern, U.K.). The sizes and potentials of liposomes were measured in deionized water with a sample refractive index of 1.59 and a viscosity of 0.89. Liposomes of coconut amphiphiles 1 and control dilauroyl amphiphile 2 were complexed with DNA in plain DMEM for size and potential measurements of lipoplexes. The system was validated by using the 200 nm þ 5 nm polystyrene polymer (Duke Scientific Corps. Palo Alto, CA). The diameters of liposomes and lipoplexes were calculated by using the automatic mode. The zeta potential was measured using the following parameters: viscosity, 0.89 cP; dielectric constant, 79; temperature, 25 °C; F(Ka), 1.50 (Smoluchowski); maximum voltage of the current, V. The system was validated by using DTS0050 standard from Malvern, U.K. All the size measurements were done 10 times in triplicate with the zero field correction and values represented as the average of triplicate measurements. The potentials were measured 10 times and represented as their average values as calculated by using the Smoluchowski approximation. FRET Study. The membrane fusogenicities were measured for all the liposomes with the same composition used for the transfection studies, i.e., lipid/chol (1:1 mol ratio) as described previously.20 NBD-PE and N-Rho-PE (Avanti-Polar Lipids, USA) were used as the donor and acceptor fluorescent lipids, respectively. The model liposome formulation which acts as a biomembrane mimic was prepared using DOPC/DOPE/DOPS/ Chol (45:20:20:15, w/w ratio, the total lipid concentration used was 0.5 mM). This model biomembrane formulation was labeled with 0.005 mM NBD-PE and N-Rho-PE lipids (i.e., 1% with respect to the total biomembrane mimicking formulation content). The total lipid concentrations used for all liposomes were the same as that of the biomembrane micking lipid formulation (0.5 mM). Labeled model biomembrane liposomal formulations were placed in a FLX 800 Microplate Fluoroscence Reader (BioTek Instruments Inc., U.K.) at room temperature and an equimolar amounts of liposomes of coconut amphiphiles 1 and control dilauroyl amphiphile 2 with chol/lipid (1:1 mol ratio) were added. Fluorescence intensities were recorded as a function of time with excitation at 485 nm and emission at 595 nm. Fusion (100%) was determined from the Rho-PE fluorescence intensity of the labeled biomembrane liposomal formulation in the presence of 1% Triton X-100. AFM Analysis of Liposomes. Atomic force microscopy (AFM) was employed to analyze the morphology of the liposomes. Briefly, a glass piece (0.5 0.5 mm2) is placed onto one side of a double-sided tape and a mica sheet was then attached

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onto the other side of the tape. The mica sheet with a cellophane tape stuck onto it was pulled out several times until a smooth surface of mica sheet was obtained. 10 μL aliquots of the 0.5 mM liposome sample (prepared using equimolar ratio of individual lipids and cholesterol) was uniformly dispersed onto freshly cleaved mica, and the mica surface was then dried with a thin stream of nitrogen followed by aerial drying in a dust-free zone for 12 h. The AFM images were observed in the tapping mode in air using Digital Nanoscope IV (Veeco Instruments, Santa Barbara, CA). The imaging was carried out with the vibrationdamped microscope. Commercial phosphorus (n) doped silica tips on a I-tape cantilever with a length of 115-135 lm and resonance frequency of about 260 kHz were used. Cellular Uptake Studies by Epifluorescence Microscopy. For fluorescence microscopy experiments, 10 000 cells were seeded in each well of a 96-well plate (Corning Inc., Corning, NY) 12 h in 100 μL of growth medium such that the well became 30-50% confluent at the time of transfection. Rhodamine-PE labeled coconut amphiphiles 1 and the control control dilauroyl amphiphile 2 were complexed with pCMV-SPORT-β-gal (0.3 μg/well) at 2:1 lipid/DNA charge ratio in a total volume of 100 μL DMEM for 15-20 min. The complexes were then added to the cells. After 4 h incubation, cells were washed with PBS (2 100 μL) and fixed with 3.8% paraformaldehyde in PBS at room temperature for 10 min. The red fluorescent cells were detected under an inverted fluorescence microscope (Nikon, Japan). In Vivo Studies. Biodistribution experiments were performed in accordance with the Institutional Bio-Safety and Ethical Committee Guidelines using an approved animal protocol. One day before injection, lipoplexes of coconut amphiphiles 1 and control dilauroyl amphiphile 2 were prepared by incubating the dried lipid films with the pCMV-Luc plasmid precomplexed with commercially available PLUS Reagent (Invitrogen, USA) in 5% aqueous glucose solution for 12 h. 1 μL of PLUS Reagent was used to complex 1 μg pCMV-Luc plasmid. Each of the 68-week-old male Balb/c mice (∼20-22 g) was injected in the tail vein with 300 μL of either lipoplex at lipid/DNA charge ratio of 4:1 or DNA/PLUS Reagent complex alone (without any liposomes) in 5% w/v glucose solution using a 26.5 gauge syringe needle. Mice were sacrificed 8 h postinjection, and organs were harvested and washed in cold saline. 600 μL of lysis buffer (0.1 M Tris-HCl, 2 mM ETDA, and 0.2% Triton X-100, pH 7.8) was added to each organ and homogenized using a mechanical homogenizer. The homogenates were centrifuged at 14 000 rpm for 10 min at 4 °C, 10 μL supernatant was assayed using the Promega Luciferase assay kit (Madison, WI, USA) in Microplate Luminometer (FLx800, Bio-Tek Instruments, USA). Protein concentration of each tissue extract was determined by the modified Lowry procedure,35 and the luciferase activity in each organ was expressed as the relative light units (RLU) per mg of extracted protein.

’ RESULTS Syntheses. The fatty acid mixtures I obtained from alkaline hydrolysis of commercially available food-grade coconut oil were converted to tertiary amine mixtures II by reacting the intermediate fatty acyl chlorides with N-methyl-N,N-diethanolamine (Scheme 1, Part A). The tertiary amine mixture II, upon quaternization with 2-iodoethanol followed by ion exchange chromatography over Amberlite IRA 400 chloride resin afforded the target coconut amphiphiles 1 (Scheme 1, Part A). Importantly, 502



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cell viability assay19 in representative CHO cells using lipoplexes (lipid/DNA complexes) of 1 and 2 revealed strikingly less cytotoxic nature (>90% cell viability) of both the coconut amphiphiles and the control dilauroyl cationic amphiphile across the entire range of lipid/DNA charge ratios 8:1-0.5:1 (Figure 3). Thus, higher cytotoxicity of 2 than that of 1 was clearly ruled out as the origin of relatively poor transfection efficiencies of 2. Biomembrane Fusogenicities and Cellular Uptake Studies. Toward gaining mechanistic insights into the origin of higher transfection efficiency of the coconut amphiphiles 1 than that of the control amphiphile 2 (Figure 1, Part A), relative biomembrane fusogenicities of the liposomes of both 1 and 2 containing equimolar amounts of cholesterol as colipid) were assessed by fluorescence resonance energy transfer (FRET) assay.30 The percent of normalized fluorescence recovery was found to be ∼65% when the double-fluorophore-labeled biomembranemimicking liposomal formulations was incubated with liposomes of coconut amphiphiles 1 for 30 min, while it was ∼45% when biomembrane-mimicking liposomes were incubated with the liposome of the control lipid 2 for the same time period (Figure 4, Part A). Atomic force microscopy revealed significantly more nonlamellar character for liposomes of coconut amphiphiles 1 than that for liposomes of control symmetric amphiphiles 2 (Figure 4, Part B). Consistent with the above-mentioned high biomembrane fusibility of the liposomes of coconut amphiphiles 1, many Rho-PE labeled CHO cells were detected in the inverted position when cells were incubated with Rho-PE labeled lipoplexes of coconut amphiphiles 1 in the cellular uptake study using epifluorescence microscopy (Figure 5). Contrastingly, few Rho-PE labeled CHO cells were detected upon similar treatment of the cells with Rho-PE labeled lipoplexes of the control amphiphile 2 (Figure 5). Thus, poor cellular uptake of liposomes of the control dilauroyl amphiphile 2 is likely to play a major role behind its compromised transfection efficacies. In Vivo Studies. All the above-mentioned promising features of the liposomes of coconut amphiphiles 1, namely, higher cellular uptake, higher biomembrane fusogenicity, strong DNAbinding characteristics, and least cellular cytotoxicity, finally prompted us to evaluate their In Vivo potential. Since the transfection-efficient coconut amphiphiles 1 showed optimal in vitro gene transfer efficacies in combination with equimolar amount of cholesterol (Figure 1, Part A), the In Vivo potentials were also measured using equimolar amounts of cholesterol as colipid. An important issue is worth discussing at this point. Simple lipoplexes prepared by mixing aqueous solutions of p-CMV-luc plasmid and liposomes of amphiphiles 1 and 2 failed to transfect any mouse organs (data not shown). However, the use of p-CMV-luc plasmid precomplexed with commercially available PLUS reagent revealed lung selective transfection characteristics of the coconut amphiphiles 1 (Figure 6). Initial experiments using lipoplexes of varying lipid/DNA (() charge ratios (4:1-16:1) revealed 4:1 as the optimal In Vivo charge ratio (data not shown). Preliminary In Vivo studies revealed that onset of In Vivo gene expression starts around 6 h post injection, with the degree of gene expression remaining at maximum values during 8-24 h post injection and then decreasing gradually after 24 h (data not shown). Thus, in all the subsequent In Vivo experiments, transgene expressions were monitored 8 h post injections. The lipoplexes of the coconut amphiphiles 1 showed remarkably lung selective transfection efficiency (Figure 6). Importantly, the lung transfection efficiency of the lipoplexes of coconut amphiphiles 1 were 4-fold higher than that for the control dilauroyl amphiphile

the fatty acid compositions of both the coconut oil and the coconut amphiphiles 1 were found to be grossly similar as measured by gas chromatographic analysis of the corresponding fatty acid methyl esters. The lauroyl group was found to be the predominant alkyl chain in both coconut oil and coconut amphiphiles 1 (∼40-45%, Table 1). ESI-MS of the coconut amphiphiles 1 as well as that of its tertiary amine precursor II showed the presence of all the expected aliphatic hydrocarbon chains (C8-C18) in coconut oil (Figures S2 and S4, Supporting Information). Toward comparing gene transfer efficiencies of the coconut amphiphiles 1 with hydrophobic chain asymmetry with that of the symmetric cationic amphiphile containing two lauroyl chains (the major aliphatic hydrocarbon chain of coconut oil), the control dilauroyl amphiphile 2 was also synthesized following the same synthetic route as used for preparing coconut amphiphiles 1 (Scheme 1, Part B). Physicochemical Characterization of Liposomes and Lipoplexes. Electrostatic binding interactions between the plasmid DNA and the cationic liposomes of coconut amphiphles 1 and those between the plasmid DNA and the liposomes of the control symmetric dilauroyl amphiphile 2 were measured by the conventional gel retardation assay across the lipid/DNA charge ratios 0.5:1-8:1. Findings in the gel retardation assay revealed both the coconut amphiphiles 1 and the control dilauroyl amphiphile 2 to be strongly DNA binding at lipid:DNA charge ratios higher than 2:1 (Figure S10A, Supporting Information). Interestingly, while the sizes (measured by dynamic laser light scattering technique) of the cationic liposomes of both coconut amphiphiles 1 and the control dilauroyl amphiphile 2 were found within the range 200-250 nm, those for the lipoplexes of coconut amphiphiles 1 decreased from ∼400 nm to ∼200 nm as the lipid/DNA charge ratio decreased from 8:1 to 0.5:1 (Tables S1 and S2, Supporting Information). The lipoplex sizes for the less transfection efficient amphiphile 2 did not change significantly from ∼400 nm (Table S2, Part A, Supporting Information). Surface potentials of the lipoplexes of transfection competent coconut amphiphiles 1 were found to be higher than those for transfection incompetent amphiphile 2 at lipid/DNA charge ratio range 4:1-1:1 (Table S2, Part B, Supporting Information). Thus, higher lipoplex size and lower surface potentials for the lipoplex of amphiphile 2 could be playing some important role in its poor transfection efficiency. Transfection Biology and Cytoxicity. In vitro gene delivery efficacy of the coconut amphiphiles 1 as well as that of the control dilauroyl analogue 2 were evaluated in multiple cultured animal cells including COS-1 (SV 40 transformed African green monkey kidney cells), CHO (Chinese hamster ovary cells), HepG2 (human hepatocarcinoma cells), and B16F10 (murine melanoma cells) using p-CMV-SPORT-β-gal plasmid DNA as the reporter gene encoding the enzyme β-galactosidase as described previously.19 Cationic liposomes of the coconut amphiphiles 1 prepared in combination with equimolar cholesterol as colipid showed high gene transfer efficiencies (comparable to that of LipofectAmine 2000, one of the most efficient commercially available liposomal transfection kits) in all 4 cells while liposomes of the symmetric dilauroyl analogue 2 exhibited much less transfection efficiencies across the lipid/DNA charge ratio (() range 4:1-0.5:1 (Figure 1). Similarly, the efficiencies of the coconut amphiphiles 1 in transfecting HepG2 and CHO cells were found to be superior to those of the control dilauroyl amphiphiles 2 when the reporter gene encoded GFP, green fluorescent protein (Figure 2). 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based 503



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Figure 2. Epifluorescence microscopic images of representative HepG2 (A) and CHO (B) cells transfected with lipoplexes of coconut amphiphiles 1 (I-III), control dilauroyl amphiphile 2 (IV-VI), and pR5GFP. Lipid/DNA charge ratios in all the lipoplexes were maintained at 2:1. Phase contrast bright field images (I, IV), fluoroscent images (II, V) overlay images (III, VI).

2 (Figure 6). Toward probing whether the PLUS reagent used in precomplexing DNA had any contribution behind the observed remarkably lung selective transfection property of the coconut amphiphiles 1, a control In Vivo experiment was performed using p-CMV-luc plasmid precomplexed with PLUS reagent alone (without liposomes). In the absence of liposomes, p-CMV-luc plasmid precomplexed with only PLUS reagent (without using any liposomes) was found to be essentially transfection incompetent (Figure 6, labeled as Plus DNA).

’ DISCUSSION Numerous structure-activity studies have demonstrated in the past that gene delivery efficiencies of cationic amphiphiles, the widely used nonviral gene carriers, crucially depend on various structural parameters including the chain length of the aliphatic hydrocarbon tails, the nature of the hydrophilic polar heads, the linker group tethering the polar head and the nonpolar hydrophobic tails, and others.11,12,15-21 More recent structureactivity studies have revealed the dramatic influence of hydrophobic chain asymmetry in modulating gene delivery efficacies of synthetic cationic amphiphiles.22-24 Koynova et al. showed for the first time remarkably high in vitro gene transfer efficiency of a cationic amphiphile with asymmetric hydrocarbon chains, namely, oleoyldecanoyl-ethylphosphatidylcholine (C18:1/C10EPC).22 Most recent structure-activity studies by Koynova et al,23 and Nantz et al.24 have provided further evidence for

Figure 3. MTT-assay-based percent cell viabilities of representive CHO cells treated with lipoplexes of coconut amphiphiles 1 and control dilauroyl amphiphile 2 with lipid/DNA charge ratios 8:1-0.5:1. The cell viability values shown are the average of triplicate experiments performed on same day.

superior transfection properties of cationic amphiphiles with asymmetric hydrophobic chains to their analogues with symmetric chains. These recent reports prompted us to undertake the present investigation. Since food-grade coconut oils (triacyl 504



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Figure 4. (A) Biomembrane fusogenicities of liposomes of cholesterol/coconut amphiphiles 1 and cholesterol/control dilauroyl amphiphile 2. Fusion was induced by adding the liposomes to the double fluorophore labeled biomembrane-mimicking DOPC/DOPE/DOPS/Chol (45:20:20:15, w/w) liposomal formulation. The values shown are representative of three independent measurements. (B) Atomic force microscopic images of liposomes prepared from coconut amphiphiles 1 (I) and the control dilauroyl amphiphile 2 (II) with cholesterol as colipid (at a lipid/cholesterol mole ratio of 1:1). Bar corresponds to 100 nm.

glycerols) contain fatty acyl chains of varying lengths, we envisaged that essentially three easy chemical steps (shown in Scheme IA) should be able to convert food-grade coconut oil (in principle, any other food-grade edible oil) into a mixture of nontoxic cationic amphiphiles containing natural fatty acyl chain asymmetry of coconut oil. Because of their hydrophobic chain asymmetry and because of being synthesized from a mixture of natural fatty acids of coconut oil, the resulting catonic amphiphiles are expected to be transfection competent and less cytotoxic. To begin with, we first ensured that the fatty acid compositions of coconut oil did not get grossly affected upon its conversion to coconut amphiphiles. Gas chromatographic analysis of the fatty acid methyl esters indeed revealed grossly similar composition for the fatty acyl chains in both coconut amphiphiles 1 and the precursor coconut oil. In both, the lauroyl group (C12) was found to be present as the major aliphatic chains (Table 1). Since pure synthetic cationic amphiphiles with hydrophobic

chain asymmetry have been demonstrated to be more transfection efficient than their symmetric counterparts,23,24 for the sake of comparison, we also synthesized the symmetric dilauroyl analogue 2 (Scheme 1B) following the same synthetic procedure adopted for synthesizing coconut oil amphiphiles 1. Consistent with the higher transfection efficacies of pure synthetic cationic amphiphiles with hydrophobic chain asymmetry than that for their symmetric analogues,23,24 the transfection efficiencies of coconut amphiphiles 1 were found to be remarkably higher than those of their synthetic pure dilauroyl amphiphiles 2 (Figures 1 and 2). These findings demonstrate for the first time that the transfection-enhancing influence of asymmetric hydrocarbon chains in pure synthetic cationic amphiphiles containing two defined hydrocarbon chains of different lengths also works for cationic amphiphiles designed with natural, asymmetric fatty acyl chains of a food-grade oil. As for examples of prior reports on syntheses of cationic transfection lipids starting from oil, cationic 505



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Figure 5. Epifluorescence microscopic images of representative HepG2 (A) and CHO (B) cells transfected with lipoplexes of Rhodamine-labeled coconut amphiphiles 1 (I-III) and lipoplexes of Rhodamine-labeled control dilauroyl amphiphile 2 (IV-VI) with a lipid/DNA charge ratio of 2:1. Phase contrast bright field images (I, IV), fluoroscent images (II, V), overlay images (III, VI).

amphiphiles with in vitro gene transfer properties have been synthesized from vernonia oil, a natural epoxidized triglyceride.25-27 Examples of previously reported asymmetric synthesis of cationic lipids for use in gene delivery include asymmetric synthesis of dialkyloxy-3-alkylammonium cationic lipids28 and asymmetric synthesis of 2,3-acyloxy-1-propylammonium salts.29 However, to the best of our knowledge, the present study is the first report on the synthesis of lung-selective cationic transfection lipid designed with the hydrophobic chain asymmetry of food-grade coconut oil. After finding that the lipoplexes of both coconut amphiphiles 1 and its pure synthetic dilauroyl analogue 2 are remarkably less cytotoxic across the entire range of lipid/DNA ratios of 0.5:18:1 (Figure 3), we measured the membrane fusogencities of the two liposomes toward gaining mechanistic insights into the origin of higher transfection efficiencies of coconut amphiphiles 1 using fluorescence resonance energy transfer (FRET) assay.30 This technique depends upon the interactions that occur between two fluorophores when the emission band of one (the energy donor) overlaps with the exicitation band of the second (the energy acceptor) and when such fluorophores are in close physical proximity. Liposomes, whose membrane fusogenicities are to be measured, are incubated with a popular biomembranemimicking liposomal formulation (dioleyol-phosphatidylcholine/dioleyol-phosphatidylethanolamine/dioleyol phosphatidylserine/cholesterol at 45:20:20:15, w/w). The conditions of close physical proximity of the energy donors and energy acceptors are

satisfied when the biomembrane-mimicking liposomal formulations are prepared with both the donor and the acceptor fluorophores such as N-(7-nitro-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) and Rhodamine Red-x-1,2-dihexadecanoyl-sn-glycero 3-phosphoethanolamine (Rho-PE), respectively. In such liposomes, the energy from a photon absorbed by the energy donor, NBD-PE, is transferred to the energy acceptor, Rho-PE, causing the latter to fluoresce as if it is excited directly. Since the efficiency of FRET between two such fluorophores critically depends upon their spatial separation, any fusion event of such double-fluorophore-containing biomembrane-mimicking liposomes with cationic liposomes of coconut amphiphiles 1 and the control dilauroyl amphiphile 2 (devoid of any fluorophore) decreases the efficiency of resonance energy transfer. In other words, decrease in percent of normalized fluorescence recovery (i.e., maximum fusion corresponding to Rho-PE fluorescence emission intensity at 595 nm observed when the double-fluorophore-labeled biomembrane liposomal formulation is completely disrupted with 1% Triton X-100) provides evidence for reduced membrane fusion. The observed higher biomembrane fusogenicity for the liposomes of coconut amphiphiles 1 compared to those for the liposomes of pure synthetic dilauroyl amphiphile 2 (Figure 4A) support the notion that higher transfection efficiencies of coconut amphiphiles 1 partly originate from their higher cell membrane fusion characteristics. 506



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Figure 6. Selective targeting of genes to mouse lung by lipoplexes of coconut amphiphiles 1. pCMV-Luc plasmid was precomplexed with commercially available PLUS reagent in 5% aqueous glucose solution, and such precomplexed DNA was incubated for 12 h with dried lipid film to prepare lipoplexes. PLUS reagent induced precomplexed pCMV-Luc plasmid (labeled as Plus DNA) was used as control. 6-8-week-old Balb/C mice were intravenously injected with lipoplexes of both coconut amphiphiles 1 and control dilauroyl amphiphile 2 using 50 μg of pCMV-Luc precondensed with PLUS reagent at lipid/DNA charge ratio of 4:1. Luciferase activities were estimated 8 h post injection. Details of the experiment are described in the text. Data shown are the mean values ( SD (n = 3).

(Figure 6). However, these findings do not shed insight into why the efficiency of the coconut amphiphiles 1 in transfecting mouse lung is so much higher than its efficacies in transfecting the other organs. Perhaps, the lipoplex sizes for the transfection-efficient coconut amphiphiles 1 under circulation are large enough to get filtered first in the lung (the first pass organ) and nothing passes into circulation beyond the lung. Consistent with the in vitro findings (Figures 1 and 2), the control lipoplex of the pure synthetic dilauroyl analogue 2 was found to be significantly less efficient than coconut amphiphiles 1 (by ∼4-fold) in transfecting mouse lung. Intravenous injection of 5% aqueous glucose solution of naked plasmid DNA failed to transfect mice body organs to any significant extent (data not shown). An important issue deserves further elaboration at this point of discussion. In the In Vivo experiments, we used commercially available PLUS reagent to precondense the plasmid DNA, since lipoplexes prepared by mixing aqueous solutions of p-CMV-luc plasmid and liposomes of amphiphiles 1 and 2 (without using PLUS reagent) failed to transfect any mouse organs. Given that the interactions of lipoplexes with circulation proteins under In Vivo settings are likely to make membrane fusion difficult, one might argue that condensation of the nucleic acids followed by lipoplex formation are more important than the enhanced membrane fusogenic nature of the coconut amphiphiles 1 for their In Vivo lung transfection property. Clearly, toward probing the relative contributions of hydrophobic chain asymmetry and DNA precondensing agent behind the observed lung transfection property of the coconut amphiphiles 1 under In Vivo settings, further In Vivo experiments need to be conducted in the future using lipoplexes of additional control amphiphiles containing asymmetry in hydrophobic chains. Taking the usually observed toxicity of commercially available LipofectAmine 2000 under In Vivo settings into consideration, the lung-selective transfection property of coconut amphiphiles 1 is promising. Once the preclinical formulation parameters are further optimized (for instance, transgene expression in the

Koynova et al. previously demonstrated more fusogenic nature of nonlamellar liposomes.22 Thus, the observed higher biomembrane fusogenicity of the liposomes of coconut amphiphiles 1 compared to that for liposomes of the control amphiphile 2 (Figure 4A) was consistent with the nonlamellar structures for the liposomes of coconut amphiphiles 1 found in the atomic force microscopic (AFM) study (Figure 4B). Currently believed lipofection (cationic lipid mediated gene transfer) pathways include the following: (a) endocytotic cellular uptake of the lipid/DNA complex (lipoplex); (b) release of DNA from the resulting endosomes into the cell cytoplasm; and (c) nuclear transport of the endosomally released DNA followed by transcription and gene expression.31-33 Improved biomembrane fusogenicity of cationic liposomes is expected to mediate not only enhanced cellular uptake of the cationic liposome associated DNA (step a), but also the efficacy of step b. This is because efficient release of DNA from endosomes into cell cytoplasm (step b) is likely to depend on how efficiently the cationic lipids can fuse with the anionic lipid components of the endosomal membranes. Wang and MacDonald have also found a similar correlation between membrane fusogenicity and transfection efficacies of cationic liposomes.34 Enhanced cell membrane fusibility of the lipoplexes of the coconut amphiphiles 1 became most evident in the cellular uptake study using Rho-PE labeled liposomes. In both HepG2 and CHO cells, the number of fluorescently labeled cells were found to be significantly higher when cells were incubated with Rho-PE labeled lipoplexes of the coconut amphiphiles 1 for 4 h (Figure 5). Thus, higher cellular uptake resulting from the more fusogenic nature of the liposomes of coconut amphiphiles 1 are likely to play a dominant role in making the coconut amphiphiles transfection competent. The remarkable cellular uptake of the lipoplexes of coconut amphiphiles 1 (Figure 5) plus its less cytotoxic nature (Figure 3) finally prompted us to evaluate its In Vivo potential. Importantly, the degree of transgene expression in lung was found to be 2-3 orders of magnitude higher than in kidney, heart, spleen, and liver 507



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present study gradually diminished after 24 h post injection; data not shown), the presently described coconut amphiphiles 1 may find future use in nonviral gene therapy of lung diseases.

’ CONCLUSIONS We have demonstrated for the first time that cationic amphiphiles designed with the natural fatty acyl chain asymmetry of food-grade coconut oil are less cytotoxic and deliver genes selectively to mouse lung. Despite lauroyl chains being the major fatty acyl chains of coconut oil, both the in vitro and In Vivo gene transfer efficiencies of such cationic amphiphiles were found to be remarkably superior (>4-fold) to those of their pure dilauroyl analogue. Our findings demonstrate for the first time that the transfection enhancing influence of asymmetric hydrocarbon chains previously observed in pure, synthetic cationic amphiphiles containing two defined aliphatic hydrocarbon chains of different lengths also works for cationic amphiphiles designed with the natural fatty acyl chain asymmetry of a food-grade oil. Mechanistic studies involving the technique of fluorescence resonance energy transfer (FRET) revealed higher biomembrane fusibility of the cationic liposomes of the coconut amphiphiles than that of the symmetric dilauroyl analogue. AFM study revealed pronounced fusogenic nonlamellar structures of the liposomes of coconut amphiphiles. Findings in the FRET and cellular uptake study, taken together, support the notion that the higher cellular uptake resulting from the more fusogenic nature of the liposomes of coconut amphiphiles 1 is likely to play a dominant role in making the coconut amphiphiles transfection competent under in vitro conditions. However, further In Vivo experiments using additional control amphiphiles with hydrophobic chain asymmetry need to be carried out in the future to confirm the transfection-enhancing role of hydrophobic chain asymmetry under systemic settings.

Abbreviations: Chol,cholesterol; DCM,dichloromethane; DMEM,Dulbecco’s Modified Eagles Medium; MEM,Minimum Essential Medium; DMF,N,N-dimethyl formamide; FBS,fetal bovine serum; ONPG,o-nitrophenyl-β-D-galactopyranoside; PBS,phosphate buffered saline; GFP,green fluorescent protein.

’ REFERENCES (1) Check, E. (2003) Harmful potential of viral vectors fuels doubts over gene therapy. Nature 423, 573–574. (2) Bell, P. C., Bergsma, I. P., Bras, W., Stuart, M. C. A., Rowan, M. C., Feiters, M. C., and Engberts, J. B. F. N. (2003) Transfection mediated by gemini surfactants, engineered escape from the endosomal compartment. J. Am. Chem. Soc. 125, 1551–1558. (3) Martin, B., Sainlos, M., Aissaoui, A., Oudrhiri, N., Hauchecorne, M., Vigneron, J, P., Lehn, J. M., and Lehn, P. (2005) The design of cationic lipids for gene delivery. Curr. Pharm. Des. 11, 375–94. (4) Kirby, A. J. (2003) et al. Gemini surfactants: new synthetic vectors for gene transfection. Angew. Chem., Int. Ed. Engl. 42, 1448–1457. (5) Prata, C. A. H., Zhao, Y., Barthelemy, P., Li, Y., Luo, D., McIntosh, T. J., Lee, S. J., and Grinstaff, M. W. (2004) Charge-reversal amphiphiles for gene delivery. J. Am. Chem. Soc. 126, 12196–12197. (6) Desigaux, L., Sainlos, M., Lambert, O., Chevre, R., LetrouBonneval, E., Vigneron, J. P., Lehn, P., Lehn, J. M., and Pitard, B. (2007) Self-assembled lamellar complexes of siRNA with lipidic aminoglycoside derivatives promote efficient siRNA delivery and interference. Proc. Natl. Acad. Sci. U.S.A. 104, 16534–16539. (7) Unciti-Broceta, A., Holder, E., Jones, L. J., Stevenson, B., Turner, A. R., Porteous, D. J., Boyd, A. C., and Bradley, M. (2008) Tripod-like cationic lipids as novel gene carriers. J. Med. Chem. 51, 4076–4084. (8) Lynn, D. M., Anderson, D. G., Putman, D., and Langer, R. (2001) Accelerated discovery of synthetic transfectionvectors: Parallel synthesis and screening of a degradable polymer library. J. Am. Chem. Soc. 123, 8155–8156. (9) Choi, J. S., Joo, D. K., Kim, C. H., Kim, K., and Park, J. S. (2000) Synthesis of a barbell-like triblock copolymer, poly(L-lysine) dendrimerblock-poly(ethylene glycol)-block-poly(L-lysine) dendrimer, and its self-assembly with plasmid DNA. J. Am. Chem. Soc. 122, 474–480. (10) Luo, D., Haverstick, K., Belcheva, N., Han, E., and Saltzman, W. M. (2002) Poly (ethylene glycol)-conjugated PAMAM dendrimer for biocompatible, high-efficiency DNA delivery. Macromolecules 35, 3456–3462. (11) Srinivas, R., Samanta, S., and Chaudhuri, A. (2009) Cationic amphiphiles: promising carriers of genetic materials in gene therapy. Chem. Soc. Rev. 38, 3326–3338. (12) Bhattacharya, S., and Bajaj, A. (2009) Advances in gene delivery through molecular design of cationic lipids. Chem. Commun 31, 4632– 4656. (13) Singh, R. S., Mukherjee, K., Banerjee, R., Chaudhuri, A., Hait, S. K., Moulik, S., Ramadas, Y., Vijayalakshmi, A., and Rao, N. M. (2002) Anchor dependency for non-glycerol based cationic lipofectins: mixed bag of regular and anomalous transfection profiles. Chem.—Eur. J. 8, 900–909. (14) Kukowska-Latallo, J. F., Bielinska, A. U., Johnson, J., Spinder, R., Tomalia, D. A., and Baker, J. R. (1996) Efficient transfer of genetic material into mammalian cells using Star burst polyamidoamine dendrimers. Proc. Natl. Acad. Sci. U.S.A. 93, 4897–4902. (15) Byk, G., Dubertret, C., Escriou, V., Frederic, M., Jaslin, G., Rangara, R., Pitard, B., Crouzet, J., Wils, P., Schwartz, B., and Scherman, D. (1998) Synthesis, activity, and structure-activity relationship studies of novel cationic lipids for DNA transfer. J. Med. Chem. 41, 224–235.

’ ASSOCIATED CONTENT

bS

1

H NMR and ESI-MS mass spectral characterizations for coconut amphiphiles 1 and Control dilauroyl amphiphile 2 as well as for their tertiary amine precursors (Figures S1-S8), reverse-phase HPLC chromatograms and HPLC conditions for control dilauroyl amphiphile 2 in two mobile phases (Figure S9), DNA binding assay and DNase I sensitivity assay (Figure S10), size and surface potential data (Tables S1 and S2) (total 17 pages). This material is available free of charge via the Internet at http://pubs.acs.org. Supporting Information.

’ AUTHOR INFORMATION Corresponding Author

*To whom correspondence should be addressed. E-mail: [email protected], Tel: 91-40-27193201, Fax: 91-40-27160757.

’ ACKNOWLEDGMENT This work was supported by the Council of Scientific and Industrial Research, Government of India, New Delhi (NWP0036). We thank N. M. Rao, Centre for Cellular and Molecular Biology, Hyderabad, India for providing us p-CMV-SPORT-βGal; VC thanks University Grants Commission, MS and KR thank CSIR, Government of India, New Delhi, for providing doctoral research fellowships. 508



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Cationic Amphiphiles with Fatty Acyl Chain Asymmetry of Coconut Oil

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