Novel cationic lipids based on malonic acid amides backbone: Transfection efficacy and cell toxicity properties

Article abstract of DOI:10.1021/bc9004624

Gene delivery using nonviral approaches has been extensively studied as a basic tool for intracellular gene transfer. Despite intensive research activity, the aim of creating a vector which meets all necessary demands has still not been reached. One possibility to solve the nonviral vector associated problem of low transfection efficacy is the development of new cationic amphiphiles. Therefore, the non-glycerol-based cationic lipids 1-9 have been synthesized and tested for in vitro gene delivery experiments. The backbone structure of the lipids consists of a malonic acid diamide with two long hydrophobic chains. The degree of saturation of the hydrophobic chains and the structure of the polar cationic headgroup were varied. The preparation follows an easy process and facilitates the trouble-free insertion of different alkyl chains. By studying in vitro gene delivery an increase of transfection efficacy was observed when using at least one unsaturated alkyl chain in the hydrophobic part and lysine or bis(2-aminoethyl)aminoethylamid as hydrophilic headgroup. This leads to cationic lipids exhibiting comparable or even higher transfection efficacies compared to the commercially available LipofectAmine and SuperFect.

Full text of DOI:10.1021/bc9004624

696
Bioconjugate Chem. 2010, 21, 696–708
Novel Cationic Lipids Based on Malonic Acid Amides Backbone:
Transfection Efficacy and Cell Toxicity Properties
Martin Heinze,† Gerald Brezesinski,‡ Bodo Dobner,† and Andreas Langner*,†
Institute of Pharmacy, Department of Biochemical Pharmacy, Martin-Luther-University, Wolfgang-Langenbeck-Strasse 4,
06120 Halle (Saale), Germany, and Max Planck Institute of Colloids and Interfaces, Am Muehlenberg 1,
14476 Potsdam, Germany. Received November 11, 2009; Revised Manuscript Received December 23, 2009
Gene delivery using nonviral approaches has been extensively studied as a basic tool for intracellular gene transfer.
Despite intensive research activity, the aim of creating a vector which meets all necessary demands has still not
been reached. One possibility to solve the nonviral vector associated problem of low transfection efficacy is the
development of new cationic amphiphiles. Therefore, the non-glycerol-based cationic lipids 1-9 have been
synthesized and tested for in vitro gene delivery experiments. The backbone structure of the lipids consists of a
malonic acid diamide with two long hydrophobic chains. The degree of saturation of the hydrophobic chains and
the structure of the polar cationic headgroup were varied. The preparation follows an easy process and facilitates
the trouble-free insertion of different alkyl chains. By studying in vitro gene delivery an increase of transfection
efficacy was observed when using at least one unsaturated alkyl chain in the hydrophobic part and lysine or
bis(2-aminoethyl)aminoethylamid as hydrophilic headgroup. This leads to cationic lipids exhibiting comparable
or even higher transfection efficacies compared to the commercially available LipofectAmine and SuperFect.
genes into cells, especially by the usage of N-methyl-4-
(dioleyl)methylpyridiniumchloride (SAINT-2) or 1-[2-(oleoy-
loxy)ethyl]-2-oeyl-3-(2-hydroxyethyl)imidazoliniumchloride
(DOTIM) (19, 20).
For a number of these and other cationic amphiphiles, it was
shown that hydrophobic chains as well as the polar head and
even the connecting backbone have a great influence on the
transfection efficiency (16, 19, 21–23). Several groups have been
working on establishment of a structure-function relationship
to define the requirements for safe and efficient gene transfer
with cationic lipids (16, 19–26).
We have now designed a new class of cationic lipids
consisting of the diamide structure of malonic acid as anchor
group for the hydrocarbon chains as well as the cationic
headgroup inclusive spacer molecules. The idea for the prepara-
tion of these new substances is mainly based on three charac-
teristics: First, the new compounds are easy to produce starting
from cost-effective materials, and the syntheses facilitate the
trouble-free insertion of different alkyl chains. Second, it may
be accepted that the compounds should have biocompatibility
due to the amide bond as a connection between polar headgroup,
linker moiety, and partially the hydrophobic chains. Third, a
positive effect may result from the ability of the malonic diamide
structure to build an H-bonded network, which could stabilize
the complex formation especially under neutral conditions.
INTRODUCTION
Without doubt, gene therapy is a very promising method for
the treatment of cancer and genetic diseases which are still
insufficiently treatable (1–3). Although a lot of work has been
done creating efficient gene shuttles and progress has been made
over the past years, there exists no satisfying gene transfer
system. In addition to the application of viral vectors, the design
and synthesis of new cationic lipids is a very promising tool
for gene therapy. While the transduction with modified viruses
is very efficient especially for in vivo gene transfer, viruses have
eminent disadvantages due to their immunogenic potential and
their possibility to induce cancer (4–7). Nonviral gene delivery
systems have, on one hand, been demonstrated as standard tools
for in vitro transfection with currently more than 60 com-
mercially available kits (8). On the other hand, they are regarded
asapromisingapproachforthetreatmentofgeneticdiseases(9,10)
and cancer (11–13). However, nonviral gene delivery suffers
from low transfection efficiency. Surely this is the main reason
that these gene delivery systems have just a marginal stake in
clinical trials (14). For this reason, it is of great importance to
advance nonviral gene transfer systems. One possibility is the
development of new, efficient, and nontoxic cationic lipids.
There are mainly three structures of cationic amphiphiles used
for gene delivery: glycerol derived lipids and cholesterol-based
and non-glycerol-based compounds. The group of glycerol
derived lipids includes in addition to N-[1-(2,3-dioleyloxy)pro-
pyl]-N,N,N-trimethylammoniumchloride (DOTMA)1 , first de-
scribed by Felgner in his pioneering work (15), 1,2-dioleoyloxy-
3-(trimethylammonio)propane (DOTAP), 1,2-dimyristyloxypropyl-
3-dimethyl(hydroxyethyl)ammoniumbromide (DMRIE), and 2,3-
dioleyloxy-N-[2-(spermincarboxamido)ethyl]-N,N-dimethyl-1-
propane-ammoniumtrifluoracetate (DOSPA) (16–18). Substances
that exhibit no glycerol backbone are also capable of transfering
1Abbreviations: DOTMA, N-[1-(2,3-dioleyloxy)propyl]-N,N,
N-trimethylammoniumchloride; DOTAP, 1,2-dioleoyloxy-3-(trimethylam-
monio)propane; DMRIE, 1,2-dimyristyloxypropyl-3-dimethyl(hydro-
xyethyl)ammoniumbromide; DOSPA, 2,3-dioleyloxy-N-[2-(spermincar-
boxamido)ethyl]-N,N-dimethyl-1-propane-ammoniumtrifluorace-
tate; SAINT-2, N-methyl-4-(dioleyl)methylpyridiniumchloride; DOTIM,
1-[2-(oleoyloxy)ethyl]-2-oeyl-3-(2-hydroxyethyl)imidazoliniumchlo-
ride; EEDQ, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydrochinoline; BOP,
(benzotriazol-1-yloxy)-tris-(dimethylamino)-phosphoniumhexafluoro-
phosphate; DCC, N,N′-dicyclohexylcarbondiimid; ONPG, O-nitrophe-
nyl-ꢀ-galactopyranoside; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoe-
thanolamine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide.
* Corresponding author footnote. Phone: +49-(0)345-5525080. Fax:
+49 -(0)345-5527018. E-mail: andreas.langner@pharmazie.uni-halle.de.
† Martin-Luther-University.
‡ Max Planck Institute of Colloids and Interfaces.
10.1021/bc9004624  2010 American Chemical Society
Published on Web 03/23/2010

Novel Cationic Lipids Based on Malonic Acid Amides
Bioconjugate Chem., Vol. 21, No. 4, 2010 697
The characteristic of the compounds is the specific feature
of hydrophobic chain bound to malonic acid amides backbone.
One alkyl chain is bound to C2 of the malonic acid, whereas
the second hydrophobic chain was linked via an amide bond to
one of the carboxylic groups of the diacid. The two alkyl chains
that are necessary for satisfying gene delivery vary in the degree
of saturation. Starting from two C16 chains, we replaced them
by successive substitution with the oleyl residue whereby the
chain was elongated. According to the generally accepted finding
that spacer molecules enhance the efficacy of transfection (27),
at first we used only ethylene diamine and tris(aminoethyl)amine
as spacer molecules. These compounds were connected with
the malonic acid backbone by a second amide bond. Lipids 1-3
with the ethylene amine spacer itself were capable of transfering
DNA into cells in culture, admittedly to a minor degree.
However, this molecule residue was introduced for binding a
proper cationic headgroup. The primary amino group was then
transformed into a further amide function with boc-lysine
resulting in two protonable amine residues after elimination of
the protective groups. On the other hand, the tris(aminoethyl)-
amine comprises both the spacer and the headgroup resulting
from the two aminoethyl residues at the tertiary amine. This
branched structure is suitable for the creation of dendrimer-
like headgroups (28).
(30 mL). Then, potassium hydroxide (2.92 g, in 30 mL ethanol)
was added dropwise during 1 h into the solution with stirring.
The mixture was stirred for further 3 h and was then allowed
to stand overnight at room temperature. The mixture was heated
for a short time to reflux, and the hot solution was filtered to
remove bis-potassium salt. From the cooled mixture, the
monopotassium salt of hexadecyl malonic acid monoethyl ester
was separated by filtration. The salt was dissolved in water and
acidified with equimolar amount of 0.1 M hydrochloric acid.
The crude monoester was then extracted three times with 30
mL ether. The combined etheral solutions were washed with
water and brine, dried over sodium sulfate, and evaporated to
dryness. The compound was purified by crystallization from
heptane or by column chromatography using silica gel 60.
In the case of IIb, no salt precipitates. Therefore, the mixture
was concentrated under vacuum to the half of the volume and
acidified after cooling. Compound IIb was then extracted 3
times with ether, and the combined etheral phase was washed
and dried as described above. IIb was purified by column
chromatography.
Hexadecyl Malonic Acid Monoethyl Ester (IIa). The
experimental data were in agreement with the literature (29).
(9Z)-Octadec-9-enyl Malonic Acid Monoethyl Ester (IIb).
Yield: 68%, colorless oil, C23H42O4, 382.57 g/mol, EIsMS: 382
(M+).1H NMR (400 MHz, CDCl3) δ: 0.85 (t, 3H, [-CH3]),
1.20-1.90 (m, 29H, [H3C(CH2)6CH2CHdCHCH2(CH2)7-],
[-OCH2CH3]), 1.95-2.05 (m, 4H, [-CH2CHdCHCH2-]),
3.38-3.40 (t, 1H, [-CH-]), 4.22 (q, 2H, [-OCH2CH3]), 5.30-5.39
(m, 2H, [-CHdCH-]). Anal. Calcd.: C, 72.20; H, 11.06. Found:
C, 71.92; H, 11.17.
The main focus of attention for the new lipids was concen-
trated on transfection efficacy and toxicity properties compared
with LipofectAmine and SuperFect, as they are the frequently
used nonviral gene delivery systems. In first tests with these
new transfection reagents, we have observed enhanced gene
transfection activity with regard to commercially available
vectors.
2-[(Alkylamino)carbonyl]octadecanoic Acid Ethyl Ester
IIIa,b. Procedure 1. Compound IIa (0.014 mol) and 10 mL
thionyl chloride were given into a flask, and the mixture was
allowed to stand overnight at room temperature until a clear
solution appears. Then, the excess thionyl chloride was removed
under vacuo. The residue was dried under vacuum over
potassium hydroxide. After that, the crude malonic ester chloride
was dissolved in 50 mL dry CHCl3 and cooled down to 0 °C.
Hexadecylamine or oleylamine (0.013 mol) and triethyl amine
(1.3 g, 0.013 mol), dissolved in 10 mL dry CHCl3, were dropped
into the solution with stirring at the same temperature. After
2 h stirring at room temperature, the CHCl3 solution was washed
three times with ice water and dried over sodium sulfate. The
crude product was crystallized from acetone.
Procedure 2. A suspension of compound II (0.014 mol),
hexadecylamine or oleylamine (0.014 mol), and 3.5 g (0.014
mol) EEDQ in 100 mL ethanol was heated for 24 h at 50 °C.
The solvent was then reduced to half of the volume, diluted
with 100 mL CHCl3, and washed with water. After drying the
organic layer over sodium sulfate, the solvent was evaporated
and the residue recrystallized from acetone.
2-[(Hexadecylamino)carbonyl]octandecanoic Acid Ethyl
Ester (IIIa). Yield: 91% (procedure 1), 86% (procedure 2),
C37H73NO3, 580.0 g/mol, mp: 72-73 °C. ESIsMS: (M++Na).1H
NMR (400 MHz, CDCl3) δ: 0.85 (t, 6H, 2[-CH3]), 1.23-1.86
(m, 61H, [-OCH2CH3], [H3C(CH2)15-], [H3C(CH2)14CH2NH-]),
3.14-3.18 (m, 2H, [-CH2NHCO-]), 3.22-3.28 (m, 1H,
[-CH-]), 4.16 (q, 2H, [-OCH2CH3]), 6.52-6,55 (m, 1H,
[-NH-]). Anal. Calcd.: C, 76.62; H, 12.69; N, 2.41. Found: C,
76.58; H, 12.70; N, 12.37.
2-{[(9Z)-Octadec-9-enylamino]carbonyl}octadecanoic Acid
Ethyl Ester (IIIb). Yield: 83% (procedure 1), 83% (procedure 2),
C39H75NO3, 606.03 g/mol, mp: 45-47 °C. ESIsMS: (M++H). 1H
NMR (400 MHz, CDCl3) δ: 0.85 (t, 6H, 2[-CH3]), 1.18-1.86 (m,
57H, [H3C(CH2)15-], [H3C(CH2)6CH2CHdCHCH2(CH2)6CH2NH-],
[-OCH2CH3]), 1.90-2.04 (m, 4H, [-CH2CHdCHCH2-]), 3.14-3.27
(m, 3H, [-CH2NHCO-], [-CH-]), 4.17 (q, 2H, [-OCH2CH3]),
EXPERIMENTAL PROCEDURES
General Procedures and Materials. All materials and
reagents were purchased from SigmaAldrich Co. Ltd. unless
otherwise stated. All solvents were analytically pure and dried
before use. The reactions were performed under argon. TLC
was carried out on aluminum sheets precoated with silica gel
60 F254 (Merck) and developed with bromothymol blue dip.
Column chromatography was performed using silica gel 60
(0.04-0.063 mm) for middle-pressure liquid chromatography
(MPLC, Bu¨chi, Germany) or silica gel 60 (0.063-0.200 mm)
for normal-pressure procedure. The ESI-mass spectra were
recorded with a Finnigan MAT 710 C (Thermo Separation
Products, San Jose, USA) with electron spray ionization energy
of 4.5 kV in negative and positive modus. EI-mass spectra
were prepared with an AMD 402 (70 eV, AMD Intecta GmbH,
1
Harpstedt, Germany). The H NMR and the 13C NMR spectra
were recorded on a Varian Gemini 2000. For performing the
elemental analysis, a Leco apparatus was used. The final
products were fully characterized by 1H NMR, 13C NMR, mass
spectrometry, and elemental analysis. p-CMV-SPORT-ꢀ-Gal
plasmid and LipofectAmine reagent were purchased from
Invitrogene Life technologies (Germany). SuperFect and plasmid
isolation kit were purchased from QIAGEN (QIAGEN, Hilden,
Germany). LLC PK1 cells (ATCC CLs101) were purchased
from the American Type Culture Collection (ATCC USA) and
A549 cells from German Collection of Microorganisms and Cell
Cultures (DSMZ GmbH). Cell culture media, fetal bovine serum,
and phosphate buffered saline were supplied from PAA
Laboratories (PAA Laboratories GmbH Germany). Antibiotics
and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) were purchased from Sigma (Sigma Aldrich,
Germany). DOPE was purchased from Fluka (Switzerland), and
cholesterol was supplied from Roth (Carl Roth GmbH Ger-
many).
Alkyl Malonic Acid Monoethyl Ester IIa,b. The alkyl
malonic acid diethyl ester I (0.045 mol) was dissolved in ethanol

698 Bioconjugate Chem., Vol. 21, No. 4, 2010
Heinze et al.
5.30-5.35 (m, 2H, [-CHdCH-]), 6.53-6.56 (m, 1H, [-NH-]).
Anal. Calcd.: C, 77.29; H, 12.47; N, 2.31. Found: C, 76.96; H, 12.43;
N, 2.37.
tris(aminoethyl)amine (20 mmol), and BOP (0.442 g, 1 mmol)
in CH2Cl2 (20 mL). The mixture was stirred at room temperature
overnight. The solution was filtered, washed with water, dried
over sodium sulfate, and evaporated. The residue was purified
by crystallization from heptane.
2-{[(9Z)-Octadec-9-enylamino]carbonyl}-(11Z)-icos-11-
enoic Acid Ethyl Ester (IIIc). Yield: 83% (procedure 2),
C41H77NO3, 632.04 g/mol, EIsMS: 632 (M+, 100), mp: 32-35
N-(2-Aminoethyl)-N′,2-dihexadecyl Propane Diamide (Va).
1
°C. H NMR (400 MHz, CDCl3) δ: 0.87 (2t, 6H, 2[-CH3]),
Yield: 65% (procedure 1), 83% (procedure 2), white substance,
1
594.01 g/mol, mp: 74-76 °C. ESIsMS: (M++H), H NMR
1.17-1.88 (m, 51 H, [H3C(CH2)6CH2CHdCHCH2(CH2)7-],
[H3C(CH2)6CH2CHdCHCH2(CH2)6CH2NH-], [-OCH2CH3]),
1.90-2.05 (m, 8H, 2[-CH2CHdCHCH2-]), 3.15-3.19 (m, 2H,
[-CH2NHCO-]), 3.20-3.27 (m, 1H, [-CH-]), 4.19 (q, 2H,
[-OCH2CH3]), 5.30-5.40 (m, 4H, 2[-CHdCH-]), 6.50-6.54
(m, 1H, [-NH-]). Anal. Calcd.: C, 77.91; H, 12.28; N, 2.21.
Found: C, 77.83; H, 12.38; N, 2.30.
(400 MHz, CDCl3/CD3OD) δ: 0.87 (t, 6H, 2[-CH3]), 1.00-1.57
(m, 58H, [H3C(CH2)15-], [H3C(CH2)14CH2NH-]), 2.72 (m,
2H, [H2NCH2-]), 2.90-3.10 (m, 4H, [-CH2NHCO-],
[H2NCH2CH2NHCO-]),3.25-3.31(m,1H,[-CH-]),4.15-4.20
(m, 2H, 2[-NH-]). Anal. Calcd.: C, 74.81; H, 12.73; N, 7.07.
Found: C, 74.75; H, 12.69; N, 7.05.
Saponification of IIIa-c. General Procedure. Potassium
hydroxide (0.56 g, 0.01 mol) was added to a suspension of
compound III (2.9 g, 0.005 mol) in ethanol (50 mL), and the
mixture was heated under reflux for 5 h. After cooling to room
temperature, the precipitated potassium salt was filtered by
suction, washed with ether, and dried in vacuum. The salt (3.0
g, 0.0049 mol) was then suspended in 50 mL water containing
1.25 mL conc. HCl under cooling with ice and stirring. After
1 h, the acid IV was thrice extracted with CHCl3. The combined
CHCl3 layers were dried and evaporated and the residue was
crystallized from heptane.
2-[(Hexadecylamino)carbonyl]octadecanoic Acid (IVa).
Yield: 85%, white crystals, C35H69NO3, 577.96 g/mol, mp:
74-76 °C. ESIsMS: (M-H).1H NMR (400 MHz, CDCl3/
CD3OD) δ: 0.86 (t, 6H, 2[-CH3]), 1.23-1.95 (m, 58 H,
[H3C(CH2)15-], [H3C(CH2)14CH2NH-]), 3.09-3.18 (m, 2H,
[-CH2NHCO-]), 3.26-3.35 (m, 1H, [-CH-]), 5.89-5.94 (m,
1H, [-NH-]). Anal. Calcd.: C, 76.16; H, 12.60; N, 2.54. Found:
C, 76.16; H, 12.67, N, 2.54.
2-{[(9Z)-Octadec-9-enylamino]carbonyl}octadecanoic Acid
(IVb). Yield: 82%, white crystals, C37H71NO3, 577.96 g/mol,
mp: 74-76 °C. ESIsMS: (M-H).1H NMR (400 MHz, CDCl3/
CD3OD) δ: 0.86 (t, 6H, 2[-CH3], 1.18-1.89 (m, 54H,
[H3C(CH2)15-], [H3C(CH2)6CH2CHdCHCH2(CH2)6CH2NH-]),
1.92-2.05 (m, 4H, [-CH2CHdCHCH2-]), 3.14-3.30 (m, 3H,
[-CH2NHCO-], [-CH-]), 5.30-5.36 (m, 2H, -CHdCH-]),
6.45-6.50 (m, 1H, [-NH-]). Anal. Calcd.: C, 76.89; H, 12.38;
N, 2.42. Found: C, 76.91; H, 12.36; N, 2.42.
N-(2-Aminoethyl)-2-hexadecyl-N′-[(9Z)-octadec-9-enyl]-
propane Diamide (Vb). Yield: 68% (procedure 1), 80%
(procedure 2), white substance, C39H77N3O2, 620.05 g/mol, mp:
68-71 °C. ESIsMS: (M++H).1H NMR (400 MHz, CDCl3/
CD3OD) δ: 0.85 (t, 6H, 2[-CH3]), 1.20-1.88 (m, 54H,
[H3C(CH2)15-], [H3C(CH2)6CH2CHdCHCH2(CH2)6CH2NH-]),
1.92-2.03 (m, 4H, [-CH2CHdCHCH2-]), 2.79-2.81 (m,
2H, [H2NCH2-]), 2.94-3.23 (m, 4H, [-CH2NHCO-],
[H2NCH2CH2NHCO-]),3.26-3.30(m,1H,[-CH-]),5.29-5.35
(m, 2H, [-CHdCH-]), 5.77-5.80 (m, 2H, 2[-CONH-]).
Anal. Calcd.: C, 75.55; H, 12.52; N, 6.78. Found: C, 75.43; H,
12.37; N, 6.69.
N-(2-Aminoethyl)-N′,2-di[(9Z)-octadec-9-enyl]propane Di-
amide (Vc). Yield: 82% (procedure 2), white substance,
C41H79N3O2, 646.06 g/mol, ESIsMS: (M++H). 1H NMR (400
MHz, CDCl3) δ: 0.86 (t, 6H, 2[-CH3]), 1.20-1.90 (m, 48H,
[H3C(CH2)6CH2CHdCHCH2(CH2)7[-H3]C, (CH2)6CH2CHdCHCH2-
(CH2)6CH2NH-]), 1.92-2.05 (m, 8H, 2[-CH2CHdCHCH2-]),
2.78-2.82 (m, 2H, [H2NCH2CH2NH-]), 3.18-3.22 (m, 4H,
-CH2NHCO-], [H2NCH2CH2NHCO-], 3.25-3.32 (m, 1H,
[-CH-]), 5.25-5.40 (m, 4H, 2[-CHdCH-]), 5.80-5.89 (m,
2H, 2[-CONH-]). Anal. Calcd.: C, 76.22; H, 12.32; N, 6.50.
Found: C, 76.22; H, 12.38; N, 6.42.
N-[Bis(2-aminoethyl)amino]ethyl-N′,2-dihexadecylpro-
pane Diamide (Vd). Yield: 49% (procedure 1), 76% (procedure
2), white substance, C41H85N5O2, 680.15 g/mol, mp: 84-86 °C.
1
ESIsMS: (M++H). H NMR (400 MHz, CDCl3/CD3OD) δ:
0.84 (t, 6H, 2[-CH3]), 1.22-1.79 (m, 58H, [H3C(CH2)15-],
[H3C(CH2)14CH2NH-]), 2.51-2.56 (m, 4H, 2[H2NCH2CH2-]),
2.73-2.76 (m, 4H, 2[H2NCH2CH2-]), 2.96 (t, 2H,
[-NCH2CH2NH-]), 3.13-3.20 (m, 2H, [-CH2NHCO-]),
3.21-3.35 (m, 2H, [-NCH2CH2NHCO-]), 3.60-3.63 (m, 1H,
[-CH-]), 5,77-5.89 (m, 2H, [-CONH-]). 13C NMR (100
MHz, CDCl3) δ: 14.56, 23.22, 27.48, 28.13, 29.83, 29.88, 30.00,
30.15, 30.17, 30.20, 30.24, 32.47, 33.33, 38.01, 39.59, 40.05,
40.18, 54.02, 54.87, 56.98, 172.00, 172.16. Anal. Calcd. for
C41H85N5O2 × H2O: C, 70.53; H, 12.56; N, 10.03. Found: C,
70.37; H, 12.64; N, 9.95.
2-{[(9Z)-Octadec-9-enylamino]carbonyl}-(11Z)-icos-11-
enoic Acid (IVc). Yield: 85%, white waxy substance,
C39H73NO3, 604.00 g/mol, mp: 70-72 °C. ESIsMS: (M-H).1H
NMR (400 MHz, CDCl3) δ: 0.80-0.89 (t, 6H, 2 [-CH3]),
1.20-1.90 (m, 50H, [H3C(CH2)6CH2CHdCHCH2(CH2)7-],
[H3C(CH2)6CH2CHdCHCH2(CH2)6CH2NH-]), 1.92-2.08 (m,
8H, 2[-CH2CHdCHCH2-]), 3.08-3.30 (m, 3H, [-CH2NHCO-],
[-CH-]), 5.34-5.40 (m, 4H, 2[-CHdCH-]), 6.05-6.15 (m, 1H,
[-NH-]). Anal. Calcd.: C, 77.54; H, 12.18; N, 2.32. Found: C, 77.44;
H, 12.36; N, 2.30.
Reaction of IVa-c with the Spacer Molecules to Va-f.
Procedure 1. Compound IV (1 mmol) was dissolved in 50 mL
CH2Cl2 and 140 mg (1.2 mmol) N-hydroxysuccinimide were
added. Then, 288 mg (1.4 mmol) DCC was added to the slurry,
and the mixture was allowed to stir for 12 h at room temperature.
After that time, the precipitate of dicyclohexyl urea was filtered
off and ethylene diamine or tris(aminoethyl)amine (20 mmol)
was added to the solution. The mixture was stirred for 4 h at
room temperature. The organic layer was then filtered, washed
with water and brine, and dried over sodium sulfate. After
evaporation of the solvent, the crude product was purified by
column chromatography on silica gel 60 and CHCl3/methanol
using gradient technique.
N-[Bis(2-aminoethyl)amino]ethyl-2-hexadecyl-N′-[(9Z)-oc-
tadec-9-enyl]propane Diamide (Ve). Yield: 49% (procedure 1),
71% (procedure 2), white waxy substance, C43H87N5O2, 706.18
1
g/mol, mp: 35 °C. ESIsMS: (M++H). H NMR (400 MHz,
CDCl3/CD3OD) δ: 0.85 (t, 6H, 2[-CH3], 1.20-1.89 (m, 54H,
[H3C(CH2)15-], [H3C(CH2)6CH2CHdCHCH2(CH2)6CH2NH-]),
1.95-2.05 (m, 4H, [-CH2CHdCHCH2-]), 2.50-2.56 (m, 4H,
2[H2NCH2CH2-]),2.79-2.81(m,4H,2[H2NCH2CH2-]),2.94-3.23
(m, 4H, [-CH2NHCO-], [-NCH2CH2NHCO-]), 3.26-3.30 (m,
1H, [-CH-]), 5.29-5.35 (m, 2H, [-CHdCH-]), 5.77-5.86 (m,
2H, 2[-CONH-]). 13C NMR (100 MHz, CDCl3) δ: 14.31, 23.14,
25.84, 27.48, 27.67, 28.00, 29.69, 29.77, 29.79, 29.84, 29.91, 30.01,
30.10, 30.14, 30.19, 30.24, 30.28, 31.04, 31.40, 32.42, 33.05, 37.72,
37.97, 40.20, 52.35, 54.19, 54.39, 54.56, 130.14, 130.34, 172.12,
Procedure 2. Triethylamine (0.152 g, 1.5 mmol) was added
to a solution of compound IV (1 mmol), ethylene diamine or

Novel Cationic Lipids Based on Malonic Acid Amides
Bioconjugate Chem., Vol. 21, No. 4, 2010 699
172.24. Anal. Calcd. for C43H87N5O2 × H2O: C, 71.31; H, 12.38;
[H3C(CH2)6CH2CHdCHCH2(CH2)6CH2NH-], [-(CH2)3CH2NH2]),
1.90-2.05 (m, 8H, 2[-CH2CHdCHCH2-]), 2.80-2.85 (m, 2H,
[H2NCH2-]), 3.05-3.10 (m, 1H, [-CH-]), 3.15-3.25 (m, 2H,
[-CH2NHCO-]), 3.25-3.40 (m, 5H, [-CONHCH2CH2NHCO-],
[-CONHCH2CH2NHCO-], [-COCH(NH2)CH2-]), 5.30-5.40 (m,
4H, 2[-CHdCH-]), 6.60-6.65 (2 m, 2H, 2[-CONH-]). 13C NMR
(100 MHz, CDCl3/CD3OD) δ: 13.66, 22.38, 26.67, 26.92, 27.21,
28.94, 29.01, 29.14, 29.22, 29.41, 29.47, 30.62, 31.62, 32.29, 34.32,
38.58, 39.32, 40.35, 53.99, 54.47, 129.31, 129.50, 170.67, 171.70.
Anal. Calcd. for C47H91N5O3 × H2O: C, 71.25; H, 11.83; N, 8.84.
Found: C, 71.37; H, 11.57; N, 8.86.
Preparation of Liposomes. A CHCl3/methanol solution (9/
1, v/v) of the pure cationic lipid was combined with a CHCl3
solution of DOPE or cholesterol. The mixtures were dried under
reduced pressure to remove the organic solvent, and the dried
film was vacuum-desiccated for 3 h. The lipid film was dissolved
in distilled water at room temperature to a final concentration
of 2 mg lipid/mL. The film was allowed to swell in a water
bath at 40 °C for at least 15 min. Subsequently, the lipid
formulations were vortexed and sonicated to clarity in a bath
sonicator. The samples were stored in the refrigerator at 4 °C
before experiments.
N, 9.67. Found: C, 71.03; H, 12.45; N, 9.52.
N-[Bis(2-aminoethyl)amino]ethyl-N′,2-di[(9Z)-octadec-9-
enyl]propane Diamide (Vf). Yield: 74% (procedure 2), white waxy
substance, C45H89N5O2, 732.22 g/mol. ESIsMS: (M++H). 1H NMR
(400 MHz, CDCl3) δ: 0.87 (t, 6H, 2[-CH3]), 1.20-1.90 (m, 50H,
[H3C(CH2)6CH2CHdCHCH2(CH2)6CH2NH-], [H3C(CH2)6CH2CHd
CHCH2(CH2)7-), 1.90-2.05 (m, 8H, 2[-CH2CHdCHCH2-]),
2.49-2.6 (m, 4H, 2[H2NCH2CH2N-]), 2.80-2.92 (m, 6H,
2[H2NCH2CH2N-], [-NCH2CH2NH-), 3.16-3.30 (m, 4H,
[-CH2NHCO-], [-NCH2CH2NHCO-]), 3.50-3.59 (m, 1H,
[-CH-]), 5.30-5.38 (m, 2H, [-CHdCH-]), 5.80-5.89 (m,
2H, [-CONH-]). 13C NMR (100 MHz, CDCl3) δ: 13.93, 22.50,
26.81, 27.05, 27.50, 29.00, 29.13, 29.23, 29.27, 29.31, 29.34,
29.36, 29.43, 29.48, 29.52, 29.59, 31.72, 32.41, 32.66, 37.70,
39.32, 39.41, 53.40, 54.49, 56.46, 129.36, 129.38, 129.53,
129.55, 170.78, 171.11. Anal. Calcd. for C45H89N5O2 × H2O:
C, 72.04; H, 12.22; N, 9.34. Found: C, 72.38; H, 12.17; N, 9.25.
Reaction Va-c to Compounds VIa-c. General Procedure.
0.84 mmol of compound V was dissolved in 30 mL CH2Cl2.
0.9 mmol (0.4 g) diboc-lysin-N-hydroxysuccinimid ester was
given to this solution. The mixture was stirred for 1 h at room
temperature and then for another 10 h under reflux. After that
time, the solvent was evaporated, the residue was chromato-
graphed using a short column and dry silicagel with CHCl3/
methanol (98:2, v/v) as eluent. The so-purified product was
dissolved in 15 mL CH2Cl2, and 1 mL trifluoro acetic acid was
added. The mixture was allowed to stir for 4 h, then mixture
was diluted with 60 mL CHCl3. 100 mL diluted ammonia was
poured into the mixture, which was shaken until two clear phases
evolved. The organic layer was separated, dried over sodium
sulfate, and evaporated. The crude products VI were then
purified by column chromatography using silicagel 60 and
CHCl3/methanol/NH3 with gradient technique.
N′-2[(2,6-Diamino-1-oxohexyl)amino]ethyl-2,N-(dihexade-
cyl)propane Diamide (VIa). Yield: 54%, white waxy substance,
C43H87N5O3, 722.18 g/mol, mp: 111-112 °C. ESIsMS:
(M++H).1H NMR (400 MHz, CDCl3) δ: 0.86 (t, 6H, [-CH3]),
1.18-1.88 (m, 64H, [H3C(CH2)15-], [H3C(CH2)14CH2NH-],
[-(CH2)3CH2NH2]), 2.82-2.87 (m, 2H, [-CH2NH2]), 3.05-3.09
(m, 1H, [-CH-]), 3.20-3.38 (m, 5H, [-CONHCH2CH2NHCO-],
[-CONHCH2CH2NHCO-], [-COCH(NH2)CH2-]), 6.58-6.70 (m,
2H, 2[-CONH-]). 13C NMR (100 MHz, CDCl3/CD3OD) δ: 13.11,
22.03, 22.08, 22.11, 26.32, 26.85, 28.61, 28.73, 28.85, 28.96, 29.00,
29.03, 29.07, 30.18, 31.16, 31.24, 31.32, 34.03, 38.26, 38.42, 38.95,
40.00, 53.62, 54.26, 170.51, 171.35. Anal. Calcd. for C43H87N5O3 ×
H2O: C, 69.77; H, 12.12; N, 9.46. Found: C, 70.05; H, 12.16; N, 9.39.
Preparation of Plasmid DNA. pCMV Sport ꢀ-Gal was
isolated from Escherichia coli DH5R (Invitrogene life technolo-
gies) using a Quiagen plasmid mega kit (Quiagen) following
the manufacturer’s instructions (30). DNA with an OD260/OD280
) 1.94 was used for experiments.
Cell Culture. LLC PK1 cells (porcine kidney epithelial cells)
were cultured in 75 cm2 tissue culture flasks in Medium 199
adjusted to contain 2.2 g/L sodium bicarbonate, 10% fetal bovine
serum (FBS), and 0.05 mg/mL gentamycin at 37 °C and 5%
CO2. The cells were grown confluently and were regularly split
twice a week. Only cells that had undergone fewer than 30
passages were used for transfection and MTT experiments.
A549 cells (human lung carcinoma cells) were cultured in
75 cm2 tissue culture flasks in Dulbecos’s modified eagle
medium (DMEM) adjusted to contain 4.5 g/L glucose, 10%
FBS, and 0.05 mg/mL gentamycin at 37 °C and 5% CO2. The
cells were grown confluently and were regularly split twice a
week. For experiments, only cells in the range of passages
20-60 were used.
Transfection Biology. 16-24 h before transfection, cells
were seeded into a 96 well plate at a density of 8000-10 000
cells/well by usage of LLC PK1 and 18 000-20 000 in the case
of A549 cells. Lipoplex mixtures were prepared by combining
plasmid DNA (0.1 µg per well ) 2.5 µg/mL) with varying
amounts of cationic liposome suspension in the absence or
presence of 10% serum in Medium 199 or DMEM. The samples
were incubated for 15 min at room temperature (total volume
40 µL/well). The N/P ratios were varied from 1:1 to 4:1 or even
higher in cases when the results of experiments foreshadow
increasing transfection efficacies at higher charge ratios. During
this time, cells were washed once with phosphate buffered saline
(PBS). Then the complexes were added to the cells. After 4 h
of incubation, 160 µL of Medium 199 or DMEM was added to
the cells in a way that the final concentration of FBS reached
10%. The medium was refreshed after 24 h, and the reporter
gene activity was estimated after 48 h. Therefore, the cells were
washed with PBS lysed for 15 min in lysis buffer (5 mM Chaps
in 50 mM Hepes buffer). The solution was taken out of the
wells, reunited in safe-lock tubes (Eppendorf), centrifuged, and
stored on ice. It was pipeted again in a 96 well plate and
substrate solution [1.33 mg/mL of O-nitrophenyl-ꢀ-galactopy-
ranoside (ONPG), MgCl2 2 mM, ꢀ-mercaptoethanol 100 mM
in 0.2 M sodium phosphate pH 7.3] was added and incubated
for 30 min at 37 °C. The reaction was stopped with 1 M sodium
carbonate. Absorption at 405 nm was converted to ꢀ-galactosi-
N′-2-[(2,6-Diamino-1-oxo-hexyl)amino]ethyl-2-hexadecyl-
N-[(9Z)-octadec-9-enyl]propane Diamide (VIb). Yield: 56%,
white waxy substance, C45H89N5O3, 748.22 g/mol, mp: 104-105 °C,
ESIsMS: (M++H). 1H NMR (400 MHz, CDCl3) δ: 0.85 (2t, 6H,
[-CH3]), 1.20-1.80 (m, 60H, [H3C(CH2)15-], [H3C(CH2)6CH2CHd
CHCH2(CH2)6CH2NH-], [-(CH2)3CH2NH2]), 1.92-2.04, (m, 4H,
[-CH2CHdCHCH2-]), 2.80-2.85 (m, 2H, [-CH2NH2]), 3.05-3.10
(m, 1H, [-CH-]), 3.22-3.43 (m, 5H, [-CONHCH2CH2NHCO-],
[-CONHCH2CH2NHCO-], [-COCH(NH2)CH2-]), 5.33-5.41 (m,
2H, [-CHdCH-]), 6.55-6.65 (2m, 2H, 2[-CONH-]). 13C NMR
(100 MHz, CDCl3/CD3OD) δ: 13.99, 22.38, 26.66, 26.91, 27.20,
28.94, 29.01, 29.06, 29.16, 29.22, 29.31, 29.36, 29.40, 29.47, 31.63,
31.91, 32.28, 34.32, 38.58, 39.32, 40.30, 53.97, 54.45, 129.29, 129.51,
170.68, 171.70. Anal. Calcd. for C45H89N5O3 × H2O: C, 70.54; H,
11.97; N, 9.14. Found: C, 70.71; H, 11.63; N, 9.17.
N′-2-[(2,6-Diamino-1-oxo-hexyl)amino]ethyl-2,N-di[(9Z)-
octadec-9-enyl]propane Diamide (VIc). Yield: 53%, white pasty
substance, C47H91N5O3, 774.26 g/mol, mp: 109-111 °C. ESIsMS:
(M++H). 1H NMR (400 MHz, CDCl3) δ: 0.86 (t, 6H, 2[-CH3]),
1.15-1.88 (m, 56H, [H3C(CH2)6CH2CHdCHCH2(CH2)7-],

700 Bioconjugate Chem., Vol. 21, No. 4, 2010
Heinze et al.
Scheme 1. Synthesis of Lipids 1-9 Starting from Alkyl Malonic Acid Diethyl Ester
dase units using a calibration curve constructed with pure
commercial ꢀ-galactosidase enzyme. Protein concentration was
detected with bichinonic acid reaction (31). Absorption at 570
nm was converted by using a calibration curve made of bovine
serum albumin. All experiments were carried out sixfold. Every
experiment was repeated three to five times on different days.
were prepared by following the instructions used for transfection
experiments and measured with 2.5 µg DNA/mL sample. The
z-average and PDI were calculated by using the automatic mode.
Every sample was measured by three spectroscopic runs
consisting of ten consecutive scans, each of 10 s in duration.
Results shown are the average of these three values.
Toxicity Assay. Cytotoxicity of the lipoplexes was assessed
by the MTT reduction assay (32). The cytotoxicity assays were
performed in 96 well plates by maintaining the same ratio and
number of cells and lipoplexes as used in the transfection
experiments. MTT was added after refreshing the medium (24
h after addition of lipoplexes to the cells) and incubated for 3 h
at 37 °C. Afterward, the cells were lysed with a mixture of 10%
sodium dodecyl sulfate in acetic acid/dimethyl sulfoxide.
Absorption was measured at 570 nm. The results were expressed
as percent viability ) [A570 (treated cells) - background]/[A570
(untreated cells) - background] × 100.
X-Gal Staining. To estimate a successful transfection, cells
were histochemically stained with 5-bromo-4-chloro-3-indolyl-
ꢀ-D-galactopyranoside (X-Gal). ꢀ-Galactosidase expressing cells
can be indicated by blue color. The experiments were carried
out in 24 well plates with a density of 40 000-50 000 cells per
well and DNA concentration of 2.5 µg/mL (1.25 µg/well in 24
well plate). 48 h after transfection, cells were washed two times
with PBS (2 × 500 µL) and fixed with 0.2% glutaraldehyde
and 2.0% formaldehyde in PBS for 10 min at room temperature.
Afterward, the cells were washed with PBS again (2 × 500
µL) and were stained subsequently with 1 mg/mL X-gal in PBS
containing 2 mM MgSO4 and 4 mM K3[Fe(CN)6] and 4 mM
K4[Fe(CN)6] for 3 h at 37 °C.
Size Measurements. The sizes of liposomes and lipoplexes
were measured in distilled water by photon correlation spec-
troscopy on a HPPSsET (Malvern, U.K.) with sample refractive
index of 1.25 and a viscosity of 0.8872 at 25 °C. Lipoplexes
Statistical Analysis. All measurements were collected (n )
3-9) and expressed as means ( standard deviation. One-way

Novel Cationic Lipids Based on Malonic Acid Amides
Bioconjugate Chem., Vol. 21, No. 4, 2010 701
ANOVA was used in conjunction with Bonferoni’s multiple
comparison test to assess the statistical significance.
In Vitro Transfection and Cell Toxicity of Malonic
Acid Amides Cationic Liposomes. To form liposomes for gene
transfer experiments, cationic amphiphiles are usually mixed
with colipids. Most frequently, 1,2-dioleoyl-sn-glycero-3-phos-
phoethanolamine (DOPE) and cholesterol are used (26, 35–37).
Their influence on transfection is ascribed to their tendency to
adopt the inverted hexagonal HII phase that seems to be
necessary for the DNA release from the endosomal compart-
ment (36, 38–42). For that reason, all of the lipids 1-9 were
mixed in different molar ratios with DOPE or cholesterol to
determine the most auspicious combination. To identify the kind
of structure/activity relationship for the malonic acid amides,
nearly 60 samples were prepared and tested for their ability to
transfect cells in culture.
Figure 1 summarizes the relative in vitro gene delivery
efficiencies of lipid 8 mixed with colipids. Lipoplex (cationic
liposome/DNA complex) mixtures were prepared by combining
plasmid DNA (0.1 µg per well ) 2.5 µg/mL) with varying
amounts of cationic liposome suspension in plain Medium 199.
The N/P ratios were varied from 1:1 to 4:1. Lipid 8 has been
proven to be successful for gene transfer into cells in culture.
The majority of the analyzed samples exhibit high transfection
efficacies. All tested combinations of this lipid with cholesterol
show equal or higher transfection efficacy than LipofectAmine
and SuperFect. Merely the maximum values were found at
different N/P ratios. The use of DOPE as colipid decreases the
transfection efficiencies. Only lipid 8/DOPE 1:1 (n/n) is
comparable to commercially available gene transfer kits.
Table 1 summarizes the results of all lipids 1-9 in combina-
tion with the used helper lipids. It shows the most auspicious
combination of any lipid with any colipid in gene transfer
experiments. Furthermore, the N/P ratio, the MTT based percent
cell viability, and the results of size measurements are listed.
Apart from four samples, for all new lipids transfection was
detected, but only lipid 6, lipid 8, and lipid 9 exhibit transfection
efficacies that make them comparable to commercially available
vectors. Liposomes that consist of lipid 6 or lipid 8 and
cholesterol as colipid lead to increasing transfection efficacies.
As shown in Figure 2, this effect is statistically significant
compared to LipofectAmine (*) or SuperFect (∧), whereas the
effects on cell survival are similar.
RESULTS
Synthesis of Malonic Acid Amides Cationic Lipids. The
synthesis of the compounds is shown in Scheme 1. Starting from
long-chain monoalkylated malonic acid esters, which are easy
to prepare by alkylation reaction of malonic acid diesters (33)
with the corresponding alkyl halides or methansulfonates, one
of the ester groups was saponified according to published
methods (34). We have also studied the preparation of the
monoester starting from malonic esters with different alcoholic
residues followed by a selective saponification. Because of the
simplicity of the first reaction, this procedure was used.
Compounds IIa,b were then transformed into the 2-alkylmalonic
acid ester amides IIIa-c by reaction with hexadecyl or oleyl
amine using condensing reagents. The most effective reagents
were 2-ethoxy-1-ethoxycarbonyl-1,2-dihydrochinoline (EEDQ)
and the benzotriazole derived compounds known from peptide
synthesis, like (benzotriazol-1-yloxy)-tris-(dimethylamino)-phos-
phoniumhexaflourophospahte (BOP), leading to the products in
high yield.
For the synthesis of IIIa,b, the activation via the correspond-
ing acid chloride was also a high-yield procedure, but in the
case of the unsaturated chain of IIb, we used only the modern
condensation agents. The saponification of the ester with
potassium hydroxide yields the acids. For the second coupling
reaction with ethylene diamine or tris(aminoethyl)amine to
Va-f, N-hydroxysuccinimide/N,N′-dicyclohexylcarbondiimid
(DCC) or better BOP was used as condensing reagent. We found
that a 20-fold excess of the di- or triamine is necessary. This
ensures yields of more than 70%. On the other hand, the reaction
with only a 5- to 10-fold excess leads only to a yield of 20%,
also in the case of BOP as the most effective condensing reagent.
The introduction of a lysine residue into compounds Va-c was
realized in good yields using the commercially available diboc-
lysine-N-hydroxysuccinimide ester. The crude products were
purified to some extent by filtration using silica gel. For the
removal of the protective groups, trifluoroacetic acid in meth-
ylene chloride was used. The yields after the two steps were
more than 50%.
Figure 1. In vitro transfection efficacies of lipid 8 in LLC PK1 cells using cholesterol and DOPE as colipid (at different lipid/cholesterol or
lipid/DOPE molar ratios). Micro units of ꢀ-galactosidase related to cell protein concentration were plotted against the varying N/P ratios.
The transfection efficiencies of the liposome samples were compared to those of the commercially available LipofectAmine and SuperFect.
The experiments were performed as described in the text. The transfection values shown are the average of at least three experiments carried
out on three different days.

702 Bioconjugate Chem., Vol. 21, No. 4, 2010
Heinze et al.
Table 1. In Vitro Transfection Efficacies, Size Measurements, and MTT Assays of Lipids 1-9 in LLC PK1 Cells Using DOPE and Cholesterol
as Colipid (at Different Lipid/Colipid Molar Ratios)a
sample
N/P ratio z-average ( s [nm] liposomes z-average ( s [nm] lipoplexes activityꢀ-gal/mprotein ( s [µE/µg] viability ( s [%]
Lipid 1/DOPE 2:1 (n/n)
Lipid 1/Cholesterol 1:1 (n/n)
Lipid 2/DOPE 1:1 (n/n)
Lipid 2/Cholesterol 1:1 (n/n)
Lipid 3/DOPE 2:1 (n/n)
Lipid 3/Cholesterol 2:1 (n/n)
Lipid 4/DOPE 1:2 (n/n)
Lipid 4/Cholesterol 1:2 (n/n)
Lipid 5/DOPE 3:1 (n/n)
Lipid 5/Cholesterol 1:1 (n/n)
Lipid 6/DOPE 2:1 (n/n)
Lipid 6/Cholesterol 2:1 (n/n)
Lipid 7/DOPE 1:1 (n/n)
Lipid 7/Cholesterol 1:1 (n/n)
Lipid 8/DOPE 1:1 (n/n)
4:1
3:1
4:1
1:1
4:1
1:1
3:1
6:1
6:1
2:1
6:1
7:1
2:1
2.5:1
3:1
4:1
2:1
2:1
50 ( 1
147 ( 5
99 ( 10
108 ( 3
120 ( 1
151 ( 2
104 ( 7
120 ( 7
112 ( 50
100 ( 3
87 ( 16
88 ( 17
108 ( 1
139 ( 1
131 ( 46
90 ( 4
185 ( 1
330 ( 11
205 ( 2
136 ( 1
171 ( 2
681 ( 33
423 ( 6
189 ( 6
113 ( 1
116 ( 1
189 ( 1
134 ( 1
1132 ( 114
642 ( 12
268 ( 7
137 ( 1
583 ( 2
258 ( 1
no transfection
no transfection
no transfection
352 ( 191
276 ( 157
29 ( 13
86 ( 11
69 ( 9
91 ( 7
93 ( 17
85 ( 13
94 ( 13
84 ( 14
94 ( 11
92 ( 6
102 ( 13
77 ( 15
82 ( 8
84 ( 9
83 ( 7
89 ( 11
85 ( 14
86 ( 10
90 ( 8
190 ( 69
16 ( 7
156 ( 38
31 ( 25
335 ( 104
1018 ( 206
no transfection
117 ( 37
633 ( 263
1294 ( 511
309 ( 211
692 ( 190
Lipid 8/Cholesterol 1:1 (n/n)
Lipid 9/DOPE 2:1 (n/n)
Lipid 9/Cholesterol 1:1 (n/n)
121 ( 2
94 ( 1
a The transfection and viability values listed are the average of at least three experiments carried out on different days.
Figure 2. In vitro transfection efficacies and cell viability values of lipids 6, 8, and 9 in LLC PK1 cells using cholesterol or DOPE as colipid (at
different lipid/cholesterol or lipid/DOPE molar ratios). Micro units of ꢀ-galactosidase related to cell protein concentration and cell viability in %
were plotted against different samples. The transfection efficiencies of the liposomes were compared to that of commercially available LipofectAmine
and SuperFect. Percent cell viability is plotted on secondary y-axis via scatter diagram. The transfection and cytotoxicity values shown are the
average of at least three experiments carried out on three different days. Statistical analyses were carried out with p < 0.05, one-way ANOVA, and
Bonferoni’s multiple comparison tests (*∧).
Figure 3 summarizes the results of gene transfer experiments
in LLC PK1 cells with LipofectAmine (A), lipid 1 (B), and
lipid 6 (C) visualized by histochemical staining (X-Gal assay).
Whereas LipofectAmine and lipid 6/cholesterol 2:1 (n/n) lead
to considerable staining of wells, no colored cells were found
when using lipoplexes consisting of lipid 1 as gene transfer
reagent.
In the majority of cases, the cell viability fluctuates between
80% and 90% (cf. Table 1). These results suggest that the
applied liposomes have an influence on the cell survival but
that the efficacy of the gene transfer is not directly coupled with
the toxicity. For instance, lipid 1 and lipid 7 with no or low
transfection efficacy have similar or even stronger influence on
the cell viability than lipid 6, lipid 8, or lipid 9, although those
are proven to be successful for the transfection process.
Figure 4 shows the results of 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) measurements of lipid
1, lipid 7, and lipid 8 and lipid 9 lipoplexes. The addition of
lipoplexes decreases the cell viability. This effect was observed
for all lipids and all N/P ratios. Furthermore, there is no
statistically significant difference between the charge ratios of
one sample, which suggests that the cell viability is already
decreased at low lipid concentrations. An increase of cationic
lipid concentration shows little effect on the toxicity at least in
applied N/P ratios. Interestingly, the influence on cell viability
of lipid 1 in combination with cholesterol is more distinct than
that of lipid 8 or lipid 9. Liposomes, which are not qualified
for in vitro gene delivery, influence cell survival to the same
degree or even more than efficiently transfecting samples. By
comparing lipoplexes that consist of lipid 9 with those of lipid
1 and lipid 7, the differences in cell viability are statistically
significant just like the comparison of lipid 1 and lipid 8. The
results suggest that the chemical structure of the cationic lipid
and the composition of liposomes play a major role in cell
damaging characteristics, as well as in the ability to transfer
nucleic acids. It was observed that the addition of lipoplexes
leads to derogation of LLC PK1 cell survival independent of
successful or unsuccessful gene transfer. Transfection of cells
in culture is to a certain level connected with toxicity, but the
reverse is not compulsory. To look for other parameters, which

Novel Cationic Lipids Based on Malonic Acid Amides
Bioconjugate Chem., Vol. 21, No. 4, 2010 703
Figure 3. Pictures of X-Gal assay in LLC PK1 cells using lipid 1, lipid 6, and LipofectAmine. Cells were incubated with 2.5 µg/mL of DNA and stained
48 h after starting transfection. (A) LipofectAmine, magnification 1:10. (B) Lipid 1/DOPE 1:1 (n/n), magnification 1:200. (C) Lipid 6/Cholesterol 2:1
(n/n), magnification 1:10.
Figure 4. MTT-based cellular cytotoxicities of lipids 1, 7, 8, and 9 in LLC PK1 cells using DOPE or cholesterol as colipid (at a lipid/colipid molar
ratio of 1:1). The percent cell viability was plotted against the varying N/P ratios. The experiments were performed as described in the text. The
cytotoxicity values shown are the average of at least three experiments carried out on different days.
are important for cell damaging characteristics of cationic
liposomes, MTT measurements of lipoplexes consisting of lipid
8 were specified.
experiments than 100 nm lipoplexes (41, 43). For successful in
vivo gene transfer, particles should have a size less than 150
nm, because larger ones are quickly cleared from blood (44, 45).
The results of size measurements of six liposomes and
lipoplexes consisting of lipid 1, lipid 2, lipid 7, lipid 8, and
lipid 9 are plotted. They were chosen to distinguish between
well-transfecting and nontransfecting lipoplexes. All of them
disclose some similarities. The increasing particle size in
connection with increasing cationic lipid concentrations has a
maximum value at charge equalization (17). The displaced
maximum values are presumably due to varied capability to
condense DNA. By comparing lipoplexes consisting of lipid 7,
lipid 8, and lipid 9, it is conspicuous that not only the number
of amine residues and the cationic lipid concentration but also
the fluidity of liposome membranes is important for charge
equalization. Unfortunately, the differences in particle size
between transfecting and nontransfecting liposomes are mar-
ginal, so that differentiations between the samples are hardly
assessable. Certainly, it should be noted that lipoplexes, which
exhibit appreciable transfection features, have z-average values
which are only slightly larger than those of the pure liposomes.
The particle size of the majority of lipoplexes is less than 500
nm (cf. Table 1).
Figure 5 summarizes these results in LLC PK1 cells. As
shown, the N/P ratios are varied from 1:1 to 4:1 for all applied
lipoplexes. The concentration of lipid 8 is the same in all
samples and increases merely with increasing N/P ratios (in the
range 4.05-16.2 µmol/L). It is shown that lipoplexes consisting
of a larger percentage of colipids exhibit more cytotoxic
potential as lipoplexes consisting of larger percentage of lipid
8. With increasing concentration of the helper-lipid, cell survival
decreases. This effect is highly pronounced using cholesterol,
but also visible using DOPE as colipid. The concentration of
the colipid also seems to play a decisive role in cell damaging
characteristics of cationic liposomes. As the mixed liposomes
influence the shape and structure of lipoplexes, variances in the
ratio between the cationic and the helper lipids has an impact
not only on transfection efficacy, but also on compatibility and
consequently the cell viability.
Figure 6 summarizes the results of PCS measurements for
selected malonic acid amides. Since particle size seems to be
crucial for transfection efficiencies compared with lipoplex
charge or zeta potential (41), the size measurements of all
lipoplexes were used to find a correlation of transfection efficacy
and particle size. For DOTAP lipoplexes, it was shown that
particles with 500 nm sizes were more effective in cell culture
As a matter of course, lipid 6, lipid 8, and lipid 9 were
analyzed for their ability to transfer genes in another cell strain.
Figure 7 summarizes the results of in vitro gene delivery and

704 Bioconjugate Chem., Vol. 21, No. 4, 2010
Heinze et al.
Figure 5. MTT-based cellular cytotoxicities of lipid 8 in LLC PK1 cells using cholesterol and DOPE as colipid (at different lipid/colipid molar
ratios). The percent cell viability was plotted against the varying N/P ratios.
MTT based cell viability assays in A549 cells. Even if
transfection efficacy of lipid 8 is not as appreciable as in LLC
PK1 cells, it is still comparable to LipofectAmine and SuperFect.
In human lung carcinoma cells, the multivalent cationic lipids
6 and 9 with two unsaturated alkyl chains were found to transfer
DNA statistically significantly better than commercially avail-
able vectors at similar cell viability values (*).
Figure 8 summarizes the experiments of gene transfer in the
presence of FBS in LLC PK1 cells. Lipoplexes were incubated
with the cells as described in the Experimental Procedures
section, certainly the concentration of serum was adjusted to
10% from the outset of experiments and kept for 48 h.
Admittedly, total protein concentration of human serum that is
between 66 and 83 g/L (46) is just reached with a level of 60%,
but information about serum compatibility of applied lipoplexes
is still possible. A slight influence of serum was observed for
SuperFect and lipid 8/cholesterol 2:1 (n/n), whereas lipid
8/cholesterol 2:1 (n/n) offers nearly twice the amount of
transfection efficacy. Notably, for LipofectAmine, which con-
tains DOPE, a decrease of gene transfer was observed. Li-
poplexes consisting of lipid 6 and lipid 9 deteriorate in
transfection efficacy in regard to gene transfer without serum
over a period of 4 h. Nevertheless, these samples exhibit gene
transfer rates in excess of LipofectAmine that are partially
statistically significant (*).
DISCUSSION
Since their introduction as gene carriers in 1987 (15), cationic
liposomes have become well-studied nonviral vectors (47).
Lipoplex mediated gene delivery systems are currently the most
favorable means to achieve transgene expression in cells in
culture. In the meantime, about 1300 clinical trials in gene
therapy research have been carried out (14). Although hundreds
of cationic lipids have been studied (48), there is still a need to
develop new gene transfer systems.
By engineering new lipids for nonviral gene delivery, it
is important to investigate structure/activity relationships to
expedite the reasonable development of these substances. In
the case of malonic acid amides, several effects of
structure-function relationships were demonstrated. Inde-
pendent of the chemical structure of the hydrophilic molecule
part or the colipid, it was observed that the insertion of at

Novel Cationic Lipids Based on Malonic Acid Amides
Bioconjugate Chem., Vol. 21, No. 4, 2010 705
Figure 6. Photon correlation spectroscopy analyses of liposomes and lipoplex mixtures: Changes of particle size as a function of increasing lipid
concentration (concentration of DNA ) 2.5 µg/mL). The average diameters of lipoplex particles in each set of lipoplex mixtures were determined
by three spectroscopic runs comprising ten consecutive scans, each of 10 s in duration.
Figure 7. In vitro transfection efficacies and MTT based cell viability values of lipids 6, 8, and 9 in A549 cells using cholesterol as colipid. The
transfection efficacies and percent cell viability values were compared with LipofectAmine and SuperFect. Percent cell viability is plotted on a
secondary y-axis via scatter diagram. The transfection and cell viability values shown are the averages of at least three experiments carried out on
different days. Statistical analyses were carried out with p < 0.05, one-way ANOVA, and Bonferoni’s multiple comparison tests (*).
least one unsaturated alkyl chain leads to higher transfection
efficacies. Already, Felgner et al. could demonstrate that
increasing membrane fluidity often leads to increasing
transfection efficacies (16). This effect has been attributed
to an enhanced capability of fluid amphiphiles to condense
DNA (16, 49). Besides the electrostatic interaction of cationic
lipids and DNA, packing properties of the lipids seem to be
important for the condensation of DNA (40). It was shown
that the insertion of tris(aminoethyl)amine and lysine as
hydrophilic headgroup increase transfection efficacy. Al-
though lipids 1-3 are able to adsorb DNA in monolayer
experiments and protect partially nucleic acids in ethidium
bromide exclusion assay (data not shown), multivalent
headgroups seem to be more suitable for lipoplex-mediated
gene transfer. Except for monovalent substances, all lipids
exhibit higher transfection efficacies in combination with
cholesterol as colipid. Helper lipids mainly affect the tertiary
structure of the plasmid DNA in lipoplexes (8). Although
this does not affect the transfection effiency (39, 50), the
influence of colipids for gene transfection is indisputable.
As mentioned above, colipids influence the transfection
because of their tendency to adopt the inverted hexagonal
HII phase that seems to be necessary for the DNA release
from the endosomal compartment. Furthermore, DOPE
promotes the lipid exchange between liposome and the
endosome membrane, which is also crucial for the endosome
releasing process (51, 52).
The fact that cholesterol was found to be a more efficacious
colipid than DOPE is an encouraging result in regard to in
vivo gene transfer experiments. Although the application of
cationic liposomes as gene delivery systems in cells in culture
is popular, the in vivo application still has serious drawbacks
(53). Due to the chemical structure of the lipids used,
liposomes interact with different components of the human

706 Bioconjugate Chem., Vol. 21, No. 4, 2010
Heinze et al.
Figure 8. In vitro transfection efficacies of lipids 6, 8, and 9 in LLC PK1 cells in the presence of serum compared with LipofectAmine and
SuperFect. Concentration of FBS was adjusted to 10% from the outset of experiment and kept up for 48 h. N/P ratios: lipid 6, 6:1; lipid 8, 4:1 and
4:1; lipid 9, 3:1. The transfection values shown are the average of leastwise three experiments carried out on three different days. Statistical
analyses were carried out with p < 0.05, one-way ANOVA, and Bonferoni’s multiple comparison tests (*∧).
organism, especially negatively charged amphipathic proteins
and polysaccharides that are present in blood, mucus, or tissue
matrix (47). Plasma proteins like albumin adsorb on the
liposome surface and make them recognizable for cells of
the reticuloendothelial system (54). For liposomes consisting
of phospholipids, it is well-known that lipoproteins lead to
membrane disintegrations as a consequence of lipid exchange.
Mainly liposomes containing lipids with unsaturated fatty
acids interact notably with lipoproteins (55). For that reason,
DOPE is unsuitable for in vivo gene transfer (56). On the
contrary, cholesterol was found to stabilize the lipoplex
structure in blood (57, 58). To minimize disadvantageous
effects of nonviral vectors in systemic gene delivery,
PEG-lipid conjugates were successfully incorporated in
lipoplexes (59). On the other hand, lipoplexes exhibiting
certain serum stability without modification could advance
in vivo gene delivery. As shown, cationic liposomes that
consist of malonic acid amides have the ability to transfer
nucleic acids even in the presence of FBS.
Malonic acid amides describe an interesting novel group of
cationic amphiphiles. Nine substances were synthesized and
mixed with DOPE or cholesterol, and liposomes were tested
for their ability to transfect cells in culture. It was shown that
three of them transfect cells at comparable or even higher levels
than commercially available LipofectAmine and SuperFect.
Thereby, it was observed that lipids containing at least one
unsaturated alkyl chain in the hydrophobic molecule part and
multivalent headgroups are very successful in gene transfer
experiments. Along with this, the cell damaging characteristics
of lipoplexes were analyzed. Although the addition of lipoplexes
is connected with decreasing cell survival, in the majority of
cases the applied lipoplexes show remarkably high cell viability
(>80%).
we expect further developments from this kind of cationic lipids
toward successful in vivo gene transfer.
ACKNOWLEDGMENT
We thank the Max Planck Institute of Colloids and Interfaces
Potsdam for financial support.
LITERATURE CITED
(1) Rubanyi, G. M. (2001) The future of human gene therapy. Mol.
Aspects Med. 22, 113–142.
(2) Anderson, W. F. (1998) Human gene therapy. Nature 392, 25–
30.
(3) Hunt, K. K., and Vorburger, S. A. (2002) Tech.Sight. Gene
therapy. Hurdles and hopes for cancer treatment. Science 297,
415–416.
(4) El-Aneed, A. (2004) An overview of current delivery systems
in cancer gene therapy. J. Controlled Release 94, 1–14.
(5) Hacein-Bey-Abina, S., Garrigue, A., Wang, G. P., Soulier, J.,
Lim, A., Morillon, E., Clappier, E., Caccavelli, L., Delabesse,
E., Beldjord, K., Asnafi, V., MacIntyre, E., Dal Cortivo, L.,
Radford, I., Brousse, N., Sigaux, F., Moshous, D., Hauer, J.,
Borkhardt, A., Belohradsky, B. H., Wintergerst, U., Velez, M. C.,
Leiva, L., Sorensen, R., Wulffraat, N., Blanche, S., Bushman,
F. D., Fischer, A., and Cavazzana-Calvo, M. (2008) Insertional
oncogenesis in 4 patients after retrovirus-mediated gene therapy
of SCID-X1. J. Clin. InVest. 118, 3132–3142.
(6) Howe, S. J., Mansour, M. R., Schwarzwaelder, K., Bartholomae,
C., Hubank, M., Kempski, H., Brugman, M. H., Pike-Overzet,
K., Chatters, S. J., de Ridder, D., Gilmour, K. C., Adams, S.,
Thornhill, S. I., Parsley, K. L., Staal, F. J., Gale, R. E., Linch,
D. C., Bayford, J., Brown, L., Quaye, M., Kinnon, C., Ancliff,
P., Webb, D. K., Schmidt, M., von Kalle, C., Gaspar, H. B., and
Thrasher, A. J. (2008) Insertional mutagenesis combined with
acquired somatic mutations causes leukemogenesis following
gene therapy of SCID-X1 patients. J. Clin. InVest. 118, 3143–
3150.
Furthermore, some lipids proved to be successful in gene
transfer experiments notwithstanding the presence of serum.
Certainly, despite these promising results, further research is
clearly needed to clarify the complex mechanism of cationic
lipid mediated gene transfer. The presented malonic acid amides
are qualified for these requirements. The preparation facilitates
the trouble-free variation of alkyl chain length. In addition,
multivalent hydrophilic molecule parts are suited to link other
cationic residues or molecules for functionalization. Thereby,
(7) Walther, W., and Stein, U. (2000) Viral vectors for gene
transfer: a review of their use in the treatment of human diseases.
Drugs 60, 249–271.
(8) Boktov, J., Hirsch-Lerner, D., and Barenholz, Y. (2007)
Characterization of the interplay between the main factors
contributing to lipoplex-mediated transfection in cell cultures.
J. Gene Med. 9, 884–893.

Novel Cationic Lipids Based on Malonic Acid Amides
Bioconjugate Chem., Vol. 21, No. 4, 2010 707
(9) Hyde, S. C., Southern, K. W., Gileadi, U., Fitzjohn, E. M.,
Mofford, K. A., Waddell, B. E., Gooi, H. C., Goddard, C. A.,
Hannavy, K., Smyth, S. E., Egan, J. J., Sorgi, F. L., Huang, L.,
Cuthbert, A. W., Evans, M. J., Colledge, W. H., Higgins, C. F.,
Webb, A. K., and Gill, D. R. (2000) Repeat administration of
DNA/liposomes to the nasal epithelium of patients with cystic
fibrosis. Gene Ther. 7, 1156–1165.
(10) Noone, P. G., Hohneker, K. W., Zhou, Z., Johnson, L. G.,
Foy, C., Gipson, C., Jones, K., Noah, T. L., Leigh, M. W.,
Schwartzbach, C., Efthimiou, J., Pearlman, R., Boucher, R. C.,
and Knowles, M. R. (2000) Safety and biological efficacy of a
lipid-CFTR complex for gene transfer in the nasal epithelium
of adult patients with cystic fibrosis. Mol. Ther. 1, 105–114.
(11) Veelken, H., Mackensen, A., Lahn, M., Kohler, G., Becker,
D., Franke, B., Brennscheidt, U., Kulmburg, P., Rosenthal, F. M.,
Keller, H., Hasse, J., Schultze-Seemann, W., Farthmann, E. H.,
Mertelsmann, R., and Lindemann, A. (1997) A phase-I clinical
study of autologous tumor cells plus interleukin-2-gene-trans-
fected allogeneic fibroblasts as a vaccine in patients with cancer.
Int. J. Cancer 70, 269–277.
(12) Hui, K. M., Ang, P. T., Huang, L., and Tay, S. K. (1997)
Phase I study of immunotherapy of cutaneous metastases of
human carcinoma using allogeneic and xenogeneic MHC DNA-
liposome complexes. Gene Ther. 4, 783–790.
(13) Stopeck, A. T., Jones, A., Hersh, E. M., Thompson, J. A.,
Finucane, D. M., Gutheil, J. C., and Gonzalez, R. (2001) Phase
II study of direct intralesional gene transfer of allovectin-7, an
HLA-B7/beta2-microglobulin DNA-liposome complex, in pa-
tients with metastatic melanoma. Clin. Cancer Res. 7, 2285–
2291.
(14) Edelstein, M. L., Abedi, M. R., and Wixon, J. (2007) Gene
therapy clinical trials worldwide to 2007--an update. J. Gene
Med. 9, 833–842.
(15) Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan,
H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen,
M. (1987) Lipofection: a highly efficient, lipid-mediated DNA-
transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84, 7413–
7417.
(16) Felgner, J. H., Kumar, R., Sridhar, C. N., Wheeler, C. J., Tsai,
Y. J., Border, R., Ramsey, P., Martin, M., and Felgner, P. L.
(1994) Enhanced gene delivery and mechanism studies with a
novel series of cationic lipid formulations. J. Biol. Chem. 269,
2550–2561.
(25) Haas, M., Audouy, S., Muizebelt, I., Smisterova, J., Engberts,
J. B., Hoekstra, D., Storm, G., and Hulst, R. (2005) In-vivo
delivery of DNA and protein using conceptually new cationic,
sunfish’, amphiphiles. J. Controlled Release 101, 401.
(26) Kim, H. S., Moon, J., Kim, K. S., Choi, M. M., Lee, J. E.,
Heo, Y., Cho, D. H., Jang, D. O., and Park, Y. S. (2004) Gene-
transferring efficiencies of novel diamino cationic lipids with
varied hydrocarbon chains. Bioconjugate Chem. 15, 1095–1101.
(27) Karmali, P. P., Kumar, V. V., and Chaudhuri, A. (2004)
Design, syntheses and in vitro gene delivery efficacies of novel
mono-, di- and trilysinated cationic lipids: a structure-activity
investigation. J. Med. Chem. 47, 2123–2132.
(28) Ewert, K. K., Evans, H. M., Zidovska, A., Bouxsein, N. F.,
Ahmad, A., and Safinya, C. R. (2006) A columnar phase of
dendritic lipid-based cationic liposome-DNA complexes for gene
delivery: hexagonally ordered cylindrical micelles embedded in
a DNA honeycomb lattice. J. Am. Chem. Soc. 128, 3998–4006.
(29) Eisner, U., Polonsky, M. J., and Lederer, E. (1955) Bull. Soc.
Chem. Fr. 212–218.
(30) Quiagen Plasmid Purification handbook. www1.qiagen.com/
Plasmid/handbooks.aspx.
(31) Rosenberg, I. M. (1996) Protein Analysis and Prurification:
¨
Benchtop techniques, Birkenhauser, Boston.
(32) Mosmann, T. (1983) Rapid colorimetric assay for cellular
growth and survival: application to proliferation and cytotoxicity
assays. J. Immunol. Methods 65, 55–63.
(33) Schmidt, R. R., and Jankowsky, K. (1996) Newtypes of
nonionic surfactants with sugar head groups. Liebigs Ann. 6, 867–
879.
(34) Breslow, D. S., Baumgarten, E., and Hauser, C. R. (1944) A
new synthesis of ꢀ-Ketoesters of the type RCOCH2COOC2H5.
J. Am. Chem. Soc. 66, 1286.
(35) Mukherjee, K., Sen, J., and Chaudhuri, A. (2005) Common
co-lipids, in synergy, impart high gene transfer properties to
transfection-incompetent cationic lipids. FEBS Lett. 579, 1291–
1300.
(36) Ma, B., Zhang, S., Jiang, H., Zhao, B., and Lv, H. (2007)
Lipoplex morphologies and their influences on transfection
efficiency in gene delivery. J. Controlled Release 123, 184–194.
(37) Patil, S. D., Rhodes, D. G., and Burgess, D. J. (2005) DNA-
based therapeutics and DNA delivery systems: a comprehensive
review. Aaps J 7, E61-77.
(38) Koltover, I., Salditt, T., Radler, J. O., and Safinya, C. R. (1998)
An inverted hexagonal phase of cationic liposome-DNA com-
plexes related to DNA release and delivery. Science 281, 78–
81.
(39) Simberg, D., Danino, D., Talmon, Y., Minsky, A., Ferrari,
M. E., Wheeler, C. J., and Barenholz, Y. (2001) Phase behavior,
DNA ordering, and size instability of cationic lipoplexes.
Relevance to optimal transfection activity. J. Biol. Chem. 276,
47453–47459.
(40) Zuhorn, I. S., Oberle, V., Visser, W. H., Engberts, J. B.,
Bakowsky, U., Polushkin, E., and Hoekstra, D. (2002) Phase
behavior of cationic amphiphiles and their mixtures with helper
lipid influences lipoplex shape, DNA translocation, and trans-
fection efficiency. Biophys. J. 83, 2096–2108.
(17) Lasic, D. D. (1997) Liposomes in Gene DeliVery, CRC Press,
New York.
(18) Leventis, R., and Silvius, J. R. (1990) Interactions of mam-
malian cells with lipid dispersions containing novel metabolizable
cationic amphiphiles. Biochim. Biophys. Acta 1023, 124–132.
(19) van der Woude, I., Wagenaar, A., Meekel, A. A., ter Beest,
M. B., Ruiters, M. H., Engberts, J. B., and Hoekstra, D. (1997)
Novel pyridinium surfactants for efficient, nontoxic in vitro gene
delivery. Proc. Natl. Acad. Sci. U.S.A. 94, 1160–1165.
(20) Solodin, I., Brown, C. S., Bruno, M. S., Chow, C. Y., Jang,
E. H., Debs, R. J., and Heath, T. D. (1995) A novel series of
amphiphilic imidazolinium compounds for in vitro and in vivo
gene delivery. Biochemistry 34, 13537–13544.
(21) Bajaj, A., Kondiah, P., and Bhattacharya, S. (2007) Design,
synthesis, and in vitro gene delivery efficacies of novel cholesterol-
based gemini cationic lipids and their serum compatibility: a
structure-activity investigation. J. Med. Chem. 50, 2432–2442.
(22) Heyes, J. A., Niculescu-Duvaz, D., Cooper, R. G., and
Springer, C. J. (2002) Synthesis of novel cationic lipids: effect
of structural modification on the efficiency of gene transfer.
J. Med. Chem. 45, 99–114.
(41) Zuhorn, I. S., Engberts, J. B., and Hoekstra, D. (2007) Gene
delivery by cationic lipid vectors: overcoming cellular barriers.
Eur. Biophys. J. 36, 349–362.
(42) Zuhorn, I. S., Bakowsky, U., Polushkin, E., Visser, W. H.,
Stuart, M. C., Engberts, J. B., and Hoekstra, D. (2005) Nonbilayer
phase of lipoplex-membrane mixture determines endosomal
escape of genetic cargo and transfection efficiency. Mol. Ther.
11, 801–810.
(23) Sen, J., and Chaudhuri, A. (2005) Design, syntheses, and
transfection biology of novel non-cholesterol-based guanidiny-
lated cationic lipids. J. Med. Chem. 48, 812–820.
(24) Choi, J. S., Lee, E. J., Jang, H. S., and Park, J. S. (2001)
New cationic liposomes for gene transfer into mammalian cells
with high efficiency and low toxicity. Bioconjugate Chem. 12,
108–113.
(43) Simberg, D., Weisman, S., Talmon, Y., and Barenholz, Y.
(2004) DOTAP (and other cationic lipids): chemistry, biophysics,
and transfection. Crit. ReV. Ther. Drug Carrier Syst. 21, 257–
317.
(44) Kayser, O., and Kiderlen, A. F. (2004) Non Viral Gene
DeliVery Systems. In Pharmaceutical Biotechnology, Kayser and
Mu¨ller, Eds. WileyVCH, Weinheim.

708 Bioconjugate Chem., Vol. 21, No. 4, 2010
Heinze et al.
(45) Li, W., and Szoka, F. C., Jr. (2007) Lipid-based nanoparticles
for nucleic acid delivery. Pharm. Res. 24, 438–449.
(46) Schaller, J., Gerber, S., Kaempfer, U., Lejon, S., and Trachsel,
C. (2008) Human Blood Plasma Proteins, John Wiley & Sons
Ltd., Chichester-West Sussex.
(47) Gao, X., Kim, K. S., and Liu, D. (2007) Nonviral gene
delivery: what we know and what is next. Aaps J. 9, E92-
104.
(54) Oja, C. D., Semple, S. C., Chonn, A., and Cullis, P. R. (1996)
Influence of dose on liposome clearance: critical role of blood
proteins. Biochim. Biophys. Acta 1281, 31–37.
(55) Hain, I., and Pepen, G. (1990) Phospholipids: Biochemical,
Pharmaceutical and Analytical Conciderations, Plenum Press,
New York.
(56) Matsuura, M., Yamazaki, Y., Sugiyama, M., Kondo, M., Ori,
H., Nango, M., and Oku, N. (2003) Polycation liposome-
mediated gene transfer in vivo. Biochim. Biophys. Acta 1612,
136–143.
(48) Liu, D., Ren, T., and Gao, X. (2003) Cationic transfection
lipids. Curr. Med. Chem. 10, 1307–1315.
(57) Song, Y. K., Liu, F., Chu, S., and Liu, D. (1997) Characteriza-
tion of cationic liposome-mediated gene transfer in vivo by
intravenous administration. Hum. Gene Ther. 8, 1585–1594.
(58) Li, S., Tseng, W. C., Stolz, D. B., Wu, S. P., Watkins, S. C.,
and Huang, L. (1999) Dynamic changes in the characteristics of
cationic lipidic vectors after exposure to mouse serum: implica-
tions for intravenous lipofection. Gene Ther. 6, 585–594.
(59) Ambegia, E., Ansell, S., Cullis, P., Heyes, J., Palmer, L., and
MacLachlan, I. (2005) Stabilized plasmid-lipid particles contain-
ing PEG-diacylglycerols exhibit extended circulation lifetimes
and tumor selective gene expression. Biochim. Biophys. Acta
1669, 155–163.
(49) Spector, M. S., and Schnur, J. M. (1997) DNA ordering on a
lipid membrane. Science 275, 791–792.
(50) Zuidam, N. J., Barenholz, Y., and Minsky, A. (1999) Chiral
DNA packaging in DNA-cationic liposome assemblies. FEBS
Lett. 457, 419–422.
(51) Xu, Y., and Szoka, F. C., Jr. (1996) Mechanism of DNA
release from cationic liposome/DNA complexes used in cell
transfection. Biochemistry 35, 5616–5623.
(52) Farhood, H., Serbina, N., and Huang, L. (1995) The role of
dioleoyl phosphatidylethanolamine in cationic liposome mediated
gene transfer. Biochim. Biophys. Acta 1235, 289–295.
(53) Mintzer, M. A., and Simanek, E. E. (2009) Nonviral vectors
for gene delivery. Chem. ReV. 109, 259–302.
BC9004624