Vol.2, No.2, 53-62 (2011) Journal of Biophysical Chemistry
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/JBPC/
Ionization and transfection activity of
n-methyl-substituted carbamoyl-cholesterol derivatives
Samuel Ach eampong, Michalak is Savva*
Division of pharmaceuticals Sciences, Arnold & Marie Schwartz College of Pharmacy and Health Sciences, Long Island University,
New York, USA; *Corresponding Author: micsavva@gmail.com
Received 10 November 2010; revised 13 December 2010; accepted 29 December 2010.
Five novel cationic lipids, the polar head group
of which was attached to the cholesterol back-
bone via a tertiary carbamate linker, were syn-
thesized and their physicochemical properties
were compared to their transfection efficiencies.
Transfection activity of the primary amine ana-
log was highest among the series, while the qu-
aternary ammonium iodide salt was essentially
transfection incompetent. Contrary to DC-Chol,
methyl and ethyl carbamoyl derivatives of DC-
Chol mediated high levels of transfection in the
absence of DOPE. Ionization of the cationic as-
semblies in 40 mM Tris buffer pH 7.2 exactly
correlated with the competitive nature of the
inductive and steric effects of the methyl groups
on the aliphatic nitrogen of the lipids’ polar
moiety. Interestingly, the pH interaction zone of
all lipid dispersions at 25˚C was extended by ±2
pH units from the pKa, while the pKa of the cati-
onic lipids determined in mixed vesicles com-
posed of 90% DOPC and cholesterol was ap-
proximately 1.3 to 1.5 times higher than that of
pure cationic assemblies. The interaction of ca-
tionic lipids with plasmid DNA was correlated
with pKa, but not the transfection activity.
Keywords: Cationic Lipids; DC-Chol; Transfection;
Pka; Plasmid DNA; Monolayer; Surface Pressure
Cholesterol-based cationic lipids are one of the most
promising series of reagents for in vitro and in vivo gene
delivery [1-4]. Over the years, these structures were sys-
tematically varied in an effort to generate structure-
activity relationships to rationalize their design and ef-
fectively optimize their transfection efficiency [5-8].
Most of the approaches focus on attaching a hydrophilic
basic moiety capable of binding to the negatively char-
ged nucleic acid, to the membrane compatible choles-
terol moiety via a chemical linker. Quite frequently, the
polar head group of these lipids is composed of a pri-
mary, secondary, tertiary or quaternary amine and almost
always these lipids require the presence of the helper
lipid DOPE in order to mediate transfection. DOPE is a
zwitterionic amphiphil that forms reverse hexagonal
phases (HII) at physiological pH and is widely believed
to promote fusion with cell membranes, facilitate en-
dosomal escape, reduce the toxicity and increase the
transfection activity of lipoplexes [9-13].
In our recent report, the physicochemical properties of
cholesterol-based cationic lipids carrying a secondary
carbamate linker were correlated with their transfection
activity in the absence of helper lipids. The study found
that the tertiary and quaternary amine derivatives, DC-
Chol and TC-Chol (herein termed DC and TC), failed to
mediate transfection and further concluded that the high
transfection activity mediated by the primary and secon-
dary analogs AC and MC in the absence of DOPE, was
due to their increased association with plasma membrane
and endosomal escape [14]. The aim of this investigation
was to examine how methylation on the carbamoyl ni-
trogen affects the lipids’ ionization, interaction with
plasmid DNA and transfection activity.
N-methylethylenediamine 95%, N,N’-dimethylethyle-
nediamine 99%, N,N,N’-trimethylethylene-diamine 97%,
N,N-dimethyl-N’-ethylethylenediamine 98%, Cholesteryl
chloroformate 97%, iodomethane 99.5%, 2-hydroxy-
ethylmercaptan, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphen-
yltetrazolium bromide (MTT) and o-nitrophenyl-β-D-
galactopyranoside (ONPG) were purchased from Sigma-
Aldrich (St. Louis, MO). Tris-(hydroxymethyl)-amin-
omethane (ultrapure grade) and edetate disodium di-
hydrate (EDTA) were purchased from Spectrum Chemi-
cal Manufacturing Company (New Brunswick, NJ).
Murine melanoma cells B16F0 and Dulbecco’s modified
S. Acheampong et al. / Journal of Biophysical Chemistry 2 (2011) 53-62
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/JBPC/
I -
Scheme 1. Cholesterol-based cationic lipids.
Eagle’s medium (DMEM) were obtained from American
Type Culture Collection (Manassas, VA). Fetal calf se-
rum, penicillin (5 000 units)/streptomycin (5,000 μg) and
ethidium bromide solution (10 mg·mL–1) were obtained
from Invitrogen Corporation (Grand Island, NY). 1,2-
dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) was
purchased from Avanti Polar Lipids, Inc. (Alabaster, AL).
2.1. Synthesis
The cholesterol-based analogues MAC, MMC, MDC
and EDC (Scheme 1) were synthesized from N-methyle-
thylenediamine, N,N’-dimethylethylenediamine, N,N,N’-
trimethylethylene-diamine and N,N-dimethyl-N’-ethyle-
thylenediamine, respectively, at yields > 75 %, as de-
scribed elsewhere [14]. The quaternary ammonium de-
rivative MTC was synthesized by refluxing MDC in the
presence of an equimolar concentration of methyl iodide
for 2 h.
cholesterol (MAC)
Anal. Calcd for C31H54N2O2 (MW 486.42): C, 76.42;
H, 11.09; N, 5.75, Found: C, 75.44; H, 11.09; N, 5.68,
MS (Positive/Negative ES) m/z 487.43 [M+H]+; 1H
NMR (400 MHz, CdCl3, 20˚C, TMS) δ 0.68 (s, 3H,
H’18), 0.85 - 0.88 (d, 6H, H’26 - H’27), 0.90 - 0.92 (d,
3H, H’21), 1.02 (s, 3H, H’19), 1.04 - 1.59 (m, 21H, H’1,
H’9, H’11 - H’12, H’14 - H’17, H’20, H’22 - H’25), 1.81
- 2.02 (m, 5H, H’2, H’7, H’8), 2.30 - 2.35 (m, 2H, H’4),
2.83 - 2.94 (m, 5H, H2NCH2, OCON(CH3)CH2), 3.31 -
3.32 (m, 2H, OCON(CH3)CH2), 4.53 (m, 1H, H´3), 5.37
- 5.38 (d, 1H, J = 5, H’6).
cholesterol (MMC)
Anal. Calcd for C32H56N2O2 (MW 500.4): C, 76.68; H,
11.18; N, 5.59, Found: C, 75.34; H, 11.04; N, 5.49, MS
(Positive/Negative ES) m/z 501.3 [M + H]+; 1H NMR
(400 MHz, CdCl3, 20˚C, TMS) δ 0.67 (s, 3H, H´18), 0.85
- 0.88 (d, 6H, H’26-H’27), 0.90 - 0.92 (d, 3H, H’21),
1.02 (s, 3H, H’19), 1.04 - 1.63 (m, 21H, H’1, H’9, H’11 -
H’12, H’14 - H’17, H’20, H’22 - H’25), 1.78 - 2.02 (m,
5H, H’2, H’7, H’8), 2.26 - 2.38 (m, 2H, H’4), 2.46 (s, 3H,
NH(CH3)), 2.74 (m, 2H, (CH3)HNCH2), 2.93 (s, 3H,
OCON(CH3)CH2), 3.38 (m, 2H, OCON(CH3)CH2), 4.51
(m, 1H, H’3), 5.37 - 5.38 (d, 1H, J = 5, H’6).
cholesterol (MDC)
Anal. Calcd for C33H58N2O2 (MW 514.4): C, 76.92; H,
11.27; N, 5.44, Found: C, 76.93; H, 11.64; N, 5.33, MS
(Positive/Negative ES) m/z 515.3 [M + H]+; 1H NMR
(400 MHz, CdCl3, 20˚C, TMS) δ 0.63 (s, 3H, H’18), 0.82
- 0.84 (d, 6H, H’26 - H’27), 0.87 - 0.90 (d, 3H, H’21),
0.97 (s, 3H, H’19), 1.00-1.60 (m, 21H, H’1, H’9, H’11-
H’12, H’14 - H’17, H’20, H’22 - H’25), 1.75 - 1.98 (m,
5H, H’2, H’7, H’8), 2.22 (s, 6H, N(CH3)2), 2.36 - 2.40
(m, 4H, H’4, (CH3)2NCH2), 2.87 (s, 3H, OCON(CH3)
CH2), 3.20 - 3.36 (m, 2H, OCON(CH3)CH2), 4.48 - 4.52
(m, 1H, H´3), 5.36 - 5.38 (d, 1H, J = 5, H’6).
cholesterol (EDC)
Anal. Calcd for C34H60N2O2 (MW 528.5): C, 77.15; H,
11.34; N, 5.29, Found: C, 76.05; H, 11.38; N, 4.79, MS
(Positive/Negative ES) m/z 529.5 [M + H]+; 1H NMR
(400 MHz, CdCl3, 20˚C, TMS) δ 0.63 (s, 3H, H´18), 0.81
- 0.83 (d, 6H, H’26 - H’27), 0.86 - 0.88 (d, 3H, H’21),
0.97 (s, 3H, H´19), 1.08 - 1.58 (m, 21H, H’1, H’9, H’11 -
H’12, H’14 - H’17, H´20, H’22 - H’25), 1.76 - 2.00 (m,
5H, H’2, H’7, H’8), 2.20 - 2.36 (m, 2H, H’4), 2.42 (s, 6H,
N(CH3)2), 2.70 (m, 2H, (CH3)2NCH2), 2.91 (bs, 5H,
OCON(C2H5)CH2), 3.38 (m, 2H, OCON(C2H5)CH2),
4.48 - 4.52 (m, 1H, H´3), 5.36 - 5.38 (d, 1H, J = 5, H´6).
S. Acheampong et al. / Journal of Biophysical Chemistry 2 (2011) 53-62
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/JBPC/
cholesterol iodide (MTC)
Anal. Calcd for C34H61N2O2 I (MW 656.4): C, 62.16;
H, 9.45; N, 4.27, Found: C, 58.19; H, 9.37; N, 4.05, MS
(Positive/Negative ES) m/z 529.4 [cation of salt], 1H
NMR (400 MHz, CdCl3, 20˚C, TMS) δ 0.68 (s, 3H,
H’18), 0.85 - 1.74 (m, 33H, H’1, H’9, H’11 - H’12, H’14
- H’17, H’19 – H’27), 1.85 - 1.97 (m, 5H, H’2, H’7, H’8),
2.35 (m, 2H, H’4), 3.08 (bs, 3H, OCON(CH3)CH2), 3.50
(s, 9H, N(CH3)3), 3.80 - 4.04 (m, 4H, NCH2CH2N), 4.45
(m, 1H, H’3), 5.40 (d, 1H, J = 5, H’6).
2.2. Transfection and Cytotoxicity
The studies were conducted as described elsewhere
[14]. Briefly, the plasmid DNA pUC19-β-gal was propa-
gated in DH5α-competent cells using standard protocols
[15] and purified by gel permeation chromatography us-
ing Sepharose 4B as the stationary phase and 2.5 M am-
monium acetate as the mobile phase. Fractions containing
plasmid DNA were pooled and precipitated with isopro-
pyl alcohol. The precipitate was resuspended in a few mL
of 40 mM TE buffer pH 8, dialyzed for 2 - 4 h at room
temperature against 40 mM TE buffer to eliminate traces
of ammonium acetate and quantified by its absorption at
260 nm (4.63 mg·mL–1). The integrity and purity of the
pDNA was verified by agarose gel electrophoresis and a
260 nm/280 nm absorption ratio of 1.8. B16F0 cells were
cultured to confluence using DMEM supplemented with
10% fetal bovine serum, 50 units·mL–1 penicillin, 50
g·mL–1 streptomycin in a 5% CO2 at 37˚C. Approxi-
mately 50,000 cells in 0.5 mL growth media were added
to each well of 48 well plates. After 12 to 14 hours later,
the serum media were removed and the cells were trans-
fected with 250 L lipoplexes per well. The amount of
plasmid DNA was kept constant at 1 g per well, while
the amount of cationic lipid was increased appropriately
to achieve cationic lipid to DNA charge ratios of 1 : 1, 2 :
1 and 4 : 1. The cells were incubated for 4 hours, after
which time the lipoplexes were removed and the serum
free media were replaced with complete growth media.
After an additional incubation of 44 h, the cells were
washed with cold phosphate-buffered saline (PBS) pH 7.4
twice and lysed with 150 L lysis buffer (0.1 M Tris pH
7.2, 0.1% w/v Triton X) per well. Beta-galactosidase ac-
tivity was quantified by an ONPG assay. The cytotoxicity
of cationic lipids was evaluated by an MTT assay as de-
scribed elsewhere [14].
2.3. EtBr Displacement Studies
A Cary Eclipse spectrofluorometer was used to assess
the level of displacement of ethidium bromide (EtBr)
from the pDNA (pUC19). Briefly, 0.8 g of EtBr and
22.5 g of pDNA were diluted to 3 mL in either Tris
buffer pH 7.2 or in serum free media (SFM). Aliquots of
11.4 l of cationic lipid (1.2 mM) were added to the so-
lution until there was either a complete quenching of the
EtBr or no apparent change in the fluorescence intensity.
All samples were scanned at an excitation wavelength of
515 nm and the emission collected at the wavelength
between 550 - 605 nm (excitation and emission slit
widths were set at 5 nm). All experiments were con-
ducted at 22˚C. The EtBr emission intensity f, was plot-
ted against the lipid to DNA charge ratio R, and the re-
sults were fitted either linearly or parabolically within a
95% confidence level. The interaction of MTC was
shown to be a special case of cooperative interaction and
was simulated by Eq.1:
max min
where fmin and fmax are the lowest and highest normalized
fluorescence intensities, respectively, R, is the +/– charge
ratio, R½ is the charge ratio at which 50 % of plasmid
DNA is neutralized or compacted by cationic lipid and
the exponent n denotes the steepness of the curve. Data
were fitted by the method of non-linear least-squares
using the Microsoft Excel Solver function, through
minimization of the square residuals with 3 adjustable
parameters (fmin, fmax and R½) at 5% tolerance.
2.4. Gel Retardation Assay
0.8% Agarose solution was prepared in 40 mM TAE
(Tris base, glacial acetic acid and EDTA) pH 7.4, fol-
lowed by the addition of 10 L EtBr (10 mg mL–1). The
lipoplexes were prepared by keeping the pDNA amount
constant at 0.2 g while the amount of cationic lipid (0.6
mM) was varied to produce lipid to pDNA charge ratios
of 0.5, 1, 2, 4, 6 and 8. Naked pDNA (0.2 g) was used
as a control in the first and last lanes. To each sample 1
L of a 6X gel loading buffer (0.25% w/v bromophenol
blue, 0.25% w/v Xylene cyanol FF, 30% v/v glycerol in
water) was added and the volume was brought to 11 L
with 40 mM TAE buffer pH 7.4. The samples were al-
lowed to sit for 10 - 15 minutes before they were loaded
onto the wells of the agarose gel. Afterwards, an appro-
priate amount of running buffer (TAE) was used to sub-
merge the gel and the gel was subjected to an electropho-
retic field of 90 V for 35 min. The results of the gel elec-
trophoresis were assessed using a Bio-Rad Mini Transil-
2.5. Langmuir Monolayer Studies
Cationic lipid monolayers were studied at the air/water
S. Acheampong et al. / Journal of Biophysical Chemistry 2 (2011) 53-62
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/JBPC/
interface using the Langmuir film balance technique as
described elsewhere [16]. Compression isotherms of the
cationic lipids were obtained using a Langmuir film bal-
ance (model KSV 1000, KSV instruments LTD, Finland)
equipped with a Wilhelmy plate for measuring the surface
pressure. The Teflon microtrough (24,225.0 mm2) is
equipped with two barriers that allow for symmetric
compression. Each run was followed by a thorough wash
of both trough and barriers with ethanol and a final rinse
with distilled water. The Wilhelmy plate was heated over
a blue flame for about 30 seconds before each run. In a
typical experiment, 15 μL (30.0 nmol) of cationic lipid (2
mM dissolved in chloroform) was spread onto the sub-
phase (40 mM Tris buffer pH 7.2) maintained at 22˚C,
with a Hamilton® micro syringe and allowed to sit for 15
min before the start of each run. The barriers were co-
mpressed at a constant speed of 10 mm·min1. All exper-
iments were conducted at a temperature of 22˚C, while
each run was repeated at least three times for reproduci-
bility. Independent and dependent variables collected by
the instrument were imported into an Excel spreadsheet
and the compressibility modulus, K, was calculated from
the first derivative of the monolayer surface pressure-
trough Area isotherms using Eq.2:
 
Equilibrium parameters at the onset of monolayer col-
lapse were determined at the peak point of the K- iso-
2.6. pKa Studies
Buffer solutions (2 mL) made from 40 mM Tris and
40 mM MES having a pH from 1.2 to 13.2 were trans-
ferred into plastic cuvettes together with 0.1 mL of lipid
dispersions (0.6 mM for cationic lipid (CL) alone and 2
mM for CL/DOPC/Chol 0.2/0.9/0.9 molar ratio) and 6
L of TNS (0.1 mM) for CL alone and 20 L TNS (0.1
mM) for CL/DOPC/Chol 0.2/0.9/0.9 mol/mol/mol). The
lipid to probe molar ratio was kept constant at 100 : 1.
The static fluorescence intensity of these samples was
registered at an excitation wavelength of 321 nm (slit
width 5 nm) and an emission wavelength of 445 nm (slit
width 5 nm) on a Cary Eclipse fluorescence spectropho-
tometer at 25˚C. The emission intensity of TNS was plot-
ted against the pH and the results were fitted using a
modified version of the Henderson-Hasselbalch (Equa-
tion 3), using the
max min
min c (pH)
ff 
Microsoft Excel Solver through minimization of the
sum of the squared residuals with four adjustable param-
eters at a 5% tolerance level [17]. The parameter, f is the
calculated TNS fluorescence, fmax and fmin are the maxi-
mum and minimum fluorescence values of TNS and c is
a constant that adjusts the slope of the curve.
2.7. Particle Size and Electrophoretic
Mobility Studies
A Malvern Zetasizer Nano Series instrument was used
to determine the size and zeta potential of the pure cho-
lesterol derivatives and corresponding lipoplexes at +/–
charge ratios of 1 : 1, 2 : 1 and 4 : 1. Experiments were
performed in both filtered Tris buffer (using 0.2 m
membrane filter) and SFM at 22˚C. Lipoplexes were
prepared by adding pDNA into 200 L of 0.6 mM lipid
dispersion to produce the desired lipid to pDNA charge
ratios and then diluted to 1.5 mL with Tris buffer (40
mM, pH 7.2) or SFM [17].
3.1. In Vitro Transfection and Cytotoxicity
Regardless of charge ratio, the MAC mediated highest
transfection activity while the quaternary ammonium salt,
MTC, exhibited the lowest potency among its peers
(Figure 1(a)). The invariable
-gal expression levels by
MAC could be due to decreased cell viability with in-
creasing charge ratio. Impressively, unlike DC-Chol
which is transfection incompetent in the absence of
DOPE, the MDC and EDC that bear methyl and ethyl
substitution at the carbamoyl nitrogen, respectively, me-
diated significant transfection activity proportional to
charge ratio. More specifically, at +/– charge ratio of 1,
the activity mediated by the cationic lipids decreased
with polar head methyl substitution, while at +/– charge
ratio of 2 and 4 the transfection activity mediated by
MMC, MDC and EDC was essentially indistinguishable.
MDC exhibited the lowest toxicity maintaining greater
than 70% cell viability, whereas MMC was quite cyto-
toxic at all charge ratios (Figure 1(b) ).
3.2. pKa Studies
The pKa of cationic lipids is conventionally determined
in mixed lipid vesicles using the membrane potential
indicator TNS [17-21]. In this investigation, an attempt
was made to determine the apparent pKa of cationic lipids
from pure assemblies in the absence of any co-lipids. As
shown in Table 1, the pKa of the primary amine deriva-
tive MAC is slightly lower than that of the secondary
amine derivative MMC. Further methyl substitution on
the amine polar head drastically decreases the pKa by one
unit (MDC), while the apparent pKa of the ethyl carbam-
oyl tertiary amine derivative EDC, is even lower reaching
S. Acheampong et al. / Journal of Biophysical Chemistry 2 (2011) 53-62
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/JBPC/
Figure 1. (a) Transfection activities and (b) Cytotoxicity studies of MAC, MMC, MDC, MTC and EDC at dif-
ferent +/ charge ratios in mouse melanoma cells (B16F0). Results are the average of three different experi-
ments. In the control experiment, plasmid DNA was added to the cells without cationic lipids.
Tab le 1. Apparent pKa of cationic lipids determined at 25˚C in pure cationic assemblies and in the presence of
DOPC and cholesterol (CH) at a molar ratio of 2 : 9 : 9.
Composition pKa Composition pKa
MAC 7.19 ± 0.06 MAC/DOPC/CH 8.83 ± 0.055
MMC 7.24 ± 0.06 MMC/DOPC/CH 8.82 ± 0.16
MDC 6.17 ± 0.09 MDC/DOPC/CH 7.54 ± 0.07
EDC 6.04 ± 0.03 EDC/DOPC/CH 7.37 ± 0.14
DC 6.76 ± 0.075 - -
the value of 6.04. This trend, MMC > MAC > MDC >
EDC can be explained by the lipid hydration that pre-
cedes the inductive effect [22,23]. Generally, the amine
basicity is increased with methyl substitution due to the
electron donation from the methyl groups. However, in
aqueous solution, the increased electron density and ba-
sicity of the methyl substituted nitrogen is compromised
by the low solvation energy of the increasingly hydro-
phobic substituted nitrogen. Interestingly, the pKa of the
DC is much higher than that of MDC (6.76 versus 6.17)
but significantly lower than the structural isomer MMC.
Thus, moving the methyl group from the amine head to
the carbamoyl group increases the pKa by 0.5 units and
converts an ineffectual lipid into a transfection potent
As with the pure assemblies, mixed assemblies con-
taining primary and secondary amine derivatives were
more basic by 1.3 and 1.5 pH units than those containing
the tertiary amine derivatives MDC and EDC, respec-
tively (Figure 2 and Table 1). Furthermore, due to the
increased molecular separation, the pKa of all cationic
lipids in mixed vesicles composed of 90% DOPC and
charge ratio
+/– charge ratio
negative control
S. Acheampong et al. / Journal of Biophysical Chemistry 2 (2011) 53-62
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/JBPC/
Figure 2. Fluorescence intensity of TNS as a function of bulk pH in pure and mixed cationic assemblies at 25˚C.
Results are the average of three independent experiments with the error bars representing standard uncertainties.
(a) (b)
Figure 3. EtBr displacement assay at 22˚C (a) in 40 mM Tris buffer pH 7.2; (b) in serum free media (SFM) of physiological ionic
strength. Symbols are the average of 3 different experiments while solid lines represent data fitted by linear equations and parabolas
(top panel). The interaction of MTC with plasmid DNA in 40 mM Tris pH 7.2 (top panel) was simulated by Eq.2, as described in
Materials and Methods.
S. Acheampong et al. / Journal of Biophysical Chemistry 2 (2011) 53-62
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/JBPC/
DNA 0.5 1 2 4 6 8 DNA
Figure 4. Representative agarose gel retardation studies in 40
mM Tris buffer pH 7.2 of MAC, MMC, MDC, MTC and EDC
at different +/– charge ratios (no lipid,0.5, 1, 2, 4, 6, 8, no lipid).
cholesterol increased by 1.3 to 1.6 units. However, what
is really interesting is the fact that 90% ionization takes
place within ± 2 pH units from the pKa value and not 1
unit as predicted by the Henderson-Hasselbalch equation.
This expansion of the buffering region of cationic choles-
terol assemblies was not observed with double-chained
monovalent cationic lipids (results not shown) and it defi-
nitely extends the range of pH at which cationic lipid-
DNA interaction occurs.
Interestingly, the transfection efficiency decreased
with the increase in the methylation on the terminal ni-
trogen while it surprisingly increased with the increase in
alkyl chain of the carbamoyl nitrogen. This trend reveals
that higher ionization of a cationic lipid is not always
correlated with a higher transfection activity.
3.3. Monolayer studies
As shown in Table 2, the trend in the mean molecular
areas (MmA) of cationic lipids at the onset of collapse
pressure is MTC > MMC > MAC > MDC > EDC. Look-
ing at the MDC and EDC derivatives, the mean surface
area decreased from 35.7 Å2 molecule–1 for MDC to 33.7
Å2 molecule–1 for EDC. Tighter packing of EDC as com-
pared to MDC molecules is due to the increased van der
Waals forces and the lower pKa, both a consequence of
the increased hydrophobicity of the molecule which is
also reflected in lower monolayer collapse pressure and
Table 2. Monolayer properties determined at the onset of
monolayer collapse with 40 mM Tris buffer pH 7.2 as the sub-
phase at 22˚C. Standard uncertainties were calculated from
three to five independent experiments. The % ionization of
cationic lipids in the monolayers were calculated from the
measured pKa values included in Table 1.
lipid MmA (Ǻ2) (mN m–1) K (mN m–1) % ioniza-
MAC 36.6 ± 0.342.5 ± 2.4 212 ± 21 49.3 ± 3.4
MMC 37.6 ± 1 41.9 ± 0.4 233 ± 48 52.4 ± 3.4
MDC 35.7 ± 1.142.6 ± 1.7 151 ± 12 8.60 ± 2
EDC 33.7 ± 1 37.3 ± 2.1 126 ± 10 6.50 ± 0.4
MTC 44.6 ± 0.432.0 ± 0.9 123 ± 11 100
Table 3. (+/– charge ratio at which 50% of plasmid DNA is
compacted in 40 mM Tris buffer pH 7.2 and in Serum Free
Media (SFM) at 22˚C.
Cationic lipid 40 mM Tris buffer SFM
MAC 1.8 1.7
MMC 1.7 2.7
MDC 2.8 -
EDC 3.5 -
MTC 1.1 0.7
magnitude of the compressibility modulus, K.
The trend in the MmA of cationic lipids as a function
of amino group methyl substitution (MTC > MMC >
MAC > MDC) verifies that cationic lipid molecular di-
mensions are directly proportional to the pKa and degree
of ionization in Tris buffer pH 7.2. Thus, although MDC
is a bulkier tertiary amine, the increased ionization of the
primary amine MAC at pH 7.2 (49% versus 9%; Table
2), encourages greater hydration and repulsion among
the aligned molecules in the interface, thus effectively
increasing its molecular dimensions as determined just
before the monolayer collapse.
3.4. EtBr Displace ment and Exclusi on
Stud ies
To better understand the transfection activity profile of
the cationic lipids, their ability to bind and compact
pDNA was assessed using the EtBr displacement assay.
The cationic lipids displaced EtBr from pDNA in 40 mM
Tris pH 7.2 in the following order: MTC > MMC >
MAC > MDC > EDC (Figure 3(a)). Thus, the more ba-
sic the lipid and the more expanded the molecular di-
mensions, the more efficient is its interaction with plas-
mid DNA. This trend was also observed with a gel retar-
dation assay, although none of the lipids was able to
completely retard the pDNA at the exact ratio as deter-
mined in the EtBr displacement assay (Figure 4). In the
high ionic strength serum free media (SFM), the interac-
tion cooperativity observed in 40 mM Tris was lost and
the order of interaction was changed to MTC > MAC >
S. Acheampong et al. / Journal of Biophysical Chemistry 2 (2011) 53-62
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/JBPC/
MMC. MDC and EDC failed to significantly condense
the plasmid DNA (Figure 3(b)). Most importantly, the
charge ratio at which 50% of EtBr is displaced from
pDNA decreased from 1.1 in 40 mM Tris to 0.7 for MTC,
remained the same around 1.7 to 1.8 for the MAC and
increased from 1.7 to 2.7 for MMC (Table 3). Evidently,
the increased ionic strength of the media did not ad-
versely affect the electrostatic interaction of the more
hydrophilic quaternary ammonium salt MTC and pri-
mary amine derivative MAC, but it seriously reduced the
reactivity of the more lipophilic secondary and tertiary
amine derivatives MMC and MDC and EDC, respec-
tively, due to reduced hydration.
3.5. Particle Size and Electrophoretic
Mobility Studies
Cationic lipid dispersions in the absence of pDNA
were in general below 200 nm in 40 mM Tris buffer pH
7.2. The smallest particle diameter was exhibited by the
quaternary ammonium derivative MTC presumably due
to increased ionization and polar head expansion. Owing
to reduced hydration, the particle size of cationic lipid
dispersions and corresponding zeta potential increased
and decreased, respectively, in the high ionic strength
SFM (Tabl e 4). The lipoplexes exhibited a very different,
but predictable, zeta potential and particle size distribu-
tion (Table 5). Lipoplex particles were slightly bigger
than the size of cationic lipid dispersions in the absence
of pDNA in 40 mM Tris and SFM, until the point of
charge neutrality where a big jump in the lipoplex size
was observed. Specifically, as previously determined by
the EtBr displacement assay, complete charge neutraliza-
tion occurs at +/– charge ratio of 2 and 4 for MTC and
MAC and MMC, respectively. A drastic increase in lipo-
Table 4. Hydrodynamic characterization of lipid dispersions (in the absence of DNA) carried out in 40 mM Tris buffer pH 7.4 and
serum free media (SFM) at 22˚C. Standard uncertainties were calculated from the average values measured from three separate ex-
Composition Hydrodynamic Diameter (nm) PDI Zeta Potential (mV)
40 mM Tris SFM 40 mM Tris SFM 40 Mm Tris SFM
MAC 160 ± 6 258 ± 78 0.26 ± 0.02 0.15 ± 0.01 +53 ± 3 +19.5 ± 1
MMC 113 ± 7 160 ± 9 0.24 ± 0.01 0.26 ± 0.02 +55 ± 1 +24 ± 2
MDC 169 ± 14 183 ± 11 0.39 ± 0.07 0.36 ± 0.04 +45 ± 1.5 +21 ± 3
EDC 203 ± 26 302 ± 83 0.45 ± 0.03 0.40 ± 0.01 +45 ± 2.6 +22 ± 2
MTC 77 ± 5 98 ± 3 0.26 ± 0.04 0.24 ± 0.02 +31 ± 7 +23 ± 1
Table 5. Particle size and zeta potential of lipoplexes at various charge ratios in Tris 40 mM buffer pH 7.2 and in Serum Free Me-
dium (SFM) at 22˚C. Standard uncertainties were calculated from the average values measured from three independent experiments.
Cationic lipid +/– charge
ratio Hydrodynamic Diameter (nm) Polydispersity index Zeta Potential (mV)
40 mM Tris SFM 40 mM Tris SFM 40 mM Tris SFM
MAC 1/1 204 ± 4 199 ± 7 0.26 ± 0.01 0.20 ± 0.01 –52 ± 2.3 –27 ± 3
MAC 2/1 328 ± 40 247 ± 14 0.30 ± 0.03 0.22 ± 0.01 –51 ± 1.5 –31 ± 3
MAC 4/1 1173 ± 714 731 ± 266 0.71 ± 0.4 0.34 ± 0.1 –6.35 ± 31.3 –33 ± 4
MMC 1/1 200 ± 59 250 ± 25 0.25 ± 0.04 0.26 ± 0.01 –50 ± 2.6 –29 ± 2
MMC 2/1 174 ± 32 273 ± 14 0.18 ± 0.07 0.25 ± 0.02 –49 ± 1.5 –31 ± 3
MMC 4/1 1137 ± 68 658 ± 166 0.53 ± 0.04 0.32 ± 0.16 +36 ± 4 –35 ± 1.3
MDC 1/1 229 ± 23 260 ± 25 0.28 ± 0.01 0.26 ± 0.02 –55 ± 2 –30 ± 2.3
MDC 2/1 236 ± 26 304 ± 31 0.31 ± 0.05 0.32 ± 0.08 –55 ± 2.6 –31 ± 2
MDC 4/1 411 ± 26 383 ± 35 0.27 ± 0.03 0.28 ± 0.07 –52 ± 0.4 –31 ± 2
EDC 1/1 454 ± 31 418 ± 112 0.19 ± 0.03 0.26 ± 0.09 –61 ± 2.6 –29 ± 2
EDC 2/1 323 ± 78 448 ± 149 0.26 ± 0.03 0.20 ± 0.05 –60 ± 2.6 –31 ± 0.2
EDC 4/1 421 ± 172 484 ± 116 0.20 ± 0.08 0.29 ± 0.01 –59 ± 2 –33 ± 2
MTC 1/1 270 ± 97 737 ± 437 0.37 ± 0.1 0.54 ± 0.16 –38 ± 0.6 –25 ± 2
MTC 2/1 1350 ± 1077 3782 ± 925 0.70 ± 0.2 0.62 ± 0.15 –30 ± 11 –18 ± 6.5
MTC 4/1 172 ± 13 475 ± 236 0.33 ± 0.05 0.41 ± 0.06 +39 ± 1 +26 ± 1
S. Acheampong et al. / Journal of Biophysical Chemistry 2 (2011) 53-62
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/JBPC/
plex particle size was observed close to these charge ra-
tios (Table 5). Charge neutralization in lipoplexes com-
posed of these cationic lipids was verified in 40 mM Tris
pH 7.2, where the zeta potential reverses sign and be-
comes positive or it is very close to zero as the charge
ratio increases from 1 : 1 to 2 : 1 and 4 : 1.
In summary, it was found that increased hydration of
cholesterol-based cationic lipids correlates with increased
ionization, increased polar head group dimensions due to
higher repulsive forces among the lipids and greater inter-
action with plasmid DNA, as shown by EtBr displace-
ment and exclusion assays. Thus, hydration was found to
be the primary factor controlling ionization of cationic
lipids and their interaction with DNA. Factors affecting
the hydration of the cationic lipids, such as increased io-
nic strength, were shown to adversely affect primarily
the ionization and DNA interaction of the more hydro-
phobic tertiary (MDC, EDC) and secondary (MMC)
amine derivatives. Furthermore, ionization and pKa of
the cholesterol-based cationic lipids increased tremen-
dously by 1.3 to 1.5 pH units in the presence of other
lipids due to increased spacing, improved hydration and
reduced electronic overlap. No correlation was found
between the pKa of cationic assemblies and the transfec-
tion activity. Also, the similarity in lipoplex particle size
and zeta potential precludes predicting a cationic lipid
trend in mediating transfection activity. However, com-
parison of this work with our published report on secon-
dary carbamate isomers AC, MC, DC and TC [14] sub-
stantiates the following: Firstly, methylating or ethylat-
ing the carbamoyl nitrogen of DC decreases the pKa of
cationic assemblies and their interaction with plasmid
DNA, but it substantially increases their transfection ac-
tivity in the absence of the helper lipid DOPE. Secondly,
the transfection activity of the tertiary carbamate analog
MMC is significantly lower than that of the secondary
carbamate MC, presumably due to toxicity issue. Thirdly,
the transfection activity of the primary amine derivatives,
AC and MAC, was highest, while the quaternary ammo-
nium derivatives, TC and MTC were essentially transfec-
tion incompetent despite their highly efficient interaction
with plasmid DNA.
Our success of measuring the apparent pKa of pure ca-
tionic assemblies and demonstrating that membrane ioni-
zation in pure cationic assemblies is much lower than
that in mixed membranes was followed by the unfortu-
nate realization that transfection activity is not correlated
with the pKa of basic cationic lipids. The appropriate test
to build structure-activity relationships is still to be dis-
covered, but as our studies indicated, the key lies in the
three analogs MMA, DC and MDC which their physico-
chemical properties and their transfection activities
differ drastically from each other. Rather a delicate bal-
ance between the hydrophilicity and hydrophobicity of
these cationic lipids must be controlling their transfection
[1] Gao, X.A. and Huang, L. (1991) A novel cationic lipo-
some reagent for efficient transfection of mammalian
cells. Biochemical and Biophysical Research Communi-
cations, 179, 280-285.
[2] Middleton, P.G., Caplen, N.J., Gao, X., Huang, L., Gaya,
H., Geddes, D.M. and Alton, E.W.F.W. (1994) Nasal ap-
plication of the cationic liposome DC-Chol: DOPE does
not alter ion transport, lung function or bacteria growth.
European Respiratory Journal, 7, 442-445.
[3] Blagbrough, I.S., Geall, A.J. and Neal, A.P. (2003) Poly-
amines and novel polyamine conjugates interact with
DNA in ways that can be exploited in non-viral gene
therapy. Biochemical Society Transactions, 31, 397-406.
[4] Griesenbach, U., Kitson, C., Garcia, S.E., Farley, R.,
Singh, C., Somerton, L., Painter, H., Smith, R.L., Gill, D.
R., Hyde, S.C., Chow, Y.-H., Hu, J., Gray, M., Edbrooke,
M., Ogilvie, V., MacGregor, G., Scheule, R.K., Cheng,
S.H., Caplen, N.J. and Alton, E.W.F.W (2006) Inefficient
cationic lipid-mediated siRNA and antisense oligonucleo-
tide transfer to airway epithelial cells in vivo. Respiratory
Research, 7, 1-15.
[5] Vigneron, J.-P., Oudrhiri, N., Fauquet, M., Vergely, L.,
Bradley, J.-C., Basseville, M., Lehn, P. and Lehn, J.-M.
(1996) Guanidinium-cholesterol cationic lipids: Efficient
vectors for the transfection of eukaryotic cells. Proceed-
ings of the National Academy of Sciences of the United
States of America, 93, 9682-9686.
[6] Fang, N., Wang, J., Mao, H.-Q., Leong, K.W. and Chan,
V. (2003) BHEM-Chol/DOPE liposome induced pertur-
bation of phospholipid bilayer. Colloids and Surfaces B:
Biointerfaces, 29, 233-245.
[7] Ghosh, Y.K, Visweswariah, S.S. and Bhattacharya, S.
(2000) Nature of linkage between the cationic head group
and cholesterol skeleton controls gene transfection effi-
ciency. Federation of European Biochemical Societies
Letters, 473, 341-344.
[8] Bajaj, A., Mishra, S.K., Kondaiah, P. and Bhattacharya, S.
(2008) Effect of the headgroup variation on the gene
transfer properties of cholesterol based cationic lipids
possessing ether linkage. Biochimica et Biophysica Acta:
Biomembranes, 1778, 1222-1236.
[9] Esposito, C., Generosi, J., Mossab, G., Masotti, A. and
Castellano, A.C. (2006) The analysis of serum effects on
structure, size and toxicity of DDAB-DOPE and DC-
Chol-DOPE lipoplexes contributes to explain their differ-
ent transfection efficiency. Colloid Surface B Biointer-
S. Acheampong et al. / Journal of Biophysical Chemistry 2 (2011) 53-62
Copyright © 2011 SciRes. Openl y accessible at http://www.scirp.org/journal/JBPC/
faces, 53, 187-192.
[10] Koltover, I., Salditt, T. and Safinya, C.R. (1999) Phase
diagram, stability, and overcharging of lamellar cationic
lipid-DNA self-assembled complexes. Biophysical Jour-
nal, 77, 915-924.
[11] Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan,
W., Wenz, M., Northorp, J.P., Ringold, G.M. and Daniel-
sen, M. (1987) Lipofection: A highly efficient lipid-
mediated DNA transfection procedure. Proceedings of
the National Academy of Sciences of the United States of
America, 84, 7413-7417.
[12] Israelachvili, J.N., Marcelja, S. and Horn, R.G. (1980)
Physical principles of membrane organization. Quarterly
reviews of Biophysics, 13, 121-200.
[13] Gruner, S.M., Cullis, P.R., Hope, M.J. and Tilcock, P.S.
(1985) Lipid polymorphism: The molecular basis of non-
bilayer phases. Annual Review of Biophysics and Bio-
physical Chemistry, 14, 211.
[14] Kearns, M., Donkor, A.M. and Savva, M. (2008) Struc-
ture—Transfection activity studies of novel cationic cho-
lesterol-based amphiphiles. Molecular Pharmaceutics, 5,
128-139. doi:10.1021/mp700131c
[15] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Mo-
lecular cloning: A laboratory manual. 2nd Edition, Cold
Spring Harbor Laboratory Press, New York.
[16] Savva, M. and Acheampong, S. (2009) The interaction
energies of cholesterol and 1,2-dioleoyl-phosphoetha-
nolamine in spread mixed monolayers at the air-water in-
terface. Journal of Physical Chemistry B, 113, 9811-9820.
[17] Spelios, M. and Savva, M. (2008) Novel N,N-1,3-
diaminopropyl-2-carbamoyl bivalent cationic lipids for
gene delivery-synthesis, in vitro transfection activity, and
physicochemical characterization. Federation of Euro-
pean Biochemical Societies Journal, 275, 148-162.
[18] Wang, J.L. and Edelman, G.M. (1971) Fluorescent probes
for confromational states of proteins. Journal of Biologi-
cal Chemistry, 246, 1185-1191.
[19] Bailey, A.L. and Cullis, P.R. (1994) Modulation of mem-
brane fusion by asymmetric transbilayer distributions of
amino lipids. Biochemistry, 33, 12573-12580.
[20] Asokan, A. and Cho, M.J. (2003) Cytosolic delivery of
macromolecules II. Mechanistic studies with pH-sensitive
morpholine lipids. Biochimica et Biophysica Acta: Bio-
membranes, 161, 151-160.
[21] Heyes, J., Palmer L., Bremner, K. and MacLachlan, I.
(2005) Cationic lipid saturation influences intracellular
delivery of encapsulated nucleic acids. Journal of Con-
trolled Release, 107, 276-287.
[22] Marchington, A.F., Moore, S.C.R. and Richards, W.G.
(1979) The inductive effect in molecules and ions. Jour-
nal of the American Chemical Society, 101, 5529-5532.
[23] Miessler, G.L. and Tarr, D.A. (2003) Inorganic Chemis-
try, Prentice Hall, New Jersey.