Cationic amphiphiles with fatty acyl chain asymmetry of coconut oil deliver genes selectively to mouse lung

Article abstract of DOI:10.1021/bc100537r

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. Pharmaceutics2010, 7, 786-794; Koynova, R. et al. Mol. Pharmaceutics2009, 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.

Full text of DOI:10.1021/bc100537r

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
S Supporting Information
b
ABSTRACT: Recent structure-activity studies have revealed a
dramatic influence of hydrophobic chain asymmetry in enhan-
cing 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, asym-
metric fatty acyl chains of a food-grade oil. Herein, we demon-
strate 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
of cationic amphiphiles.20,21 In 2006, in a thought-provoking
structure-activity study, Koynova et al. demonstrated dramati-
cally 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 asym-
metric counterpart stearoyldecanoyl-ethylphosphatidylcholine
(C18:0/C10-EPC).22 In this report, it was demonstrated that
liposomal compositions of C18:1/C10-EPC containing two de-
fined 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 influe-
nce of hydrophobic domain asymmetry in modulating the gene
transfer efficacies of synthetic cationic amphiphiles. These impor-
tant 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
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 biosafety-
related disadvantages including adverse inflammatory and im-
munogenic 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 dendri-
mers,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
Received: December 1, 2010
Revised:
January 21, 2011
Published: February 21, 2011
r
2011 American Chemical Society
497
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Scheme 1. Synthesis of Coconut Amphiphiles 1 (A) and Control Dilauroyl Amphiphile 2 (B)
1
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 amphi-
philes, 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 unsatu-
rated 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.
spectrometer for ESI analysis. H 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-β-
gal 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-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
poly(ethylene glycol) 8000, o-nitrophenyl-β-D-galactopyrano-
side, N-methyl-N,N-diethanolamine, and cholesterol were pur-
chased 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 coco-
nut amphiphiles 1 and control dilauroyl amphiphile 2 were shown
in parts A and B, Scheme 1, respectively. Structures of all the syn-
1
thetic intermediates were confirmed by H 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
’ 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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ARTICLE
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, con-
centrated 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 com-
plete 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, N-
diethanolamine 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-dietha-
nolamine (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.
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 chromatogra-
phic purification (using 60-120 mesh silica gel and 4% metha-
nol/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).
1H 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-hydro-
xyethyl]-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 con-
firm 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
1H NMR of coconut oil derived N-methyl-N,N-diethanola-
mine 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
499
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ARTICLE
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,N-di-[O-dodecanoyl-2-hydroxyethyl]-N-methyl amine as yel-
low liquid (3.6 g, 89% yield, Rf = 0.8, 5:95 methanol/chloroform,
v/v), which solidified upon storage at 4 °C.
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 trans-
fection profiles obtained on different days were identical.
Cytotoxicity Assay. The cytotoxicities of coconut amphi-
philes 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-(4,5-
dimethylthiazol-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 (un-
treated cells) - background] ꢀ 100.
1H NMR of N,N-di-[O-dodecanoyl-2-hydroxyethyl]-N-meth-
yl amine (300 MHz, CDCl3): δ/ppm = 0.9 [t, 6H, CH3-CH2-
C9H18-]; 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)8-
CH2-CH2-CO-]; 2.4 [s, 3H, N-CH3]; 2.7 [t, 4H, N-CH2-CH2-
O-]; 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-2-
hydroxyethyl]-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 chlo-
ride 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 recrystalliza-
tion 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).
Fatty Acid Composition Profile by Gas Chromatogra-
phy. 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 per-
formed 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 composi-
tion percentages of cationic lipid as obtained by gas chromato-
graphic 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 mois-
ture-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 2-
3 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-CMV-
SPORT-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.
1H 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
2ꢀ 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
Transfection Protocol for r5GFP Plasmid. Cells were
seeded in 24-well plates (Corning Inc., Corning, NY, 4 ꢀ 104
500
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ARTICLE
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.
Table 1. Fatty Acid Composition Data of Coconut Oil and of
Coconut Amphiphiles 1
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% parafor-
maldehyde 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 6ꢀ 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.
fatty acid composition* (%)
sample no methyl ester of fatty acid coconut oil coconut amphiphiles 1
1
2
3
4
5
6
7
8
9
methyl caproate (C6)
methyl caprylate (C8)
0.55
7.29
5.61
-
8.76
6.36
methyl caprate (C10
)
methyl laurate (C12)
methyl myristate (C14
methyl palmitate (C16
45.8
20.1
8.88
39.5
)
)
16.1
10.7
methyl stearate (C18:0
methyl oleate (C18:1
methyl linoleate (C18:2
)
2.99
6.98
1.84
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 amphi-
phile 2 (using indicated lipid/DNA charge ratios in a total volu-
me of 30 μL in Hepes buffer (pH 7.4) and incubated at room
temperature for 30 min on a rotary shaker. Subsequently, the
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complexes were treated with 10 μL DNase I (at a final concen-
tration 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 super-
natants were separated, loaded (20 μL for coconut amphiphiles 1
and control dilauroyl amphiphile 2) on a 1% agarose gel (pres-
tained with ethidium bromide), and electrophoresed at 80 V for
45 h. DNase-I treated and untreated naked DNA was also inclu-
ded 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 poten-
tials 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 (Smolu-
chowski); 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, res-
pectively. The model liposome formulation which acts as a bio-
membrane 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 con-
tent). 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 pre-
sence of 1% Triton X-100.
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 vibration-
damped 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 Com-
mittee 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 6-
8-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 lipo-
somes) 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 homo-
genizer. 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 Lumin-
ometer (FLx800, Bio-Tek Instruments, USA). Protein concen-
tration 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 inter-
mediate fatty acyl chlorides with N-methyl-N,N-diethanolamine
(Scheme 1, Part A). The tertiary amine mixture II, upon quater-
nization with 2-iodoethanol followed by ion exchange chroma-
tography over Amberlite IRA 400 chloride resin afforded the
target coconut amphiphiles 1 (Scheme 1, Part A). Importantly,
AFM Analysis of Liposomes. Atomic force microscopy
(AFM) was employed to analyze the morphology of the lipo-
somes. 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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the fatty acid compositions of both the coconut oil and the
coconut amphiphiles 1 were found to be grossly similar as mea-
sured 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 amphi-
philes 1 (∼40-45%, Table 1). ESI-MS of the coconut amphi-
philes 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 Infor-
mation). 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 amphi-
philes 1 (Scheme 1, Part B).
Physicochemical Characterization of Liposomes and Li-
poplexes. Electrostatic binding interactions between the plas-
mid 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 amphi-
phile 2 to be strongly DNA binding at lipid:DNA charge ratios
higher than 2:1 (Figure S10A, Supporting Information). Inter-
estingly, while the sizes (measured by dynamic laser light scat-
tering 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 Infor-
mation). 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 Infor-
mation). 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-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based
cell viability assay19 in representative CHO cells using lipoplexes
(lipid/DNA complexes) of 1 and 2 revealed strikingly less cyto-
toxic nature (>90% cell viability) of both the coconut amphi-
philes 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 Stud-
ies. 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 bio-
membrane fusogenicities of the liposomes of both 1 and 2 contai-
ning 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 biomembrane-
mimicking 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 amphi-
philes 1 in the cellular uptake study using epifluorescence micro-
scopy (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 cel-
lular uptake, higher biomembrane fusogenicity, strong DNA-
binding characteristics, and least cellular cytotoxicity, finally
prompted us to evaluate their In Vivo potential. Since the trans-
fection-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 lipo-
plexes prepared by mixing aqueous solutions of p-CMV-luc plas-
mid 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 re-
agent 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, trans-
gene expressions were monitored 8 h post injections. The lipo-
plexes 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
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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 incom-
petent (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
Figure 3. MTT-assay-based percent cell viabilities of representive
hydrophobic tails, and others.11,12,15-21 More recent structure-
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 per-
activity studies have revealed the dramatic influence of hydro-
phobic chain asymmetry in modulating gene delivery efficacies of
synthetic cationic amphiphiles.22-24 Koynova et al. showed for
formed on same day.
the first time remarkably high in vitro gene transfer efficiency of a
cationic amphiphile with asymmetric hydrocarbon chains,
namely, oleoyldecanoyl-ethylphosphatidylcholine (C18:1/C10-
EPC).22 Most recent structure-activity studies by Koynova
et al,23 and Nantz et al.24 have provided further evidence for
superior transfection properties of cationic amphiphiles with
asymmetric hydrophobic chains to their analogues with sym-
metric chains. These recent reports prompted us to undertake
the present investigation. Since food-grade coconut oils (triacyl
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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 envi-
saged 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 composi-
tions of coconut oil did not get grossly affected upon its con-
version 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 transfec-
tion 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
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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 syn-
thesis 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 0.5:1-
8:1 (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 be-
tween 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 biomembrane-
mimicking liposomal formulation (dioleyol-phosphatidylcho-
line/dioleyol-phosphatidylethanolamine/dioleyol phosphatidyl-
serine/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 formula-
tions are prepared with both the donor and the acceptor fluoro-
phores such as N-(7-nitro-2-oxa-1,3-diazol-4-yl)-1,2-dihexadeca-
noyl-sn-glycero-3-phosphoethanolamine (NBD-PE) and Rhoda-
mine Red-x-1,2-dihexadecanoyl-sn-glycero 3-phosphoethanola-
mine (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 separa-
tion, 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 normal-
ized 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 cha-
racteristics.
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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).
Koynova et al. previously demonstrated more fusogenic
nature of nonlamellar liposomes.22 Thus, the observed higher
biomembrane fusogenicity of the liposomes of coconut amphi-
philes 1 compared to that for liposomes of the control amphi-
phile 2 (Figure 4A) was consistent with the nonlamellar struc-
tures 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 tran-
scription 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.
(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 commer-
cially 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 (with-
out 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 amphi-
philes 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 con-
ducted in the future using lipoplexes of additional control
amphiphiles containing asymmetry in hydrophobic chains. Tak-
ing 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
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
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ARTICLE
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.
Abbreviations:
Chol,cholesterol;
DCM,dichloromethane;
DMEM,Dulbecco’s Modified Eagles Medium;
MEM,Minimum Essential Medium;
DMF,N,N-dimethyl formamide;
FBS,fetal bovine serum;
’ CONCLUSIONS
We have demonstrated for the first time that cationic amphi-
philes 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 amphi-
philes 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 revea-
led pronounced fusogenic nonlamellar structures of the lipo-
somes 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.
ONPG,o-nitrophenyl-β-D-galactopyranoside;
PBS,phosphate buffered saline;
GFP,green fluorescent protein.
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’ ASSOCIATED CONTENT
Supporting Information. 1H NMR and ESI-MS mass
S
b
spectral characterizations for coconut amphiphiles 1 and Control
dilauroyl amphiphile 2 as well as for their tertiary amine precur-
sors (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.
’ AUTHOR INFORMATION
Corresponding Author
*To whom correspondence should be addressed. E-mail: arabin-
da@iict.res.in, Tel: 91-40-27193201, Fax: 91-40-27160757.
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